Marine Products for Healthcare: Functional and Bioactive Nutraceutical Compounds from the Ocean

Marine Products for Healthcare Functional and Bioactive Nutraceutical Compounds from the Ocean

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FUNCTIONAL FOODS AND NUTRACEUTICALS SERIES Series Editor

G. Mazza, Ph.D. Senior Research Scientist and Head Food Research Program Pacific Agri-Food Research Centre Agriculture and Agri-Food Canada Summerland, British Columbia

Marine Products for Healthcare: Functional and Bioactive Nutraceutical Compounds from the Ocean

(2009)

Vazhiyil Venugopal, Ph.D.

Methods of Analysis for Functional Foods and Nutraceuticals, Second Edition

(2008)

Edited by W. Jeffrey Hurst, Ph.D.

Handbook of Fermented Functional Foods, Second Edition

(2008)

Edited by Edward R. Farnworth, Ph.D.

Functional Food Carbohydrates

(2007)

Costas G. Biliaderis, Ph.D. and Marta S. Izydorczyk, Ph.D.

Functional Food Ingredients and Nutraceuticals: Processing Technologies

(2007)

John Shi, Ph.D.

Dictionary of Nutraceuticals and Functional Foods

(2006)

N. A. Michael Eskin, Ph.D. and Snait Tamir, Ph.D.

Handbook of Functional Lipids

(2006)

Edited by Casimir C. Akoh, Ph.D.

Handbook of Functional Dairy Products

(2004)

Edited by Collete Short and John O’Brien

Herbs, Botanicals, and Teas

(2002)

Edited by G. Mazza, Ph.D. and B.D. Oomah, Ph.D.

Functional Foods: Biochemical and Processing Aspects Volume 2

(2002)

Edited by John Shi, Ph.D., G. Mazza, Ph.D., and Marc Le Maguer, Ph.D.

Functional Foods: Biochemical and Processing Aspects Volume 1

(1998)

Edited by G. Mazza, Ph.D.

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Marine Products for Healthcare Functional and Bioactive Nutraceutical Compounds from the Ocean

Vazhiyil Venugopal

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2009 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-13: 978-1-4200-5263-3 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Venugopal, V. (Vazhiyil), 1942Marine products for healthcare : functional and bioactive nutraceutical compounds from the ocean / author, Vazhiyil Venugopal. p. ; cm. -- (Functional foods and nutraceuticals series) “A CRC title.” Includes bibliographical references and index. ISBN 978-1-4200-5263-3 (alk. paper) 1. Seafood--Health aspects. 2. Functional foods. 3. Marine pharmacology. I. Title. II. Series: Functional foods & nutraceuticals series. [DNLM: 1. Seafood--analysis. 2. Dietary Supplements. 3. Food, Fortified. 4. Nutritive Value. 5. Seaweed--chemistry. WB 426 V458m 2009] QP144.F56V46 2009 615’.3--dc22

2008036599

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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Contents Foreword ................................................................................................................xvii Preface ....................................................................................................................xix Author .....................................................................................................................xxi Chapter 1

Functional Foods: An Overview ..........................................................1

1.1 1.2 1.3 1.4

Introduction .......................................................................................................1 Functionality of Food and Food Components ..................................................2 Role of Diet in Health Protection ......................................................................2 Nutraceuticals....................................................................................................4 1.4.1 Definition................................................................................................4 1.4.2 Factors Influencing Consumer Selection of Nutraceuticals and Food ....................................................................5 1.5 Functional Food ................................................................................................6 1.5.1 Definition................................................................................................6 1.5.2 Classification of Functional Foods .........................................................6 1.5.3 Recent Developments .............................................................................7 1.5.4 Consumer Surveys on Functional Foods................................................9 1.5.5 Data Required for Design of Functional Foods ................................... 10 1.5.5.1 Food Consumption Pattern..................................................... 10 1.5.5.2 Food Composition Database .................................................. 10 1.5.5.3 Bioavailability of Nutrients .................................................... 11 1.5.5.4 Reference Standards for Nutrients Intake and International Recommendations ..................................... 12 1.5.5.5 Safety and Regulation of Functional Foods ........................... 12 1.5.5.6 Marketing and Trade of Functional Foods ............................. 14 1.6 Marine Products as Functional Food: An Overview ...................................... 16 References ................................................................................................................ 19 Chapter 2

Marine Habitat and Resources ........................................................... 23

2.1 Introduction ..................................................................................................... 23 2.2 Marine Environment ....................................................................................... 23 2.3 Marine Fishery Products .................................................................................25 2.3.1 Landing ................................................................................................26 2.3.2 Demand and Concerns ......................................................................... 27 2.3.3 Underutilized Fisheries ........................................................................ 29 2.3.4 Some Novel Species ............................................................................. 29 2.3.4.1 Antarctic Krill and Other Deep-Sea Fauna ........................... 29 2.3.4.2 Sea Cucumbers ....................................................................... 30 2.3.4.3 Newer Species ........................................................................ 31 v

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2.3.5 Mariculture ........................................................................................... 31 2.3.6 Contribution of Seafood to Food Security ........................................... 32 2.3.7 Global Consumption Pattern of Seafood.............................................. 33 2.3.8 Changing Trends in Consumption........................................................34 2.4 Coral Reef and Corals ..................................................................................... 39 2.4.1 Symbiotic Associations on the Reefs ...................................................40 2.4.2 Reef-Associated Fisheries ....................................................................40 2.4.3 Bleaching and Other Problems with Coral Reefs ................................ 41 2.4.4 Efforts for Restoration of Corals .......................................................... 42 2.5 Seaweed........................................................................................................... 42 2.6 Microalgae ...................................................................................................... 43 2.7 Marine Bacteria .............................................................................................. 45 References ................................................................................................................46

Chapter 3 3.1 3.2 3.3 3.4

3.5

3.6 3.7

3.8

3.9 3.10 3.11

Seafood Proteins: Functional Properties and Protein Supplements .................................................................... 51

Introduction ..................................................................................................... 51 Seafood Proteins as Dietary Component ........................................................ 51 Protein Content of Raw Fish Muscle .............................................................. 52 Functional Properties of Proteins ................................................................... 54 3.4.1 Definition.............................................................................................. 54 3.4.2 Solubility .............................................................................................. 55 3.4.3 Emulsifying Capacity ........................................................................... 55 3.4.4 Foaming Capacity ................................................................................ 56 3.4.5 Gelation ................................................................................................ 56 3.4.5.1 Rheological Properties of Gel ................................................ 57 Physical Functions of Proteins in Food........................................................... 58 3.5.1 Modification of Functional Properties of Proteins............................... 59 3.5.1.1 Chemical Modifications .........................................................60 3.5.1.2 Enzymatic Modifications .......................................................60 Functionality of Seafood Proteins .................................................................. 61 3.6.1 Postharvest Changes in Functional Properties ..................................... 62 Functionally Active Marine Protein Supplements .......................................... 63 3.7.1 Fish Meat Mince and Mince-Based Products ...................................... 63 3.7.1.1 Surimi and Surimi-Based Products ....................................... 65 Fish Protein Powders....................................................................................... 70 3.8.1 Thermostable Protein Dispersions and Powders .................................. 71 3.8.2 Other Protein Supplements .................................................................. 75 3.8.2.1 Protein from Krill .................................................................. 75 3.8.2.2 Squid Proteins ........................................................................ 75 3.8.2.3 Blood Proteins ........................................................................ 75 3.8.3 Fish Protein Hydrolyzates .................................................................... 76 Fermented Fish Products.................................................................................80 Animal Feed.................................................................................................... 81 Marine Connective Tissue Proteins ................................................................ 83

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3.11.1 Collagen................................................................................................ 83 3.11.2 Gelatin .................................................................................................. 85 3.11.2.1 Extraction of Gelatin from Marine Sources........................... 86 3.11.2.2 Gelation Characteristics and Other Properties ...................... 88 3.11.2.3 Applications............................................................................ 91 3.12 Some Seafood Products of Consumer Interest................................................92 3.12.1 Food Flavorings....................................................................................92 3.12.2 Sea Cucumber ......................................................................................92 3.12.3 Product from Jellyfish ..........................................................................92 3.12.4 Roe from Eggs and Its Powder ............................................................. 93 3.12.5 Commercial Aspects ............................................................................ 93 References ................................................................................................................94

Chapter 4

Seafood Proteins: Nutritional Value, Bioactive Peptides, Marine and Cold-Adapted Enzymes ................................ 103

4.1 Introduction ................................................................................................... 103 4.2 Dietary Protein Requirements ...................................................................... 103 4.3 Nutritive Value of Proteins............................................................................ 104 4.3.1 Methods for Evaluation of Nutritional Quality of Proteins ............... 104 4.4 Nutritive Value of Seafood Proteins ............................................................. 106 4.4.1 Influence of Processing on Nutritive Value........................................ 107 4.5 Nutritive Value of Marine Protein Supplements ........................................... 109 4.5.1 Seafood Protein Powders ................................................................... 110 4.5.2 Nutritive Value of Fish Protein Hydrolyzates .................................... 111 4.5.3 Fermented Fishery Products .............................................................. 114 4.6 Bioactive Peptides ......................................................................................... 116 4.7 Bioactive Peptides from Seafood .................................................................. 117 4.7.1 Isolation of Seafood Peptides ............................................................. 117 4.7.2 Functional Roles of Marine Peptides in Foods .................................. 119 4.7.2.1 Calcium-Binding Activity .................................................... 120 4.7.2.2 Obesity Control .................................................................... 120 4.7.2.3 Antibacterial Activity ........................................................... 120 4.7.2.4 Antioxidant Activity ............................................................. 121 4.7.2.5 Angiotensin I-Converting Enzyme Inhibitory (Antihypertensive) Activity .................................................. 122 4.7.2.6 Immunostimulant Activity ................................................... 124 4.7.2.7 Human Immunodeficiency Virus-I Protease Inhibiting Activity ................................................................ 125 4.7.2.8 Antithrombin ........................................................................ 125 4.7.2.9 Calcitonin ............................................................................. 125 4.7.2.10 Miscellaneous Physiological Functions of Marine Proteins................................................................ 125 4.8 Marine Enzymes ........................................................................................... 127 4.8.1 Isolation .............................................................................................. 127 4.8.2 Applications........................................................................................ 129

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4.9 Antifreeze Proteins ....................................................................................... 130 4.9.1 Applications of Antifreeze Proteins ................................................... 131 4.10 Cold-Adapted Enzymes ................................................................................ 131 4.10.1 Applications of Cold-Adapted Enzymes ............................................ 132 4.11 Commercial Status ........................................................................................ 133 References .............................................................................................................. 135 Chapter 5

Polyunsaturated Fatty Acids and Their Therapeutic Functions ....... 143

5.1 Introduction ................................................................................................... 143 5.2 Marine Lipids ................................................................................................ 144 5.2.1 Fatty Acids ......................................................................................... 145 5.2.2 Lipid Profile of Seafood ..................................................................... 146 5.3 Oxidation of Fatty Acids ............................................................................... 150 5.3.1 Antioxidants ....................................................................................... 152 5.3.2 Role of Antioxidants in Health Protection ......................................... 152 5.3.3 Lipid Oxidation in Marine Fishery Products ..................................... 154 5.4 Nutritional Value of Lipids ........................................................................... 155 5.4.1 Health Benefits of Omega-3 Fatty Acids ............................................ 155 5.4.1.1 Cellular Processes ................................................................ 156 5.4.1.2 Blood Pressure...................................................................... 156 5.4.1.3 Cardiovascular Disease ........................................................ 156 5.4.1.4 Cancer .................................................................................. 158 5.4.1.5 Pregnancy and Infancy ......................................................... 158 5.4.1.6 Obesity ................................................................................. 160 5.4.1.7 Asthma ................................................................................. 160 5.4.1.8 Behavioral Pattern ................................................................ 160 5.4.1.9 Diabetes ................................................................................ 161 5.4.1.10 Bone Health .......................................................................... 161 5.4.1.11 Other Benefits ....................................................................... 162 5.4.2 Mode of Action................................................................................... 163 5.4.3 Indication............................................................................................ 165 5.4.4 Some Current Intake Levels of Omega-3 PUFA ................................ 165 5.4.5 Recommended Consumption Levels of Omega-3 PUFA ................... 166 5.5 Omega-3 PUFA-Rich Oils from Marine Fish ............................................... 168 5.5.1 Extraction ........................................................................................... 168 5.5.2 Properties of Fish Oils........................................................................ 170 5.5.3 Other Sources of Omega-3 PUFA ...................................................... 173 5.6 Squalene ........................................................................................................ 174 5.6.1 Functionality of Squalene .................................................................. 174 5.7 Commercial Aspects ..................................................................................... 175 References .............................................................................................................. 178 Chapter 6

Seafood Processing Wastes: Chitin, Chitosan, and Other Compounds ..................................................................... 185

6.1 Introduction ................................................................................................... 185 6.2 Major Compounds from Shellfish Processing Wastes .................................. 185

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6.3 Chitin............................................................................................................. 186 6.3.1 Isolation of Chitin............................................................................... 187 6.3.2 Structure ............................................................................................. 190 6.3.3 Properties ........................................................................................... 190 6.3.4 Applications........................................................................................ 191 6.4 Chitosan ........................................................................................................ 192 6.4.1 Structure ............................................................................................. 192 6.4.2 Properties of Chitosan ........................................................................ 193 6.4.3 Applications........................................................................................ 194 6.4.3.1 Food ...................................................................................... 196 6.4.3.2 Antimicrobial Activity ......................................................... 197 6.4.3.3 Antioxidant Activity ............................................................. 198 6.4.3.4 Edible Films ......................................................................... 199 6.4.3.5 Role in Nutrition ...................................................................200 6.4.3.6 Medical Applications ...........................................................202 6.4.3.7 Biotechnology.......................................................................203 6.4.3.8 Water Treatment ...................................................................203 6.4.3.9 Hydrogel ...............................................................................203 6.4.3.10 Catalytic Support and Packaging .........................................204 6.4.3.11 Other Applications ...............................................................204 6.4.4 Chitin Oligosaccharides .....................................................................204 6.5 Enzymes Degrading Chitin and Chitosan ....................................................207 6.5.1 Chitinases ...........................................................................................207 6.5.2 Chitosanases .......................................................................................207 6.5.3 Safety and Regulatory Status .............................................................208 6.6 Glucosamine .................................................................................................209 6.7 Shark Cartilage and Chondroitin Sulfate ......................................................209 6.7.1 Applications of Glucosamine and Chondroitin Sulfate ..................................................................... 211 6.8 Commercial Products.................................................................................... 213 References .............................................................................................................. 214 Chapter 7 7.1 7.2 7.3 7.4

7.5

7.6 7.7

Carotenoids....................................................................................... 221

Introduction ................................................................................................... 221 General Properties ........................................................................................ 221 Units and Requirements ................................................................................ 222 Marine Sources of Carotenoids .................................................................... 223 7.4.1 Algal Sources ..................................................................................... 223 7.4.2 Marine Fishery Sources ..................................................................... 223 Isolation and Characterization ...................................................................... 223 7.5.1 Algal Sources ..................................................................................... 223 7.5.2 Fishery Sources ..................................................................................224 Bioavailability of Carotenoids ...................................................................... 227 Functional Roles of Carotenoids ................................................................... 228 7.7.1 Antioxidant Activity ........................................................................... 228

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7.7.1.1 Assay of Antioxidant Activity .............................................. 229 7.7.1.2 Antioxidant Activities of Carotenoids Containing Marine Products ................................................................... 229 7.7.2 Vitamin A Activity ............................................................................. 230 7.8 Benefits of Dietary Carotenoids .................................................................... 231 7.8.1 Hypercholesterolemic Activity ........................................................... 231 7.8.2 Anticancer and Other Activities ......................................................... 232 7.8.3 Functions of Carotenoids in Aquaculture .......................................... 232 7.8.4 Poultry Feed ....................................................................................... 233 7.8.5 Biotechnology..................................................................................... 234 7.9 Commercial Status ........................................................................................ 234 References .............................................................................................................. 235 Chapter 8

Marine Sources of Vitamins and Minerals ...................................... 239

8.1 Introduction ................................................................................................... 239 8.2 Vitamins ........................................................................................................ 239 8.2.1 Vitamin Contents of Seafood ............................................................. 241 8.2.2 Vitamins in Seaweeds ........................................................................ 243 8.2.3 Influence of Processing on Vitamins ................................................. 243 8.3 Minerals ........................................................................................................ 243 8.3.1 Mineral Contents of Seafood ............................................................. 247 8.3.1.1 Fish Bone as a Source of Minerals....................................... 249 8.3.1.2 Calcium from Fish Bone ...................................................... 250 8.3.2 Minerals from Seaweeds .................................................................... 251 8.3.3 Bioavailability of Minerals................................................................. 253 8.4 Bone Health in Human..................................................................................254 8.4.1 Functional Role of Fish Bone Components in Bone Health .............. 255 8.5 Commercial Products.................................................................................... 255 References .............................................................................................................. 256 Chapter 9

Seaweed: Nutritional Value, Bioactive Properties, and Uses ........... 261

9.1 9.2 9.3 9.4

Introduction ................................................................................................... 261 Processing of Seaweed .................................................................................. 261 Identification of Seaweed .............................................................................. 262 Proximate Composition................................................................................. 262 9.4.1 Proteins and Amino Acids .................................................................264 9.4.2 Lipids ..................................................................................................266 9.4.3 Vitamins and Minerals .......................................................................266 9.4.4 Polysaccharides .................................................................................. 267 9.5 Dietary Fiber ................................................................................................. 268 9.5.1 Definition............................................................................................ 268 9.5.2 Health Benefits ................................................................................... 268 9.5.3 Fiber from Seaweed............................................................................ 271 9.5.4 Enrichment of Fiber in Foods with Seaweed ..................................... 272 9.5.5 Seaweed as Dietary Supplements ....................................................... 272

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9.5.6 Seaweed as Sources of Bioactive Compounds ................................... 273 9.5.6.1 Antioxidant Activity ............................................................. 273 9.5.6.2 Antibacterial and Antiviral Activities .................................. 276 9.5.6.3 Platelet Aggregation ............................................................. 277 9.5.6.4 Antitumor Activity ............................................................... 277 9.5.6.5 Hyperoxaluria ....................................................................... 278 9.5.6.6 HIV Inhibition ...................................................................... 279 9.5.6.7 Enzyme Inhibition ................................................................ 279 9.6 Industrial Uses of Seaweed ........................................................................... 281 9.6.1 Agriculture ......................................................................................... 281 9.6.2 Animal Feed ....................................................................................... 282 9.6.3 Feed for Aquaculture.......................................................................... 283 9.6.4 Antifouling Agents .............................................................................284 9.6.5 Biosorption of Heavy Metals ............................................................. 285 9.6.6 Other Miscellaneous Applications ..................................................... 285 9.7 Farming of Seaweed...................................................................................... 286 9.8 Commercial Products.................................................................................... 288 9.9 Regulatory Status .......................................................................................... 289 References .............................................................................................................. 289

Chapter 10 Seaweed Hydrocolloids ................................................................... 297 10.1 10.2 10.3

10.4

10.5

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Introduction ................................................................................................ 297 General Functional Properties of Seaweed Hydrocolloids......................... 297 Agar ............................................................................................................ 299 10.3.1 Source ............................................................................................. 299 10.3.2 Extraction ....................................................................................... 299 10.3.3 Composition ...................................................................................300 10.3.4 Properties .......................................................................................300 10.3.5 Uses ................................................................................................300 Alginate ...................................................................................................... 301 10.4.1 Source ............................................................................................. 301 10.4.2 Extraction ....................................................................................... 301 10.4.3 Composition and Structure.............................................................302 10.4.4 Properties .......................................................................................302 10.4.5 Uses of Alginates in Food, Medicine, and Biotechnology.............304 Carrageenan ................................................................................................308 10.5.1 Extraction and Characterization ....................................................308 10.5.2 Structure ......................................................................................... 310 10.5.3 Properties ....................................................................................... 311 10.5.4 Analysis .......................................................................................... 311 10.5.5 Gelation of Carrageenan ................................................................ 313 10.5.5.1 Rheological Properties................................................... 313 10.5.6 Applications of Carrageenans in Food Product Development ....... 316 10.5.6.1 Modification of Textural Properties............................... 317 10.5.6.2 Reduction of Fat ............................................................. 318

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10.5.6.3 Reduction of Salt ............................................................ 320 10.5.6.4 Flavor Perception ........................................................... 320 10.5.6.5 Fortification with Fiber .................................................. 320 10.5.6.6 Control of Browning ...................................................... 321 10.5.6.7 Cryoprotective Effect ..................................................... 321 10.5.6.8 Miscellaneous Applications ........................................... 321 10.5.7 Biological Activities of Carrageenan ............................................. 322 10.5.7.1 Antimicrobial Properties ............................................... 322 10.5.7.2 As Growth Factor Antagonist ........................................ 322 10.5.7.3 Antioxidant Activity ...................................................... 323 10.5.7.4 Suppression of Immune Response ................................. 323 10.5.7.5 Anticancer Activity ........................................................ 324 10.5.7.6 Inactivation of Paralytic Shellfish Poison ...................... 324 10.5.7.7 Elicitor of Plant Defense ................................................ 324 10.5.8 Biotechnology................................................................................. 324 10.5.8.1 Immobilization of Enzymes .......................................... 325 10.5.8.2 Enzyme Purification ...................................................... 325 10.5.9 Toxicology of Carrageenan ............................................................ 325 10.5.10 Degradation of Carrageenan .......................................................... 327 10.6 Fucoidan ..................................................................................................... 327 10.6.1 Biological Activities ....................................................................... 328 10.7 Laminarin ................................................................................................... 330 10.8 Commercial Status...................................................................................... 330 References .............................................................................................................. 331

Chapter 11 Marine Microalgae, Other Microorganisms, and Corals................ 339 11.1 11.2 11.3

11.4

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Introduction ................................................................................................ 339 Marine Microalgae ..................................................................................... 339 11.2.1 Nutritional Composition .................................................................340 Major Components from Microalgae ......................................................... 341 11.3.1 Lipids .............................................................................................. 341 11.3.2 Carotenoids and Other Pigments ................................................... 343 11.3.3 Sterols and Hydrocarbons .............................................................. 343 11.3.4 Polysaccharides ..............................................................................344 11.3.5 Vitamins .........................................................................................344 11.3.6 Single Cell Proteins ........................................................................344 Bioactive Compounds from Microalgae .....................................................344 11.4.1 Antiviral Compounds ..................................................................... 345 11.4.2 Anticancer Compounds ..................................................................346 11.4.3 Antioxidant Compounds ................................................................346 11.4.4 Antimicrobial Compounds ............................................................. 347 11.4.5 Antihypertensive Peptides .............................................................. 347 11.4.6 Other Bioactive Compounds .......................................................... 347

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11.5

Cultivation of Microalgae ........................................................................... 349 11.5.1 Cultivation of Microalgae for Lipids .............................................. 350 11.5.2 Cultivation for Carotenoids ............................................................ 351 11.6 Some Specific Examples of Algae .............................................................. 352 11.6.1 Chlorella ......................................................................................... 352 11.6.2 Spirulina ......................................................................................... 353 11.6.2.1 Nutritional Benefits ........................................................ 354 11.6.2.2 Biological Functions ...................................................... 355 11.6.3 Dunaliella ....................................................................................... 357 11.7 Microalgae as Feed for Aquaculture .......................................................... 358 11.8 Marine Bacteria .......................................................................................... 359 11.8.1 Marine Bacteria as Sources of PUFA ............................................360 11.8.2 Microbial Biotechnology................................................................ 361 11.9 Coral Reefs and Corals ............................................................................... 362 11.9.1 Biological Activity ......................................................................... 363 11.10 Commercial Status......................................................................................364 References .............................................................................................................. 365 Chapter 12 Drugs and Pharmaceuticals from Marine Sources ......................... 371 12.1 12.2

Introduction ................................................................................................ 371 Prospects of Finding Drugs from Marine Organisms ................................ 371 12.2.1 Marine Secondary Metabolites and Their Functions .................... 372 12.3 Some Major Marine Drugs ......................................................................... 373 12.3.1 Anticancer Agents .......................................................................... 375 12.3.2 Tuberculosis .................................................................................... 379 12.3.3 Malaria ........................................................................................... 379 12.3.4 Osteoporosis ................................................................................... 379 12.3.5 Arthritis .......................................................................................... 380 12.3.6 Antimicrobial and Antiviral Compounds ...................................... 380 12.3.7 Analgesic and Hypotensive Drugs ................................................. 382 12.4 Marine Products Having Potential Bioactive Compounds ......................... 382 12.4.1 Corals ............................................................................................. 382 12.4.2 Marine Microorganisms ................................................................. 387 12.4.3 Marine Plants ................................................................................. 388 12.4.4 Marine Toxins as Drugs ................................................................. 390 12.4.5 Fish and Shellfish ........................................................................... 391 12.4.5.1 Sea Cucumber ................................................................ 392 12.4.5.2 Jellyfish .......................................................................... 393 12.4.5.3 Bivalves .......................................................................... 393 12.5 Marine Biotechnology ................................................................................ 394 12.6 Development of Marine Drugs ................................................................... 394 12.6.1 Problems in Marine Drug Development ........................................ 396 12.7 Global Interests and Commercial Status .................................................... 398 References .............................................................................................................. 399

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Chapter 13 Marine Nutraceuticals for Food Fortification and Enrichment ......405 13.1 13.2 13.3 13.4

Introduction ................................................................................................405 Dietary Guidelines......................................................................................405 Supplementation .........................................................................................406 Food Fortification and Enrichment ............................................................407 13.4.1 Requirements for Fortification .......................................................408 13.5 Some Examples of Food Fortification ........................................................409 13.5.1 Iodine..............................................................................................409 13.5.2 Vitamins ......................................................................................... 410 13.5.3 Minerals ......................................................................................... 411 13.5.4 Carotenoids..................................................................................... 412 13.5.5 Proteins and Amino Acids ............................................................. 412 13.5.6 Probiotics ........................................................................................ 414 13.6 Marine Ingredients for Food Fortification and Supplementation ............... 415 13.6.1 Omega-3 Fatty Acids ...................................................................... 415 13.6.1.1 Marine Oil-Fortified Products ....................................... 415 13.6.1.2 Process Optimization ..................................................... 417 13.6.1.3 Therapeutic Benefits of PUFA-Fortified Products......... 418 13.6.1.4 Regulatory Status ........................................................... 419 13.6.1.5 Marketing Campaigns.................................................... 419 13.6.2 Marine Proteins .............................................................................. 420 13.6.3 Minerals ......................................................................................... 421 13.6.4 Glucosamine ................................................................................... 421 13.6.5 Chondroitin Sulfate ........................................................................ 422 13.7 Commercial Status...................................................................................... 422 References .............................................................................................................. 425 Chapter 14 Marine Macromolecules as Nutraceutical Carriers and Biofilms ..... 429 14.1 14.2 14.3 14.4

14.5 14.6

14.7

Introduction ................................................................................................ 429 Functions of a Delivery System .................................................................. 430 Matrix Design for Delivery of Nutraceuticals ............................................ 430 Encapsulation .............................................................................................. 431 14.4.1 Classification .................................................................................. 432 14.4.2 Techniques of Encapsulation .......................................................... 432 14.4.2.1 Spray Drying .................................................................. 432 14.4.2.2 Liposomes ...................................................................... 433 14.4.2.3 Microemulsion ............................................................... 433 Some Novel Delivery Systems.................................................................... 434 14.5.1 Marine Macromolecules as Delivery Systems ............................... 434 Encapsulation of Marine Ingredients ......................................................... 437 14.6.1 Polyunsaturated Fatty Acids .......................................................... 437 14.6.2 Glucosamine and Chondroitin Sulfate ........................................... 438 Biodegradable and Edible Films................................................................. 439 14.7.1 Edible Films ................................................................................... 441 14.7.1.1 Properties ........................................................................ 441

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14.8

Marine Macromolecules as Food Coatings and Edible Films ................... 442 14.8.1 Marine Proteins .............................................................................. 442 14.8.1.1 Collagen and Elastin ......................................................444 14.8.1.2 Gelatin............................................................................ 445 14.8.2 Marine Polysaccharides .................................................................446 14.8.2.1 Chitosan .........................................................................446 14.8.2.2 Carrageenan ...................................................................448 14.8.2.3 Alginate..........................................................................448 14.8.3 Multicomponent Films ...................................................................449 14.8.4 Active Packaging ............................................................................ 451 14.8.4.1 Marine Polysaccharides for Active Packaging .............. 451 14.8.4.2 Casting of Films ............................................................. 453 14.9 Nanotechnology .......................................................................................... 454 14.9.1 Nanotechnology for Marine Polysaccharide Films and Particles ......................................................................... 454 14.10 Hydrogels and Membranes for Therapeutic Applications .......................... 455 14.10.1 Marine Macromolecules as Hydrogels and Membranes for Drug Delivery ........................................................................... 455 14.10.2 Marine Polysaccharides as Scaffolds ............................................. 457 14.11 Commercial Status...................................................................................... 459 References .............................................................................................................. 461

Chapter 15 Safety Hazards with Marine Products and Their Control............... 467 15.1 15.2 15.3

15.4

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Introduction ................................................................................................ 467 Food-Borne Hazards................................................................................... 467 Types of Hazards of Marine Products ........................................................468 15.3.1 Microbiological Hazards ................................................................ 470 15.3.1.1 Bacterial Pathogens........................................................ 470 15.3.1.2 Histamine Poisoning ...................................................... 475 15.3.2 Insects............................................................................................. 476 15.3.3 Algal Toxins ................................................................................... 476 15.3.3.1 Paralytic Shellfish Poisoning ......................................... 479 15.3.3.2 Ciguatera Poisoning .......................................................480 15.3.3.3 Puffer Fish Poisoning.....................................................480 15.3.3.4 Diarrhetic Shellfish Poisoning .......................................480 15.3.3.5 Amnesic Shellfish Poisoning ......................................... 481 15.3.3.6 Other Biotoxins .............................................................. 481 15.3.3.7 Implications of Biotoxins ............................................... 481 15.3.4 Parasites.......................................................................................... 483 15.3.5 Fungi and Others ............................................................................484 15.3.6 Chemical Hazards ..........................................................................484 15.3.7 Seafood Allergy ............................................................................. 486 Control of Hazards...................................................................................... 487 15.4.1 Control of Biotoxins ....................................................................... 487 15.4.2 Removal of Allergens ..................................................................... 488

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15.4.3 Control of Parasites ........................................................................ 489 15.4.4 Control of Chemical Hazards......................................................... 489 15.4.5 Control of Microbiological Hazards .............................................. 490 15.4.5.1 Food Irradiation ............................................................. 490 15.4.6 Hazard Analysis Critical Control Point ......................................... 493 References .............................................................................................................. 494 Appendix ............................................................................................................... 501 A.1 Some International and National Organizations Related to Marine Products........................................................................................ 501 A.2 Dietary Components and Composition of Foods ..........................................502 A.3 Food Hazards and Safety ..............................................................................502 A.4 Trade Related ................................................................................................ 503 A.5 Fish Network ................................................................................................. 503 A.6 Books.............................................................................................................504 A.7 Fish Composition and Consumption Guidelines .......................................... 505 A.8 Microbiological Standards ............................................................................508 Index ...................................................................................................................... 511

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Foreword The concept that nature imparts a health-giving and curative function to foods is not new. Herbal teas and remedies have been used for centuries and continue to be used in many parts of the world even today. With the developments in nutrition and advances in analytical techniques at the molecular level, opportunities are available to understand the biochemical structure–function relationship of myriad chemicals that are present naturally in foods and their effects on the human body. The holistic approach to link medicine and diet that began in the 1970s has now seen a renewal as we realize that certain foods, because of the presence of specific biochemical ingredients in addition to their nutritive values, can have a positive impact on an individual’s health including his physical well-being and mental state. In fact, because of the negative image of drugs and the gray area of supplements, the use of foods that are “functional” is becoming a growth area for the modern food industry. At the same time, novel technologies, including biotechnology and genetic engineering, have created an era where scientific discoveries, product innovations, and mass production will be possible as never before. The main aim of this book is to offer a comprehensive review of developments in the area of marine nutraceutical products, their functional role in healthcare and uses in food fortification and active packaging. These aspects have been adequately covered by an amazing collection of information on various marine nutraceutical/ functional products from seafood, seaweed, microalgae, and corals. A few marine nutraceuticals and functional ingredients that have been highlighted include omega3 fatty acids as a healthy oil for cardiovascular disease; chitosan as a dietary fiber for weight management; shark cartilage, gluosamine, and chondroitin sulfate to fight rheumatoid arthritis; and alginate and carrageenan acting as biopolymers. The information provided should prove to be of value to the food and pharmaceutical industries in understanding the potentials of using marine nutraingredients as unique and novel specialty products, offering a sea of opportunities for healthcare. It is my hope that this book will serve not only to review the science and market base available for the development of marine nutraceutical/functional foods, but will also be a source to stimulate more research and committed development to this emerging field. This will certainly benefit those food scientists, nutritionists, and marketers who are involved in the design of novel products from marine nutraceuticals for healthcare that are acceptable to the consumer. Finally, this book will also help us understand how consumers’ views and legal concerns will impact the kinds of products that can be made, helping us take a proper stand on nutrafunctional foods. S. V. Padgaonkar SPICA-TECH Specialties (P) Ltd. Worli, Mumbai, India

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Preface The ocean is considered Mother Nature’s medicine cabinet. The power of the ocean to provide food and healthcare has been known from time immemorial. According to Hindu mythology, the gods, interested in eternal youth, churned the ocean with the help of demons to get nectar that could serve as the panacea for all diseases. Although for centuries the ocean has provided food and livelihood to millions across the world, in modern times, the sea is being recognized as a reservoir of potent molecules that are elicited by marine organisms that help them survive in the hostile oceanic environment. Many of these compounds have been recognized as useful for human healthcare. The search for these molecules is, however, still in its infancy, although some functional and bioactive nutraceuticals such as polyunsaturated fatty acids, chondroitin, carotenoids, polysaccharides, and some drugs have attracted attention. It is felt that in a couple of decades more health-protecting compounds will be originating from the sea. Furthermore, with the advancement in technology several drugs from marine products are being identified that can have a positive influence in controlling human ailments. Some of these compounds can also serve as models for drug development. In addition, several marine macromolecules such as alginate, chitosan, and carrageenans have been found useful for biopackaging and encapsulating drugs, and also as scaffolds for tissue engineering for the regeneration of skin, bones, etc. An exciting possibility in this respect is the use of marine macromolecules for encapsulation and delivery of nutraceuticals of marine origin itself, thereby providing opportunities for total utilization of marine products for healthcare. The commercial potentials of these developments are promising and open to the ingenuity of the scientist and entrepreneur. My previous book Seafood Processing: Adding Value through Quick Freezing, Retortable Packaging and Cook Chilling (CRC Press, 2006) discussed the current and upcoming technologies for processing marine products. The aim of this book is to bring awareness about the role of marine components in health promotion. This book attempts to consolidate recent data on the functional, nutraceutical, and therapeutic potentials of diverse marine resources, which include fishery products, seaweeds, microalgae, corals, marine microorganisms, and others. The introductory chapter discusses in general terms the characteristics of functional foods, with an overview on functionality of marine products. Since fishery products are the major sources of food, detailed discussions on the nutraceutical and other functional properties of their components including proteins, lipids, carotenoids, minerals, and shell waste products have been provided in Chapters 3 through 8. This is followed by Chapters 9 and 10 that discuss seaweeds, which are useful as food supplements, additives, and sources of bioactive compounds. Microalgae and corals are rich in nutrients, pigments, and therapeutic agents, as pointed out in Chapter 11. A number of secondary metabolites of corals, particularly sponges, have potential as lifesaving drugs. This aspect is discussed in Chapter 12. Optimal benefits of marine nutraceuticals could be derived through food fortification. Furthermore, the xix

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Preface

bioactive compounds also need to be properly delivered to their physiological sites of action for maximum advantages. Therefore, Chapters 13 and 14 discuss recent developments in the areas of food fortification, packaging, and delivery of bioactive components. Emphasis has been given to various components of marine origin both as fortificants as well as materials for biopackaging, edible films, and uses as drug delivery systems, tissue regeneration scaffolds, etc. Since some marine products pose safety hazards, it is pertinent to conclude the book with a discussion on these aspects, which is provided in Chapter 15. In preparing this book I have received support and encouragement from many of my esteemed colleagues and other experts in the field. The chapter on marine drugs would not have been possible without the valuable input and advice given by Professor Jack Wekell, PhD research chemist (retired, National Oceanic and Atmospheric Administration, United States). Dr. Rupsankar Chakrabarti, principal scientist, Mumbai Research Center of the Central Institute of Fisheries Technology, has painstakingly gone through some chapters of this book. I am thankful to Dr. K. Devadasan, director, Central Institute of Fisheries Technology, Cochin, India, and Dr. S. D. Tripathi, former director, Central Institute of Freshwater Aquaculture, Bhubaneswar, India, for their interest and support. Some eminent persons in the field have shared valuable information with me while preparing this book. They are Dr. Anthony Bimbo, International Fisheries Technology, Kilmarnock, Virginia; Dr. S. Subasinghe, editor, Infofish, Kuala Lumpur, Malaysia; Dr. Mohan Joseph; Dr. N. Rajagopalan, director and principal scientist, Central Marine Fisheries Research Institute, Cochin, India; and Dr. K. N. Ganeshiah, University of Agricultural Sciences, Bangalore, India. I also thank M. Vijaykumar, secretary, NAAS, New Delhi, India; D. P. Sen; and V. Muralidharan who have provided some data while preparing this book. I have also benefited from my association with the Seafood Discussion Group of the University of California, Davis, California and some expert members of the group sharing information on many recent developments in the field. Aquinova AG, Birkenweg, Germany, has kindly sent me a picture of a drug delivery system that has been included in Chapter 14. Rajeev and Srikant provided some computer support. My thanks are due to Stephen Zollo, Rachael Panthier, and the team at the Macmillan Publishing Solutions for their valuable editorial support. I would consider my objective fulfilled if this book draws the attention of students and professionals in the area to the numerous functional and therapeutic potentials of marine organisms. Suggestions from readers to improve the contents of this book and correct any inadvertent errors are welcome. V. Venugopal [email protected] [email protected]

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Author V. Venugopal received his MSc (chemistry) from the University of Kerala, India and PhD (biochemistry) from the University of Bombay, India. He began his career at the Central Institute of Fisheries Technology, Cochin, India, and later moved to the Bhabha Atomic Research Center, Mumbai, India, where he was the head of the Seafood Technology Section of the Food Technology Division. He has been a postdoctoral research fellow at the National Institutes of Health, Bethesda, Maryland and a visiting scientist at the Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. His main interests include value addition of fishery products, radiation processing of seafoods, and marine proteins. He has published more than 120 publications in these fields. He is a fellow of the National Academy of Agricultural Sciences, New Delhi, India. His previous book Seafood Processing: Adding Value through Quick Freezing, Retortable Packaging and Cook Chilling focused on novel and emerging technologies for the value addition of fishery products from marine, freshwater, and aquaculture sources. The book attracted excellent reviews in reputable journals in the field of food and seafood technology, hailing it as a source of state-of-the-art technology.

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Foods: 1 Functional An Overview 1.1

INTRODUCTION

The twentieth century has seen significant changes in life styles of world population, essentially due to rise in purchasing power together with increased leisure and reduced physical activity. The life style changes had specific impact on food consumption patterns of general public. There was a shift toward consumption of energy-dense foods with high levels of sugar and saturated fats, which during the course of time showed detrimental effects on health, indicated by rapid global rise in chronic diseases such as obesity, coronary heart disease, diabetes, hypertension, and rheumatoid arthritis.1,2 It has been calculated that, in 2001, these diseases contributed approximately 60% of the 56.5 million total reported deaths in the world.1 The rise in chronic diseases geared up the medical profession to look for new technologies for their diagnosis and cure. Parallel to these developments, there was also increasing general awareness on the protective role of diet to combat these diseases. The 400 BC tenet of Hypocrates, “Let food be thy medicine and medicine thy food,” started attracting increasing attention by the modern world. Educational campaigns undertaken on the importance of both macro and micronutrients in food made consumers recognize the importance of diet in maintenance of health, which also made them return to natural foods, particularly with the “back-to-nature” revolution of the 1960s. The elderly population throughout the world, in particular, has a cautious approach toward foods, preferring only those having low contents of saturated lipid, sugar, and sodium. In recent times, there is also a tendency among the public, in general, to regularly check the biomarker profiles (low-density lipoprotein, blood pressure, glucose tolerance, etc.) with a view to maintain them under acceptable level to reduce the risk of cardiovascular disease, stroke, and diabetes. As a consequence, there has also been an increasing demand for functional foods and food supplements. A new self-care paradigm that foods can provide health benefits and coexist with traditional medicines to disease treatment is getting its deserving attention.3,4 The change in consumer outlook has also resulted in a shift in the operation profiles of global food industry from traditional limited objectives of preservation, quality improvement, and value addition to a wider program of development of products that can protect consumer health. The industry is being called upon to develop specific fortified and dietetic foods (also called therapeutic diets) incorporating nutraceuticals to address nutritional needs of persons, whose normal processes of assimilation/metabolism is affected. Attempts in this direction involve translating scientific advances in the field of nutrition into development of functional foods that can address problems of nutrient deficiencies in conventional diets. Table 1.1 shows top health concerns influencing purchase of functional foods.5 1

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TABLE 1.1 Top Health Concerns Influencing Purchase of Functional Foods Weight reduction Reduction in cholesterol Promotion of healthy bones and teeth Energy Boost of immune system and disease resistance Promotion of healthier gut and digestion system Source: Adapted from Arvanitoyannis, I. S. et al., Crit. Rev. Food Sci Nutr., 45, 385, 2005. With permission from Taylor & Francis Ltd. (www.informaworld.com).

1.2 FUNCTIONALITY OF FOOD AND FOOD COMPONENTS For a food product to get acceptability, two important criteria need to be satisfied, namely, its sensory and nutritional properties. The functional value of any food, therefore, should be viewed from the point of view of both nutritional functionality and sensory functionality. The definition of functionality, therefore, differs for different professionals. From a nutritionist’s point of view, functional property is the presence of certain compounds in natural or processed foods that can provide health benefits beyond basic nutrition. However, the food technologist views functional property as any property of a food or food ingredient except its nutritional ones, which affects its use.6 In arriving at true functional value, a food needs to be evaluated in terms of both these viewpoints. Recent interest in diets that can protect health has resulted in foods that have functionality from the nutritional point of view. The notion of functionality is the main driving force behind the development of new food products that promote optimal health and relieve the risk of diseases. These foods serve to promote health or help to prevent disease, and in general, the term is used to indicate a food that contains some health-promoting components beyond the traditional nutrients.7 Key issues for the twenty-first century food processing industry have indicated that health and well-being will become major driving forces to enhance consumer value, and functional foods will be important in determining the foods of the future. In addition, food safety will continue to challenge the government and food industry and biotechnology will be important in determining the foods of the future.8

1.3 ROLE OF DIET IN HEALTH PROTECTION Historically, the importance of diet in maintenance of health has been known from time immemorial in some parts of the world. In Asia, the concept is rooted in a tradition, known as Ayurveda, “the Science of Life,” which had its origin some 5000 years ago. It recognized the importance of a balanced diet that could contain some herbal products for therapeutic effects. In recent times, the application of science in nutrition started during the later parts of the last century. The developments in this field could be considered to have taken place in three distinct stages. In the first stage, up to 1970, attention was primarily focused on the identification

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of relationship between deficiencies of certain nutrients such as vitamins and minerals on the physiological functions in the human body. In the second stage, from 1970 to 1990, nutritionists and public health authorities became aware of the need for appropriate intakes of nutrients to prevent deficiency signs, since their availability from food was not always sufficient for maintenance of optimal health. For example, intake of antioxidants including vitamin C was considered to lower the risk of cancers, while vitamin K was found to help bones retain calcium to control the onset of osteoporosis. Recognition of these beneficial effects led to the dissemination of dietary guidelines, aimed to reduce the frequency of chronic nutrition-related diseases such as obesity, cardiovascular disease, hypertension, type II diabetes, osteoporosis, and several forms of cancer. In the third stage, namely, after the 1990s, attention was paid to the presence of substances in the human diet that can exert beneficial effects in the body, which are different from the classical nutrients such as vitamins, minerals, and trace elements, which are collectively termed as nutraceuticals.9 The recent scientific advances have blurred the line of demarcation between food and medicine, as scientists identify bioactive food components that can reduce the risk of chronic disease, improve the quality of life, and promote proper growth and development.3 Since the types and nature of these bioactive compounds vary in different foods, consumers are more conscious of the importance of a balanced and varied diet. In a recent survey, it was observed that 82% of European and 75% of U.S. shoppers felt that “eating from a diverse range of foods” is “important” or “very important” in maintaining a healthy diet.10 For example, a diet rich in fruits and fresh vegetables is considered to result in lowered incidence of cancer and heart attacks. The World Health Organization (WHO) and the U.S. Surgeon General have recognized potential health-promoting properties of fruits and vegetables (FAV) and these organizations are encouraging consumption of fruits, vegetables, and nuts to prevent certain chronic diseases and promote better health. The benefits have been attributed to compounds called flavonoids. A newly updated, easy-to-use United States Department of Agriculture (USDA) database documents 26 key antioxidants including flavonoids in about 400 fruits, vegetables, and other foods.11 Apart from FAV, other food components from both plants and animals may participate in human health promotion. Phenolic and polyphenolic compounds also constitute an important class of secondary plant metabolites that act as free radical scavengers, inhibitors of lowdensity lipoprotein (LDL) cholesterol oxidation, and deoxyribonucleic acid (DNA) breakage. Their role in the prevention of cardiovascular disease and certain types of cancer is well recognized. Recognition of the involvement of food in health protection led to the concepts of “nutraceuticals” and “functional foods.” The term, “functional foods” was introduced in Japan in the mid-1980s. Till date, over 100 products are approved as functional foods in Japan alone. In China, there has been attempt to fuse a Chinese medicated diet into functional food.12 In the United States, the terms “functional foods” and “nutraceuticals” are used interchangeably. However, it is generally accepted that nutraceuticals refer to “chemicals” found as naturally occurring components of food that provide health benefits, whereas functional foods are “foods” or “food ingredients” that provide a health benefit beyond the traditional nutrients they contain.4 The fastest growing functional food

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in the United States is the herbal-based nutraceutical such as ginseng, garlic, and medical mushrooms.3 The characteristics of nutraceuticals and functional foods are discussed in detail in Sections 1.4 and 1.5, followed by a general discussion on the functionality of marine foods.

1.4

NUTRACEUTICALS

1.4.1

DEFINITION

Nutraceuticals are health-promoting compounds or products that have been isolated or purified from food sources. The term “nutraceutical” is often used to refer to a food, dietary supplement, or biologically active compound that provides health benefits. A nutraceutical is defined as any substance that may be considered as a food or part of a food and provides medical or health benefits including the prevention and treatment of disease. The term was coined in 1989 by the Foundation for Innovation in Medicine, New York. Nutraceuticals may range from isolated nutrients, dietary supplements, and diets to genetically engineered “designer” foods and herbal products. Examples are flavanoids isolated from soybean, fish oil capsules, herbal extracts, glucosamine, chondroitin sulfate, lutein-containing multivitamin tablets, and antihypertensive pills that contain fish protein-derived peptides. Table 1.2 gives some examples of nutraceuticals from different sources. These ingredients are not identified as essential nutrients, but are considered (proven or not proven) as bioactive substances with a health benefit. The ongoing research in this field is likely to result in new generation of foods that could possibly reduce the demarcation between food and drug. Public health authorities consider prevention and treatment with nutraceuticals as a powerful instrument in maintaining health and to act against

TABLE 1.2 Examples of Nutraceuticals from Different Food Products Muscle Foods Conjugated linoleic acid (CLA) Eicosapentaenoic acid (EPA) Decosahexaenoic acid (DHA) Choline Lecithin Calcium Ubiquinone Selenium Zinc

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Fermented Foods

Plant Foods

Sacharomyces boulardii Bifidobacterium bifidum B. longuin Lactobacillus acidophilus Streptococcus salivarius subspp. Thermophilus

Ascorbic acid Quercetin Lutein Gallic acid Allicin δ-Limonene Lycopene Capsaicin β-Ionone α-Tocopherol β-Carotene Zeaxanthin Isoflavanones

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TABLE 1.3 Some Health Claims by Major Food Compounds that Could Find Increased Presence in Health Foods and Beverages Calcium-rich foods reduce risk of osteoporosis Low-sodium foods reduce high blood pressure Folate-rich foods reduce risk of neural tube defects Slow-digesting carbohydrates help weight management Omega-3 fatty acids provide heart health and antiageing Probiotics offer immunity and digestive health Peptides provide heart health, immunity, and weight management Carotenoids offer overall antioxidant properties and antiaging and immunity High-fiber foods help better bowel movements, reduce risk of cancer Source: Adapted from Ohr, L. M., Food Technol., June 2007, www.ift.org. With permission.

nutritionally induced acute and chronic diseases, thereby promoting optimal health, longevity, and quality of life.13,14 Table 1.3 indicates some health claims made by major food compounds that could find increased presence in health foods and beverages.

1.4.2 FACTORS INFLUENCING CONSUMER SELECTION OF NUTRACEUTICALS AND FOOD Both taste and availability of nutraceuticals for specific health benefits to be accrued through consumption determine food selection by consumers. Taste has been viewed as a particularly important variable, since many nutraceutical compounds have natural bitter, astringent, or other off-flavors.15,16 A conjoint analytic study was conducted with military and civilian consumers to assess the importance of taste and other product characteristics in the intended use of nutraceutical products. The study clearly indicated that taste was the primary driving force in selection of food by both the consumer groups. Apart from taste, the nature of the health benefit of the product also influenced consumer purchase. Other factors determining food selection from a health point of view included the source of the benefit claim, frequency of consumption to obtain the benefit, required dosage, mode of consumption (food compared with tablet or capsule form), and product type (natural compared with synthetic). These considerations were irrespective of large differences in age, gender, and physical activity level between the two populations. Interest among the people working in armed forces was greatest for products that increased muscle mass, whereas interest among civilians was greatest for products that improved mental ability. Convenience, as reflected in the desire for a low frequency of consumption (once a day or less) and in a tablet or capsule form, was an important factor that determined interest in these products by both consumer groups. It was also recognized that the benefit claims substantiated by medical authorities was highly valued by the consumers, in comparison with claims made by the manufacturer.17 Table 1.4 gives factors influencing selection of neutraceuticals.

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TABLE 1.4 Factors Influencing Selection of Neutraceuticals Awareness of nutritional significance Cultural considerations Frequency of consumption to obtain the desired effect Health consciousness Medical authorization Mode of consumption Nature of product and its ingredient Sensory properties Source (synthetic or natural) Source: Adapted from Schaafsma, G., Dietary Fibre—Bioactive Carbohydrates for Food and Feed, Wageningen Academic Publisher, The Netherlands, 2004, p. 27.

1.5

FUNCTIONAL FOOD

1.5.1

DEFINITION

The term “functional food” was coined in Japan in the mid-1980s to describe processed foods that contain ingredients such as oligosaccharides, minerals, polyunsaturated fatty acids, and fibers that address diseases such as hypertension, in addition to being nutritious.18 A food can be regarded as functional if it is satisfactorily demonstrated to beneficially affect one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either improved health or well-being and to a reduction in the risk of disease. According to recent data, a functional food must remain food and it must demonstrate its effects in amounts that can normally be expected to be consumed in the diet. It is not a pill or a capsule, but part of the normal food pattern.19 The U.S. Institute of Food Technologists Expert Panel defines functional food as food and food components that provide a health benefit beyond basic nutrition (for the intended population). These foods provide essential nutrients often beyond qualities necessary for normal maintenance, growth, development and other biologically active compounds that impart health benefits or desirable physiological effects.20 The Institute of Medicine of the U.S. National Academy of Science has defined functional foods as foods to which “one or more ingredients have been manipulated or modified to enhance their contribution to a healthful diet.”21 From a European point of view, a food may be considered functional if it contains a component (be it nutrient or not) with a selective effect of one or various biological functions, whose positive effects justify that it can be regarded as functional (physiological) or even healthy.

1.5.2

CLASSIFICATION OF FUNCTIONAL FOODS

Functional foods have been variously termed as designed foods, medicinal foods, bioactive foods, therapeutic foods, vita foods, super-foods, foodceuticals, pharmafoods, medical foods, etc.4 In most cases, the term refers to a food that has been modified in some way to become more functional. Modifications can be achieved by incorporation of nutraceuticals such as phytochemicals, bioactive peptides, omega-3

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(n-3) fatty acids, and probiotics. Such foods need to fit into current life styles providing convenience, use, good taste, and acceptable price.22 The first generation of functional foods (primary functional foods), in fact, already existed before the concept of functional food was introduced. Products like skimmed and diet margarines are examples for such foods. Fortified foods (foods with added nutrients, see Chapter 13) also belong to this category of foods. These foods are marketed with nutritional claims (not health claims), such as “low in fat,” “rich in vitamin C,” or “rich in vitamin D to strengthen the bones.” The second generation of functional foods is specifically developed to reduce the risk of chronic nutrition-related diseases or to enhance certain body functions to promote well-being. Examples are applications of nutraceuticals as food ingredients, such as probiotics, phytoestrogens, phytosterols, bioactive proteins and peptides, conjugated linoleic acid isomers, and several long-chain polyunsaturated fatty acids. Health claims connected to secondary functional foods are type A health claims (enhancement of body function) or type B (disease risk reduction).9 According to their health benefits, functional foods have been divided into various categories. Dairy products dominate the area of functional foods for gut health. Common gut health products include fermented milk and yogurt drinks. The most common gut health ingredients include probiotics, prebiotics, and synbiotics. Probiotics are beneficial bacteria that help maintain the balance of beneficial and harmful bacteria in the gut, whereas prebiotics are natural food for probiotic bacteria, thus supporting their growth. Most functional foods that claim a bone health benefit are fortified with calcium. Calcium-enriched food products such as milk and fruit juices are commercially available. Sometimes, vitamin D, essential for the absorption of calcium is also added to these products. Heart health products focus on reducing the risk factors for cardiovascular disease and also elevated blood cholesterol levels and hypertension. Omega-3 fatty acids–enriched products have a particularly protective role in heart health. Functional foods that claim to enhance the immune system have mainly been fortified with vitamins or contain prebiotics. These foods have the ability to boost the immune system, while the antioxidant vitamins A, C, and E can increase the resistance of the body to infection. Many of these products also have an effect on heart or gut health.22 Dietary supplements in the form of powders, capsules, or tablets containing various probiotics are now available in health and natural food sections of supermarkets.23 Reasons for increasing popularity of functional foods include better understanding of the nutrition–health relationship, commercial availability of nutraceuticals, scientific validation of nutritional claims, lack of side effects during consumption, among others, as shown in Table 1.5.

1.5.3

RECENT DEVELOPMENTS

Recent developments in the area of food science and technology have specific aims at improving benefits of functional foods. Application of enzymes has resulted in fats with desired composition of fatty acids, and milk protein concentrate with reduced content of lactose. Progress in membrane technology has resulted in successful modification of food composition associated with altered functionality through separation of one or more solutes in a liquid medium. This technology, in particular ultrafiltration, has helped the dairy industry to separate and concentrate

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TABLE 1.5 Reasons for Increasing Popularity of Functional Foods Better understanding of the nutrition–health relationship Commercial availability of bioactive ingredients (nutraceuticals) Increasing purchasing power of the consumers Advances in food processing and food technology Need of the industry to market value-added products Individualization of society Less leisure to attend to individual food requirements Deregulation of nutrient fortification No negative side effects at normal use No disturbance of a “healthy” eating pattern Scientific substantiation of health claims Source: Adapted from Schaafsma, G., Dietary Fibre—Bioactive Carbohydrates for Food and Feed, Wageningen Academic Publisher, The Netherlands, 2004, p. 27.

milk components such as lactoferrin, without denaturing the proteins or bioactive substances. It can also help in separation of specific peptides from marine sources (see Chapter 4), demineralization by nanofiltration and electrodialysis and removal of antinutrients (compounds perceived as negative or nonhealthy) such as fats, cholesterol, and caffeine. Supercritical fluid extraction has allowed major breakthrough in processes such as removal of caffeine from coffee, alcohol from cider, and wine and fat from snack foods. In contrast to this technology, fortification of foods is practiced to enhance the contents of functionally active compounds in foods. Advances in biotechnology, microencapsulation, ingredient synthesis/ extraction/purification, nonthermal processing, and predictive modeling support the design of “healthy” foods.24 Multifunctional foods are also being designed by adding a few complementary ingredients such as antioxidants and vitamins C and E to foods.25 Although genetic engineering cannot be defined as a processing technique, the advances in this area help in developing specific functionalities to raw material, through alteration of the genes contained in certain cells. It helps to genetically engineer food materials with improved organoleptic properties, such as tomato with improved texture and lycopene content, corn with higher content of oleic acid, and white wines with enhanced concentration of resveratrol. The technique could also have potential to produce specific foods such a hypoallergenic foods in which a specific protein or peptide has been removed. Figure 1.1 gives technological factors influencing the functionality of functional foods. Despite increasing popularity, a number of problems have been recognized that influence the future developments in functional foods. First, the lack of validated biomarkers to substantiate health benefits of functional foods. Second, while the side effects of many drugs having strong bioactivity could be tolerated to some extent, such side effects are not acceptable in the case of functional foods. Third, a need exists for the development of new techniques to measure the biological effects, namely, bioefficacy and biosafety of consumption of functional foods on a short-term basis. At present such tests are not available, although developments in the field of nutrigenomics

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9

Daily dosage

Stability

Food matrix formulation

Stability

Viability Functionality

Aseptic packaging Fermentation technology

Encapsulation

Heating technology

Drying technology

FIGURE 1.1 Technological factors influencing the functionality of functional foods. (Reprinted from Arvanitoyannis, I. S. et al., Crit. Rev. Food Sci. Nutr., 45, 385, 2005. With permission from Taylor & Francis Ltd. (www.informaworld.com).

and bioinformatics could address these problems.9 Although there are clinically demonstrated uses for many dietary supplements, certain limitations also exist. For example, recent studies demonstrate that little overwhelming evidence exists to support the widespread use of antioxidants as functional food components. The antioxidant hypothesis, proposed nearly 50 years ago, was based on the assumption that these compounds have the capacity to limit the adverse effects of oxidative damage. However, issues relating to the types and quantities of antioxidant-rich foods that need to be consumed still remains under debate today. Nevertheless, it is generally agreed that antioxidant-rich foods are important to prevent age-related diseases.26

1.5.4

CONSUMER SURVEYS ON FUNCTIONAL FOODS

A recent survey conducted in 34 countries concluded that consumers were aware of the influence of diet in controlling chronic diseases and potential medicinal effects of bioactive compounds in foods. Consumers in 19 out of 34 countries also felt that food is less safe than what it was 10 years ago. Their major concerns were pesticide residues in food and contamination of drinking water generated the highest level of perceived risks, while mad cow disease and genetically modified foods generated the lowest levels of perceived risks. In addition, consumers also recognize problems with the current healthcare system, perceiving that it is often expensive and time consuming.27 Another recent global survey on the trend of the food industry showed the importance of consumer health in food product development. An overwhelming 76% of the professionals considered health and wellness as very important, while only 20% considered these as average importance. Nearly 47% respondents identified consumers as the most important driving force behind health and wellness market; but almost 44% acknowledged that substantial effect also came from the food industry. The survey also showed that a large group of consumers was actively looking for health foods. According to the consumers, the most important health and wellness foods included probiotics (20% respondents), low fat (19%), heart health (19%), and organic (18%) foods.5 According to American respondents, fiber and whole grain (37%) appear to be the best ingredients for health and wellness. Omega-3 fatty acids (25%) and probiotics

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TABLE 1.6 Consumer Preferences of Food Categories Related to Health and Wellness Probiotics (20%) Low fat (19%) Heart health (19%) Organic foods (18%) Fiber and whole grains (37%) Omega-3 fatty acids foods (25%) Probiotics (22%) Source:

Reprinted from Hansen, C., Health and Wellness Survey, Chr. Hansen (India) Pvt. Ltd., 2006. With permission.

(22%) were the second and third, respectively. An outlook for the year 2008 indicated respondents voting probiotics (31%) as their favorite ingredient with fiber, whole grain, omega-3 fatty acids, and antioxidants, all close for the second position. The survey confirmed consumer health as the single most important challenge faced by the food producer’s body in the development of new food products.28 More specifically, consumers will look to functional foods and beverages to aid in areas such as weight management, heart health, antiageing, immunity, and digestive health. Because of this, ingredients including omega-3 fatty acids, slow digesting carbohydrates, probiotics, peptides, and fiber will find increased presence in healthful foods and beverages among others.29 According to a 1997 survey on consumer attitudes toward medicinal foods, most generations saw a connection between food and medicine. A segment comprising 13% of people of age group 40–49 said that “they strongly felt that foods can be used to reduce their use of some drugs and medicinal therapies.” The survey indicated that overall fortified foods, medicinal foods, and nutraceutical foods will continue to enjoy exceptional market opportunities with rising consumer interests among consumers.30 Table 1.6 shows important foods related to health and wellness according to global survey.

1.5.5

DATA REQUIRED FOR DESIGN OF FUNCTIONAL FOODS

1.5.5.1

Food Consumption Pattern

Successful designing and development of functional foods require information on food consumption pattern and compositions of foods that are consumed. Food consumption patterns are dynamic and are influenced by complex, interrelated biological, social, cultural, and psychological processes. Some of the effects of societal changes associated with globalization: gender, work, and family roles; materialism; information technology; and increasing longevity have influenced food consumption trends.31 1.5.5.2 Food Composition Database A typical food composition database (FCD) provides values for the amount of energy, protein, fat, vitamins, minerals, and some other specific nutrients present

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in a food item. The values are normally determined by standard chemical analyses (e.g., “official methods”) or sometimes derived, in the case of complex foods, from the nutrient composition of ingredients. FCDs have been methodically compiled over the years in many countries and provide information about the nutrients contained in most consumed foods. There are over 150 food composition tables and electronic databases, which exist worldwide. The International Directory of Food Composition Tables lists FCDs from different regions and countries.32 FCDs are generally used to assess the nutrient content of diet and to derive nutrition guidelines. Food composition tables for most foods and nutrients give estimations of the mean concentration. However, there is a limited availability of data for some bioactive components that are known to play important physiological roles. The limitation of FCDs is that information contained in FCDs is related to the amount of nutrients present in foods before ingestion but gives no clue of the actual amount that becomes available for physiological activity after absorption in the gut. Information on all intrinsic, deliberately added, and incidental components, including environmental contaminants, additives, and bioactive nonnutrients is essential to design functional foods. Organizations such as Food and Agriculture Organization (FAO) and also USDA have separate databases for nutrients, additives, and contaminants. The Second International Total Diet Study Workshop held at Brisbane in 2001 brought toxicologists and food composition specialists to discuss their common areas of activity.33,34 1.5.5.3 Bioavailability of Nutrients Bioavailability is the proportion of an ingested nutrient that is made available (i.e., delivered to the blood stream) for its intended mode of action. Bioavailability is more relevant than the total amount present in the original food. Several factors influence bioavailability such as the chemical state of the nutrient, its release from the food matrix, possible interactions with other food components, presence of suppressors or cofactors, and formation of stable compounds that are slowly metabolized. The state of the matrix of natural foods or the microstructure of processed foods may favor or hinder the in vivo nutritional response of many nutrients. The U.S. Food and Drug Administration (FDA) has defined bioavailability as the rate and extent to which the active substances or therapeutic moieties contained in a drug are absorbed and become available at the site of action. This definition also applies to nutrients.35 The relevant in vivo and in vitro methods to assess bioavailability of some nutrients, types of microstructural changes imparted by processing and during food ingestion that are relevant in matrix–nutrient interactions, and their effects on the bioavailability of selected nutrients have been discussed recently.35 Bioavailability is important in designing functional foods. The presence of a good bioavailable nutrient ensures that the food will be efficacious for the indication specified. With the introduction of health claims (as discussed later), the onus will be on food manufacturers to provide scientific substantiation based not only on the literature related to an active nutrient, but also on intervention trials that demonstrate bioavailability and efficacy of the nutrient when delivered in a specific type of food. Such an approach offers significant opportunities for product innovation as per consumer requirements. Moreover, recognizing that a particular condition such as heart or bowel health may be influenced by more than one type of nutrient, manufacturers

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can design and evaluate unique foods with appropriate combinations of nutrients to optimize health status. The transition from traditional foods and herbal remedies of uncertain value to designer foods with guaranteed health benefits could be facilitated by adopting aspects of the pharmaceutical approach to substantiation and regulation.36 The ability to manipulate the properties of ingredients allows greater creativity in the design of new food products.37 1.5.5.4

Reference Standards for Nutrients Intake and International Recommendations

The most common reference standard for nutrient intake has been the recommended daily allowance (RDA), first established by the U.S. Food and Nutrition Board in 1941, with the recent edition in 1989. The RDA is an amount to be consumed as part of normal diet. RDA is neither a minimal requirement nor an optimal level of intake but represents a safe and adequate level of intake based on current scientific knowledge. The RDA is most appropriately used as a nutrient intake guide applied to subgroups of the population, but can be used to estimate the probable risk of nutrient deficiency for an individual. Scientific evidence at present is insufficient to establish an RDA for nutrients. Therefore, estimated safe and adequate daily dietary intakes (ESADDIs) have been established for nutrients. The WHO published diet recommendations with the goal of reducing the risk of chronic diseases. The WHO recommendations are expressed as a range of average daily intakes from lower to upper levels.38 The International Food Information Council (IFIC) with other associations has developed “Guidelines for Communicating the Emerging Science of Dietary Components for Health.” Food science and nutrition communicators can use these guidelines to translate emerging research finding into understandable message to consumers39 (see Chapter 13). 1.5.5.5

Safety and Regulation of Functional Foods

Currently, there is no universally accepted regulation governing functional foods, each country has its own interpretation. The first country to have a specific regulatory definition as well as an approval process for functional foods is Japan.14 In April 2001, the Japanese government enacted a regulatory system called “foods with health claim,” which consists of “foods for specified health use” (FOSHU) and “foods with nutrient functions claims” (FNFC). The FOSHU was set up by the Ministry of Health and Welfare to encourage maintenance of health based on the consumption of functional foods with scientific evidence and to approve descriptions on a label regarding an effect of food on the human body. There are three important requirements for FOSHU approval, which are scientific evidence of the efficacy including clinical testing, safety for consumption, and the analytical determination of the effective component. At present there are about 300 items approved as FOSHU. Under FNFC, 12 vitamins (vitamins A, B1, B2, B6, B12, C, E, D, biotin, pantothenic acid, folic acid, and niacin) and two minerals (calcium and iron) are standardized.14 It has been suggested that the Japanese administration and the food industry cooperate with Association of South East Asian Nations (ASEAN) countries to work together in the development and promotion of nutrition and health claims on foods.40

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In the United States, there is currently no definition for functional foods in FDA regulation. Regulatory guidelines for consumption of functional foods are also not available in the United States at present, unlike traditional nutrients, namely, vitamins, minerals, and essential fatty acids, for which recommended intakes have been established.41 The Federal Food, Drug and Cosmetics (FD&C) Department and the U.S. Federal Trade Commission (FTC) have jurisdiction over foods. Under the FD&C Act, the distinction between foods and drugs is clearly defined. Furthermore, special dietary foods, and dietary supplements are also clearly defined. Thus, until a more specific regulatory category is defined, functional foods, which are marketed and sold as foods, are regulated as foods.4 Any food additive used in a food product must be either under a specific food additive regulation or generally recognized as safe (GRAS) under FD&C act. For approval, additives must be shown to be safe as well as beneficial. Sales, development, and health claims of food products in the United States fall under three major acts, namely, the Nutrition Labeling and Education Act (NLEA) of 1990, Dietary Supplement Health Education Act of 1994, and the FDA act of 1997.42 The U.S. FDA has considered allowing “qualified health claims” using scientific evidence. These qualified health claims were not formally codified as health claims, but were allowed under “enforcement discretion.” These include calcium (osteoporosis); sodium (high blood pressure); saturated fat (cancer); saturated fat and cholesterol (cardiovascular disease); fruits, vegetables, and cereals with dietary fiber (cancer and also heart disease); fruits, vegetables, and antioxidants (heart disease); vegetables, fruits, vitamin C, and fiber (cancer); folic acid (neural tube injury); sugar-free sugar alcohols (caries); soluble fiber from oats (reduced risk of coronary heart disease); soy protein (reduced risk of heart disease); plant sterol/ stanol ester (heart disease); and whole grain (reduced risk of heart disease and certain cancer).43,44 The Institute of Food Technologists (IFT), United States, expert panel recommends that the benefits for functional foods be based on nutritive value or through the provision of a physical or physiological effect that has been scientifically documented or for which a substantial body of evidence exists for a plausible mechanism. Examples include “calcium helps build strong bones” and “proteins help build strong muscles.”3 In the absence of specific dietary guidelines, consumer selection of functional foods essentially depends on general awareness on their nutritional properties. In Canada, the term “functional foods” is used to describe those foods with “demonstrated physiological benefits and/or reduced risk of chronic diseases, but which are similar to appearance to conventional foods and are consumed as part of the diet.”4 A study of 280 manufacturers of functional foods in Canada showed that the sector is widely heterogeneous in firm composition, markets served, and product strategies. Nevertheless, the study identified six main strategic groups within the nutraceutical and functional foods sector.45 In the European Union, the newly adopted European regulation on nutrition and health claims came into force on January 19, 2007. The law sets out conditions for the use of claims and establishes a system for their scientific evaluation. After consultation with the European Food Safety Authority (EFSA), the commission is likely to adopt a community list of permitted claims by January 31, 2010. It is meant to help the European industry, researchers, and the regulatory authorities to compile the information required for the national

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lists of health claims, and to explain how the scientific references can be presented to show the strength and consistency of the evidence.46 In the United Kingdom, an Expert Group on Vitamins and Minerals (EVM) is an independent expert advisory committee, which was set up in 1998 to advise on safe levels of intakes of vitamins and minerals in food supplements and fortified foods. Thirty-four substances were assessed in detail. Safe upper levels (SULs) were recommended for eight vitamins and minerals and guidance was issued for twenty-two.47 1.5.5.6

Marketing and Trade of Functional Foods

The future challenges to food technology are likely from the point of view of functional foods. Some of these challenges include isolation of bioactive compounds from food wastes, developing foods containing various bioactive compounds to combat diseases, strategies for enhancing stability and functions of bioactive compounds by techniques such as fortification and microencapsulation, development of novel packaging materials, which contain antimicrobial and similar agents to extend shelf life, among others. (These are highly relevant for marine products, as discussed in this book.) Functional foods, are expected to be one of the emerging markets for the food industry in the new millennium.27 The IFT expert panel identified a seven-step process that addresses critical aspects in the design, development, and marketing of functional foods, as shown in Table 1.7.48 Among the seven steps, specific requirements within each step will vary depending on the physical, chemical, and biological characteristics of the functional component.3 Recent marketing campaigns of functional foods with slogans such as “you are what you eat” emphasize multiple beneficial effects of consumptions of food products. Consumers will respond to functional foods marketing based on a range of motivation health conditions and knowledge levels.49 Across Japan, America, and Europe, customers are already buying expensive yogurts, drinks, marshmallows, and jams, which claim to contain ingredients such as collagen, enzymes, and others that could have beneficial effects on health. Some of these products include “wrinklefree lunch,” launched in Scotland, which claims contents of ingredients known for their antiageing properties, a Japanese marshmallow with collagen, and a French antiwrinkle jam that contains essential fatty acids, antioxidants, lycopene, and vitamins E and C. Table 1.8 presents some of the recently marketed functional foods and their claims.50 The current world market for functional foods and nutritional supplements is highly dynamic and is estimated to be U.S.$100 billion, with an annual growth potential of 20%.51,52 The global nutraceuticals market is growing at a rate of 9.9% and is projected to reach $74.7 billion by 2007.53 Japan is leading the market for functional foods, with an estimated sale of U.S.$11.7 billion in 2003. In Japan, more than 200 functional foods are being marketed under the FOSHU legislation.22 The European functional food market was worth U.S.$3.49 billion in 2007; the largest segment being gut health-specific products.51,52 The U.S. market for functional foods as a percentage of the total food market is expected to increase from 4% in 2002 to 5.4% in 2007, showing a potential to reach $37.7 billion by 2007 with an average annual growth rate of 13.3%.53 Table 1.9 gives global functional food markets in 2004–2006.54

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TABLE 1.7 Seven-Step Process to Address Critical Aspects in Design, Development, and Marketing of Functional Foods Steps 1

Process

Evaluation Parameters

2

Identify relationship between food component and health benefit Demonstrate efficacy and determine intake level necessary to achieve desired effect

3

Demonstrate safety at efficacious levels

4

Develop suitable food vehicle for bioactive component Demonstrate scientific sufficiency of evidence for efficacy

5

6 7

Communicate benefits to consumers Conduct in-market confirmation of efficacy and safety

Identify bioactive component(s) Assess stability and bioavailability of component(s) Demonstrate efficacy using biological endpoints and biomarkers Estimate intake by population subgroups Consider prior GRAS and food additive uses Assess safety if component is new to food use Address potential allergenicity, if necessary Assess stability and bioavailability of components Conduct independent peer review panel Submit evidence to FDA for claim approval, if necessary Employ Step 2 parameters Monitor efficacy Monitor intake Monitor safety Employ Step 3 parameters

Source: Adapted from Clydesdale, F. M., Food Technol., 58(12), 34, 2004; Sloan, A. E., Food Technol., 4, 16, 18, 2004.

TABLE 1.8 Some of the Functional Foods Marketed Recently and Their Claims Product

Manufacturer

“Wrinkle-free lunch” The product contains roasted Gressingham duck with berry sauce “Antiwrinkle jam” containing seaweed and green tea

Rufflets Country House Hotel, Scotland

“V&T” (“Life and Tea” in Spanish) Marshmallow, sweet

Coca-Cola, Spain

Laboratoires Noreva, France

Eiwa, Japanese sweet maker

Benefit Claims Presence of selenium (in the duck meat) and antioxidants (in berry) offer antiageing properties and youthfulness Essential fatty acids, antioxidants, lycopene, noreline, and vitamins E and C The antioxidants present offer antiageing and other health benefits Collagen offers skin benefits

Source: Adapted from Times of India, August 15, 2006.

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TABLE 1.9 Global Functional Food Markets in 2004–2006 Functional Food Category Digestive Cardiovascular Brain and nervous system Immune system Bone health Beauty benefits Others

2004

2005

2006

166 115 33 44 17 16 833

252 156 68 53 23 24 840

656 268 92 78 69 46 1059

Source: Adapted from Suzuki, M., The Future of Functional Foods, International Union of Food Science and Technology, Oakville, Ontario, Canada, 2007. With permission.

1.6 MARINE PRODUCTS AS FUNCTIONAL FOOD: AN OVERVIEW Marine products, due to their phenomenal biodiversity, are attractive not only as nutritious food items, but also as treasure house of novel, biologically active compounds. Extensive scientific research in the recent past has documented the numerous health benefits of eating fish, especially fatty marine fish species. Fish is an excellent source of lean protein, omega-3 fatty acids, antioxidants, and vitamins. Fish oil has long been recognized as a functional food because of its ability to reduce blood pressure and lower the risk of cardiovascular disorders such as abnormal heartbeat and blockage of blood vessels by cholesterol. The health benefits from seafood consumption could be considered with respect to two groups of population, namely, women, infants, and young children, who derive benefits of omega-3 fatty acids, and adults, who can prevent or delay chronic diseases through seafood consumption. The amount and composition of dietary fat is arguably the most important influencing factor on risk due to coronary heart disease and stroke, the major dangers to human. A significant body of consistent evidence indicates that a decrease in dietary saturated to unsaturated fat ratio (polyunsaturated + monounsaturated) and an increased intake of long-chain n-3 polyunsaturated fatty acids (omega-3 fatty acids) found in fish, is cardio-protective.53 In addition to omega-3 fatty acids, marine fish and other products are also rich sources of nutraceuticals, which include gelatin, peptides, glucosamine, and other cartilage products providing health benefits. Dietary advices to general public from several regulatory bodies have indicated that seafood should be a component of healthy diet, particularly as it can displace other protein foods that are high in saturated fat. Several agencies such as Dietary Guidelines Advisory Committee of American Heart Association and USDA have recommended consumption of two to three servings of fish per week. On October 15, 2007, the State of Alaska, United States, issued new fish consumption guidance for people who catch and eat fish from Alaska waters.55 The document, entitled “Fish Consumption Advice for Alaskans: A Risk Management Strategy to Optimize the Public’s Health,” observes that a balanced diet that includes fish can lower the risk of

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heart disease, diabetes, and stroke. Fish should be also an important part of diet for pregnant and nursing women, and young children as the omega-3 fatty acids in fish improve maternal nutrition and brain development in unborn and young children. Women who are or can become pregnant, nursing mothers, and children aged 12 years and under should continue unrestricted consumption of fish from Alaska waters (that are low in mercury), which include Alaska salmon, pacific cod, walleye pollock, black rockfish, pacific ocean perch, halibut, and lingcod. The National Oceanic and Atmospheric Administration (NOAA), with support from the FDA, sponsored an independent study titled, “Seafood Choices: Balancing Benefits and Risks.” The study concludes that “seafood is a nutrient-rich food that makes a positive contribution to a healthful diet” and therefore advises all Americans to eat seafood regularly.56 In view of depletion of stocks of several marine fish species (see Chapter 2), the WHO has observed that “recommending the increased consumption of fish is an area where the feasibility of dietary recommendations needs to be balanced against concerns for sustainability of marine stocks and the potential depletion of this important marine source of high quality nutritious food. Added to this is the concern that a significant proportion of the world fish catch is transformed into fish meal and used as animal feed in industrial livestock production and thus is not available for human consumption.”1 Over the past two decades, more than 3000 new compounds have been isolated from various marine organisms that include seaweed, corals, and microorganisms. Organisms such as corals provide a diverse array of drugs and other rare bioactive compounds (Chapter 12). Some of these compounds have been employed in clinical therapies. Many compounds derived from seaweeds, which possess biological activities for medicinal uses have been reported.57,58 Numerous investigations have been reported that crude seaweeds or their extracts have antiproliferating activities against human cancer cell lines in vitro.58–60 In recent years, several nutraceuticals have been isolated from marine products.61 Japan is in the forefront of biotechnology intended for production of marine nutraceuticals. An overview of recent research on marine natural products by Australian and New Zealand scientists indicates the research being carried out for isolation of natural products from marine microalgae, macroalgae, microorganisms, ascidians, bryozoans, corals and sponges, and other products. The compounds under investigation include marine lipids, fish oils, chemical inhibitors of marine biofouling, natural sunscreens, and coral sperm attractants.62 Many of these products could also be developed through aquaculture.63 In addition to specific nutraceuticals, marine foods also offer a number of valuable food ingredients for product development. These include seafood proteins, enzymes, hydrocolloids, among others.64 Utilization of marine products as sources of various nutraceuticals and bioactive compounds lead to their sustainable and efficient uses.65–68 Major functional and nutraceutical compounds from marine sources are shown in Table 1.10. The potential uses of bioactive compounds as medicine embrace the domain of pharmaceutical industry. The industry, by what is termed as “biomining,” examines different organisms including marine organisms, bacteria, and plants for exploring their natural products for use as drugs to treat various diseases. The pharmaceutical industry is tightly overseen by government agencies such as the FDA of the

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TABLE 1.10 Some Major Functional and Nutraceutical Compounds from Marine Sources Source Marine fish

Marine shellfish

Corals, ascidians, bryozoans, etc.

Seaweeds

Microalgae

Products Protein supplements Bioactive peptides (various bioactivities) Protein hormones Cartilage products such as shark cartilage and chondroitin sulfate Antioxidants, for example, carotenoids and peptides Enzymes including cold-adapted enzymes Antifreeze proteins (cryoprotectants) Polyunsaturated fatty acids (various functions) Drugs Gelatin, collagen Enzymes Chitin, chitosan, and related compounds Glucosamine Carotenoids Glue from mussel Enzymes Antioxidants Antimicrobials Drugs and other bioactive compounds Specific bioactive compounds Enzymes Biochemicals Calcium Mineral sources Proteins Hydrocolloids (food additives, fibers, and bioactive functions) Carotenoids Fine chemicals

United States. However, as discussed earlier, the nutraceutical industry makes use of these compounds to enhance the nutraceutical and functional properties of the food by techniques such as fortification and encapsulation (see Chapters 13 and 14). The nutraceutical industry has little oversight for the “therapeutic” claims of the compound. Nevertheless, because of increasing consumer interests in recent times, many pharmaceutical firms are engaging themselves in the production of neutraceutical and functional foods. The global markets for these marine biotechnology products and processes are estimates at U.S.$2.4 billion in 2002, a 9.4% increase from 2001. The non-U.S. market in 2002 was $1.6 billion and is projected to rise in the next five years at 6.4%, faster than 4.7% growth predicted for the U.S. markets for the same period. The U.S marine biotechnology market is projected to surpass $1 billion in 2007.69 Table 1.11 presents some future challenges to food technology with respect to development of functional foods.70

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TABLE 1.11 Some Future Challenges to Food Technology Foods and diet will become the primary focus of disease prevention Bioactive compounds will become key elements for products Microcapsules will be used to protect flavor compounds, bioactive compounds, etc. Flavor compounds will be released into foods in microcapsules for release at the time of consumption Microsensors and biosensors will rapidly detect harmful agents in foods Active packaging will be introduced in a big way to extend shelf life Waste materials will become resources of bioactive compounds Bioactive compounds will be incorporated into the products for delivery to appropriate sites during metabolism of the foods Organic foods Source: Adapted from Heldman, D. R., IFT in 2006–07, Newsletter, March 19, 2006, Institute of Food Technologists, Washington, DC.

In conclusion, consumers’ perception of food is changing with a clear leaning toward health-promoting functional foods and nutraceuticals. Marine products are important in this respect because of the presence of several neutraceuticals. Regular consumption of marine foods has been recognized to be useful for better healthcare. Details on functional and bioactive properties of marine ingredients including drugs are discussed in subsequent chapters.

REFERENCES 1. WHO, Diet, nutrition and prevention of chronic diseases, Report of a Joint FAO/WHO Expert Consultation, Technical Report Ser. 916, World Health Organization, Geneva, 2003. 2. WHO, Fact sheet—Obesity and Overweight, World Health Organization, Geneva, Switzerland, 2004. 3. Clydesdale, F. M., Functional foods; opportunities and challenges, Food Technol., 58(12), 34, 2004. 4. Schmidt, R. H. and Turner, E., Functional foods and nutraceuticals, in Food Safety Handbook, Schmidt, R. H. and Rodrick, G. E., Eds., Wiley, New York, 2003, p. 673. 5. Arvanitoyannis, I. S. et al., Functional foods: a survey of health claims, pros and cons, and current legislation, Crit. Rev. Food Sci. Nutr., 45, 385, 2005. 6. Cherry, J. P., Protein Functionality in Foods, American Chemical Society, Washington, DC, 1981, p. 1. 7. Berner, L. A. and Dannell, J. A., Functional foods and health claims legislation: application to dairy foods, Int. Dairy J., 8, 355, 1998. 8. Kuzuminski, L. N., Issues and pressures for food and beverage research and development in the 21st century, Crit. Rev. Food Sci. Nutr., 39, 1, 1999. 9. Schaafsma, G., Health claims, options for dietary fibre, in Dietary Fibre— Bioactive Carbohydrates for Food and Feed, van der Kamp, J. W. et al., Eds., Wageningen Academic Publisher, The Netherlands, 2004, p. 27. 10. Anonymous, Superfood and drinks: consumer attitudes to nutrient rich products, Information Store Datamonitor Reports, IFIS Publishing, http://www.foodsciencecentral. com/fsc/ixid14899, August 2007.

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11. USDA, U.S. Department of Agriculture, Release 2.1, 2007. www.ars.usda.gov/ nutrientdata/flav, accessed on November 2007. 12. Xu, Y., Perspectives on the 21st century development of functional foods bridging Chinese medicated diet and functional foods, J. Food Sci. Technol., 36, 229, 2001. 13. Audlauer, W. and Furst, P., Nutraceuticals: a piece of history, present status and outlook, Food Res. Int., 35, 171, 2002. 14. Ohr, L. M., Nutraceuticals: health foods stand at forefront. Food Technol., June 2007, www.ift.org. 15. Drewnowski, A. and Gomez-Carneros, C., Bitter taste, phyto-nutrients, and the consumer: a review, Am. J. Clin. Nutr., 72, 1424, 2000. 16. Reineccius, G. A., Flavoring systems for functional foods, in Essentials of Functional Foods, Schmidl, M. K. and Labuza, T. B, Eds., Aspen Publishing, Gaithersburg, MD, 2000, p. 89. 17. Cardello, A. V. and Schutz, H. G., Factors to consumer interest in nutraceutical products: civilian and military comparisons, J. Food Sci., 68, 1519, 2003. 18. Arai, S., Studies on functional foods in Japan—state of the art, Biosci. Bitech. Biochem., 60, 9, 1996. 19. Tsau, R. and Akthar, A. M. H., Current international regulatory status, Food Agri. Env., 3, 18, 2005. 20. Hasler, C. M., Functional foods, their role in disease prevention and health promotion, J. Food Technol., 52, 63, 1998. 21. Uzzan, M., Nechrebeki, J., and Labuza, T. P., Thermal and storage stability of nutraceuticals in a milk beverage dietary supplement, J. Food Sci., 72, E109, 2007. 22. Makhal, S., Mandal, S., and Kanawijia, S. K., Development of bioactive fermented dairy products with special reference to cheese: scope and challenges, Ind. Food Ind., 23, 25, 2004. 23. Anantharaman, G., Recent trends in functional food, Presented at Fourth International Food Convention, CFTRI, Mysore, India, 1998, Souvenir, p. 170. 24. Senoranas, F., Ibanez, E., and Cifuentes, A., New trends in food processing, Crit. Rev. Food Sci. Nutr., 43, 507, 2003. 25. Chandan, R. C., Role of functional foods and probiotics in human health, Fourth International Food Convention, CFTRI, Mysore, India, 1998, p. 172. 26. IFIS, Antioxidants and 21st century nutrition, http://www.foodsciencecentral.com/fsc/ ixid13735, accessed on September, 2005. 27. Tucker, M., Waley, S. R., and Sharp, J. S., Consumer perception of food-related risks, Int. J. Food Sci. Technol., 41, 135, 2006. 28. Hansen, Chr., Health and Wellness Survey, Newsletter, Institute of Food Technologists, Washington, DC, July 12, 2006. 29. Urala, N. and Lahteenmaki, L., Consumers’ changing attitude towards functional foods, Food Qual. Pref., 18, 1, 2007. 30. Anonymous, Nutraceutical trend takes root despite definitional changes, Nutr. Bull. J., August, 1–3, 15, 1997. 31. Worsley, A., Food and consumers: where are we heading? Asia Pacific J. Clin. Nutr., 9, S103, 2000. 32. Food and Agriculture Organization of the United Nation, Rome, http://www.fao. org/infoods/directory_en.stm. 33. Pakkala, H., Reinivuo, H. and Ovaskainen, M. L., Food composition on the world wideweb: a user-centred perspective. J. Food Compos. Anal., 19, 231, 2006. 34. Pennington, J. A. T., Food composition databases for bioactive food components, J. Food Comp. Anal., 15, 419, 2002. 35. Paraba, J. and Aguilera, J. M., Food microstructure affects the bioavailability of several nutrients, concise review, J. Food Sci., 72, R27, 2007.

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36. Howe, R. P., How to make a functional food, functional? Asia Pacific Clin. Nutr., 9, S108, 2000. 37. Rastall, R., Tailor-made food ingredients: enzymatic modulation of nutritional and functional properties, IFIS Publ., http://www.foodsciencecentral.com/fsc/ixid3729. 38. Read, M., The health promoting diet throughout life: adults, in Handbook of Food and Nutrition, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 299. 39. Reinharadt, W., Backpage, Food Technol., 58(12), 128, 2004. 40. Shimizu, T., Newly established regulation in Japan: foods with health claims, J. Clin. Nutr., 11, S94, 2002. 41. Roberts, W. A., Jr., Function takes form, Prep. Foods, 174, 23, 2005. 42. Sedo, http://www.functionalfoods.nu/file/dyn/0000m/381i/dyn381.asp, accessed on September 2007. 43. U.S. FDA, Center for Food Safety and Applied Nutrition, http://www.cfsan.fda.gov/ ~dms/flg-6c.html, accessed on September 2007. 44. Geiger, C. J., Food labeling: food and dietary supplements, Ch. 14, Handbook of Nutrition and Food, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 393. 45. Cloutier, L. M. and Salvesa, A.-L., Functional eating and strategic groups in Canada, Can. J. Agri. Econo., 50, 569, 2002. 46. IFIS, Guidelines for an evidence-based review system for the scientific justification of diet and health relationships under Article 13 of the new European legislation on nutrition and health claims, http://www.foodsciencecentral.com/fsc/ixid14747, April 23, 2007. 47. Food Standards Agency, U.K., http://www.food.gov.uk/, accessed on September 2007. 48. Sloan, A. E., The heart of the matter: functional foods and nutraceuticals, Food Technol., 4, 16, 18, 2004. 49. Teratanavat, R. and Hooker, N. H., Consumer valuation and preference heterogeneity for a novel functional food, J. Food Sci., 71, S533, 2006. 50. Times of India, August 15, 2006. 51. Kumara, S., Chidambara, N., and Subbiah, V., Regulatory and marketing strategies for novel nutraceutical products: an industry perspective, Ind. Food Ind., 24, 46, 2005. 52. Aluko, R., Functional foods and nutraceuticals, IFIS Publishing, April 2006, http://www.ifis.org/fsc/ixid14335. 53. IFIS, Dietary fat composition and cardiovascular disease, IFIS Publishing, Functional Foods, http://www.foodsciencecentral.com/fsc/ixid14369, June 8, 2006. 54. Suzuki, M., The Future of Functional Foods, International Union of Food Science and Technology, Oakville, Ontario, Canada, 2007. 55. State of Alaska, Bulletin, Fish consumption advice for Alaskans: A risk management strategy to optimize the public’s health, State of Alaska, http://www.epi.alaska. gov/bulletins/docs/b2007_29.pdf. 56. Anonymous, Seafood affirmed as healthy food choice, IFT Weekly Newsletter, Institute of Food Technologists, Washington, DC, October 18, 2006. 57. Faulkner, D. J., Marine pharmacology, Antonie van Lecuwenhoek, 77, 135, 2000. 58. Noda, H. et al., Studies on the anti-tumor activities of marine algae, Nippon Suisan Gaikkaishi, 55, 1259, 1989. 59. Schwartsmann, G. et al., Marine organisms as a source of new anticancer agents, Lancet Oncol., 2, 221, 2001. 60. Cragg, G. M., Newman, D. J., and Weiss, R. B., Coral reefs, forests and thermal vents: the worldwide exploration of nature for novel anti-tumor agents, Semin. Oncol., 24, 156, 1997. 61. Pszczola, D. E., Choosing new alternatives to alternative ingredients. Food Technol., 57(10), 54, 2003.

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62. Volkman, J. K., Australasian research on marine natural products: chemistry, bioactivity and ecology, Marine Freshwater Res., 50, 761, 1999. 63. Anonymous, Foods from aquaculture, Food Technol., 45(9), 87, 1991. 64. Alasalvar, C. and Taylor, T., Seafoods—Quality, Technology and Nutraceutical Applications, Springer-Verlag, Heidelberg, Germany, 2002, p. 175. 65. Blanco, M. et al., Towards sustainable and efficient use of fishery resources: present and future trends, Trends Food Sci. Technol., 18, 29, 2007. 66. Gildberg, A., Enhancing returns from greater utilization, in Safety and Quality issues in Fish Processing, Bremner, H. A., Ed., Woodhead Publishing, Cambridge, England, 2002, p. 425. 67. Kanazawa, A., Recent advances in aquatic food technology and nutrition, Keynote addresses: 5th and 6th Asian Fisheries Forums. 5th Asian Fisheries Forum, Chiang Mai (Thailand), 11–14 1998, AFS Special Publication, Liao, I., Ed., Bangkok, No. 11, p. 81, July 2001. 68. Faulkner, D. J., Highlights of marine natural products chemistry, 1972–1999, Nat. Prod. Rep., 17, 1, 2000. 69. BCC Research, Evolving Neutraceutical Business. The global nutraceuticals market is growing at a rate of 9.9% and is projected to reach $74.7 billion by 2007. Report ID: FOD013B, p. 135, 2003, www.bccresearch.com. 70. Heldman, D. R., IFT in 2006–07, Newsletter, March 19, 2006, Institute of Food Technologists, Washington, DC.

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Habitat 2 Marine and Resources 2.1 INTRODUCTION The ocean is the richest reservoir of living and nonliving resources, besides influencing global climate and functioning as a means of transport and communication. The food resources of the oceans are potentially greater than those of the land because of their larger area and hence the ability to absorb significant amounts of solar radiation for food production through photosynthesis by the microalgae. The algal photosynthesis provides food for the higher organisms including fishes, which, in turn, function as food to several organisms including human beings. From the point of view of food, the term “marine” brings into the mind of general public the various fish and shellfish, which serve as a rich bowl of delicate food containing digestible proteins, lipids, and also a variety of micronutrients including vitamins and minerals. Apart from providing fishery products, the oceans also serve as dwelling places for various species of seaweed, reef corals, and several microorganisms including bacteria. Fish, crustaceans, and mollusks from marine sources have been traditionally exploited as food, whereas there are other constituents of the sea, such as seaweed, coral reefs, corals, and microorganisms, which can generate a wide variety of nutraceuticals, drugs, novel enzymes, and bioactive and industrial compounds for food and healthcare. Recent scientific pursuit has thrown newer insights into the nutritional and therapeutic values of bioactive components from these marine organisms. The databases with this information, such as those on carotenoids, flavonoids, omega-3 fatty acids, β-carotene, phytosterol, and plant sterols are expected to boost the search of novel resources including those from the marine environment.1 This chapter provides an overview on different marine resources, which will help understand their potential to deliver functional foods and nutritional and therapeutic ingredients, which will be discussed in the subsequent chapters.

2.2 MARINE ENVIRONMENT The marine ecosystem, the largest on the planet, has been divided into photic, pelagic, benthic, epipelagic, and aphotic zones, the depths of which vary from 200 to 10,000 m. There are more than 40,000 different species of phytoplankton, which are divided into major classes, namely, cyanobacteria, chlorophyta, cryptophyta, rhodophyta, heterokontophyta, dinophyta, haptophyta, and euglenophyta.2 The dominant autotrophs (living organisms capable of producing energy) are single-celled microscopic plants of various groups of algae, which form the first stage of the marine food chain. Much of the primary productivity in the open oceans, even at significant depths, is 23

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due to the photosynthetic activities of these microalgae—the aerobic prokaryotes that contain chlorophylls a and b.2–4 The primary production, that is, photosynthetic fixation of carbon dioxide is limited by nutrients and therefore living cells are comparatively less in the ocean than in the freshwater ecosystem. Inshore ocean areas are typically more nutritionally rich than open waters and therefore support more dense populations of phototropic microorganisms. Significant numbers of prokaryotic cells in the range 105–106 per milliliter are suspended in open ocean waters. In addition, about 104 cells/mL of very small eukaryotic organisms are present. In tropical and subtropical oceans, the planktonic filamentous marine cyanobacterium Trichodesmium form tufts of filaments that constitute significant fraction of the biomass suspended in the waters. This organism is capable of nitrogen fixation and therefore is thought to be a major link in the nitrogen cycle in the marine environment. Very small phototropic algae (Ostreococcus), measuring only about 0.7 µm in diameter (which is smaller than a cell of Escherichia coli), are also involved in nitrogen fixation. Many prokaryotes in the photo zone (up to 300 m) of the ocean contain a form of the visual pigment, rhodopsin, which the cells use to convert light energy into adenosine triphosphate (ATP).5 Marine microbiological communities have significant influence on the marine food chain. Marine bays and inlets receiving sewage or industrial waste can have very high phytoplankton and bacterial population. This, in turn, supports higher densities of chemotrophic bacteria and aquatic animals such as fish and shellfish.5 Organisms that inhabit the deep sea are faced with three major environmental extremes, namely, low temperature, high pressure, and low nutrient levels. Below depths of about 100 m, ocean water remains at a constant temperature of 2–3°C. Pressure increases by 1 atm for every 10 m depth. Thus, organisms growing at 5000 m must be able to withstand pressures as high as 500 MPa. These extreme conditions result in reduction in microbial levels with increasing depth. Thus, compared to about 3 × 105 cells/mL of surface waters, the cell counts may be as low as 3000 per milliliter at a depth of 2000 m. A temperature below 0°C exists in the Arctic and Antarctic oceans, whereas temperatures exceeding 100°C are found in the hydrothermal vents in the ocean bottoms. Salinities as high as 6 N have been found in salt marshes and mines. Because of these diverse environmental conditions, there is an immense biodiversity of marine organisms in the ocean. Each organism has a different metabolism adapted to such conditions. Cold-adapted enzymes from fish living at temperatures above the freezing point of seawater and thermoresistant enzymes from organisms including crustacean, living in the hydrothermal vents have been reported.6 Dense, thriving animal communities, supported by the activities of microorganisms, cluster around thermal springs in deep-sea waters.7 Seasonal and annual environmental characteristics such as temperature, chlorophyll content, salinity, microbial water quality, and algal lipid composition have significant influence on the living creatures with respect to their meat content, shell size, and lipid compositions.8 Despite obvious differences, marine biodiversity patterns in environmental conditions of the various oceanographic regions show a worldwide consistency. Several nonedible species including sponges, crustaceans, and other animals live in deep marine environment.9 The sea is also a rich source of minerals. These include those from sedimentary deposits underlying the continental shelves, inshore deposits of continental

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shelves, and those present in seawater. A 1000 g of seawater usually contains 35 g of dissolved salts, which consists of six major inorganic ions that include chloride (55%), sodium (30.6%), sulfate (7.6%), magnesium (3.69%), calcium (1.16%), and potassium (1.1%). Seawater is the major source of magnesium bromide. Seawater is slightly alkaline, having a pH in the range 7.5–8.4. The nutrient contents of open seawater are often very low when compared to freshwater environments. This is especially true in the case of key inorganic nutrients such as nitrogen, phosphorus, and iron. Iron is a key micronutrient in the oceans, whose bioavailability influences both the extent of primary production (photosynthesis) and the plankton community structure. The 1990s have been dubbed “The iron age of oceanography.” The accumulation of mineral and organic remains on the seafloor vary widely in composition and characteristics as a function of water depth, distance from land, and environmental characteristics.10 The phenomenon of global warming is believed to have disastrous effects on the marine environment. The causative factors of global warming are the greenhouse gases, namely, carbon dioxide, methane, ozone, and nitrous oxide. Projections from global warming models indicate a possibility of nearly continuous rise in temperature on the order of 0.5°C per decade for every decade of this century. The effects of global warming on the sea are the rise in seawater temperature, salinity, and sea level; drop in sea surface pH; and changes in the current upwelling, water mass movement, and El Nino and La Nina events. This could in turn affect the abundance and distribution of marine organisms including fish species. For instance, a rise in temperature as small as 1°C can have important effects on the mortality of some organisms and their geographic distributions. Such changes would result in varying and novel mixes of organisms in a region, leaving species to adjust to new predators, prey, competitors, and parasites. It has been pointed out that climate changes may increase the acidity of the oceans by bleaching corals where fish breed. Preliminary analysis indicates that the distribution of fish species such as sardines having comparatively smaller generation times may show rapid demographic responses to temperature changes. Some pelagic species such as mackerel show shift in the depth of distribution necessitating changes in patterns of fishing operations for their harvest. Owing to the sea level rise and inundation, the coastal fishing communities could also be affected. The mean sea level (MSL) has increased at the rate of 0.705–3.77 mm/year in the Arabian Sea, Bay of Bengal, and Andaman Sea during the period 1992–2005.11 In addition to global warming, during the past few decades, an adverse impact on marine ecosystems due to human activities leading to pollution, overfishing, etc. has also been noted.12

2.3 MARINE FISHERY PRODUCTS Seafood is the major food product from the oceans. The term “seafood” generally refers to groups of biologically divergent animals consisting of not only fish, but also shellfish, which include crustaceans and mollusks. The crustaceans comprises crayfish, crab, shrimp, and lobster, whereas the mollusks could be bivalves such as mussel, oyster, scallop; univalve creatures such as abalone, snail, and conch; and cephalopods, which include squid, cuttlefish, and octopus. It has been estimated that

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the ocean is inhabited by about 13,000 species of fish, 50,000 species of mollusks, and 1,000 species of cephalopods. Seafood is nutritionally important because their proteins are highly digestible and contain all the essential amino acids. The lipid content of seafood is primarily in the form of triglycerides or triacylglycerols and is the only major source of highly unsaturated fatty acids. Seafood accumulate omega-3 fatty acids through phytoplankton—the primary producers of omega-3 fatty acids.13

2.3.1

LANDING

According to The State of World Fisheries and Aquaculture 2006, published by the Food and Agriculture Organization of the United Nations (FAO), Rome, capture of marine fishes in 2004 was 95 million tons (mt). The capture fisheries together with aquaculture supplied the world with 106 mt of food fish in 2004, providing an apparent per capita availability of 16.6 kg (live weight equivalent).14 The provisional total world fish landings in 2005 were 141.6 mt. Of these, marine fishery resources were 84.2 mt, and aquaculture of marine fisheries contributed to 18.9 mt, totaling to 103.1 mt of marine fishery products for the year. The total amount of fish utilized for human consumption was 107.2 mt in 2005, maintaining a per capita consumption of 16.6 kg, as in 2004. Table 2.1 shows total world fish production (in million tons) during the years 2000 to 2006.15 It is apparent that the availabilities of both captured and aquacultured fish are showing a declining trend. Table 2.2 shows top 10 species of marine fish captured in 2004.16 Fisheries provide a vital source of food, employment, trade, and economic wellbeing for people throughout the world. According to the FAO, the current international trade in fish products is about U.S.$ 71.5 billion.14 The trade has helped developing countries gain significantly, with their net earnings increasing from U.S.$ 3.4 billion in 1980 to U.S.$ 20.4 billion in 2004. China alone exported seafood worth U.S.$ 6.6 billion in 2004.14 Japan is the world’s largest importer of fish and fish products worth U.S.$ 14.6 billion. Shrimp is the most popular internationally traded

TABLE 2.1 Total World Fish Production (in Million Tons) and Utilization during the Period 2000–2005 Products

2000

2002

2004

2005

Marine capture Inland capture Inland aquaculture Marine aquaculture Total marine capture Total aquaculture Human consumption

86.8 8.8 21.1 14.3 101.1 35.5 96.9

84.5 8.8 23.9 16.5 101.0 40.4 100.2

85.8 9.2 27.2 18.3 104.1 45.5 105.6

84.2 9.6 28.9 18.9 103.1 47.8 107.2

Source: Adapted from FAO, The State of World Fisheries and Aquaculture, Food and Agriculture Organization of the United Nations, Rome, 2006. With permission.

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TABLE 2.2 Top 10 Species of Marine Fish Captured in 2004 Species Anchovita Alaska pollock Blue whiting Skipjack tuna Atlantic herring Chub mackerel Japanese anchovy Chilian jack mackerel Largehead hairtail Yellowfin tuna

Capture (mt) 10.7 2.7 2.4 2.1 2.0 2.0 1.8 1.8 1.6 1.4

Source: Adapted from FAO, FAO Yearbook of Fishery Statistics, Food and Agriculture Organization of the United Nations, Rome, 2006. With permission.

commodity accounting to 16.5% of the total value of internationally traded fishery products in 2004. The United States imported 50,000 t shrimp worth U.S.$ 3.7 billion in 2004. Ground fish, which included hoki, cod, haddock, Alaska pollock, tuna, and salmon, followed shrimp at 10.2% of the total value of the trade in 2004.14 During the period 2006–2007, India exported an amount of 612,641 t seafood worth U.S.$ 1.85 billion, up 12.7% over the previous year. The European Union (EU) continued to be the major market for Indian marine products by value, followed by Japan and the United States. Of these, frozen shrimp was 54% of the total value. The United States followed by the EU was the major importers of Indian shrimp.16

2.3.2

DEMAND AND CONCERNS

During 2004 and 2006, a total of 68.9 and 69.0 mt of fishery products were used for human consumption, with a per capita food fish supply of 13.6 and 13.4 kg, respectively.14 World’s total demand for fish and fishery products is projected to increase to 183 mt by 2015, at an annual growth rate of 2.1%. The growth rate was 2.7% in the past decade, which indicated a declining trend in seafood catch. It has been predicted that the share of pelagic and demersal species in total fish output would decline from 30.8 and 16.2% in 1999/2001 to 24.5 and 12.7% by 2015, respectively. The deficit of all types of fish combined would amount to 9.4 mt by 2010 and to 10.9 mt by 2015.14,17 The likely global trends for fish supply, demand, and consumption have been forecast by the International Food Policy Research Institute (IFPRI) in collaboration with the World Fish Center, which have projected 130.1 mt production (with 41% share from aquaculture) for the year 2020. The forecast predicts that apart from rising population, the demand for fishery products is likely to rise partly due to changing food habits and the increasing purchasing power of consumers in several developing countries.18

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The increasing demand for seafood has caused excessive fishing pressure, leading to overfishing and wasteful use of stocks.19 As a result of competitive trawling and introduction of gears such as purse seines, resources in the 0–50 m depth zone were significantly exploited. Some of the exploited species were sciaenids, silver bellies, elasmobranchs, pink perch, lizard fish, goatfish, threadfins, and eels, whereas resources such as catfish, whitefish, ghol, and flatheads declined in production. This was mainly due to the destruction of juveniles as well as the trampling of the bottom habitat. Another example is of tuna stocks in the Indian Ocean that provides over 1.5 mt, or nearly one-third of the total present in the world, dominated by yellowfin and skipjack tuna. Of these, skipjack stocks appear to be unaffected, whereas yellowfin and bigeye tuna stocks are fully exploited.20 The FAO voiced “serious concern” on overexploitation of stocks of a number of marine fish species and called for their better monitoring and management. It was observed that 17% of fish stocks are overexploited, 7% depleted, and 1% recovering from depletion. Although stocks have been fairly stable for the past 15 years, more than 50% of the stocks of highly migratory sharks and 66% of straddling fish stocks—such as hakes, Atlantic cod, halibut, orange roughy, basking shark, and bluefin tuna—have been ranked as either overexploited or depleted.14 The National Ocean and Atmospheric Administration in its annual report “2006 Report of Status of U.S. Fisheries” observed that out of the 187 fish stocks and multispecies groupings between 3 and 200 mi off U.S. coasts, 47 were overfished and 48 were subject to overfishing.21 In Australia, out of a total of 67 target species, 11 species are classified as overfished. These species include southern bluefin tuna, brown tiger shrimp, grooved tiger shrimp, southern scallop, tropical rock lobster, orange roughy, among others.22 In the west coast of India, out of a total of 34 major species, 13 are overexploited. These include Bombay duck, pomfrets, mackerel, sardines, and lobster (Deshmukh, V. D., Central Marine Fisheries Research Institute, Mumbai, Personal communication, 2007). It has been generally recognized that governments worldwide have failed to prevent overfishing. In a recent analysis of 1519 main species of the FAO world fisheries catch database,14 it was found that 366 fisheries collapsed, although the number of collapses has been stable since 1950s indicating no improvement in the overall fisheries management. Three typical patterns emerged from the analysis of catch series during the period preceding the collapses: smooth collapse (33%), that is, a long regular decline; erratic collapse (45%), that is, a fall after several ups and downs; and a plateau-shaped collapse (21%), that is, a sudden fall after a relatively long and stable persistence of high level of catches.23 Reef fishes are highly vulnerable to overfishing as they need 5–10 years before they reach breeding age. Large parts of the reefs in the Philippines, Indonesia, and Malaysia are becoming depleted of marine life as a result of overfishing and use of unsustainable fishing methods. Recently, the UN World Conservation Union issued a warning that 20 species of reef fishes were threatened with extinction unless appropriate conservation measures were introduced.24 A recent analysis by the Malaysia-based World Fish Center and the IFPRI cautioned that within the next 20 years, fish, which currently accounts for about 7% of global food supplies, would deprive 1 billion people in developing countries of

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their source of protein. The center observed that almost three-quarters of the 130 mt landed in 2000 came from fish stocks already depleted, overfished, or fully exploited. The situation is alarming particularly due to the annual increase in the world population by about 90 million and increasing consumer interests in fishery products. In addition, health-related demands on some fish—such as salmon for their lipids—could add pressure to the already vulnerable fisheries.25 It was observed that although there is appreciable growth in fish farming, it could only partially save the world from a critical situation of shortage of fishery products.26 The United Nations Environment Program (UNEP) in its Global Environment Outlook Yearbook 2007 observed that commercial fish stocks would be depleted by 2050 if overfishing and climate change are not immediately addressed. It was suggested that the number of marine-protected areas should be expanded to circumvent the situation. The governments, which participated in the World Summit on Sustainable Development (WSSD) in 2002, endorsed a plan to develop a network of marine reserves by 2012. The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), administered by the UNEP, suggests corrective steps to address environmental threats ranging from habitat destruction, climate changes, and unrestrained commercial harvesting with a view to protect endangered species.27

2.3.3

UNDERUTILIZED FISHERIES

Although the supply of several commercially important fish species is dwindling, a significant amount of fish, caught as bycatch of shrimp trawling, which despite good nutritional value remains underutilized. Several varieties of pelagic, demersal, and unconventional fish species are not fully utilized as human food.14 Out of a total production of 22.5 mt of demersal and 37.6 mt of pelagic fish, only 13.7 and 18.8 mt, respectively, are used for human consumption. The rest are used as fish meal or discarded in the ocean (Deshmukh, V. D., Central Marine Fisheries Research Institute, Mumbai, Personal communication). Many of the currently underutilized fish have potential as human food.28 These fish, in the order of their possible food value, include anchovy, barracuda, Bombay duck, catfish, croaker, flying fish, garfish, gray mullet, hake, herring, horse mackerel, jewfish, leatherjacket, mackerel, pony fish, ray, rock cod, sardine, scad, Spanish mackerel, spotted bat, tilapia, and others. The underutilized bottom water species include blue ling, roundnose, grenadier, black scabbard, and various small sharks. The global trends in low-cost fish catch and need for their better utilization for human consumption have been discussed extensively.28–31

2.3.4

SOME NOVEL SPECIES

Some seafood items that are not well exploited or offer novel products, offer scope for use as functional food and as sources of nutraceuticals. 2.3.4.1

Antarctic Krill and Other Deep-Sea Fauna

Krill is a herbivore and is the main food for whales, seals, penguins, salmons, and squids and sustains large number of fauna of the Antarctic sea. The main species of

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Antarctic krill are Euphausia superba and Meganyctiphanes norvegica. An adult krill has a body length of 4–6 cm with an average weight of 6 g. The tail of krill, containing 13% protein, 2.8% fat, and 80% moisture, is the only edible part suitable for human consumption.32 Atlantic krill is found in Norwegian coastal waters, and is also abundantly available in Northern Pacific and Antarctic regions. The fact that abundance of krill can go a long way in satisfying the need for alternate protein has led to the worldwide interest in exploitation and utilization of the species.33,34 The stock of krill has been estimated as 360–1400 mt and annual landing has been 300,000–500,000 t during the 1980s, which declined to about 100,000 in the 2000s. The total current global production is about 150,000 t annually.35,36 In a recent exploration, about 600 species of crustaceans, carnivorous sponges, and hundreds of new worms were discovered in the dark waters as deep as 20,000 ft around Antarctica, suggesting that these depths can be sources of abundant marine life. The deeper parts of the Southern Ocean exhibit some unique environmental features, including a very deep continental shelf and a weakly stratified water column, and are the source for much of the deep water in the world ocean. These features suggest that deep-sea faunas around the Antarctic region may be related both to adjacent shelf communities and to those in other oceans. Unlike shallow-water Antarctic benthic communities, however, little is known about life in this vast deep-sea region. Many species were similar to those found around the world, notably in the Arctic, whereas several others were unique to Antarctica. The latter organisms included gourd-shaped carnivorous sponge called Chondrocladia, free-swimming worms, and 674 species of isopod—a diverse order of crustaceans that includes wood lice, sea lice, or sea centipedes. Other exploratory studies conducted during 2002 and 2005 have also led to the detection of marine organisms in water and sediment from 748 to 6348 m in the deep Weddell Sea and adjacent areas. The Weddell Sea is an important source of deep water for the rest of the ocean. These results also suggested that species can enter the depths of the Weddell Sea from shallower continental shelves.37,38 A number of these species could be potential sources of useful compounds, such as sponges (see Chapter 12).39 2.3.4.2

Sea Cucumbers

Sea cucumbers or holothurians are spiky-skinned animals of the phylum Echinodermata (class Holothuroidea). Related species are the sea lily, sea urchin, starfish, and sand dollars. Sea cucumbers are soft, wormlike marine animals commonly found in shallow-water areas of the sea to deep ocean floors and amid corals. Most sea cucumbers are deposit feeders, living on organic matter and associated microorganisms. Sea cucumbers have cylindrical-shaped body with leathery skin, invertebrate endoskeleton just below the skin. It has a life span of 5–10 years and feed on decaying matter that floats on water or that is found in the sand. Although more than 1400 species of sea cucumber exist throughout the world, only about 30 species are considered commercially important. For processing, fresh cucumber is slit and entrails squeezed out. It is then boiled and sun-dried or sometimes smoked. The product formed is one of the most important and highly priced seafood products in the international market and is marketed as beche-de-mer (meaning processed sea slug or sea cucumber). The product is said to cure low blood pressure, kidney disorders,

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and impotence and prevents ageing.40 The high demand and premium value of sea cucumber products have resulted in overexploitation of the resource in some producing countries, leading to its extinction in several habitats resulting in its listing as endangered species of wild fauna and flora. The most expensive sea cucumbers are from the Gulf of Mannar, which include Holothuria scabra (commonly called as sand fish), Holothuria atra, and the Stichopus hermanni (warty sea cucumber). Aquaculture of sea cucumber is being practiced in Japan, China, and Australia.41,42 A process for large-scale seed production of sea cucumber (Holothuria spinifera) consists of rearing 48 h old larvae for 12 days under ideal conditions of temperature of 20–32°C, salinity of 35 ppt, and pH of 7.8, which gives maximum survival of the larvae. The bioactive compounds obtained from sea cucumber are discussed in Chapter 12. 2.3.4.3 Newer Species There is good potential for identifying species from underutilized fish that can have food value and consumer acceptability. Recently, a research program was undertaken to identify newer species that can have high consumer acceptability. Of these species examined, silver smelt (Argentina silus) and blue whiting (Micromesistius poutassou) are two white-fleshed species with good quotas and reasonable stocks. The silver smelt fish is low in fat content (approximately 0.5% in the flesh) and has an excellent white color on cooking. The small bones that could militate against consumption as fillets could be removed from fish mince. Fresh fish, and also mince and gels made from fresh silver smelt, had a good water holding capacity (WHC). These fish, as fillets and value-added products, received good consumer acceptance. Silver smelt fillets (from frozen whole fish) received higher odor, aroma, and preference scores than block frozen fillets, or block frozen mince after 235 days of storage at –28°C. Blue whiting was tested as freeze-chilled fillets packed in a modified atmosphere (MAP; 30% O2, 40% CO2, and 30% N2) or in air. Freezing offered logistic benefits in terms of streamlining production and enabling the products to reach distant markets. When chilled at 2–4°C, the shelf life is 3–5 days, whereas the MAP storage extended the shelf life of the fish to 5–8 days.43,44

2.3.5

MARICULTURE

Mariculture, the aquaculture of marine species, is widely proposed as a significant method to supplement marine fishery products. Southeast Asia has a very active aquaculture sector, with extensive hatchery production, and the contribution of aquaculture to global supplies of fish, crustaceans, and mollusks and other aquatic animals is increasing every year. Total aquaculture production increased from 27.1% in 2000 to 32.4% in 2004, growing at an annual average rate of 8.8% since 1970. This growth rate is much higher compared to a value of 1.2% for capture fisheries and 2.8% for terrestrial farmed meat production system over the same period. The FAO has predicted that by 2015, 39% of all fish for human consumption will come from aquaculture or sea ranching.17 In 2004, global production by mariculture was 30.2 mt, representing 50.9% of the total aquaculture production. Fish is the major group of aquacultured products constituting 47.4% of total production. Mollusks and

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crustaceans are the third and fourth in production volumes, accounting to 22.3% and 6.2%, respectively. The prominent farmed shellfish are black tiger prawn (Penaeus monodon), white leg shrimp (Penaeus vannamei), and scallop (Pecten yesoensis). The major producers of aquacultured fishery products included China (30.6 mt), India (2.4 mt), Vietnam (1.2 mt), Thailand (1.1 mt), Japan (0.7 mt), Norway (0.6mt), and the United States (0.6 mt).14,45 Although aquaculture has been taken up to augment the limited supply of marine fishery products, certain limitations of fish farming have been recognized. Relatively, only a few species are suitable for farming based on current knowledge. Furthermore, many consumers regard farmed fish as inferior to its counterpart obtained from their natural habitat.46 Transgenic fish have many potential applications in aquaculture, but also raise concerns regarding the possible deleterious effects of escaped or released transgenic fish on natural ecosystems.47 A paper that conceptualizes a program to triple the current value of aquaculture in the United States by 2025 to achieve objectives set by the Department of Commerce for the national industry has been prepared recently. It discusses the spatial impact of aquaculture on the marine environment, including marine sanctuaries and marine protected areas in federal and state waters. The paper also identifies plans to increase per capita consumption of seafood, marketing seafood, among others.48 Table 2.3 depicts prospects of seafood production and future scenario.

2.3.6

CONTRIBUTION OF SEAFOOD TO FOOD SECURITY

All over the world, fish has made significant contribution to food security, particularly in the developing countries. Seafood, particularly, marine fish contributes to national food self-sufficiency through direct consumption and through trade and exports. Fishery products constitute a major part of the diet of people, particularly in the coastal belts of developing countries even in small quantities. Countries with low per capita gross domestic product tend to consume generally low-priced fish, which satisfy their animal protein consumption. Globally, about 1 billion people rely on fish as their main source of animal proteins, and in coastal areas the dependence on

TABLE 2.3 Prospects of Seafood Production and Future Scenario Developing countries (particularly Asian countries) will dominate food fish production, from both capture fisheries and aquaculture Overfishing will remain a major concern. Sustainability concerns will increase and motivate environmental regulations and institutions Stocks that are not fully exploited will be fished more heavily Fish will become an increasingly high-value commodity and the shift, in traded products, from frozen low-grade whole fish to value-added products will continue Fisheries and aquaculture technology will address new challenges for reducing and mitigating the environmental impacts of intensive aquaculture Issues concerning safety of marine products including environmental pollution will receive more attention worldwide

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fish is usually higher. About 20% of the world’s population derives at least 20% of animal protein from fish, and some small island nations depend almost exclusively on fish. Seafood products are considered cheap as compared with those from land animals, and hence this makes more than 1 billion people worldwide to rely on fish as an important source of animal proteins. Traditional fish products such as dried or otherwise cured fish can be conveniently stored and transported, making them more readily available to weaker sections of consumers. Because of these reasons, the share of fish in human diets exceeds 25% in many developing countries and much higher in isolated parts of coastal or inland areas in some countries.46 The relative contribution of fish to total animal protein varies greatly from country to country, being the highest (15–25%) in Egypt, Morocco, Oman, and Yemen.14 There is also evidence suggesting that fish can play an important role in maternal, fetal, and neonatal nutrition. Eating fish two or three times a week is being encouraged as part of a healthy balanced diet both for child-bearing women and the family as a whole.49,50 Fish can contribute up to 180 kcal per person per day as a substantial share of the dietary energy.14 The nutritive and functional roles of fish proteins, lipids, and other components have been discussed in Chapters 3 through 5.

2.3.7

GLOBAL CONSUMPTION PATTERN OF SEAFOOD

To understand the nutritional benefits that are derived from seafood consumption (as will be discussed in the subsequent chapters), it is important to know the detailed consumption patterns of seafood products. Global per capita fish consumption has increased from 9.0 kg in 1961, 12.5 kg in 1980 to 16.5 kg in 2003. In 2004, a combined 105.6 mt was used for human consumption, averaging 16.6 kg per person per year. The world average per capita consumption is expected to increase to 18.4 kg in 2010 and 19.1 kg in 2015.14 It is further expected to rise between 19 and 21 kg by 2030.51 It may be noted that the preceding values are higher than the per capita consumption of 11 kg recommended by the World Health Organization for nutritional security. There were variations in consumption in different parts of the world. In 1997, the per capita food fish supply was higher in Oceania with 19.9 kg, followed by Europe (18.5 kg), Asia (17.9 kg), North and Central America (16.7 kg), South America (10.0 kg), and Africa (7.1 kg). In industrialized countries, where diets are generally more diversified as far as animal proteins are concerned, supply has increased from 13.2 to 26.7 mt, with an implied per capita supply progressing from 19.7 to 27.7 kg between 1961 and 1997. Per capita fish consumption values (in kilograms) for some countries for 2003 were 10.9, Australia; 23.6, Indonesia; 5, Iran; 26.2, Myanmar; 52, Republic of Korea; 2, Pakistan.17 Consumption of seafood in the United Kingdom has been increasing over the years. Consumption of fatty fish has risen by 10% since 2004, and the number of people eating shellfish has risen by 8%. Seafood consumption among children in the United Kingdom has also been shown to increase. In the course of the past four decades, however, the share of fish proteins to animal proteins has exhibited a slight negative trend due to a faster growth in consumption of other animal products. Per capita demand for finfish would account for 13.7 kg in 2010 and 14.3 kg in 2015, respectively, whereas demand for shellfish and other aquatic animals would be 4.7 kg and 4.8 kg, respectively.17 An analysis of consumption pattern showed that fresh fish (53.7%) was the most preferred item, followed

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TABLE 2.4 Per Capita Food Fish Supply by Continent and Economic Grouping in 2003 Region World Africa North and Central America China Europe Oceania

Per Capita Food Fish Supply (Kilograms per Year) 16.5 14.2 18.6 25.9 19.9 23.5

Source: Adapted from FAO, The State of World Fisheries and Aquaculture, Food and Agriculture Organization of the United Nations, Rome, 2006. With permission.

by frozen (25.7%), canned (11.0%), and cured fish (9.0%).17 An amount of 45 mt of marine finfish accounted for 75% of the per capita fish consumed in 1997. Shellfish (crustaceans, mollusks, and cephalopods) shared the remaining 25%. Demersal fish are highly preferred in North Europe and North America. In these countries, as much as 60% of all the fish consumed is either fillet or value-added product. Cephalopods are consumed in certain Mediterranean and Asian countries, and to a much lesser extent in other continents. Crustaceans are highly priced commodities and their consumption is limited to the affluent countries.52,53 Table 2.4 indicates per capita consumption of fish in some countries.54

2.3.8

CHANGING TRENDS IN CONSUMPTION

Marketing campaigns launched for some fish products tend to affirm that consumption of fish is an appropriate means of satisfying the consumer’s need for variety and for nutritious, tasty, healthy, and fashionable foods. These campaigns together with changes in consumer lifestyles have contributed to increased demands for fishery products. An important trend in consumer lifestyle is healthy eating; consumers preferring food items that are low in calorie, fat, sugar, and sodium; and are capable of protecting health. Furthermore, modern consumers are also aware of health hazards associated with food, such as the presence of pathogenic microorganisms, parasites, viruses, and industrial pollutants. Consumers expect a positive assurance that the food product including seafood should be safe, tasty, easy and quick to prepare, low in calories, easy to digest, and nutritive. It has been noted that the modern consumers prefer two distinct types of seafood products. The first type includes fresh, chilled products that are conveniently packaged, processed, and ready-to-cook, such as salmon steaks or hoki loin fillets. The second type includes processed, chilled, ready-to-eat seafood products, such as cold smoked salmon or hot smoked mussels. In both types, a need for convenience and easy handling has been focused.

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Generally, consumers relate freshness of fishery products to the inherent quality of the freshly caught fish. They consider that the product retains its original characteristics only if the time lapse after harvest is short. This may not always be correct since eating quality is a subjective attribute. The flavor of cooked cod, for example, has the strongest intrinsic characteristics after 2 days of storage in melting ice. Many fatty species such as salmon, ocean perch, and halibut improve much in terms of flavor, taste, and texture during the first 2–4 days in ice. This is mainly due to the redistribution of fat, and the development of flavor components such as amino acids, nucleotides, or sugars, which are produced by autolytic processes occurring during rigor mortis.55 Butterfish, cod, crab, flounder, haddock, hake, lake perch, mussel, oyster, pollock, scallop, sole, whitefish, and whiting have delicate texture. Bluefish, crayfish, lobster, mackerel, orange roughy, salmon, sardine, shrimp, and tilapia are species having moderate texture. Clams, catfish, grouper, halibut, mahi-mahi, marlin, monkfish, octopus, salmon, sea bass, seer fish, shark, snapper, squid, swordfish, tilefish, tuna, and wolffish are characterized by hard texture. Table 2.5 presents tentative classification of some seafood according to their flavor. Modern consumers prefer processed foods that are more convenient to handle, store, and prepare. The consumers insist that such products also possess high quality, freshness, nutrition, and health. They would also appreciate flavorsome food items produced by more ethical methods, including environmental friendly processes and economically acceptable behavior. The emergence and growth of supermarkets facilitate a greater penetration of such seafood products in accordance with consumer interests.56 Modern seafood technology is also aiming to address these changing consumer interests.57 The trends in seafood consumption in the United States have been shown by a number of recent surveys conducted by professionals.58–60 According to the U.S. National Oceanic and Atmospheric Administration (NOAA), overall seafood consumption in 2002 was 7.1% of the total food consumption, with an annual per capita purchase of 5 kg fish, consisting mostly of fresh and frozen items. A survey conducted by the National Fisheries Institute showed that elderly people preferred seafood to red meat since these people were aware of the nutritive merits of seafood. People in the age group of 50–64 are 71% more likely to eat fish, whereas those above the age of 65 ate 41 times a year. It was observed that the per capita consumption of fish might reach about 27–31 kg in the next 15 years in the United States.59

TABLE 2.5 Tentative Classification of Some Seafood According to Their Flavor Mild

Moderate

Strong

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Cod, crab, flounder, grouper, haddock, hake, halibut, lobster, monkfish, orange roughy, pollock, scallop, seer fish, sole sea bass, shrimp, snapper, squid, tilapia, tilefish, white pomfret, wolffish Black pomfret, butterfish, catfish, crayfish, lake perch, lobster, mahi-mahi, octopus, shark, sturgeon, orange roughy, shrimp, tilapia, tuna, whitefish, whiting Bluefish, clams, Indian salmon, mackerel, marlin, mussel, oyster, salmon, sardine, swordfish

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Consumers’ preferences for value-added seafood products were shown in another survey. Grilled seafood were more popular, particularly, preseasoned, ready-to-grill items, whereas boil-in-bag products were less preferred. Fried products were less attractive, whereas boneless fillets were highly popular. Traditional battered and breaded items, which once formed 70–80% of the products consumed, decreased to a consumption level of 50%. Minimally processed products such as salmon portions made up the rest.60 Convenience is the driving force behind daily food choices in the United States.60,61 Salmon was bought mainly due to recognized health benefits associated with consumption of the fish; taste and flavor were secondary in this respect. Trout is another preferred species, fresh fish commanding increased acceptance than frozen samples.62 There was an overall downward trend in the U.S. per capita seafood consumption from the mid-1980s, which was due to insufficient supply, lack of convenient products, high prices, lack of perceived value, and lack of general promotion campaigns. Table 2.6 indicates general trends in global patterns of fish consumption. According to the report “Fisheries of the United States” prepared by the U.S. National Marine Fisheries Service (2005), the total consumer expenditures for fishery products in 2005 was $65.2 billion.63 The U.S. Department of Agriculture projects that seafood/fish will record 26% gain in consumption between 2000 and 2020. In terms of protein preferences, 65 million Americans over 55 years of age significantly favor fish/seafood over beef and chicken. The interest in seafood has been triggered by the Dietary Guidelines issued by the U.S. Food and Drug Administration (FDA) that favors consumption of the commodity. This is coupled with the awareness of research data on the role of omega-3 fatty acids in health protection and calorie consciousness and obesity control. Shrimp rose to 40% in chain menu entrees over the past 5 years. Tuna, salmon, pollock, catfish, cod, crab, tilapia, clams, and scallops formed the top 10 most frequently consumed fish items in 2003. Sales of nonbreaded frozen fish rose by about 16%, frozen nonbreaded shrimp by 8.5%, and frozen seafood meals/entrees by more than 9%. Fresh refrigerated seafood was the ninth fastest growing supermarket category in 2003. Seafood consumption at home has also increased. More than 28% of people ate fish two or more times a week at home, 32% at least once a week, and 17% more than once a month. During the past decade,

TABLE 2.6 General Trends in Global Fish Consumption Pattern The amount of fish caught in developing countries that is shipped to developed countries is increasing Per capita fish supplies in developing countries are low and often stagnating or even decreasing Fish supplies for urban consumption in developing countries are increasing, which is detriment to the rural consumption Per capita fish consumption differs widely among different urban income groups, the lower income groups consuming less but spending a larger percentage of their income on fish Supply is shifting in favor of the more expensive species and away from cheap species Fish prices are rising faster than prices of meat, especially poultry

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growing consciousness of the environment, sustainable harvesting practices, and the overfishing of key species has caused a shift in preference toward farm-raised fish such as red mullet, salmon, catfish, halibut, trough, red snapper, shrimp, and carp (Nelson, P., Personal communication, February 20, 2007). Table 2.7 presents consumer preference of seafood in the United States in 2004.64 Seafood, however, has recently attracted some concerns with respect to consumption in the United States. There has been a fear regarding the presence of higher levels of mercury in some fish such as swordfish, shark, king mackerel, and tilefish. Contamination of seafood with pathogens has been another problem. The U.S. FDA has cautioned women who are or those who may become pregnant, or are breastfeeding, and children up to 12 years to limit their intake of some seafood and completely avoid certain others.65 The FDA has also advised consumers to avoid eating raw oysters harvested in the Pacific Northwest as a result of increased reports of illnesses associated with the naturally occurring bacteria Vibrio parahaemolyticus known to cause gastrointestinal illness. Consumers were advised to cook oysters before consumption to reduce the risk of infection from bacteria that may be found in raw oysters. Despite these problems, most Americans have felt that the health advantages of eating seafood generally outweigh the risks. The report by the Institute of Medicine (IOM) issued at the request of a division of U.S. Department of Commerce could help lay to rest outgoing fears that contamination from pollutants such as methyl mercury and microorganisms including viruses make seafood consumption unsafe. According to the IOM, an average person can consume more fish than they do. While the benefits of consumption of seafood are many, selecting fish species or consuming a mixture of species may avoid any potential risks posed

TABLE 2.7 Consumer Preference of Seafood in the United States in 2004 Seafood

Preference

Shrimp Tuna Salmon Pollock Catfish Tilapia Crab Cod Flatfish

4.2 3.3 2.2 1.3 1.1 0.7 0.6 0.6 0.3

Note: The grading is on a maximum of 5. Source: Adapted from Otwell, S., National Academy of Sciences Report, Seafood Choices, 30th Annual Seafood Science and Technology Society of the Americas Conference, St. Antonio, TX, November 13–16, 2006. With permission.

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by environmental contamination of fishery products.66 Table 2.8 gives guidelines for consumption of seafood by different population segments given by the IOM (seafood affirmed as healthy food choices). A recent survey has shown that 50% of U.S. consumers are aware of these dietary guidelines. However, only 15% of the consumers have changed their diet to include more seafood.60 The interests in seafood consumption have increased in other countries too. In Japan, during 1965–1998, demand for fish paralleled with the increase in average income. Elderly people favored sashimi or sushi products, whereas the younger generation preferred cooked or grilled steaks and fillets of tuna. Quantities of fish consumed in restaurants as ready-to-eat products also increased substantially in the country.56 Consumption of fish and seafood in Europe is predicted to increase in all major European markets. This is attributed to a number of factors including consumers’ attitudes and lifestyles, recent fear over meat safety, and increased opportunities for value addition of fish and fish products. It was shown that the main species presently consumed in Europe are mussel and cod followed by tuna, herring, cephalopods, sardines, salmon, shrimp, and trout.55 Recently, the EU has backed a collaborative seafood project entitled “SEAFOODplus” to investigate the benefits of seafood for the consumer as well as related issues in aquaculture, the environment,

TABLE 2.8 Guidelines for Consumption of Seafood Population Segment

Consume

Avoid

Women who are or may become pregnant or who are breast-feeding Children aged 12 and below

Reasonable amount—two 3 oz servings per week, but can safely consume up to 12 oz/week

Large predatory fish such as shark, swordfish, tilefish, and king mackerel

As mentioned earlier except that serving sizes should be age-appropriate Two 3 oz servings per week. If more than two servings per week, choose a variety of seafood to reduce the risk of exposure to contaminants from a single source Two 3 oz servings per week. There may be additional benefits by selecting seafood high in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), although supporting evidence is limited

Large predatory fish such as shark, swordfish, tilefish, and king mackerel

Adolescent and adult males, and women who will not become pregnant

Adult men, and females who are at risk of coronary heart disease

—

Source: Adapted from Institute of Medicine, Seafood affirmed as healthy food choice, http://www8. nationalacademies.org/onpinews/newsitem.aspx? RecordID=10172006 (Newsletter, October 18, 2006, Institute of Food Technologists, Washington, DC).

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and the economy.67 Total fish production and consumption profiles in the 13 new countries joining the EU are also available.68

2.4

CORAL REEF AND CORALS

Coral reefs are massive deposits of calcium carbonate in the oceans, which harbor a rich and diverse ecosystem of animals. They are produced primarily by corals with minor additions from calcareous algae and other organisms that secrete calcium carbonate. The skeletal remains of corals and plants on the reef may be considered as marine equivalent of tropical rain forests. The carbonate in the corals has two distinct mineral forms—calcite and aragonite, the latter containing significant amounts of magnesium. The reefs are unique among marine associations in that they are built up entirely by biological activity. Coral reefs are widespread and are also found in the clean coastal waters of the tropics and subtropics, which give optimal conditions such as moderate temperature and good sunlight favoring the growth of reef-forming organisms. It has been estimated that coral reefs occupy about 600,000 mi2 of the earth’s surface, representing about 0.17% of the total area of the planet. On the continental shelves of northern and western Europe, extensive reefs are formed, at depths of 60–2000 m. The age of reefs extends to thousands of years; the Great Barrier Reef of Australia is said to be more than 9000 years old. Coral reefs occur in many different sizes and shapes, resulting from particular hydrological and geological situations that recur in different areas of the tropics. The reefs, in general, are grouped into one of the three categories: atolls, barrier reefs, and fringing reefs. Atolls are usually easily distinguished because they are the remodified, ring-shaped reefs that rise out of very deep waters far from land and enclose a lagoon. With few exceptions, atolls are found only in the Indo-Pacific area. Barrier reefs and fringing reefs, however, tend to grade into each other and are not readily separable. Some major physical factors that limit coral reef development are temperature, depth, light, salinity, sedimentation, and emergence into air. Significant contributions to CaCO3 deposits on reefs are made by mollusks of different types including various giant clams, sea urchins, sea cucumbers, starfish, and feather stars.2,69,70 Corals, the major organisms that form the basic reef structure, are members of the phylum Cnidaria, class Anthozoa, and order Madreporaria, which include diverse forms such as jellyfish, hydroids, the freshwater Hydra, and sea anemones. There is a bewildering array of other organisms associated with reefs. Corals secrete an external calcium carbonate skeleton, whereas anemones do not. The rate of growth of different tiny corals varies widely. For example, members of the genera, Acropora (stag horn coral) and Pocillopora grow rapidly and they represent a considerable proportion of tropical coral reefs. Stony corals are the foundation of coral reef ecosystems. Coralline algae (algae that secrete calcium carbonate often resembling corals) contribute to the calcification of many reefs. These red algae precipitate CaCO3 as the corals do, but they encrust and spread out in thin layers. Shallow-water corals owe their beautiful colors in part to the symbiotic algae, which live inside the coral cells.2,70 Sponges are abundant on reefs, but they have little to do with reef construction. About 27 species of sponges have been found on the reef flats. Siliceous sponges (Demospongiae) may however be important in holding coral and rubble together. The

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important genera of sponges are Callyspongia, Oceanapia, Haliclona, Axinella, and Sigmadocia spp.2,69,70 Marine sponges are the most primitive multicellular animals and contain many new metabolites, many of which have shown to possess diverse biological activities (see Chapter 12). These organisms are difficult to classify due to the few available useful morphological characteristics. Recently, a comprehensive taxonomy was published, which provides the state of the art.71

2.4.1

SYMBIOTIC ASSOCIATIONS ON THE REEFS

Corals generally form associations with other reef species. Symbioses between photosynthesizing organisms (e.g., cyanobacteria and dinoflagellates, and diatoms and algae) and several invertebrate corals including poriferans, cnidarians, ascidians, and mollusks have been reported. These associations are particularly operating in tropical coral reefs. Symbiosis is also influenced by physical and environmental factors such as depth-dependent light and temperature, and seasonal fluctuations in these parameters.72–74 Many of these associations may be ecologically important and play a role in maintaining the health and diversity of reef systems, rendering it critical to understand the influence of symbiotic organisms in mediating responses to perturbation.75 For instance, the importance of symbiotic association with crabs in reducing adverse effects of sediments deposited on corals has been indicated. Mortality rates of two species of branching corals were significantly lowered by the presence of crabs. All out-planted corals with crabs survived, whereas 45–80% of corals without crabs died within a month. In surviving corals that lacked crabs, growth was slower and tissue bleaching and sediment load were higher.76

2.4.2

REEF-ASSOCIATED FISHERIES

Several hundred fish species are found on the reefs, which contribute immensely to biological structure and integrity of the reefs, the notable ones being butterfly fishes (Chaetodontidae), parrot fishes (Scaridae), clown and damsel fishes (Pomacentridae), and lion and scorpion fishes (Scorpaenidae).2,77 According to the FAO, reef-associated fisheries make up about 10% of total world marine fishery landings. Reef fisheries provide food, livelihood, and income for millions of people in the tropics and subtropics; 20–25% of all reef fishes considered globally are caught in developing countries. Mollusks alone contributed about 30% of total reef fauna. Favia, Porites, Acropora, Tubipora, and Montipora spp. were found to contribute about 80% of the total coral populations. Sepia spp. was found to be the vulnerable molluskan species from this area.78 Reef fishes are nutritionally and economically significant and contribute to food security, whereas biologically they are vulnerable to both overexploitation and degradation of their habitat. Relative to other global fisheries, reef fisheries are undermanaged, underfunded, undermonitored, and as a consequence, poorly understood or little regarded by national governments. Patterns of changes in reef fishery ecosystem during the past 30 years in 16 tropical countries have been recently recognized. It was pointed out that sustaining reef fish fisheries and conserving biodiversity can be complementary and is important in poverty alleviation programs.46,79

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2.4.3

41

BLEACHING AND OTHER PROBLEMS WITH CORAL REEFS

Coral reefs have been recognized to be vulnerable to destruction. A number of reasons have been found responsible for the situation. These include the action of bioeroding organisms, coastal pollution, overfishing, coral mining, and recreational activities, among others. Bioeroding organisms, consisting of lionfish, butterfish, wrasses, gastropods (Lambis spp.), and bivalves (Tridacna spp.) have been found to be involved in coral destruction. In addition, coastal pollution can have a detrimental effect on coral species. The dumping of fly ash, for example, has shown to affect the recolonization of Acropora formosa. The release of sewage has affected the corals by spreading through the massive sediments. Infectious diseases are recognized as significant contributors to the drastic loss of corals observed worldwide. However, the causes of increased coral disease prevalence and severity are not well understood. The effect of nutrient enrichment on the progression of black band disease (BBD) has been recognized. The increased use of commercial fertilizer and resulting availability of nutrients resulted in doubling of BBD progression and coral tissue loss in the common reef framework coral Siderastrea siderea. These findings provide evidence that the impacts of this disease on coral populations are exacerbated by nutrient enrichment. Curtailing excess nutrient loading may be important for reducing coral cover loss due to BBD.71,80,81 Coral reef fisheries started to decline a few centuries ago. During the past few decades, increasing fishing pressures have resulted in marked decline in several species, ranging from groupers in the tropical western Atlantic to the bump head parrot fish and others. About 50 coral reef fishes are listed as threatened and these make up 60% of all marine fish species assessed.47,81,82 Extensive coral mortality can be attributed to natural stresses such as coral bleaching, catastrophic low-tide events, and storms.83 Pollution, overfishing, coral mining, higher localized incidence of ciguatoxic fishes, and recreational activities are other problems that threaten the very existence of coral reefs reducing its resilience and ability to recover in the face of natural or man-made catastrophes. Globally, more than 40% of coral reefs habitats are degraded, which in turn have adversely affected fisheries that are economically important to coastal communities.47,84 Since corals have a narrow range of temperature tolerance, the increased ocean temperature as a result of global warming can have adverse impact in terms of bleaching and mortality. When ocean temperatures exceed 28–30°C, corals become stressed and eject the algae that live inside them and give them their color. Without the food that the algae provide them through photosynthesis, the corals appear to be bleached and could starve.85,86 Recently, scientists from the United States and Australia launched a joint effort to understand the influence of factors such as sea surface temperatures, ultraviolet light exposure, turbidity, and weather changes on the bleaching of corals with a view to reduce stresses such as recreational fishing and boating. The program uses satellite and expert system observations from the NOAA with on-site data from two Australian research institutes. It was pointed out that except the Central Pacific Ocean, an El Nino event in 1998 resulted in extensive bleaching of coral in every major reef area, with mortality as high as 90% in parts of the Indian Ocean.87 Founded in 1996, the Reef Check Foundation

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is an international nonprofit organization dedicated to the conservation of coral reefs (http://www.reefcheck.org/).

2.4.4

EFFORTS FOR RESTORATION OF CORALS

In light of the deteriorating state of coral reefs worldwide, the necessity of restoring corals has been seriously felt, resulting in worldwide efforts during 1994–2004. Several approaches in this area include construction of artificial reefs (ARs), mariculture, and restocking. ARs have been used to control beach erosion, mitigation of detrimental impacts on habitats, conservation of biodiversity, and test ecological theories.88 The ability of benthic AR communities, mainly filter feeders such as bryozoans, bivalves, sponges, and tunicates, to resemble those of a natural reef is of great use in rehabilitation and restoration of degraded marine habitats. Construction of AR makes use of low-profile structures such as shipwrecks in the seabed to mimic natural reef. Despite the efforts in this direction, understanding of the interactions between artificial and natural reefs is poor and doubts exist on the ability of ARs to mimic adjacent natural reef communities, performance of ARs and their possible effects on the natural surroundings. Furthermore, distinct differences in coral species count, living cover, and diversity were found between the artificial and its neighboring natural reef. Although the species composition on ARs may resemble that of natural reefs after approximately 20 years, obtaining a similar extent of coral cover may require a full century.89 Restocking is an approach used in some areas of Southeast Asia as an attempt to restore overexploited reef fish and invertebrate populations. Mostly these initiatives involve the release of hatchery-produced or small wild-caught fish, which include grouper (Serranidae), rockfish (Scorpaenidae), and snapper (Lutjanidae). Effective restocking will require careful studies to determine appropriate species, timing and location of release, and follow-up monitoring. Marine protected areas are widely advocated as an appropriate fishery management tool for coral reef-associated fisheries. Currently, these techniques have found only limited success. Fisheries development and management needs to be based on clear objectives that address both food production and ecosystem maintenance.47 In addition, a gardening concept, where coral materials (nubbins, branches, and spats) are maricultured to a size that is suitable for transplantation, has also been examined. The use of nubbins (down to the size of a single or few polyps) has been suggested and employed as a unique technique for mass production of coral colonies. Substrate stabilization, developing colonies on a three-dimensional structure, and the use of molecular/biochemical tools are a part of novel technology approaches developed recently.90 In the past 10 years, Reef Watch Marine Conservation (RWMC; www.reefwatchindia.org) has become one of the leading organizations in India working on marine and coastal conservation issues.

2.5 SEAWEED Seaweed (also known as kelp forests) are huge collections of brown macroalgae, which resemble thick forests and are collectively called “seaweed”—considered one of the commercially important marine living renewable resources. Kelps are attached

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to the substrate by a structure called holdfast rather than by true roots. The stem arises from the holdfast, which ends in one or more broad, flat blades. Kelps obtain their nutrients directly from the sea. Luxuriant growth of seaweed occurs in the shallow and deep waters of sea up to 150 m depth and also in estuaries and backwaters. In contrast to most algae, which are small extending to a few centimeters, the major kelps are giants, with lengths equivalent to that of trees on land. Such massive plants grow upward from the bottom and spread their blades at the surface of the water where they obtain maximum amount of light. Kelp beds are dominated by the genera Macrocystis, Laminaria, Pterygophora, Nereocystis, and Ecklonia spp. The Pacific coast of both North and South America is dominated by Macrocystis, whereas Laminaria is dominant in Atlantic waters and in Japan. On the basis of pigmentation, seaweeds are generally classified into four main groups, namely, green algae (Chlorophyceae), blue-green algae (Cyanophyceae), red algae (Rhodophyceae), and brown algae (Phaeophyceae). Red and brown algae are found almost entirely in marine environments and are commercially important. The green and blue algae are more common in freshwater and land.91 Brown seaweed is one of the most abundant seaweed groups of economic importance. Within this group, seaweed belong to Sargassum are widely distributed in tropical and subtropical regions. More than 250 species have been identified under the genus.91 Brown algae are multicellular and most are macroscopic, some growing as long as 45 m or more. Like all photosynthetic eukaryotes, the brown algae possess chlorophyll a. Carotenoids such as fucoxanthin give characteristic color to these algae. The red algae, Rhodophyta, is a large morphologically diverse group of algae consisting of more than 700 genera and 6000 species, which are found at a maximum depth of 200 m. Other than red, rhodophytes can also be black, brownish, violet, yellow, or green. The thallus is usually red to violet due to the pigment phycoerythrin, some species also contain the blue pigment phycocyanin, and all species contain chlorophyll a but not chlorophyll b. Rhodophytes use chlorophyll in conjunction with accessory pigments including phycocyanins, phycoerythrins, and allophycocyanins (phyco in Greek means seaweed). Among the red algae, the genus Gracilaria, consisting of more than 40 species contributes to about 70% of the raw materials required for the production of hydrocolloid agar. Red algae are an important source of commercial colloids including agar used for various purposes.70,91–93 World aquatic plant production in 2004 reached 13.9 mt (worth U.S.$ 6.8 billion), most of which came from China, the Philippines, Republic of Korea, and Japan at quantities of 10.7, 1.2, 0.55, and 0.48 mt, respectively. Japanese kelp (Laminaria japonica) showed the highest production of 4.5 mt followed by 2.5 mt of Wakame (Undaria pinnatifida), and 1.3 mt of Nori (Porphyra tenera).14 Developing countries have good scope for commercial production of seaweed.94 In India, which has a coastal line of 7000 km, 770 species are harvested. However, despite such a huge diversity, no single seaweed species is being commercially exploited in the country.95,96

2.6 MICROALGAE As mentioned earlier, the marine microalgae is the largest primary biomass, which covers almost three-quarters of the earth’s surface to a depth of up to 200 m, and forms the base of the marine food web through their photosynthetic activity.97 In the

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wild the microalgae is invisible to the naked eye, although under certain conditions, they can actually be seen even from a space satellite. Microalgae are the primary producers of oxygen in aquatic environments, which are probably among the first cellular living entities that have played a significant part in the biology and geology of the oceans. Diatoms (class Bacillariophyceae) are a major group of microalgae, and are one of the most common types of phytoplankton. Most diatoms are unicellular, although some form chains or simple colonies. Diatoms may occur singly or in chains. The diatoms are enclosed within unique glass (pillbox or frustule) and have no visible form of locomotion. Each box is composed of two parts or valves fitting over another. The living part of the diatom is within the box, which is made up of silicon dioxide. These frustules show a wide diversity in form, some are quite beautiful and ornate, but usually consist of two asymmetrical sides with a split between them, hence the group name. Diatoms such as Chaetoceros calcitrans, C. gracilis, C. muelleri, and Skeletonema costatum are commonly used as live feeds for bivalve mollusks, crustacean larvae, and zooplankton.2 Dinoflagellates (Dinophyceae) form a significant part of primary planktonic production in both oceans and lakes. They are microscopic, usually unicellular, flagellated, often photosynthetic protists, and are commonly regarded as algae. They have two flagella and lack extended silicon cover, rarely form chains, and reproduce by simple fission like diatoms. The dinoflagellates could be autotrophs, mixotrophs, osmotrophs, phagotrophs, or parasites. Organisms in this phylum have remarkable morphological diversity including nonflagellate amoeboid, coccoid, palmelloid, or filamentous. Approximately 130 genera with about 2000 living and 2000 fossil species have been described, most of them belonging to the marine habitat. They are characterized by a transverse flagellum that encircles the body and a longitudinal flagellum oriented perpendicular to the transverse flagellum. This imparts a distinctive spiral to their swimming motion. Both flagella are inserted at the same point in the cell wall. The cell wall of many dinoflagellates is divided into plates of cellulose. These plates form a distinctive geometry/topology known as tabulation, which is the major means for classification. If dinoflagellates become extremely abundant (2–8 m/L), significant toxin formation occurs, which has a devastating effect. Such extreme concentrations are called “red tide” and responsible for causing mortality in fish and invertebrates (see Chapter 15). Cryptophytes, a major species of phytoplankton, are unicellular flagellates with 12–23 genera comprising 200 species. Cells have a flattened asymmetrical shape with two anterior flagella. They are distributed both in freshwater and marine environments. A few species are colorless heterotrophs, but most of them possess various colored plastids with chlorophylls, carotenoids, and phycobiliprotein. Alloxanthin is a xanthophyll that is unique to cryptomonads. The phylum Heterokontophyta is the most diverse algal group with huge commercial and biotechnological potentials. They range in size from microscopic single cells to giant kelp averaging several meters. They are primarily characterized by the similarities in their ultrastructural and biochemical characteristics. The phylum Euglenophyta encompasses unicellular flagellate organisms and comprises 40 genera and 900 species. The chloroplast originating from the green algae contains chlorophylls a and b and carotenoids such as neoxanthin, diadinoxanthin, and β-carotene. Owing to difficulties in culturing, no

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direct economic significance has been associated with this phylum. The phylum Haptophyta is a group of unicellular flagellates having brownish or yellowish-green color due to the presence of chlorophylls a and c1/c2, and carotenoids such as β-carotene, fucoxanthin, and others. The cells are commonly covered with scales that aremainly made up of carbohydrates or calcium bicarbonate, and hence many species produce calcified scales. About 70 genera and 300 species have been isolated to date, most being tropical marine species forming food for aquatic communities.2 Although not algae in true sense, cyanobacteria, commonly referred to as bluegreen algae, are oxygenic photosynthetic prokaryotes that show large diversity in their morphology, physiology, ecology, biochemistry, and other characteristics. Currently, more than 2000 species of cyanobacteria are recognized. These organisms are distributed widely not only in saltwater but also in freshwater, brackish water, polar areas, and hot springs. Cyanobacteria are generally considered to be associated with marine plants and animals. The cyanobacteria of the genus Prochlorococcus are the smallest (0.6 μm diameter) and most numerous of the photosynthetic marine organisms. It has been estimated that a drop of seawater contains up to 20,000 cells of organisms belonging to Prochlorococcus. Prochlorococcus and another marine pelagic Synechococcus contribute largely to global oxygen production. Their associations range from casual encounters to obligate symbioses, providing unique opportunities for bacterial adaptation. Some also exist in symbiotic association with sponges, ascidians, echiuroid worms, planktonic diatoms, and dinoflagellates in marine environments. The associations have helped these organisms survive in highly stressful growth conditions such as high salinity, high and low temperatures, and limiting nutrient conditions. Their metabolic adaptability has resulted in the production of several secondary metabolites, thereby making them important sources of drugs and other bioactive compounds.2,3,97

2.7 MARINE BACTERIA These organisms may be autotrophs or heterotrophs. Autotrophs live on inorganic material and use CO2 as the sole source of carbon obtaining their energy from sunlight (phototrophs) or from chemical reactions (chemoautotrophs). Marine heterotrophic bacteria are abundant in sediments and as colonizers of settling particulate matter following plankton blooms. Marine bacteria are involved in nutrient cycling and the degradation of marine organic matter. The role of bacteria in marine food webs has two aspects: First, as primary food sources and second, as components of the microbial communities of marine animals.98,99 Generally, they form part of a symbiotic association with hosts such as algae. The majority of these marine microbial organisms cannot be cultured under artificial laboratory conditions and is thus not accessible for detailed taxonomical and physiological characterizations. However, advanced molecular techniques have altered the perspective on naturally occurring diversity and distribution of such marine microorganisms. Marine microorganisms as sources of several important nutraceuticals will be discussed in Chapter 12. Figure 2.1 depicts various marine ecosystems that are useful and hence of human interest. To summarize, the sea is a rich reserve of a multitude of resources. Although the food potential of several fishery items has been well realized, a number of species,

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Marine Products for Healthcare Mammals Recreation (tourism, hunting, and angling)

Birds Fish Mollusks

Species number

Increasing

Crustaceans

Food

Other commodities (ornamental species, shells, etc.)

Sponges Bryozoa Other invertebrates Macrophytes

Natural products (pharmaceuticals, antifouling compounds, glues, etc.)

Phytoplankton Microbes (bacteria, fungi)

FIGURE 2.1 Diagram showing various classes of marine organisms that are of human interest.

presently considered as underutilized, do possess nutritional properties comparable to those of popular species. In addition, ocean reserves such as seaweed, corals, microalgae, and microorganisms have not been fully exploited either as food or sources of nutraceutical and bioactive compounds. The usefulness of these organisms for human and animal healthcare is being unraveled by research. These will be discussed in the subsequent chapters.

REFERENCES 1. Pennington, J. A. T., Food composition databases for bioactive food components, J. Food Comp. Anal., 15, 419, 2002. 2. Nybakken, J. W., Marine Biology: An Ecological Approach, 4th Ed., Addison-Wesley, Reading, MA, 1997. 3. Matsunaga, T. et al., Marine microalgae, Adv. Biochem. Eng./Biotechnol., 96, 165, 2005. 4. Steele, R. L., Comparison of marine and terrestrial ecosystems, Nature, 313, 355, 1985. 5. Madigan, M. T. and Martinko, J. M., Biology of Microorganisms, 11th Ed., Pearson, London, 2005. 6. Haard, N. F., Specialty enzymes from marine organisms, Food Technol., 52(7), 64, 1998. 7. McEdward, L. R., Ed., Ecology of Marine Invertebrate Larvae, CRC Press, Boca Raton, FL, 1985. 8. Khan, M. A. et al., Effects of environmental characteristics of aquaculture sites on the quality of cultivated Newfoundland blue mussels (Mytilus edulis), J. Agric. Food Chem., 54, 2236, 2006. 9. Irigoien, X. et al., Global biodiversity patterns of marine phytoplankton and zooplankton, Nature, 429, 863, 2004. 10. Turner, D., Chemistry takes center stage in marine science, Chem. Int., 28, 4, 2006.

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65. Sloan, A. E., A ‘Fab’ future for fish and shellfish, Food Technol., 59(3), 52, 2005. 66. Santerre, C. R., Farmed salmon: caught in a number game, Food Technol., 58(2), 108, 2004. 67. Institute of Medicine, Seafood affirmed as healthy food choice, http://www8.nationalacademies.org/onpinews/newsitem.aspx? RecordID=10172006 (Newsletter, October 18, 2006, Institute of Food Technologists, Washington, DC). 68. Anonymous, SEAFOODplus’ promises safer, healthier and better seafood products, Infofish Int., 3, 82, 2004. 69. Kuzebski, E., Expansion of the EU: new opportunities for export, Infofish Int., 4, 30, 2001. 70. Sakthivel, M., Ramathilagam, G., and Pushparaj, A., Field study on corals and coral living organisms in Van Tivu, in the Gulf of Mannar, Fishery Technol., 42, 11, 2005. 71. Scott, T. A., Concise Encyclopedia Biology, Walter de Gruyter, Berlin, 1996, p. 320. 72. Hooper, J. N. A. and van Soest, R. W. M., Systema Porifera: A Guide to the Classification of Sponges, Kluwer Academic, New York, 2002. 73. Douglas, A. E., Coral bleaching—how and why? Review, Mar. Pollut. Bull., 46, 385, 2003. 74. Shick, J. M. and Dykens, J. A., Oxygen detoxification in algae invertebrate symbioses from the Great Barrier, Reef Ecologia., 66, 33, 1985. 75. Cerrano, C. et al., Are diatoms a food source for Antarctic sponges? Chem. Ecol., 20, 57, 2004. 76. Trench, R. K., Microalgae–invertebrate symbioses: a review. Endocytobiol. Cell Res., 9, 135, 1993. 77. Steward, H. L. et al., Symbiotic crabs maintain coral health by clearing sediments, Coral Reefs, 25, 609, 2006. 78. Sadovy, Y., Trouble on the reef: the imperative for managing vulnerable and valuable fisheries, Fish Fish., 6, 167, 2005. 79. Michael, S. M., Reef Fishes, Vol. 1, Scott M. Michael, Microcosm, Charlotte, Vermount, 2001. 80. Santhanam, R. and Venkataramanjuam, K., Impact of industrial pollution and human activities on coral resources of Tuticorin (South India) and methods for conservation, Proceedings of the International Coral Reef Symposium, Panama, 1996, p. 177. 81. Voss, J. D. and Richardson, L. L., Nutrient enrichment enhances black band disease progression in corals, Coral Reefs, 25, 569, 2006. 82. World Resources Institute, 2005, http://www.wri.org, accessed September 2007. 83. Pauly, D. et al., Towards sustainability in world fisheries, Nature, 418, 689, 2002. 84. Wilkinson, C. R., Executive summary, in Status of Coral Reef of the World, Wilkinson, C. R., Ed., Australian Institute of Marine Science, Townsville, 2000, p. 7. 85. Kohler, S. T. and Kohler, C. C., Dead bleached coral provides new surfaces for dinoflagellates implicated in ciguatera fish poisonings, Env. Biol. Fish, 35, 413, 1992. 86. Brown, B. E. et al., Marine ecology: bleaching patterns in reef corals, Nature, 404, 142, 2000. 87. Regoli, F. et al., Seasonal variability of pro-oxidant pressure and antioxidant with measurements of the total ROS scavenging capacity, in the Mediterranean demosponge Petrosia ficiformis, Mar. Ecol. Prog. Ser., 275, 129, 2004. 88. Anonymous, Coral reef project aims to pinpoint hot spots, Env. Sci. Technol., 33, 270, 1999. 89. Baine, M., Artificial reefs: a review of their design, application, management and performance, Ocean Coast. Manage., 42, 241, 2001. 90. Perkol-Finkel, S., Shashar, N., and Benayahu, Y., Can artificial reefs mimic natural reef communities? The roles of structural features and age, Mar. Env. Res., 61, 121, 2006.

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91. Rinkevich, B., Conservation of coral reefs through active restoration measures: recent approaches and last decade progress, Environ. Sci. Technol., 39, 4333, 2005. 92. Anonymous, Uses and markets for seaweed products—Malaysia and Thailand, Infofish Int., 4, 22, 1996. 93. Chapman, V. J. and Chapman, D. J., Seaweeds and Their Uses, 3rd Ed., Chapman & Hall, London, 1980, p. 95. 94. Wong, P. F. et al., Proteomics of the red alga, Gracilaria changii (Gracilariales, Rhodophyta), J. Phycol., 42, 113, 2006. 95. McHugh, D. J., Prospects for Seaweed Production in Developing Countries, FAO Fisheries Circular No. 968 FIIU/C968. Food and Agriculture Organization of the United Nations, Rome, 2002, p. 2. 96. Kaliaperumal, N., Seaweed resources, in India—Status, Problems and Management Strategies, Vol. 2, Edward, J. K. P., Murugan, A., and Patterson, J., Eds, SDMRI Research Publ., Tuticorin, 2002, p. 139. 97. Falkowski, P. G., The ocean’s invisible forest, Sci. Am., 287 (July), 54, 2002. 98. Kurano, N. and Miyachi, S., Microalgal studies for the 21st century, Hydrobiologia, 512, 27, 2004. 99. Nichols, D. S., Prokaryotes and the input of polyunsaturated fatty acids to the marine food web, FEMS Microbiol. Lett., 219, 1, 2003.

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Proteins: 3 Seafood Functional Properties and Protein Supplements 3.1 INTRODUCTION Proteins are fundamental and integral food components, both functionally and nutritionally. Proteins are the major structural component of all cells in the body. They also function as enzymes, in membranes; transport carriers; and hormones. The amino acids of proteins serve as precursors for nucleic acids, hormones, vitamins, and other important molecules. From a technological point of view, they determine the physicochemical and sensory properties of proteinaceous foods and hence are increasingly being utilized to perform functional roles in food formulations. The major functional properties of proteins in foods include solubility, gelation, emulsification, and foaming. From nutritional point of view, proteins are a source of energy and amino acids, which are essential for growth and maintenance. Many dietary proteins possess specific biological properties, which make them potential ingredients in health-promoting foods.1 This chapter discusses the functional properties of proteins and protein supplements from seafood, whereas Chapter 4 will discuss their nutritive value and physiological functions.

3.2 SEAFOOD PROTEINS AS DIETARY COMPONENT Proteins from marine sources are excellent sources of functionally active and nutritive proteins that can significantly contribute to human needs.2 Fish has always been recognized as a cheap source of animal protein. Countries with low per capita gross domestic product (GDP) tend to have a higher proportion of fish protein in their animal protein consumption. The share of fish protein in total animal protein expenditure is higher for lower income groups, and poor people consume mostly low-price fish. This shows the importance of low-priced fish as a primary source of protein among poor households in developing countries. For example, the proportion of animal protein derived from marine products in the diet of population in West Africa is as high as 63% in Ghana, 62% in Gambia, and 47% in Senegal.3,4 In many countries, marine fish is mostly used to improve the palatability of diets, which in turn increase total food intake, thereby improving the nutritional status of the consumer.5 Fish can be used to improve the overall protein content of cereal-based diets, which generally lack the essential amino acid, lysine. However, during the past four decades, the share of fish proteins to animal proteins has exhibited a slight negative trend due to a faster growth in consumption of other animal products.3 51

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3.3 PROTEIN CONTENT OF RAW FISH MUSCLE The protein contents of most raw finfish flesh are in the range of 17–22%, with an average of 19%, whereas some fish such as tuna when cooked may contain as much as 30% protein.6 Crustacean (crab, shrimp, and oysters) flesh is slightly higher in proteins. The contents of proteins in crustaceans and cephalopods are as follows: shrimp, 17.0–22.1%; scallop, 14.8–17.7%; squid, 13.2–19.6%; crab, 15–18.4%; lobster, 18.2–19.2%; krill, 12–13%; mussel and oyster, 8.9–11.7%. The amino acid pattern of fish is comparable to that of red meat. The stroma proteins of muscle consist of collagen, elastin, and gelatin. As compared with red meat, fish meat contains only 3% stroma proteins, except sharks, rays, and skates, which may contain up to 10%. The nonprotein nitrogen compounds of fish muscle can influence palatability. The content of nonprotein nitrogen (NPN) is normally higher than that of terrestrial animals, and ranges between 10 and 40%. The NPN contains amino acids, small peptides, trimethylamine oxide (TMAO), trimethylamine, creatine, creatinine, and nucleotides. Shrimp, lobster, crab, squid, and other shellfish generally contain larger amounts of amino acids, which include arginine, glutamic acid, glycine, and alanine, than finfish. The higher contents of these amino acids during the winter season make squids more palatable as compared with those harvested in summer. Demersal fish generally contains larger quantities of TMAO than pelagic fish, and its contents vary from 19 to 190 mg%.7 The structural proteins (myofibrillar proteins, or proteins that make the muscle structure) of fish and shellfish muscles are functionally important components, and their contents range between 65 and 75%, whereas sarcoplasmic (soluble proteins including enzymes) are in the range of 20–35%. Of the structural proteins, myosin constitutes 50–58% in fish and is very important in determining functionality of food products including protein supplements. A myosin molecule is a long rod with two globular heads at one end and tail portion, which has a total length of 155–160 nm. The molecular weight of myosin is approximately 500 kDa. It consists of two large (molecular weight of 200,000 Da, each) and four small (20,000 Da, each) subunits, which have abilities to bind calcium. The tail portion of myosin consists of two polypeptide chains in the form of α-helical coil, which constitutes about 70% of the total α-helix. Figure 3.1 shows the structure of myosin. Myosin forms natural complexes with actin. Molecules of myosin

LMM 90 nm

HMM 60 nm Trypsin

Rod 140 nm 150 nm

LC-2 (DTNB) LC-1 (Alkali) Papain S-2 50 nm

LC-3 (Alkali) LC-2 (DTNB) 10 nm

S-1

FIGURE 3.1 Myosin structure.

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(up to 400) aggregate to form fi laments. Similar to mammalian myosins, fish myosins also possess adenosine triphosphatase (ATPase) activity, regulated by the presence of calcium and magnesium ions. Myosin can be hydrolyzed by trypsin into functional fragments called light meromyosin (LMM) of 150 kDa and heavy meromyosin (HMM) of 350 kDa. The HMM catalyzes ATP hydrolysis and builds a complex with actin, whereas LMM does not have ATPase activity. The HMM after longer incubation with trypsin can be split into two further fragments, S1 and S2; S1 contains ATPase region, actin-binding sites, and two sites for binding light chains. The head region of myosin exhibits appreciable surface hydrophobicity, which influences gelation and emulsification of the protein. The regulatory proteins, which are involved in the contractile mechanism, include actins (F and G types), tropomyosin and troponin, and are present in the thin fi laments. Actin has a molecular weight of 42 kDa and has no ATPase activity. Myosin contains a large amount of aspartic and glutamic acid residues and a few amount of histidine, lysine, and arginine. In postmortem muscle, myosin and actin exist as actomyosin complex. Actomyosin consists of a complex of long fi laments of actin and myosin together with tropomyosin and troponin. The structure of fish muscle has been discussed by several authors.8–10 The muscle compositions of different fish species have been examined. For example, the molecular weights of structural proteins of Japanese sting fish (Sebastes inermis) were as follows: myosin, 200 kDa; actin, 42 kDa; HMM, 125 kDa; and light meromyosin, 66 and 77 kDa. Ca–ATPase activity of HMM was higher than that of myosin, whereas EDTA–ATPase and Mg–ATPase activities were similar to those of myosin. The KCl-dependent enzymatic activity was similar for both myosin and HMM. HMM was more stable than myosin during storage at 4°C. The transition temperatures of each protein as determined by differential scanning calorimetry were as follows: myosin, 40.9°C; actin, 61.1°C; HMM, 40.9°C and 59.3°C; and light meromyosin, 62.2°C.9,11 Although there is a slight difference in the composition of amino acids, the myosins of all vertebrates such a rabbit, chicken, and fish (e.g., cod and tilapia) are similar.7,9 Compared to carcass meat, fish myosins are unstable, being more sensitive to denaturation, coagulation, degradation, or chemical changes, which can adversely affect functional properties, suggesting need for care in handling these proteins from fishery resources. Paramyosin is a protein found in striated muscles of invertebrates and is involved in the catch contraction of bivalves. The contents of paramyosin in scallop, squid, and oysters are 3, 14, and 19%, respectively. In the white adductor muscle of some oysters and clams, 38–48% of the myofibrils are paramyosins, which form a core with a surface layer of myosin. The protein has a molecular weight of 200–258 kDa, consisting of two subunits of 95–125 kDa with glutamic acid contents as high as 20–23%. The comparative biochemistry of paramyosins including those from mollusks has been reviewed.12 In a recent study, it was reported that in comparison with the actomyosin from striated muscle of scallop, the actomyosins of mollusk, squid, and smooth and striated muscles of scallop have a significant higher contents of paramyosin, but a lower proportion of myosin.13 Table 3.1 depicts composition of proteins in muscle.

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TABLE 3.1 Composition of Proteins in Muscle Proteins

Percentage

Myofibrillar proteins Myosin Actin Connectin Tropomyosins Troponins (C, I, T) Actinins (α, β, γ) Myomesin Desmin, etc. Sarcoplasmic proteins Hemoglobin Myoglobins Enzymes (glycolytic enzymes, creatine kinase, etc.) Other extracellular proteins Connective tissue proteins Collagen Elastin Mitochondrial proteins (including cytochrome c and others)

60.5

29

10.5

Source: Xiong, Y. L., Food Proteins and their Applications, Marcel Dekker, New York, 1997, p. 34. With permission from Taylor & Francis Ltd. (www.informaworld.com).

3.4 FUNCTIONAL PROPERTIES OF PROTEINS The functional properties of proteins, in general, are discussed, followed by detailed discussion on these properties with respect to seafood proteins.

3.4.1

DEFINITION

Functional properties of food macromolecules including proteins are defined as a set of physicochemical characteristics that contribute to the structural, mechanical, and other physicochemical properties and determine the behavior of food systems during processing, storage, preparation, and consumption.14 Proteins present different surface activity, related to their conformation and ability to unfold at interfaces determined by molecular factors (flexibility, conformational stability, distribution of hydrophilic and hydrophobic residues in the primary structure) and external factors (pH, ionic strength, temperature, possible competitive adsorption of other proteins or lipids in the interface). These characteristics are also related to the intrinsic, physicochemical, and structural properties of the macromolecules. These include size, shape, amino acid composition and sequence, net charge and distribution pattern

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of charges, hydrophobicity/hydrophilicity ratio, secondary structure, tertiary and quaternary structural arrangements, inter-/intrapeptide cross-links, molecular rigidity in response to changes in environmental conditions, and the nature and extent of interactions of proteins with other food components.15 Functional property has also been defined as any property of a food or food ingredient except its nutritional ones, which affects its use.16 Following are the salient functional properties of proteins.

3.4.2

SOLUBILITY

Solubility is often considered to be a prerequisite for the performance of a protein in food applications. Solubility and related properties of proteins such as wettability, dispersibility, thickening, foaming, emulsification, and gelling are determined by the extent of interactions with water. In muscle food, a significant amount of water remains bound to proteins. The bound water can be in one or more of six basic forms: structural water (which is unavailable for chemical reactions), hydrophobic hydration water, monolayer water, nonfreezable water, capillary water, and hydrodynamic water. The extent of these interactions determines solubility of the proteins, helping them maintain their structural integrity so as to render them functionally active. Under a given set of environmental conditions, these properties are the thermodynamic manifestations of the equilibrium between protein–protein and protein–solvent interactions, which are related to the net free energy charge arising from the interactions of hydrophobic and hydrophilic residues with surrounding aqueous environment.17,18 Water-soluble proteins generally contain 25–30% of hydrophilic amino acid residues and a higher percentage of charged residues. Environment and processing conditions influence solubility of proteins by alteration in the ionic, hydrophilic, and hydrophobic interactions at the protein surface. The insolubility of most proteins at their isoelectric pH is due to neutralization of charge repulsion among protein molecules, leading to aggregation of proteins. Certain salts exert ionspecific effects on the solubility characteristics.17–19

3.4.3

EMULSIFYING CAPACITY

Food emulsions generally are of three types: (i) oil-in-water or water-in-oil emulsions; (ii) foam, in which air (gas) bubbles are dispersed in an aqueous medium; and (iii) sol, which is small solid particles dispersed in liquid medium. In both oil-in-water and water-in-oil emulsions, the dispersed phase is distributed in the form of droplets and hence such systems are often called colloidal dispersions. In oil-in-water emulsions, an aqueous medium is the continuous phase and oil is the dispersed phase. Most food emulsions, including mayonnaise, fall under this category. In the case of water-in-oil emulsions, the oil is the continuous phase and water is the dispersed phase, such as margarine. Food emulsions and foams are essentially colloidal dispersions, that is, the continuous phase of these systems does not have the thermodynamic ability to wet the dispersed phase.20–22 The emulsification properties of proteins are influenced by their surface hydrophobicity. Generally, emuslifying capacities of muscle proteins vary as myosin > actomyosin > sarcoplasmic proteins > actin. During emulsification, myosin is taken up at fat–water interface. Heat treatment of globular proteins invariably causes polymerization via sulfhydryl–disulfide interchange reactions influencing

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their emulsifying properties.23 Several studies have shown that mild heat treatment (such as surimi gelation, see Section 3.7.1.1) that does not result in protein insolubilization can improve emulsification properties of proteins through increase in surface hydrophobicity. Food proteins isolates contain several protein components in varying amounts. Depending on their molecular properties these compounds might selectively or differentially adsorb to the oil–water interface during emulsification. Diverse protocols are found in literature to determine emulsifying properties. These protocols use different operational conditions and the values obtained are defined with different units. This involves a problem in data comparison in diverse methods.17

3.4.4

FOAMING CAPACITY

Proteins are the main surface-active agents required to stabilize the gaseous dispersed phase in food products. Foaming requires a large interfacial area to facilitate the incorporation of air to the liquid phase for the formation of foams. Foaming capacity is determined by the ability of the protein to reduce the surface tension, the molecular flexibility, and physico-chemical properties, namely, hydrophobicity, net charge and charge distribution, and hydrodynamic properties. Good foaming proteins must (i) rapidly adsorb during whipping and bubbling; (ii) have a rapid conformational change, rearranging at the air–water interface with reduction of surface tension; and (iii) form a viscoelastic cohesive film through intermolecular interactions. Foaming capacity (or whippability and foam expansion) can be determined by direct measure of the foam volume produced after whipping or aeration of a protein solution or by indirect methods such as conductivity. Foam stability, measured as time required for a 50% reduction in foam volume, indicates the ability of foam to stabilize against gravitational and mechanical stresses.21

3.4.5

GELATION

A gel is an intermediate between solid and liquid, in which strands of chains of proteins or carbohydrates are cross-linked to form a continuous three-dimensional network. Proteins are more efficient gelling agents than carbohydrates because large molecules are capable of forming crosslinks in three dimensions. Gelation is favored by the protein size, since large molecules form extensive networks by crosslinking in three dimensions, and by the ability of the proteins to denature.24 To form gels, the protein is subjected to partial denaturation by mild heating, which results in unfolding of the tertiary structure giving long chains without breakage of covalent bonds. Other factors favoring partial denaturation are pH, ionic strength, reducing agents, urea, temperature, the presence of nonprotein components, and the mechanical forces applied to the system. The partially unfolded proteins are allowed to aggregate under appropriate conditions to form a three-dimensional network. In food-processing operations, the partial denaturation is achieved by mild heating, causing unfolding of α-helices in the tail portion of myosin molecules. It must be noted that control of temperature is important since high temperatures fully denatures the proteins. During association step of the partially denatured proteins, water, oil, and flavoring compounds can be entrapped in the gel matrix, which are held together by hydrophobic and hydrogen bonds. Rigidity of the gel can be suitably changed by incorporating

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ingredients such as salt, starch, oil, polyphosphate, or proteins from other sources into the gel matrix. The gelled products have appealing textural properties.25 Protein–protein binding is important in restructured meat products. The binding capacity of crude myosin begins to increase at about 50ºC, and increases linearly up to 80ºC. Adhesion and cohesion forces participate in meat binding. Salt-soluble proteins increase binding capacity. Actin does not have binding capacity, but in presence of myosin, it supplements formation of gelled structure. Gel matrix development of myofibrillar proteins can be directly influenced by chemical interactions among the nonmuscle proteins and the myofibrillar proteins as well as indirectly by changes in the molecular environment (total protein concentration, water state and availability, ionic strength and types, and pH) brought out by the presence of nonmuscle proteins.26 Protein lipid and protein-flavor interactions play several important roles in biological and technological processes. Protein lipid interactions are responsible for the correct organization of a large number of crucial biological structures such as membranes, organelles, cells, tissues, and entire organisms. In food technology, these interactions are important in product development, which, however, can be manipulated by physico-chemical techniques such as heating, mixing, shearing, and addition. Proper holding of proteins in these interactions is very important to get ideal functional requirement whether in biological system or product development.27 3.4.5.1

Rheological Properties of Gel

Rheology is the study of deformation and flow of matter. In foods, deformation is a measure of mouth feel, while flow is associated with viscosity.24 Rheological properties are important in deciding the functional properties of food. One of the important properties of protein gels, including fish protein gels, is their viscoelastic character, which make them behave as elastic solids and exhibit viscous flow. These properties have direct relations with the texture of gel-derived products. Measurement of viscoelasticity involves identification of responses of the gels to both large and small stresses. The response to large stress is usually determined by “texture profile analysis” using texturometers such as the Instron Universal testing machine.28 Although behavior toward large stress at failure, as obtained by texturometers, is comparatively easy to study, the data may be subject to variations depending upon the conditions. A major advance in this field is dynamic measurements of small deformations in the gel under either constant or sinusoidal oscillating stress.29 The controlled-stress approach, where the measurement is based on displacement (rotational speed) in response to an applied torque (stress), provides subtle changes in the gel indicative of its viscoelasticity. The controlled stress rheometers can measure viscosity versus rate of shear, creep, stress relaxation, the bulk modulus, and storage modulus, etc. Dynamic rheological tests based on controlled stress are widely used to study heatinduced gelation of surimi. However, it has also been cautioned that the fundamental rheological properties measured at low strain may bear no relation to the behavior and texture at high strains.14 Multidisciplinary approaches for rapid determination of fish protein quality have been evaluated based on various biochemical and rheological methods. A positive correlation between gel deformation and ATPase activity was found, whereas an inverse correlation was obtained between storage moduli G′ for gelling point and gel deformation. However, lower correlations were found

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TABLE 3.2 Some Basic Rheological Expressions Expression Viscosity Apparent viscosity Elasticity Viscoelasticity Newtonian flow

Pseudoplastic behavior Yield value

Bingham system

Casson relation Thixotropy Dilatancy

Description The flow behavior of a substance under the influence of stress to become irreversibly deformed Viscosity of non-Newtonian fluids Property of a material to recover its original size and shape immediately after removal of force causing deformation Attributes of substances that are both elastic and viscous. Viscoelastic substances save a part of the deformation energy Viscosity at a constant temperature and pressure is constant regardless of the applied shear rate and time. The shear rate is directly proportional to the shear stress. Therefore, a single viscosity measurement will give its true value The shear rate is not directly proportional to the shear stress. Therefore, several viscosity measurements against different shear rates are necessary to establish a rheological profile Minimum shear rate needed to induce flow. Expressed as N.m−2 or Pa. Above this value, materials exhibit plastic flow behavior A type of plastic flow behavior, where there is no movement below a characteristic yield value. Above this yield value, the system acts like a Newtonian liquid Another type of plastic flow involving a special form of relationship between shear stress, shear rate, and yield value Viscosity decreases under constant shear stress. If the stress is removed, the viscosity increases again Viscosity increases with rising shear rate. Dilatant behavior (shear thickening) is opposite of pseudoplasticity

with other gel tests. These include tests between storage moduli G′ at its initial increase and gel-breaking force; between storage moduli G′ at gelling point and gel deformation; between storage moduli G′ at gelling point and gel-breaking force.30 Table 3.2 gives some basic rheological expressions that are relevant for determination of quality of surimi and surimi-based products and also protein dispersions.

3.5 PHYSICAL FUNCTIONS OF PROTEINS IN FOOD The domain of physical functions associated with the presence of proteins in a food system typically include (i) increased hydration and water binding, which affect viscosity and gelation; (ii) modification of surface tension and interfacial activity, which control emulsification and foaming stability; and (iii) chemical reactivity leading to altered status of cohesion/adhesion and a potential for texturization. These properties of proteins to a great extent provide foods their particular characteristics. In a food system, some of the components such as lipids do not mix with each other and may exist as different phases within the food matrix. Besides, some

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TABLE 3.3 Functional Roles of Proteins in a Food System Function

Mechanism

Food

Solubility Viscosity

Hydrophilic character Water binding, hydrodynamic size and shape Hydrogen bonding, ionic hydration Water entrapment, immobilization, network-formation Hydrophilic, ionic, hydrogen bonding Hydrophobic bonding, sulfide crosslinks Adsorption and film formation at interfaces Interfacial adsorption and film formation

Beverages Salad dressings, dessert Meat, sausage, bread, cake Meat, sausage, bread, cake, cheese

Whey proteins Gelation

Meat, sausage, bread, cake, cheese Meat, bakery

Muscle and egg proteins, milk proteins Muscle, cereal proteins

Sausages, bolognas, soup, cakes Whipped toppings, ice cream, cakes, desserts Low-fat bakery products, desserts

Muscle and egg proteins, milk proteins Egg and milk proteins

Water-holding capacity Gelation

Cohesion–adhesion Elasticity Emulsification Foaming

Fat-flavor bonding

Hydrophobic bonding and entrapment

Protein Types

Muscle and egg proteins Muscle and egg proteins, milk proteins

Milk and cereal proteins

Source: Adapted from Damodaran, S. and Paraf, A., Food Proteins and their Applications, Marcel Dekker, New York, 1997. With permission.

proteins and polysaccharides, although soluble in the aqueous phase, often exhibit incompatibility of mixing. Small molecular weight organic substances such as flavors separate between the aqueous and the oil phases depending on their relative solubility in these two phases. These phenomena profoundly influence the sensory properties, especially the textural properties of foods.8,15,31 Table 3.3 shows functional roles of food proteins in a food system.32

3.5.1

MODIFICATION OF FUNCTIONAL PROPERTIES OF PROTEINS

Processing of foods involves physicochemical and thermal treatments, which affect both nutritional value and functional properties of proteins. Most native proteins of fresh foods show functional properties, which are characteristic of the foods. While drastic processing could adversely affect protein functionality, functional properties of proteins are amenable to enhancement under controlled processing conditions, which offers scope for development of novel foods by food-processing industries. Therefore, modification of proteins for improving the nutritional value and functional properties has been an active area of research. Such modifications result in both structure and conformations of the proteins as well as optimal characteristics of size, surface, charge, ratio of hydrophobicity to hydrophilicity, and molecular flexibility. Table 3.4 indicates factors that influence the functional properties of proteins in foods.

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TABLE 3.4 Factors that Influence the Functional Properties of Proteins in Foods Intrinsic Amino acid composition Conformation Structure

3.5.1.1

Environmental

Processing Treatment

Salts Water Carbohydrates Lipids Food additives Oxidation–reduction status pH

Chilling Coating Drying Freezing Heating High pressure Modified atmosphere

Chemical Modifications

Chemical modifications are commonly used in studies performed to characterize the relationships between structure, stability, and functional properties of protein isolates.19 One of the aims of these studies is to develop newer protein-based ingredients that can impart novel properties to the food and also effective utilization of unutilized or low-utilized proteins such as those from low-cost fish. Chemical modification can be achieved through alkylation, oxidation, acylation, and esterification of amino acids. Most studies performed with food proteins are based on the derivatization of the amino group of lysine residues.19 Recently, the potential of glycosylation to improve the functional properties of tuna byproducts has been reported.33 Phosphoproteins are abundant in nature and some, for example, milk casein and egg white albumin are part of the regular human diet. Phosphorylation with POCl3 (in the absence and presence of essential amino acids) could be a promising tool for improving functional and nutritional properties of food proteins (e.g., yeast protein, zein, and soybean protein). The amount of phosphorus covalently bound to proteins can reach up to 3·9%, but is usually in the order of 1–2%. The in vitro digestibility of food proteins is not adversely affected by phosphorylation. The in vivo digestibility (using the Tetrahymena thermophili bioassay) has been studied in the case of casein and zein. Although digestibility was not affected in the case of casein, the growth rate of the microorganism showed an 11-fold improvement on modified zein over that of the original zein.19,34 3.5.1.2

Enzymatic Modifications

Enzymes, because of their specificity in action, safety in use, and low energy requirements, are ideal tools for protein modifications and food formulations. Enzyme-modified food proteins with altered fat and water-binding properties have the ability to replace fats and carbohydrates in food products to provide texture, viscosity, mouth feel, and flavor. Proteases are providing tools to improve the palatability of reformulated low-carbohydrate-high protein foods. Protease treatment can also improve health and safety of foods by removing antinutritive factors from products such as soybean and also stabilize foaming and gelation capacities

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and stability of flours.10,19,34–38 Treatment with transglutaminase has been recognized to improve the textural properties of protein foods.36,37 The various applications of enzymes in seafood processing have been reviewed recently.10 Apart from improvement of functional and nutritional properties, additional advantages of these modifications are delaying of deterioration reactions, removal of toxic or inhibitory compounds, better utilization of by-products as well as development of nutritional supplements.19

3.6 FUNCTIONALITY OF SEAFOOD PROTEINS Structural proteins of fish muscle display important functionalities. Fish myosin such as mammalian myosins exhibits three important functional properties. As mentioned earlier, it is an enzyme having ATPase activity, it forms natural complexes with actin and myosin molecules, and interact with each other and build filaments/ complexes. Fish myosin and actomyosin are soluble in aqueous salt solutions of high ionic strength, and the extractability in these solutions is considered as an index of the fish quality, since denaturation can result in loss of solubility. These proteins are also soluble in water at very low ionic strength.39 The myofibrillar proteins, particularly myosin, are responsible for functional properties such as oil-emulsification capacity, whippability, gel-forming ability, and chewability.40 Gel formation is an important property that determines consumer acceptance of fish protein-based products. The myosins are able to form a wider range of cross-links to form gels having different characteristics. For production of gels, the important properties of these proteins are their flexibility, including their ability to denature and give extended chains, and to form extensive networks by cross-links. The gel formation process involves partial denaturation of the proteins with some loss of their α-helix structure followed by aggregation of the proteins through intermolecular associations. Such gels are opaque and are usually made by mild heating. As the temperature increases, the molecules partly unfold, exposing hidden reactive groups, which then react at intermolecular levels to form a continuous network. The gelation of fish myosin is through the interactions of hydrogen bonds, electrostatic bonds, hydrophobic, and covalent bonds (disulfide).41 Table 3.5 presents typical functional properties performed by fish muscle structural proteins. Proteins from a number of fish species have been examined for their functionality, particularly to correlate them with palatability. The oil-absorption capacities of the fish products are generally in the range of 130–370%; water-absorption capacities, 280–404%; and gelation capacities, 6–10%. The physicochemical and functional properties of myofibrillar proteins from scallop, bivalve mollusk, and squid have been investigated. Dynamic viscoelastic behavior of actomyosin of green mussel (Perna viridis) revealed the ability to form network during heating; however, the strength of network appeared to be weak. The flow behavior of actomyosin solution indicated pseudoplastic behavior at different concentrations and temperatures. Casson and Herschel-Bulkley models were examined to evaluate flow behavior of actomyosin, which revealed maximum yield stress value at 30 mg/mL at 40°C. Amino acid profile of actomyosin revealed a relatively high proportion of glutamic acid, alanine, tryptophan, and aspartic acid. The viscosities of actomyosin from

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TABLE 3.5 Typical Functional Properties Performed by Fish Muscle Structural Proteins Functional Property

Mode of Action

Product

Solubility Water absorption and binding Viscosity Gelation

Protein solvation Entrapment of water through hydrogen bonding Thickening Protein matrix formation

Cohesion– adhesion Elasticity

Adhesion

Emulsification Fat binding

Formation of fat emulsion Hydrophilic bonding and entrapment Adsorption, entrapment, release Entrapment of air and film formation

Dispersion, soup Surimi, surimi-based products, sausage, fish balls Gravies, soups Surimi, surimi-based products, sausage, patties Surimi, surimi-based products, sausage Surimi, surimi-based products, sausage Sausage, fish balls, soup Sausage, fish balls

Flavor-binding Foaming

Disulfide bonds

Seafood analogs Protein hydrolysate, prepared products

striated and smooth adductor muscle of scallop were significantly lower than those of squid and mollusk. Surface hydrophobicity and emulsifying capacity of actomyosins from striated muscle of the mollusk were lower than those corresponding actomyosin of the other species.42,43 The highest emulsification capacity was observed in extracts of squid mantle.13

3.6.1

POSTHARVEST CHANGES IN FUNCTIONAL PROPERTIES

Postharvest handling of fish affects the functional properties of proteins. The functional properties of fish can be lost when they are stored in ice. Holding pink perch (Nemipterus japonicus) in ice before mincing and freezing resulted in significant loss of protein solubility, emulsifying capacity, water-binding capacity, cooking loss, thaw drip, and texture scores. Changes in functional properties correlated with protein solubility. Significant decreases in protein functionality, texture scores, and thaw drip values during frozen storage were also observed.44 Similar changes have also been reported for Atlantic salmon and lizard fish.45,46 Frozen storage of oil sardine mince stored for 150 days at −20°C resulted in a decrease in protein solubility, emulsifying capacity, relative viscosity, and increases in cooking loss, peroxide value, and free fatty acids. Water-holding capacity in terms of absorbed and retained water decreased when the fish was stored up to 120 days. Significant correlations existed among these parameters with storage period. In general, sensory attributes of cooked meat were rated acceptable up to 90 days.47

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It may be mentioned that the loss of functionality of proteins also occurs in the case of freshwater fish. The effect of iced storage on functional properties such as solubility, emulsion activity index (EAI), viscosity, and foaming of muscle proteins from fresh water Rohu (Labeo rohita) was evaluated recently. Myofibrillar protein solubility showed an increasing trend up to 11 days of storage followed by a decrease. A 44% drop in EAI was observed in the case of myofibrillar proteins on storage. These proteins showed foam volume stability of 94%. Viscosity of sarcoplasmic proteins remained relatively stable throughout the duration of study, whereas myofibrillar proteins showed an increasing trend during the first week and then decreased. There were changes in reactive sulfhydryl groups and hydrophobicity of myofibrillar fraction during storage.48 The loss of functionality during frozen storage has been attributed to intermolecular aggregation of proteins through hydrogen, hydrophobic, and disulfide bonds, resulting in loss of solubility, viscosity, and ATPase activity of the myosin.49 Physicochemical changes of muscle from croaker, lizardfish, threadfin bream, and bigeye snapper during storage up to 24 weeks at −18°C were investigated. Ca2+–ATPase activity decreased, whereas Mg2+–EGTA–ATPase activity increased throughout the storage. However, no marked changes in Mg2+–Ca2+–ATPase and Mg2+–ATPase activities were observed. Among all species tested, lizardfish muscle was the most susceptible to these changes. Disulfide bond formation with the concomitant decrease in sulfhydryl group was found in all species. However, croaker and lizardfish contained higher disulfide bonds as storage time increased compared to other species. Surface hydrophobicity increased in all species with the sharp reduction observed in lizardfish after 12 weeks. For all species, α-glucosidase and β-N-acetyl-glucosaminidase activities increased in association with the increased expressible drip. The results showed that extended frozen storage caused the denaturation of proteins as well as the cell disruption in all species, but the degree of changes was dependent upon species.45,50

3.7 FUNCTIONALLY ACTIVE MARINE PROTEIN SUPPLEMENTS The favorable functionality of marine fish proteins has encouraged industrial research in marine fish processing with a view to develop a variety of functionally active protein supplements that can be more varied and far-reaching than poultry or meat products.51 The versatility of these products arises from the possibility of making protein ingredients from diverse low cost fish species offering several economically viable protein products having diverse functional and nutritional properties.52

3.7.1

FISH MEAT MINCE AND MINCE-BASED PRODUCTS

Recovery of meat mince, perhaps, is the ideal means of utilizing many underutilized and low-cost fish species, particularly pelagic fish as a protein source. Fish frames and trimmings from filleting operations, which contain appreciable amounts of meat are also sources of proteins. The technology of meat mince collection has been discussed recently.10,53 It is also possible to recover up to 60% of minced meat from the filleted frames. Products incorporating fish mince include patties, balls, wafers, loaves, burgers, fish fingers, fish fritters, dehydrated salt minces, breaded patties, cutlet, and picked products. It is important that fresh fish or fresh frames are

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used for mince production for the secondary products. This is because mechanically deboned fish meat is more susceptible to quality deterioration during storage than the intact muscle tissue, since the mincing operation causes tissue disruption and exposure of the flesh to air, which accelerates lipid oxidation and autolysis. Such reactions adversely affect the color and odor of the products. Functional properties of interest in fish mince are gelation, water absorption, cohesion–adhesion (simulated meats), emulsification and fat absorption (sausages), flavor binding (simulated meats), viscosity, and solubility (soups). Rheological and flavor properties of fish proteins (which are important in restructured products from surimi) depend on their ability to form gel, which in turn, is influenced by intrinsic molecular properties such as size, shape, and conformation, as well as processing conditions such as temperature, pressure, pH, and salt contents. If the fish mince is kept frozen before use, lipid oxidation can be a significant problem on the surface layers of frozen blocks of fish mince. The oxidized lipids interact with proteins causing denaturation, drip loss, and changes in the functional properties. TMAO present in appreciable amounts in gadoid fish undergoes enzymatic cleavage to dimethylamine and formaldehyde, the latter influencing protein denaturation and toughening. Denaturation of proteins and resulting loss of functionality could be prevented by incorporation of cryoprotectants such as polyphosphates and other salts, sugars, and hydrocolloids such as alginate, carrageenan, carboxymethyl cellulose, guar gum, and xanthan gum. Carbohydrates such as sorbitol and sucrose used as cryoprotectants stabilize the proteins thermodynamically through their interaction with the surrounding water. It is important to note that the quality of fish mince, as reflected by its color, flavor, and functional properties, is also dependent on the species, season of harvest, handling, as well as the method of processing. Fish caught during and after spawning are high in moisture and relatively low in lipid and protein contents, as compared with those caught during intense feeding periods. The mince quality is also influenced by the quality of fish used for the process. Prolonged ice storage of fish before deboning or frozen storage of the mince before secondary processing can adversely affect protein functionality including texture, and cooking loss due to drip formation, which in turn adversely affect the functional quality of the secondary products.10,54 Fish ball is a popular and nutritious fish jelly product in Malaysia. It is made from fish meat mince, ground with salt to a smooth sticky paste. Other ingredients, namely, 2% whey protein concentrate and 0.5% carrageenan are added to enhance the texture and flavor of the paste, which is then shaped and cooked. Mince of fresh threadfin bream (Nemipterus tolu) is a common raw material. Production of fish balls in Thailand has grown to an industrial scale requiring approximately 35 t of raw materials per day. Another popular product in Malaysia is fish crackers, made from fish species such as sardine and jewfish along with tapioca and sago flours. The type of fish used did not influence sensory characteristics. Spiced minced fish cakes that are ready to fry or can be used in stews were produced from trawler by-catch. The cakes were found to be organoleptically acceptable to Nigerian consumers for a minimum period of three months at a frozen storage temperature of −20ºC.55 Precooked frozen burgers from sardine meat has been prepared by chopping the fish mince with 14% emulsion curd, 8% bread crumbs, 3% soybean protein, 1.5% salt, 2% sugar, and small amounts of bicarbonate, polyphosphates, and spices.

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The seasoned sardine meat was fried in soybean oil at 165ºC for 3 min. Mince from whole sprat gave acceptable fish balls, the quality of which was improved by prewashing the mince and incorporation of food additives.10 In a recent work, a low-fat protein product with good gel strength properties from catfish fillets was produced on a pilot plant scale. The fish fillets were minced and solubilized in dilute acid (pH 3.0) at 1.9% concentration. Insolubles were removed by centrifugation and the soluble protein was recovered by isoelectric precipitation (pI approximately 5.4). The preparation having 78% moisture and 2% NaCl were stuffed into casings, cooked, and chilled. Analyses of proximate composition, color, cook yield, water-holding ability, and texture profile analysis (TPA) were performed. The solubilization process removed 74% of the fat of catfish fillets. The gels from treated protein were less red and yellow, whereas there was no difference in whiteness. The cook yield of the gel was 94.6% and a water-holding capacity of 0.73 g/g protein. The texture analysis indicated significantly higher hardness, springiness, chewiness, and cohesiveness.56 Marinbeef is a dehydrated low-fat product with good water holding capacity. The process of production consists of extruding fish mince, containing about 3% salt, into alcohol. This results in denaturation of the proteins and separation of the bound water. The product is then dehydrated to give highly deodorized protein powder, having up to 92% protein and negligible fat. It has superior nutritional quality such as protein efficiency ratio, net protein utilization, and net protein ratio and biological value with excellent protein digestibility (see Chapter 4). The product can be prepared from both lean as well as fatty fish including Alaska pollock (Theragra chalcogramma), blue whiting (Gadus poutassou), sardine (Sardinops melanosticta), and Pacific mackerel (Scomber japonicus). When using fatty fish, the meat needs to be initially washed with aqueous bicarbonate solution to remove adhering lipids from the meat. Marinbeef from lean fish can replace animal meat (beef) to a substantial extent in different preparations. Balls made of a mixture of hydrated marinbeef and minced beef gives a texture similar to beef mince balls. Marinbeef products have also been prepared from Indian marine fishes, which included threadfin bream, catfish, ribbonfish, barracuda, and jewfish. The products had 85–88% protein, 1–3% fat, and up to 10% moisture contents. Dehydrated fish protein-rich foods incorporating carbohydrates have been developed in several parts of the world. These products have been named as fish-macaroni, fricola, fish-potato flakes, and fish wafer. The fish used in these products are mostly low-cost species from marine sources, although low cost and freshwater fishes are also being examined for their suitability.57,58 The product from threadfin bream had all the essential amino acids in levels higher than those prescribed by the FAO/WHO. The protein-efficiency ratio (see Chapter 4) of the product was 2.5–2.9%, with a net protein utilization (see Chapter 4) value of 83% at 10% protein level. Several scientific and technological aspects of fish product development have been recently discussed.59 Figure 3.2 gives a general scheme for utilization of fish mince for the development of various products.60 3.7.1.1

Surimi and Surimi-Based Products

“Surimi” is the Japanese name for washed, preserved fish meat. In the native fish muscle, several low-molecular-weight compounds and enzymes are adhered to myosin

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Minced fish (underutilized)

Extrusion

Hydrolysis

Washing

Surimi Hydrolysate Starch-based foods (peptones, amino acids plasteins, flavour enhancers)

Traditional products (cakes, balls, patties, loaves, breaded products, saltdried items, canned products, mince blocks, restructured and composite files)

Structural protein gels Soluble matters (animal feed)

Spinning

Fibers

Functional protein powder

Kamaboko and hybrid products

Fermentation

Sauces, pastes flavourings

FIGURE 3.2 A general scheme for utilization of fish mince for the development of various products. (Reprinted from Venugopal, V. and Shahidi, F., Crit. Rev. Food Sci. Nutr., 35, 431, 2005. With permission from Taylor & Francis Ltd. (www.informaworld.com))

and actomyosin, which hinder their interactions with water, and hence poor solubility of the structural proteins. Washing removes the soluble components such as pigments, enzymes, and lipids adhering to the proteins liberating polar sites for interactions. The resulting change in electrostatic balance causes unfolding of the myosin molecules bringing the buried nonpolar side chains into contact with water, favoring improved functionality.61 The most important functional quality of surimi is its gelforming ability, since this property dictates versatility of surimi to give required texture in diverse products. Gel formation is essential for proper binding of ingredients required to modify the textural attributes. Fish actyomyosin sol forms a fine translucent gel at about 40ºC (high-temperature setting) or at a slower rate during prolonged storage at refrigerated temperatures (low-temperature setting). Further heating to 80–90ºC results in a stronger structure. Surimi paste that has initially been set (setting is called “suwari” in Japanese) at 40–50ºC gives a stronger gel, if subsequently heated to 80–90ºC. However, at 60–70ºC, the gel softens, a phenomenon, termed as “modori” in Japanese. Optimum gel formation in surimi is assisted by 2–3% sodium chloride, which enhances water uptake and protein solubility. The salt improves the binding ability of proteins by increasing the amount of salt-extractable proteins as well as altering the ionic strength and pH of the medium, facilitating the formation of a coherent three-dimensional structure during the process of heating the mince.

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Polyphosphate increases the binding of meat particles in surimi.41,62 The strength of surimi gels, measured in terms of viscoelastic properties,14,28 differs depending upon the fish species. Of the white-flesh varieties, those having the appreciable gel strength include croaker, barracuda, threadfin bream, lizardfish, cutlass fish, stripped mullet, leather jacket, sea bream, frigate mackerel, puffer, and red big eye. Among the dark flesh species, Pacific blue marlin has the highest gel-forming capacity, followed by flying fish, dolphin fish, scads, horse mackerel, yellow fin tuna, Pacific mackerel, and skip jack tuna. Among sharks, dog shark (Scoliodon walbeehmi) has good ability to form gel. Salmon surimi has not been preferred due to its poor gel formability compared with pollock surimi.40 Alaska pollock (T. chacogramma) has been the fish widely used for surimi making because of soft muscle tissue, low fat content, and white flesh. The process of surimi production from Alaska pollock is shown in Figure 3.3. Fresh Alaska pollock, Raw materials Water

100% Washing

Heading/gutting 60% Washing

Deboning/mincing 47%

Water Washing

45% Refining

Dehydration 20%

Additives

Waste water

Mixing

Shaping/packing

Frozen surimi 24%

FIGURE 3.3 Surimi process from Alaska pollock. (Reprinted from Ohshima, T., Suzuki, T., and Koizumi, C., Trends Food Sci. Technol., 4, 157, 1993. With permission from Elsevier.)

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which is caught by mid-water and bottom trawler are ideal for best quality product. After harvest, the fish is stored in crushed ice or in refrigerated seawater. The mince, collected by mechanical deboning of the fish, is subjected to washing in three or four separate tanks, each filled with cold (<10ºC) fresh water to remove soluble compounds. Gentle stirring for 9–12 min, with 3–4 repeated washings in separate tanks are commonly employed. The washed meat is passed through a refiner to remove any remaining small bones, skin tissues, dark muscle tissues, and scales. The refiner is a high-speed rotary spiral surrounded by a screen with many small pores of 1.2–3.2 mm in diameter. Excess water in the washed mince is removed using a screw press, to reduce the moisture content of the mince to 90%. It is then mixed well with 4% sucrose or 4–5% sorbitol in addition to 0.2–0.3% polyphosphate, as cryoprotectants. Sucrose may impart a sweet taste and hence sorbitol is preferred. The yields of mince and surimi from the fish were 47 and 24%, respectively, based on the weight of whole fish. The surimi is then packed in a freezing pan and frozen quickly to below −5ºC using a contact freezer.41 A decline in the landing of Alaska pollock has led to testing the feasibility of other species for surimi production. About 60 different species from tropical and cold waters have been identified for the purpose. These include hoki, threadfin bream, croaker, blue whiting, Pacific whiting, Atlantic cod, menhaden, sardine, mackerel, lizardfish, eel, barracuda, leather jacket, and capelin, among others.40,45,50 Sardine is a plausible alternate. By washing the sardine mince the yield decreased three times, from 27 to 21%, although the treatment removed 80% of the lipid and improved the texture and color of the prepared kamaboko.63,64 Surimi, because of its characteristic ability to form gels, can be used to develop products that simulate the appearance, flavor, and texture of expensive products such as imitation breaded scallops, lobster tail, imitation breaded crab claws, scallop, sushi products, sushi sticks, imitation crab shreds, minced sticks, and filament sticks. For production of seafood analogs, the surimi blocks are chopped to create a paste. The paste is combined usually with additional amounts of cryprotectants and other additives such as salt, soy protein, starch, egg white, and alginate to promote cohesion among the protein molecules and thereby to improve the texture and flavor of the finished product. For chopping the frozen blocks, it is ideal to use a vacuum mixer, which helps to disintegrate the mince and make the proteins available for binding with the ingredients. A vacuum mixer also removes any air that could be introduced in the chopped product, which can result in uneven heating during cooking. The chopped paste is extruded as a flat sheet (approximately 1–2 mm thick), molded into desired shapes, and set by placing on a cooking belt, where it is heated. Heating is done at 90–93ºC for 30–100 s on a stainless steel belt drum. Final texture is developed during thermal pasteurization, which is performed after bundling, cutting, and packaging. The pasteurization step eliminates bacterial pathogens that might grow during the storage of the product. Generally, surimi seafood should be cooled from 60 to 21.1ºC or below within 2 h and to 4.4ºC or below within the next 4 h and should be kept at 4.4ºC or below throughout storage and distribution process. These seafood analogs possess the accepted texture, flavor, and appearance of the authentic products. Figure 3.4 shows flow chart for the production of surimi crab leg. A patented process for production of shrimp analogs consists of mixing

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Surimi ↓ Mixing of ingredients (surimi base, water, wheat starch, vegetable oil, egg white, flavors, natural colorants) to a smooth paste ↓ Pumping, extrusion and cooking ↓ Drying ↓ Rolling and coloration ↓ Film wrapping ↓ Cutting ↓ Vacuum packaging ↓ Pasteurization ↓ Chilling ↓ Boxing ↓ Storage at 1°/ +41°C

FIGURE 3.4

Flow chart showing the production of surimi crab leg.

surimi with glucomannan, carrageenan, protein, and starch, extruding the mixture and cutting the extruded material to suitable sizes, and heating the pieces in hot water bath followed by chilling. Many products have evolved from the original crabmeat stick analog. The main types are flakes, chunks, and combo, all of which are ready for use and have a more irregular shape than the original stick. There are structural differences among these products, since some are filament style and consist of aligned fibers, whereas others are described as solid meat but in fact consist of random fibers.65 Fresh surimi-based products have been familiar to the Japanese for many centuries such as kamaboko, chikuwa, and satsuma-age, which differed in shape and preparation and were steamed, baked, or fried. These products are prepared by grinding the washed mince in presence of salt, sugar, and possibly glutamate, alginate, and potato and cornstarch for 30–40 min. The paste is then spread on a wooden plate made usually from pine or cedar wood. It is then steamed for 20–90 min to attain an inner core temperature of 75ºC. The treated mince undergoes setting at ambient temperature, resulting in a translucent product that maintains its shape. Whereas traditional kamaboko is processed by steaming, broiled and fried surimi products are called chikuwa and tempura, respectively. Kamaboko accounts for over 60% of the fish paste market in Japan. Surimi produced from croaker, shark, or threadfin bream are highly in demand for manufacture of best-quality kamaboko. Products may also be shaped into fish balls and related products, which are set by soaking in water before cooking to prevent their shape changing in the air. Kamaboko is generally prepared from marine fish, since the products from freshwater species

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have poor acceptability. Ice storage and washing of the mince before processing kamaboko influence the yield and sensory quality of the product. Surimi, because of its high functionality, is also an ideal raw material for sausage manufacture. Sausages including Vienna sausages, frankfurters, and bologna are complex mixtures of muscle tissue, solubilized proteins, binders, spices, salt, and water. The products are stabilized through an emulsion of protein film around fat globules. Usually, animal or vegetable fat is mixed with fish mince or surimi. The ingredients are mixed with the fish mince by using a silent cutter. The duration of the mixing operation is completed in 10–12 min and the temperature of the fish paste is maintained at 15–16ºC during mixing by adding crushed ice. The fish paste is stuffed into krehalon or some other casing and sealed with aluminum wire using a ringing machine. The product is heat processed at 90 ± 2ºC in a constant temperature water bath and then cooled in chilled water. The sausage is further heated at 98–100ºC for 30 s to remove wrinkles on the surface. The heat-processed products are stored at chilled temperature (6 ± 2ºC). Smoking can also be employed to enhance flavor. The sausage has moisture content of 68%, protein 17%, and fat 5.5% with a gel strength of 250 g.cm and expressible water above 6%.66

3.8 FISH PROTEIN POWDERS Early worldwide interests in the use of fish meat as protein supplements led to development of a product, known as fish protein concentrate (FPC). FPC type A is a completely deodorized protein powder, prepared by solvent extraction of fish meat employing either ethanol or isopropanol. The final product is light brown or creamy powder with insignificant fishy odor and taste. The product, however, failed to make commercial impact due to various reasons such as high cost of production, presence of residual solvent, and poor functional properties.67 Some attempts have been made to increase functional properties of FPC by proteolytic digestion. Another comparable product is FPC type B, which, unlike FPC type A, possesses fishy odor. It is prepared by cooking, dehydration, and grinding of fish meat, and did not employ any solvent extraction. This product has also poor functional properties.68 Recent interests in functionally active fish protein powders for use as binders, emulsifiers, and gelling agents in foods have caused renewed research in the field. A major effort in this direction was in the year 1993, when the Association of Danish Fish Processing Industries and Exporters (DFE) commercially produced fish-based protein powders to enhance water binding and stability properties of frozen foods. Thus, the powders developed from herring and arrowtooth were high-quality proteins with desirable functional properties. Soluble protein powders were also made from the byproducts of processing of Alaska pollock, namely, viscera, liver, heads, trimmings, and frame. These powders also possessed appreciable functional, nutritional, and rheological properties.69–72 A spray dried fish powder made from saithe (Pollachius virens) by-products was also developed. The powder had low lipid content, exhibited antioxidant activity in a model system of linoleic acid emulsion. Storage of the powder at 0°C retained its functional properties.73

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THERMOSTABLE PROTEIN DISPERSIONS AND POWDERS

A general method to prepare thermostable water dispersions of fish meat has been developed, which is beneficial for products such as powders and edible protein films. The dispersion is usually made using mechanically deboned fish mince. Alternatively, headed, eviscerated boneless fish meat cut into pieces of 4–5 g each could be used. The fish mince or meat pieces are repeatedly washed in cold water as in conventional preparation of surimi. A three-step washing is recommended for meat pieces. In the first step, the pieces are held overnight in a cold room in excess (usually three times the weight of meat) of cold (<10°C) water. If mince is used, it has to be soaked in cold water for 1–2 h. For low-fat fish such as threadfin bream, washing is repeated twice with fresh cold water, keeping the holding time of 1 h for each wash. In the case of shark species such as the Indian dog shark (Scoliodon laticaudus), the skinned meat pieces (4–5 g, average size) are subjected to repeated washing in cold water. In the case of fatty fish such as mackerel and herring, it is advisable to use 0.5% aqueous solution of sodium bicarbonate in the second washing to remove lipids adhering to the meat. The alkaline pH of the bicarbonate solution significantly aids removal of the lipids. Fish mince that has an unappealing dark color such as that of capelin may be decolorized by an initial washing with 0.5% aqueous solution of sodium chloride. The washing process enhances water uptake by the proteins enhancing their solubility in water. The washed meat is homogenized in fresh cold water, usually at a ratio of 1:1. Few drops of glacial acetic acid are added to the aqueous slurry with gentle stirring, to bring down its pH to 3.5 to induce gelation of the myofibrillar proteins. Usually an amount of acid equivalent to 0.5% of the slurry is sufficient for the purpose. The acidified dispersions are heated to 50ºC to get low-viscosity protein dispersions, in which all the proteins are soluble.74 Figure 3.5 Fresh fish ↓ Evisceration, heading ↓ Mechanical/manual deboning ↓ Washing in excess of cold water, decanting ↓ Washing in 0.5% sodium bicarbonate solution (for fatty fish), decanting ↓ Washing in excess of cold water, decanting ↓ Lowering of pH to 3.5 by dropwise addition of acetic acid ↓ Gel ↓ Homogenisation of gel in water ↓ Heating 45−50°C ↓ Gel dispersion

FIGURE 3.5 General process for preparation of acetic acid-induced gel and thermostable gel dispersion of fish meat in water.

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TABLE 3.6 Soluble Proteins in Thermostable Dispersions of Mince from Different Fish Species

Fish species Atlantic mackerel Atlantic herring Indian mackerel Capelin Shark

Protein Content of Unacidified Fish Mince Homogenate in Water (%)

Protein Content of Gel Dispersion (%)

Solubility of Proteins in Dispersions (%)

2.40 1.30 1.50 2.50 2.94

2.35 1.28 1.30 2.10 2.16

98.0 99.0 86.0 84.0 73.0

Source: Reprinted from Venugopal, V., Trends Food Sci. Technol., 8, 271, 1997. With permission from Elsevier.

gives a general process for preparation of acetic acid-induced gel and thermostable gel dispersion of fish meat in water. Weak acid (e.g., acetic acid) induced fish meat gel dispersions are low-viscosity fluids in which the proteins are highly thermostable. Although mild acidification and heating decreased the viscosity of gel dispersions, the proteins were highly soluble in the heated dispersions. Almost all of the proteins in the washed meat were retained in the dispersions. Furthermore, the proteins in the dispersions were not precipitated by heating even at 100ºC for 15 min followed by centrifugation (up to 135,000 × g), suggesting remarkable stability of the proteins in water. Table 3.6 shows the solubility of proteins in thermostable dispersions prepared from different fish species.74 Nevertheless, almost all proteins in the dispersions were precipitated when they were heated after increasing their pH from 3.5 to 7.0. In addition, presence of salts such as NaCl and CaCl2 adversely affected the stability of proteins in the dispersions. The stability is achieved through gel formation of the protein under mild acidic conditions, which is different in several respects from the conventional surimi gelation, as summarized in Table 3.7.74,75 The possibility of preparation of free-flowing thermostable water dispersions of fish meat rendered preparation of functionally active protein powders suitable by spray drying of the dispersion. The spray dried protein powders have up to 90% and superior functional characteristics such as solubility and oil-emulsification capacity. Such powders have been prepared from fish species including capelin, threadfin bream, and shark.74 The powder obtained from threadfin bream was colorless and odorless and had a protein-efficiency ratio comparable with that of casein. Its oil-emulsification capacity and water solubility were 2–3 times higher than those of conventional protein powder prepared by drying and grinding fish meat.69 Table 3.8 gives the proximate composition and properties of spray dried threadfin bream protein powder.74 A major disadvantage of the process is that, unlike in the case of milk powder, the solid contents in the dispersion have to be maintained below 3% for successful spray drying. It may be mentioned that apart from spray drying other less

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TABLE 3.7 Comparison of Conventional Surimi Gelation with Weak Acid–Induced Gelation of Fish Muscle Structural Proteins Characteristics

Conventional Surimi Gelation

Weak Acid-Induced Gelation

Gelation pH Agents for gelation

Neutral or slightly alkaline Mild heat in presence of NaCl

Chemical changes

Formation of covalent (disulfide) and noncovalent linkages, degradation of myosin heavy chain, decrease in α-helix content and increase in hydrophobicity of myosin Good Poor Viscoelastic nature

Acidic pH in the range of 3.5–4.0 Weak organic acids such as acetic or lactic acid Formation of covalent (disulfide) and noncovalent linkages, degradation of myosin heavy chain, decrease in α-helix content and increase in hydrophobicity of myosin Good Good Viscoelastic nature so far reported only in shark and Alaska pollock Adversely affect gel characteristics Rapid fall in viscosity of gel giving free-flowing dispersion, solubility of proteins in dispersion not affected Restructured products, edible coating, spray dried protein powder, sauce, etc.

Water-holding capacity Microbial stability of gel Rheological characteristics

Influence of ionic compounds on the gel Influence of heat on rheological properties

Gel characteristics not affected

Applications

Restructured products, edible films (but not a microbial barrier)

No significant change

Source: Reprinted from Venugopal, V., Fish. Technol. (India), 40, 61, 2003. With permission from the Society of Fishery Technologists (India).

expensive methods could be examined to make protein powder, making use of the properties of the dispersion such as instability of dissolved proteins in the presence of traces of salt.74–79 Making use of weak acid induced gelation of fish proteins, restructured shark meat products have also been developed. The washed shark meat is converted into a gel by lowering the pH of shark meat slurry in water to a value of 3.5 using acetic acid, which results in formation of a hard gel. The gel is molded and steamed to get restructured products. The texture of the product is dependent upon the moisture content of the gel, which can be varied by changing the proportions of washed shark meat in the slurry. The product is stable against microbial growth, when stored at 10°C even up to 2 months, due to the presence of small amounts of acetic acid. Before consumption, the product is deacidified and salted to taste by dipping for 20 min in an equal volume of aqueous solution of sodium bicarbonate and common salt at concentrations of 5% each. The salted product can be breaded, battered, and shallow fried in vegetable oil and kept frozen. Consumer studies indicated good

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TABLE 3.8 Proximate Composition and Other Characteristics of Spray Dried Protein Powder from Threadfin Bream Color Odor Density Moisture (%) Protein (%) Lipid (%) Ash (%) Protein-efficiency ratio Trypsin digestibility (%) Pepsin digestibility (%) Trypsin + pepsin digestibility (%)

Colorless Odorless 0.13 5.4 93.1 1.1 2.6 3.5 54.1 48.4 81.9

Source: Adapted from Venugopal, V., Trends Food Sci. Technol., 8, 271, 1997. With permission.

acceptability of the product. Alternately, the shark gel cut into desired shapes could be used as paneer. The process can be extended to other species belonging to elasmobranchs. Since elasmobranchs contain significant amount of urea, the washing process removes urea in addition to other soluble components to give a product devoid of their characteristic odor. The gel could also be kneaded with other fish meat to develop composite restructured products.76 Application of surimi to prepare functionally active powder has been reported. Such powders are prepared by drying surimi from fish species such as lizardfish, threadfin bream, and purple spotted big eye fish. The resulting powders contained 73% protein and 17% carbohydrate (from the cryoprotectant added during surimi preparation). Functional properties such as solubility, gelation, water-holding capacity, emulsification and foaming properties, and color varied depending upon the type of fish used.80 A process to prepare functional protein isolates from fatty fish has also been reported, based on the differential solubility of the muscle proteins. The muscle proteins are first solubilized at low pH (3.5) to form a solution of low viscosity, which is centrifuged to remove oil- and the lipid-containing membranes. Protein in the interphase is then precipitated by adjustment of pH, followed by centrifugation to remove the low-molecular-weight water-soluble impurities. The recovered protein has low-lipid content, good functional properties, and can be obtained in high yields.81 Fish meat powders could be pressed into blocks to minimize air in between the grains of the product, followed by anaerobic packaging in 300 gauge polyethylene to avoid rancidity development. It has a storage life of about 6 months. Alternately, the powder together with binders could be made into tablets of 250 mg each. The product had 4.2% moisture, 48% protein, and 3.4% fat.82 It has been, however, cautioned that these protein supplements may not provide a universal solution to alleviate world hunger or protein malnutrition, due to reservations of some segments of global populations to accept fish as a source of food. Therefore, nutritional interventions using

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fish-based protein products should be aimed at populations to whom the products are acceptable in harmony with their social characteristics.81,83,84

3.8.2

OTHER PROTEIN SUPPLEMENTS

3.8.2.1 Protein from Krill The use of abundantly available Antarctic krill as a source of marine protein has been proposed due to its high harvest potential and scope for production of various functional agents and chemical constituents. The use of Antarctic krill is presently limited to meat extracts, dried products, fishing bait, aquaculture feed, krill oil, and carotenoid pigments. Instead of whole krill, the meat from the tail could be collected employing meat-separating machines followed by freezing and packaging. The packages should be frozen in freezers on board the vessel. Alternately, the meat mince could be converted into surimi by washing and mixing with additives (5% sorbitol, 0.3% polyphosphate, and 0.1% dried egg powder by weight of meat), packaged and frozen. Krill fingers have been prepared from frozen krill meat, which is cut into portions and coated, as in the case of commercial fish fingers. The coating batter consists of a mixture of wheat flour, starch, salt, and water. After the addition of breading, the fingers are fried for 20 s in soybean oil at 180ºC. The fried product is frozen at −23 to −26ºC after wrapping in polyethylene pouches.83 The Antarctic krill can be used as additive to color meat extracts, dried products, fishing bait, aquaculture feed because of its carotenoid pigments. Krill protein hydrolyzates prepared from the krill has also been found useful as a cryoprotectant. It has been recommended that handling and processing/freezing of krill should be done on board of a fishing vessel to avoid rapid quality deterioration.85–87 3.8.2.2

Squid Proteins

Over 80% of the squid protein is extractable in alkaline medium, salt solution, or by isoelectric precipitation. Up to 70% of the protein can be extracted in water at pH 7.0. Maximum proteins are extracted using 4% NaCl or NaOH at pH 11, employing an extraction time of 45 min; temperature, 22ºC; and solvent to squid ratio, 10:1. The extract was subjected to membrane desalination in the case of salt extract followed by isoelectric precipitation at pH 5.0 and spray drying of the precipitated proteins.88 3.8.2.3

Blood Proteins

Although cattle and swine blood and their fractions have been well used, there is relatively little information about fish blood and its fractions as food ingredients. Fish blood or its fractions contain high-quality protein and heme iron and may be useful in nutrition. Fish blood proteins also exhibit good functional properties and could have potential use in food formulations. The nutritional and functional properties of protein fractions from rainbow trout blood were investigated with a view of using these proteins as food ingredients. The physicochemical and functional characteristics of plasma and red cell fractions from rainbow trout were investigated. Solubility, textural properties, thermal stability, foaming, and emulsifying properties were determined and compared with those of swine blood fractions. Although differences

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were observed, the fish blood fractions exhibited sufficient functionality to be used as food ingredients. However, the high potential for spoilage through lipid oxidation and the susceptibility of fish hemoglobin to autooxidation are issues that need to be addressed for commercial utilization.89

3.8.3

FISH PROTEIN HYDROLYZATES

Hydrolyzates are defined as proteins that are chemically or biologically broken down to peptides of varying sizes. Fish protein hydrolyzate (FPH) is prepared by digestion of fish meat by proteolytic enzymes and has been considered as an alternative approach for converting underutilized fish biomass into edible protein products, instead of animal feed or fertilizer. The process of preparation of FPHs consists of proteolytic digestion of fish and fish waste at optimal conditions of pH and temperature required by the enzymes. The enzymes can be from plant (papain, ficin), animal (trypsin, pancreatin), or microbial (pronase, alcalase) sources.90 Liquefaction of the proteins takes place within a few hours. The hydrolyzate is then decanted and centrifuged to remove scales and bones. The soluble fraction is concentrated by evaporation and is suitably dried, preferably by spray drying, giving a yield of about 14% of the raw material. Fish protein hydrolyzates are characterized by their high solubility in water, high protein contents as well as low fat and ash contents. By using different fish species, enzymes, and digestion conditions, a wide range of hydrolyzates can be produced. Lean fish species are ideal raw material for FPH. Fatty fish such as herring may require initial treatment with ethanol to remove lipids before enzymatic hydrolysis. Uncontrolled or prolonged proteolysis results in the formation of smaller and highly soluble peptides completely lacking the functional properties of native proteins, such as water-holding capacity, emulsification capacity, and foaming ability. By careful control of hydrolysis, it is possible to suitably modify the functional properties, which are useful in food formulations. The degree of hydrolysis, which is expressed as the percentage of α-amino nitrogen in the soluble fraction, is important in optimizing the process parameters. The functional potential of FPH may be modified by the use of specific enzymes and by choosing a defined set of hydrolysis conditions such as temperature, pH, and time. Several authors have discussed commercial aspects of FPH production.90–102 Hydrolyzates from several fish species have been prepared employing a variety of enzymes and treatment conditions. The marine fish species include capelin,8,69,92,94 herring, red salmon and dogfish, barracuda, catfish, jew fish, large spine flat head, lizard fish, milk fish, perch, ribbon fish, sole, and threadfin bream and also their processing wastes such as frames, liver, and that from roe industry.69–72,92–98 The effects of different proteolytic enzymes and different reaction conditions on yield, functional and nutritional properties have been evaluated.67 Yield of hydrolysate as dry soluble from miscellaneous fish species ranged between 4 and 14%. The enzyme used for hydrolysis has influence on the properties of FPH. Alcalase hydrolyzed defatted herring meat to a higher degree than papain. Papain hydrolysates were bitter compared with alcalase-treated samples. Color and nonenzymatic browning measurements indicated darkening of the product during three months of storage at 20ºC.95,103 Functional fish protein hydrolyzate, denoted as “Biocapelin” was prepared

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by hydrolyzing capelin (Mallotus villosus) using alcalase or cod pepsin. The product, with a degree of hydrolysis of 12, had up to 80% soluble proteins after dehydration. The functional properties of biocapelin include fat absorption (171%), water adsorption (6.4 at 48% relative humidity for 48 h), cooking loss reduction at 3% addition (54.7%), emulsifying capacity (51%), emulsion stability (92%), whippability (90%), and foam stability (120% for 0.5 min). The product had also exceptionally good amino acid composition and a PER value in the range of 2.61–3.11.90 Backbones and liver from farmed cod (Gadus morhua) were hydrolyzed using either commercial enzymes or cod muscle endogenous enzymes. Enzymes from the latter source gave higher yield. Centrifugation was important for separation of oily fractions, but not for separation of protein fractions. The oil and emulsion fractions consisted mainly of triglycerides, whereas phospholipids were found mostly in the protein-containing fractions. The insoluble sludge had 30% lipids including 46% phospholipids.98 FPH was prepared from scraps of five marine species by treating a mixture of the material with equal proportion of water with Bacillus subtilis endoprotease at pH 8.0 and temperature 60ºC for 2 h. After the treatment, the pH was lowered to 6.0 by malic acid and enzyme hydrolysis was continued using Aspergillus oryzae exoprotease level at 60ºC for 2 h. After raising the pH to 7.0 with 1 N NaOH, the sample was ultrafiltered to get the FPH. The products prepared from the five fish wastes had protein contents ranging from 82 to 86%.99 Roe herring is a waste product from the roe industry was hydrolyzed using an endopeptidase preparation from the bacterium Bacillus licheniformis. At 36% hydrolysis, the herring hydrolysate presented good emulsifying stability (>120 min) and an adequate foam expansion (142%) as compared with the soluble fraction from the unhydrolyzed control herring. The lipid content of the product was less than 1%, whereas the protein content was 77%. The emulsification capacity and fat adsorption capacities of arrowroot and herring powders were higher than those of soy protein concentrate. The emulsion stability of whole herring powder was lower than that of egg albumin, but greater than soy proteins. These studies showed that protein-rich seafood by-products possessed dynamic properties, which could be useful as food binders, emulsifiers, and gelling agents.69–72 Emulsion capacity (1 mL oil emulsified per 200 mg protein) and emulsion stability of pollock protein powders ranged from 29 to 34.65% and 65 to 78%, respectively. Highest and lowest fat absorption values were observed for the protein from the fish frame and viscera (10.6 and 4.1 mL of oil per gram of protein, respectively). The emulsions made with the soluble protein powders exhibited viscoelastic characteristics. A comparison with FAO/WHO 1990 recommendations showed that these powders were good sources for all the essential amino acids. The lysine contents of the powders were above 60 mg/g protein, although the values were lower for protein powders from arrowroot by-products. The studies concluded that the protein powders from the fish by-products could be used as ingredients in food formulations.69–72 Degree of hydrolysis values for the 75-min digestion ranged from 6.4 to 16.7%. Oil yield (4.9–10.6%) from red salmon heads was affected by the enzyme used. Protein hydrolysate powders were yellowish and contained 62.3–64.8% protein with high levels of essential amino acids. Increased degree of hydrolysis values were weakly correlated with increased hydrolysate solubility. Maximum emulsion stability and fat adsorption were observed for the

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dried hydrolysate generated in the 25-min reaction time. Water adsorption of hydrolysate powders ranged from 1.0 to 3.3 mL water/g of dried hydrolysate.69–72 Salmon mince slurry in water having 7.5% protein was treated with one of four commercial alkaline proteases or endogenous-digestive proteases. Reaction conditions were controlled at pH 7.5. Protein contents for the hydrolysates ranged from 71.7 to 88.4%, with a nitrogen recovery range from 40.6 to 99.7%. Emulsification ranged between 75 and 29 mL of oil emulsified per 200 mg protein; and some were better than soy protein concentrate; however, lower than that of egg albumin. Emulsion stability for fish protein hydrolystes was similar to or lower than those of egg albumin or soy protein concentrate. Fat absorption was greater for 5 and 10% degree of hydrolysis than for 15% hydrolyzed sample. It was concluded that FPH at 5% degree of hydrolysis compared with reference proteins and other FPHs studied and the high functionality of the product could be attributed to the unique hydrophilic nature of the hydrolyzate.91 Table 3.9 indicates various products that can be generated by proteases treatment of seafood and seafood wastes.10,104,105

TABLE 3.9 Products from Protease Treatment of Seafood and Seafood Waste Type of Waste

Product

Enzyme Used

Various fish Head and viscera Red meat of tuna Filleting wastes Shellfish waste

Hydrolyzates Proteins hydrolyzate Proteins hydrolyzate, oil Proteins hydrolyzate Proteins hydrolyzate, Chitin Flavor

Fish liver

Proteins as hydrolyzate Oil Reduced viscosity solution, useful for protein powder Proteins as hydrolyzate Guanine

Pepsin, trypsin, and other proteases Papain Alcalase, papain Papain, bromelain Trypsin, chymotrpsin, pepsin, alcalase, microbal, alcalase, pepsin Papain, pepsin, trypsin

Stick water (fish meal processing) Small underutilized fish Fish scales from carp, herring, etc. Various fish

Fish sauce

Capelin, etc.

Roe

Seafood waste

Silage

Papain, pepsin, alcalase Papain, Aspergillus niger protease Pepsin Cathepsin A, C and trypsin-like enzymes Cathepsin D and aminopeptidasedependent amino acid release Pepsin, cathepsin D, and aminopeptidases

Source: Adapted from Chakraborti, R., Food Biotechnology, 2nd ed., CRC Press, Boca Raton, FL, 2006; Haard, N. F., J. Aquat. Food Prod. Technol., 1, 17, 1992; Venugopal, V., Seafood Processing: Adding Value Through Quick Freezing, Retortable Packaging and Cook-chilling, CRC Press, Boca Raton, FL, 2006; Raghunath, M. R. and McCurdy, A. R., J. Agric. Food Chem., 38, 45, 1990.

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FPHs are amorphous powders having reddish brown to creamy yellow color, hygroscopic nature, and bitter taste. As discussed earlier, they also have improved functional properties. They generally contained coagulable nitrogen, proteoses, petones, and free amino acids and will cake rapidly especially at higher ambient temperatures and relative humidities.101 Their proximate compositions are 1–8% moisture, 81–93% protein, 0–5.0% fat, and 3–8% ash.67,102 Bitterness is a common characteristic of fish hydrolyzate that restricts a wide range of its applications. Bitterness is due to presence of several l-amino acids including arginine, praline, leucine, phenyl alanine, tryptophan, and isoleucine, which are bitter to taste. Peptides having hydrophobic residues such as the dipeptides, glycyl-l-leucine and l-leucyll-phenylalanine are also characterized by bitter taste.106 By using specific proteases and digestion conditions the formation of bitter peptides can be reduced. The traditional methods are by masking bitterness with additives such as polyphosphate, gelatin, gelatinized starch, skim milk, acidic oligopeptides, flavors, and sweet amino acids (l-alanine and l-serine). Other additives are glutamic acid or glytamyl-rich peptides. Another method is by removing peptides through suitable gel filtration techniques. The bitterness can also be reduced under controlled conditions by allowing formation of nonbitter larger polypeptides by the action of some proteolytic enzymes such as pepsin, papain, and α-chymotrypsin, by a process called “plastein reaction,” which results in peptide–peptide condensation. Significant amount of plastein synthesis was observed in autolyzed fish waste by pepsin at pH values ranging from 54 to 8.106,107 The plastein reaction also improves the nutritional quality and physical properties of the hydrolyzates. A protease derived from Aspergillus has been recognized to be beneficial for the purpose.93 Immobilization of proteases can simplify the debittering process.103 The commercial aspects of FPH production have been discussed.102 The cost of production of FPH based on a yield of 14% of the raw material is more than that of fish meal. The economic advantage of FPH as a milk replacer has to be considered in this respect.102 The shortcomings of most of those studies are that they completely lack comparison data among different enzymes at the same activity level and often fail to characterize the final degree of hydrolysis of the product that is subject to functional analysis. Protein hydrolyzates are generally used for the modification of functional properties of foods for its use in dietetic foods as a source of small peptides. Enzymatic fish protein hydrolyzates can be used as protein supplements to cereals proteins such as those of wheat, rice, and corn. As discussed earlier, enzymatic modification of food proteins by controlled proteolysis can enhance their functional properties over a wide pH range, and other processing conditions. Choosing the right proteolytic enzyme, environmental conditions, and degree of hydrolysis, the functional properties of proteins can be improved.93 As a result of its high solubility and amino acid balance, FPHs have obvious advantages over dried products such as fish protein concentrate or even human grade fish meal. FPHs are used in dietetic foods as a source of small peptides and amino acids. Its high dispersability makes it suitable as a replacer for milk proteins and as an additive to cereal foods, soups, and bread and crackers.108 FPHs have other applications also besides as nutritional supplement. FPH in lower concentrations can function as a cryoprotectant to prevent denaturation of proteins. FPH suppressed dehydration-induced denaturation of fish meat during

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frozen storage, which was attributed to the stabilization of the hydrated water surrounding the myofibrils.99,109 FPH from Antarctic krill and oyster meat has also been used as a cryoprotectant.85,108 Another use is as a fertilizer. Scrap fish or fish waste can be converted into a stable liquid fertilizer, and can be used as a well-balanced fertilizer for a wide variety of agricultural crops. The material is highly nutritious since it contains desirable macro and microelements from the viscera and head portions. The use of the product as fertilizer can help bacteria and other organisms grow and build soil, providing micronutrients for healthy growth of plants, easiness of application, and reduction of overall costs based on yields and profits. The product has been successfully tested for cranberries, cherries, and applies.110

3.9 FERMENTED FISH PRODUCTS Fermented fishery products including sauces constitute a component of general diet in East and Southeast Asian countries, which include Burma, Indonesia, Kampuchia, Laos, Malaysia, Philippines, Thailand, and Vietnam. In the eastern parts of India a few traditional fermented fish products, designated as hentak and ngari, based on small-sized freshwater fish species are popular. These products are mainly in the form of a sauce or a paste and may be high in salt content, filtered products, unfiltered sauces, Shiokara paste (involving hydrolysis of meat with or without grinding/ drying), and narezushi. The sauces are generally amber colored liquid, having salty taste and cheese-like aroma, whereas pastes are reddish brown with salty taste. Some of the other popular sauces are ngapi, ketjap-ikan, mam-pla, nuoc-mam-gau-ca, among others. These products are used as condiments, which facilitate consumption of rice and hence are important source of nutrition for consumers from the poor strata of the society. Several authors have discussed the processes for production, quality aspects, and consumption pattern of these fermented products.67,90,111–118 Fermented products may be classified broadly into two types of formulations, namely (i) fish and salt and (ii) fish, salt, and carbohydrates. In the former category, fermentation results from autolytic enzymes present in the tissue, while high levels of salt (>20%) prevent microbial deterioration of the meat. Biochemically, fish sauce is salt-soluble protein in the form of amino acids and peptides. It is developed by fermentation with salt-tolerant (halophilic) bacteria, which are principally responsible for flavor and aroma. The marine fish species used for their production include anchovies, mackerel, lizard, clupeids, shad, among others, although freshwater fish are used for sauces such as muoc-mam and mam-pla. Manufacturing methods of fish sauce, factors affecting its quality, nutritional values, microorganisms involved with fermentation, flavor aspects, and methods for their quality evaluation have been discussed.112,113 In general, pastes are fermented for a shorter period of time than sauces. The usually long fermentation time of sauces (5–23 months) may be reduced by the addition of exogenous proteases. Exoproteases have been examined to enhance sauce production. Sardine was incubated along with salt at 30°C for 6 months in presence of carboxy- and aminopeptidases. Carboxy peptidase activity disappeared from the salt/fish mixture within a few days after the start of incubation, whereas aminopeptidase was highly active for the first 2 months. The addition of dipeptidyl peptidase to

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capelin mince was shown to accelerate the fermentation and yield of a product with a desirable taste.103,115 The lactic acid fermentation of fish/carbohydrate mixtures in the presence of small amounts of salt (6–10%) provides possibilities for developing a various products from underutilized marine fish species. The principal carbohydrate used in such fermentations is cooked rice. Sometimes, small amounts of cassava flour or cooked millet may be added. The organisms used for fermentation generally belong to the genera Lactobacillus, Streptococcus, Pediococcus, or Leuconostoc.90 Fermentation of kamaboko (prepared from surimi) has been attempted. Fermentation by different lactobacilli gave a sour taste to the product compared with commercial samples.114 Fermented sausages employing lactic acid fermentation of cod and haddock had 17–36% moisture and 21–35% fat contents.116 Fermentation can be used to prepare hygienic fish sauces.117 There are possibilities for development of mold-fermented products. These products include Asian mold-fermented foods (shoyu, miso, katsuobushi, tempeh, etc.) and also European mold-fermented items (cheese and meat products). Although there are also potentials for use of several mold species, certain problems could be encountered such as possible mycotoxin formation depending on the characteristics of such as some Penicillin spp. used as starter cultures.118 Dehydrated fish protein-rich foods containing carbohydrates have been developed in several parts of the world. These products have been variously named such as fish-macaroni, fricola, fish-potato flakes, and fish wafer. The fish used in these products are mostly low-cost species from marine sources, although freshwater fish are also being examined for their suitability.112 Potential use of functionally active powders to enhance protein quality of food formulations has been suggested.71 The health benefits of consumption of fermented foods including fishery items are that Lactobacillus, Streptococcus, Pediococcus, or Leuconostoc, etc., which cause fermentation are considered as microbial nutraceuticals or probiotics, because of their therapeutic or health benefits. These organisms, by their presence or by antimicrobial substances (e.g., bacteriocins and lactoperoxidase system) control the proliferation of undesirable bacteria in the gut. Other benefits that have been associated with specific strains of these organisms include enhancing immune function, prevention of infantile diarrhea, anticholesterolemic properties, prevention of urinary tract infections, among others (see also Chapter 13).

3.10 ANIMAL FEED Fish silage is a product obtained by acid preservation of commercially unimportant fish and fish offal and is used as an animal feed. The product is required to have a pH of 3.7–4.0 and shelf life of 6 months at 15–30ºC. For preparation of ensilage, formic acid is added to the raw material to lower its pH to about 4.5 to suppress growth of spoilage causing bacteria; however, at this pH muscle proteases are active which degrade the proteins to peptides and amino acids. Preparation of ensilage from rainbow trout (Oncorphymchus mykiss) has been studied. In presence of formic acid, both endo- and exoproteases broke down the proteins to amino acids. There were significant amounts of shorter peptides also.107 Silages from fish waste such as those from silver bellies (Leiognathus spp.), jewfish (Pseudosciaeina spp.), sole fish (Cynoglossus semifasciatus), and tuna have been prepared. The wastes were

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incubated with molasses and sweet potatoes at pH 5.0 in presence of Lactobacillus spp., usually L. plantanum or mixture culture from butter milk together with papaya skin (which contained proteolytic enzymes). Fermentation was carried out at ambient temperature. The products had a storage life of 6 months; beyond this period, there was reduction in amino acid contents. The silage is used as a cattle fodder, because of nutritional quality of its protein as well as contents of vitamins A, D, B1, and B2. These vitamins remain grossly unchanged during silaging; however, some loss of vitamin B12 has been reported.67 Dried tuna silage has a dry matter content of 28.3%; crude protein, 19.7%; ash, 4.9%; and lipid, 5.5%.119 Aquaculture production of certain fish species such as salmon (Salmo spp.) has increased significantly. Recovery of fillets leads to significant amounts of waste, which has otherwise useful food and nutraceutical value. In Norway, the salmon waste is used for silage production. In the well-practiced process, all wastes generated during filleting operation are directly removed by vacuum suction to the silage plant. The waste is minced well and stabilized against microbial spoilage by reducing pH to 3–4 using lactic acid. After one day, the liquid is transferred to a storage tank, where autolysis proceeds due to enzyme activities. The mince separates into three phases, namely, fat, the aqueous protein phase, and a phase of undissolved bones. The top fat layer is separated and heated to 95°C to separate and refine the oil. The salmon oil is used for margarine and for technical use as a component in tyres, candles, paper chemicals, and textile production aids. The protein fraction is evaporated at 80º–85ºC resulting in concentrates having 17–30% protein. The product is used in feed formulation for pigs, cattle, and furry animals and for farmed fish other than Atlantic salmon. These proteins can also be used as nutraceuticals and pharmaceutical products. The bone fraction can be a source of calcium for human consumption. When used in animal feed, the bone fraction gives better utilization of the feed and better growth. The technology can be extended to other aquacultured fish such as Pangasius, the production of which has increased to more than 800,000 t in Vietnam. Besides other products such as gelatin could also be recovered from skin wastes.118 The shrimp industry generates 30–40% waste, consisting of shell and head, which is a good source of protein and other nutrients. The shrimp head can be sun dried for its use as poultry feed. The dried heads contain 69% protein, 17% ash, 5% calcium, and 1% phosphorus. On the contrary, shrimp shells contain 23% protein, 31% ash, 27% chitin, 11% calcium, and 3% phosphorus. This can be used in livestock feed formulations, including diets for farmed shrimp, since it contains essential amino acids. A process has been described to prepare low fiber, low-chitin protein from shrimp head. For this the shrimp heads used must be of good quality and should have been kept in ice from the time of harvest of the shellfish, since storage at ambient temperature would result in the enzymatic degradation of the head proteins and hence poor quality of the product. The process involves cooking shrimp heads to inactivate enzymes and denature proteins. The product is then pressed to eliminate water before submitting to the meat/bone separator. The wet pulp obtained can be frozen or dried for further processing or storage. The pulp has 12.5% protein, and relatively low ash, and 6.5% chitin. The product can find use in shrimp aquaculture, since it is readily accepted by the shellfish brood stock. Its amino acid content is similar to that of commercial brood stock diet.120

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The shrimp waste is a good raw material for silage. Liquid silage from shrimp head is prepared by enzymatic action enhanced by the addition of organic acids such as formic, propionic acids, which have antiseptic and bacteriostatic properties. Instead, lactic acid fermentation has also been employed. During fermentation, the shrimp waste turns to a liquid state within 2–3 days, the liquid representing about 70% of the silage. The product had the following composition: net protein, 16.5%; lipid, 6.4%; ash, 8.5%; and moisture, 67.5%. Typically, about 85% of chitin of the shrimp shell waste remains in the sediment, and the small amount of fat present appears as a scum on top of the liquid. The liquid can be separated by centrifugation, which is dried to give an animal feed, whereas the chitin is used separately for various purposes including preparation of chitosan. The silage could be mixed with substances such as malt wastes, rice husk, corn, bananas, and other carbohydraterich substances to get a paste. The product can be used directly for animal feeding. It can also be used as poultry feed and also as feed in shrimp aquaculture.67,118–122 The acid needs to be neutralized before it can be fed to animals. Studies conducted at the Industrial Technology Institute, Sri Lanka revealed that supplementation of pig feed with 25% of tuna silage gave a feed that was better than conventional pig feed which contained fish meal. Shrimp head and shell waste can be used for production of yeast biomass. The chitin hydrolysate could be used as a substrate for production of Saccharomyces cerevisiae K1V-116 in batch and continuous fermentations.120,123 The production of fish meal and (also fish oil) for aquaculture utilizes a significant proportion of inedible fish, resource caught as by-catch. The present level of catch is sufficient to meet the required production level of around 6.5 mt fishmeal and 1.3 mt fish oil for feed purpose. By the year 2010, total use of fish meal for aquaculture is estimated at 3.5 mt, which represents around 60% of world production. Production of fish oil needs around 1.2 mt, which represents around 92% of world production. With some demand for fish oil for pharmaceutical and nutraceutical products (up to 10% of world production), supplies will be barely adequate as 2010 approaches.124

3.11

MARINE CONNECTIVE TISSUE PROTEINS

3.11.1

COLLAGEN

Collagen consists of three peptide chains, which can be different or identical depending upon the source. The three peptide chains each of which has a helical structure forms together a triple-stranded helix of three almost identical polypeptide chains consisting of repeating triplets (Glycine–X–Y)n, where X and Y are often proline (Pro) or hydroxyproline (Hyp). This basic structural unit of collagen fiber is called tropocollagen. It has a molecular weight of approximately 30 kDa, with a length of about 280 nm and diameter 1.4–1.5 nm. Tropocollagen fibers associate in specific ways to form collagen fibers. During maturation and ageing, collagen fibers strengthen and are stabilized by covalent cross-linkages giving mechanical strength (through enzymatic oxidation of lysine and hydroxyl lysine to aldols and aldimines). Collagen swells in water but does not solubilize. Enzymatically it can be hydrolyzed to various extents using collagenases. A vertebrate animal collagenase, which is a metal proteinase, splits a special bond in native collagen. Heating of collagen

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results in its shrinking. The shrinkage temperature (Ts) is different for different collagen. For fish collagen, Ts is 45ºC and for mammals, the value is 60–70ºC. Heating destroys triple helix structure to random coils, which are soluble in water and are known as gelatin.125,126 Collagens from land-based animals (bovine and porcine) are traditionally used in functional food, and also in cosmetics and pharmaceuticals. Emergence of bovine spongiform encephalopathy (BSE), foot-and-mouth disease (FMD) crisis has resulted in apprehensions in the use of collagen and collagen-derived products from land-based animal skin.125 Seafood processing wastes, which include filleting frames and scales are good source of collagen that can be put to various uses. Backbone wastes from processing of Atlantic cod (G. morhua) account for approximately 15% of the initial wet weight of the fish. The extracellular organic matrix of bone constitutes 35–40% of the tissue by weight, the remaining being mostly minerals. Type I collagen accounts for 90–95 of the extracellular matrix with other noncollagenous proteins.127 Extraction of collagen from scales consists of decalcification and disaggregation followed by treatment with pepsin digestion. The yields of collagen from different fish scales are sardine, 51%; red sea bream, 37.5%; and Japanese sea bass, 41%. These scale collagens are heterotrimers with a chain composition of (α1)2 α2. The denaturation temperature of the collagen was lower than land-animal collagen.125 The fish collagens have characteristic amino acid pattern of that of calf collagen; however, there are differences in the contents of imino acids and amino acids with hydroxyl groups in the side chain. The influence of the imino content on the stability of the α-chain helix in the secondary structure of the protein is believed to be through restricted rotation imposed by imino acids in peptide linkage. The tertiary structure of the collagen molecule is stabilized by both hydrogen bonds and intramolecular covalent cross-links. The number and nature of the cross-links have a profound effect on the stabilization of the collagen and their physical properties.128 An extraction procedure has been developed for isolation of collagen from fish bone (and also pig skin) for use in cosmetic materials. The bone is initially treated with acetone for 12 h at room temperature and dried to remove lipids. The treated fish bone was decalcified using a 10-fold 0.6 M hydrochloric acid at room temperature for 24 h, to get collagen. After decalcification, the supernatant was treated with B. subtilis protease at pH 8 for 1 h at 60°C. After hydrolysis with the protease, the product was filtered and lyophilized. A 1% solution of the powder in water was fractionated by ultrafiltration to separate collagen peptides. The peptides in the hydrolyzate had angiotensin-converting enzyme (ACE) inhibiting activity, giving 50% inhibition at 0.6–2.8 mg/mL. The activity was maximal for the fraction having molecular weight of 10,000.128 Collagen from fish and mammals have been compared.129 Collagen from acetic or citric acid extracts of skins of fish such as Baltic cod (G. morhua) could be precipitated using 0.1% κ-carrageenan. At 0°C, the yield of precipitated collagen was higher than at 20°C.130 Fish collagen from black drum (Pogonia cremis) skin is an excellent source of collagen, which is comparable to land-based collagen. Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were isolated from the bones and scales of black drum and also sheepshead seabream (Archosargus probatocephalus) caught in the Gulf of Mexico. The fish bone and scale collagens were typical type-I collagens. The

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molecular weights of the collagen subunits were about 130 kDa for α1 and 110 kDa for α2. The amino acid composition of the PSCs was closer to that of calf skin ASC than that of cod skin ASC. The melting temperatures of ASC and PSC were above 34°C. Intrinsic viscosity of the PSCs was similar to the intrinsic viscosity of collagen from fish species such as hake, cod, and catfish. Transition temperature of black drum ASC is 36.3ºC, similar to that of calf hide ASC. The black drum collagen has shown potential therapeutic effects in terms of anti-inflammatory activity, inhibition of angiogenesis, and for tissue engineering.131 ASC and PSC contents in the skin of channel catfish (Ictalurus punctaus) were 25.8 and 38.4%, respectively, on a dry weight basis. The catfish collagens were composed of two distinct α chains, which are similar to the porcine type I collagen. The collagens contained more than 23% glycine as the most abundant amino acid. The denaturation temperature of ASC was 32.5°C, about 5°C lower than that of the porcine skin collagen.132 The hake (Merluccius merluccius L.) skin collagenous material showed higher functionality. The values for protein solubility, apparent viscosity, and water-binding capacity were maximum at pH between 2 and 4. The functional values were also found to be higher as compared with collagen from trout.133 Figure 3.6 shows isolation of collagen from black drum.125 Skin and frame byproducts from Pacific whiting surimi manufacturing are good resources for collagen extraction.134 Dried shark fins are in great demand for making ceremonial dish called shark fin soup. It is a major export item from India.

3.11.2

GELATIN

Gelatin is the hydrolyzed form of collagen and is an important industrial biopolymer because of its utility, particularly as a food ingredient. It has many functional applications in food formulations including water holding, thickening, colloid stabilization, crystallization control, film formation, whipping, and emulsification. Gelatin is primarily known for its thermoreversible gelation behavior. The bloom strength,

Fish skin Alkaline treatment neutralization extraction with 0.5 M CH3COOH

Supernatant

Collagen 1

Collagen 2

Residue Acid/pepsin extract Collagen 3

Supernatant

Residue

Salt precipitation

Collagen 1

Collagen 2

Collagen 3

FIGURE 3.6 Isolation of collagen from black drum. (Reprinted from Losso, J. N. et al., Paper presented at the 30th Annual Seafood Science and Technology Society of the Americas, Conference, St. Antonio, TX, November 13–16, 2006. With permission.)

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which is equivalent to the gel strength referred in industry, varies from less than 100 or more than 300 bloom for most commercial gelatins. Most commercial gelatins have a viscosity between 15 and 75 mP. The melting point of gelatin gels is usually lower than the human body temperature. This melt-in-the-mouth property is one of the important characteristics of gelatin gels, responsible for its wide applications in food and pharmaceutical industries. In comparison with gelatin, agar, and carrageenan (which also form thermally reversible gels such as gelatin), have melting temperature higher than the human body temperature. Gelatin is useful for encapsulation of thermolabile pharmaceuticals and in coating of photographic paper. Being devoid of tryptophan, gelatin is not a complete protein and has poor biological value. Researchers have sought to develop gelatin derivatives or modified gelatins such as coldwater-soluble gelatin, hydrolyzed gelatin-based bioactive peptides, and esterified gelatin.135–137 Most of the current production of gelatin is from porcine and bovine hides, demineralized bones, and hooves. 3.11.2.1 Extraction of Gelatin from Marine Sources Gelatins from marine fish skins and bones could be substitutes to mammalian gelatins. A number of seafood and seafood-processing wastes have been examined as sources of gelatin. Further selection of fish species as raw material and optimization of extraction conditions could help in development of gelatins with varied functional properties such as bloom value, viscosity, and solubility.138 The head waste in cod fisheries, which is a major by-product fraction yielding about 20% of the fish weight, could be an excellent source of collagen. About 250,000 t of cod by-products such as heads, backbones, viscera, and skin are yielded annually and most of these are either discarded at sea or sold at a low price for feed production. Cod head contains about 55% muscle, 20% bone, 15% gill, 5% skin, and 4% eyes, with an average protein content of 15%. A simple process to isolate gelatin from cod head involves successive extraction of the raw material at room temperature in dilute NaOH (pH 11) and HCl (pH 2–2.6), which gives a yield of 12%. The gelatin extracted from cod head had similar molecular weight, viscosity, and gel strength as that from cod skin. The fish bone could also be used as raw material for gelatin.139 Although warm temperature and acidity are required to extract gelatin from the bones, higher temperatures and stronger acidity resulted in more hydrolyzed gelatin with poor gelling properties, however viscosity was not affected.139 Figure 3.7 gives process for extraction of gelatin from cod head.140 The viscosity and gel strength of gelatin extracts from cod head are given in Table 3.10.140 Optimal extraction conditions for gelatin from yellowfin tuna (Thunnus albacares) skin were concentration of NaOH (1.89%), treatment time (2.9 days), extraction temperature (58ºC), and extraction time (4.7 h) to get a maximum of 89.7% gelatin having a gel strength (429 bloom). The gel strength of yellowfin tuna skin gelatin (426 bloom) was higher than bovine and porcine gelatins (216 and 295 bloom, respectively), whereas gelling and melting points were lower. Dynamic viscoelastic properties of yellowfin tuna skin gelatin did not change at 20°C, but increased at 10°C similar to bovine and porcine gelatins.141 Instead of strong alkali and acid, gelatin could also be extracted using weak acids such as acetic (50 mM) or lactic (25 mM) acid. These extractants were used for Dover sole (Solea vulgaris) skin-gelatin extraction. The resultant gelatins were

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100

1. Extraction pH 11.0–10.6 2. Extraction pH 11.0–10.8

pH 6.6 Precipitation pH 7.0

3. Extraction pH 2.0–2.6 Precipitate

Supernatant

34.7

12.8

Bone

Soft solid tissues

Gelatine from soft connective tissues

Gelatine from bone 5.7

6.3 Residual bones

Residual soft tissues

4.3

28.5

FIGURE 3.7 Flow sheet for extraction of gelatin from cod head. (Reprinted from Arnesen, J. A. and Gildberg, A., Proc. Biochem., 41, 697, 2006. With permission from Elsevier.)

TABLE 3.10 Viscosity and Gel Strength of Gelatin Extracts from Cod Head Source

Viscosity at 60ºC (mps)

Soft connective tissue Bone, extraction I Bone, extraction II Bone, extraction III

31.2 ± 3.6 24.0 ± 3.7 25.2 ± 3.0 24.3 ± 3.3

Gel Strength (g) at 4ºC 123.2 ± 2.9 90.9 ± 1.6 63.2 ± 1.4 45.6 ± 1.0

Source: Adapted from Arnesen, J. A. and Gildberg, A., Proc. Biochem., 41, 697, 2006. With permission from Elsevier.

comparable in terms of yield, amino acid composition, molecular weight distribution, gel strength, viscoelastic properties, ability to refold into triple helical structures, and aggregation phenomena. Increasing the concentration of lactic acid to 50 mM resulted in a highly hydrolyzed gelatin, with lower folding ability, gel strength,

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and viscoelastic properties.142 Uses of ionic salt solutions were not recommended for fish skin extraction. Washing skin with NaCl, KCl, or MgCl2 adversely affected molecular weight distribution, and consequently gel strength and rheological properties.143 Microbial transglutaminase (MTGase) was examined for extraction of gelatin from the skin of bigeye snapper (Priacanthus macracanthus) and brownstripe red snapper (Lutjanus vitta). The enzyme treatment gave yields of 6.5 and 9.4%, respectively, on the basis of wet weight. MTGase-treated gelatin showed a decrease in band intensity of β- and γ-subunits, suggesting MTGase-induced cross-linking of these components.144 Recovery of muscle proteins from minced backbone wastes was undertaken using a mild proteinase hydrolysis, with subsequent heating to recover gelatin and a protein–calcium fraction from the purified bone fraction. The gelatin recovered had lower molecular weight fractions (15 and 30 kDa) than standard gelatin preparations (>100 kDa).139 The use of high pressure at 250 and 400 MPa, for 10 or 20 min, to extract fish gelatin from skin at 45°C has been recently shown to be a useful alternative. The treatment reduced extraction time and yielded a product having appreciable gel-forming properties.145 Gelatins from two species of snapper had high protein and hydroxyproline. The bloom strength of gelatin gel from brownstripe red snapper skin gelatin (218.6 g) was greater than that of bigeye snapper skin gelatin (105.7 g). Extraction conditions of Alaska pollock gelatin have also been optimized.146 North Sea horse mackerel (Trachurus trachurus) skin was extracted using sodium hydrogen carbonate (0.125%), sodium hydroxide (0.2%), sulfuric acid (0.2%), and citric acid (0.715%) in distilled water, followed by filtration and deionization. The proximate composition of the extracted gelatin indicated that the ash, moisture, color, molecular weight (195.8 kDa), and bloom strength (230) compared well with that of commercial gelatin. The imino acid and hydrophobic amino acids profile of extracted horse mackerel gelatin were closer to those of the commercial warm water tilapia gelatin rather than the nongelling cod gelatin.146 The fish skin for gelatin extraction could be kept dried for convenient processing. Keeping the fish skin of Dover sole (S. vulgaris) under dried conditions did not affect the rheological properties of extracted gelatin. The fish skins were air-dried by using ethanol, ethanol–glycerol mixture, and marine salt, and stored at room temperature for 160 days. Although drying involved a slight decrease in viscoelastic properties as well as gelling and melting points, gel strength was not affected.146 Instead of drying, frozen storage of fish skin may not be advisable, since gelatin from skins frozen at −12°C had lower gel strength when compared to that of fresh skins. Gel electrophoresis of gelatin from fresh skins showed clear bands corresponding to α-, β-, and γ-components, whereas gelatin from frozen skins showed less α- and β-chains but more bands corresponding to lower-molecular-weight fragments; γ-components were less evident in both cases but especially in the case of skins frozen at −12°C.147,148 3.11.2.2 Gelation Characteristics and Other Properties Fish gelatin is heterogeneous in molecular compositions and contains α- and β-chains, similar to animal gelatins. Gelation of fish gelatin, such as mammalian

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gelatins, involves electrostatic interactions, which are important in the stabilization of the gelatin gel network. In comparison with the animal gelatins, fish gelatin gels generally have considerably lower storage modulus, gelling (4–5°C), and melting temperature (12–13°C). Imino acids, proline, and hydroxyproline contribute in stabilization of the gel network. In a comparative study on characteristics of animal (pork) and fish (tilapia) gelatins, it was observed that for both the gelatins, the gel melting temperatures decreased and melting regions narrowed. Increasing gelatin concentration resulted in a higher melting temperature and a broader melting region for all gelatin gels. This is probably due to the lower content of proline and hydroxyproline in fish gelatins. G′ (storage modulus) was found to be a power law function of concentration of gelatins. The storage modulus for fish gelatin at 10% at various ionic strengths showed that loss modulus, G′, increased at low ionic strengths, whereas decreased at higher salt concentrations.149 The tuna gelatin contains a higher percentage of peptides larger than 10,000 Da. This has favored good gelling properties and potential to be used as a gelling agent in the food industry.119 The gelling and melting temperatures could be enhanced using additives such as potassium chloride and carrageenan. In the presence of 1% carrageenan and 20 mM KCl, maximum gel strength was obtained at 2% concentration of fish gelatin. Presence of KCl increased elastic moduli and reduced turbidity. The mixed gels were much stronger at 4°C than at 22°C.145 A mixture of horse mackerel gelatin and egg albumin in the ratio of 3:10 gave a gel that had superior rheological properties. It was suggested that the gelling properties and compatibility with egg proteins made the horse mackerel gelatin a potential replacer of porcine and bovine gelatin in desserts and bakery products.149 Gelatins from skins and bones of young and adult Nile perch were compared. Total gelatin yield, extracted sequentially at 50, 60, 70, and 95°C was in the order adult fish skins > young fish skins > adult fish bones > young fish bones. Nile perch skin and bone gelatins had turbidity units of 20.5–158 and 109–517 and ash contents of 0.5–1.7 and 4.4–11.2%, respectively. Gelatin from adult Nile perch skins exhibited higher viscosity and lower setting time than bone and the young fish skin gelatins. Skin gelatins were found to exhibit higher film tensile strength but lower film percent elongation than bone gelatins. Bone and skin gelatins had similar amino acid composition, with a total imino acid content of about 21.5%. Nile perch skin gelatins had a higher content of polypeptides larger than β compared to bone gelatins. Both bone and skin gelatins also contained low-molecular-weight (<α) peptides. The differences in functional properties between the skin and bone gelatins appeared to be related to differences in molecular weight distribution of the gelatins.150 Table 3.11 shows amino acid composition of collagens and gelatins from various sources. [Regenstein, J. M., Personal communication]. The oil emulsification capacity of fish gelatin was examined with respect to different molecular weight, namely 55 and 120 kDa. Emulsions of 20% corn oilin-water (pH 3.0), with monomodal particle size distributions and small mean droplet diameters (0.35–0.71 μm) could be produced at protein concentrations ≥4.0 wt%. The surface activity of fish gelatin was that of globular proteins such as β-lactoglobulin, as indicated by the presence of some large droplets in the gelatin emulsions after homogenization. Emulsions stabilized by 58 kDa gelatin

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TABLE 3.11 Amino Acid Composition of Collagens and Gelatins from Various Sources

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

Alaska Pollock Skin Gelatin

Alaska Pollock Skin Collagen

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

108 51 51 0 74 358 8 6 55 11 20 26 16 12 95 63 25 0 3 18 150 1000

Cod Skin Gelatin 96 56 52 0 78 344 8 6 50 11 22 29 17 16 106 64 25 0 3 18 156 1001

Tilapia Skin Gelatin

Pork Skin Gelatin

123 47 48 0 69 347 6 8 79 8 23 25 9 13 119 35 24 0 2 15 198 1000

112 49 46 0 72 330 4 6 91 10 24 27 4 14 132 35 18 0 3 26 223 1003

Source: Courtesy of Regenstein, J. M., Personal communication.

contained a bigger population of large droplets than 120 kDa gelatin emulsions, but were more stable to creaming. Fish gelatin stabilized emulsions remained moderately stable to droplet aggregation and creaming after they were subjected to changes in holding temperature (30 or 90°C for 30 min), salt concentration (NaCl ≤ 250 mM), and pH (3–8). The study demonstrated that fish gelatin could have some limited use as a protein emulsifier in oil-in-water emulsions.151 The flocculation, coalescence, and creaming properties of oil-in-water emulsions prepared using fish gelatin, sodium caseinate, and whey proteins were compared. It was shown that where fish gelatin is intended as a replacement for milk protein, there was a need in optimizing the protein/oil ratio to avoid the presence of large droplets, which may be susceptible to coalescence, especially at high ionic strength.36,135–137,152 Table 3.12 shows amino acid composition of collagens and gelatins from various sources [Regenstein, J. M., Personal communication].

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TABLE 3.12 Chemical and Physiochemical Properties of Gelatins from Different Sources Property Hydroxyproline content (%) Bloom strength (bloom) Gel melting temperature (°C) Viscosity (mP)

AG

TG

PG1

PG2

7.3 ± 0.3 98 ± 2 21.2 ± 0.2 120 ± 2

10.6 ± 0.2 273 ± 4 25.4 ± 0.2 38 ± 1

13.3 ± 0.2 110 ± 3 27.5 ± 0.3 22 ± 1

13.1 ± 0.3 240 ± 5 31.2 ± 0.1 47 ± 1

Note: AG, Alaska pollock gelatin; TG, tilapia gelatin; PG1, pork gelatin with low bloom value; PG2, pork gelatin with high bloom value (mean ± standard deviation). Source: Courtesy of Regenstein, J. M., Personal communication.

3.11.2.3

Applications

Marine gelatins have the potential to replace mammalian gelatins in several areas. The cod gelatin is of particular interest for certain applications for use as an additive in cold-stored products, where gelling is not desirable. It has a low melting point of 10ºC. Hence thermolabile compounds such as certain drugs, can be encapsulated at a lower temperature when cod gelatin is used. After gentle drying, the gelatin forms a protective membrane that stays intact until it is solubilized by water in the body.135–137,139 One upcoming use of fish gelatin is for the development of biodegradable films and coatings (see Chapter 13). Gelatin coating can enhance shelf life of muscle foods. Gelatin–chitosan coating increased shelf life and elasticity of the patties, whereas adding powdered chitosan to the patty mixture increased the other rheological parameter values, however shelf life was not affected.151 In a recent study, a 20% bovine gelatin solution was spray-coated onto beef tenderloins, pork loins, salmon fillets, and chicken breasts, which were then packaged in an 80% O2- and 20% CO2-modified atmosphere and stored. All of the gelatin-coated fresh meat products showed a reduction in purge due to barrier to water loss. The gelatin coat reduced color deterioration by acting as a barrier to oxygen. Fish gelatins could also be used for similar applications. Gelatin also influenced rheological properties of food products.153,154 The protein could increase the G′ and G′′ values and decrease the denaturation temperature of myosin. Therefore, it is advisable to add gelatin into chicken meat comminuted products to improve the rheological attributes.154 Other uses of gelatin have also been reported. Mixtures of enzymatic hydrolystes of casein and gelatin are used in slimming diets. Gelatin hydrolyzates are administered along with other products to compensate certain deficiencies during childhood and adolescence, pregnancy, and lactation. They can also be used for treatment of osteoporosis. Gelatin hydrolyzates strengthen the hair by penetrating the hair cuticle and depositing in the cortex, encouraging its use in shampoos and haircare. In addition, peptides in gelatin hydrolyzates can have antioxidant and other biological activities (see Chapter 4). Other industrial applications of fish gelatin are in the areas of light-sensitive coatings, low set-time gels, and as an active ingredient in shampoo with protein.117,133

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3.12

Marine Products for Healthcare

SOME SEAFOOD PRODUCTS OF CONSUMER INTEREST

3.12.1

FOOD FLAVORINGS

Seafood flavors are in high demand for use in products such as artificial crabmeat and fish sausage. Shrimp flavor is used as an additive in surimi-based products and cereal-based extrusion products. Proteolytic enzymes including those from bacteria can aid in the extraction of flavor compounds from shells and other materials, which are then concentrated up to 50–60% dry matter.155 Hydrolyzate from red hake (Urophycis chuss) mince and frame was prepared as a flavoring agent by treating the raw material with commercial proteases. Addition of small amounts of sodium chloride and sodium tripolyphosphte improved the flavor of the hydrolyzate. The umami taste of the product was due to high content of glutamic acid.156

3.12.2

SEA CUCUMBER

Sea cucumbers are popular in China and Singapore, under the name beche-de-mer. Dried sea cucumber is a popular product, prepared by slitting the body, squeezing out the entrails, boiling, and drying. Processing involves boiling sea cucumber in seawater till the body gets swelled, removal of the body by slitting along the back, and further boiling till a rubber-like hardness is reached. They are then drained, guts removed, smoked, and dried over coconut husk fire for 2–3 days, followed by sundrying.157 In Japan, the name for the processed product is iriko, whereas in Malaysia and the Philippines, it is called as trepang. For consumption, the dried product must first be soaked in water, then heated, and finally left at 20°C for 24 h; this procedure being repeated several times in preparation for cooking. After full-scale soaking twice, its weight increases by seven times to give a softened product. However, during the soaking process some components such as minerals, glycosaminoglycan, and collagen of the sea cucumber are eluted into the soaking water. Instead of potable water, a solution of potassium carbonate has found to be useful for soaking, swelling being faster in the carbonate solution than in water.157,158

3.12.3

PRODUCT FROM JELLYFISH

Cured jellyfish in semidried form has export demand in south East Asian countries viz. Japan, Indonesia, Hong Kong, Korea, and Malaysia. The process includes treating the fish with NaCl and alum. In the first stage, the fish is dipped in salt and alum concentrations at 10 and 4%, respectively. This is followed by the second stage when the salt and alum concentrations were 15 and 2%, whereas in the third stage 20% salt is used. The product having a yield of 9% has 17% salt, 6% protein and 77% moisture contents. Jellyfish possess a unique capacity to retain water and control its moisture level. Observation of this function led to successful development of novel absorbent materials such as diapers. The available commercial products have two weaknesses, namely, their nondegradable character and poor absorption capacity, when the liquid to be absorbed contain salt or body fluids. The product based on jellyfish consists of a network of polysaccharides and protein that create a structure that binds water. This structure appears to play significant role in retaining liquid and also overcomes the shortfalls of commercial products. The new material, named

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as Jellysorb, has been applied for the development of female health products. Apart from this, jellyfish can also be a source of hyaluronic acid, used in eye surgery and plastic surgery or cartilage-like substances that could be extracted from the fish. The oral arms and the bell or umbrella of jellyfish is used for dyeing [Devadasan, K., Personal communication].

3.12.4

ROE FROM EGGS AND ITS POWDER

Processed roe from eggs of fish such as capelin, salmon, and trout are good edible products. For processing, the egg sacks have to be removed, which can be done employing proteases, including fish pepsins. Treatment of the roe with the enzyme for a few minutes at ambient temperature results in hydrolysis of the supportive sack tissue that envelops the roe. The treated roe is washed in salt solution, which is usually pickled salt and vinegar to get an edible product. Alaska walleye pollock roe is processed into tarako (salted roe) or mentaiko (roe seasoned with salt, sugar, and monosodium glutamate) and can be used as an ingredient in salad dressing, soups, and sauces. The grade of the pollock roe is determined by color, size, maturity, and firmness. There are significant quantities of immature pollock roe, harvested annually, that are discarded or processed into fish meal. Immature pollock roe can be a potential raw material for the manufacture of protein powders with distinct nutritional characteristics. Freeze-dried protein powders have been prepared from the immature roe. The product had 81.7% protein and 9.2% lipid, the major protein having a molecular weight of 103 kDa. Palmitic acid, DHA (C22:6ω3), and EPA (C20:5ω3) (see Chapter 6) were the three most abundant fatty acids in the product. The powder could be utilized as a nutritional supplement [Devadasan, K., Personal communication]. In addition, various innovative products from bone, gills, vertebral column, skin, and fish gut lining (maw) of large sized fishes such as ghol (Protonibea diacanthus), sailfish (Istiophorus platypterus), skate, and rays have been developed. Trials on the use of dried fish gills for human consumption have been carried out. Fresh gills are soaked for one day in water for cleaning. They are sun dried on racks and which takes 3–4 days giving a yield of 15%. Half gill from big fishes is cut and made into a set of two pieces. The product from India is exported to several East Asian countries. Fish maws or isinglass (dried air bladder from fish such as ghol) is pure collagen and can be used as a substitute for gelatin in clarification of wine and beer. A technology has been developed at the Central Institute of Fisheries Technology, Cochin, India, for production of fine grade isinglass without any preservative that can be directly used for clarification. Fish species used includes sailfish (Istiophorus platypterus), ghol (Protonaebea diacanthus), marlins, and rays. Fresh gills from these fish are soaked for one day in water for cleaning. They are sun dried on racks and dried for 3–4 days to give a yield of 15% on the basis of wet gills [Devadasan, K., Personal communication].

3.12.5

COMMERCIAL ASPECTS

At present, commercial preparations of marine proteins are rare in the market. Products such as fermented fishery products and fish protein hydrolyzates are commercially available. A commercial freeze-dried marine protein product having 75% protein has been developed from whole sardines and anchovies. The fish are

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cooked, pressed, freeze-dried, ground into powder, and encapsulated. Consumption of five capsules per day provides 5 g of protein, equal to ~10–15% of the adult daily protein requirement plus 246 mg of omega-3 fatty acids. According to the manufacturer, a nine capsule serving provides the following amino acids (mg): isoleucine, 199; lysine, 454; phenylalanine, 215; tryptophan, 58l; leucine, 402; methionine, 151; threonine, 156; valine, 198, cystine, 51; histidine 167; arginine, 315; glycine, 227; aspartic acid, 308; serine, 163; glutamic acid, 596; proline, 200; hydroxyproline, 14; alanine, 291; and tyrosine, 181.159 In summary, several protein products could be prepared from marine fishery resources, which may go a long way in alleviation of world protein scarcity for human and animals. These products are rich sources of various functionally active proteins that can be used as additives for food product developments and also other uses. Their functional qualities are grossly retained during processing. In addition to conventional fishes, there is scope for new protein sources such as krill, blood proteins, and immature roe. Proteins from seafood-processing waste are also an excellent feed for animals and aquaculture purposes. The U.S. market for protein ingredients from different sources was worth almost $2.3 billion in 2005 and climbed to $2.4 billion in 2006. At an average annual growth rate of 3.5%, this market is expected to reach $2.8 billion by 2011 (www.bccresearch.com). Marine proteins can contribute significantly to the market in the coming years. The potential of providing bioactive compounds such as peptides also make fish proteins important in human nutrition and health management, which will be discussed in Chapter 4.

REFERENCES 1. Lee, T. C., Sensory and nutritive qualities of food, J. Food Sci., 66, 485, 1983. 2. Clarkson, P. M. and Rawason, E. S., Nutritional supplements to increase muscle mass, Crit. Rev. Food Sci. Nutr., 39, 317, 1999. 3. FAO, State of World Fisheries and Aquaculture, Vol. 95, Food and Agriculture Organization of the United Nations, Rome, Italy, 2002. 4. Bene, C. and Neiland, A. E., Fisheries development issues and their impact on the livelihood of fishing communities in west-Africa—an overview, Food Agric. Environ., 1, 128, 2003. 5. Teutscher, F., Fish, food and human nutrition, Food and Nutr. (FAO), 12, 2, 1986. 6. Venugopal, V. and Shahidi, F., Structure and composition of fish muscle, Food Rev. Int., 12, 175, 1996. 7. Konasu, S. and Yamaguchi, K., Trimethylamine contents in fishery products, in Chemistry and Biochemistry of Marine Food Products, Martin, R. E. et al., Eds., AVI Publishing, Westport, CT, 1982. 8. Kijowski, J., Muscle proteins, in Chemical and Functional Properties of Muscle Proteins, Sikorski, Z. E., Ed., Technomic, Lancaster, PA, 2001, p. 233. 9. Skaara, T. and Regenstein, J. M., Structure and composition of fish muscle, J. Muscle Foods, 1, 269, 1990. 10. Venugopal, V., Seafood Processing: Adding Value through Quick Freezing, Retortable Packaging and Cook-chilling, CRC Press, Boca Raton, FL, 2006, Ch. 15. 11. Nagai, T. et al., Properties of myofibrillar proteins from Japanese stingfish (Sebastes inermis) dorsal muscle, Food Res. Int., 32, 401, 1999. 12. Kantha, S. S., Watabe, S. and Hashimoto, K., Chemistry and biochemistry of paramyosins, J. Food Biochem., 14, 61, 1990.

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13. Mignino, L. A. and Paredi, M. E., Physico-chemical and functional properties of myofibrillar proteins from different species of mollusks, LWT Food Sci. Technol., 39, 35, 2006. 14. Hamann, D. D., Rheological studies of fish proteins, in Food Hydrocolloids, Nishinaki, K. and Doi, E., Eds., Plenum Press, New York, 1994, p. 225. 15. Damodaran, S., Functional properties, in Food Proteins: Properties and Characterization, Nakai, S. and Modler, H. W., Eds., Marcel Dekker, New York, 1996, p. 167. 16. Cherry, J. P., Protein Functionality in Foods, American Chemical Society, Washington, DC, 1981, p. 1. 17. Kinsella, J. E., Functional properties of proteins in foods: A survey, Crit. Rev. Food Sci. Nutr., 7, 219, 1976. 18. Kuniz, I. D. and Kauzmann, W., Hydration of proteins and polypeptides, Adv. Protein Chem., 28, 239, 1974. 19. Moure, A. et al., Functionality of oilseed protein products: a review, Food Res. Int., 39, 945, 2006. 20. Damodaran, S., Amino acids, peptides, and proteins, in Food Chemistry, Fennema, O. R., Ed., Marcel Dekker, New York, 1996, p. 321. 21. Walstra, P. and de Roos, A. L., Proteins at air-water and oil-water interfaces: static and dynamic aspects. Food Rev. Int., 9, 503, 1993. 22. Hill, S. E., Emulsions, in Methods for Testing Protein Functionality, Hall, G. M., Ed., Blackie Academic & Professional, London, UK, 1992, p. 153. 23. van Hippel, P. H. and Schleich, T., The effects of neutral salts on the structure and conformal stability of macromolecules in solution, in Structure and Stability of Biological Macromolecules, Timasheff, S. N. and Fasman, G. D., Eds., Marcel Dekker, New York, 1959, p. 417. 24. Oakenfull, D., Pearse, D. J. and Burley, R. W., Protein gelation, in Food proteins and their Applications, Damodaran, S. and Paraf, A., Eds., Marcel Dekker, New York, 1997, p. 111. 25. Gaithersburg, M. D. et al., A review of physical and chemical protein gel induction, Int. J. Food Sci. Technol., 37, 589, 2002. 26. Mandigo, R. W., Restructuring muscle foods, Food Technol., 40, 85, 1986. 27. Chobert, J.-M. and Haertle, T., Protein lipid and protein-flavor interactions, in Food Proteins and their Applications, Damodaran, S. and Paraf, A., Eds., Marcel Dekker, New York, 1997, p. 143. 28. Bourne, M. C., Food Texture and Viscosity: Concept and Measurement, Academic Press, Orlando, FL, 1982. 29. Holcomb, D. N., Rheology, in Encyclopedia of Food Science and Technology, Francis, F. J., Ed., Wiley, New York, 1991, Vol. 4, p. 1349. 30. Kok, T. N. et al., Multidisciplinary approaches for early determination of gelation properties of fish proteins, J. Aquat. Food Prod. Technol., 16, 5, 2007. 31. Xiong, Y. L., Structure—function relationship of muscle proteins, in Food Proteins and their Applications, Damodaran, S. and Paraf, A., Eds., Marcel Dekker, New York, 1997, p. 34. 32. Damodaran, S. and Paraf, A., Food Proteins and their Applications, Damodaran, S. and Paraf, A., Eds., Marcel Dekker, New York, 1997. 33. Arbdeya, J. C. et al., Improving functional properties of tuna byproducts by nonenzymatic glycosylation. Paper presented at the 2nd Joint Trans-Atlantic Fisheries Technology Conference, Quebec City, October 29 to November 1, 2006. 34. Matheis, G., Phosphorylation of food proteins with phosphorus oxychloride— improvement of functional and nutritional properties: a review, Food Chem., 39, 13, 1991. 35. Birschbach, P. et al., Enzymes: tools for creating healthier and safer foods, Food Technol., 58(4), 20, 2004.

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36. Dickinson, E. and Lopez, G. M., Comparison of the emulsifying properties of fish gelatin and commercial proteins, J. Food Sci., 66, 118, 2001. 37. Kuraishi, C. et al., Applications of transglutaminase for food processing, Hydrocolloids, 2, 281, 2000. 38. Venugopal, V. et al., Enzymes in fish processing, biosensors and quality control: a review, Food Biotechnol., 14, 21, 2000. 39. Wu, M. C., Atallah, M. T. and Hultin, H. O., The proteins of washed minced fish muscle have significant solubility in water, J. Food Biochem., 15, 209, 1992. 40. Lanier, T. C., Functional properties of surimi, Food Technol., 40, 107, 1986. 41. Stone, D. W. and Stanley, A. P., Gelation of fish muscle proteins, Food Res. Int., 25, 381, 1992. 42. Ogunlade, I., Olaofe, O., and Fadare, T., Chemical composition, amino acids and functional properties of selected seafoods, J. Food Agric. Environ., 3, 130, 2005. 43. Binsi, P. K., Shamasundar, B. A., and Dileep, A. O., Some physico-chemical, functional and rheological properties of actomyosin from green mussel (Perna viridis), Food Res. Int., 39, 992, 2006. 44. Reddy, G. V. and Srikar, L. N., Pre-processing ice storage of fish mince proteins, J. Food Sci., 56, 965, 1991. 45. Benjakul, S., Visessanguan, W. and Tueksuban, J., Changes in physiochemical properties and gel-forming ability of lizardfish during post-mortem storage in ice, Food Chem., 80, 535, 2003. 46. Wang, H. et al., Physico-chemical properties of muscle and natural actomyosin extracted from farmed Atlantic salmon (Salmo salar) stored at 4ºC, J. Food Biochem., 29, 71, 2005. 47. Verma, J. K. et al., Effect of frozen storage on lipid freshness parameters and some functional properties of oil sardine (Sardinella longiceps) mince, Food Res. Int., 28, 87, 1995. 48. Mohan, M., Ramachandran, D., and Sankar, T. V., Functional properties of Rohu (Labeo rohita) proteins during iced storage, Food Res. Int., 39, 847, 2006. 49. Shenouda, S. Y. K., Theories of protein denaturation during frozen storage of fish, Adv. Food Res., 26, 275, 1980. 50. Benjakul, S. et al., Comparative study of physico-chemical changes of muscle proteins from some tropical fish species, Food Res. Int., 36, 787, 2003. 51. Giese, J., Proteins as ingredients: types, functions and applications, Food Technol., 48, 50, 1994. 52. Rustad, T. and Falch, E., Making the most of fish catches, Food Sci. Technol., 16, 36, 2000. 53. Hall, G. M. and Ahmad, N. H., Surimi and fish mince products, in Fish Processing Technology, Hall, G. M. Ed., Blackie Acad. Professional, Glasgo, UK, 1994. 54. Grantham, G. J., Minced fish technology: a review. Fisheries Technical Paper 216, Food and Agriculture Organization of the United Nations, Rome, Italy, 1981. 55. Yu, S. Y., Utilization of whey protein concentrate and carrageenan in fish ball processing. FAO Fisheries Report 514, Suppl., 1995, p. 225. 56. Dewitt, C. A. M. et al., Pilot plant scale production of protein from catfish treated by acid solubilization/isoelectric precipitation, J. Food Sci., 72, E351, 2007. 57. Suzuki, T., Heat textured fish protein concentrate (marinebeef), in Fish and Krill Protein: Processing Technology, Applied Science Publisher, London, UK, 1981, p. 14. 58. Shenoy, V. A. et al., Textured meat from low cost fish, Fish. Technol., 25, 124, 1988. 59. Kasapis, S. et al., Scientific and technological aspects of fish product development, Part I: Handshaking instrumental texture with consumer preference in burgers, Int. J. Food Prop., 7, 449, 2004. 60. Venugopal, V. and Shahidi, F., Crit. Rev. Food Sci. Nutr., 35, 431, 2005.

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61. Suzuki, T., Fish and Krill Protein Processing Technology, Elsevier, London, 1981. 62. Ohshima, T., Suzuki, T., and Koizumi, C., New developments in surimi technology, Trends Food Sci. Technol., 4, 157, 1993. 63. Roussel, H. and Cheftel, J. C., Characteristics of surimi and kamaboko from sardines, Int. J. Food Sci. Technol., 23, 607, 1988. 64. Chakraborti, R. and Gupta, S., Characteristics of gel from the meat of twelve species of fish from Visakhapatnam coast, Fish. Technol. (India), 37, 5, 2000. 65. Moller, A. B., Studies on seafood value addition. FAO/GLOBEFISH, Special Market Study, Food and Agriculture Organization of the United Nations, Rome, Italy, 2003, p. 93. 66. Raju, C. V., Shamasundar, B. A., and Udupa, K. S., The use of nisin as a preservative in fish sausage stored at ambient (28 ± 2°C) and refrigerated temperatures, Int. J. Food Sci. Technol., 38, 171, 2003. 67. Sen, D. P., Advances in Fish Processing Technology, Allied Publisher, New Delhi, 2005, p. 305. 68. Venugopal, V., Chawla, S. P., and Nair, P. M., Spray dried protein powder from threadfin bream: preparation, properties and comparison with FPC type-B, J. Muscle Foods, 7, 55, 1996. 69. Sathivel, S. et al., Functional and nutritional properties of red salmon enzymatic hydrolyzates, J. Food Sci., 70, 401, 2005. 70. Sathivel, S. et al., Biochemical and functional properties of herring (Clupea harengus) by-product hydrolysates, J. Food Sci., 68, 2196, 2003. 71. Sthivel, S. and Bechtel, P. J., Properties of soluble protein powders from Alaska pollock (Theragra chalcogramma), Int. J. Food Sci. Technol., 41, 520, 2006. 72. Sathivel, S. et al., Properties of protein powders from arrowtooth flounder and herring by-product, J. Agric. Food Chem., 52, 5040, 2004. 73. Bragadottirm, N. et al., Stability of fish powder made from saithe (Pollachius virens) as measured by lipid oxidation and functional properties, J. Aquat. Food Prod. Technol., 16, 115, 2007. 74. Venugopal, V., Functionality and potential applications of thermostable water dispersions of fish meat, Trends Food Sci. Technol., 8, 271, 1997. 75. Venugopal, V., Gel formation of fish structural proteins under mild acidic conditions: comparison with conventional surimi gelation and applications. Reprinted from Fish. Technol. (India), 40, 61, 2003. 76. Venugopal, V. et al., Restructured shelf stable steaks from shark meat gel, Lebensm. Wiss. Technol., 35, 185, 2002. 77. Venugopal, V. et al., Rheological and solubility characteristics of washed capelin (Mallotus villosus) mince in water, J. Food Biochem., 19, 175, 1995. 78. Venugopal, V. et al., Gelation of shark meat under mild acidic conditions: physicochemical and rheological characterization of the gel, J. Food Sci., 67, 2681, 2002. 79. Venugopal, V., Doke, S. N., and Nair, P. M., Gelation of shark myofibrillar proteins by weak organic acids, Food Chem., 50, 185, 1994. 80. Nurul, H., Amiah, A., and Salam, B. A., Functional properties of surimi powder from three Malaysian marine fish, Int. J. Food Sci., Technol., 36, 401, 2001. 81. Hultin, H. O., Recovery of functional protein isolates from fat fish. Paper presented at the 5th Joint Meeting, Atlantic Fisheries Technology Conference & Tropical and Subtropical Seafood Science and Technology Society of the Americas, Florida, November 10–14, 1999. 82. Nurul, H., Amiah, A., and Salam, B. A., Functional properties of surimi powder from three Malaysian marine fish, Int. J. Food Sci., Technol., 36, 401, 2001. 83. Nair, L. et al., Nutritional value of edible meat powder and meat from three fatty deepsea fishes, Fish. Technol. (India), 28, 67, 1991.

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84. Pariser, E. R. and Walterstein, M., Fish protein concentrate: lessons for future food supplements, Food Policy, 5, 295, 1980. 85. Kuwano, K. and Mitamura, T., On the Antarctic krill protein concentrate (KPC)-I, Nippon Suisan Gakkaishi, 43, 559, 1977. 86. Rehbein, R., Composition and properties of krill fingers, in Advances in Fish Science and Technology, Connell, J. J., Ed., Fishing News Books, Surrey, England, 1980, p. 311. 87. Zhang, N., Yamashita, Y., and Nozaki, Y., Effect of protein hydrolysate from Antarctic krill meat on the state of water and denuaturation by dehydration of lizard fish myofibrils, Fish. Sci., 68, 672, 2000. 88. Kahn, L. N. et al., Squid protein isolate: effect of processing conditions on recovery and yield, J. Food Sci., 39, 592, 1974. 89. Saguer, E., Fort, N., and Regenstein, J. M., Fish (rainbow trout) blood and its fractions as food ingredients, J. Aquat. Food Prod. Technol., 15, 19, 2006. 90. Loffler, A., Proteolytic enzymes: sources and applications, Food Technol., 40(1), 63, 1986. 91. Quaglia, G. B. and Orban, E., Influence of degree of hydrolysis on the solubility of protein hydrolyztes from sardines, J. Sci. Food Agric., 35, 271, 1987. 92. Owens, J. D. and Mendoza, L. S., Enzymatically hydrolysed and bacterially fermented fishery products, J. Food Technol., 20, 273, 1985. 93. Kristiansson, H. G. and Rasco, G. A., Fish protein hydrolysates: production, biochemical, and functional properties, Crit. Rev. Food Sci. Nutr., 40, 43, 2000. 94. Shahidi, F., Han, Z.-Q., and Synowiecki, S., Functional fish protein hydrolyzate, in Seafood Safety, Processing and Biotechnology, Shahidi, F., Jones, Y., and Kitts, D. D., Eds., Technomic, Lancaster, PA, 1997, p. 15. 95. Panyam, D. and Kilara, A., Enhancing the functionality of food proteins by enzymatic modification, Trends Food Sci. Technol., 7, 120, 1998. 96. Diniz, F. M. and Martin, A. M., Effects of the extent of enzymatic hydrolysis on functional properties of shark protein hydrolysate, LWT Food Sci. Technol., 30, 266, 1997. 97. Hoyle, N. T. and Merritt, J. H., Quality of fish protein hydrolysate from herring, Food Sci., 59, 76, 2003. 98. Liceaga-Gesualdo, A. M. and Li-Chan, E. C. Y., Functional properties of fish protein hydrolysat from herring (Clupea harengus), J. Food Sci., 64, 1000, 1999. 99. Hoyle, N. T. and Merritt, J. H., Quality of fish protein hydrolysate from herring, J. Food Sci., 59, 76, 2003. 100. Slizyte, R. et al., Hydrolysis of cod (Gadus morhua) by-products: influence of initial heat inactivation, concentration and separation conditions, J. Aquat. Food Prod. Technol., 13, 31, 2004. 101. Khan, M. A. et al., Effect of enzymatic fish protein hydrolyste from fish scrap on the state of water and denaturation of lizard fish (Saurida wanieso) myofibrils during dehydration, Food Sci. Technol. Res., 9, 257, 2003. 102. Merritt, J. M., Assessment of the producton costs of fish protein hydrolysates, Animal. Feed Sci. Technol., 7, 147, 1989. 103. Damle, M. V., Jamdar, S. N., and Harikumar, P., Enzymatic process for debittering of protein hydrolysate using immobilized peptidases’. Int. Patent No. PCT/In2006/00025, WO/080599, 2007. 104. Chakraborti, R., Enzymatic bio-processing of tropical seafood wastes, in Food Biotechnology, 2nd ed., Shetty, K. et al., Eds., CRC Press, Boca Raton, FL, 2006, 1605. 105. Haard, N. F., A review of proteolytic enzymes from marine organisms and their application in the food industry, J. Aquat. Food Prod. Technol., 1, 17, 1992. 106. Raksakulathai, R. and Haard, N. F., Exoproteases and their applications to reduce bitterness in food: a review, Crit. Food Sci. Nutr., 43, 401, 2003.

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107. Hall, G. M. and Ahmad, N. H., Functional properties of fish protein hydrolysates, in Fish Processing Technology, Hall, G. M., Ed., Blackie Academic & Professional, New York, 1992, Ch. 11, p. 24. 108. Frokjaer, S., Use of hydrolysates for protein supplementation, Food Technol., 48(10), 86, 1994. 109. Darmanto, Y. S. et al., Effect of protein hydrolyste of oyster meat on the state of water and denaturation of fish myofibrils during dehydration, Nippon Suisan Gakkaishi, 63, 378, 1997. 110. Pigott, G. M., Enzyme hydrolysis of fish waste for animal feed and fertilizer, in Seafood Safety, Processing and Biotechnology, Shahidi, F., Jones, Y., and Kitts, D. D., Eds., Technomic, Lancaster, PA, 1997, p. 249. 111. Steinkraus, K. H., Ed., Handbook of Indigenous Fermented Foods, Marcel Dekker, New York, 1983, p. 95. 112. Lee, C. H., Steinkraus, K. H., and Reilly, P. J. A., Eds., Fish Fermentation Technology, Yurim Publication, Seoul, South Korea, 1989. 113. Sarojnalini, C. and Singh, W. V., Composition and digestibility of fermented fish food of Manipur, J. Food Sci. Technol., 25, 349, 1988. 114. Venugopal, V. and Shahidi, F., Value-added products from underutilized fish species, Crit. Rev. Food Sci. Nutr., 35, 431, 1995. 115. Lopetcharat, K. et al., Methods of production and nutritional quality of fish sauces, Food Rev. Int., 17, 65, 2001. 116. Kanoh, S. et al., Preparation of fermented kamaboko using lactic acid bacteria, J. Jap. Soc. Food Sci. Technol., 39, 519, 1992. 117. Hwang, J. W. et al., Preparation of fermented sausages from underutilized fish and meat sources, J. Food Proc. Preserv., 13, 187, 1989. 118. Leistner, L., Mold-fermented foods: recent developments, Food Biotechnol., 4, 433, 1990. 119. Sultanbawa, Y. and Aksnes, A., Tuna process waste—an unexploited resource, Infofish Int., 3, 37, 2006. 120. Barratt, A. and Montano, R., Shrimp heads—a new source of protein, Infofish Int., 4, 21, 1986. 121. Sorensen, N. K., Turn Pangasius by-products into cash! Infofish Int., 4, 35, 2007. 122. Javed, A. and Mahendrakar, N. S., Growth and meat quality of broiler chicks fed with fermented viscera silage, Int. J. Animal Sci., 11, 1, 199. 123. Ferrer, J. et al., Acid hydrolysis of shrimp-shell wastes and the production of single cell protein from the hydrolysate, Bioresource Technol., 57, 55, 1996. 124. GAFTA, Fishmeal from sustainable sources, A summary of documentary evidence. Fish meal Information Network/Grain and Feed Trade Association, London, www. iffo.com. 125. Losso, J. N. et al., Comparative biochemical properties of collagen from skin and bones of black drum, Pogonia cremis, Presented at 30th Annual Seafood Science and Technology Society Conf., St. Antonio, TX, November 13–16, 2006. 126. Gildberg, A., Utilization of cod backbone by biochemical fractionation, Proc. Biochem., 38, 475, 2002. 127. Morimura, S. et al., Development of an effective process and evaluation for utilization of collagen contained in livestock and fish waste, Proc. Biochem., 37, 1403, 1999. 128. Nagai, T., Izumi, M., and Ishi, M., Fish scale collagen. Preparation and partial characterization, Int. J. Food Sci. Technol., 39, 239, 2004. 129. Hofman, K., Hall, B., and Avery, N., Fish and mammalian skin collagen—similarities and Differences. Paper presented at the 29th Annual Seafood Science and Technology Society of the Americas Conf., Norfolk, VA, November 7–9, 2007.

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130. Sadowska, M. and Kolodziejska, I., Optimisation of conditions for precipitation of collagen from solution using κ-carrageenan. Studies on collagen from the skin of Baltic cod (Gadus morhua), Food Chem., 91, 45, 2005. 131. Ogawa, M. et al., Biochemical properties of bone and scale collagens isolated from the subtropical fish black drum (Pogonia cromis) and sheepshead seabream (Archosargus probatocephalus), Food Chem., 88, 495, 2004. 132. Liu, H. Y. and Guo, S., Studies on collagen from the skin of channel catfish (Ictalurus punctaus), Food Chem., 101, 623, 2007. 133. Moneto, P., Gómez-Guillén, M. C., and Borderías, A. J., Functional characterisation of muscle and skin collagenous material from hake (Merluccius merluccius L.), Food Chem., 65, 55, 1999. 134. Kim, J. S. and Park, J. W., Characterization of acid-soluble collagen from Pacific whiting surimi processing byproducts, J. Food Sci., 69, 637, 2004. 135. Wasswa, J., Tang, J., and Gu, X., Utilization of fish processing by products in the gelatin industry, Food Rev. Int., 23, 159, 2007. 136. Ward. K. B., Wang, Z., and Xu, S., Gelatin: a valuable protein for food and pharmaceutical industries, Crit. Rev. Food Sci. Nutr., 41, 481, 2001. 137. Baziwane, D. and He, Q., Gelatin: the paramount food additive, Food Rev. Int., 19, 423, 2003. 138. Lu, X., Chapman, K. W., and Regenstein, J. M., Characterization of several fish gelatins, in Seafood Safety, Processing and Biotechnology, Shahidi, F., Jones, Y. and Kitts, D. D., Eds., Technomic, Lancaster, PA, 1997, p. 18. 139. Anonymous., Gelatin from cod skin, Infofish Int., 5, 71, 2007. 140. Arnesen, J. A. and Gildberg, A., Extraction of muscle proteins and gelatin from cod head, Proc. Biochem., 41, 697, 2006. 141. Cho, S. M., Gu, Y. S., and Kim, S. B., Extracting optimization and physical properties of yellowfin tuna (Thunnus albacares) skin gelatin compared to mammalian gelatins, Food Hydrocoll., 19, 221, 2005. 142. Gimenez, B. et al., Use of lactic acid for extraction of fish skin gelatin, Food Hydrocoll., 19, 941, 2005. 143. Gimenez, B. et al., The role of salt washing of fish skins in chemical and rheological properties of extracted gelatin, Food Hydrocoll., 19, 951, 2005. 144. Jongjareonrak, A. et al., Skin gelatin from bigeye snapper and brownstripe red snapper: chemical compositions and effect of microbial transglutaminase on gel properties, Food Hydrocoll., 20, 1216, 2006. 145. Gómez-Guillén, M. C., Giménez, B., and Montero, P., Extraction of gelatin from fish skin by high pressure treatment, Food Hydrocoll., 19, 923, 2006. 146. Zhou, P., Mulvaney, S. J., and Regenstein, J. M., Properties of Alaska pollock skin gelatin: a comparison with tilapia and pork skin gelatins, J. Food Sci., 71, C313, 2006. 147. Gimenez, B. et al., Storage of dried fish skins on quality characteristics of extracted gelatin, Food Hydrocoll., 19, 258, 2005. 148. Fernandez-Díaz, M. D. et al., Effect of freezing fish skins on molecular and rheological properties of extracted gelatin, Food Hydrocoll., 17, 281, 2004. 149. Badii, F. and Howell, N. K., Fish gelatins: structure, gelling properties and interaction with egg albumen proteins, Food Hydrocoll., 20, 630, 2006. 150. Muyonga, J. H., Cole, C. G. B., and Duodu, K. G., Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin, Food Hydrocoll., 18, 581, 2004. 151. Surch, J. and Decker, E. A., and McClements, D. J., Properties and stability of oilin-water emulsions stabilized by fish gelatin, Food Hydrocoll., 20, 596, 2006. 152. Choi, S. S. and Regenstein, J. M., Physicochemical and sensory characteristics of fish gelatin, J. Food Sci., 65, 194, 2000.

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153. Lopez-Caballero, M. E. et al., A chitosan–gelatin blend as a coating for fish patties, Food Hydrocoll., 19, 303, 2005. 154. Yang, Y. L. et al., Rheological properties of myosin-gelatin mixtures, J. Food Sci., 72, C270, 2007. 155. Pan, B. S., Recovery of shrimp waste for flavor, in Advances in Fishery Technology and Biotechnology for Increased Profitability, Voigt, M. N. and Botta, J. R., Eds., Technomic, Lancester, PA, 1990, p. 4. 156. Imm, J. Y. and Lee, C. M., Production of seafood flavor from red hake (Urophycis chuss) by enzymatic hydrolysis, J. Agric. Food Chem., 47, 2360, 1999. 157. Bruce, C., Sea cucumbers-extraordinary but edible all the same, Infofish Int., 6, 19, 1983. 158. Fukunaga, T. et al., Effects of soaking conditions on the texture of dried cucumber, Fish. Sci., 70, 329, 2004. 159. http://www.seagateproducts.com/marine-protein.html, Accessed on December 2007. 160. Raghunath, M. R. and McCurdy, A. R., Influence of pH on the proteinase complement and proteolytic products in rainbow trout viscera silage, J. Agric. Food Chem., 38, 45, 1990.

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Proteins: 4 Seafood Nutritional Value, Bioactive Peptides, Marine and ColdAdapted Enzymes 4.1 INTRODUCTION Chapter 3 discussed the various protein products and supplements obtained from marine fishery sources. This chapter discusses the nutritive value and physiological functions of marine proteins. Proteins are essential for human nutrition. Proteins are required for growth, building up of muscle mass, and efficient working of body functions. Inadequate protein in the diet may lead to a variety of health concerns, such as loss of muscle mass, growth failure, weakening of the basic bodily functions, and even death. In addition, they trap indigestible substances and toxins in the digestive tract and expel them through feces. Proteins also play an important role in lowering cholesterol and blood pressure and in weight management.1 Digestion of protein through enzymatic hydrolysis produces a wide range of peptides, which are then absorbed into the blood circulatory system. These peptides also play physiological roles other than being sources of metabolic energy and essential amino acids. Ingestion of such peptides either as part of a food, drink, or drug has been shown to cure a number of disorders such as high blood pressure in hypertensive patients.2

4.2 DIETARY PROTEIN REQUIREMENTS The human protein requirement varies depending on age, gender, and physical activity. The need for a dietary protein is both for essential amino acids and dietary nitrogen because of high turnover of tissue protein (approximately 240 g/day) accompanied by an insufficient utilization of the amino acids, which are lost from the body as metabolic products such as urea. Dietary amino acids are required to keep the body in a state of nitrogen balance in adults or during periods of growth (infancy, childhood, pregnancy, physical training, or during diseases) in a positive nitrogen balance. Otherwise, the body will use its own tissue protein as a nitrogen source. The recommended dietary allowance for protein is 0.8 g/kg body weight per day. Athletes require a greater amount of protein, above 2 g/kg body weight per day. The generally accepted dietary intake guidelines recommend that women 103

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aged 19–70 years consume 46 g of protein per day, whereas men of the same age group will require about 15% more due to their larger muscle mass. The amount of protein required is higher for pregnant and lactating women and for those recovering from the postoperative trauma. The recommended amino acid scoring pattern for proteins for children who are 1 year and older is as follows (in milligrams per gram of protein): isoleucine, 25; leucine, 55; lysine, 51; methionine + cysteine, 25; phenylalanine + tyrosine, 47; threonine, 27; tryptophan, 7; valine, 32; and histidine, 18. Supply of functionally and nutritionally important proteins to meet the requirement of the rising population is one of the major aims of agriculture and food technology. Several vegetables and legumes are rich sources of proteins, whereas they are generally nutritionally weak as compared to animal proteins because of deficiency in one or more essential amino acids.3–11

4.3 NUTRITIVE VALUE OF PROTEINS The nutritive values of proteins vary and are governed by source, amino acid composition, ratios of essential amino acids, effects of processing and interaction with other components of the diet, susceptibility to digestion, and physiological utilization of specific amino acids after digestion and absorption. In general, protein quality depends on the concentration and ratios of constituent amino acids making up a specific protein. The amino acids in a protein have been divided into three categories based on their importance in protein synthesis in vivo in the case of human nutrition. These are essential or indispensable amino acids, namely, histidine, isoleucine, leucine, lysine, threonine, phenylalanine, methionine, and valine; conditionally indispensable, namely, arginine, cysteine; and dispensable, which include alanine, aspartic acid, glutamic acid, glycine, proline, and serine. The greater the ratio of indispensable amino acids in a protein, the greater is the biological value (BV) or quality. Proteins that are deficient in one or more amino acids are of poor quality. For example, tryptophan and lysine are limiting in corn, lysine in wheat and other cereals, and methionine in soybeans and other legumes. From a nutritional point of view, protein requirement can be fulfilled by an intake of free amino acid, protein hydrolyzates, or by intact protein assuming the content of essential amino acids and the bioavailability is the same. Protein hydrolyzates are highly soluble and unlike whole proteins, do not form viscous solutions, and have relatively acceptable flavor. Therefore, they are used as the sole or partial nitrogen source in specialized adult nutritional products and supplements. The gastrointestinal absorption of hydrolyzates, especially di- and tripeptides, seems to be more effective compared to both intact protein and free amino acids, especially under conditions of impaired digestive functions.3–5

4.3.1

METHODS FOR EVALUATION OF NUTRITIONAL QUALITY OF PROTEINS

Biological methods used to evaluate the nutritional value of food proteins are based on animal feeding studies or chemical estimations. The objective of biological methods is to estimate the amount of biological utilization of proteins. These include the nitrogen balance method based on protein digestibility, nitrogen conversion factor, plasma amino acid ratio, protein efficiency ratio (PER i.e., weight gained per gram of protein consumed), and estimation of relative nutritive value in comparison with

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TABLE 4.1 Methods for Evaluation of Protein Quality Method Amino acid availability (%) Amino acid or chemical score Digestibility

Limiting amino acid

NPU Apparent digestibility coefficient (ADC) NBI Nitrogen conversion factor Nitrogen requirement for humans PER Reference protein

Description (The total intake of amino acid − fecal excretion of amino acid)/(total intake of amino acid) × 100 (Milligrams of amino acid in 1 g of test protein)/(milligrams of amino acid in a reference protein) (Nitrogen (N) consumed (test animals) − N excreted in feces (test group) + metabolic N in feces)/N consumed (test animals) × 100, where N excreted in feces (protein-free animals) × food intake (test group)/food intake (protein-free animals) An essential amino acid in a protein that shows the greatest difference in concentration from the same amino acid in a reference, highquality protein (N retained/N intake) × 100 that is, 100 × [R − (R0 − I0)]/I 100 × (I − F)/I 100 × (B − B0)/A Factor used to convert N to protein, ranges from 5.18 to 6.38; 6.25 for seafood proteins Endogenous N in urine (U) + endogenous N in feces (F) + N lost as sweat, skin, and integument (S) + N required for growth (G) (Weight gain of a test group)/(total protein consumed) A protein of high BV such as casein containing a specified pattern of amino acids

Note: I, I0 = N intake of animals with and without proteins, respectively; F, F0 = N in feces of animals fed with and without proteins, respectively; B, B0 = N balance in animals fed with and without proteins respectively; R, R0 = N of whole animals fed with and without proteins, respectively; A = I − (F − F0). Source: Adapted from Friedman, M., J. Agr. Food Chem., 44, 6, 1996. With permission from American Chemical Society.

a protein of high quality such as casein. Table 4.1 presents methods for evaluation of protein quality.7 Most of the methods make use of animal feeding experiments. Generally, a PER value below 1.5 denotes a protein of low or poor quality; between 1.5 and 2.0, intermediate quality; and above 2.0, good to high quality. A BV of 100% denotes the highest quality of protein. These methods may not be regularly used for quantitatively estimating the effectiveness of food to meet human protein needs or for nutritional labeling. Others are amino acid availability, amino acid or chemical score, and determination of limiting amino acids such as lysine.5 Availability of amino acid refers to a chemical integrity of the amino acid and its influence to processing by heat, high pH, oxidation, etc. Digestibility of proteins indicates the susceptibility of its peptide bonds to hydrolysis, which varies with proteins. Since proteins form a component of food, another parameter, that is, protein/energy ratio

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is also a useful criterion for establishing the nutritional quality of proteins. When properly used, they measure the ability of a given food to meet protein requirements if consumed in sufficient quantity to meet energy needs. At any given level of dietary protein, the addition of energy improves the nitrogen balance of adults until a plateau is reached, reflecting an adequate protein intake. This beneficial effect of energy requires sufficient protein of adequate quality to permit achievement of nitrogen balance.5,6

4.4 NUTRITIVE VALUE OF SEAFOOD PROTEINS The nutritive value of marine fish proteins is equal to or better than that of casein and red meat proteins because of their favorable essential amino acid pattern. Fish proteins are rich in all the essential amino acids (particularly methionine and lysine) in contrast to most proteins from plant sources, which lack adequate amounts of one or more essential amino acids. There are no significant differences in the amino acid composition of freshwater and marine fish. However, certain marine fish such as mackerel and tuna, may be exceptionally rich in the amino acid histidine. Fish proteins are highly sensitivity to proteolytic digestion, with a digestibility of more than 90%. The in vivo digestibility of proteins of raw fish meat is in the range 90–98%, and that of shellfish is about 85%. The enhanced digestibility is mainly due to the absence of strong collagenous fibers and tendons in fish muscle, which are common in land animals. PER of fish proteins is slightly above that of casein—the major milk protein. The PER values of most fish range from 3.1 to 3.7. The relative PER of raw flesh of fish and shellfish are as follows: mackerel, 149; cod, 113; rockfish, 103; surf clam, 102; and squid, 99. The NPU of fish flesh is 83, as compared to values of 80 and 100 for red meat and egg, respectively.12,13 Evidently, the protein quality of most fish may exceed that of meat or are equal to that of an ideal protein such as lactalbumin.5 Amino acid scores of several shellfish ranged from 68 for abalone to 95 for crab shell, with widely consumed scallop (above 71) and clams (up to 87) having intermediate values. In contrast to fish, cephalopods in general contain 20% more protein, 80% less ash, and 50–100% less lipid.14–16 Cephalopods have a high BV, Octopus vulgaris giving a value of 83.5 ± 1. Amino acid composition of krill protein is similar to the muscle proteins of other crustaceans. The essential amino acids of krill protein constitute about 45% of the total amino acids; lysine and tyrosine contents of krill proteins are higher than those of fish and shrimp. Relatively large amounts of alanine, glycine, proline, arginine, and lysine have also been reported. Lipid content ranges between 1 and 7% depending on the size of krill, generally containing 45% saturated, 33% monosaturated, and 22% polyunsaturated fatty acids. The nutritive value of krill protein in terms of PER, NPU, and BV is slightly lower than that of whole egg protein, but equal to that of casein. No difference was apparent between the digestibility of krill and that of whole egg. In adult men who were fed boiled krill for 21 days, the NBI was 0.55 and NPU was 55, and the corresponding values for whole egg were 0.61 and 61, respectively.17 The nutritional quality of raw, precooked, and canned tuna was assessed by determining the total amino acids, in vitro protein digestibility, computed protein efficiency

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ratio (C-PER), tetrahymena relative protein value (RPV), and fluorodinitrobenzene reactive lysine, together with selected vitamin and mineral assays. Amino acid composition, in vitro protein digestibility, C-PER, and tetrahymena RPV were the same for the raw, precooked, and canned tuna. The percent retention of FDNBreactive lysine was 91% for the precooked tuna and 80–85% for canned tuna. Significant amounts of thiamin were lost during canning of tuna, whereas niacin and riboflavin were retained up to 73% and 50%, respectively. The values for Cu, Fe, K, and Ca were significantly lower in canned tuna.18 The amino acid composition and nutritive quality of pearl oyster proteins have been reported. The growth rates of rats fed with these proteins were better than those fed with casein. Consumption of pearl oysters also significantly reduced plasma cholesterol from 82 mg/dL in the case of casein-fed rats to 57 mg/dL with oyster-fed rats. The shellfish provide a high quality of protein, with the first limiting amino acids being either leucine or valine.19 The nutritive value of diets high in protein from two species of sea urchin (Paracentrotus lividus and Echinus esculentus) has been studied as compared to casein. In addition, the effects of these diets on intestinal and hepatic leucine aminopeptidase and gamma-glutamyltranspeptidase were also examined. The test was carried out on three groups of male rats fed with these diets for 23 days. The result indicated that the nutritional value of sea urchin E. esculentus was significantly lower than that of casein. In contrast, the nutritive parameters of sea urchin P. lividus were similar to those determined for casein except for digestibility and NPU. The essential amino acids of Penaeus kerathurus are comparable with those of egg proteins.20 In general, the studies suggested that these marine species are a good source of nutritive protein comparable to casein.20,21

4.4.1 INFLUENCE OF PROCESSING ON NUTRITIVE VALUE Processing treatment can limit availability and digestibility (especially lysine, methionine, threonine, and tryptophan), the two important parameters in determining the nutritional value of proteins. Chilling of fish and shellfish in ice can lead to leaching of some nutrients, whereas prolonged frozen storage is associated with changes in their texture and flavor. Sun drying of fish results in only minor changes with respect to the contents of amino acids, sulfhydryl groups, and available lysine as well as digestibility. The hydroperoxides formed as a result of oxidation of lipids can interact with amino acids, such as available lysine, resulting in loss of nutritive value. Furthermore, increase in the intensity of brown color of dried products is due to Maillard reaction between carbonyl compounds generated during lipid oxidation with ε-amino groups of amino acids such as lysine present in the fish proteins. Drying at above 60°C causes appreciable damage to proteins. Drying of threadfin bream (Nemipterus japonicus) at 50, 60 and 70°C for 12 h caused a decrease in the sulfhydryl group contents from 27.3 to 20.3 for 11.0 and 5.2 mol/g dry matter, respectively. The digestibility of the fish proteins decreased from 77.8 to 71.1 after drying at 60°C for 12 h.22–24 Exposure of food proteins to certain processing conditions induces two major chemical changes, namely, racemization of l-amino acids to d-isomers and concurrent formation of cross-linked amino acids such as lysinoalanine. Racemization of

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l-amino acids residues to d-isomers varies depending on the amino acid, presence of other proteins, pH, processing conditions, and temperature. Therefore, the diet contains both processing-induced and naturally formed d-amino acids. d-amino acids are present in microorganisms, plants, and marine invertebrates. Racemization impairs digestibility and nutritional quality. The nutritional utilization of different d-amino acids varies widely in animals and humans since some d-amino acids may be both beneficial and harmful. The wide variation in the utilization of d-amino acids is illustrated by the fact that d-methionine is largely utilized as a nutritional source of the l-isomer, whereas d-lysine is totally devoid of any nutritional value. Understanding of the effects of d-amino acids against cancer, schizophrenia, and infection, and overlapping aspects of the formation, occurrence, and biological functions of d-amino acids should lead to better foods and improved human health.25 Cooking, in general, enhances the digestibility of fish proteins. Mild cooking causes little loss of protein with only a slight decrease in available lysine, whereas drastic heating can significantly reduce the protein quality. Boiling has little effect on the composition of shellfish. In a detailed study, the effects of different cooking methods on the protein quality of hagfish (Eptatretus burgeri) flesh were investigated. Fish were eviscerated, skinned, boned, and cut into 6 cm pieces before cooking by one of the following methods: boiling in tap water at 97°C (5, 10, or 15 min), steaming at 100°C (5, 10, or 15 min), microwave treatment at 2450 MHz (2, 3, or 4 min), and convection oven cooking at 220°C (5, 10, or 15 min). In vitro protein digestibility of flesh was not significantly affected by cooking. The cooked flesh had a digestibility of 81.3–83.5% as compared to a value of 82.9% for the raw flesh. However, steaming flesh at 110°C for 15 min gave a digestibility as high as 86.3%. Convection oven cooking (220°C, 15 min) resulted in a higher trypsin indigestible substrate with a value of 49.2 mg/g solid compared to a value of 38.9 mg/g solid for raw flesh. The free amino acids content of raw flesh was reduced after boiling for 10 min. Both convection oven cooking and microwave cooking for 3 min decreased the available lysine from 4.9 to 3.8 to 4.1 g/16 g N. In vivo apparent protein digestibility of hagfish flesh was similar for raw and cooked flesh, with a value of 92.4%, but was lower than casein, which gave a value of 94.3%. The PER (3.7–4.1) and NPR (3.7–4.9) of cooked flesh were significantly higher than those of raw flesh (3.3 and 3.6, respectively) and casein (2.5 and 2.6, respectively), despite their lower in vivo protein digestibility. The results, summarized in Table 4.2, suggested that cooking hagfish flesh at optimum conditions, particularly steaming, has a positive effect on in vitro and in vivo protein qualities.26 Cooking did not adversely affect protein digestibility of fish flesh, but could be beneficial if proper cooking such as steaming is employed.26 It has been concluded that some nutritional losses as a result of processing need not pose concerns and consumption of processed food including seafood need not necessarily amount to compromising with their nutritional value.27 Surimi is a washed concentrate of fish myofibrillar proteins, as discussed in Chapter 3. The processing conditions on the protein quality of surimi prepared from Alaska pollock was investigated in terms of protein digestibility, PER, trypsin inhibitor content, and protein solubility. Steamed kamaboko from Alaska pollock had good protein quality measured in terms of PER and protein digestibility. The protein

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TABLE 4.2 Effect of Cooking Methods on Protein Quality of Hagfish Quality Parameters Protein digestibility (%) Trypsin indigestible substrate (mg/g) Available lysine (g/16 g N) In vivo apparent protein digestibility (%) PER NPR

Hagfish Flesh (Raw)

Hagfish Flesh (Cooked)

ANRC Casein

82.9 86.3 (by steaming) 38.9 4.9 92.4 3.7–4.2 3.7–4.9

81.3–83.5

—

49.2 3.8–4.1 92.4 3.3 3.6

— — 94.3 2.5 2.6

Source: Adapted from Hwang, E.-Y. et al., Nutr. Food, 7, 287, 2002.

digestibility of all the products ranged from 86 to 89%. Of the different processing methods, steaming did not give any significant advantage with respect to digestibility of proteins, although a higher PER was observed. A two-stage steaming process, the first at 40°C for 20 min followed by a second steaming at 95°C for 10 min was found to be the most effective for the protein quality of kamaboko. PER values of marketed Korean surimi products ranged from 2.8 to 2.9, which were superior to that of casein, having a PER value of 2.5. The surimi prepared from Alaska pollock also showed a balance of essential amino acids, with an overall score of 101, as compared to beef, pork, chicken, and turkey, which had scores of 99, 104, 101, and 100, respectively.28 Leaching of soluble proteins is a major drawback of surimi preparation. A method to recover these proteins has been reported recently. Soluble proteins from surimi wash water were precipitated using a chitosan–alginate (chi–alg) complex and recovered by centrifugation followed by freeze-drying. Analysis showed that the preparation had a crude protein content of 73.1% and a high concentration of essential amino acids, including 3% histidine, 9.4% lysine, 3.7% methionine, and 5.1% phenylalanine. In a rat-feeding trial, the preparation showed higher modified PER and net protein ratio than the casein control. Blood chemistry analysis revealed no deleterious effect from the full protein substitution or the chitosan in the product. Therefore, this preliminary study revealed that proteins recovered from surimi wash water using the chi–alg complex could be used in feed formulations.29

4.5 NUTRITIVE VALUE OF MARINE PROTEIN SUPPLEMENTS Seafood proteins can have a range of dynamic properties making use of which protein supplements could be developed for use in foods, which can also find applications as binders, emulsifiers, and gelling agents, as discussed in Chapter 3. This section summarizes the nutritional quality of some major protein supplements from seafood. Early studies on nutritive value of fishery products started with fish protein concentrate (FPC), developed during the 1950s to solve the problem of malnutrition among poor sections of world population, as discussed in Chapter 3.

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FPC has been well examined as a protein supplement in a variety of foods. These data, in general, indicated that replacing one-third of daily protein intake by FPC resulted in efficient utilization of the protein, and also absorption of calcium and phosphorus. The data were also comparable to that of dietary protein intake given as meat. When 37% of conventional dietary protein was replaced with FPC, the metabolic balance of calcium, phosphorus, nitrogen, and fluoride was not affected, suggesting feasibility of replacing one-third of daily protein with FPC. However, the availability of fluoride from FPC was high.30 Because of commercial failure of FPC, newer products with improved functional and nutritive properties were developed (see Chapter 3).

4.5.1

SEAFOOD PROTEIN POWDERS

Soluble protein powders were also made from by-products of processing of Alaska pollock, namely, viscera, liver, heads, trimmings, and frame and were evaluated for their functional, nutritional, and rheological properties. The products had protein contents ranging from 65 to 79%. A comparison with Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) 1990 recommendations showed that these powders were good sources of all the essential amino acids.31–33 Table 4.3 indicates amino acid composition of soluble protein powders from head, frame, and trimmings of Alaska pollock.32 Protein powders from fish such as threadfin bream, lizardfish, and purple-spotted bigeye fish were prepared by drying surimi from these fish species. The resulting products contained 73% protein and 17% carbohydrate (from the cryoprotectant added during surimi preparation, see Chapter 3). The powder obtained by spray drying of the solution had 65% protein and as much as 24% carbohydrate.33 The biochemical composition and nutritional value of Antarctic krill has been examined with a view to use it as a protein supplement. The product has been found to possess comparable nutritive value as conventional FPC. However, krill should be processed soon after landing to avoid problems of high activities of muscle proteases, melanosis (blackening), and formation of unpleasant odor due to the formation of cis-4-heptenal, affecting the quality of the products.34,35 Table 4.4 shows the amino acid composition of hydrolyzates from Kuruma prawn and Antarctic krill.36 The spray-dried protein powder obtained from threadfin bream has a PER of 3.5, comparable to that of casein. The digestibilities of the powder using tryspin and pepsin were 54% and 48%, respectively, with a total digestibility of 82% with both the enzymes. The powder also contained all the essential amino acids.37 Marinebeef is a dehydrated low-fat product with good water-holding capacity. The threadfin bream marinebeef had all the essential amino acids in levels higher than those prescribed by the FAO/WHO. The PER of the product was 2.5–2.9%, with an NPU value of 83% at 10% protein level.17 A process has been described to prepare low-fiber, low-chitin protein from goodquality shrimp head. The pulp has been found to have 12.5% protein, and relatively low ash and 6.5% chitin. The product can find use in shrimp aquaculture since it is readily accepted by the shellfish brood stock and has an amino acid content similar to that of commercial brood stock diet.38

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TABLE 4.3 Amino Acid Composition of Soluble Protein Powder from Head, Frame, and Trimmings of Alaska Pollock Amino Acid Hydroxyproline Aspartic acid Threoninea Serine Glutamic acid Proline Glycine Alanine Valinea Methioninea Isoleucinea Leucinea Tyrosine Phenylalaninea Histidinea Lysinea Arginine Total essential amino acids (TEAA) TEAA, Percentage of total amino acids a b

Head

Frame

Trimmings

Essential Amino Acid Requirement as per FAO/WHO 1990

34.9 85.5 36.7 54.9 132.5 70.0 139.5 77.6 38.6 27.7 28.3 55.9 23.8 34.6 16.0 61.0 92.2 298.8

22.4 93.6 36.8 47.3 162.3 47.0 99.4 73.0 38.8 32.1 31.5 64.4 24.0 34.0 16.3 73.9 79.6 327.8

28.5 87.9 30.0 43.3 172.3 45.9 119.9 81.0 29.6 29.9 25.5 59.7 17.3 23.0 10.9 77.2 88.9 285.8

— — 9 — — — — — 13 17b 13 19 — — — 16 — —

30.0

33.1

29.0

—

Essential amino acid. Methionine + cysteine.

Note: Values are in milligram per gram of protein. Source: Adapted from Sathivel, S. and Bechtel, P. J., Int. J. Food Sci. Technol., 41, 520, 2006. With permission from Blackwell Publishing.

4.5.2

NUTRITIVE VALUE OF FISH PROTEIN HYDROLYZATES

The preparation of fish protein hydrolyzate (FPH) has been well dealt with in Chapter 3. It is known that hydrolysis of food proteins results in changes in their chemical, physical, biological, nutritive, and immunological properties. As a result of its high solubility and amino acid balance, FPH has obvious nutritional advantages over products such as FPC or even human-grade fish meal. Information on these aspects of FPH not only helps in the optimization of its production, but also in the isolation of therapeutically important biomolecules from the product.39,40 Proximate compositions of FPHs are in the range 1–8% moisture, 81–93% protein, 0–5% fat, and 3–8% ash. The amino acid composition of the product is comparable to that of the original unhydrolyzed fish muscle. Evaluation of hydrolyzates using nitrogen

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TABLE 4.4 Amino Acid Composition of Hydrolyzates from Kuruma Prawn and Antarctic Krill Amino Acid Aspartic acid Glutamic acid Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Cystine Tryptophan

Kuruma Prawn

Antarctic Krill

7.36 12.35 3.25 5.36 2.59 6.31 2.34 4.99 3.42 2.23 1.87 1.70 1.28 4.90 2.97 8.47 0.34 —

6.12 8.56 2.30 3.66 2.40 4.85 1.67 3.72 2.79 1.97 1.16 1.21 0.87 3.37 2.26 3.44 0.14 —

Note: Values in gram per 100 g of dried matter; —, not reported. Source: Adapted from Zhang, N., Yamashita, Y., and Nozaki, Y., Fish. Sci. 68, 672, 2002. With permission from Blackwell Publishing.

balance studies and growth experiments on rats established high nutritional values for the product. The PER of FPH depended on the extent of enzymatic digestion and its amino acid profile. The PER values are usually in the range 2–3.19 FPH prepared from deboned whole hake had a PER value of 3.44 as compared to a value of 3.0 obtained for casein, suggesting increased weight gain due to FPH. The digestibility of FPH from rainbow trout was tested in larval diet, utilizing the product both as a binding agent and a source of protein.41 Six protein hydrolyzates of minced fish, obtained by distinct enzymatic systems were characterized with respect to the extent of hydrolysis, peptide molecular size distribution, chemical composition, and amino acid profile. Hydrolysis resulted in solubilization of 63.4–94.2% of proteins according to the enzymes used and process variables. The hydrolyzates had amino acids as required by the dietary reference intakes for essential amino acids established for adults and infant nutrition. The ratio between the concentration of branched chain and aromatic amino acids (Fischer ratio) was above 3.5 indicating that the hydrolyzates obtained could be useful for dietetic management of patients with chronic liver diseases. The hydrolyzates were composed of peptides with fairly defined molecular weights, particularly the hydrolyzate obtained using pepsin and a protease from Streptomyces griseus, which

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showed a high percentage (57%) of peptides smaller than 3 kDa suitable for use in hypoallergenic formulae.42 Squid protein hydrolyzates (SPHs) prepared from four squid species by proteinase treatment are highly nutritive and functional proteins.43 Chemical analyses of the hydrolyzate from Chilean hake (Merluccius gayi) showed that the product contained about 63% protein. All the essential amino acids were present in the product in greater amounts than those established for man in the 1973 FAO/WHO amino acid scoring pattern, with the only exception of threonine, which is in slight deficit. Lysine amounted to 11.89 (g/16 g N) of which 75% was biologically available. The adjusted PER values (casein = 2.5) were consistently higher than or at least equal to the standard, suggesting excellent biological quality of the hydrolyzate.3 Composition, functional properties, and antioxidative activity of protein hydrolyzate prepared from defatted round scad (Decapterus maruadsi) mince, using Flavourzyme, having a degree of hydrolysis (DH) of 60%, were determined. The brownish yellow protein hydrolyzate had 58% protein, 24.6% ash, and contained a high amount of essential amino acids (48%) with arginine and lysine as the dominant amino acids. The protein hydrolyzate had an excellent solubility (99%) and possessed functional properties that were governed by their concentrations. The protein hydrolyzate exhibited antioxidant activity (see Section 4.7.2.4). During storage at 25°C and 4°C for 6 weeks, the antioxidant activities and the solubility of round scad protein hydrolyzate slightly decreased.44 Antioxidant activities of protein hydrolyzates from yellow stripe trevally (Selaroides leptolepis) prepared using Alcalase 2.4 L (HA) and Flavourzyme 500 L (HF) with a DH of 15% by pH-stat method were determined. Both protein hydrolyzates exhibited the antioxidant activities in a concentration-dependent manner. The antioxidant activities of both hydrolyzates were stable when heated at 90°C for 10 and 30 min and subjected to a wide pH range (2–12). However, α-tocopherol at 200 ppm showed the higher antioxidant activity in the system.45 Hydrolysis of raw herring using a bacterial endopeptidase did not change the amino acid composition of the product as compared to untreated herring meat. The lysine contents of the powders were above 60 mg/g of protein, although the values were lower for protein powders from arrowroot by-products. The fish protein powder samples were also good sources of potassium, phosphorus, magnesium, and amino acids.31,32 Freeze-dried FPH from herring containing 77–87% proteins with a DH ranging from 10 to 18% was reported to have a significant antioxidant activity.44 The high dispersability of FPH makes it suitable as a replacement for milk proteins. It has been used as a milk replacer for calves and lambs in certain countries. Substitution of milk solids by FPH at 33% did not affect the growth of calves. For producing a milk substitute for animals, particularly for neonatal animals, FPH either as a liquid hydrolyzate or spray-dried powder was mixed with a fat homogenate, lactose, and other ingredients.46 Protein hydrolyzates can have better value as protein supplements to cereal foods, soups, and bread.3,47,48 Supplementation of cereals with the hydrolyzate at the levels of 2, 4, 6, 8, and 10% resulted in an increase in both quantity and quality of dietary protein, suggesting that the fish hydrolyzate can be used as a protein supplement for use of populations that ingest low-protein diets such as those based on wheat, rice, or corn. Others include its use as a microbiological growth media, as a fertilizer to increase crop growth and yield,

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and as a stimulator for expression of many commercially valuable phytochemicals. Hydrolyzed fish can be stabilized for long periods of time and utilized as a highly nutritional feed supplement for animals and also as a binder for fish diets. It can also be used in agriculture.41,48 Soluble protein concentrate from tuna liver could be used as a single substitute for peptone, beef extract, and yeast extract for the cultivation of sulfite-reducing Clostridia.48

4.5.3

FERMENTED FISHERY PRODUCTS

Fermented products are nutritionally important products. The main benefit of fermentation is enhancing the digestibility of proteins and making essential amino acids available, especially lysine in the products. Lactic acid bacteria (LAB), many of which are considered as probiotics, are often used in food fermentation processes. Probiotics may be defined as “live microbial feed supplement, which beneficially affects the

TABLE 4.5 Some Probiotic Bacteria and Their Reported Health Benefits Strain

Health Benefits

L. casei

Prevents intestinal disturbances, balances intestinal bacteria, lowers fecal enzyme activities Treats lactose intolerance, produces bacteriocins, lowers fecal enzyme activity Lowers fecal enzyme activity, decreases fecal mutagenicity, prevents radiotherapy-related diarrhea, reduces constipation Balances intestinal microflora, protects against traveler’s diarrhea, enhances immunity Controls rotavirus diarrhea, prevents antibioticassociated diarrhea, reduces cystic fibrosis symptoms, alleviates intestinal inflammation in children having atopic eczema/dermatitis syndrome (AEDS) and cow’s milk allergy (CMA) Adhere to human intestinal cells, balances intestinal microflora, enhances immunity, adjuvant in treatment of Helicobacter pylori–associated gastritis and peptic ulcers Various health benefits

L. acidophilus L. acidophilus NCFB 1748

L. acidophilus La-5 L. rhamnosus GG (ATCC 53013)

L. johnsonii La-1

Bifidobacterium spp. (B. bifidum, B. longum, B. infantis, B. breve, B. adolescentis) Leuconostoc spp. Ln. lactis Ln. mesenteroides subsp. cremoris Ln. mesenteroides subsp. dextranicum Streptococcus spp. S. salivarius subsp. thermophilus

Various health benefits

Various health benefits

Source: Reproduced from Shrivastava, S. and Goyal, G. K., Ind. Food Ind., 26, 41, 2007; Schmidt, R. H. and Turner, E., Food Safety Handbook, Wiley, New York, 2003.

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host animal by improving its intestinal microbial balance.” The characteristic features of probiotics are that they should be capable of being prepared in a viable manner, have the ability to maintain viability during storage and use of the product, and also have the ability to survive in the intestinal ecosystem to provide benefits to the host animal. The benefits include combating disorders such as diarrhea, lactose intolerance, prevention of pathogenic microorganisms, and enhanced cellular immunity, among others. Therefore, probiotics help maintain a healthy gut microflora. Probiotic cultures might also decrease the exposure to chemical carcinogens by detoxifying ingested carcinogens and also control blood pressure. Studies indicate that specific Lactobacillus spp. reduce the growth of cancer cells and the activity of fecal carcinogenic enzymes implicated in the development of colon cancer.49 Table 4.5 shows some probiotic bacteria and their reported health benefits.49,50 Many traditional fermented foods including fermented fishery products may be classified as probiotic foods. The nutritional benefits of these products, their availability in different countries, and processes of their preparation with particular references to microorganisms have been discussed.51 Fish silage is usually added to pig, cattle, poultry, and aquafeed to substitute fish meal. The value of the feed is determined not only by its protein, but also by the contents of vitamins A, D, B1, and B2, which remain grossly unchanged. However, loss of vitamin B12 during silage

TABLE 4.6 Amino Acid Composition of Tuna Silage Amino Acid

Content (g/100 g Protein)

Aspartic acid Glutamic acid Hydroxyproline Serine Glycine Histidine Arginine Threonine Alanine Proline Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Lysine Cysteine/cystine Tryptophan

8.3 11.4 3.5 3.9 10.9 3.2 7.8 4.0 7.2 6.6 2.9 4.4 2.6 3.5 6.1 3.5 6.3 1.4 0.7

Total

98.2

Source: Adapted from Sultanbawa, Y. and Aksnes, A., Infofish Int., 3, 37–40, 2006. With permission from Infofish.

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preparation has been reported.19,41 Table 4.6 gives the amino acid composition of tuna silage.52 The results presented summarize the significant nutritional value of seafood protein products. Because of their superior nutritional quality, these protein products from marine sources, such as powders offer scope for applications as protein supplements in cereal-based products to enhance their nutritional value.53 In addition, there are various physiological advantages of peptides generated during digestion of proteins, as discussed in the following section.

4.6

BIOACTIVE PEPTIDES

During digestion of food proteins, a wide range of bioactive peptides is produced. These peptides can serve as biological messengers, stimulating or suppressing a wide array of physiological responses. Specific peptides derived from food proteins play a significant role in maintaining health and prevention of diseases of the cardiovascular, nervous, immune, or nutritional system, besides being sources of metabolic energy and essential amino acids. Such proteins or their precursors may occur naturally in raw food materials, exerting their physiological action directly or on enzymatic hydrolysis in the digestion system of the body or during food-processing operations. Early work on bioactive peptides was initiated with milk, which was fractionated into tryptophan-rich α-lactalbumin that was shown to ameliorate sleep disorders. Dairy whey protein fractions (sold as whey powder) contains compounds that can boost natural immunity, decrease the risk of cancer, reduce the severity of muscle tissue degeneration associated with liver diseases, and lower the susceptibility to diarrhea. Other milk peptides isolated were antimicrobial peptides such as lactoferricines formed by enzymatic degradation of the milk protein lactoferrin by pepsin and bacteriocins produced during milk fermentation by LAB.54 Another important source of bioactive peptides is soy proteins. Because of its rich protein content (about 50% on a dry weight basis), soybean is a good source of bioactive peptides. These peptides have been shown to be capable of preventing the production of cholesterol by liver cells, leading to lower cholesterol levels in the blood. Consumption of 25 g or more soy proteins a day can have beneficial effects in this respect.55 The large volume of information on bioactive peptides has encouraged compilation of data sets on their structures and properties and activities. The information has been especially useful for antimicrobial, angiotensin I-converting enzyme (ACE) inhibitory, and bitter-tasting peptides, but could easily be expanded to other areas within food research.56 Table 4.7 summarizes major bioactivities of peptides.

TABLE 4.7 Major Bioactivities of Peptides Antihypertensive (ACE inhibitory) activity Antioxidant activity Antimicrobial activity Antihypoallergenic activity Obesity control Cell immunity

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Protein synthesis Cellular growth Secretion of digestive enzymes Ripening of fermented foods Influences food taste Antifreeze proteins

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4.7 BIOACTIVE PEPTIDES FROM SEAFOOD Recent research has shown that marine organisms including fishes, sponges, ascidians, mollusks, sea anemones, and seaweeds are rich sources of bioactive peptides. These are formed either by the action of endogenous proteases or during the action of exogenous proteolytic enzymes. In fresh seafood, the concentration of peptides may be low, whereas their contents may increase during storage due to proteolytic degradation of muscle. In a study on the formation of peptides, acid-soluble fractions in fresh fish stored up to 2 weeks in ice were separated by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by electrospray ionization-mass spectrometry (MS). At least 25 polypeptides with molecular weights of 2–32 kDa were identified; those with molecular weights of 3.9, 11.4, and 32.8 kDa decreased during chilled storage, whereas those with molecular weights of 12.5 and 16.5 kDa increased. The increase in peptide profiles during storage correlated with changes in total aerobic and anaerobic counts in the fish, suggesting that the peptide analysis could offer an index of spoilage.57 Peptides are also formed from fish muscle structural proteins during ripening of salted anchovies. Using SDS-PAGE, chemical changes in the muscle proteins during the ripening were found to relate to the degradation of the myofibrillar structure of the muscle. Hydrolysis of muscle proteins was significant during the first 6 weeks, with proteins of molecular weight >35 kDa being more likely to be hydrolyzed. Myosin heavy chains were the most sensitive myofibrillar protein, whereas actin, α-actinin, and tropomyosin were more resistant to enzymatic degradation.58

4.7.1

ISOLATION OF SEAFOOD PEPTIDES

Bioactive peptides can be produced in vitro by enzymatic hydrolysis of food proteins. These peptides are inactive within the sequence of the parent protein, whereas they become active on hydrolysis. Usually, pancreatic enzymes, generally trypsin, and other enzymes or a combination of enzymes including proteinases, alcalase, chymotrypsin, and pepsin, as well as enzymes from bacterial and fungal sources have been utilized to generate bioactive peptides. After hydrolysis, the peptides in the hydrolyzate are fractionated and enriched using chromatographic, isoelectric focusing, and other suitable methods. Ultrafiltration membranes have been successfully used to enrich specific peptides fractions. Apart from hydrolysis with proteolytic enzymes, physical removal, chemical modification, and heat denaturation are some of the other strategies to remove epitopes.59,60 Label-free liquid chromatographymass spectrometry (LC-MS) profiling is a powerful quantitative proteomic method to identify relative peptide abundances between two or more biological samples.61 Figure 4.1 shows a flow diagram for the production and separation of bioactive peptides from food proteins.55 Seafood processing wastes and low-cost fish can be good sources of bioactive peptides.59,60,62 A mild procedure for obtaining lipipeptic and peptidic fractions from abundantly available sardine (Sardina pilchardus) has been reported recently.63,64 Partially digested protein hydrolyzate produced by the action of commercial proteinase, alcalase, on cooked sardine (S. pilchardus) meat could be a source of peptides. The peptides in the hydrolyzate can be analyzed by techniques such as

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Precursor protein⫹enzyme

Hydrolysis

Ultrafiltration (30 – 3 kDa)

Permeate 1

Retentate 1

Concentration • Nanofiltration (500 Da) • Ion exchange membrane

Permeate 2

Retentate 2

Freezedrying

Enriched peptide fraction

FIGURE 4.1 Flow diagram for the production and separation of bioactive peptides from food proteins. (Reprinted from Philanto, A. and Korhonen, H., Adv. Food Nutr. Res., 47, 175, 2003. With permission from Elsevier.)

gastrin radioimmunoassays, calcitonin gene-related peptide (CGRP) radioreceptor assays, and analysis of mitogenic activity. Gastrin and CGRP were detected in all hydrolyzates produced, but only some fractions exhibited a growth factor–like activity. Exclusion chromatography results indicated that degree of biological activity in hydrolyzate fractions was associated with the size of molecules; bioactivity appeared in molecules of about 650 Da in size.65 Limited hydrolysis of by-products of cod generates peptides for potential use in the development of food and feed supplements. Cod muscle was subjected to hydrolysis using Alcalase 2.4 L under different conditions (temperature, pH, time, and enzyme concentration). Depending on the conditions, the hydrolyzates yielded biologically active peptides such as growth factors; gastrin and cholecystokinin (molecules exhibiting a large spectrum of activities ranging from the stimulation of protein synthesis to the secretion of digestive

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enzymes) occurred in several fractions under optimum conditions.66 World tuna catch is approximately 4 mt of which 40–60% results in process discards. Processing discards from the tuna canning industry are estimated at 450,000 t annually. The tuna industry, therefore, can provide a large volume of by-products for the recovery of important nutraceuticals for the food, feed, and pharmaceutical industries. A number of peptides possessing structural and functional characteristics of gastrin and cholecystokinin or cellular growth factors have been detected in tuna stomach hydrolyzates prepared using Alcalase.52 Apart from myofibrillar proteins, stroma proteins can also be sources of biological active peptides. A method has been described for producing a peptide from fish skin gelatin. The method comprises the following steps: washing fish skin with 0.1–5% (preferably 0.5–2%) saltwater and freshwater, extraction of gelatin from the skin using fresh hot (50–100°C) water, and subjecting the water extract to enzymatic digestion. The enzyme digest is concentrated, passed through activated carbon, and drying the resultant product. The fish skin is preferably from white flesh fish, particularly pollock, such as Alaskan pollock, or Pacific cod.52 Table 4.8 presents peptide composition of gelatin isolated from tuna waste.52

4.7.2

FUNCTIONAL ROLES OF MARINE PEPTIDES IN FOODS

Recent advances in research on bioactive peptides show much promise in deriving multifarious health benefits from these biomolecules. The biological activities are discussed in this section with particular reference to marine peptides. Bioactive peptides play an important role in flavor development in protein-rich foods. Wellknown examples are FPHs, and other products such as soy sauce, ripened cheeses, and fermented or cured meat products, from which peptide fractions with different tastes have been isolated. The peptides, Glu-Asp-Glu, Asp-Glu-Ser, and Ser-Glu-Glu found in FPH have savory properties similar to sodium glutamate. The bitter taste TABLE 4.8 Tuna Gelatine Peptide Composition Peptides (Molecular Weight) (Da) Range >70,000 70,000–60,000 60,000–50,000 50,000–40,000 40,000–30,000 30,000–20,000 20,000–10,000 10,000–5,000 5,000–1,000 1,000–100

Percentage 20.59 61.25 Nil Nil 4.78 4.20 5.02 1.81 0.43 1.74

Source: Adapted from Sultanbawa, Y. and Aksnes, A., Infofish Int., 3, 7, 2006. With permission from Infofish.

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is due to the formation of low-molecular-weight peptides consisting of 2–23 amino acids in the molecular range 500–3000 Da, composed of mostly hydrophobic amino acids.67 The bioactive peptides can also work indices of fish quality.57 Fish collagen peptide, when orally administrated, can repair damaged cartilage and can have synergistic effects with N-acetyl glucosamine.68 4.7.2.1

Calcium-Binding Activity

Binding of minerals such as calcium and iron can help in their absorption in the gut. Skeleton from industrial processing of hoki was digested by heterogeneous enzyme extracted from intestine of bluefin tuna. The tissue enzyme could biodegrade the hoki bone matrices composed of collagen, noncollagen proteins, carbohydrate, and minerals. A fish bone phosphopeptide (FBP) containing 23.6% of phosphorus was isolated. The FBP had a molecular weight of 3.5 kDa and could bind calcium without the formation of insoluble calcium phosphate. It was suggested that the product could be used as a nutraceutical with a potential calcium-binding ability.69 Peptides from collagen and gelatin hydrolyzates can also have functional properties. A fish commercial collagen hydrolyzate, Peptan F, has been found to enhance protein quality and flavor characteristics of beverages.11 Gelatin peptide from fish waste can have antioxidant activity.70 4.7.2.2 Obesity Control Certain bioactive peptides have been shown to contribute to weight management. A specific casein peptide, glycomacropeptide (GMP) plays a significant role in appetite suppression. It stimulates the production of cholecystokinin, an intestinal hormone, which induces the sensation of satiety.71 4.7.2.3

Antibacterial Activity

Antimicrobial peptides that are widely distributed in nature are involved in host defense. Various antimicrobial peptides have been used to reduce pathogens in foods and to extend the shelf life of many perishable foods. The antimicrobial peptides have been used to prevent growth of Clostridium botulinum spores in foods such as cheese. Recently, biopeptide has been shown to inhibit the growth of Listeria monocytogenes on the surface of cooked meat products.72 Bacteriocins are common antimicrobial peptides formed during the fermentation of foods by LAB, such as Lactococcus lactis, L. brevis, L. plantarum, L. acidophilus, and Pediococcus acidolactis. Lactoferricine formed through the degradation of the milk protein lactoferrin by pepsin is another antimicrobial peptide.73 These antimicrobial peptides have potential to function as food preservative agents because of their simplicity in use, broad activity spectra, and bacterial resistance over known preservative agents. Protamine, extracted from fish milt, demonstrated antimicrobial activity against a range of gram-negative and gram-positive bacteria, yeasts, and molds. This 30 amino acid peptide is believed to disrupt the cytoplasmic membrane. In recent studies, this peptide was evaluated for its efficacy against L. monocytogenes and Escherichia coli. The peptide showed better activity against gram-negative bacteria, particularly at alkaline pH values in a medium containing gelatin.56 Antimicrobial activity of

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histones from hemocytes of the Pacific white shrimp has been reported.74 Antibacterial proteins have also been reported to be present in rainbow trout and rockfish.75,76 4.7.2.4

Antioxidant Activity

In the 1950s, scientists discovered that many diseases—including heart disease, strokes, cancer, diabetes, cataracts, arthritis, and neurodegenerative disorders such as Parkinson and Alzheimer were linked to damage caused by highly reactive free radicals. Free radicals are compounds with unpaired electrons that stabilize themselves by oxidizing other molecules—including proteins, carbohydrates, lipids, and deoxyribonucleic acid (DNA). In the process they often create more free radicals, sparking off a chain of destruction, leading to oxidative damages responsible for most, if not all, diseases. Antioxidant hypothesis suggests that these compounds (reducing agents) have the capacity to prevent oxidation damage and to reduce the risks of diseases and also ageing. The failure of antioxidant defense mechanisms is implicated in damage of DNA, lipid, and proteins. This, in turn, poses increased risk of chronic diseases including cancer and cardiovascular disease.77 Much physiological damage may be directly imputable to the hydroxyl radical because of its high reactivity. Many hydroxyl radicals produced in vivo react at or close to their sites of formation. Presence of antioxidative compounds in biological systems is important to combat the influences of prooxidants (see also Chapters 5 and 8). Many peptides including those from marine fishes have been reported to have antioxidant activities against peroxidation of lipids or fatty acids.78,79 Meat from Alaska pollock frame, a by-product, was hydrolyzed with mackerel intestine crude enzyme. A fraction having a molecular weight below 1 kDa exhibited the highest antioxidative activity. This fraction was further purified using Sephadex and HPLC. The purified peptide fraction had an amino acid sequence, Leu-Pro-His-Ser-Gly-Tyr, with a molecular weight of 672 Da. The purified peptide scavenged 35% hydroxyl radical at 53.6 μM concentration.79 A heptapeptide, mussel-derived radical scavenging peptide (MRSP) having a molecular weight of 962 kDa was purified from fermented marine blue mussel (Mytilus edulis). The peptide was found to be highly effective for radical scavenging. The MRSP could scavenge superoxide, hydroxyl, carbon-centered, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals up to 98% at 200 μg/mL concentration. The concentrations for 50% scavenging activities of these radicals were found to be 21, 34, 52, and 96 μM, respectively. In addition, MRSP exhibited a strong lipid peroxidation inhibition at 54 μM, which was higher than α-tocopherol. Furthermore, it also exhibited high Fe2+ chelating activity. These results identified MRSP as a potent natural antioxidant, which performs its activity via different mechanism of actions.80 An antioxidant peptide has been recently isolated from sea cucumber and prawn.80–82 Antioxidant active of peptides from fish gelatins has been reported. Peptides derived from tryptic hydrolyzate of hoki (Johnius belengerii) skin gelatin exhibited significant scavenging activities on superoxide, carbon-centered DPPH radicals as assessed by electron spin resonance spectroscopy. Following consecutive chromatographic separations of tryptic hydrolyzate, the peptide sequence His-Gly-Pro-LeuGly-Pro-Leu (797 Da) was found to act as a strong radical scavenger under the studied conditions. Furthermore, this peptide could also function as an antioxidant

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against linoleic acid peroxidation, and the activity was closer to the highly active synthetic antioxidant, butylated hydroxytoluene (BHT). In addition, antioxidative enzyme levels in cultured human hepatoma cells increased in the presence of this peptide and it was presumed to be the peptide involved in maintaining the redox balance in the cell environment. The data indicated that free-radical-scavenging activities of hoki skin gelatin peptides substantially contribute to their antioxidant properties measured in different oxidative systems. In addition, these peptides have also shown to accelerate absorption of dietary calcium in animal models increasing calcium bioavailability.70 In another study, Alaska pollock gelatin was serially hydrolyzed with alcalase, pronase, and collagenase using a three-step recyclic membrane reactor. Pronase-E hydrolyzate was composed of peptides from 1.5 to 4.5 kDa and showed high antioxidative activity. The different peptides having high antioxidative activities were isolated by chromatographic techniques. The isolated peptides were composed of 13 and 14 amino acid residues, both containing a glycine residue at the C-terminal and repeating motif Gly-Pro-Hyp. P2 peptide had potent antioxidative activity on peroxidation of linoleic acid. Cell viability of cultured liver cells was significantly enhanced by this peptide, suggesting that the purified P2 peptide is a natural antioxidant.83 Elastin is a major protein component of elastic tissues such as arterial wall, ligament, and skin, as discussed in Chapter 3. The insoluble elastin was rendered soluble by pepsin digestion or treatment with hydrochloric acid. The pepsin-solubilized elastin (PSE) and acid-solubilized elastin (ASE) were effective inhibitors of oxidation of oleic acid. The antioxidative activity of PSE and ASE were enhanced in the presence of citric acid as synergist, suggesting the potential of hydrolytic products of elastin as effective antioxidants.84 4.7.2.5 Angiotensin I-Converting Enzyme Inhibitory (Antihypertensive) Activity ACE is an exopeptidase (a dipeptidyl carboxypeptidase, EC 3.4.15.1) that plays a fundamental role in blood pressure homeostasis as well as fluid and salt balance in mammals. ACE, which is a zinc metallopeptidase, cleaves dipeptides from the C-terminal of various peptide substrates. It cleaves angiotensin-I into a potent vasoconstrictor octapeptide angiotensin II and changing depressor (bradykinin) to inert peptide, which results in the increase in blood pressure. ACE inhibitors are therefore antihypersensitive compounds, which are widely used for the treatment of high blood pressure. Currently, specific inhibitors of ACE are used as pharmaceuticals to treat hypertension, congestive heart failure, and myocardial infarction. Although synthetic inhibitors have been designed such as Captopril, they are known to have side effects such as cough and skin rashes. However, ACE inhibitors derived from food proteins have not shown these side effects. Peptides inhibiting the ACE have been intensively studied and are recognized as blood pressure–lowering nutraceuticals. ACE inhibitory peptides are rarely present as such in foods. They must be released from the parent protein preferably by controlled enzymatic hydrolysis. Various ACE inhibitors have been found in the enzymatic hydrolyzates of food proteins. At least 40 different food-derived peptides have been shown to produce antihypertensive effect.

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Peptides, ranging from 2 to 30 amino acids, which inhibit ACE have been produced by the enzymatic hydrolysis of casein and whey proteins. Other proteins include zein, gelatin, yeast, corn, and fish. Associated advantages of these peptides include treatment of cardiovascular diseases, cancer, diabetes, osteoporosis, hypertension, gastrointestinal disorders, and renal function. These peptides have a potential as antihypertensive components in functional foods or nutraceuticals. Information on ACE inhibitory peptides obtained by enzymatic hydrolysis of protein from meat, fish, and invertebrates (including oysters) has been summarized.85,86 The first marine ACE inhibitory peptide was isolated from sardine, which was hydrolyzed by Denazyme AP, a protease from Aspergillus oryzae. The IC50 (concentration of sample that inhibits 50% of the ACE activity) value of the sardine peptide was 3.79 mg/L.87 Later, ACE inhibitory peptides have been isolated from hydrolyzates of various other fishery sources. These include salmon, sardine, oyster, wakame, yellowfin sole, and dried bonito.88–94 Salmon muscle hydrolyzate has been observed to be a potent inhibitor of ACE and exhibited an antihypertensive effect when administered orally to spontaneously hypertensive rats.88 Sequence of amino acids in the peptides has influence on the ACE inhibitory activity. For instance, of the six ACE peptides that were identified in salmon hydrolyzate, one peptide, Phe-Leu, noncompetitively inhibited the enzyme, showing 50% inhibition at a concentration of 13.6 μM. The reverse sequence dipeptide, namely, Leu-Phe, exhibited competitive inhibition and was less effective than Phe-Leu. Of the other peptides examined from salmon muscle hydrolyzate, those with Trp as the C-terminal residue, Ala-Trp, Val-Trp, Met-Trp, Ile-Trp, and Leu-Trp showed noncompetitive inhibition. However, reversed sequence peptides with Trp at the N-terminal were competitive inhibitors, except Trp-Leu.88 Treatment of salmon processing waste by heating under pressure resulted in appreciable extraction of collagen and other proteins. The extract also significantly inhibited ACE activity, the degree of inhibition being about seven times higher than that of cartilage and skin extract. Besides, the cartilage extract also inhibited oxidation of linoleic acid, and was active against all reactive oxygen species, such as superoxide anion, hydroxyl, and DPPH radicals. The data indicated its potential to suppress hypertension and inhibit oxidative processes, which are important in controlling lifestyle-related diseases such as cancer, cardiovascular diseases, and diabetes.89 An alkaline protease-derived ACE-inhibitory peptide from sardine showed an IC50 (concentration for 50% inhibition) value of 1.63 μM for inhibition of ACE. Comparable values for peptides from bonito intestine and Indonesian dry salted fish were 320 μM and 31.97 μM, respectively. The former was generated during autolysis of bonito intestine, whereas the latter was obtained by pepsin treatment of the dry fish.55 In another study, the enzymatic hydrolyzate of fish waste showed strong angiotensinconverting enzyme inhibiting activity. Concentrations required for 50% inhibition (IC50) were 0.6–2.8 mg/mL.95 In some cases, enzymatically hydrolyzed fish skin gelatin have shown better biological activities compared to the peptides derived from fish muscle proteins to act as antioxidants and antihypertensive agents.96 Adequate dietary intake of fish, which leads to hydrolytic release of these peptides, could therefore be therapeutically useful. Animal feeding studies conducted at Norway suggested the effect of FPH as a cardioprotective nutrient. The fish protein treatment

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reduced the plasma cholesterol level; gave higher content of high-density lipoprotein (HDL); and altered the fatty acid composition in liver, plasma, and triglycerol-rich lipoproteins in obese zucker rats.97 The activity of peptide from pearl oyster was indicated by the fact that consumption of the oyster significantly reduced plasma cholesterol. The plasma cholesterol was 82 mg/dL in the case of casein-fed rats, which reduced to 57 mg/dL in rats fed with oyster.19 As a result of ACE inhibitory activity of peptides generated during digestion, dietary protein intake has been recognized as inversely related to blood pressure. A dietary protein intake of 39 g results in 3.55 mm Hg lower systolic blood pressure. Vegetarians having high fiber intake has a lower average blood pressure. Higher intake of fiber is also associated with lower diastolic and systolic blood pressure in whites.98 The relationship between vegetable and animal protein in blood pressure, however, is unclear. C-reactive protein (CRP) is a blood plasma protein produced by the liver. It has a role in improving immunity and levels go up when there is an acute inflammation in the body. Normally, CRP levels remain below 1 mg/L. It has been shown that every for 1% increase in energy intake from saturated fat, CRP level changes by 0.14 mg/L. Values above 3 indicate atherosclerosis. Presence of CRP-related molecules in protein hydrolyzates from industrial fish wastes have been identified suggesting antihypoallergenic activity of these peptides.99 A hydrolyzed offal was produced by enzyme treatment. Chemical analysis showed that the hydrolyzate contained 16.0% protein, 4.21% fat, 76.28% moisture, and 3.39% ash. The amino acid profiles of the hydrolyzate was comparable to those recommended by the FAO/WHO. Animal experiments revealed that the hydrolyzed offal protein inhibited mice anemia caused by an injection of cyclophosphamide. The hydrolyzed offal protein could significantly inhibit the decrease in red blood cells, hemoglobin, and platelets. (The antianemia action of the hydrolyzed offal protein might also be due to nutritional functions of minerals, calcium, phosphorus, and iron.) The results indicated potential for effective utilization of fish offal as a health supplement.100 Besides ACE inhibitory activity, peptides from cod, salmon, and trout proteins also inhibited prolyl endopeptidase (EC 3.4.21.26) isolated from pork muscle. Peptide fractions from both fish hydrolyzates and autolyzates were effective in inhibiting hydrolysis of Z-Gly-Pro-amido-methylcoumarin by prolyl endopeptidase, suggesting that the hydrolyzates and autolyzates from the three fish species contained inhibitory peptides for the enzyme. The peptides were of different molecular mass and apparent hydrophobicity.101 4.7.2.6

Immunostimulant Activity

In vitro and in vivo studies have shown that certain peptide fractions in FPHs may stimulate the nonspecific immune defense system. Both fish sauce and fish silage are protein hydrolyzates with immune stimulating properties. Generally, fish sauce is regarded as a typical Asian product made from tropical fish species, but ancient literature reveals that fish sauce was a common food product in southern Europe more than 2000 years ago. Recent studies have shown that it can also be made from cold water species.59

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Human Immunodeficiency Virus-I Protease Inhibiting Activity

The human immunodeficiency virus (HIV) is the retrovirus that causes acquired immune deficiency syndrome (AIDS). Thermolysin hydrolyzate from oyster has been reported to have HIV-protease activity.102 4.7.2.8 Antithrombin Antithrombin inactivates thrombin almost instantaneously in the presence of heparin, but only slowly when heparin is absent. Antithrombins from Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss Walbaum) have been isolated, which functions in vitro as does its human counterpart. The inactivation of thrombin by salmonid antithrombin, which was dependent on heparin concentration was maximal at pH 7.8–8.4 and maximal at concentrations between 1.5 and 6 units/mL. Unlike the human system, the salmonid thrombin–antithrombin interaction functions over a wide range of temperatures and was present at temperatures as low as 3°C.103 4.7.2.9

Calcitonin

Osteoporosis, a crippling disease marked by a wasting away of bone mass, is affecting significant number of elderly population. Osteoporosis is responsible for 1.5 million fractures of the hip, wrist, and spine in people above 50 years, and about 50,000 deaths, causing an economic loss of about $10 billion a year, according to the U.S. National Osteoporosis Foundation. The disease could be treated using a hormone called calcitonin that is obtained from salmon. The hormone helps regulate calcium and decreases bone loss. For osteoporosis patients, taking salmon calcitonin, which is 30 times more potent than that secreted by the human thyroid gland, inhibits the activity of specialized bone cells called osteoclasts that absorb bone tissue. This enables bones to retain more bone mass. Calcitonin inhibits osteoclast-mediated bone resorption. The parathyroid hormone stimulates calcitonin synthesis and also bone-calcium absorption and renal-calcium conservation. Nevertheless, other proteins such as albumin (which carries calcium in blood) and casein α-lactalbumin are involved in calcium metabolism, influencing bone health. Nowadays, salmon calcitonin is also being made synthetically. It is similar to salmon calcitonin and offers an economical way to create large quantities of the product.104 The U.S. Food and Drug Administration (FDA) has approved the first drug based on salmon calcitonin in 1975. An oral version of salmon calcitonin is in clinical trials currently. Salmon calcitonin is approved only for postmenopausal women who cannot tolerate estrogen, or for whom estrogen is not an option.105 4.7.2.10 Miscellaneous Physiological Functions of Marine Proteins Peptides might help diabetes by improving absorption of glucose into the cells of the body.1 Peptides in fish hydrolyzates could be beneficial when used as feed components in aquaculture. Small peptides from cod or shrimp hydrolyzates can enhance disease resistance of aquacultured salmon. Medium-size (3–10 kDa) peptides found in hydrolyzates of cod stomach and head could stimulate the growth of mouse fibroblasts. Scientists from The University of Auckland, New Zealand reported that

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a protein from mussel could heal human wounds. The protein, in the natural environments, forms strong glue that sticks the shellfish to rocks and functional biomolecules from seafoods. However, some protein-rich foods such as shellfish (and milk proteins) can trigger allergic reactions in susceptible individuals. Specific amino acid sequences (epitopes) present in the food proteins interact with cells of the immune system to trigger allergic response (see Chapter 15). Some peptides can function as antifreeze compounds for cryostabilization of myofibrils in meat products. Effect of SPH (which contained peptides as high as 84–88%) on stabilization and dehydration-induced denaturation of frozen lizard fish (Saurida wanieso) myofibrillar protein was investigated. Stabilization effects of 5% SPH on the fish myofibrils were evaluated in terms of Ca2+-ATPase inactivation and the presence of unfrozen water. Myofibrillar proteins with added SPH were found to contain higher levels of monolayer and multilayer sorption water, resulting

TABLE 4.9 Bioactivity of Peptides from Marine Sources Bioactivity Antihypersensitive action through inhibition of ACE activity, results in increase in HDL

Antioxidant activity

Calcium-binding oligophosphopeptide Antifreeze proteins (AFPs) (cryostabilization) Gastrin and CGRPs

HIV-I protease inhibiting activity Prolyl endopeptidase inhibition

Stimulation of nonspecific immune defense system

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Source

Reference

Pacific hake Sardine Salmon Oyster Wakame Fish bone collagen Dried bonito digest FPH Alaska pollock, sea cucumber Saithe Round scad Mussel Tuna gelatin Hoki gelatin Fish bone from hoki Protein hydrolyzate from Antarctic krill, salmon Atlantic/Greenland cod Winter flounder Cold-tolerant microbes Sardine Industrial seafood waste Oyster Cod Salmon Tout Hydrolyzate of chub mackerel and other fish

42, 63, 88, 90, 92, 93, 94, 95, 97

44, 69, 79, 80, 81, 96

69 43, 124 64, 65

102 101

59, 94, 96

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TABLE 4.10 Further Research Needs for Peptides from Seafood Sources Screening for potential bioactivity among different seafood proteins Development of novel fractionation and purification methods Study of interaction of bioactive proteins/peptides with other food components and effects on bioavailability Basic research on the transgenic products of bioactive proteins and the potential side effects such as allergenicity and toxicity Evaluation of the efficacy of the peptides in animal model and human clinical trials per se and in food system Source: Adapted from Philanto, A. and Korhonen, H., Adv. Food Nutr. Res., 47, 175, 2003. With permission from Elsevier.

in decreased water activity and rate of Ca2+-ATPase inactivation. The amount of unfrozen water in myofibrillar proteins with SPH increased significantly, suggesting that the peptides of SPH stabilized water molecules on the hydration sphere of myofibrillar proteins and therefore suppressed dehydration-induced denaturation. The stabilization effect of SPH was less than that of sodium glutamate.43 Table 4.9 summarizes bioactive functions of peptides from marine sources. Further research needs in the area of marine peptides include screening for detection of potential bioactivities among diverse marine sources, development of novel techniques for their isolation and purification, studies on their interaction with other food components and evaluation of their safety, and identification of transgenic products for bioactive proteins.55 Table 4.10 indicates further research needs for peptides from seafood sources.55

4.8 MARINE ENZYMES An important characteristic feature of marine organisms is their adaptation to diverse extreme environmental conditions, such as high salt concentration, low or high temperature, high pressure, and low nutrient availability that may be present in habitats such as hydrothermal vents and oceanic caves. Such an adaptation would not have been possible without the help of enzymes present in these organisms. These enzymes may differ from those of terrestrial organisms in their properties such as salt tolerance, stability to high temperatures and pressure, and ability to adapt to extreme cold temperatures. Therefore, a marine enzyme may be a unique protein molecule not found in any terrestrial organism or it may be a known enzyme from a terrestrial source with novel properties.106

4.8.1

ISOLATION

Marine enzymes can be extracted from marine fish species, prawns, crabs, algae, and also from plants, fungi, bacteria and actinomycetes, and other organisms. Seafood and seafood processing wastes can be one of the economically viable options for marine enzymes. Fish muscle contains a variety of proteases, which in the live tissue are involved in protein degradation and synthesis depending on nutritional status, diet, biological

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age, spawning, exercise, water salinity, and hydrostatic pressure. Inhibitors of serine and cysteine have also been identified in fish muscle. Apart from the muscle cells, the endogenous proteases are also present in extracellular matrix and connective tissue surrounding muscle cells and digestive and other organs. Proteases in the intestine of fish are normally secreted from the pyloric ceca or pancreas depending on the species. These include trypsin, trypsinlike enzymes, chymotrypsin, collagenase, elastase, carboxylpeptidase, and carboxylesterase. In the muscle tissue, catheptic and other hydrolytic enzymes are housed in two types of lysosome—a subcellular organelle. One species is within the muscle fiber and the other is in the extracellular matrix, originating from macrophages and connective tissue cells. Lysosomes contain cathepsins, a family of endopeptidases and exopeptidases. Cathepsins (classified as A, B, C, D, etc.) generally possess acidic pH optima, although some are most active at neutral pH. Cathepsin D is an aspartic protease, whereas all the others found in muscle thus far are serine or cysteine proteases.106–108 During commercial processing of finfish species, a large amount of offal wastes are accumulated. Of these, fish viscera are a rich source of digestive enzymes, which include proteases, chitinase, alkaline phosphatase, hyaluronidase, and acetyl glycosaminidase, among others.107,109 A range of proteolytic enzymes including pepsin, trypsin, chymotrypsin, and collagenases are commercially extracted from marine fish viscera in large scale. Procedures have been developed for the isolation of both acidic and alkaline proteases from salmon viscera and acidic proteases from cod and mackerel viscera using centrifugation, polyacrylic acid and ammonium sulfate precipitation, ultrafiltration, and batch ion exchange chromatography. A large-scale process for the recovery of enzymes in wastewater from the shrimp processing industry has been reported. The water used in thawing frozen raw shrimp is flocculated by ferric chloride, concentrated by cross-flow ultrafiltration, and then freeze-dried. Alkaline phosphatase, hyaluronidase, β-N-acetylglucosaminidase, and chitinase have been recovered from shrimp shell waste in good yield. Pepsins and gastricins have been isolated from fish gastric mucosa, trypsins and chymotrypsins from pyloric ceca, and trypsinlike enzymes from hepatopancreas. Trypsin and chymotrypsin purified from cod viscera are commercially available. Collagenase prepared from crab hepatopancreas has been used for skinning of squid (Loligo spp.). Lysozyme has been recovered from commercial processing wastes of Arctic scallop and clamshell, which has a potential for application as a preservative in refrigerated foods. “Caviar” is riddled and cured roe (eggs) separated from the roe sack (ovary). Caviar production is a somewhat laborious process and is either carried out manually or mechanically. Cold-active fish pepsins from Atlantic cod and orange roughy have been used for caviar production from the roe of various species including orange roughy and salmon.110,111 Besides finfish, other marine sources of proteases include sponges, crabs, bacteria, and fungi. Many commercially important enzymes have been isolated from marine phytoplanktons. Haloperoxidases such as vanadium bromoperoxidase could become valuable products because halogenation is an important process in the chemical industry. Japanese researchers have developed methods to induce a marine alga to produce large amounts of the enzyme superoxide dismutse, which is used in enormous quantities for a range of medical, cosmetic, and food applications. Marine microorganisms are good sources of marine enzymes. However, the

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TABLE 4.11 Extracellular Gastric Enzymes from Fish and Aquatic Invertebrates Proteases

Source

Pepsin Pepsinogen Gastricin Trypsin Alkaline proteinases Neutral proteinases Neutral proteinase (calpains) Chymotrypsin Collagenases Elastase

Sardine, capelin, Atlantic cod, Greenland cod, salmon, mackerel, orange roughy, bluefin tuna, marine crab Rainbow trout, bluefin tuna, shark Hake, Atlantic cod Sardine, capelin, Greenland cod, Atlantic cod, different salmon species, anchovy, Atlantic croaker, carp, hybrid tilapia, krill, crayfish, oyster White croaker, chum salmon, tilapia Crucian carp Tilapia Capelin, herring, Atlantic cod, spiny dogfish, rainbow trout, scallop, abalone, white shrimp, grass carp Fiddler crab, freshwater prawn, crayfish, Atlantic cod, king crab Carp, catfish, Atlantic cod

Source: Adapted from Shahidi, F. and Janak Kamil, Y. V. A., Trends Food Sci. Technol., 12, 435, 2001; Haard, N.F., Food Technol., 52(7), 64, 1998.

symbiotic nature (microorganisms living in association with marine sponges, corals, and other species) and their habitats necessitate sophisticated isolation and culture conditions for production of these enzymes.112–115 Dimethyl sulfide (DMS), the most abundant volatile sulfur compound emitted from oceans, is primarily formed by the action of dimethylsulfoniopropionate (DMSP) lyase, which cleaves DMSP, an algal osmolyte, to equimolar amounts of DMS and acrylate. The enzyme has been isolated from a marine bacterium, identified as an Alcaligenes spp., a salt marsh bacterial organism and also from a sulfate-reducing bacteria and other marine organisms.106 Table 4.11 gives various extracellular gastric enzymes from fish and aquatic invertebrates.107,109

4.8.2

APPLICATIONS

Enzymes have been used in a wide variety of foods for centuries and provide alternate approach to food protection. They are well accepted by the consumers than chemical additives such as antioxidants. For example, glucose oxidase, produced by molds such as Penicillium notatum and Aspergillus niger, has been shown to control spoilage of fishery products. The preservative action of the enzyme is due to the generation of gluconic acid, which lowers the surface pH, thereby retarding bacterial spoilage. When sufficient glucose levels are available, glucose oxidase and catalase completely remove oxygen from an enclosed system thereby preventing off-flavor production.116 The food industry has already benefited from several enzymes that are produced using genetically modified production hosts to reduce the cost or enhance the functionality of the enzyme.60 The characteristic properties of marine fish proteinases such as higher catalytic efficiency at low temperatures, lower sensitivity to substrate concentrations,

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TABLE 4.12 Applications of Proteases for Fish Processing Preparation of hydrolyzates from fish and shellfish Debittering of hydrolyzate Scaling of fish Peeling and deveining of shrimp Removal of clam viscera Recovery of protein from filleting waste Removal of protein from shellfish waste Isolation of pigment from shellfish waste Ripening of salted fish Acceleration of fish sauce production Membrane removal from cod liver Membrane removal from roe Seafood flavorings Tenderization of squid Tenderization of fish meat Viscosity reduction of fish meal stickwater during drying Prevention of curd formation in canned salmon Source: Adapted from Venugopal, V., Seafood Processing, CRC Press, Boca Raton, FL, 2006.

and greater stability at broader pH range have been useful for their applications in many food-processing operations.107,108 They can also be utilized to produce bioactive components such as biopeptides on a large scale. Several attempts to obtain crude enzyme mixtures from internal organs of some fish species and to utilize them for various purposes have been summarized.117–119 Other applications of endogenous proteases are roe processing, fish sauce production, silage, and hydrolyzates. Table 4.12 summarizes the applications of proteases for fish processing.13 Enzyme biotechnology is continually providing new and modified enzymes. Creative use of these enzymatic tools will allow a new generation of tailored food ingredients with enhanced nutritional and functional properties. As is so often the case, it is the cost considerations that are currently restricting industrial scale developments in this area. New developments at a research level are likely to increase the demand for modified ingredients producing the drive to optimize processes and reduce costs.60,120,121 Functions and application of marine enzymes isolated from various animal sources include uses of proteases from marine sponges and crabs to degrade casein and hide powders, use of choline esterases from bivalves as biomarkers for aquatic pollution, Na, K-ATPases from spiny lobster for generation of osmolyte gradients, α-N-acetyl galactosaminidase from sea squirt in structural analysis of carbohydrate epitopes, and catalase from marine mussel as antioxidant enzyme for uses in toxicological studies, among others.106

4.9

ANTIFREEZE PROTEINS

AFPs help several marine fishes survive in the extremely cold habitats they dwell in. The effect of AFPs is due to depression of the freezing point of the blood of these

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organisms through inhibition of ice crystal formation. The mechanisms for inhibition of ice crystal formation include lowering the freezing point, the so-called thermal hysteresis effect, and modification of ice crystallization by accumulating at the ice–water interface. The AFPs are generally glycopeptides, which have molecular weights in the range 2.4–36 kDa and remain in the colloidal fraction of the blood. They are structurally diverse; each is radically different from the other proteins in its primary, secondary, and tertiary structures. The ice-binding sites of AFPs tend to be less polar and more hydrophobic than other AFP surfaces. The antifreeze glycoprotein-producing fishes include Atlantic cod, Greenland cod, winter flounder, Atlantic wolffish, and sculpins. Antifreeze glycoproteins from different fish species have a similar chemical structure with a molecular weight of 3–26 kDa. Flounder (Pseudopleuronectes americanus) provides a good source of AFPs, which are alanine-rich and have a molecular weight ranging from 3.3 to 4.5 kDa. Some AFPs have a few disulfide bridges. AFP isolated from Newfoundland ocean pout (Macrozoarces americanus), having a molecular weight of 5–6.7 kDa, contains no cysteine residues. The AFP of Atlantic herring (Clupea harengus) is unique in its requirement of Ca2+ for antifreeze activity.122,123 Certain cold-tolerant microorganisms also produce AFPs, although in a limited quantity. Recombinant DNA approach was used to make yeast (Pichia pastoris) wild-type strain X-33 or protease-deficient strain secrete the herring AFP. Both the yeast strains secreted the recombinant proteins properly folded and functioning as the native herring AFP into the culture medium. The expression at a lower temperature increased the yield of the recombinant protein drastically. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring AFP required for structural and functional studies.124

4.9.1

APPLICATIONS OF ANTIFREEZE PROTEINS

AFPs can have applications in food preservation. These could be applied to foods such as dairy products to help control water crystallization. Owing to their ability to cause depression of freezing point, they have potential for use in frozen foods as normal ice modulators. They can be used in combination with chilling, facilitating reduction of chill temperature without freezing of the muscle tissue. On a molar basis, their effect in depression of freezing point is 200–300 times more than that of sodium chloride. Usually, the consumption of AFPs does not impart any toxicologically significant effect. However, cost aspects need to be considered in such applications.125,126

4.10

COLD-ADAPTED ENZYMES

The Antarctic marine environment is characterized by challenging conditions for the survival of native organisms. Fishery products, microorganisms, and other living creatures such as corals, which have adapted to cold marine environment such as the Antarctic having an average temperature of about −1°C, are sources of cold-adapted enzymes having unique properties. These organisms have successfully developed adaptations enabling them to thrive at low temperatures. In a frozen environment, organisms would have to avoid dehydration and freezing of the intracellular space. The role of AFPs in protecting the organisms has been discussed earlier. In addition,

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nutrient fluxes will be limited and the secreted enzymes should be protected against cold denaturation. This adaptation to cold involves modifications of the cytoplasmic membrane so as to maintain the appropriate permeability and other adjustments to secure feasible rates of transcription and translation. Proteins and peptides constitute a substantial portion of the organic nutrients present in the deep-sea sediments as well as suspended particulate matter. Therefore, proteases, particularly, extracellular proteases are likely to play a pivotal role in making nutrients available in cold environments.127,128 Enzymes in aquatic organisms living in cold habitats have bestowed themselves with certain unique characteristics to help these organisms adapt to the environments. These enzymes may manifest alterations in isoenzyme distribution, substrate binding, substrate turnover rate, thermal stability, physiological efficiency, and thermodynamic properties. In the cold environment, the low temperature significantly enhances the viscosity of water, which contributes to slowdown in reaction rates. To facilitate better turnover of products with limited available quantity of enzymes, cold-adapted enzymes have usually higher specific activity, with only a few reported exceptions, than those of their mesophilic counterparts, with differences sometimes exceeding by a factor of 10. In addition to high specific activities, there could be structural variations in the enzymes to facilitate cold adaptation, such as unique amino acid sequence, thermal stability, reaction thermodynamics, and heat inactivation characteristics. This has been shown with respect to l-glutamate dehydrogenase (GDH) of Antarctic fish Chaenocephalus aceratus. The enzyme showed dual coenzyme specificity and was inhibited by guanosine triphosphate (GTP), although adenosine diphosphate (ADP) and adenosine triphosphate (ATP) activated the forward reaction. The complete primary structure of C. aceratus GDH, in comparison with homologous mesophilic enzymes, showed a less compact molecular structure with a reduced number of salt bridges. The structural modification resulted in higher catalytic efficiency at lower temperatures and was indicative of a high extent of protein flexibility.129 Digestive proteolytic enzymes from cold-adapted aquatic organisms possess unique properties compared to mammalian proteases.130,131 There is appreciable scope in studying the strategies of the adaptation of microorganisms to cold. Marine symbiotic microorganisms growing in association with animals and plants were shown to produce enzymes of commercial interest, such as proteases, carbohydrases, and peroxidases. Presently, only two genomes from marine organisms from Antarctic seawaters have been sufficiently analyzed to understand specific adaptations to cold. Furthermore, genome sequences are needed to detect whether there are some general trends in cold adaptation or, if in contrast, each microorganism has its own specific strategy.132,133

4.10.1 APPLICATIONS OF COLD-ADAPTED ENZYMES As cold-adapted enzymes display a high specific activity associated with a relatively high thermosensitivity and lower free energies of activation, there is particular scope to make use of these enzymes for possible applications in several fields. These areas include food processing, biomass conversion, molecular biology, environmental biosensors, bioremediation, cleaning of contact lenses, and several other processes.

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The lower free energy is useful in various processing applications requiring lowtemperature treatment. For example, there is an increasing industrial trend to treat foodstuffs under mild conditions to avoid adverse changes in properties including nutritive value. These properties could be particularly useful in those industrial processes that require limited time enzymatic treatment followed by rapid termination of the reactions so as to prevent excessive or deleterious changes. Examples are use of cold-adapted proteases for low-temperature proteolysis, caviar production, and extraction of carotenoprotein for use as colorants or flavorants in food or feed. Being heat labile, such cold-active enzymes can be inactivated by modest increases in temperature, without adverse impact on the food. Other examples are meat tenderizing in which excessive protease action could detrimentally affect the meat texture, and stone washing in the textile industry in which the excessive action of cellulases could lead to the loss of mechanical resistance of the cotton fibers. The problem of lactose intolerance in children can be addressed by treating milk with cold-active β-galactosidases, which hydrolyzes lactose to glucose and galactose, thereby removing lactose in the milk. Its high specific activity at low temperature helps treatment of milk at refrigerated temperatures, particularly during transport. Heat-sensitive β-galactosidases have been isolated and characterized from several psychrophilic microorganisms.134 Cold-active lipases could be useful for the development of various tastes and flavors owing to their high specific activity and unique specificities. These enzymes could also be useful in the development of animal feed having improved digestibility and assimilation potential. They can also find uses in the synthesis of various valuable peptides, fatty acids, and polysaccharides by reverse hydrolysis in low water conditions. This process can also be extended to pharmaceutical and chemical industries for the production of compounds with high added value.132,133 AFPs, fluorescent proteins, antitumor peptides, antibiotics, and hormones have already been cloned and overexpressed in microorganisms for innovative applications in food processing and genetic engineering. The expected impact of cloning fish proteins in different fields of technology has been pointed out.107,135 Marine enzyme biotechnology can assist in the development of cold-adapted enzymes, particularly with respect to their molecular biology and bioprocessing. Novel techniques in molecular biology applied to assess the diversity of chitinases, nitrate, nitrite, ammonia-metabolizing, and pollutantdegrading enzymes have been identified. Genes encoding chitinases, proteases, and carbohydrases from microbial and animal sources have been cloned and characterized.136,137 Table 4.13 gives the functions and applications of enzymes isolated from some marine sources.106

4.11 COMMERCIAL STATUS Presently, commercial production of peptide products from nonmarine sources is void. In recent years, a few fermented dairy products with naturally occurring antihypertensive peptides have been launched in both Japanese and Finnish markets. A beverage containing antihypertensive dodecapeptide from milk digests has been introduced in the Japanese market. Ingredia, a French dairy company has developed “Prodiet F200,” a milk protein hydrolyzate that contains a bioactive peptide with relaxing properties. The patented product has an antistress effect proven by clinical

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TABLE 4.13 Functions and Applications of Enzyme Isolated from Some Marine Sources Enzyme Class Hydrolases

Enzyme Protease

Marine crab Scylla serrata

Amylase

Sparus aurata, Scophthalmus maximus, and Sebastes mentella Bivalves and other species

Choline esterases

Transferase

Oxidoreductase

Lyase

Source

Adenosine monophosphate (AMP) deaminase Na–K ATPase

Teolost sea scorpion

Spiny lobster

ATP–N-glycosidase

Marine sponge

Citrate synthase, pyruvate kinase Transglutaminase Catalase, superoxide dismutase

Northern krill Red sea bream liver Marine mussel

Application/Function Collagenolytic metalloproteases Digestive enzymes in marine fishes Biomarker for aquatic pollution Purine nucleotide

Generation of osmolyte gradient Conversion of ATP into adenine and ribose-5-phosphate Metabolic key enzymes

Monooxygenase

Sea bass

Phenol oxidase

Marine mussel

β-Hydroxyesteroid dehydrogenase Phospholipases A2

Japanese eel

Protein cross-linking Antioxidant properties, potential use in toxicological studies Biomarkers of polycyclic aromatic hydrocarbon exposure Oxidation of phenolic substrates Key steroidogenic enzyme

Sea snake

Novel purification

Source: Adapted from Debashish, G., Malay, S., Barindra, S., and Joydeep, M., Adv. Biochem. Eng. Biotechnol., 96, 189, 2005. With permission of Springer Science and Business Media.

studies. A sour milk drink product that contains bioactive peptides is widely sold in Japan and some European countries. In the United States, a milk powder that contains antihypertensive peptides is also available as a functional food for blood pressure reduction. The in situ production of bioactive peptides in fermented dairy products such as yogurt and cheese has now been conceptualized as a novel approach to improve the functional value of the products.138 Peptides from marine sources are not commercially available. Consumption and uses of some of these products as protein supplements could be therapeutically beneficial to the consumers. Nisin, an antimicrobial compound originating from bacteria, is so far the only U.S. FDAapproved peptide.56 However, commercial production of peptides from marine sources has not been reported.

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The global use of enzymes in 2000 was U.S.$1.5 billion.139–141 Marine products are excellent sources of enzymes and could contribute to the total available enzymes in world markets. Hydrolytic enzymes such as proteases, amylases, esterases, and lipases occupy a major share in the industrial enzyme market. In recent times, lipases have emerged as key enzymes, owing to their multifaceted properties and applications in food technology, detergent, chemical industry, and biomedical sciences.142,143 A digestive protease from North Atlantic cod designated as Penzim is commercially available in the United States. It is a very powerful psychrophilic proteolytic enzyme. An isolate of krill enzymes known as Neptune Krill Enzymes is commercially available, which contains proteases, phosphatases, and phosphohydrolases, combined with peptides. Another product, Neptune Aquatein, which contains various enzymes including phospholipases, alkaline phosphatase, esterase, trypsin, hyaluronidases, and others is a dry fraction extracted from krill. The cold-adaptive enzymes are unique in their activities, as discussed earlier. Fiskeriforskning, a Norwegian biotechnology firm, has isolated another cold-active lysozyme chlamysin with antimicrobial activity from the viscera of the marine bivalve Chlamys islandica and has also encoded a complementary DNA (cDNA) gene that actuates the enzyme production in scallops.106 Some additional areas to be examined for commercial applications of marine peptides include technology for obtaining sufficient quantity of the peptides and better isolation procedures. Incorporation of fish-based biopeptides, however, may require safety assessment to rule out any possibility of allergenic, toxic, or carcinogenic effects.56 A fish-based skincare product has been developed jointly by the Norwegian University of Science and Technology and the University of Bergen, which shows promising results in the treatment of psoriasis and eczema. The cream contains enzymes, particularly zonase found in fish eggs, which is responsible for the juveniles emerging from the eggs. This enzyme can break down skin cells without damaging live cells. The product also contains gelatin. Used in psoriasis treatment, the cream helps to peel off dead skin and stimulates the growth of new skin cells.143 Commercial products in the form of capsules containing 75% protein and also omega-3 fatty acids prepared from sardine and anchovies are available. Nine capsules provide about 5 g protein, appreciable amounts of all the essential amino acids, and 246 mg omega-3 fatty acids.144 In conclusion, proteins from marine resources are highly nutritive. Hydrolyzates of marine proteins and also fermented fishery products are rich sources of bioactive peptides, which can display a variety of physiological functions including cardiovascular and antioxidant activities. Therefore, consumption of adequate quantities of seafood can help humans derive significant health benefits. Furthermore, several marine resources from cold habitats are sources of unique molecules such as AFPs and cold-adapted enzymes, which are ideal for specific applications in food science and biotechnology.

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49. Shrivastava, S. and Goyal, G. K., Therapeutic benefits of pro- and pre-biotics: a review, Ind. Food Ind., 26, 41, 2007. 50. Schmidt, R. H. and Turner, E., Functional foods and nutraceuticals, in Food Safety Handbook, Schmidt, R. H. and Rodrick G. E., Eds., Wiley, New York, 2003. 51. Farnworth, E. T. R., The beneficial health effects of fermented foods—potential probiotics around the world, J. Nutra. Funct. Med. Foods, 4, 93, 2004. 52. Sultanbawa, Y. and Aksnes, A., Tuna process waste—an unexploited resurce, Infofish Int., 3, 37, 2006. 53. Plante, S. et al., Protein powders from fish processing byproducts. The 2nd Joint TransAtlantic Fisheries Technology Conference, Quebec City, October 29–November 1, 2006. 54. Salinder, H. and Onen, Z., Bioactive Peptides in Dairy Products and their Functionality, IFIS, http://www.foodsciencecentral.com/fsc/ixid14786, accessed September 2007. 55. Philanto, A. and Korhonen, H., Bioactive peptides and proteins, Adv. Food Nutr. Res., 47, 175, 2003. 56. Pripp, A. H. et al., Quantitative structure activity relationship modelling of peptides and proteins as a tool in food science, Trends Food Sci. Technol., 16, 484, 2005. 57. Al-Omirah, H.-F. and Alli, I., Proteolytic Degradation Products as Indicators for Quality Assessment in Fish, Abstracts, Institute of Food Technologists, Annual Meeting, 1996, p. 80. 58. Hernandez-Herrero, M. M. et al., SDS-PAGE of salted anchovies (Engraulis encrasicholus L) during the ripening process, Eu. Food Res. Technol., 212, 26, 2000. 59. Gildberg, A., Enzymes and bioactive peptides from fish waste related to fish silage, fish feed and fish sauce preparation, J. Aquatic Food Prod. Technol., 13, 3, 2004. 60. Birschbach, P. et al., Enzymes: tools for creating healthier and safer foods, Food Technol., 58(4), 20, 2004. 61. Meng, F. et al., Quantitative analysis of complex peptide mixtures using FTMS and differential mass, J. Am. Soc. Mass. Spectr., 18, 226, 2007. 62. Bhakuni, D. S. and Rawat, D. S., Bioactive Marine Natural Products, Springer, Netherlands, 2005, Vol. XV, p. 400. 63. Dumay, J. et al., Mild procedure for obtaining lipipeptic and peptidic fractions from sardine (Sardina pilchard). The 2nd Joint Trans-Atlantic Fisheries Technology Conference, Quebec City, October 29–November 1, 2006. 64. Ravellec, P. R. et al., The presence of bioactive peptides in hydrolyzates prepared from processing waste of sardine (Sardina pilchardus), J. Sci. Food Agr., 81, 1120, 2001. 65. Martinez-Alvarez, O. et al., Presence of CGRP related molecules in fish protein hydrolyzates from industrial origin, Paper presented in 2nd Joint Trans-Atlantic Fisheries Technology Conference, Quebec City, October 29–November 1, 2006. 66. Ravellec, P. R. et al., Influence of the hydrolysis process on the biological activities of protein hydrolyzates from cod (Gadus morhua) muscle, J. Sci. Food Agr., 80, 2176, 2000. 67. Nogochi, M. et al., Isolation and identification of acidic oligopeptides occurring as flavor potentiating fraction from FPH, Agr. Food Chem., 231, 49, 1975. 68. Sabiro, M. et al., Effect of cartilage regeneration by GlcNAc and fish collagen peptide, Chitin Chitosan Res., 12, 184, 2006. 69. Wong-Kyo, J. et al., Preparation of hoki (Johnius belengeri), bone oligophosphopeptide with a high affinity to calcium by carnivorus intestine crude proteinase, Food Chem., 91, 333, 2005. 70. Mendis, E., Rajapakse, N., and Kim, S.-K., Antioxidant properties of a radicalscavenging peptide purified from enzymatically prepared fish skin gelatin hydrolyzate, J. Agr. Food Chem., 53, 581, 2005. 71. Brody, E. P., Biological activities of bovine glycomacropeptide, Brit. J. Nutri., 84, S39, 2000.

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72. King, S., US Patent, Antibacterial composition for control of gram positive bacteria in food formulations, #6,620,446, 2003. 73. Clare, D. A. and Swaisgood, H. E., Bioactive milk peptides: a prospectus, J. Dairy Sci., 83, 1187, 2000. 74. Patat, S. A. et al., Antimicrobial activity of histones from hemocytes of the Pacific white shrimp, Eur. J. Biochem., 271, 4825, 2004. 75. Smith, V. J. et al., Antibacterial proteins in rainbow trout, Fish Shellfish Immunol., 10, 243, 2000. 76. Nagashima, Y. et al., Purification and characterization of an antibacterial protein in the skin secretion of rock fish, Comp. Biochem. Physiol. Part C, 136, 63, 2003. 77. Howes, R., The free radical fantasy: a panoply of paradoxes, Ann. NY Acad. Sci., 1067, 22, 2006. 78. Chen, K. M. and Dekker, E. M., Endogeneous muscle antioxidants, Crit. Rev. Food Sci. Nutr., 34, 403, 1994. 79. Je, J., Park, P. J., and Kim, S.-K., Antioxidant activity of a peptide isolated from Alaska pollack (Theragra chalcogramma) frame protein hydrolyzate, Food Res. Int., 38, 45, 2005. 80. Rajapakse, N. et al., Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties, Food Res. Int., 38, 175, 2005. 81. Mamaeiona, J. et al., Phenolic acid and antioxidant capacity of sea cucumber, The 2nd Joint Trans-Atlantic Fisheries Technology Conference, Quebec City, October 29–November 1, 2006. 82. Suetsuna, K., Antioxidant peptides from protease digest of prawn (Penaeus japonicus) muscle, Mar. Biotechnol., 2, 5, 2000. 83. Kwon-Kim, S. et al., Isolation and characterization of antioxidative peptides from gelatin hydrolyzate of Alaska pollock skin, J. Agr. Food Chem., 49, 1984, 2001. 84. Hattori, M. et al., Antioxidative activity of soluble elastin peptides, J. Agr. Food Chem., 46, 2167, 1998. 85. Vercruysse, L., Camp, J. and van Smagghe, G., ACE inhibitory peptides derived from enzymatic hydrolyzates of animal muscle protein: a review, J. Agr. Food Chem., 53, 8106, 2005. 86. Li, G. H. et al., Angiotensin-converting enzyme inhibitory peptides from food proteins and their physiological and pharmacological effects, Nutr. Res., 24, 469, 2004. 87. Suetsuna, K. and Osajima, K., The inhibitory activity of angiotensin I-converting enzyme of basic peptides from sardine and hair tail meat, Bull. Jap. Soc. Sci. Fish., 52, 1981, 1987. 88. Ono, S. et al., Isolation of peptides with angiotensin I-converting enzyme inhibitory effect derived from hydrolyzate of upstream chum salmon muscle, J. Food Sci., 68, 1611, 2003. 89. Nagai, T. et al., Antioxidative activities and angiotensin I-converting enzyme inhibition of extracts prepared from chum salmon (Oncorhynchus Keta) cartilage and skin, Int. J. Food Prop., 9, 813, 2006. 90. Matsumoto, K. et al., Separation and purification of angiotensin I-converting enzyme inhibitory peptide in peptic hydrolyate of oyster, Nippon Shokuhin Kogyo Gakkaishi, 41, 589–594, 1994. 91. Ohba, R. et al., Physiological functions of enzymatic hydrolyzates of collagen or keratin contained in livestock and fish waste, Food Sci. Technol. Res., 9, 91, 2003. 92. Sato, M. et al., Angiotensin I-converting enzyme inhibitory peptides derived from wakame (Undaria pinnatifida) and their antihypertensive effect in spontaneously hypertensive rats, J. Agri. Food Chem., 50, 6245, 2002. 93. Jung, W. K. et al., Angiotensin-I lowering enzyme inhibitory peptide from yellowfin sole (Limanda aspera) protein and its antihypertensive effect in spontaneously hypertensive rats, Food Chem., 94, 26, 2006.

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94. Yokoyama, K., Chiba, H., and Yoshikawa, M., Peptide inhibitor for angiotensin I converting enzyme from thermolysin digest of dried bonito, Biosci. Biotechnol. Biochem., 56, 1541, 1992. 95. Morimura, S. et al., Development of an effective process and evaluation for utilization of collagen contained in livestock and fish waste, Proc. Biochem., 37, 1403–1412, 1999. 96. Kim, S. K. and Mendis, E., Bioactive compounds from marine processing by-products—a review, Food Res. Int., 39, 383, 2006. 97. Wergedah, H. et al., Fish protein hydrolyzate reduces plasma cholesterol, increases the proportion of HDL-cholesterol and lowers acyl-coA-cholesterol acyltransferase activity in liver of zucker rats, J. Nutr., 134, 1320, 2004. 98. Prisant, M., Nutritional treatment of blood pressure: Non-pharmacologic therapy, in Handbook of Food and Nutrition, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 961 99. Kristiansson, H. G. and Rasco, G. A., Fish protein hydrolyzates: production, biochemical, and functional properties, Crit. Rev. Food Sci. Nutr., 40, 43, 2000. 100. Shang-gui, D. et al., Amino acid composition and anti-anaemia action of hydrolyzed offal protein from Harengula Zunasi Bleeker, Food Chem., 87, 97, 2004. 101. Sorensen, R. et al., Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase, Nahrung, 48, 53, 2004. 102. Lee, T. G. and Maruyama, S., Isolation of HIV-I protease inhibiting peptides from thermolysin hydrolyzate of oyster protein, Biochem. Biophys. Res. Commun., 253, 604, 1998. 103. Salte, R., Norberg, K. K. and Ødegaard, O. R., Some functional properties of teleost antithrombin, Thrombosis Res., 80, 193, 1995. 104. Palacios, C., The role of nutrients in bone health from A to Z, Crit. Rev. Food Sci. Nutr., 46, 621, 2006. 105. Henkel, J., FDA Consumer Henkel, Consumer Magazine, U.S. FDA, Washington, DC, January–February 1998. 106. Debashish, G., Malay, S., Barindra, S., and Joydeep, M., Marine enzymes, Adv. Biochem. Eng. Biotechnol., 96, 189, 2005. 107. Shahidi, F. and Janak Kamil, Y. V. A., Enzymes from fish and aquatic invertebrates and their application in the food industry, Trends in Food Sci. Technol., 12, 435, 2001. 108. Raghunath, M. R., The activity and stability of digestive proteinases in nine species of marine fish, in Nutrients and Bioactive Substances in Aquatic Organisms, Devadasan, K. et al., Eds., Society of Fishery Technologists (India), Kochi, 1994, p. 112. 109. Haard, N. F., Specialty enzymes from marine organisms, Food Technol., 52(7), 64, 1998. 110. Kim, S. K. et al., Enzymatic recovery of cod frame proteins with crude proteinase from tuna pyloric caeca, Fish. Sci. (Tokyo), 63, 421, 1997. 111. Myrnes, B. and Johansen, A., Recovery of lysozyme from scallop waste, Prep. Biochem., 24, 69, 1994. 112. Joo, H. S. et al., Bleach resistant alkaline protease produced by a Bacillus sp. isolated from the Korean polychaete, Periserrula leucophryna, Proc. Biochem., 39, 1441, 2004. 113. Suzuki, S. and Odagami, T., Low-temperature-active thiol protease from marine bacterium Alteromonas haloplanktis, J. Biotechnol., 5, 230, 1997. 114. Kumar, G. C. and Takagi, H., Microbial alkaline proteases: from bio-industrial view point, Biotechnol. Adv., 17, 561, 1999. 115. Raghukumar, C., Damare, S., and Muraleedharan, C., Patent filed, A process for production of low temperature-active alkaline protease from a deep-sea fungus, # NF 271/2003. 116. Kantt, C. A. et al., Glucose oxidase/catalase solution for on-board control of shrimp microbial spoilage: model studies, J. Food Sci., 58, 104, 1993.

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117. Stefansson, G., Enzymes in the fishing industry, Food Technol., 42(3), 64, 1998. 118. Sikorski, Z. E., Gildberg, A., and Ruiter, A., Fish products, in Fish and Fishery Products—Composition, Nutritive Properties and Stability, Ruiter, A., Ed., CAB International, Wallingford, 1995, p. 315. 119. Kim, S. et al., Recovery of fish bone from hoki (Johnius belengeri) frame using a proteolytic enzyme isolated from mackerel intestine, J. Food Biochem., 27, 255, 2003. 120. Rastall, R., Tailor-made food ingredients: enzymatic modulation of nutritional and functional properties, IFIS Publishing, http://www.foodsciencecentral.com/fsc/ ixid3729, December 2001. 121. IFIS, Natural preservation of foods using bacterial metabolites and live addition of bacteria, http://www.foodsciencecentral.com/fsc/ixid14740, IFIS Publishing, March 2007. 122. Jia, Z. and Davies, P. L., Antifreeze proteins—an unusual receptor–ligand interaction, Trends Biochem. Sci., 27, 101, 2002. 123. Davis, P. L., Fletcher, G. L., and Hew, C. L., Fish antifreeze protein genes and their use in transgenic studies, in Oxford Surveys on Eukaryotic Genes, Vol. 6, Maclean, N., Ed., Oxford University Press, Oxford, UK, 1989, p. 89. 124. Zhengjun, L. et al., Low-Temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris, Protein Express. Purif., 21, 438, 2001. 125. Li, B. and Sun, D. W., Novel methods for rapid freezing and thawing of foods—a review, J. Food Eng., 54, 175, 2002. 126. Mishra, V. and Pattnaik, P., Anti-freeze proteins: prospects and perspectives in food sector, Indian Food Ind., 18, 238, 1994. 127. Smith, D. C. et al., Intense hydrolytic enzyme activity on marine aggregates and implications for rapid particle dissolution, Nature, 359, 139, 1992. 128. Damare, S. et al., Deep-sea fungi as a source of alkaline and cold tolerant proteases, Enz. Microbiol. Technol., 39, 172, 2006. 129. Ciardiello, M. A. et al., Primary structure, function and thermodynamic characterisation: relationship with cold adaptation, Biochim. Biophys. Acta (Prot. Stru. Mol. Enz.), 1543, 11, 2000. 130. Diaz-Lopez, M. and Garcio-Carreno, F. L., Application of fish and shellfish enzymes in food and feed products, in Seafood Enzymes, Haard, N. F. and Simpson, B. K., Eds., Marcel Dekker, New York, 2000, p. 571. 131. Asgeirsson, B. and Bjarnason, J. B., Properties of elastase from Atlantic cod, a coldadapted proteinase, Biochim. Biophys. Acta, 1164, 91, 1993. 132. Marx, J.-C. et al., Cold-adapted enzymes from Antarctic marine microorganisms, Invited Article, Mar. Biotechnol., 1, 1, 2006. 133. Siddiqui, K. S. and Cavicchioli, R., Cold-adapted enzymes, Annu. Rev. Biochem., 75, 403, 2006. 134. Nakgawa, T., Fujimoto, Y., and Ikehata, R., Purification and molecular characterization of cold-active galactosidase from Arthrobacter psychrolactophilus strain F2, Appl. Microbiol. Biotechnol., 72, 720, 2006. 135. Macouzet, M., Simpson, B. K., and Lee, B. H., Cloning of fish enzymes and other fish protein genes, Crit. Rev. Biotechnol., 19, 178, 1999. 136. Ghosh, D. et al., Marine enzymes, Adv. Biochem. Eng. Biotechnol., 96, 189, 2005. 137. Ohgiya, S. et al., Biotechnology of enzymes from cold-adapted microorganisms, in Biotechnological Aspects of Cold-Adapted Organisms, Margessi, R. and Schinner, F., Eds., Springer, Berlin, 1999, p. 17. 138. Makhal, S., Mandal, S., and Kanawijia, S. K., Development of bioactive fermented dairy products with special reference to cheese: scope and challenges, Ind. Food Ind., 23, 25, 2004. 139. Kork, O. et al., Industrial enzyme applications, Curr. Opin. Biotechnol., 13, 345, 2000.

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140. Ktidyinddon, C. H., Functional bioactive peptides from hydrolyzed aquatic food proteins, in Marine Nutraceuticals and Functional Foods, Barrow, C. and Shahidi, F., Eds., CRC Press, Boca Raton, FL, 2007 (in press). 141. Losso, J. N., Sato, K., and Mazza, G., Functional Proteins, Peptides and Amino Acids, CRC Press, Boca Raton, FL, 2008. 142. Gupta, R., Gupta, N., and Rathi, P., Bacterial lipases: an overview of production, purification and biochemical properties, Appl. Microbiol. Biotechnol., 64, 763, 2004. 143. Anonymous, Fish based skin cream, Infofish Int., 3, 71, 2006. 144. http://www.seagateproducts.com/marine-protein.html, accessed October 2007.

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5 Polyunsaturated Fatty Acids and Their Therapeutic Functions 5.1 INTRODUCTION Lipids contribute to food quality by providing flavor, aroma, color, texture, taste, and nutritive value. From the nutritional point of view, lipids function as sources of metabolic energy, carrier of fat-soluble vitamins (e.g., A, D, E, and K), and contribute to the formation of cell and tissue membranes. In addition to their contribution in meeting energy needs, intakes of dietary fat must be sufficient to meet the requirements of essential fatty acids (EFA) and fat-soluble vitamins. The minimum intake consistent with health varies throughout a person’s life and among individuals. Sufficient intake of dietary fat is particularly important prior to and during pregnancy and lactation. Increasing the availability and consumption of dietary fats is often a priority for overcoming the problems of protein-energy malnutrition. Recommendations to populations concerning desirable ranges of fat intakes may vary according to prevailing conditions, especially dietary patterns and the prevalence of diet-related noncommunicable diseases. For most adults, dietary fat should supply at least 15% of their energy intake. Women of reproductive age should consume at least 20% of their energy from fat. Both the amount and quality of dietary fat consumed can affect child growth and development. These influences are mediated through energy levels and through the action of specific fatty acids and various nonglyceride components of the fat. Breast milk provides between 50 and 60% energy as fat, and during the weaning period (i.e., the transition from full breast-feeding to no breast-feeding), care needs to be taken to prevent dietary fat intakes from falling too rapidly or below the required levels. The use of fat, especially vegetable oils, in the foods fed to weanling infants and young children is an effective way to maintain the energy density of their diets. The consumption of adequate amounts of EFA is also important for normal growth and development.1 For the marine ecosystem, lipids are very important in the physiology and reproductive processes of marine animals and reflect the special biochemical and ecological conditions of the marine environment. The interests in marine lipids are essentially due to the fact that they contain significant amounts of long-chain polyunsaturated fatty acids (designated as ω-3 PUFA/ω-3 PUFA), which have been recognized to be important in human health and nutrition. Initial studies on marine lipids involved characterization of their components, facilitated by advent of methods such as gas-liquid chromatography for fatty acid analysis and radioisotope tracer techniques, which have led to the understanding of the molecular biodiversity and identification 143

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of important sources of ω-3 PUFA.2 During the last few decades, investigations on the nutritional aspects of marine lipids particularly, ω-3 PUFA have opened up great vistas for these compounds in health protection. The work was kindled in the 1970s with the recognition of the role of diet in the health of native Greenland Eskimos. It was observed that the longevity and coronary health of Eskimos was related to their diet, which contained an average 450 g fatty fish per day.3 High fish consumption is believed to contribute to several health benefits to the Japanese, who eat about 80 g of fish and shellfish per day, providing approximately 1000–2000 mg/day of ω-3 PUFA.3 Because of the recognized health benefits, fatty fish species, which contain significant amounts of ω-3 PUFA were considered as functional food.4,5 However, recent decline in certain fisheries together with preference of some sections of the populations to the foods of vegetable origin initiated search on alternate sources of these fatty acids, such as transgenic plants and microalgae. It is said that the food processors are locked in a “fish oil arms race”; many entrepreneurs are interested in development of genetically modified crops that could challenge the supremacy of fish as the best source of ω-3 fatty acids.6,7 Nevertheless, the supremacy of marine products as sources of PUFA is difficult to challenge at least in the near future. This chapter will essentially discuss the nutritional importance of PUFA from marine sources, with particular reference to fatty fishes.

5.2 MARINE LIPIDS The relative proportion of lipids and fatty acids in marine organisms is characteristic of their genus and species, and also depends on environmental conditions. The principal producers of marine lipids in the marine environment are microalgae, which support both pelagic and benthic food webs. Marine lipids are composed of neutral lipids comprising triacylglycerols, phospholipids, sterols, wax esters, and some unusual lipids such as glyceryl esters, glycolipids, sulfolipids, and hydrocarbons. Most of the variations in lipid are found in the triacyl glycerol fraction, whereas the phospholipids show fewer variations. The triacyl glycerol functions as a reserve of fatty acids that provide energy through oxidation and also help conversion into phospholipids, which are present in cell membranes. The phospholipids of fish muscle contain generally more phosphatidylcholine than phosphatidylethanolamine. The phospholipids of tropical fish are more saturated than fish from temperate waters. The neutral lipids have a lower specific gravity than seawater, therefore the roles in regulating buoyancy has often been postulated, especially for wax esters.2 Marine fish are commonly classified according to the fat content of their fillets. They are grouped as lean (under 3% fat), medium (3–7% fat), and high fat (over 7% fat). Lipids in fatty fish are mostly subcutaneous in nature, whereas in lean fish they are deposited in the liver, muscle tissue, and mature gonads. Lean fish such as sole are usually whitish, whereas, fish with higher fat content (e.g., cod, haddock, halibut, and pollock) are white to off-white. The flesh of high fat fish (e.g., herring, sardine, anchovy, and salmon) is usually pigmented (e.g., yellow, pink, and grayfish). In an individual fish, lipid content increases from tail to head, with higher level of fat deposition in the belly flap and dark muscle. The amounts of lipids in fish may vary widely from 0.2 to 24%, depending on anatomical position, sex, location in body,

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age, season, and diet. In many pelagic fish, lipid contents ranging from 12 to 20% are found during winter when compared with 3–5% during summer. The fat levels in some fish correlate with spawning cycles. For instance, anadromous fish store fat prior to migration to freshwater for spawning. A variety of seafood items including clams, cod, flounder, grouper, haddock, halibut, northern lobster, mahi-mahi, monk fish, perch, pike (Northern eye), pollock, orange roughy, scallop, shrimp, red snapper, sole, squid, tuna (skip jack), tuna (yellow fin), and whiting contain less than 2.5 g total fat in 3 ounce cooked portions. However, the same amount of cooked portions of butterfish, herring, Spanish mackerel, salmon (Atlantic, coho or sockeye), lake trout, bluefin tuna, and whitefish provide 5–10 g total fat.8–11 The marine steroids are composed of cholesterol, which is present in a concentration of 50–90 mg per 100 g fish meat. In some pelagic fish species such as anchovy, bluefin tuna, pilchards, and different mackerels, cholesterol may be up to 150 mg per 100 g meat, and may be as high as 50–650 mg in roe and liver. Shellfish tend to contain slightly higher amounts of cholesterol. Thus crustaceans (crab, lobster, and shrimp) contain 69–100 mg per 100 g. Squid and octopus may contain 250 and 120 mg of the steroid per 100 g, respectively.8–11 Crustaceans and some mollusks require dietary sources of sterol for growth and survival because of the absence of de novo sterol-synthesizing ability.12

5.2.1

FATTY ACIDS

The polar lipids of marine organisms, whether the glycolipids that predominate in the thylakoid membranes of unicellular photosynthetic organisms that constitute phytoplankton, or the phosphoglycerides that predominate in the cell membrane bilayers of animals, are all composed of highly unsaturated fatty acids. Fatty acids are straight chain carboxylic acids. Fatty acids with chain length of 10 carbon atoms or less are referred to as short-chain fatty acids, and they are all saturated. Fatty acids having up to 14 carbon atoms are medium-chain fatty acids and those with more than 14 carbon atoms are long-chain fatty acids, which may be saturated or unsaturated. The position of the first double bond is given by the (n-x) notation, counting the number of carbon atoms from the methyl end, according to the international nomenclature. For example, ω-3 and ω-6 (also referred as n-3 and n-6 fatty acids) denote fatty acids, in which the first double bond starts at 3 and 6 carbons from the methyl end, respectively. The symbol, 18:4 ω-3 identifies a fatty acid, with 18 carbon atoms and four double bonds, the first double bond occurring after the third carbon atom. The human diet contains long-chain PUFA belonging to the n-6 and n-3 families. Major dietary n-6 PUFA include linoleic, C18:2w6 (18:2); γ-linolenic, C18:3w6 (18:3); and arachidonic, C20:4w6 (20:4) acids, whereas major dietary ω-3 PUFA include α-linolenic, C18:3w6 (18:3); eicosapentaenoic acid (EPA), C20:5w3 (20:5); docosahexaenoic acid (DHA), C22:6w3 (22:6); and to some extent, docosapentaenoic acid. Linoleic, α–linolenic acids (ALA), and γ-linolenic acids are present in large quantities in foods of plant origin, such as oils from corn, maize, sunflower seed, cottonseed, soybean, linseed, and canola. Arachidonic acid (AA) originates from muscle and organ meats, or alternatively may be synthesized within the body by successive desaturation and chain elongation of linoleic acid (LA). EPA and DHA are synthesized by desaturation

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TABLE 5.1 Terms and Symbols Designating Major Fatty Acids in Foods Common Name

Chain Double Symbol Symbol Lengtha Bonds Ib IIc

Caproic Caprylic Capric Lauric Myristic Palmitic Palmitoleic Stearic Oleic Linoleic γ-Linoleic α-Linolenic Gadoleic Arachidonic EPA DHA a b

c

d

6 8 10 12 14 16 16 18 18 18 18 18 20 20 20 22

0 0 0 0 0 1 0 1 2 3 3 1 4 5 6

C5:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:1 C18:0 C18:1 C18:2 C18:3 C18:3 C20:1 C20:4 C20:5 C22:6

C16:1w7 C18:1w9 C18:2w6 C18:3w6 C18:3w6 C20:1w9 C20:4w6 C20:5w3 C20:6w3

Symbol IIId 6:0 8:0 10:0 12:0 14:0 16:0 16:1 n-7 18:0 18:1 n-9 18:2 n-6 18:3 n-6 18:3 n-9 20:1 n-9 20:4 n-6 20:5 ω-3 20:6 ω-3

Systematic Name n-Hexanoic n-Octanoic n-Decanoic n-Dodecanoic n-Tetradecanoic n-Hexadecanoic cis-9-Hexadecanoic n-Octadecanoic cis-9-Octadecanoic cis, cis-9,12-Octadecadienoic All cis-6,9,12-octadecatrienoic All cis-9,12,15-Octadecatrienoic n-11-Eicosenoic All cis-5,8,11,14-Eicosatetraenoic cis-5.8.11.14.17-Eicosapentaenoic cis-4,7,10,13,16,19-Docosahexaenoic

Carbon atoms are numbered starting from the carboxyl group which is number 1. In the case of unsaturated fatty acids, the symbol I is sometimes used to indicate points of unsaturation. For example, C16:2 for designating LA. Carbon atoms are numbered from the methyl group, “w” indicates the first carbon where point of unsaturation is found. The letter n gives the position of the first Carbon atom where a point of unsaturation is found, starting from the methyl group.

Source: Adapted from Venugopal, V., in Encyclopedia in Food Microbiology, Academic Press, New York, 2000, p. 1743. With permission.

and chain elongation of ALA within the human body. When present in equimolar concentration, LA and ALA compete for conversion to their respective longer chain products, AA and eicosapentaenoic acid.4 Table 5.1 shows the nomenclature and systematic names of major fatty acids in foods.13 Assays of fatty acids are commonly carried out by gas chromatography, after conversion of the lipid material into corresponding methyl esters (fatty acid methyl esters [FAME]) through suitable derivatization reactions. Quantitative derivatization depends on the type of catalyst and processing conditions employed, as well as the solubility of the sample in the reaction medium.14 A convenient rapid method has been developed recently for the preparation of FAME.15

5.2.2

LIPID PROFILE OF SEAFOOD

The fatty acid composition of seafood is fundamentally different from meat, vegetable, and dairy products and shows marked variability within and between species and also according to environmental variables such as diet and their habitats.

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The nature of fatty acids essentially determines the quality of lipids. The fatty acids are present in fish lipid as triglycerides, which are prone to hydrolysis by lipases with the formation of free fatty acids. Lipid hydrolysis is more in ungutted than in gutted fish, probably due to the involvement of lipases present in digestive enzymes. Cellular phospholipases are also known to hydrolyze the lipids, particularly, the phospholipids (which leads to increased oxidation of the hydrolyzed lipids). Marine oils are rich sources of long-chain PUFA of the ω-3 type. The two fish oils, which have been studied most extensively, are the ω-3 fatty acids, 20 carbon EPA (C20:5w3, cis-5.8.11.14.17-EPA), and the 22-carbon DHA (C22:6w3, cis-4,7,10,13,16,19-DHA). EPA contains five double bonds and DHA, six double bonds. In this chapter, the term “PUFA” used with respect to marine products should be considered as “long chain ω-3 polyunsaturated fatty acids,” unless otherwise stated. From a structural point of view, these fatty acids in natural state are cis isomers, whereas processing may give rise to the formation of large quantities of trans isomers. Other ω-fatty acids such as linoleic and linolenic acids are present in fish oil, although to a minor level. They are, however, present in high quantities in vegetable oils (canola, soybean, and sunflower) and nuts such as peanuts and almonds. These fatty acids are collectively referred to as EFAs. Table 5.2 shows ω-3 fatty acid content in some seafood.16 The fatty acids, which have attracted particular attention from therapeutic point of view, are EPA and DHA. Unlike terrestrial organisms, seafood lipids contain significant amounts of EPA and DHA. The biogenesis of these fatty acids in fish has been studied. Long-chain PUFAs containing 20 or more carbon atoms such as AA and EPA, and 22 carbon atoms DHA are formed from the 18-carbon LA and ALA by the process of chain elongation and desaturation. Fish have the ability to synthesize the saturated and monounsaturated fatty acids de novo, and also to selectively absorb

TABLE 5.2 Contents of ω-3 Fatty Acids in Some Seafood Content (g/100 g meat) ≤0.5 Atlantic cod Atlantic pollock Catfish Haddock Oil sardine Pacific cod Pacific halibut Rockfish Skipjack tuna Sole Yellow perch

0.6–1.0

≥1.0

Atlantic mackerel Channel catfish Indian mackerel Red snapper Silver hake Spiny dogfish Swordfish Torbot Trout

Anchovy Atlantic herring Atlantic salmon Bluefin tuna Pacific mackerel Pacific herring Pink salmon Rainbow trout

Source: Reproduced from Venugopal, V. and Shahidi, F., Food Rev. Int., 12, 175, 1996. With permission from Taylor & Francis Ltd. (www.informaworld.com).

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and metabolize dietary fatty acids including long-chain PUFA. Mammals and fish are generally incapable of de novo synthesis of C18:2 (n-6), C18:3 (ω-3), C20:4 (n-6), C20:5 (ω-3), and C22:6 (ω-3), and dietary sources of these fatty acids are likely to be essential for them. This is because marine fish lack or have a very low activity of desaturase enzyme for the synthesis of the fatty acid. Thus, marine finfish accumulate large amounts of DHA and EPA, which are synthesized by phytoplankton of the marine food chain.4,17 Fatty acid composition and its origin in seafood from different locations has been the subject of detailed study. The fatty acids, triacylglycerol oils from northern hemisphere fish are generally dominated by C16:0, C18:1 (n-9), C20:1 (n-9), and C22:1 (n-11) fatty acids. The C20:1 (n-9) and C22:1 (n-11) acids originate from the corresponding fatty alcohols in zooplanktonic wax esters, these esters commonly account for more than 50% of the dry weight of the zooplankton. These acids are major sources of metabolic energy in fish and are oxidized by conventional mitochondrial β-oxidation pathways along with peroxisomal β-oxidation. The glycolipids of photosynthetic organisms contain a range of PUFAs including C16:2 (n-7), C16:3 (n-4), C16:4 (n-1), C18:4 (ω-3), C18:5 (ω-3), and C20:5 (ω-3). Whereas, C22:6 (ω-3) is present in unicellular, photosynthetic organisms such as prymnesiophytes and dinoflagellates, it is present in phosphatidylcholine. The commonly held view that this is an adaptation to low environmental temperatures is not supported by experimental evidence. Thus, the phosphoglycerides of the warm blooded tuna are unusually rich in C22:6 (ω-3).18 Table 5.3 shows levels of TABLE 5.3 Levels of ω-3 Fatty Acids as Percent of Total Fatty Acids, In Various Commercial Fish Oils Species Anchovy Atlantic menhaden Sardine/pilchard Gulf menhaden Pollock Capelin Sand eel Mackerel Blue whiting Herring Tuna Norway pout Whitefish spp. Salmon, wild Salmon, farmed Sprat Tilapia, farmed Catfish, farmed

18:3

18:4

20:5

22:5

22:6

EPA + DHA

1 1 1 2

2 3 3 3 2 3 5 4 3 3 1 3 2 1 3

22 14 16 13 15 8 11 7 7 6 6 9 9 8 9 6

2 2 2 3

9 12 9 8 4 6 11 8 8 6 22 14 13 11 11 9 5 3

31 26 25 21 20 14 22 15 15 12 28 23 22 19 18 15 5 4

1 1 1 1 2 1 1 1 2 1 2 2 1

1

1 1 1 1 2 1 2 4 2 1 3 1

Source: Bimbo, A. P., Lipid Technol., 19, 176, 2007. With permission from Wiley-VCH Verlag Gmbh, Weinheim.

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ω-3 fatty acids in various commercial fish oils. It can be seen that the total amounts of EPA and DHA are about 20% of total fatty acids. Some fish such as tuna, Norway pout, whitefish spp., and salmon contain more DHA than EPA. Over 200 fish species from Australian waters have been analyzed for fatty acid composition and the data have been compiled into a handbook.19 In comparison to wild-caught fish, farmed finfish such as salmon can provide 3–10 times more ω-3 PUFA, as shown in Table 5.4. This can be achieved by using special feeds and also by farming transgenic fish capable of synthesizing these acids. Salmon oil is an excellent source of EPA (18%) and DHA (12%) and is commercially available.20,21 Influence of substitution of sunflower oil with PUFA-rich flaxseed oil on the fatty acid profiles of Nile tilapia (Oreochromis niloticus) was examined. The main fatty acids detected were palmitic, stearic, oleic, linoleic, and α-linolenic in all the treatments. The 30 day–fed fish presented the highest values for total ω-3 fatty acids, with a prominence of ALA, showing that the flaxseed oil as well as the feed supply time influenced the fatty acid profiles.22 Lipids are very important food reserve, in particular in the oocytes of mollusks, which assures viability of the larvae. The lipid composition of mollusks can be affected by external factors such as seasonal fluctuations in the environmental conditions and availability of phytoplanktons. Mollusks having good access to phytoplankton, accumulate a high proportion of EPA and DHA. These fatty acids have been reported to be essential for optimal growth of several juvenile bivalves. Accumulation of the PUFA has also been observed in other bivalves including

TABLE 5.4 Contents of ω-3 PUFA in Wild and Farmed Australian Seafood Products Wild Fish Fish Shellfish Prawn Lobster Farmed Striped perch Atlantic salmon Barramundi Silver perch Others Beef Chicken Turkey Pork

ω-3 Long-Chain PUFA Contents (mg per 150 g wet weight) 350 225 180 160 3700 2985 2960 1200 30 30 30 0

Source: Adapted from Nichols, P. D., Long-chain ω-3 oils in wild and farmed Australian seafood IfIS Publishing, April 10, 2006, http://www.foodscience central.com/fsc/ixid14325. With permission.

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oysters, patella, clams, and scallops. In scallops, the highest level of EPA and DHA was found in the adductor muscle, the major edible part of scallop. Aquaculture employing special diets such as microalgae-supplemented feeds can yield mollusks rich in EPA and DHA. In crabs, EPA is effective in maintaining survival, whereas DHA plays an important role in accelerating the intermolt period and produces a wider carapace width in swimming crab larvae. High concentrations of ω-3 PUFA have also been reported in squid. The digestive gland of squid is a rich source of EPA and DHA, indicating that this discard could be a cheap raw material for production of the fatty acids.4 The fatty acid composition of triglycerides and phospholipid fractions along with hydrophilic, lipophilic, and enzymatic antioxidants were compared in the muscle tissues of 21 species of teleosts, 3 species of cephalopods, and 6 species of crustaceans, caught from the Mediterranean Sea. The fatty acid composition, enzymatic activities, and the levels of low-molecular-weight antioxidants showed marked interspecies differences. The results showed that the total PUFAs (21.7–61.5%) were the highest, followed by saturated (16.9–41.3%) and monounsaturated (9.1–42.8%) fatty acids. The total ω-3 fatty acid contents (16.6–57.1%) were found to be higher than the total n-6 fatty acid content (4.1–10.6%). All of the species studied had an ω-3/n-6 ratio of more than 1, pointing out the importance of fish and shellfish as a significant dietary source of ω-3 PUFA.23 Extraction conditions are important in determining the lipid profile of seafoods, since variations in the conditions could influence yield of fatty acids. For example, the concentrations of myristic, palmitic, stearic, linoleic, arachidonic, and EPA in sea cucumber Stichopus chloronotus found in Malaysia differed significantly depending on the extractants. Phosphate buffer saline (PBS) extraction resulted in a much higher content of EPA (25.69%) compared to 18.89% in ethanol, 7.84% in distilled water, and only 5.83% in methanol, whereas no DHA was detected in ethanol extractions.24 Influence of processing on PUFA contents of fishery products has been reported. Coho salmon (Oncorhynchus kisutch) fillets were processed using smoking, canning, freezing, acidifying, or salting. Salmon preserved by smoking, canning, or freezing retained higher values of total fatty acids, including EPA and DHA. Salting and acidifying (pickling) treatments resulted in a significant decrease in PUFA content.25

5.3

OXIDATION OF FATTY ACIDS

Fatty acids yield energy by β-oxidation in the mitochondria of all cells. Saturated short, medium, and long-chain fatty acids undergo the first step of β-oxidation with different dehydrogenases. The process yields successive acetyl CoA molecules, which enter the tricarboxylic acid cycle or other metabolic pathways. Acetate is the eventual product from fatty acids with an even number of carbon atoms. Oxidation of unsaturated fatty acids, including LA is as fast as or faster than that of palmitic acid. Long-chain (>14 carbon atoms) fatty acids are preferentially oxidized by peroxisomes. Peroxisomal oxidation is energetically less efficient than mitochondrial oxidation and yields more heat. This type of oxidation can be induced by diets, which are high in fat as well as by a variety of xenobiotics.4 Discussion on in vivo lipid metabolism is not within the purview of this chapter.

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Autooxidation is the main mechanism by which oxidation of lipids occurs in foods and is also important in limiting the shelf life of lipids extracted from marine organisms. Autooxidation results in the formation of hydroperoxides, which subsequently give several secondary products such as aldehydes, ketones, and alcohols. Several factors influence oxidation, such as oxygen, temperature, pH, pressure, and trace metals such as iron and copper, and is caused by the high reactivity of free radicals.26 Fatty acids, particularly polyunsaturated acids, are extremely sensitive to oxidation, which involves a complex set of chemical reactions involving initiation (5.1), chain elongation (5.2), and termination (5.3), as follows: RH + initiator → R•

(5.1)

ROOH → ROO• R• + O2 → ROO•

(5.2)

ROO• + RH → RO2H + R• R• + R• → R–R

(5.3)

ROO• + R• → ROOR The process is initiated by removal of a proton from the central carbon of unsaturated fatty acid, usually a pentadiene moiety of the fatty acid, with the formation of a lipid radical. The latter reacts quickly with atmospheric oxygen and forms a peroxy radical (ROO•), a major radical, belonging to a group collectively referred to as “reactive oxygen species” (ROS). Other ROS include superoxide anion (O2•−), hydroxyl radical (HO•), alkoxy (RO•), hydroperoxy (HOO•) and nitric oxide (NO•) derived radicals. Nonradical derivatives are hydrogen peroxide (H2O2), ozone (O3), and singlet oxygen (1O2). These ROS are formed either enzymatically or chemically or photochemically during aerobic metabolism of food in the body. They are also formed by irradiation of food. Hydroxy radical is the most reactive, followed by singlet oxygen.27 Unsaturated lipids are easily oxidized by ROS, which results in low-molecular volatile aldehydes, alcohols, and hydrocarbons. These products include undesirable volatile compounds and carcinogens, and the secondary reactions also result in destruction of essential nutrients, and changes in the functionalities of proteins, lipids and carbohydrates. Extensive research has demonstrated that ROS are formed in the human body. They cause oxidative damage to DNA, block cellular signal transduction, modulate gene expression and enzyme activity. ROS reaction with proteins, sugars, and vitamins results in cross-linking or cleavage of proteins, which leads to loss of their functionality; production of undesirable volatile compounds; and destruction of essential fatty acids, amino acids, and vitamins. Interaction of ROS with DNA, cell membranes, proteins, and other cellular constituents leads to their damage and favors induction of serious human diseases including atherosclerosis, rheumatoid arthritis, muscular dystrophy, cataracts, neurological disorders, cancer, as well as ageing. The role of ROS in the etiology of cancer, cardiovascular disease, and neurogenerative processes has been an area of increasing investigation and controversy. ROS-induced

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reactions are favored by activation of hemoproteins and increase in free iron, whereas antioxidants negatively influence the oxidation.28–30 No single analytical method is available to give a satisfactory description of lipid oxidation status. High-resolution NMR spectroscopy techniques have been successfully used to establish possible correlations with traditional analytical methods and to study oxidation of DHA. Correlations were found between primary oxidation products (peroxide value and conjugated dienes) and the appearance of the 1H NMR spectra.31

5.3.1

ANTIOXIDANTS

Antioxidants may be defined as any substance, which is capable of delaying, retarding, or preventing the development of rancidity or other flavor deteriorations. Antioxidants can inhibit or retard oxidation either by scavenging the free radicals that initiate oxidation, or by breaking the oxidative chain reactions. The mechanisms also involve binding of metal ions, scavenging of oxygen, converting hydroperoxides to nonradical species, deactivating singlet oxygen, and thereby suppressing the generation of free radicals and reducing the rate of oxidation. Primary antioxidants are butyl hydroxyanisole (BHA), α-tocopherol, flavanoids, gallates, etc. BHA, butylhydroxyl toluene (BHT), tertiary butyl hydroxyquinone (TBHQ), and esters of gallic acid, for example, propyl gallate, are the major synthetic antioxidants. They are used generally in concentration up to 0.02% of the fat or oil content, and sometimes in combinations for synergistic effects. Some of the secondary antioxidants include peroxide decomposers such as thioethers, methionine, metal chelaters, glutathione peroxidase, and catechins. Antioxidants such as tocopherol, BHA, BHT, TBHQ, gallate esters, and ascorbyl palmitate are lipid soluble; whereas sulfur dioxide, ascorbic acid, and cysteine are water soluble. Natural antioxidants are carotenoids, α-tocopherol, flavanoids, etc. Polyvalent acids such as tartaric, malic, gluconic, oxalic, succinic acids, sodium triphosphate, pyrophosphate, and phytic acids were reported to exhibit synergistic effects in lipid oxidation. Ascorbic acid has been demonstrated to be an effective radical scavenger of superoxide, hydrogen peroxide, hydrochlorite, peroxyl radical, and singlet oxygen. The growing consumer demand for food devoid of synthetic antioxidants has focused efforts on the discovery of new natural antioxidants, which are presumed to be safe since they occur in foods naturally.32,33 Measures of the antioxidant capacities of 277 selected foods have been released by the U.S. Department of Agriculture. The database provides access to antioxidant values of a wide variety of foods, many of which may be excellent sources of healthful compounds.33

5.3.2

ROLE OF ANTIOXIDANTS IN HEALTH PROTECTION

Reactive oxygen and nitrogen species (ROS, RNO) are continuously produced in the human body. Most of the time, their overproduction is controlled by antioxidants. However, failure of antioxidant defense mechanisms is implicated in damage of DNA, lipids, and proteins. Damage to the biomolecules, in turn, is associated with increased risk of chronic diseases including cancer and cardiovascular disease.34 Antioxidant hypothesis suggests that reducing agents (i.e., antioxidants) have the capacity to prevent oxidation damage and thus increased level will also reduce

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the risk of chronic diseases. The common dietary antioxidants, which include vitamin E (tocopherol), vitamin C, polyphenols including flavanoids, and carotenoids such as β-carotene and lycopene, augment cellular defense mechanisms and protect components of the cell from oxidative damage. In doing so, they protect human body against onset of several diseases induced by ROS. There is compelling epidemiological evidence linking consumption of antioxidant-rich diets with reduced risk of degenerative diseases. Studies conducted on humans demonstrated higher intake of foods rich in dietary antioxidants are associated with reduced morbidity and mortality.35 For instance, a daily intake of antioxidants in soft gel capsules was linked to a drop in systolic and diastolic blood pressures after 8 weeks of supplementation.36 The oxidative modification of low-density lipoprotein (LDL) may be a key early step in the pathogenesis of atherosclerosis. When LDL is oxidized, numerous chemical changes occur. The concentration of PUFA is reduced with increase in lipid peroxides and aldehydes, which may be cytotoxic to endothelial cells. Oxidized LDL also affects secretion of various growth factors and cell signals that can promote atherosclerosis. Several evidences have been accumulated, which implicate oxidative modification of LDL in the early stages of atherosclerosis. Antioxidant nutrients have been shown to decrease the susceptibility of LDL to oxidation in vitro. For instance, the antioxidant nutrients, α-tocopherol, ascorbic acid, and β-carotene have been shown to inhibit LDL oxidation in vitro. Because plasma level of these nutrients can be increased by dietary supplementation with minimal side effects, they may be useful in the prevention of coronary artery disease.37–39 There is ample evidence to suggest that people whose diets are rich in fruits and vegetables have a lower incidence of heart disease, diabetes, dementia, stroke, and certain types of cancer. The knowledge on the beneficial effects of antioxidants has resulted in an industry that produces a variety of health supplements. Most supplements labeled as antioxidants contain at least one of the aforementioned compounds, often as a pure chemical or sometimes as a concentrated plant extract. β-carotene has attracted special attention as health supplement. Natural antioxidants extracted from plants such as rosemary, sage, tea, soybean, citrus peel, sesame seed, olives, carob pod, and grapes can be used as alternatives to the synthetic antioxidants because of their equivalent or greater effect on the inhibition of lipid oxidation. The human intake of green tea decreases total cholesterol, increases the high-density lipoprotein (HDL) fraction, and decreases lipoprotein oxidation.40 The 50-year old antioxidant hypothesis, however, has faced some obstacles based on recent research. Since 1990s, a number of double-blind randomized controlled intervention trials have raised apprehensions over the antioxidant therapy. Widespread use of antioxidants has failed to quell the current pandemic of cancer, diabetes, and cardiovascular disease, or to stop or reverse the ageing process.41 Despite good evidence that vitamin E is a powerful antioxidant in vitro, a serious doubt prevails in its ability to protect oxidation of LDL in the body. It has been pointed out that vitamin E exists in eight different forms in nature, all of which function as antioxidants in vitro. However, the body uses only one form, α-tocopherol, which is easily bound by a specific protein in the liver, making it unavailable to function as antioxidant. Similarly, a consistent body of evidence

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from clinical trials suggests that β-carotene supplements do not decrease the risk of prostate, colon, or breast cancer. In fact, in the case of smokers there can be even an increase in their risk of lung cancer.42–44 These studies have raised doubts regarding the efficacy of antioxidant supplements. It has to be mentioned that while these compounds could offer protection against free-radical mediated diseases, they are unable to function as drugs to cure the diseases. It is likely that certain factors such as the relative presence of some foods and the absence of other foods are more important than the level of individual nutrients consumed. Further, as pointed out earlier, lack of agreement on the positive benefits of antioxidants could be due to lack of knowledge about oxidative mechanisms in vivo.45

5.3.3

LIPID OXIDATION IN MARINE FISHERY PRODUCTS

Fish lipids, rich in ω-3 PUFA, are very susceptible to oxidation, giving rise to ω-3 baldheads that cause distinctive oxidative off-flavors. Enzymes such as lipoxygenase, peroxidase, and microsomal enzymes from animal tissues can potentially initiate lipid peroxidation producing hydroperoxides. Lipoxygenases are concentrated in the skin tissue and remain active for up to 48 h of chilled storage of fish. Lipoxygenasedependent oxidative activity has been detected during chilled storage of fatty fish species such as sardine (Sardina pilchardus) and herring (Clupea harengus). Lipid oxidation results in formation of compounds that influence fish flavor. Some of the most abundant degradation products of the hydroperoxides formed from DHA (also arachidonic acid) that influence flavor include cis-4-heptenal, trans-2-heptenal, trans-2-cis-4-heptadienal and also 1,5-octadiene-3-ol, 1-octene-3-ol and hexanal.46 The extent of lipid oxidation can be suppressed by glutathione peroxidase, which reduces unstable lipid hydroperoxides to nonradical, stable products, which are inactive in the oxidative chain propagating mechanism. Other enzymes useful in this respect are superoxide dismutase and catalase, which remove superoxides from the peroxidation mechanism.47 Current evidence suggests that EPA is oxidized in fish by mitochondrial β–oxidation, whereas peroxisomal β-oxidation is necessary for the catabolism of DHA.15 Formation of fluorescent compounds resulting from the interaction between lipid oxidation products and biological amino constituents has been noticed during chilled (0–2°C) storage. Lipid oxidation is comparatively more during frozen storage than during chilled storage. Oxidative stability of lipids from several marine sources was compared. On the basis of peroxide formation, total lipids from squid viscera or squid muscle with skin were most stable, followed by those in trout egg, bonito oil, and tuna, respectively. Since squid viscera contained more than 25% of total lipids, they may be used as a good resource of functional lipids rich in EPA and DHA.48–50 The fatty acid patterns of triglyceride and phospholipid fractions, their sensitivities to oxidative damage and protection by endogenous antioxidants (see Section 5.3.2) were determined in fresh muscle tissue of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax) during ageing. Lipid peroxidation and accumulation of oxidized proteins during in vivo ageing were linked with an age-dependent decline of lipophilic antioxidants, coenzyme Q (CoQ), reduced form of Coenzyme Q (CoQ H 2), vitamin E,

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and vitamin C contents in muscle tissues, whereas ageing in fish was not linked to a decline in antioxidant enzymes and reduced glutathione levels. Lipophilic antioxidant and vitamin C levels represented a reliable marker of oxidative stress during ageing.51

5.4

NUTRITIONAL VALUE OF LIPIDS

As mentioned in the introduction, lipids play important roles in human nutrition and disease management. It has been recognized that in order to meet the nutritional requirements of an adult, an ideal fat should help in maintenance of health and prevention of diseases. In early 1920s, the nutritional significance of EFAs was recognized. The important EFAs, such as LA, ALA, and AA are found mostly in dairy products or meat products derived from ruminant animals (cow, goats, and sheep), vegetable oils and plants. EFAs occur in many vegetable oils especially safflower, flaxseed, sunflower, and corn oils and therefore use of these oils in diets is recommended. EFA deficiency in humans and animals cause restrictive growth, abnormality of skin and hair, damage of reproductive system, and abnormal composition of serum and tissue fatty acids. EFAs are shown to improve glucose tolerance, which is associated with type 2 diabetes and also reduces development of adipose fat. Linoleic and linolenic acids also provide increased benefits to the cardiovascular system. Two recent studies showed benefits of ALA in cardiovascular disease. Women who reported consuming diets rich in oils containing ALA seemed to have a lower risk of heart disease and sudden cardiac death than women whose diets were low in the plant derived fatty acid.51 The beneficial effects could be due to favorable changes in vascular inflammation and endothelial dysfunction.52 Oleic acid is a monounsaturated fatty acid, most commonly present in olive oil. Experiments using breast cancer cell lines showed that the fatty acid dramatically cut the levels of an oncogene, called Her-2/neu, which occurs in high levels in more than a fifth of breast cancer patients and associated with highly aggressive tumors.53 Conjugated linoleic acid (CLA) derived from ruminant animal sources (e.g., beef, lamb, and dairy) and also plant (e.g., safflower) sources, have been reported to contain antioxidant and anticancer properties. CLA is found in seafood, but to a lesser extent. A majority of research to date has been focused to study the biological effects of the cis-9, trans-11 and trans-10, cis-12 CLA isomers. Dairy derived CLA, for example, has been shown to inhibit carcinogenesis in experimental animals and also help control obesity.54 ALA can be converted to ω-3 fatty acids, EPA, and DHA in the body. An European Union has funded research aimed at identifying the scientific basis for improving health through diet, with a focus on understanding how PUFA from fish can be a protective component of diet against metabolic syndromes such as obesity and type 2 diabetes.8 The major health benefits recognized in EPA and DHA will be discussed in the following section.

5.4.1

HEALTH BENEFITS OF OMEGA-3 FATTY ACIDS

The beneficial effects of ω-3 fatty acids can be classified into two main areas. First, these fatty acids sustain normal healthy life through the reduction of blood pressure and plasma triglycerides and cholesterol, together with increased blood coagulation

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time. EPA and DHA are important for maintenance of normal blood flow as they lower fibrinogen levels and prevent platelets from sticking to each other. Second, they alleviate certain diseases such as blood vessel disorders and inflammatory diseases, and control an overactive immune function resulting in alleviation of autoimmune disease, such as arthritis and some types of dermatitis. Deficiency of these compounds causes several disorders such as restrictive growth, abnormality of skin and hair, damage of reproductive system, and abnormal composition of serum and tissue fatty acids. These benefits have been pointed out by several recent reviews summarizing research in the field.55–58 The advantages of consumption of PUFA in protecting health and addressing certain individual disease are briefly discussed. 5.4.1.1

Cellular Processes

Polyunsaturated fatty acids are essential components in higher living organisms that confer fluidity, flexibility, and selective permeability to cellular membranes. ω-3 PUFA are involved in many cellular and physiological processes in animals and plants, which include modulation of ion channels, endocytosis/exocytosis, pathogen defense, chloroplast development in plants, activity of membrane associated enzymes, and pollen formation. DHA is a vital component of the phospholipids of human cellular membranes, especially those in the brain and retina, as will be discussed later.2 5.4.1.2

Blood Pressure

Fish oil supplementation has been beneficial to control high blood pressure. A decrease of diastolic pressure by 3 mm Hg and systolic pressure by 6 mm Hg by regular consumption of EPA and DHA has been reported in a population-based intervention trial. The study, h performed a meta analysis of 40 studies testing the impact of ω-3 PUFA on blood pressure, which reported an intake of nearly 3 g EPA and DHA per day (which is equivalent to 6–10 capsules of commercial fish oil supplements or two servings of 100 g portion of fish rich in ω-3 PUFA). The overall change in blood pressure was significant for systolic blood pressure only (a reduction of 1.0–1.5 mm Hg). However, the decline in blood pressure in the range of 3.5–5.5 mm Hg in hypertensive patients was significant for both systolic and diastolic reduction. Nevertheless, this was not a function of the dose of ω-3 PUFA, duration of treatment, type of intervention (food versus oil capsule), or age of participants.59,60 A recent study on 4000 men and women in the age group between 40 and 59 in Japan, China, and the United Kingdom, has shown that certain foods which are high in ω-3 fatty acids—such as salmon oil and also ground flaxseed and walnuts—may help to lower blood pressure.61 5.4.1.3

Cardiovascular Disease

As mentioned earlier, the first recognition of the beneficial effect of these fatty acids on cardiovascular disease came from the observations on the longevity of Eskimos, which was later attributed to the high contents of fish-derived EPA and DHA in

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their diets.62,63 Since then, the effects of the ω-3 fatty acids in alleviating the clinical symptoms of cardiovascular heart disease have been well examined. These studies conclusively showed that diets rich in fish and fish oils are associated with a reduced risk of coronary heart disease (CHD). Further, epidemiological studies suggested that individuals at risk of CHD benefited from the consumption of plant and marinederived ω-3 fatty acids. More evidences have shown that fish consumption favorably affected CHD mortality, especially nonsudden death from myocardial infarction. These benefits were essentially due to lowering of the blood viscosity, inhibition of the platelet aggregation, reduction of the plasma fibrinogen, reduction of fasting serum triglyceride levels, and decreasing the blood pressure by EPA and DHA. These fatty acids are also important for the maintenance of normal blood flow as they lower fibrinogen levels and prevent aggregation of platelets. In addition, the ω-3 fatty acids prevent atherosclerosis by retaining the strongly protective HDL, which removes the harmful LDL and excess total cholesterol from the peripheral tissues. HDL also prevents the lipoprotein oxidation.11,63–66 In a randomized study, 58 elderly nursing home residents received 2 g of fish oil capsules daily. They were followed up on alternate days for a period of 6 months with 6-min measurements of heart rate variability (HRV), a measure of cardiac autonomic function. It was concluded that daily supplementation with 2 g of fish oil was well tolerated and was associated with a significant increase in HRV.67 An openlabel, randomized trial was conducted involving 52 patients receiving ≥3 active antiretrovirals, who had fasting triglyceride levels of more than 200 mg per deciliter. The patients received nutritionist-administered dietary and exercise counseling with or without fish oil supplementation for 16 weeks. Patients who received fish oil experienced a 25% mean decline in fasting triglyceride levels after 4 week. The study showed that supplementation with ω-3 fatty acids in combination with dietary and exercise counseling was well tolerated and reduced fasting triglyceride levels in patients receiving antiretrovirals.68 In another study, 20,551 U.S. male physicians, who were between 40 and 84 years of age and free of myocardial infarction, cerebrovascular disease, and cancer at baseline regularly consumed fatty fish up to 11 years. Consumption of fish at least once per week could reduce the risk of sudden cardiac death in men.69 The effect of fish oil supplements by themselves or a combination of fish oils and garlic powder on serum lipids was examined. Fifty men with moderately elevated cholesterol level were assigned to one of four treatment groups and followed for 12 weeks. The fish oil used in this study was a natural triacylglycerol, and those receiving fish oil took 12 g containing 30% of a mixture of EPA and DHA in a 1.5 ratio for a total of 2.16 g of EPA and 1.44 g of DHA, daily. One group received fish oil and garlic powder, another group received fish oil and a placebo powder, a third group received garlic powder and a placebo oil, and the remaining group was given placebo oil and a placebo powder. The fish oil group registered a 3.73% lowering of serum triglycerides, no significant change in total cholesterol and an 8.5% increase in LDL cholesterol. The fish oil and garlic powder group were found to have a 34.3% lower triglycerides, a 12% lowering of total cholesterol, a 9.5% decrease in LDL cholesterol, a 16% decrease in the total cholesterol over HDL-cholesterol ratio and a 19% decrease in the LDL cholesterol over HDL-cholesterol ratio. However, the garlic group showed no change in the serum triglyceride value, and 11.5% decrease

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in total cholesterol, a 14% decrease in LDL cholesterol, a 12.5% decrease in the total cholesterol to HDL-cholesterol ratio and a 15% decrease in the LDL-cholesterol ratio. No change in HDL cholesterol was observed in the fish oil group. A slight nonsignificant increase in HDL cholesterol was noted in the garlic group.70 Recent studies have further established an inverse relationship between fish intake and CHD death. In addition, fish intake was associated with a reduced progression of coronary artery atherosclerosis in postmenopausal women with CHD.71–73 DHA was recently shown to reduce cholesterol particle size in children. Researchers at the University of California, San Francisco, found that in participants taking DHA supplements, the amount of large, buoyant LDL particles was significantly increased and the amount of small, dense LDL particles was reduced. The study suggested that taking DHA supplements could reduce the risk of children with high cholesterol from developing heart disease in later life.73 5.4.1.4

Cancer

Several studies have shown the effects of ω-3 PUFA against cancer. An inverse relationship has been noted between blood levels of EPA and DHA and the risk of prostate cancer and adenocarcinoma. Scientists at the Paterson Institute for Cancer Research, Manchester, United Kingdom, found that the fatty acids found in salmon, mackerel, and fresh tuna can help prevent the spread of prostrate cancer to other parts of the body, which was attributed to ω-3 fatty acids.74 In addition, EPA and DHA have also been reported to act positively against cancer effects such as cachexia (abnormal weight loss) or survival rate in end-stage cancer.2 A research team at the St. George’s Hospital in South London, found that the consumption of the fish oil capsule rich in ω-3 fatty acids restored cell production to normal levels in bowel cancer patients. In the study, 80% of patients who were given concentrated fish oil capsules showed a reduced risk of developing the disease. In another population-based study involving 61,433 women without previous diagnosis of cancer, scientists from the Karolinska Institute, Sweden, found that consumption of fish with high content of ω-3 fatty acids reduced the risk of renal cell carcinoma (RCC), a common form of kidney cancer, in women by 74%, compared to those who did not take fatty fish. During a mean of 15.3 years of follow-up between 1987 and 2004, incidence of 150 RCC cases were diagnosed, which showed an inverse association of fatty fish consumption with the risk of RCC. It was observed that women who consumed one or more servings of fatty fish per week had a decreased risk of RCC by 44% compared with women who did not consume any fish. Women who reported consistent long-term consumption of fatty fish at baseline had a 74% lower risk 10 years later.75 Judicious selection of dietary fat has been suggested to prevent colon cancer.76 In spite of these reports, recent analysis of a large body of literature spanning numerous cohorts from many countries with different demographic patterns, apprehension has been raised on significant association between ω-3 fatty acids and cancer incidence.77 5.4.1.5

Pregnancy and Infancy

DHA is an important structural component of the membranes of the brain, nervous tissue, and eye. In mammals, DHA is present at very high levels in the phospholipids of

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the brain and neurological tissues and is, therefore, important in normal development of the central nervous system of the infant. The content of DHA in human brain increases almost four times during the first 3 months of pregnancy as well as postnatal life. A normal adult human brain contains more than 20 g DHA, which contributes to improved memory functions. DHA is also high in retina, and testes and sperm. Distribution, therefore, suggests that DHA is important for vision and nervous functioning in humans. Deficiency in DHA has been associated with visual impairment and delayed cognitive development. Recent research has shown that DHA display a variety of beneficial effects in fetal development. During pregnancy, EFAs, especially, AA and DHA, play an important role in maternal health and neonatal development. An adequate intake of DHA and EPA is particularly important during pregnancy and lactation. During pregnancy, and 2 years of postnatal life, the infant gets DHA and also EPA through mother, since the infant is unable to synthesize these EFAs.78 Both LA and ALA can serve as precursors for DHA, their ratio should be around 7:1, as generally found in human milk. Studies on human volunteers have shown that conversion of ALA to EPA can occur, whereas conversion of ALA to DHA is restricted, indicating a need for supplementation of DHA in the diet. It has been suggested that DHA is energetically expensive molecule to be synthesized in the body since it requires a complex biochemical route. Furthermore, it is prone to oxidation because of the high level of unsaturation. The blood of a formula-fed infant will contain less than half of the DHA of the blood of a breast-fed baby. The situation is worse in the case of prematurely delivered babies. Several studies have clearly indicated significantly higher levels of intellectual capabilities (in terms of IQ) in children who have received DHA during their initial years. Children deficient in DHA may exhibit behavioral problems. A relationship between hostility and consumption of whole fish, ω-3 and ω-6 fatty acids has been indicated by a recent study. Using a sample of ~3600 adolescents, it was found that consuming DHA and whole fish were independently related to lower hostility rates compared to those who had not consumed DHA or fish. However, it may not be valid to equate whole fish consumption with fish oil consumption since fish generally are a much better source of DHA. While fish has a DHA:EPA ratio of 3.0:4.1, the contents of DHA is lower in fish oil, with a DHA:EPA ratio of 1:2.79 Attention-deficit hyperactivity disorder (ADHD) is characterized by hyperactivity, emotional instability, poor coordination, short attention span, poor concentration, impulsiveness, and learning disorders. Initial studies have linked ADHD to a deficiency in plasma and red blood cells of PUFA including AA, EPA, and DHA, possibly due to inadequate breastfeeding during early childhood. Fish-derived PUFA, particularly DHA are beneficial against mental disorders such as schizophrenia, ADHD, Alzheimer’s disease, and dementia. Consumption of fish prevented such neurological disorders.17 Regular consumption of fish, at least twice a month, significantly reduced depression too.80 According to a study conducted at Scotland, two thirds of children are likely to have fatty acid deficiency, which may be responsible for some of the behavioral patterns and symptoms of autism. Supplementation of diet of these children with fish oil can result in improvement in their behavior.4 In modern times, increased incorporation of DHA into margarines and baby foods has been promoted to enhance brain memory development. Feeding milk

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containing a combination of AA and DHA in the ratio 1:5 with a DHA content of 0.4% has been recommended for the purpose.81 An amount of 500–600 mg of DHA per day has been recommended for prospective mothers. Eating fish twice or thrice a week could provide the required DHA. Supplementation of mother’s diet with sardines and other fish oils at a level of 2.6 g ω-3 fatty acids per day resulted in an increase in DHA in maternal red blood cells from 4.6 to 7.2%, with a corresponding increase in maternal plasma. This subsequently enhanced DHA level in infant red blood cells.82 5.4.1.6 Obesity The prevalence of overweight and obesity has increased over the past few decades. Experimental evidence supports the role of ω-6 fatty acids as being potent promoters of both adipogenesis in vitro and adipose tissue development in vivo during the gestation/lactation period. It was proposed that unnoticed changes in fatty acid composition of ingested fats over the last decades have been important determinants in the increasing prevalence of childhood overweight and obesity. Eating oily fish regularly could help fight obesity. Small doses of oil containing ω-3 fatty acids, combined with moderate exercise such as walking can control obesity. However, taking the oils without exercise, or exercising without taking oils, does not result in weight loss.83 5.4.1.7

Asthma

Fish oil or fish containing more than 2% fat has been found to have a reduced risk of airway hyperresponsiveness. Children who regularly eat fresh, oily fish have a four times lower risk of developing asthma than children who rarely eat fish. Supplementation of diet with ω-3 fatty acids confirmed their benefit in the reduction of breathing difficulties and other symptoms in asthma patients. More recently, it has been demonstrated that PUFA are also beneficial in the treatment of other lung diseases such as cystic fibrosis and emphysema. The increase in contents of ω-3 fatty acids in cell membrane during the treatment takes place at the expense of AA resulting in the competitive inhibition of pro-inflammatory group 2 icosanoid production and production of anti-inflammatory group 3 icosanoid.4,84,85 ω-3 Fatty acid supplements were recently shown to protect against exercise-induced bronchoconstriction (EIB) in asthma sufferers. EIB is a temporary narrowing of the airways that can be triggered by vigorous exercise. Sixteen asthmatic patients with documented EIB received either fish oil capsules containing 3.2 g of EPA and 2.0 g DHA or placebo capsules daily for 3 weeks. The fish oil diet improved pulmonary function to below the diagnostic EIB threshold, with a concurrent reduction in bronchodilator use. It was concluded that fish oil supplementation might represent a potentially beneficial nonpharmacologic intervention for asthmatic subjects with EIB.86 5.4.1.8

Behavioral Pattern

PUFAs appear to be a major determinant of membrane fluidity in brain cells, and this could play a major role in the maintenance of normal cognition and mood, as shown in a study involving 24 patients with a history of substance abuse, some of whom

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exhibited aggressive behavior. The adult male subjects were randomly assigned into two groups, one receiving 3 g of ω-3 fatty acids (2250 mg EPA, 500 mg DHA, and 250 mg others) in the form of purified fish oil in capsules, whereas the other received a placebo. In order to assess changes in anger level, a modified version of profiles of mood states (POMS) questionnaire was administered at baseline and every month thereafter for a period of 3 months. Thirteen patients who received the fish oil showed a clinically significant and progressive decrease in their POMS anger subscale scores, whereas in the other patients no change was observed. The study revealed that low levels of ω-3 EFAs, particularly EPA and DHA, played a significant role in the pathophysiology of anger as well as depressive, suicidal, and aggressive behavior.87 5.4.1.9

Diabetes

Epidemiologic studies have reported a lower prevalence of impaired glucose tolerance and type 2 diabetes in populations consuming large amounts of ω-3 PUFA. Preliminary evidence also suggests that increased consumption of ω-3 PUFAs with reduced intake of saturated fat may reduce the risk of conversion from impaired glucose tolerance to type 2 diabetes in obese persons. Expected health benefits and public health implications of consuming 1–2 g ω-3 PUFA per day as part of lifestyle modification in insulin resistance and type 2 diabetes have been reported. Controlled clinical studies have shown that consumption of PUFAs has cardio protective effects in persons with type 2 diabetes without adverse effects on glucose control and insulin activity. Other benefits include lower risk of primary cardiac arrest; reduced cardiovascular mortality, particularly sudden cardiac death; reduced triglyceride levels; increased HDL levels; improved endothelial function; reduced platelet aggregation; and lower blood pressure. Reported improvements in homeostasis, slower progression of artery narrowing, subclinical inflammation, oxidative stress, and obesity, however, require additional confirmation.5 5.4.1.10

Bone Health

The beneficial effects on bone health in 23 subjects, who consumed one of three specific PUFA-rich diets for 6 weeks, were examined recently by determining their serum concentrations of N-telopeptides (NTx) and bone-specific alkaline phosphatase (BSAP) as markers. The diets were (i) average American diet, consisting of 34% total fat, 13% saturated fatty acids, 13% monounsaturated fatty acids, 9% PUFA (7.7% LA, 0.8% ALA); (ii) LA diet (37% total fat, 9% saturated fatty acid, 12% monounsaturated fatty acid, and 16% PUFA [12.6% LA, 3.6% ALA]), and (iii) ALA diet containing 38% total fat, 8% saturated fatty acids, 12% monounsaturated fatty acid, 17% PUFA (10.5% LA, 6.5% ALA). Walnuts and flaxseed oil were the predominant sources of ALA. NTx levels were significantly lower in subjects who consumed the ALA diet (13.20 + 1.21 nM), in comparison to those who received average American diet (15.59 + 1.21 nM). There was no change in levels of BSAP across the three diets. Concentrations of NTx were positively correlated with the pro-inflammatory cytokine TNF-α. The results indicated that dietary ω-3 PUFA could have a protective effect on bone metabolism through a decrease in

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bone resorption in the presence of consistent levels of bone formation.88 The study employed walnuts and flaxseed, whereas the potential exists for extrapolation of the study to consumption of fatty fish. 5.4.1.11

Other Benefits

Recent literature on randomized controlled trials, meta-analyses, population studies, and case reports, which were used to compile data and identify trends in pertinent clinical applications of fatty acid therapy, showed that there are a myriad of disorders and maladies that seem to be controlled by intake of ω-3 fatty acids. Besides the various benefits accrued from consumption of ω-3 fatty acids, as discussed above, other functions include anti-inflammatory and immune-modulating properties. Consumption of high dose of ω-3 supplements daily has been reported to decrease the severity of symptoms associated with ankylosing spondylitis (ASA), a chronic disease that mostly affects joints of the spine and hips.89 Fish oil supplementation reduces overweight, and in patients with inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disorders, supplementation result in significant relief, due to improved joint tenderness and grip strength. Consumption of ω-3 PUFA could also offer protection against blindness resulting from abnormal blood vessel growth in the eye, according to a study on the influence of ω-3 and ω-6 PUFAs on vascular loss, vascular regrowth after injury, and hypoxia-induced pathological neovascularization in a mouse model that had acquired oxygen-induced retinopathy. It was shown that increasing ω-3 PUFA tissue levels by dietary or genetic means decreased the vascular area of the retina by increasing vessel regrowth after injury, thereby reducing the hypoxic stimulus for neovascularization.90,91 The protective effect of ω-3 PUFAs and their bioactive metabolites was mediated, in part, through suppression of tumor necrosis factor-α. Increasing the sources of ω-3 PUFA or their bioactive products reduced pathological angiogenesis. Western diets are often deficient in ω-3 PUFA, and premature infants lack the important transfer of ω-3 PUFA from the mother to the infant, which normally occurs in the third trimester of pregnancy. Supplementing ω-3 PUFA intake may be of benefit in preventing retinopathy.92 In view of the several therapeutic advantages discussed earlier, marketing campaigns have been launched for many marine fish products, which tend to affirm that consumption of fish or supplements containing ω-3 PUFA is an appropriate method to satisfy consumer’s need for a variety of nutritive foods. Factors underlying the popularity of ω-3 oils, particularly fish oils, their recommended daily indices, clinical trials showing their benefits on cardiovascular health, and prevention of arthritis, inflammation, and allergy, child development, mental alertness, cognitive function and mood have been pointed out in these campaigns. Improvement in the flavor of fish oils (e.g., development of various deodorized fish oils), development of various innovative delivery systems for fish oils in foods (e.g., emulsions, sirups, and gels) and government level support of health claims for ω-3 fatty acids and fish oils in Europe and the United States, have also been pointed out. Recognition of the beneficial effects of PUFA has resulted in positive changes in consumer attitude toward seafood.91,92 Table 5.5 summarizes suggested nutraceutical potentials of ω-3 fatty acids.93

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TABLE 5.5 Nutraceutical Potentials of ω-3 Fatty Acids Prevention from atherosclerosis Protection against arrhythmias Reduce blood pressure Beneficial for diabetic patients Fight against manic-depressive illness Reduce symptoms in asthma patients Protection against chronic obstructive pulmonary diseases Alleviation of symptoms of cystic fibrosis Prevent relapses in patients with Crohn’s disease Prevent various cancers Provide bone health Improve brain functions in children Source: Adapted from Narayan, B., et al., Food Rev. Int., 22, 291, 2006; Swansson, M. A. and Evenson, P., in Food Additives, Marcel Dekker, New York, 2002, 225; Berge, J.-P. and Barnathan, G., Adv. Biochem Engn/Biotechnol., 96, 49, 2005.

5.4.2

MODE OF ACTION

The mode of action of EPA and DHA in health is considered to function through their ability to give rise to a class of pharmacologically important groups of compounds such as prostaglandins, prostacyclins, thromboxanes, and leukotrienes (collectively called as eicosanoids). These metabolites bind to specific G-protein-coupled receptors and signal cellular responses and modulate many biological processes. These eicosanoids that are present in the body, are highly specific in their function to elicit a biological response, and are under remarkably tight regulation. The eicosanoids are formed from LA, AA, EPA, and DHA. Formation of eicosanoids from LA is through the intermediate formation of AA. Formation of eicosanoids from AA is shown in Figure 5.1. Many of the eicosanoids have a direct influence on biological responses associated with immune function. These include the inflammatory response as well as induction of macrophage and production of antibodies in response to some challenge by the organism. In addition, AA is easily oxidized to linear or cyclic peroxides by the enzymes—lipoxygenase and cyclooxygenase, respectively—which participate in the form of hydroxy fatty acids to form leukotrienes. However, unlike AA, EPA and DHA cannot be oxidized by cycloxygenase and hence their presence reduces the synthesis of leukotrienes. In general, availability of sufficient quantities of EPA and DHA helps to alleviate the problems caused by AA-generated eicosanoids. Prostaglandins synthesized from ω-3 acids also have additional therapeutic uses including control of blood pressure and relieving bronchial asthma.2 DHA is found in close association with membrane proteins of the 7-transmembrane structure (7-Tm), G-protein-coupled receptors (i.e., serotonin receptors, acetylcholine receptors, and rhodopsin), and certain ion channels such as calcium channel regulation in cardiac cells. Thus elevation of intracellular calcium level stimulates

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18:2n-6

∆6 Desaturase

18:3n-6

Elongase

20:3n-6

Eicosanoid series generation COX LOX 20:3n-6 1 3 20:4n-6 2 4 20:5n-3 3 5

∆5 Desaturase

HETE HPETE LT

20:4n-6

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

Dietary fat

PL

PLA2

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

COX 20:5n-3 ∆ Desaturase 5

18:3n-3

∆6 Desaturase

18:4n-3

Elongase

PG TBX

20:4n-3

FIGURE 5.1 Biosynthesis of eicosanoids from the EFAs. PL, phospholipids; PLA2, phospholipase, A2; COX, cyclooxygenae; LOX, lipoxygenase; PG, prostaglandins; TBX, thromboxanes; HETE, hydroxyl-eicosatetraenoic acids; HPETE, hydroperoxyeicosatetraenoic acids; LT, leukotrienes. Dietary lipids provide EFAs and preformed substrates for the COX/ LOX pathways. Dietary fatty acids such as 20:3 (n-6), 20:4 (n-6), and 20:5 (n-3) are direct precursors, whereas 18:2 (n-6) and 18:3 (n-3) must be elongated and desaturated prior to their conversions to eicosanoids. (From Berge, J.-P. and Barnathan, G., Adv. Biochem Engn/ Biotechnol., 96, 49, 2005. With permission from Springer Science and Business Media.)

a calcium-dependent phospholipase, which in turn cleaves off free DHA from the DHA-rich membranes. The resulting high concentration of free DHA then closes off the calcium channel, reducing the calcium inflow, and the internal calcium levels drop. In this respect, DHA inhibits ischemia-induced cardiac arrhythmias. It is therefore believed that DHA may play a greater role in biological control mechanisms.91,92 Another mechanism is suggested to be operating through control of gene expression. EFAs, particularly ω-3 PUFAs have a general effect on expression of genes in lipogenic tissues. High levels of PUFAs result in a decrease of activity of liver enzymes involved in lipogenesis. PUFA regulation of gene expression in nonlipogenic tissues has also been reported. Some studies have suggested that the action of ω-3 PUFAs may be found, both at the level of transcription and during the stabilization of the mRNA. A better understanding of the involvement of PUFAs in gene regulation, especially with respect to lipogenesis and the production of intracellular antioxidants would further throw light on the functional role of the fatty acids in diets.94

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165

INDICATION

Fish oils may primarily be indicated to lower triglyceride levels in those with hypertriglyceridemia. Another important indication may be to prevent death of people who have suffered myocardial infarctions. Fish oils are used to decrease clotting tendencies of the blood. They may also be indicated to lower blood pressure, prevent restenosis following coronary angioplasty, alleviate some of the symptoms of rheumatoid arthritis and ulcerative colitis, and help prevent relapse of Crohn’s disease. They may help stabilize mood in bipolar disorder and may have beneficial effects in IgA nephropathy. There is evidence that they may help to prevent rejection in renal transplant patients, and they are used to feed various patient categories. Table 5.6 presents examples of improvement of clinical conditions with supplementation of diet with EFAs. Fish oil supplements should be used by children, pregnant women, and nursing mothers only if recommended and monitored by a physician. Because of the possible antithrombotic effect of fish oil supplements, hemophilics, and those taking warfarin (Coumadin) should exercise caution in their use.95

5.4.4

SOME CURRENT INTAKE LEVELS OF OMEGA-3 PUFA

The dietary intake of total ω-3 PUFA has been estimated in some countries. Currently, in the United Kingdom, the average intake of long-chain ω-3 PUFA is less than 0.2 g per day, which is less than half the current conservative recommendation of a minimum of 0.45 g per day.96

TABLE 5.6 Examples of Improvement of Clinical Conditions with Supplementation of Diet with EFAs Class

Clinical Conditions

Neurological

Zelweger’s Syndrome Batten’s disease Schizophrenia Alzheimer’s disease Bipolar depression Dyslexia ADD/ADHD

Cardiovascular

Elevated triglycerides Low HDL Hypertension Asthma Rheumatoid arthritis Diabetic neuropathy Premenstrual syndrome

Other

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Supplementation Results Improved visual and physical outcome; remyelination in brain Arrested natural course of disease Significant improvement in schizophrenic symptoms Improvement in mental function Significant improvement of vision Improvement in night vision Improvement of attention Reduction of triglycerides/elevation of HDL Elevation of HDL Reduction of blood pressure Improved forced expiratory volume Reduced morning stiffness Improvement of nerve conduction velocity Reduction of global symptoms of PMS

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The estimates, however, do not distinguish between plant and marine ω-3 PUFA. In the United States, the dietary intake of total ω-3 PUFA has been reported to be 1600 mg/day, of which 100–200 mg/day is EPA and DHA.63 The major food sources contributing to ω-3 PUFA intakes include seafoods (71%), meats (20%), and eggs (6%). For individuals or populations who consume no seafood or muscle or organ meats, such as vegetarians, ALA is the only potential source of ω-3 PUFA. However, the extent of conversion of ALA to long-chain ω-3 PUFA is modest, and it is uncertain as to whether substantial amounts of conversion occur to support normal growth and development.94

5.4.5

RECOMMENDED CONSUMPTION LEVELS OF OMEGA-3 PUFA

The dietary recommendations for long-chain ω-3 PUFA are still a matter of debate. Recommendations vary depending on desired disease prevention. Daily ranges for EPA and DHA begin from 180 (for healthy adults) to 500 mg (to decrease the prevalence of heart disease) to 1000 mg (to decrease the prevalence of mental illness).97 Various levels of consumption of fatty fish or fish oil have been recommended by different agencies to derive health benefits. The World Health Organization recommends consumption of 1–2 servings of fish, containing 200–500 mg of EPA and DHA, per week.63 The American Heart Association recommends healthy individuals to consume 2–3 oz serving of fatty fish per week and that the persons diagnosed with cardiovascular diseases consume 1 g each of the fatty acid per day.98 Australian Nutrient Reference Values (previously known as Recommended Dietary Intakes), have set adequate intake at 190 mg/day. A recent study reported an average daily intake of 189 mg of marine ω-3 PUFA (20:5, 22:5, and 22:6 at 56, 26, and 106 mg/ day, respectively) in the diets of Australians.4 Some of the fish species as sources of ω-3 fatty acids include sardine, mackerel, anchovy, cod, Atlantic herring, salmon, bluefin tuna, red snapper, swordfish, and silver hake. Aquacultured fish generally are not good sources of ω-3 fatty acids. However, it is possible to rear fish such as salmon to have significant levels of ω-3 PUFAs by selective feeding techniques. Aquaculture of mussel has been optimized for maximum content of DHA and EPA.99 In case, regular consumption of fatty fish is not possible, its oil may be administered in capsules or other foods may be enriched with the oil. An amount of 0.3–0.5 g per day of EPA and DHA and 0.8–1.1 g per day of LA has been recommended to control CHDs.81,100 A daily serving of 8 oz (227 g) of the fish provides five times the effective adequate intake of C20:5 and C22:6 (0.14 g/day and 0.13 g/day, respectively) for pregnant or lactating women. The U.S. Food and Drug Administration (U.S. FDA) and U.S. Environment Protection Agency stand behind their 2004 recommendation regarding public advisory note for seafood consumption by pregnant and nursing mothers.101 Britain’s Food Standards Agency recommends that people eat at least two portions of fish in a week, one of which should be oily. But prospective mothers are advised to avoid eating shark, marlin, and swordfish.95 Table 5.7 gives daily intake of long-chain ω-3 fatty acids recommended by various bodies. The U.S. FDA in 2004 has allowed a qualified health claim about EPA and DHA as dietary supplements to reduce risks of CHD. The revised, 2005 dietary guidelines recognize that “limited evidence suggests an association between consumption of

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TABLE 5.7 Recommended Daily Intake of Long-Chain ω-3 Fatty Acids Organization National Health and Medical Research Council British Nutrition Foundation, Task Force U.K. Department of Health European Academy of Nutritional Science American Heart Association American Heart Association American Heart Association National Institutes of Health, United States

Recommended Daily Dose (mg) of EPA and DHA

Target Consumers

190

General population

500–1000 200

People at risk of CVD General population

200 1000 Oily fish twice Oily fish, >3 g, daily

General population People at risk of CVD General population To reduce triglyceride level

300

Pregnant and lactating women

Source: Reproduced from Garg, M. L. et al., J. Food Sci. 71, R66, 2006. With permission from Blackwell Publishing.

fatty acids in fish and reduced risks of mortality from cardiovascular disease for the general population” (S. Zhu, personal communication, 2007). The United Kingdom has become the first country outside the United States to grant ω-3 fish oil health claim that manufacturers throughout Europe have begun applying to their products. The claim, issued by the Joint Health Claims Initiative, made up of consumer protection groups, food law enforcers, and members of the food industry, states: “Eating 3 g weekly, or 0.45 g daily, long-chain ω-3 PUFA, as part of a healthy lifestyle, helps maintain heart health.”5 A number of other countries including Australia, Canada, and Japan and North Atlantic Treaty Organization have made formal population-based dietary recommendations for ω-3 fatty acid consumption. It has been recognized recently, for major health outcomes among adults, based on both the strength of the evidence and the potential magnitude of effects, the benefits of fish intake exceed the potential risks (such as contamination with heavy metals). For women of childbearing age, benefits of modest fish intake, excepting a few selected species, would outweigh the risks.102 Given that most Western populations fall well short of recommended oily fish servings per week, food formulators are working hard to develop other ways of increasing fish oil intake, and wide range of products including eggs, breads, crackers, milks, cheeses, and juices are expected to carry the claim in the near future. Means of delivering recommended levels of long-chain ω-3 PUFA in human diets have been discussed recently. These include food such as emulsions, sirups, and gels.5 Ideally, the ratio of n-6 PUFA to ω-3 PUFA should not exceed 4 to 1 in order to optimize the bioavailability, metabolism, and incorporation into membrane phospholipids.2,4 In 1997, the U.S. FDA has given the oil from menhaden fish (Brevoortia spp.), generally recognized as safe (GRAS) status. The oil is a rich source of ω-3 PUFA. EPA and DHA make up ~20% by weight of the oil, when it is not used in combination

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with other added oils that are significant sources of the ω-3 fatty acids. This allows use of the oil as a human food ingredient. The U.S. FDA has later affirmed the GRAS status of menhaden oil (21 CFR 184.1472) provided that the combined intake of EPA and DHA from consumption of menhaden oil does not exceed 3 g per person per day (g/p/d) (U.S. FDA, November 24, 2006, GRN #000200). The nutrition claims allowed to be used in U.S. labels are as follows: when at least 32 mg of combined EPA and DHA per serving (e.g., in the case of black cod and tuna), the following may be used “an excellent source of ω-3 EPA and DHA,” or “high in ω-3 EPA and DHA,” or “rich in ω-3 EPA and DHA.”103 The U.S. FDA has also allowed the following health claim on unsaturated fats, namely, “Replacing saturated fat with similar amounts of unsaturated fats may reduce the risk of heart disease. To achieve this benefit, total daily calories should not increase.”104

5.5 OMEGA-3 PUFA-RICH OILS FROM MARINE FISH Recognition of the presence of EPA and DHA in marine fatty fish species in significant amounts and their health benefits promoted isolation of ω-3 PUFA from these resources at commercial scale. Major fish species used in the production of fish oil include anchovies, capelin, Atlantic cod, Atlantic herring, Atlantic mackerel, Atlantic menhaden, salmonids, and sardines, and also Antarctic krill. In such fishes, the oil content varies and can reach up to 21% as in herring and 18% as in sardines. Several industries specialize in production and purification through cold pressing, further concentration, chilling and use other technologies.95,105–107 Some commercial fish oils and their contents of EPA and DHA have already been pointed out (see Table 5.3).

5.5.1 EXTRACTION Generally, oil is extracted from the processing wastes, particularly liver, which is rich in oil content. Methods of isolation of PUFA from these sources include processes such as molecular and fractional distillation, solvent and supercritical extraction. Enzymatic methods involve treatment of the raw material with proteases for release of the bound oil, for further separation and purification. A simple, inexpensive such as thin layer chromatography could be employed to enrich ω-3 PUFA from marine lipids (e.g., menhaden oil, dogfish liver oil, sea scallop lipids, herring lipids, whale oil, and catfish oil) and other sources. The method also allows detection of polyunsaturates down to less than 1% and to determine oxidative stability of ω-3 PUFA in commercial products.108 Oil from herring (Clupea harengus) was produced from frozen herring fish available on the market, using chemical and physical analysis and refining the oil by degumming, neutralizing, drying, and decolorizing. An amount of 41% of oil was produced from dried fish during an extraction period of 5 h. The extracted herring fish oil contained EPA and DHA.109 The potential recovery of lipids, particularly ω-3 PUFA, from processing wastes of cod (Gadus morhua), saithe (Pollachius virens), haddock (Melanogrammus aeglefinus) and tusk (Brosme brosme) caught in the Barents Sea or North Sea off

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Norway are good sources of fish oil. Lipid composition in a single species varies with season and also depends on the tissues being analyzed, with liver and viscera containing 43–69% and 2–9% of total lipids, respectively. Fish livers are the ideal source of lipids, and PUFA in particular. Although variations between species can be observed. A significantly higher yield of ω-3 PUFA can be obtained from haddock liver followed by cod or saithe livers. The liver lipids comprised ∼90% triacyl glycerols whilst higher levels of phospholipids were present in other tissues, reaching levels of up to 60% in flesh and gonad tissues.110 Discards from the global tuna canning industry are estimated at 450,000 t annually. This could be a rich source of unsaturated oils. Tuna with a total lipid content of 22.4% contains 26.4 g ω-3 PUFA per 100 g of extracted oil, with 19.7 and 3.9 g of DHA and EPA in 100 g of the oil. Oil of the n-6 type constitutes 3.8 g%, whereas monounsaturated fatty acids comprises of 23.3%.109 Mackerel processing waste comprising skins, viscera, and muscle tissue was evaluated for concentrating ω-3 PUFA by urea complexation. Fish oil was extracted using either chloroform/methanol or hexane/isopropanol. The mean oil yields varied between 9 and 38% for viscera, muscle, and skin, respectively. The mean iodine value was 134, which increased to 296 after urea complexation. Mackerel skin was most desirable because of its high oil content.110 Treatment of salmon head with commercial proteases (Alcalase, Neutrase, and Flavourzyme) for 2 h yielded 17% oil, which was close to that obtained by the chemical extraction method (20%). Lipolysis of the oil was carried out with a commercial lipase, Novozym SP398, to obtain a mixture of free fatty acids and acylglycerols (24 h, 45% hydrolysis). The mixture was filtered on a hydrophobic membrane to discriminate between high melting saturated fatty acids and low melting acylglycerols. The content of ω-3 PUFA increased from 41.6% in the crude oil to 46.5% in permeates. The DHA content increased from 9.9 to 11.6%, whereas the EPA changed from 3.6 to 5.6%.111 A new approach for extracting lipids from several cod by-products has been reported. This employs a prehydrolysis step with large spectrum proteases in order to disrupt tissues and cell membranes. Extraction of yields for total lipids, phospholipids, EPA, and DHA have been compared to those obtained by organic extraction.112 Shark is another major source of oil. The liver of shark is 22–30% of its body weight and the oil content in the liver may be as high as 90% or above. Oils from liver of black shark (Galeus glucas), the Mako shark (Isurus glucas) and hammerhead shark (Sphyma spp.) are rich in vitamin A and D.113 Liver oils of some deep-sea sharks mainly Centrophorus spp. that are found at a depth of 300–3000 m in the Pacific, North Atlantic, and Indian Ocean contain about 85–90% unsaponifiable matter, mainly squalene. The shovenose dogfish liver oil contains 60% hydrocarbons, consisting mainly squalene and pristane, and 25% diacyl glyceryl ether. The recovery of oil from shark consists of natural decomposition of liver, acid ensilage in presence of formic acid, alkali digestion, and steam rendering (90°C for 30 min). Traces of antioxidant, such as TBHQ protect the unsaturated fatty acids against oxidation. The liver oil recovered is degummed, bleached, and deodorized. Shark liver oil is rich in vitamins A and D and squalene.114 A method for extraction of oil from shark liver, developed in India, involved removal of the liver, chopping it into pieces, heating to 80°C by dipping in 2% caustic soda solution for 30–40 min

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TABLE 5.8 Shark Liver Oil Production, Composition and Uses Process of Extraction Natural decomposition (incubation at 30°C) Acid ensilage Alkali digestion Steam rendering (90°C for 30 min) Composition Hydrocarbons, squalene, and pristine—65% Diacetyl glyceryl ether, selacyl, chimyl and butyl alcohols—26% Triglycerides—6% Uses Squalene has several therapeutic activities such as antitumor activity Squalene and squalane are used as moisturizers in cosmetics Diacayl glyceryl ethers have bacteriostatic action Used as surfactants Wound healing Source: Adapted from Buranudeen, F. and Rajadurai, P. N. R., Infofish Marketing Dig., 1, 42, 1986.

in an open kettle. This resulted in separation of the oil on the surface, which was skimmed off. Water was removed by treating with anhydrous sodium sulfate, and subjected to vacuum distillation. The low boiling fraction that boils at 125°C was first collected. The major fraction was distilled at 240°C and the residue was discarded. The squalene that was extracted as the major fraction was filled in bottles in inert atmosphere for storage.115 Table 5.8 shows shark liver oil, production, composition, and uses. Antarctic krill (Euphausia superba) is one of the most abundant and successful animal species on Earth (see Chapter 2). Krill oil contains ω-3 fatty acids, phospholipids and is also a rich source of natural pigments, vitamins, and other components. The only disadvantage of using krill as an oil source is that the lipid in the krill is about 3% of the body weight, and hence a large amount of krill needs to be processed for oil recovery.116 Krill oil, however, has therapeutic effects. It has benefits in premenstrual syndrome (PMS) and hyperlipidemia. A double-blind, controlled, randomized trial on 70 women showed that krill oil improves all emotional PMS symptoms, including feeling overwhelmed, anxiety, stress, irritability, and depression Oil derived from krill is sold as dietary supplement for reported benefits such as health of the heart.18,117 Many psychiatric disorders, particularly schizophrenia and major depressive disorder (MDD), have shown positive results when supplementation has been used as an adjunct to standard pharmacotherapy.118–120 Figure 5.2 shows commercial process for production of fish oil. Figure 5.3 gives world fish oil production.

5.5.2 PROPERTIES OF FISH OILS Commercial fish oils are characterized by a large number of fatty acids from 12 to 26 carbon atoms having 0–6 double bonds. The bulk of the fatty acid chains are

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Refined and winterized fish oils production process Whole fish

Neutralized oil

Cooking

Purification with activated carbon and filtration

Pressure Neutralized and purified oil Fish meal Liquid fraction Bleaching with clays and filtration Centrifugation Water+ impurities Crude fish oil

Neutralization by saponification of the free fatty acids and centrifugation

Neutralized and purified bleached oil

Winterization : cooling, oil crystallization and filtration

Fish stearin Acid fish oils Neutralized, purified, bleached and winterized oil

FIGURE 5.2 Process for fish oil production. Winterization is filtration under cold temperature. (Courtesy of Winterization Europe, www.winterisation.fr.)

contributed by saturated (15–25%), monoenes (35–60%) and polyenes (25–40%). In contrast with other fats and oils, marine fish oils contain large amounts of EPA and DHA, in the range of 14–19% and 5–8%, respectively. Some fish oils such as those from tuna and salmon contain more DHA than EPA (Table 5.3). Saturated fatty acids

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Marine Products for Healthcare Japan Scandinavia

Thousand tonnes

USA Russ. Fed.

Rest

1505

1600 1400

Chile Peru

1336 1237

1200

1381

1384

1422

1194

1142

1120

1042

1000

865

800 600 400 200 0 1991

1992

1993

1994

1995

1996 1997 1998 1999 2000 Year World fish body oil production  major producers (IFFO)

Japan Scandinavia 956

1000

956

USA Chile

Peru Rest 988

969 900

857

900

786

Thousand tonnes

800 700

2001

669

665

600

540

525

500 400 300 200 100 0 1991

1992

1993

1994

1995

1996 Year

1997

1998

1999

2000

2001

World marine oils and fats exports by major exporters (IFFO)

FIGURE 5.3 World fish oil production, according to The International Fishmeal and Fish Oil Organization. (Reprinted from Berge, J.-P. and Barnathan, G., Adv. Biochem Engn/ Biotechnol., 96, 49, 2005. With permission from Springer Science and Business Media.)

contain 12–24 carbon atoms, which are mostly linear together with some branched chains. In fish tissues, the composition of lipids is determined by diet composition and lipid metabolism. Commercial cod liver oil is a complex mixture of more than 50 different fatty acids, forming triacyl glycerols, of which there is usually 8–9%

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each of EPA and DHA, and 22–24% of all ω-3 PUFAs. To reach the proposed health requirement, a much greater consumption of fish or the use of fish oil in foods is required. Therefore, depending on the oil, there may be a need to enrich PUFA content in the oil.121 Cod liver oil has been shown to help slowdown the destruction of joint cartilage in patients with osteoarthritis. A trial showed that 86% of preoperative patients with arthritis who took cod liver oil capsules (1000 mg) twice a day had significantly reduced levels of the enzyme that cause cartilage damage, compared to 26% of those given a placebo oil capsule.122 The oxidative stability of long-chain ω-3 PUFA in fish (and also algae oils) varies widely according to their fatty acid composition, the physical and colloidal states of the lipids, the contents of tocopherols and other antioxidants, and the presence of metals.123 Free ω-3 PUFA autooxidized more readily than isolated ester types.46 It is important to note that highly oxidized oil could be toxic. Oxidized sardine oil (and also lard) when administered in mice caused tumor. Therefore these oils, particularly reused oils, are harmful to health because of the increased risk of liver carcinogenesis related to the formation of 8-hydroxy-deoxyguanosine.124 In view of high sensitivity of ω-3 PUFA to oxidation, it is important to stabilize them against oxidation in foods and associated flavor changes. Oxidation can be minimized by refining and deodorizing the oil, and packaging in an inert gas such as nitrogen. Natural and synthetic antioxidants such as tocopherols and ascorbyl palmitate are commonly used to help prevent oil oxidation. Plant extracts such as rosemary leaves and extra virgin olive oil (EVO) can be used as sources of natural phenolic antioxidants. Methanol extracts from oregano and rosemary could retard oxidation of long-chain PUFAs, EPA and DHA in menhaden oil. The antioxidant activity of the rosemary extract was greater than that of oregano extract, but was sensitive to heat. The rosemary extract also demonstrated higher DPPH (2,2’-diphenyl-1-picrylhydrazyl) free radical-scavenging capability, which was approximately 3 times higher than oregano extract.125 Apart from their sensitivity to oxidation, there are certain other problems associated with fish oils, which include unappealing taste and odor. These problems could be solved by techniques such as deodorization and micro encapsulation (see Chapter 13).

5.5.3 OTHER SOURCES OF OMEGA-3 PUFA In view of increasing human population, overfishing and depletion of marine resources such as cod, marine captured fish can be considered as a sustainable source of ω-3 PUFA in the long run. Since microalgae form the primary food web, there have been attempts to isolate ω-3 PUFA from microalgae and also from other microorganisms including certain species of thraustochytrids. Algae can produce 50–100 times more oil per acre than oil crops such as soybean, corn, cotton, hemp, mustard seed, sesame, safflower, rice, sunflower, peanuts, rapeseed, olives, coconut, and palmoil. The production of oil from algae per hectare has been suggested to be about 55,000 kg (see Chapter 11). Several research groups are working toward the development of ω-PUFA-rich terrestrial plants using gene manipulation (GM) approaches. Until such gene manipulations are successfully implemented and their

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safety established, it is imperative that extraction from the current sources are optimized. Nevertheless, some chemical methods have been identified for preparation of ω-3 PUFA. Chemical modification of fats and oils is a novel strategy for production of PUFA. Chemically modified fats and oils are designated generally as structured lipids (SLs), which are triacylglycerols (TAGs) that have been modified to change the fatty acid composition or their positional distribution in glycerol backbone by chemically, or enzymatically catalyzed reactions and genetic engineering. SLs provide an effective means to produce tailor-made lipids with desired physical characteristics, chemical properties, and nutritional benefits. The production, commercialization outlook, medical, and food applications of SLs have been reviewed.126,127 For production of structured lipids, interesterification based on exchanging the fatty acid components of a triglyceride or a mixture of triglycerides with either free fatty acids (acidolysis), or fatty acids of other triglycerides or monoesters (transesterification) have been used successfully. Traditional chemical modification processes for fats and oils generally involve quite drastic conditions. The ω-3 PUFAs are highly labile and may be destroyed by oxidation or cis–trans isomerization during processes that involve extreme pHs and high temperatures.108 However, enzymatic methods, being mild, do not cause undesired chemical changes in the product. Enzymatic fat modification is performed using lipases (triacylglycerol acylhydrolase); mostly derived from microbial sources. Lipases catalyze the hydrolysis of triglycerides, diglycerides, and monoglycerides in the presence of excess of water, but under water-limiting conditions ester synthesis (reverse reaction, termed as acidolysis) can be achieved. Lipase-catalyzed acidolysis has been used to incorporate EPA and DHA into vegetable and fish oils to improve their nutritional properties.126–128

5.6 SQUALENE Liver oils of some deep-sea sharks mainly Centrophorus spp. found at a depth of 300–3000 m in the Pacific, North Atlantic, and Indian Ocean contain about 85–90% unsaponifiable matter, which is essentially the hydrocarbon, squalene (C30H50, 2,6,10,15,19,23-hexamethyl, 2,6,10,14,18,22-tetracosahexaene). Squalene is colorless but becomes pale yellow and thick when remain untouched due to oxidation. The physical (refractive index, density, and viscosity) and chemical characteristics (squalene content, unsaponifiable matter, and iodine value) showed significant correlation with squalene content among various shark species off the Portuguese coast.129 Table 5.9 gives comparative properties of shark liver squalene with commercial squalene.

5.6.1

FUNCTIONALITY OF SQUALENE

Squalene has remarkable antioxidant activity. In addition, this compound has been reported to possess antilipidemic and membrane-stabilizing properties. When adult males in phase I trial were given 860 mg squalene daily for 20 weeks showed that oral squalene is safe and tolerable.122,130,131 The protective effect of squalene on membrane function and mineral status was examined in isopreterenol-induced myocardial infarction in male albino rats. Pretreatment with squalene at 2% level

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TABLE 5.9 Comparative Properties of Shark Liver Squalene with Commercial Squalene Parameters

Shark Squalene

Commercial Squalenea

0.853 1.492 30 342 240–243°C

0.853 1.493 30 344 240–245°C

Specific gravity Refractive index Saponification value Iodine value Boiling point a

Sigma Chemical Co., United States.

Source: From Thankappan, T. K., in Seafood Safety, Society of Fisheries Technologists, India; Surendran, P. K. et al., eds., Cochin, India, 2003, p. 173. With permission.

along with feed significantly reduced the isopreterenol-induced rise in the levels of plasma diagnostic marker enzymes, Alanine amino transferase (ALT), Aspartate amino transferase (AST), lactate dehydrogenase (LDH), and creatine phosphokinase (CPK). The treatment also counteracted lipid peroxidation in plasma and heart tissue and maintained the level of reduced glutathione in the heart tissue at near normalcy. Supplementation of squalene also exerted membrane-stabilizing action against induced myocardial infarction by maintaining the activities of membranebound ATPases in heart tissues and the mineral status (Na, K, and Ca) in plasma and heart tissues. It was suggested that the cardio protective effect of squalene might be ascribable to its antioxidant action and membrane-stabilizing properties.131 The hypercholesterolemia activities of pure squalene and shark liver oil have been examined in hamsters. Diet was supplemented with squalene at the levels of 0.05, 0.1, and 0.5%, whereas shark liver oil was added in the diet at 0.05% by weight. When compared with the control group, serum total cholesterol was elevated by 32% in the 0.05% squalene group, 23% in the 0.10%, 35% in the 0.5% squalene groups; and by 19% in the 0.05% shark liver oil group, respectively. Similar trend was observed for serum triglycerides. Squalene and shark liver oil feeding also elevated hepatic cholesterol by 97–133% in the four tested groups compared with the control hamsters. In addition, supplementation of squalene and shark liver oil in diets caused significant accumulation of squalene in the liver and adipose tissue. The results suggested that squalene and shark liver oil are hypercholesterolemic at least in hamsters. Caution has to be taken when these are routinely consumed as health supplements.132 Table 5.10 indicates the influence of squalene on level of plasma diagnostic marker enzymes and lipid peroxidation of normal and isoproterenolinduced myocardial infarction in rats.133

5.7

COMMERCIAL ASPECTS

The world production of fat and oil in 2003–2004 was approximately 128.5 mt with an average consumption of about 20 kg per capita, whereas the average annual world

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TABLE 5.10 Influence of Squalene on Level of Plasma Diagnostic Marker Enzymes and Lipid Peroxidation of Normal and Isoproterenol-Induced Myocardial Infarction in Rats

Markers ALT AST LDH CPK Peroxide level in plasma Peroxide level in heart tissue

Control Rat

Myocardial Infarction-Induced Rat Fed with Control Diet

Myocardial InfarctionInduced Rat Fed with 2% Squalene-Supplemented Diet

102 84 198 138 1.8 0.9

3862 343 397 375 4.21 1.99

156 126 231 168 1.95 1.03

Note: Myocardial infarction was induced by intraperitonial injection of isoproterenol (11 mg dissolved in physiological saline per 100 g body weight) for 2 days after 45 days of feeding with standard diet. Results are mean values for 6 animals. Values expressed for ALT, AST, and LDH, micromole pyruvate per hour per liter; CPK, micromole creatine liberated per hour per liter. Peroxides, n mole per milliliter. Source: Adapted from Farvin, K. H. S. et al., J. Clin. Biochem. Nutr., 37, 55, 2005. With permission.

production of fish oil during the decade between 1991 and 2001 was 1.25 mt.134 At present, about a third of total marine catch of fish is being used for production of oil and meal. Chile, Peru, Scandinavia, United States, and Japan are the main suppliers of fish oil. The major uses of fish oil is as a component of aquaculture feed followed by its use as nutraceutical and dietary supplement. Generally, 1–2% (w/w) fish oil is incorporated in the feed. The International Fishmeal and Fish Oil Organization (IFFO) is the nongovernmental trade organization representing more than 200 member companies producing fish oil and meal producers worldwide. The IFFO estimates that the use of fish oil for aquaculture operations would rise to 1.1 mt in 2010 and the industry may find difficult to meet the future demand for rising aquaculture.2 The predicted uses of fish oil in fish feed are given in Table 5.11.135 Different forms of fish oil are commercially available. Pharmaceutical grade ω-3 fish oils containing either EPA or DHA (up to 20% each) or mixtures of EPA and DHA (ranging 12–18% each) are commercially available (www.winterisation.fr). The natural EPA and DHA are chemically TAGs and are found in fish and phytoplankton. These are the forms most commonly available at present as dietary supplements. More concentrated forms of EPA and DHA are their ethyl esters and free (i.e., unesterified) EPA and DHA. The pharmacokinetics of these forms is similar. A recent trend survey has shown that 4 in every 10 U.S. adults want more ω-3 fatty acids in their food.136 In the United Kingdom, fish oils account for ~29% (U.S.$ 140 m) of total annual market for nutraceuticals.137 The use of fish oil is bound to increase as a dietary supplement including its application in infant formulae (see Chapter 14). An increased requirement for EFAs and mucopolysaccharides is important for joint

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TABLE 5.11 Predicted Use of Fish Oil in Fish Feed Fish Oil in Feed (%)

Carp Catfish Milk fish Shrimp Eel Marine fish (flounder, turbot, halibut, sole, cod, and hake) Marine fish (bass, bream, yellow tail, grouper, jacks, and mullet) Salmon Total

Fish Oil Production (’000 t)

2000

2010

2000

2010

— 1 2 2 5 10

0.5 — 2 3 8 12

— 5 6 30 17 23

103 — 11 73 23 156

20

15

226

335

25

20

307

379

716

1209

Source: Courtesy of International Fishmeal and Fish Oil Organization, www.iffo.com.

health and functioning. Maintaining joint health is a matter of paying attention to diet and exercise. Sufficient intake of minerals and vitamin C through diet is important for synthesis of healthy connective tissue. Glucosamine sulfate, ω-3 fish oil, vitamin C, and magnesium are some of the important nutrients to consider supplementing when joint repair is required. Some of the commercial products to address joint health are “Omega 3 1000 mg” and “Jointex” (with glucosamine). The shark liver oil from deepwater sharks is considered important in many industries.129 Crude shark liver oil is processed in Japan for the preparation of cosmetic products. The content of squalene has a major influence on the physical and chemical properties, which determines the commercial value of the oil. Squalene is used as a health food, and its hydrogenated product, squalane is used as a lubricant, bacteriocide, and pharmaceutical base. Squalane is an excellent moisturizer and carrier of fragrances. It also protects against radiation and aid in the healing of wounds and inhibits tumor growth. Squalene is a skin-rejuvenating agent. It is mild on human skin and imparts softness without oily appearance. In cosmetic products it is incorporated to enhance skin permeability for the passage of active ingredients. Squalene is also useful in preventing formation of nitrosamines. It can also function as a stimulant to enhance production of hormones in the body.114 In conclusion, clinical and nutritional researchers continue to demonstrate the benefits of ω-3 long-chain PUFAs. Marine oils, which are rich in EPA and DHA, have several biological functions in the body system, as discussed in this chapter. EPA and DHA lower elevated triglyceride levels, afford protection to the cardiovascular system, have anti-inflammatory and immune-modulating properties and are beneficial for the musculoskeletal and gastrointestinal systems. Fish-derived PUFA, particularly DHA are beneficial in infant nutrition and development. Furthermore, beneficial effects of these fatty acids on obesity, kidney and liver function, diabetes,

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and asthma have been indicated. A number of international bodies have realized the importance of these fatty acids in health protection. With rapid rise in understanding the mechanisms of their functions, there will be significant demand of PUFAs for healthcare in the future.138 There is a need to develop functional foods that can provide the recommended levels of long-chain ω-3 PUFA to offer various health benefits. Fortification of foods with PUFA will go a long way in this regard, which will be discussed in Chapter 13.

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20. Ohr, L. M., Revisiting omega-3s, Food Technol., March 2007, 57, www.ift.org. 21. Nichols, P. D., Long-chain ω-3 oils in wild and farmed Australian seafood, IfIS Publishing, http://www.foodsciencecentral.com/fsc/ixid14325, April 10, 2006. 22. Passi, S. et al., Fatty acid composition and antioxidant levels in muscle tissue of different mediterranean marine species of fish and shellfish, J. Agric. Food Chem., 50, 7314, 2002. 23. Fredalina, B. D. et al., Fatty acid compositions in local sea cucumber, Gen. Pharmacol., 33, 337, 1999. 24. Bower, C. K., Malemute, C. L., and Oliveira, A. C. M., Preservation methods for retaining n-3 polyunsaturated fatty acids in Alaska Coho salmon products, J. Aquatic Food Prod. Technol., 16, 45, 2007. 25. Pryor, W. A., The role of free radical reactions in biological systems, in Free Radicals in Biology, Pryor, W. A. Ed., Academic Press, New York, 1976, p. 1. 26. Ozilgen, S. and Ozilgen, M., Kinetic model of lipid oxidation in foods, J. Food Sci., 55, 498, 1990. 27. Clemans, R. and Pressman, P., Oxidative stress: defense or disease, Food Technol., November 2007, 17. www.ift.org. 28. Choe, E. and Min, D. B., Chemistry and reactions of reactive oxygen species in foods, Crit. Rev. Food Sci. Nutr., 46, 1, 2006. 29. St. Angelo, A. J., Lipid oxidation in foods, Crit. Rev. Food Sci. Nutr., 36, 437, 1996. 30. Falch, E. et al., Correlation between 1H NMR and traditional methods for determining lipid oxidation of ethyl docosahexaenoate. J. Am. Oil Chem. Soc., 81, 1105, 2004. 31. Dimitrios, B., Sources of natural phenolic antioxidants, Trends Food Sci. Technol., 17, 505, 2006. 32. Gordon, M. H., The development of oxidative rancidity in foods, in Antioxidants in Food—Practical Applications, Woodhead Publishing, England, 2001, p. 22. 33. U.S. FDA, http://www.ars.usda.gov/nutrientdata/ORAC, accessed November 2007. 34. Gutteridge, J. M., Free radicals in disease processes: a compilation of cause and consequence, Free Radical Res. Commun., 19, 141, 1993. 35. Sen, C. K. and Packer, L., Antioxidant and redox regulation of gene transcription, FASEB J., 10, 709, 1996. 36. Ohr, L. M., Nutraceuticals, Food Technol., June 2006, 187, www.ift.org. 37. Jiala, I. and Fuller, C. J., Oxidativity modified LDL and atheroscelorosis: an evolving plausible scenario, Crit. Rev. Food Sci. Nutr., 36, 341, 1986. 38. Willcox, J. K., Ash, S. L., and Catignani, G. L., Antioxidants and prevention of chronic disease, Crit. Rev. Food Sci. Nutr., 44, 275, 2004. 39. IFIS, Antioxidants and 21st century nutrition, http://www.foodsciencecentral.com/fsc/ ixid13735, June 5, 2005. 40. Roberts, D. C., Nutrition and metabolism: antioxidants, the food matrix and methodological considerations, Curr. Opin. Lipidol., 16, 111, 2005. 41. Howes, R., The free radical fantasy: a panoply of paradoxes, Ann. NY Acad. Sci., 1067, 22, 2006. 42. Anonymous, The antioxidant myth: a medical fairy tale—health, New Scientist, 2563, August 5, 2006, 40. 43. Gupta, H., President, can support, Times of India, February 7, 2007. 44. Bjelakovic, G. et al., Mortality in randomized trials of antioxidant supplements for primary and secondary prevention, J. Am. Med. Assoc., 297, 842, 2007. 45. Lichtenstein, A. H., Robert, M., and Russell, R. M., Essential nutrients: food or supplements? Where should the emphasis be? J. Am. Med. Assoc., 294, 351, 2005. 46. Spinelli, J., Factors relating to fish flavor, odor and quality changes, in Marine and Freshwater Products Handbook, Martin, R. E., et al., Eds., Technomic, PA, 2000, p. 819.

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47. Hultin, H. O., Oxidation of lipids in seafoods, in Seafoods: Chemistry, Processing Technology and Quality, Shahidi, F. and Botta, J. R., Eds., Blackie Academic and Professional, Glasgow, 1994, p. 47. 48. Aubourg, S.-P., Fluorescence study of the pro-oxidant effect of free fatty acids on marine lipids, J. Sci. Food Agri., 81, 385, 2001. 49. Cho, S. Y. et al., Oxidative stability of lipids from squid (Todarodes pacificus) tissues, Fish. Sci. (Japan), 67, 738, 2001. 50. Passi, S. et al., Fatty acid pattern, oxidation product development, and antioxidant loss in muscle tissue of rainbow trout and Dicentrarchus labrax during growth, J. Agric. Food Chem., 52, 2587, 2004. 51. Albert, C. et al., Protection from sudden cardiac death in women may be linked with a diet rich in alpha-linolenic acid, News Release, November 8, American Heart Association, Dallas, TX, 2004. 52. Zhao, G. et al., Dietary linolenic acid reduces inflammatory and lipid cardiovascular risk factors to hyper-cholesterolemic men and women, J. Nutr., 134, 2991, 2004. 53. Mandez, J. A. et al., Forfication, Annal. Oncol., 16, 359, 2005. 54. Ip, C. et al., Conjugatead linoleic acid-enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats, J. Nutr., 129, 2135, 1999. 55. Minihane, A. M. and Lovegrove, J. A., Health benefits of polyunsaturated fatty acids (PUFAs), in Improving the Fat Content of Foods, Williams, C., Ed., Woodhead Publishing, United Kingdom, 2006, p. 560. 56. IFIS, Dietary fat composition and cardiovascular diseases, http://www. foodsciencecentral.com/fsc/ixid/14369. 57. Broek, A. and Gerritsen, J., Ω-3 fish oil: improved powder opens up new markets, Nutraceuticals-Now, 26, 2004. 58. Lands, W. E. M., Fish, ω-3 and Human Health, 2nd ed., CRC Press, Boca Raton, FL, 2005, p. 235. 59. Bonna, K. H. et al., Effect of EPA and DHA on blood pressure and hypertension. A population based intervention trial from Tromoso study, New Eng. J. Med., 322, 795, 1999. 60. Prisant, M., Nutritional treatment of blood pressure: non-pharmacologic therapy, in Handbook of Food and Nutrition, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 961. 61. Ueshima, H., Food omega-3 fatty acid intake of individuals (total, linolenic acid, longchain) and their blood pressure: INTERMAP study, Hypertension, 50, 313, 2007. 62. Lands, W. E. M., Fish, ω-3 and Human Health, 2nd ed., CRC Press, Boca Raton, FL, 2005, p. 2. 63. Kromhout, D., Bosschieter, E. B., and de Lezenne Coulander, C., The inverse relation between fish consumption and 20-year mortality from coronary heart disease, New Eng. J. Med., 312, 1205, 1985. 64. Carvalho, A. P. and Xavier-Malcata, F., Preparation of fatty acid methyl esters for gas-chromatographic analysis of marine lipids: insight studies, J. Agri. Food Chem., 53, 5049, 2005. 65. Kris-Etherton, P. M., Harris, H., and Appel, L. J., Fish consumption, fish oil, ω-3 fatty acids and cardiovascular disease, Circulation, 106, 2747, 2002. 66. Stone, N. J., Fish consumption, fish oil, lipids and coronary heart disease, Circulation, 94, 2337, 1996. 67. Holguin, F. et al., Cardiac autonomic changes associated with fish oil vs. soy oil supplementation in the elderly, Chest, 127, 1102, 2005. 68. Wohl, D. A. et al., Randomized study of the safety and efficacy of fish oil (ω-3 fatty acid) supplementation with dietary and exercise counseling for the treatment of antiretroviral therapy-associated hypertriglyceridemia, Clin. Infect. Dis., 41, 1505, 2005.

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69. Albert, C. M. et al., Fish consumption and risk of sudden cardiac death, J. Am. Med. Assoc., 279, 23, 1998. 70. Adler, A. J. and Holub, B. J., Effect of garlic and fish-oil supplementation on serum lipid and lipoprotein concentrations in hypercholesterolemic men, Am. J. Clin. Nutr., 65, 445, 1997. 71. Erkkila, A. T. et al., Fish intake is associated with a reduced progression of coronary artery atherosclerosis in postmenopausal women with coronary heart disease, Am. J. Clin. Nutr., 80, 626, 2004. 72. Hu, F. B. et al., Fish and ω-3 fatty acid intake and risk of coronary heart disease in women, J. Am. Med. Assoc., 287, 1815, 2002. 73. Engler, M. M. et al., Effect of DHA on lipoprotein subclasses in hyperlipidemic children (the early study), Am. J. Cardiol., 95, 869, 2005. 74. Anonymous, Oily fish prevents prostrate cancer, Infofish Int., 4, 71, 2006. 75. Wolk, A. et al., Long-term fatty fish consumption and renal cell carcinoma incidence in women, J. Am. Med. Assoc., 296, 1371, 2006. 76. Reddy, B., AGFD 9, Novel approaches for colon cancer prevention by types of dietary fat, pterostilbene and other food components, AGFD 9, National Meeting, American Chemical Society, Chicago, March 25–29, 2007. 77. Catherine, H. et al., Effects of ω-3 fatty acids on cancer risk: a systematic review, J. Am. Med. Assoc., 295, 403, 2006. 78. Crawford, M. A., The role of essential fatty acids in neural development, implications for perinatal nutrition, Am. J. Clin. Nutr., 57, 703S, 1993. 79. Inbarren, C. et al., Dietary intake of ω-3, n-6 fatty acids and fish: relationship with hostility in young adults—the CARDIA study, Eur. J. Clin. Nutr., 58, 24, 2004. 80. Adams, P. B. et al., Arachidonic acid to eicosapentaenoic acid ratio in blood correlates positively with clinical symptoms of depression, Lipids, 31, 157S, 1996. 81. Fleith, M. and Clandenin, M. T., Dietary PUFA for pre-term and term infants: review of clinical studies, Crit. Rev. Food Sci. Nutr., 45, 205, 2005. 82. Elvevoll, E. O. and James, D. G., Potential benefits of fish for maternal, fetal and neonatal nutrition, a review of the literature, Food Nutri. Agric., 27, 28, 2000. 83. Allihaud, G. and Guesnet, P., Fatty acid composition of fats is an early determinant of childhood obesity: a short review and an opinion, Obesity Rev., 5, 21, 2004. 84. Lands, W. E. M., ω-3 Fatty acids in lung disease, Am. J. Clin. Nutr., 71(Suppl.), 393S, 1989. 85. Biswas, A. K. and Sharma, B. D., Dietary ω-3 fatty acids and human health, Proc. Food Ind. (India), 6, 17, 2003. 86. Mickleborough, T. D. et al., Protective effect of fish oil supplementation on exercise induced bronchoconstriction in asthma, Chest, 129, 39, 2006. 87. Anonymous, Study finds fish oil supplements reduce anger, Infofish Int., 3, 71, 2006. 88. West, S. G. et al., An increase in dietary omega-3 fatty acids decreases a marker of bone resorption in humans, Nutr. J., 6, 2, 2007. 89. Sundstrom, B. et al., Supplementation of omega-3 fatty acids in patients with ankylosing spondylitis, J. Rheumatol., 35, 359, 2006. 90. Trsondsen, T. et al., Consumption of seafood—the influence of overweight and health beliefs, Food Qual. Pref., 15, 361, 2004. 91. Swansson, M. A. and Evenson, P., Nutritional additives, in Food Additives, Brown, A. L., Ed., Marcel Dekker, NY, 2002, p. 225. 92. Anonymous, IFIS Publishing, http://www.foodsciencecentral.com/fsc/ixid14325, accessed November 2007. 93. Narayan, B., Miyashita, K., and Hosaka, K., Food Rev. Int., 22, 291, 2006. 94. Anonymous, Dietary fat composition and cardiovascular disease, Functional Foods, IFIS Publishing, http://www.foodsciencecentral.com/fsc/ixid14369, June 8, 2006.

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95. Ruxton, C. H. et al., The health benefits of omega-3 polyunsaturated fatty acids: a review of the evidence, J. Hum. Nutr. Diet, 17, 449, 2004. 96. Krauss, R. M. et al., AHA dietary guidelines: revision 2000: a statement for healthcare professionals from the Nutrition Committee of the American Heart Association, Circulation, 102, 2284, 2000. 97. Khan, M. A. et al., Effects of environmental characteristics of aquaculture sites on the quality of cultivated Newfoundland blue mussels (Mytilus edulis), J. Agric. Food Chem., 54(6), 2236–2241, 2006. 98. Dallongeville, J. et al., Fish consumption is associated with lower heart rates, Circulation, 108(7), 820, 2003. 99. Ohr, L. M., Nutraceuticals and functional foods—functional fatty acids, Food Technol., 59(4), 63, 2005. 100. Santerre, C. R., Farmed salmon: caught in a number game, Food Technol., 58(2), 108, 2004. 101. Mozaffarian, D. and Rimm, E. B., Fish intake, contaminants, and human health evaluating the risks and the benefits, J. Am. Med. Assoc., 296, 1885, 2006. 102. U.S. FDA, Health claim notification for the substitution of saturated fat in the diet with unsaturated fatty acids and reduced risk of heart disease, IFT Newsletter, Institute of Food Technologists, Washington, DC, http://www.fda.gov/ohrms/dockets/dockets/ dockets.htm, May 30, 2007. 103. Wildman, R. E., ω-3 fatty acid concentrates—a review of production technologies, in Handbook of Nutraceuticals and Functional Foods, CRC Press, Boca Raton, FL, 2001, pp. 157–174, 542. 104. Haraldsson, G. G. and Hjaltason, B., PUFA production from marine sources for use in food, in Modifying Lipids for Use in Foods, Gunstone, D., Ed., Woodhead Publishing, 2006, p. 500. 105. Nichols, P. D., Bakes, M. J., and Elliott, N. G., Oils rich in docosahexaenoic acid in livers of sharks from temperate Australian waters, Mar. Freshwater Res., 49, 763, 1998. 106. Alasalvar, C. and Taylor, T., Ω-3 fatty acid concentrates—a review of production technologies, in Seafoods—Quality, Technology and Nutraceutical Applications, Springer-Verlag, Heidelberg, Germany, 2002, p. 157. 107. Adeniyi, O. D., Herring fish (Clupea harengus) oil production and evaluation for industrial uses, J. Disp. Sci. Technol., 27, 537, 2006. 108. Falch, E., Rustad, T., and Aursand, M., By-products from gadiform species as raw material for production of marine lipids as ingredients in food or feed, Proc. Biochem., 41, 666, 2006. 109. Sultanbawa, Y. and Aksnes, A., Tuna process waste—an unexploited resource, Infofish Int., 3, 37, 2006. 110. Zuta, C. P. et al., Concentrating PUFA from mackerel processing waste, J. Am. Oil. Chem. Soc., 80, 933, 2003. 111. Linda, M. et al., Proteolytic extraction of salmon oil and PUFA concentration by lipases, Mar. Biotechnol., 7, 70, 2005. 112. Dumay, J. et al., How enzymes may be helpful for upgrading fish byproducts: enhancement of fat extraction, J. Aquat. Food Prod. Technol., 13, 69, 2004. 113. Kresuzer, A. and Ahmed, A., Shark Utilization and Marketing, FAO of the United Nations, Rome, Italy, 1971. 114. Buranudeen, F. and Rajadurai, P. N. R., Shark liver oil, Infofish Mark. Dig., 1, 42, 1986. 115. Thankappan, T. K., Isolation of squalene from shark liver oil, in Seafood Safety, Society of Fisheries Technologists (India), Surendran, P. K. et al., Eds., Cochin, India, 2003, p. 173. 116. Bimbo, A. P., Current and future sources of raw materials for the long-chain omega-3 fatty acid market, Lipid Technol., 19, 176, 2007.

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117. Bunea, et al., Evaluation of the effects of Neptune krill oil on the clinical course of hyperlipidemia, Altern. Med. Rev., 9, 420, 2004. 118. Lee, S. et al., Invited review: current clinical applications of omega-3 and n-6 fatty acids, Nutr. Clin. Pract., 21, 321, 2006. 119. Diaz-Lopez, M. and Garcia-Carreno, F. L., Application of fish and shellfish enzymes in food and feed products, in Seafood Enzymes, Haard, N. F. and Simpson, B. K., Eds., Marcel Dekker, NY, 2000, p. 571. 120. Frankel, E. N. et al., Oxidative stability of fish and algae oils Containing long-chain polyunsaturated fatty acids in bulk and in oil-in-water emulsions, J. Agri. Food Chem., 50, 2094, 2002. 121. Sadakane, K. et al., Oxidized lard and dietary oils increase liver carcinogenesis and formation of 8-hydroxy-deoxyguanosine, Paper No. AGFD 170, American Chemical Society 232nd Symposium, San Francisco, September 10–14, 2006. 122. Chan, P. et al., Effectiveness and safety of low dose pravastatin and squalene, alone and in combination in elderly patients with hypercholesterolemia, J. Clin. Pharmacol., 36, 422, 1996. 123. Bhale, S. D. et al., Oregano and rosemary extracts inhibit oxidation of long-chain n-3 fatty acids in menhaden oil, J. Food Sci., 72, C504, 2007. 124. Williams, C., ed., Improving the Fat Content of Foods, Woodhead Publishing, New York, 2006, p. 560. 125. Osborn, H. T. and Akoh, C. C., Structured lipids—novel fats with medical, nutraceutical, and food applications, Compr. Rev. Food Sci. Safety, 1, 110, 2002. 126. Langholz, P. et al., Application of a specificity Mucor miehei lipase to concentrate Docosahexaenoic acid (DHA), J. Am. Oil Chem. Soc., 66, 1120, 1989. 127. Tocher, D. R., Webster, A., and Sargent, J. R., Utilization of porcine pancreatic phospholipase A2 for the preparation of marine fish oil enriched in omega-3 poly-unsaturated fatty acids, Biotechnol. Appl. Biochem., 8, 675, 1986. 128. Wanasundara, U. N. and Shahidi, F., Lipase assisted concentration of ω-3 poly-unsaturated fatty acids in acylglycerols form from marine oils, J. Am. Oil Chem. Soc., 75, 945, 1997. 129. Batista, L. and Nunes, M. L., Characterisation of shark liver oils, Fisheries Res., 14, 329, 1992. 130. Ko, T. F., Weng, T. M., and Chiou, R. Y., Squalene content and antioxidant activity of Terminalia catappa leaves and seeds, J. Agri. Food Chem., 50, 5343, 2002. 131. Farvin, K. H. S. et al., Effect of squalene on tissue defense system in isoproterenolinduced myocardical infarction in rats, Pharmacol. Res., 50, 231, 2004. 132. Zhang, Z. et al., Effect of squalene and shark liver oil on serum cholesterol levels in hamsters, Int. J. Food Sci. Nutr., 53, 411, 2002. 133. Farvin, K. H. S. et al., Protective effect of squalene against isopreterenol-induced myocardinal infarction in rats, J. Clin. Biochem. Nutr., 37, 55, 2005. 134. www.cyberlipid.org, accessed December 2007. 135. International Fishmeal and Fish Oil Organization, www.iffo.com. 136. U.S. HealthFocus Survey, January 2007 (IFT Newsletter, Institute of Food Technologists, Washington, DC, May 2, 2007. 137. www.healtheries.co.nz, accessed December 2007. 138. Ruxton, C. H. et al., The health benefits of omega-3 polyunsaturated fatty acids: a review of the evidence, J. Hum. Nutr. Diet., 17, 449, 2004.

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Processing 6 Seafood Wastes: Chitin, Chitosan, and Other Compounds 6.1

INTRODUCTION

Processing of seafood generates tremendous amounts of waste. Recent estimates reveal discards from the world fisheries exceed 20 mt equivalent to 25% of the total production of marine capture fisheries. Trawl fisheries for shrimp and demersal finfish account for over 50% of the total estimated discards, representing approximately 22% of total landings.1 Processing discards consisting of heads, exoskeleton, cephalothorax, and carapace of crab, shrimp, and lobster constitute a gigantic proportion of marine wastes. The rising global shrimp aquaculture adds significantly to this waste and has become an environmental problem. Utilization of seafood processing waste can be a major solution of environmental pollution besides providing economic benefits. Since these wastes are rich sources of industrially important ingredients, there is an immense potential for the marine industry to bioprocess them to generate products that are of practical applications.2–6 This chapter discusses the utilization of marine processing waste, particularly chitin and chitosan from shellfish wastes and their uses in food and health products.

6.2 MAJOR COMPOUNDS FROM SHELLFISH PROCESSING WASTES Table 6.1 shows the global availability of crustacean waste.7 Proximate analyses of the waste have shown that three major compounds can be isolated from the shell waste, namely, chitin, protein, and carotenoids. On an average, shrimp head and carapace contain about 17% chitin, 41% protein, and 148 μg carotene per gram on a wet weight basis.6,8–10 Approximately 35–45% by weight of shrimp raw material is discarded as waste when processed into headless shell-on products. The industrial waste from Xiphopenaeus kroyeri shrimp, one of the most important commercial species found along the Brazilian coastline has an average of 38% protein and 20% chitin as well as 5 mg astaxanthin per 100 g of waste. In another detailed analysis, cooked shrimp waste was found to contain 94.6% protein and 4.2% fat on a dry basis. Analysis of the protein indicated 17 amino acids (Asp, Glu, Ser, Thr, Arg, Gly, Ala, Pro, Val, Met, Leu, Ile, Phe, Cys, Lys, His, and Tyr; proline being the most abundant) and 7 sugars (ribose, xylose, fructose, mannose, glucose, glucosamine, and galactosamine; ribose being the most abundant).9 Table 6.2 indicates the chitin contents of selected crustacean and molluskan organisms.7,11 185

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TABLE 6.1 Global Availability of Crustacean Waste Resource

Total Landing

Waste Availablea

Dry Wasteb

Chitin Contentc

1,292,476d 398,219 943,826 150,000e

516,990 99,531 482,744 60,000

129,475 24,882 144,823 15,000

32,311 1,244 28,964 3,750

Shrimp Squid Crabs Krill a b c d e

Assuming 40% is waste. Multiplication factor 0.25, for calculating dry waste from wet waste. Multiplication factor 0.25, for calculating chitin from dry waste. Total landing during 2002. Average annual global krill landing.

Source: Adapted from Subasinghe, S., Infofish Int., 3, 58, 1999.

TABLE 6.2 Chitin Contents of Selected Crustacean and Molluskan Organisms Species Shrimp head Shrimp shell Commercial shrimp waste Cancer crab Carcinus crab Blue crab Crangan (shrimp) Alaska shrimp Nephrops (lobster) Clam shell Oyster shell Squid, skeleton pen Krill, deproteinized shell a b c

Chitin Contents (%) 11a 27a 12–18a 72.1b 64.2c 14.0a 69.0 28.0 69.8 6.1 3.6 41.0 40.0

Wet body weight. Organic weight of the cuticle. Dry body weight.

Source: Adapted from Pan, B. S., Advances in Fishery Technology and Biotechnology for Increased Profitability, Technomic, Lancester, PA, 1990, p. 437; Subasinghe, S., Infofish Int., 3, 58, 1999.

6.3

CHITIN

Chitin represents the second most abundant natural biopolymer, which is present in the exoskeletons of crustaceans and also in the cell walls of fungi, insects, and marine diatom. Chitin was first discovered in mushrooms by Henri Braconnot in France in

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1811. In the 1830s, it was isolated from insects and named chitin. The name is derived from the Greek word chiton meaning a coat of nail. Chitin is an aminopolysaccharide that is a major bioresource, with an estimated annual potential availability of 100 billion t. Marine organisms, which include lobster, crab, cuttlefish, shrimp, and prawn, are richer in chitin compared to terrestrial organisms such as insects and fungi. In crustaceans, it forms the outer protection coatings in a covalently bound network with proteins and dihydroxy phenylalanine together with some metals and carotenoids. In its deacetylated derivative, chitosan is a polycationic long-chain biopolymer with a natural affinity for the normally negatively charged biological membranes. According to the SciFinder Scholar 2001 database (as of June 21, 2005), the interests in these products are shown by approximately 22,600 publications related to chitin and chitosan during the past 100 years.12 A Japanese journal (J. Chitin & Chitosan Res.) is devoted to research developments in these products.

6.3.1

ISOLATION OF CHITIN

Global chitin production has been estimated at 150,000 mt.7 Potential industrial sources of chitin are wastes from shrimp, crabs, squid, cuttlefish, krill, squid, clams, and oysters. Since chitin in the raw material is contaminated with other compounds such as proteins and minerals, harsh treatments are necessary for its isolation. In a dehydrated and deproteinized shell waste, minerals and chitin are present in nearly equal amounts. Generally, the isolation process consists of three steps, namely, demineralization, deproteinization, and bleaching.10 Usually, demineralization is carried out by the treatment of the shell waste with dilute hydrochloric acid for a period of 1–3 h. However, harder extractants such as 90% formic acid, 22% HCl, 6 N HCl, or 37% HCl have also been employed.13,14 Because of the significant amount of minerals, particularly calcium, the demineralization step gives rise to an appreciable amount of calcium chloride (which may be used in the pulp and paper manufacturing industry).6 For deproteinization, the demineralized material is treated with 4–5 M sodium or potassium hydroxide at 65–100°C at a concentration of 10% for periods ranging from 0.5 to 6 h. After the treatment, the chitin is washed and dried. Sun drying of the chitin can result in bleaching of the carotenoids giving an almost colorless preparation. If desired, pigments could be removed by solvent extraction employing acetone or ethanol.12,13 Figure 6.1 shows the process for extraction of chitin and protein. The conditions for chitin extraction are determined by the required application of the prepared product. For example, removal of salt by drastic demineralization with acid can result in some deacetylation of chitin. Harsh alkali conditions for protein removal can cause depolymerization and deacetylation of chitin. Furthermore, the alkali-extracted protein could be of limited use since undesirable reactions between amino acids occur in a strongly alkaline medium, besides racemization of the amino acids.10 Nevertheless, the protein recovered in the form of hydrolyzates may have some use as a flavoring agent and as a supplement to fish-based foods or feed for aquaculture.10 Because of the problems with the traditional extraction process, proteolytic enzymes have been employed for extraction. Treatment of demineralized material with pepsin, papain, trypsin, pronase, or alkalase can avoid the cleavage of glycosidic linkages in chitin, which may happen during alkali treatment. The mild enzymatic treatment removes about 90% of the protein and carotenoids from shrimp

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Mixing Alcalase 4 M NaOH Hydrolysis (pH-stat controlled) 55°C pH 8.5 4 M HCl Enzyme inactivation 55°C pH 4

Centrifugation 15 min 4000 ⫻ g

Water

Charcoal Decolorization 55°C pH 4

Washing Ethanol Liquid Washing Acetone

Filtration 4 M NaOH

Solid

Washing

Neutralization pH 7

Drying

Lyophilization

Crude chitin

Protein hydrolyzate

FIGURE 6.1 Extraction of chitin and protein. (Reprinted from Synowiecki, J. and Al-Khateeb, N. A., Crit. Rev. Food Sci. Nutr., 43, 145, 2003. With permission from Taylor & Francis Ltd. (www.informaworld.com).

processing waste. Deproteinization of shrimp shells by alcalase led to the isolation of chitin containing about 4.5% of protein impurities and recovery of protein hydrolyzate.10 Despite these developments, little work has focused on a single process to the recovery of all the three main components, namely, chitin, protein, and carotenoid pigments from crustacean waste.10 Furthermore, most processes described in the literature were poorly designed for the commercial recovery of the main components of shrimp waste because either very dilute material was obtained or they

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were not compatible with astaxanthin extraction.15 Recovery of chitin, protein, and astaxanthin from industrial waste of shrimp (X. kroyeri) using enzymatic treatment with alcalase and pancreatin was reported recently.3 The enzymatic hydrolysis of the shell waste using alcalase allowed 65% protein recovery in the form of hydrolyzates, in addition to providing suitable conditions for the recovery of astaxanthin and chitin.3 The recovery, however, was dependent on the degree of hydrolysis (DH) of protein by the enzymes. An increase in DH from 6 to 12% resulted in the recovery of 26–28% protein. Alcalase was more efficient than pancreatin, increasing the recovery of protein from 57.5 to 64.6% and of astaxanthin from 4.7 to 5.7 mg/100 g of dry waste, at a DH of 12%. Figure 6.2 shows the flow diagram of the process for protein, chitin, and astaxanthin recovery from shrimp waste by enzyme hydrolysis.3 The enzymatic hydrolyzate, besides its use as a protein supplement, can also be a

Wastewater 1-1 pH adjustment Alcalase or pancreatin Hydrolysis (pH 8.5) Enzyme inactivation (90°C/5 min) Centrifugation (16,000 ⫻ g /4°C/15 min) Supernatant Insoluble fraction Freeze-drying Extraction with ether:acetone:water (15:75:10)

Filtration

Evaporation Rotaevaporator/40°C

Extraction with soy oil (heating and stirring)

Hydrolyzed protein

Centrifugation (12,000 ⫻ g/4°C/10 min)

Insoluble fraction Demineralization (2.5% HCl/2 h/room temperature)

Supernatant

Filtration Pigmented lipids

Aqueous fraction Pigmented oil

Neutralization (pH 7) Drying (60°C/16 h)

Chitin

FIGURE 6.2 Flow diagram of the process for protein, chitin, and astaxanthin recovery from shrimp waste by enzyme hydrolysis. (Reprinted from de Holanda, H. D. and Netto, F. M., J. Food Sci., 71, C298, 2006. With permission from Institute of Food Technologists, United States.)

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source of biologically active peptides, with considerable potential in pharmacology or as growth-stimulating agents in animal feeds (see Chapter 4). The abundantly available Antarctic krill (Euphausia superba) is a good source of chitin. However, there is a problem of the production of colorless chitin from this shellfish due to the pigments from the eyes of krill that impart an intense pink color to the product, which cannot be removed by acetone extraction. A modified process for the extraction of chitin from krill involves demineralization before deproteinization when the eyes form a sticky substance that retains the pigment. The mass is attached to the walls of the reactor and can be separated with ease from the suspension of solid shell residue. Treatment of these residues with acetone followed by deproteinization yields colorless chitin.16 Isolation of chitin from crustacean shell waste demands necessary care to avoid environmental problems. The effluent containing alkali, protein, and protein degradation products must be treated before being discarded.

6.3.2

STRUCTURE

Chitin is a cationic polysaccharide formed by units of N-acetyl-d-glucosamine (GlcNAc), joined by β bonds (1–4). The structure of chitin is β(1–4)-N-acetyl-dglucosamine, which is β(1–4)-N-acetyl-2-amino-2-deoxy-d-glucose. It may also be regarded as a derivative of cellulose, in which, the C-2 hydroxyl group is substituted by an acetyl amino group. Chitin occurs in three polymorphic forms, α, β, and γ, which differ in their arrangement of the molecular chains. Its most common form is α-chitin, where the unit cell is made up of two N,N-diacetyl-chitobiose units in an antiparallel arrangement. The final structure has extensive intermolecular hydrogen bonding, with the exclusion of water, leading to great stability. A less common form is β-chitin, in which the unit cell is made up of one N,N-diacetyl-chitobiose unit, giving a final structure of parallel poly-N-acetylglucosamine chains, extensively hydrogen bonded in sheets, but without the intersheet hydrogen bonds of α-chitin. Chemical differences in α and β chains have been reported to affect the viscosity of chitosan prepared from the chitin.17 Figure 6.3a shows the structure of chitin.

6.3.3

PROPERTIES

Chitin is a very light, white or yellowish, powdery/flaky product. It is insoluble in water, in almost all common organic solvents, and in acidic and basic aqueous solutions. Chitin swells in cold alkali when some deacetylation takes place. After a simple treatment in caustic soda, chitin may be solubilized with carbon disulfide and reprecipitated as a filament or film in the form of viscose rayon. The natural chitin shows one or more of the following features, namely, a variable degree of crystallinity, varying amounts of deacetylation, and cross-linking with other molecules. Chain lengths of chitin and degree of acetylation differ according to isolation conditions and sources. However, chitin found in shrimp, crab, and lobster shell is generally thought to be good for all practical purposes. Chemical derivatives of chitin having variations in chemical properties can be prepared. These include carboxymethyl chitin, hydroxyethyl chitin, ethyl chitin, chitin sulfate, glycol chitin, and glucosylated

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H

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NH

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H

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CH3

(b)

FIGURE 6.3 Structures of (a) chitin and (b) chitosan.

chitin. Chitinase enzyme, which degrades chitin, has been found in several organisms, and is discussed in Section 6.5.1.7,18,19

6.3.4

APPLICATIONS

Chitin is surely an undisputed biomolecule of great potential.13 Chitin and its deacetylated product chitosan as well as their derivatives have diverse applications in agriculture, biotechnology, chemistry, cosmetics, dentistry, food product development, medicines, textiles, and veterinary sciences. The development of technologies based on the utilization of chitin derivatives is dictated by their polyelectrolyte and surface properties; the presence of reactive functional groups; gel-forming ability; high adsorption capacity; biodegradability; and bacteriostatic, fungistatic, and antitumor activities.8,10 The applications include nonwoven artificial skin for burns, biodegradable sutures, and as the vibrating panel in audio speakers.18 Chitin has found applications in agriculture and biotechnology. In agriculture, chitin contributes to the retention of nutrients in the soil. It could control plant pathogens and pathogenic nematodes, and provoke the development of host plant resistance against these pathogens. Under natural conditions, fungi, arthropods, and nematodes are the major contributors of chitin in the soil. When chitin decomposes it produces ammonia, which takes part in the nitrogen cycle. Chitin has been used in animal feed for its growth-promoting effect in broiler chickens. Increase in average live weight, dressed weight, and decrease in wastage during dressing in broiler chicken fed on a diet containing 0.5% chitin have been reported.20 The feeds containing chitin and also glucosamine could also be used in aquaculture.19 In biotechnology, chitin (and also chitosan) has been found to be useful as a matrix for immobilization of various enzymes for processing products ranging from wine, sugar and synthesis of organic

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compounds, wastewater treatment, and construction of sophisticated biosensors for both in situ measurements of environmental pollutants and metabolite control in artificial organs.21 The use of chitin as an immobilization matrix for seal gastric protease (SGP) has been reported. The average degree of immobilization was 20% and the immobilized SGP exhibited optimum performance at a pH of 2. The half-life of the immobilized enzymes with continuous operation for hemoglobin hydrolysis at 22°C was 90 h. The immobilized SGP could be used for clotting of milk in cheesemaking.22,23 Chitin and chitosan are of great value as chromatographic supports in ion exchange, gel, chelation, thin layer, and high-pressure liquid chromatography.13

6.4

CHITOSAN

Chitosan is a collective name representing a family of de-N-acetylated chitins deacetylated to different degrees. It was discovered by C. Rouget in 1859. However, interests in the uses of chitosan were noted only in the 1930s and early 1940s. The 1970s evinced renewed interest due to a need for better utilization of shellfish shells.7,17 Chitosan is produced by deacetylation of chitin using 30–60% (w/v) sodium or potassium hydroxide at 80–140°C. The characteristics of the product in terms of molecular weight and the extent of deacetylation depend on the treatment conditions. Increasing temperature enhances deacetylation, but also results in fragmentation of the chitosan, which affects its final applications. After the deacetylation process, the chitosan is dried to produce flakes of chitosan. The prepared chitosan is purified by dissolving it in dilute acetic acid, reprecipitation with alkali, followed by washing and drying. In India, a few entrepreneurs are producing chitin and chitosan on a commercial scale under the technical guidance of the Central Institute of Fisheries Technology, Cochin. Although currently the chitin is deacetylated by harsh alkali treatment that can cause adverse impact on the quality of the product, there is potential for deacetylation under mild conditions using the enzyme chitin deacetylases from Mucor rouxii, M. mechei, and Aspergillus niger. Such enzyme-treated chitosan has better functional properties. However, fungal chitin deacetylases are able to perform heterogeneous deacetylation of solid substrate to a limited extent only. Kinetic data show that only 5–10% of the N-acetylglucosamine residues are deacetylated rapidly. The extent of deacetylation can be enhanced by improving the particle size of chitin. Chitin was dissolved in specific solvents followed by fast precipitation, which gave crystallized chitin the small particle size. The crystallized chitin, after pretreatment with 18% formic acid, was amenable to 90% deacetylation by the fungal deacetylase. The formic acid treatment reduced the molecular weight of the polymer chain from 2 × 105 Da in chitin to 1.2 × 104 Da in the chitosan product.24 A method for the determination of the degree of acetylation has been reported.25

6.4.1

STRUCTURE

Chitosan is β-(1–4)-linked-d-glucosamine, that is, poly-(β-(1–4)-linked-2-amino-2deoxy-d-glucose), which is a deacetylated chitin (DAC). It has a free amino group and two free hydroxyl groups for each glucose ring. The difference between chitosan and chitin is only in the functional group situated at C-2 of the monomeric unit.18 Figure 6.3b shows the structure of chitosan.

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193

PROPERTIES OF CHITOSAN

Chitosan is a cationic polyelectrolyte, white solid. It is insoluble in pure water, but unlike chitin is soluble in weakly acidic aqueous media. A minimum deacetylation of 70% is required for chitosan that is acceptable for various purposes. The molecular weight of natural chitosan is higher than 1 mDa and that of commercially available chitosan is about 0.1–1.2 mDa.26 Decolorization treatment of chitosan during preparation can result in significant loss of quality in terms of viscosity, probably due to changes in molecular weight as a result of the treatment. It may be mentioned that decolorization may not be essential for an acceptable chitosan in terms of various parameters. Chitosan derivatives in the form of acetate, ascorbate, lactate, malate, and others are water-soluble. The pKa value for the positively charged ammonium group is approximately 6.2. When the pH is raised to approximately 6.5, chitosan precipitates in the form of a gel. Because of its cationic nature, chitosan is incompatible in solution with most anionic water-soluble gums such as alginates, pectate, sulfated carrageenan, as well as carboxymethyl cellulose. In contrast, the acid solution of chitosan is compatible with nonionic water-soluble gums such as starch, dextrins, glucose, polyhydric alcohols, oils, fats, and nonionic emulsifiers. Chitosan can form films that are tough, flexible, and transparent. The film can be extruded from an acidic solution of chitosan in a 70°C coagulating bath containing caustic soda and sulfonic acid esters of high-molecular-weight alcohols. Chitosan is also biodegradable by a specific enzyme chitosanase. There can also be some digestion by nonspecific activity of some digestive enzymes such as amylases and lipases. By enzymatic treatment, soluble chitosan can also be obtained in the oligosaccharide form.7,19,27 Table 6.3 shows the physicochemical properties of chitosans prepared from crawfish shell.28 Chitosan gives a highly viscous solution in 1% acetic acid forming an ionic salt of chitosan acetate. The viscosity increases with the increase in molecular weight of chitosan. The viscosity is also proportional to the concentration of chitosan. Viscosity of 1% chitosan in 1% acetic acid is usually taken to determine the quality of the product. Higher the viscosity, the better the product. It has been reported that based

TABLE 6.3 Physicochemical Properties of Different Chitosans Prepared from Crawfish Shell Chitosan Type DPMCA DPMA DMCA DMA

Molecular Weight (kDa)

Deacetylation (%)

Viscosity

Nitrogen

454 1462 950 1054

86.7 86.1 84.6 84.2

35 1164 259 1054

7.35 7.20 7.22 7.36

Note: DPMCA—deproteinized, demineralized, decolorized, and deacetylated; DPMA— deproteinized, demineralized, and deacetylated; DMCA—deproteinized, decolorized, and deacetylated; and DMA—demineralized and deacetylated. Source: Adapted from Natarajah, N. et al., J. Food Sci., 71, 33, 2006. With permission.

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on viscosity, deacetylation of chitin with equal amount of aqueous 40% caustic soda for 2 h at 100°C gives an ideal chitosan preparation. In terms of viscosity, squilla (Oratosquilla spp.), a by-catch of shrimp trawling in Indian Ocean, gave a better chitosan compared to that prepared from shrimp shell. A 1% solution of chitosan from squilla in 1% acetic acid had a viscosity of 340 cP against a value of 180–200 for the shrimp product. The elastic hardness of chitosan gels increase linearly with the chitosan concentration; the maximum values for elastic hardness and breaking point were 2.8 × 106 dyn/cm2 and 7.5 × 106 dyn/cm2, respectively.29,30 Chitosan in the microcrystalline form is a special multifunctional polymeric material that can have several applications. Microcrystalline chitosan is characterized by several advantages when compared to standard chitosan. These include high stability, water retention, biodegradability, higher film-forming capacity, and high-chelating properties. Microcrystallization enhanced film formation. The product is manufactured by the aggregation of glucosamine macromolecules from solution. It occurs as a gelatinous aqueous dispersion characterized by high water retention values ranging from 500 to 5000%. In the powder form, it has lower water retention values ranging from 200 to 500%. The former has a polymer content of up to 0.1–10 units and an average particle dimension of 0.1–100 μm, whereas the latter has a polymer content of 85–95 units and a size of 0.1–50 μm. The properties of microcrystalline chitosan allows the formation of film on every type of surface, including glass.31 Various saccharide derivatives of chitosan can be prepared as shown in Figure 6.4.13 These derivatives can be prepared by employing Maillard-type reactions to yield products having improved solubility at neutral or basic pH. The modified chitosans had significantly greater solubility than native chitosan. For example, the chitosan–maltose derivative remained soluble at a pH close to 10. The chitosan– fructoseconjugate displayed the highest solubility at 17.1 g/L, but the chitosan– glucosamine derivative displayed the best overall characteristics, including high chelating capacity and relatively high antibacterial activity against Escherichia coli and Staphylococcus aureus. It was suggested that these water-soluble chitosans produced using the Maillard reaction could be a promising commercial substitute for acid-soluble chitosan.32 Table 6.4 presents the specifications for chitin and chitosan.7

6.4.3

APPLICATIONS

The application potential of chitosan is multidimensional, such as in food and nutrition, biotechnology, material science, drugs and pharmaceuticals, agriculture and environmental protection, and recently in gene therapy as well. The net cation charge as well as the presence of multiple reactive functional groups in the molecule makes chitosan a sought-after biomolecule. The latter offers scope for manipulation for preparing a broad spectrum of derivatives for specific end-use applications in diversified areas. The biomedical and therapeutic significance of chitin/chitosan derivatives is a subject of significant concern to many all over the world. Its antimicrobial, antioxidant, texturizer, and barrier properties make it a popular additive for various applications in food product development and preservation. As a polycationic coagulant it is also used in water purification, fruit juices, whey, in the immobilization of enzymes, etc. Through encapsulation, it is being used as a vehicle for nutraceutical

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OH O OH

O

I

OH

O

195

O

O

O

OH

O

O

NH OH O OH

O

O

H N H

C

HO Cu OH

R

OH Removal of toxic metals

Emulsifier NH R Cosmetics R CH2

NH Organic synthetic C O intermediate CH3

O

N-acetylation O

Metal chelation

OH

O

Hydroxypropylation

OH

Protein NH immobilizer

O N

Photoactivation

OH

OH O

O

Cl F

OH

CH2COOH

O

OH O

CHO Depolymerization (low-molecular weight chitosan) O

O -acylation

Cyanoethylation Schiff’s base

O O O S OH

NH

O Adsorbant of uranium from seawater or NO2 R explosive compound

OC R O

Sulfation

O

OCH2CH2CN O OH

O O

O

NH2 Microfiltration membrane

OH O O

OH

OH

O

NH2 Chitosan

Phosphorylation or nitration

R P OH R

O

Moisture retainer NH (skin care C O products)

Deamination

OH O O

O-/N-carboxy alkylation

OH O

O N3

OH

Deoxyhalogenation

C CH3 OH O

OCH2COOH O

O

OH

O

OH

O

NH2 or NH emulsifier C O R

O

NH2 anticoagulant

Immobilization NH system CH R

FIGURE 6.4 Derivatives of chitosan. (From Tharanathan, R. N. and Kuttur, F. S., Crit. Rev. Food Sci. Nutr., 43, 61, 2003. With permission from Taylor & Francis Ltd. (www.informaworld.com).

compounds and pharmacological compounds including anticancer agents (see Chapter 14). The applicability of chitosan is dependent on the extent of polymerization and degree of deacetylation.12–14,21,26,33 Chitosan on hydrolysis with a mineral acid such as hydrochloric acid gives oligosaccharides and finally glucosamine: Oligosaccharides are also produced by controlled enzymatic hydrolysis of polysaccharides including chitosan. Certain nutritional and functional benefits of oligosaccharides have been noted. Although they are not digested by enzymes of the gastrointestinal tract, they modify the viscosity and freezing point of foods, affect emulsification and gel formation, possess bacteriostatic properties, and act as humectant and control moisture. They are less sweet (typically 0.3–0.6 times less than sucrose) and have a low calorific value. About 20 different types of nondigestible oligosaccharides are on the world market, which are either extracted from natural sources (e.g., raffinose and soybean oligosaccharides),

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TABLE 6.4 Specifications for Chitin and Chitosan Chitosan Pharmaceutical Grade

Liquid Chitosan Technical Grade Clear yellow liquid Odorless, tasteless — <0.5 <0.5 >90 50 <5.5 <10

Properties

Chitin Food Grade

Appearance

White/yellow flake

Odor and taste Moisture (%) Ash (%) Protein (%) Deacetylation (%) Viscosity (0.5% solution; cP) pH Heavy metals (arsenic and lead; ppm)

Odorless, tasteless <10 <2.5 <1.0 Nil 600

White/yellow powder or flake Odorless, tasteless <10 <0.2 <0.3 70–100 <5

7.9 <10

7.9 <10

Source: Adapted from Subasinghe, S., Infofish Int., 3, 58, 1999. With permission from Infofish.

produced by enzymatic hydrolysis of polysaccharides, or produced by enzymatic transesterification of monomers. The Ministry of Health and Welfare, Japan has licensed eight different kinds of oligosaccharides as food for specific health use (FOSHU). These compounds include fructooligosaccharides, galactooligosaccharides, raffinose, stachyose and soybean oligosaccharides, and xylooligosaccharides.34 Applications of chitosan and its oligosaccharide derivatives with particular reference to food and biomedical area are discussed in Sections 6.4.3.1 and 6.4.3.6. 6.4.3.1 Food Chitosan is a versatile additive in food technology. Its versatility is attributed to its diverse biological activities and functional properties. The functional role of chitosan as a food additive could stem from its ability to function as an antimicrobial and antioxidant agent and its ability to convert itself into a film having poor oxygen permeabilities. The mechanism of antioxidant action could derive from its function as a chelant on metal ions and also its ability to form a complex with lipids. The antioxidant property would serve a twofold purpose, namely, as a health factor for the consumer and as a means of preventing rancidity in the product with which it is mixed. A recent review has focused on the application of chitosan for the improvement of quality and shelf life of various foods from agriculture, poultry, and seafood origin.35 In addition, ability to interact with food macromolecules such as lipids, proteins, and starch enables it to function as a texturizing and emulsifying agent.36 Chitosan, depending on the type and concentration, can enhance rheological properties of texturized food such as surimi. The effect could be due to its influence

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on the endogenous transglutaminase activity.37–39 Other applications of chitosan gels in food technology include clarification of wine and vinegars and in wastewater treatment. Chitosan and also chitin can also be used as food additives in cookies, noodles, and bread for improvement of texture. Chitosan has been found to control the black discoloration of crustaceans including shrimp. The preferred concentration ranges from 0.1 to 2.0% depending on the requirements.40,41 6.4.3.2

Antimicrobial Activity

Antimicrobial activities of chitosan against several microorganisms including Aeromonas hydrophila, Bacillus cereus, B. licheniformis, B. subtilis, Clostridium perfringens, Brochothrix spp., Lactobacillus spp., Listeria monocytogenes, Pseudomonas spp., Salmonella typhimurium, S. enteritidis, Serratia liquifaciens, and others including yeast (Candida spp. and Saccharomyces spp.) and mold (Aspergillus spp., Penicillium spp., and Rhizopus spp.) in a variety of foods have been summarized.12 The microbial susceptibility depends on the type and concentration of chitosan. Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria.12,23,42,43 The mechanism of action appears to derive in part from the ionic interaction between the cationic groups of the chitosan molecules and the anionic groups of the microbial cell membrane, which can rupture the cell membrane.44,45 Chitin and chitosan are capable of forming complexes with many of the transition metals and some of those from groups 3 to 7 of the periodic table. Cupric ions appear to form one of the strongest metal complexes with chitosn in the solid state. In this way, it could interfere with microbial growth and toxin formation.45 Chitosan can also function as an antifungal agent. The action is via the formation of gas-permeable coats, interference with fungal growth, and stimulation of many defense processes including accumulation of chitinases, production of proteinase inhibitors, and stimulators of callous synthesis.46 N-carboxymethyl chitosan inhibited iron-activated auto-oxidation by chelating action.45 The agglomeration capacity of the polycation for the anionic microbes is also possible. It also inhibits the growth of molds at low levels of sodium chloride in pickled vegetables and soy sauces. These properties make chitosan an antibacterial additive for a diverse number of food items including red meat products such as sausages and fishery products such as patties and surimi.41 Chitosan can be used as a preservative for extended chilled storage of Atlantic herring and cod,27 pork sausage,42 and fish patties.47 The antimicrobial properties could be advantageously used for the development of processes in combination with other techniques such as chilling, modified atmosphere packaging, or high pressure to extend the shelf life of meat and chilled products and also active packaging.48 Combination treatments involving high pressure and chitosan as an antibacterial additive have been reported. Chitosan, because of its antibacterial action, enhanced the inhibitory effect of high pressure on microbial growth. Cod sausage was produced at chilled temperature (7°C) and high pressure (350 MPa) for 15 min to produce a chitosan (1.5%)-enriched product having an extended shelf life.47 The combination of chitosan with transglutaminase has been successfully employed for achieving optimum gel strength of surimi.37,38

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Similar to chitosan, chitosan oligosaccharides have also been shown to possess antimicrobial activity. A 5% hydrolyzate showed appreciable inhibition on pure cultures of B. cereus, Lactobacillus brevis, Leuconostoc mesenteroides, Micrococcus varians, S. aureus, Acinetobacter spp., E. coli, Pseudomonas aeruginosa, S. typhimurium, and S. liquefaciens. High-molecular-weight chitosan oligosaccharides inhibited the growth of yeasts (Candida albicans, C. versatiles, Rodotorula glutinis, S. cerevisiae, and Trichosporon pullulans), whereas the products showed only a weak inhibition of A. niger. Treatment with 3000 ppm of chitosan hydrolyzates resulted in 75% inhibition of Mucor mucedo and 95% inhibition of Rhizopus stolonifer.49 Addition of 1% chitosan to various seafood products increased their shelf life from 5 to 9 days.50,51 Water-soluble chitin derivatives, partially DAC, N-trimethyl derivative of DAC (NTM-DAC), and chitosan oligomers were examined to determine their inhibition of bacterial growth and activation of macrophages. Sulfuryl chitin, phosphoryl chitin, and some chitin derivatives prepared by nitrous acid deamination of DAC inhibited bacterial growth and increased cytotoxicity of a macrophage cell line. NTM-DAC had higher bacterial inhibition activity than carboxymethyl chitosan. However, none of these compounds stimulated macrophage respiratory burst. These results indicated that chitin and chitosans, although are useful antimicrobial compounds, are not effective as medications.26 6.4.3.3

Antioxidant Activity

Foods containing significant amounts of unsaturated fatty acids become sensitive to oxidation (see Chapter 5). Warmed-over flavor is developed in cooked poultry and uncured meat on storage, resulting in loss of freshness. Chitosan and its derivatives can reduce lipid oxidation by 50% in cod and herring muscle depending on the concentration (50–200 ppm) and the type of chitosan (having viscosity ranging between 14 and 360 cP). Similarly, addition of 1% chitosan to meat resulted in 70% reduction in lipid oxidation after 3 days of storage at 4°C. N-carboxymethyl chitosan 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 temperatures.50–52 The antioxidant activity could be attributed to the ability of chitosan to chelate metals and combine with lipids.53 This activity could be compared with that of propyl gallate in dried milk.54 A novel method to enhance the antioxidant property has been reported. Irradiation of chitosan at 25 kGy resulted in a sixfold increase in its antioxidant activity, as measured by β-carotene bleaching assay and measurement of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity. However, the antioxidant activity in terms of carotene bleaching assay increased from 24 to 681 and in terms of DPPH it increased from 9.5 to 60.8. Table 6.5 shows the influence of gamma irradiation of chitosan on its characteristics including antioxidant activity. However, there was significant loss of viscosity of the polysaccharide, limiting its use as a texturizer.55,56 Derivatives of chitosan, namely, N,O-carboxymethyl chitosan, N,O-carboxymethyl chitosan lactate, N,O-carboxymethyl chitosan acetate, and N,O-carboxymethyl chitosan pyrrolidine carboxylate inhibited lipid oxidation at a maximum of 46.7%, 69.9%, 43.4%, and 66.3%, respectively.6

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TABLE 6.5 Influence of Gamma Irradiation of Chitosan on Its Characteristics Properties Molecular weight (Da) Viscosity (cP) β-Carotene bleaching assaya DPPH scavenging activityb a b

Unirradiated Chitosan

Irradiated (25 kGy) Chitosan

1.8 × 106 1200 24.0 9.5

1.75 × 10 4 16 681.7 60.8

Expressed as antioxidant activity coefficient. Expressed as percentage DPPH scavenging.

Source: Adapted from Kanatt, S., Chander, R., and Sharma, A., Int. J. Food Sci. Technol., 39, 997, 2004. With permission from Blackwell Publishing.

6.4.3.4

Edible Films

Films prepared from chitosan have a low oxygen permeability suggesting their potential to act as good gas barriers. These films, when used on foods, including fruits and vegetables, prolong their shelf life by acting as barriers against air and moisture and also because of the antimicrobial activity of chitosan. Making use of its antibacterial and antioxidant activities and also permeability characteristics of films, chitosan can be used for coating of food products.6,12,41,47,57 These properties, however, depend on the source and extraction process employed for chitosan and solvent types used for filmmaking. Mixed films consisting of chitosan and lauric acid have lower water permeability, providing improved moisture barriers. Microcrystalline chitosan has superior film-forming properties.12,28,31 Different viscosity chitosans (14, 57, or 360 cP) could be used to extend the shelf life of refrigerated fish such as cod and herring to control rancidity.26,50,51 Coating with chitosan film in combination with gamma irradiation could enhance the refrigerated shelf life of meat products. The coating could help in the control of microbial contamination and rancidity development in the product, whereas gamma irradiation reduced the initial microbial load of the product. It was observed that no viable bacteria or fungi were detected in chitosan-coated, irradiated intermediate moisture meat products. In contrast, similar products that were not subjected to gamma radiation showed visible fungal growth within 2 weeks. The chitosan-coated products also showed lower rancidity during storage up to 4 weeks, which could be attributed to low-barrier properties of the film, suggesting potential use of chitosan coating for the preparation of safe and stable irradiated intermediate moisture meat products.56 Irradiation of meat products is also known to enhance lipid oxidation. Since gammairradiated chitosan possessed enhanced antioxidant activity, as mentioned earlier, this property was used for the preservation of lamb meat. Addition of irradiated chitosan to the meat before radiation processing suppressed rancidity development during postirradiation storage at 0–3°C. 2-thiobarbituric acid (TBA) value of irradiated meat containing irradiated chitosan was 88% lower in the leg portion and 54% in the rib portion as compared to corresponding samples devoid of chitosan. Furthermore, after

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TABLE 6.6 Applications of Chitosan in Food Products Product

Benefits

Bread Egg Fruits and vegetables

Fruit juices Mayonnaise Meat (chilled meat, sausages, patties, and intermediate moisture items) Seafood products Dairy products

Chitosan coating controls moisture loss and oxidation, helps control starch retrogradation by inhibiting microbial growth Chitosan films protect moisture and CO2 loss and internal quality, shelf life increase Controls respiration. Antifungal activity helps control of decay. The coating also helps to incorporate antioxidants, flavors, colors, antimicrobial agents, and other additives. Helps control browning Clarification of apple, lemon, grape, and orange juices. Controls acidity and inhibits yeasts Enhances oil-in-water emulsion formation and stability Antioxidant and antimicrobial activities extend shelf life of chilled meat, patties, sausages, and intermediate moisture products. Irradiated (25 kGy) chitosan has superior antioxidant activity. Improves color and therefore can reduce nitrite in sausage at least partially Antioxidant and antimicrobial properties extend chilled shelf life Antimicrobial activity extends shelf life of milk and flavored milk products

Source: Adapted from No, H. K. et al., J. Food Sci., 72, R87, 2007. With permission.

storage for a week, development of rancidity was reduced by 39% and 59% in the leg and rib portions, respectively, of the samples treated with chitosan.55 However, it should be noted that irradiated chitosan has limitation for use in other roles such as a texturizer since irradiation drastically reduced the viscosity and molecular weight of chitosan from 1200 cP and 1.8 × 106 Da to 16 cP and 1.75 × 104 Da, respectively. Therefore, where the viscosity of chitosan is important, unirradiated natural chitosan is recommended. Table 6.6 presents the benefits of using chitosan in food products and Table 6.7 presents the functional roles of chitosan in muscle food products.35 6.4.3.5

Role in Nutrition

To date chitosan would appear to be well tolerated clinically; however, its effect in prolonged diets needs to be monitored to ensure that it does not disturb the intestinal flora or interfere with the absorption of micronutrients, particularly lipid-soluble vitamins and minerals, or have any other negative effect.58 The positive-charge nature of chitosan and its oligomers govern most of their biological activities. As identified by many researchers, chitosan and its oligomers are effective in reducing low density lipoprotein (LDL) cholesterol level in liver and blood. Chitosan reduces lipid absorption by trapping neutral lipids such as cholesterol and other sterols, by means of hydrophobic interactions. Because of this inhibitory activity on fat absorption, these molecules act as fat scavengers in the digestive tract and remove fat and cholesterol via excretion. Apart from chitosan, chitosan oligomers having

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TABLE 6.7 Functional Roles of Chitosan in Muscle Food Products Product/System Cod, herring, salmon, beef, pork, irradiated lamb meat Cod, Atlantic herring Surimi from barred garfish Edible coating for muscle foods including sausages and patties Lamb meat Sausage

Functional Activity

Reference

As edible coating or dip provides protection against lipid oxidation (antioxidant activity) Chitosan film extends quality As texturizing agent, chitin and chitosan influence transaglutaminase activity and cross-linking of surimi Antibacterial action against spoilage causing bacteria Disrupts the barrier properties of the outer membrane of gram-negative bacteria Gamma-irradiated chitosan has enhanced antioxidant activity. Can be used as edible coating Addition of chitosan reduces requirement of nitrite as curing agent

26, 51, 55 27 37–39 28, 42, 43, 47

55 41

average molecular weights of 10,000 Da could significantly enhance fecal excretion of neutral steroids. It can also enhance lysozyme activity in blood and tissues. The polysaccharide also favors lactose metabolism. Chitosan oligosaccharide presents activity as a stimulant of selective growth of lactobacilli and bifidobacteria. It is important that to be nutritionally active, chitosan needs to be introduced solubilized in the food, or as a powder, which becomes soluble with the acidic pH.18 From a nutritional point of view, chitosan has been considered as a dietary fiber. The characteristics of accepted dietary fibers include nondigestibility in the upper gastrointestinal tract, high viscosity, and high water-binding ability in the lower gastrointestinal tract. From a physiological standpoint, the prime function of a dietary fiber is to reduce intestinal lipid absorption favoring lower cholesterol and loss of body weight (by reduction of lipid absorption) (see Chapter 9). Chitosan satisfies the requirements.18 Chitosan is not highly amenable to hydrolysis by digestive enzymes, is nontoxic, and hence clinically tolerable. In vivo toxicity studies have indicated that the chitosan obtained from prawn shells with a molecular weight of 126 kDa is nontoxic and inert, causing neither hemolysis nor favoring microbial growth. In the stomach, chitosan is found to attach itself to fat before being metabolized, thus trapping the fat and preventing its absorption by the digestive tract. The fat binds to the chitosan fiber becoming a large mass, which the body cannot absorb, and hence eliminated unabsorbed. This helps in capturing the fat including triglycerides, cholesterol, and bile acids that afterward form an insoluble complex in the intestine as a consequence of the alkaline pH and is excreted. The binding of negatively charged molecules such as fats, fatty acids, other lipids, and biliary acids is through the amine groups of chitosan that bind hydrogen ion in the acid fluids of the stomach, forming a positively charged tertiary amine group. These electrostatic and hydrophobic bonds cause the formation of long polymeric compounds, which are weakly attacked during digestive processes in the organism. In the intestine, the fat/chitosan emulsion immediately changes to an insoluble gel owing to the pH of the medium. The fat droplets are not amenable

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to digestion by pancreatic or intestinal enzymes.18 The effect is hyporcholesterolemic activity of chitosan. This was shown in an animal experiment study. Male albino Wistar rats were fed diets supplemented with chitin, partially hydrolyzed chitin (PHC), and glucosamine hydrochloride (0.5% of the diet). After feeding for 13 weeks, the muscle, kidney, and heart of the rats were tested for total cholesterol contents. Cholesterol levels were significantly low in all the three experimental groups. This effect was more pronounced in the chitin and PHC-fed groups.59 The functional role of chitosan as a dietary fiber also helps in obesity control.17 In view of the functional properties, especially its role as a fiber, chitosan has been recommended as a dietary supplement, particularly for the aged people.19 However, its prolonged use as fiber in diets needs to be monitored to ensure that it does not disturb the intestinal flora or interfere with the absorption of micronutrients, particularly lipid-soluble vitamins and minerals, or have any other negative effect.58 6.4.3.6

Medical Applications

The medical applications of chitosan cover diverse fields that include its use as hemodialysis membranes, artificial skin, hemoperfusion columns, drug delivery systems, and also as a hemostatic agent. Chitosan-coated hydroxyapatite microspheres and granules reduce bleeding and hasten healing with hard tissue growth in dental and orthopedic applications. Chitosan has been found to encourage even nerve growth. The sulfated derivatives of chitin and chitosan have anticoagulant and lipolytic activities in animal blood. Sulfated derivatives of chitin and carboxymethyl chitin inhibit the tumor induced metastasis 5′-methylpyrrolidinone. Chitosan promotes the dental osteoconduction. N-hexanoyl and N-octanoyl derivatives of chitosan have an antithrombogenic activity, and are highly compatible with animal blood and tissues. Because of its antibacterial and antifungal activities chitosan prevents bacterial and fungal infections. Chitosan implanted in animal tissues heals wounds. Skin lesions, especially associated with loss of tissue, prompts a number of reactions leading to complete recovery. The spontaneous healing process, however, does not always lead to functional normality in the injured areas. Besides, diabetes, hypertension, and other diseases lead to incomplete and prolonged healing reactions. It is possible to modulate the wound-healing process using N-carboxybutyl chitosan, which favored an ordered reconstruction of dermal architecture, whereas collagen provided a valid scaffold for organizing cell and stromal matrix.33,60 Chitosan is used in tropical skin ointments as a wound-healing agent. It has been shown that chitosan and its derivatives can reduce scar tissue by inhibiting fibrin formation and affecting macrophage activity. Chitosan is a suitable candidate for the formulation of transdermal films, which slowly release drugs into the blood. Matrix diffusion–controlled transdermal delivery of atenolol (a selective β-1 blocker) from chitosan film prepared from prawn shell has been reported.61 As discussed earlier, oral administration of chitosan suppresses serum cholesterol level and hypertension. Chitin/chitosan oligosaccharides, when intravenously injected, enhances the lysozyme activity in animal blood and also the antitumor activity by activating the macrophages. Chitosan oligomers also possess antitumor activities tested both in vitro and in vivo.27 A process for the production of absorbable surgical sutures has been developed by the Central Institute of Fisheries Technology, Cochin.7,19

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Biotechnology

Chitosan is used as a matrix for immobilization of enzymes and also as whole microbial cells. Almost 160 papers have been reviewed on 63 immobilized enzymes for multiplicity of applications ranging from wine, sugar, and fish industry to organic compounds removal from wastewaters to sophisticated biosensors for both in situ measurements of environmental pollutants and metabolite control in artificial organs.20 Another area is as a flocculating agent in separation processes. Potential for production of hydrogen by fermentation of marine (shrimp and lobster shells) and also agricultural (corn fiber) chitin wastes has been reported. Clostridium paraputrificum M-21 can produce 1.5–2.2 mol of H2 from 1 mol GlcNAc at pH 6. The strain efficiently degraded and fermented ball-milled raw shrimp and lobster shells within a period of 12–14 h to produce H2. The gas evolution was enhanced twofold by employing acid and alkali pretreatment. Chitinases (see Section 6.5.1) were detected in the culture, suggesting the role of these enzymes in the degradation of chitinous materials.62 6.4.3.8

Water Treatment

Chitosan and its derivatives carboxymethyl chitosan and cross-linked chitosan have been successfully used in water treatment by the removal of lead, copper, and cadmium from drinking water owing to complex formation of the amino group and heavy metal ions. In comparison with activated charcoal, it is more efficient in the removal of polychlorinated biphenyls from contaminated water. The hydroxamic acid derivatives of chitin and chitosan are most efficient in removing lead and copper.63 The possibility of complete removal of mercury from water has also been reported. Chitosan is presently employed in domestic sewage treatment systems in conjunction with other settling aids such as alum or bentonite clay to promote coagulation and settling of colloidal and other suspended solids. The polyelectrolyte is added at the rate of 1–2 ppm, but can also be employed alone without alum when the concentration is raised to approximately 10 ppm. Being positively charged, it is very effective to agglomerate the negatively charged sludge particles.19,64 6.4.3.9 Hydrogel Hydrogels are three-dimensional and hydrophilic polymer networks capable of swelling in water or biological fluids and retaining a large amount of fluids in the swollen state. The water content in the equilibrium of swelling affects different properties of the hydrogels, namely, permeability, mechanical properties, surface properties, and biocompatibility. The utility of hydrogels as biomaterials lies in the similarity of their physical properties with those of living tissues. Hydrogels can be prepared from a wide variety of materials of natural origin obtained from plants and animals, as well as from materials prepared by the modification of the aforementioned natural structures and from synthetic polymeric materials. Among the natural polymers, proteins such as collagen and polysaccharides such as chitosan or hyaluronic acid (HA) are used. Synthetic polymers have also found application in this area.65 Gelatinous dispersion form of chitosan seems to be very effective in several applications, including industrial wastewater treatment, bacteriostatic viscose-chitosan fibers, and modified cosmetic preparations.31

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6.4.3.10 Catalytic Support and Packaging Chitosan finds use as a support for catalysts for the synthesis of fine chemicals. Both degree of deacetylation and molecular weight are two important factors that influence the surface properties of chitosan. Increase in these properties could lead to increase in polarity of the chitosan, although the former seemed to influence the surface properties greater.66 The high surface hydrophilicity of chitosan has allowed the synthesis of an efficient, selective, and stable palladium-supported aqueous-phase catalyst for organic synthesis.67 Chitosan can be used as an adhesive for surfaces such as paper, rayon, cellophane, wood, leather, rubber, and glass to form waterresistant bonds. Its use in laminated corrugated fiberboard for packaging of frozen seafood has been suggested.68 6.4.3.11

Other Applications

Chitosan is used in products such as shampoo, hair sprays, nail polish, body cream, mouth washes, liquid soap, toothpaste, dental cream, personal hygiene, as humectant in hair and skin cosmetics, as antibacterial agents, preservation of vegetable pickles, chewing gum, hypocholesterolemic foods, cookies, noodles, chitosan vinegar, audio flat panel speaker, and others.10 Microcrystalline chitosan, especially in the form of gelatinous dispersion, seems to be a promising auxiliary agent for the modification of cosmetic preparations such as shampoo or liquid soap. It serves several functions such as thickner and viscosity regulator, softener, and skin or hair-protecting agent.31 In the textile industry, chitosan finds application as a sizing material because of its chelating and adhesive properties. Chitosan citrate has been successfully evaluated as nonformaldehyde durable press finish to produce wrinkle-resistant and antimicrobial properties of cotton fabrics. The carboxylic groups in chitosan citrate were used as active sites for its fixation on cotton fabrics. Fixation was done by the padding of chitosan citrate solution onto cotton fabrics followed by dry curing. The finished fabric showed adequate wrinkle resistance, whiteness, high tensile strength, and antimicrobial properties compared to untreated cotton fabric.69 The development of organic light-emitting diodes (OLEDs) are promising and gaining popularity because of their ease in fabrication, cost efficiency, and safety to the environment on disposal as opposed to traditional inorganic LEDs. OLEDs are ideal for optical communication because of their tendency not to refract or reflect light. OLED fabrication that employs chitosan has been reported recently.70

6.4.4

CHITIN OLIGOSACCHARIDES

As mentioned earlier, chitin is insoluble in water, organic solvents, or mineral acid, whereas chitosan is water-insoluble and highly viscous in dilute acids. These solubility properties have encouraged the search for the development of oligosaccharides from chitin and chitosan as functional ingredients that are more compatible with physiological conditions. Chitin and chitosan oligomers are not only water-soluble, but their solutions also have low viscosity and possess various biological activities; the oligomers with high degrees of polymerization, especially those with six residues or more, show strong physiological activities. Chitooligosaccharides therefore

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are of interest in nutritional and food preservative applications.51 The oligosaccharides are be prepared by controlled acid or enzymatic hydrolysis of chitin or chitosan. Enzymatically, low-molecular-weight chitosans (LMCs) could be prepared by depolymerization of chitosan with A. niger pectinase (polygalacturonase) at pH 3 and 37°C. The LMCs were in the range 5,000–20,000 Da. Drying of the product at elevated temperatures resulted in loss of water solubility of the LMC presumably due to changes in chain conformation.71 However, unlike chitin and chitosan, the oligosaccharides may be absorbed in the human intestine. Table 6.8 indicates the general applications of chitin, chitosan, and their derivatives.7,10 Since it is important to control the extent of degradation to obtain specific oligosaccharides, a novel method using hydrogen peroxide at low concentrations has been developed to partially degrade chitin from shrimp and squid. The treatment resulted in the breaking of β-1,4 glycosidic linkages resulting in the decrease in average

TABLE 6.8 General Applications of Chitin, Chitosan, and Their Derivatives Compound Chitin

O-carboxymethyl chitin and O-hydroxypropyl chitin N-acetylchitohexasaccharide Chitosan

N-hexanoylchitosan and N-octanoylchitosan

N-carboxybutyl chitosan 5′-Methylpyrrolidinone chitosan Sulfates of chitin and chitosan

Applications Wound dressing In vivo absorbable sutures Drug delivery Dialysis membrane Cosmetic ingredient Structural component of liposomes Antitumor agent Agriculture (seed, fruit cover, fungicide) Biotechnology (immobilization of enzymes) Catalytic supports for organic synthesis Food additive (antimicrobial, antioxidant, thickening agent, edible films, etc.) Medicine (wound dressing, artificial skin, immunostimulation, molecular recognition and entrapment of growth factor, etc.) Nutrition (dietary fiber, weight reduction, hypocholesterolemic agent, drug carrier, etc.) Textiles and other industries Water treatment (removal of metals, radioisotopes, pesticides, etc.) Cosmetics (shampoo, skin products) Antithrombogenic material for artificial blood vessels Contact lenses Blood dialysis membranes Artificial organs Wound dressing A material for promoting dental osteoconduction Anticoagulant and lipolytic agents

Source: Adapted from Subasinghe, S., Infofish Int., 3, 58, 1999; Synowiecki, J. and Al-Khateeb, N. A., Crit. Rev. Food Sci. Nutr., 43, 145, 2003.

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TABLE 6.9 Some Novel Applications of Chitosan Chitosan nanofiber as a scaffold for nerve tissue regeneration Controlled release of drugs from chitosan hydrogel Chitin and chitosan hydrogels Chitosan–polyarylamide gel graft Chitosan-based protein absorbents Chitosan–citric acid complex film for wound healing Acetylated chitosan membrane Source: Adapted from Yoshiharu, K. et al., Chitin Chitosan Res., 12, 96, 2006.

TABLE 6.10 Future Research Needs in Commercial Applications of Chitosan Area Process standardization with respect to deproteinization, demineralization, decolorization, and deacetylation Simpler and novel processes for chitosan and chitosan oligomers Improvements in film casting techniques, incorporation of plasticizers and antimicrobial additives Removal of astringent and bitterness by techniques such as ozone technology Quality standards

Brief Description The traditional methods influence molecular weight, degree of deacetylation, viscosity, fat and water absorption, and hydrophilicity Cost-effective processes can encourage applications Better stability against humidity and antimicrobial properties Better food applications Various applications reported so far have used chitosan having diverse properties. A need exists for common standards for universal applications

Source: Adapted from No, H. K. et al., J. Food Sci., 72, R87, 2007; No, H. K. and Meyers, S. P., J. Aquat. Food Prod. Technol., 4, 27, 1999.

molecular weights of the substrate in accordance with first-order kinetics. The formation of glucosamine and chitooligosaccharides depended on the concentration of H2O2, temperature, and the physicochemical properties of chitin and chitosan substrates. Degradation rates were faster than those from ultrasonic degradation, and were comparable to enzymic hydrolysis of chitosan. Chain-end scissions occurred after chitosan was severely degraded and produced significant amounts of oligosaccharides at a temperature of 80°C or above. Trace amounts of transition metal ions and amino groups in chitosan influenced the breakdown.72 The various applications of chitosan oligomers have been summarized.51 Table 6.9 suggests some novel applications of chitosan and Table 6.10 indicates future research needs in commercial applications of chitosan.12,35,73

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6.5 ENZYMES DEGRADING CHITIN AND CHITOSAN 6.5.1

CHITINASES

Chitinase (EC 3.2.1.14) hydrolyzes N-acetyl-d-glucosaminide (1–4)-β-linkages in chitin. Chitinase can be exo- or endochitinases. Exochitinase catalyzes diacetyl-chitobiose units from the nonreducing ends of chitin chains, and endochitinases randomly catalyze hydrolysis of N-acetyl glucosaminide (1–4)-β-linkages of chitin chains. Related enzymes are N-acetyl glucosaminidases (EC 3.2.1.30), some of which can hydrolyze the terminal, nonreducing N-acetylglucosamine residues of chitin; and lysozymes (EC 3.2.1.17), which can act slowly as endohydrolases. The activity of the enzymes depends on the nature of chitin. Several bacterial, plant, and fungal chitinases have been studied.18 Chitinases have been purified from a number of sources. A crude preparation of chitinase from A. hydrophila H-2330 was used for hydrolysis of crab shell chitin at 17°C for production of GlcNAc. Yields of GlcNAc from α-chitin were 66–77% after 10 days. The GlcNAc could be used as a functional food supplement or as a therapeutic agent.74 The extracellular enzyme from Rhodotorula gracilis reduced the size of chitin having a molecular weight of 36,000 Da to one-fourth of its size without further hydrolysis.75 A chitinase from alkalophilic Bacillus sp. BG-11 has been characterized. The bacterium produced 76 units of chitinase per milliliter in liquid batch fermentation after 72 h of incubation at 50°C using chitin-enriched medium. It has a molecular weight of 41 kDa and optimal activities at pH 8.5 and 50°C. The pH and thermal stability of the enzyme was enhanced by its immobilization on chitosan. The immobilized chitinase was stable between pH 5 and 10. GlcNAc was found to be the major end product.76 Two chitinases were purified from culture supernatant of Streptomyces griseus HUT 6037. Both enzymes had estimated molecular weights of 27,000 Da. The enzymes were active in the pH range 4.5–6.0 and at 55°C. The chitinases hydrolyzed chitin, colloidal chitin, glycol chitin, carboxymethyl chitin, 53% deacetylated chitosan, and (GlcNAc)-3-6 (N-acetylglucosamine), but did not hydrolyze 96% deacetylated chitosan and glucosamine. Oligosaccharides isolated had N-acetylglucosamine as the nonreducing end and N-acetylglucosamine or glucosamine as the reducing end residues. Results indicated that enzymes cleave both the N-acetyl-β-d-glucosaminidic and the β-glucosaminidic linkages in partially N-acetylated chitosan molecules.77 Chitinase from a marine Bacillus spp. has a molecular weight of 50 kDa. It was most active at pH 7 and 35°C.78 Chitinases can have practical uses. The enzyme can find application in the release of bound proteins from the shellfish. Endogenous chitinases are already used in the deshelling of shrimp. The use of chitinases in the bioconversion of shellfish wastes to single-cell protein has been suggested. Chitinases have found use in the preparation of protoplasts from fungi—a technique of increasing importance in biotechnology. As chitinases are a natural part of a plant’s defense mechanism, there has been much interest in boosting resistance of plants to fungal attack by getting them to express cloned chitinases.18

6.5.2

CHITOSANASES

Chitosanases (3.2.1.132) are useful to hydrolyze chitosan to produce di-, tri-, and tetra-chitooligosaccharides. Chitosanase has been isolated from Streptomyces from

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soil. The enzyme is inducible by chitin or d-glucosamine. In culture filtrates, chitosanase accounted for 50–60% of total extracellular medium. The enzyme cannot tolerate a degree of acetylation up to 60% in chitosan. The enzyme did not degrade chitin or carboxymethyl cellulose. The enzyme showed an endosplitting type of activity and the end product of chitosan degradation contained a mixture of dimers and trimers of d-glucosamine. Maximum activity of the enzyme was at 65°C and at pH 5.5. The enzyme was isolated from the soil actinomycete N174. It cannot degrade chitin or carboxy methylcellulose.79,80 Cells of Paenibacillus fukuinensis D2 secrete chitosanase into the surrounding medium in the presence of colloidal chitosan or glucosamine. The gene encoding the P. fukuinensis chitosanase was cloned, sequenced, and subjected to site-directed mutation and deletion analyses. The nucleotide sequence indicated that the chitosanase was composed of 797 amino acids with a molecular weight of 85,610 Da. Expression of the cloned gene in E. coli revealed β-1,4-glucanase activity as well as chitosanase activity.81 A biotechnological process for continuous hydrolysis of chitosan to prepare highly deacetylated chitosan using chitin-immobilized chitosanase has been reported. In the process, 0.5% chitosan (89% deacetylated) solution was fed into a glass column reactor containing 2 g (dry weight) of chitin-immobilized chitosanase at a rate of 10 mL/h at a temperature of 30°C, which gave optimum production. The enzyme treatment resulted in hydrolyzates having 5–30% hydrolysis. A higher DH gave a higher amount of chitobiose but lower amounts of chitotetrose and chitopentose, whereas chitotriose increased slightly and glucosamine did not change during hydrolysis.49 Chitin deacetylases are found in several fungi and insects. They hydrolyze chitin by acting on the N-acetamido bonds to produce chitosan. The use of this enzyme provides a biotechnological method to produce chitosan.82

6.5.3

SAFETY AND REGULATORY STATUS

Being naturally present in living organisms, chitin and also its derivative chitosan are considered safe. The available literature on chitin/chitosan suggests a low order of toxicity based on chemical structure and animal studies. Like several highmolecular-weight food additive polymers of natural origin such as cellulose, carrageenan, and guar gum, chitin/chitosan are not expected to be digested or absorbed from the human gastrointestinal tract. The human gastrointestinal tract does not have the ability to degrade the β-1–4 glycosidic linkage. In addition, the glucosamine backbone of chitin/chitosan can be considered innocuous. Chitosan has received the “generally recognized as safe (GRAS)” status from the U.S. Food and Drug Administration (FDA). The U.S. Environmental Protection Agency has approved the use of commercially available chitosan in wastewater treatment up to a maximum level of 10 mg/L.51 Japan’s health department approved the use of chitin and its derivatives as functional food ingredients. Based on its definition of functional foods, chitin and chitosans possess most of the required attributes related to enhancement of immunity, prevention of illness, delaying of ageing, and recovery from illness.7 Although it can be used as a fertilizer and animal-feed additive, use of chitin and chitosan as ingredients in foods or pharmaceutical products will, however, require standardization of identity, purity, and stability. The manufacturers should consider filing

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petitions with agencies such as Food Chemical Codex, U.S. Pharmacopeia, European Pharmacopeia, and Japan Pharmacopeia. These organizations establish methods to identify specific products and standards of purity for pharmaceutical and drug use. Such standards will be needed for future expansion of the use of chitin/chitosan.83

6.6 GLUCOSAMINE Glucosamine is a natural amino sugar found in large concentrations in certain foods such as milk, eggs, liver, yeast, and molasses. It is synthesized in the body from l-glutamine and glucose. Glucosamine is also the end product of hydrolysis of chitosan. If it is produced from chitin, it has to be deacetylated. Treatment of chitin (10–200 mg) with 1 mL of 10 M HCl under vacuum for 10 min followed by heat treatment at 140°C for 60 min can give glucosamine.84 Glucosamine could be absorbed easily into the human intestine.85 The degradation products formed when glucosamine is heated in water have been identified. Thus, when an aqueous solution of glucosamine is heated at 150°C at a pH of 4–7 for 5 h it results in the formation of furfurals. At pH 8.5, additional flavor components are generated, including pyrazines, 3-hydroxypyridines, pyrrole-2-carboxaldehyde, furans, acetol, and several other compounds. As ammonia is liberated from glucosamine, it initiates the ring-opening of furfurals to form 5-amino-2-keto-3-pentenals. Intramolecular condensations of these intermediates between the amino group and the carbonyl groups lead to the formation of 3-hydroxypyridines and pyrrole-2-carboxaldehyde. The results are important with respect to applications of glucosamine.86 Presence of glucosamine in food can be correlated with fungal contamination of food products since it is a component of fungal cell wall chitin.87 Chitin determination of the fungal cell wall is often conducted as a means of assessing the extent of fungal infection of foods. Since chitin is not soluble in water and most solvents, hydrolysis with acid, alkali, or enzymes to yield glucosamine for chemical determination is necessary. Glucosamine content can also be used as an index of solid-state fermentation of agricultural wastes by fungi.88 Modified chemical procedures for the rapid and reliable determination of glucosamine have been reported recently. The methods are applicable for determining chitin purity in foods and biological materials.89,90 A multiple-laboratory collaborative study was conducted for the determination of glucosamine in various products and dietary supplements. Average recoveries at spike levels of 100 and 150% of the declared amount were 99 and 101% with reproducible results among laboratories.91

6.7 SHARK CARTILAGE AND CHONDROITIN SULFATE Cartilage is a connective tissue, which consists of chondrocytes and a highly complex extracellular matrix consisting of collagen, elastin, glycosaminoglycans, fibronectin, laminin, and water, which form a cushion between bones and joints. There are two main types of cartilage: hyaline and elastic. Hyaline cartilage found in articular joint surfaces withstands repetitive loading and provides a low-friction interface, whereas

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COO⫺ O OH

CH2OR6 O

R4O O

OR2

O NHCOCH3

n

FIGURE 6.5 Chondroitin sulfate.

elastic cartilage found in rib, tracea, pharynx, ears, and nose is flexible to maintain the shape of these organs. An essential component of cartilage is chondroitin sulfate (CS), which is a complex natural heteropolysaccharide distributed among all organisms. CS is composed of an alternating sequence of sulfated or unsulfated d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine (GalNAc) residues linked through alternating β(1–3) and β(1–4) bonds (Figure 6.5). Some GlcA residues are epimerized into l-iduronic acid (IdoA); the resulting disaccharide being referred to as dermatan sulfate. Each monosaccharide in chondroitin may be left unsulfated, sulfated once, or sulfated twice. Most commonly, the hydroxyls of the 4 and 6 positions of the N-acetyl-galactosamine are sulfated, with some chains having the 2 position of glucuronic acid. Sulfation in these different positions, which is mediated by specific sulfotransferases, confers specific biological activities to chondroitin. Although most chondroitin is made from extracts of trachea of cow and ear and nose of pig, shark cartilage is a good source of chondroitin. Chondroitin is commonly sold with glucosamine. The dosage of oral chondroitin used in human clinical trials is 800–1200 mg per day. CS is quite stable to heating. Exposure to 121°C for 2 min could inactivate only about 10% of the material. CS is the major structural component of proteoglycans (PGs), the main macromolecular complex of the extracellular matrix, but is also localized at the cellular level as intracellular components. CS plays an important role in the elasticity and function of articular cartilage and is mainly attached covalently to core proteins in the form of PGs. Evidence derived from recent glycobiology studies in the fields of biochemistry and cell and developmental biology suggests that PGs and CSs participate in and regulate many cellular events and physiological processes suggesting the use of CS in the treatment of osteoarthritis and dermatan sulfate in the prevention of thrombosis. Current estimates for osteoporosis on a global scale are that 1 in 3 women and 1 in 12 men above 55 years will suffer from osteoporosis during their lifetime. In the United Kingdom, approximately 3 million people suffer from osteoporosis. The rise of osteoporotic hip fractures worldwide to 6.26 million in the year 2050 (compared to 1.66 million in 1990) suggests the future economic impact of osteoporosis will be phenomenal.92 The entire endoskeletons of shark are composed of cartilage and hence they are rich sources of CS. Sea mussel also has a high level of chondroitin. A process for the preparation of CS from skate (Raja avirostris) cartilage is available.93 Recently, a method for quality evaluation of shark cartilage powders has been reported by analyzing the CS content. The method involves the analysis of unsaturated disaccharides

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after treatment with enzyme chondroitinase. The amount of CS in the shark cartilage products ranged from 0 to 28.9% and had an average molecular weight of approximately 40 kDa, as determined by agarose gel electrophoresis and assessment of disaccharide compositional patterns.94

6.7.1

APPLICATIONS OF GLUCOSAMINE AND CHONDROITIN SULFATE

Arthritis indicates inflammation of the joints, whereas rheumatism means aches and pains in bones, muscles, and joints. There are about 200 types of arthritis and rheumatic diseases affecting the young and old, each one having a different cause. Osteoarthritis, also referred to as rheumatoid arthritis or degenerative joint disease, is a condition in which the cartilage begins to wear out, causing pain in the joints and thereby restricting movements. Osteoporosis affects the bones leading to bone fracture due to an imbalance between bone resorption and formation, whereas osteoarthritis impacts the joints. Osteoarthritis typically strikes the hands, knees, hips, feet, and back. In the knees of a person with osteoarthritis, the cartilage protecting the ends of bones deteriorates, and the joint fluid, called synovial, also breaks down. Glycosaminoglycans, a group of polysaccharides, that include CS, dermatan sulfate, and HA, are used by the body for the biosynthesis of compounds called PGs. The PGs have a very strong affinity for water molecules in the joints to give a lubricant that fills the space between the joints. Over time, the knee joint loses its cushioning and lubrication, and bone begins to rub against bone. This can result in pain, stiffness, and loss of range of movement. Depending on its stage, osteoarthritis can be very mild or so severe that it limits ones everyday activities. It affects 70% of people above the age of 75, although the symptoms may be visible above the age of 50. The prevalence of arthritis is more in women. In the United States, half (52%) of the population above the age of 65 is said to have arthritis, with an estimated 21 million Americans, mostly middle-aged and older, suffering from osteoarthritis. High incidences of obesity, sedentary life style, and poor diet have been held responsible for the disease.95 Glucosamine is a recognized nutraceutical for joint pain relief, by helping the repair and maintenance of cartilage. It is thought to promote the formation and repair of cartilage, and therefore it is a popular medicine in the treatment of arthritis (see also Chapter 4).19 Ageing people seem to loose their ability to produce sufficient amount of glucosamine. Glucosaminoglycans and glycoproteins allow cells in tissues to hold together. They are necessary for contraction and maintenance of virtually all connective tissues and lubricating fluids in the body. In particular, N-acetylglucosamine is the final form, which together with glucoronic acid is polymerized to lubricant HA. Presence of sufficient hyaluronic acid avoids pain in the joints. A European study showed that glucosamine sulfate was more effective than the over-the-counter painkiller acetaminophen for joint pain. In this study, 318 patients with knee osteoarthritis took oral glucosamine sulfate soluble powder (1500 mg once a day), acetaminophen (1000 mg three times a day), or placebo over a period of 6 months. Results showed that both glucosamine sulfate and acetaminophen were more effective in reducing pain than the placebo. In addition, patients taking glucosamine sulfate exhibited more relief than those taking acetaminophen. The study concluded that 1500 mg oral dose of glucosamine sulfate once a day may be the

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preferred treatment for knee osteoarthritis.96,97 Oral glucosamine supplementation has demonstrated effectiveness in combating osteoarthritis. Potential of supplementing beverages with glucosamine in hydrochloride or sulfate forms has been examined with respect to their taste, consumer acceptability, and marketing potential.98 Glucosamine has also found application in cosmetics. A placebo control doubleblind study of long-term intake of glucosamine HCl (1500 mg/day for 6 weeks) was conducted among 32 women who tended to have dry and rough skin. The results indicated that glucosamine HCl significantly reduced dryness, improved smoothness, and increased the moisture content of the skin.99 Glucosamine is comparable also to other joint-health nutraceuticals, which include HA, CS, and methylsulfonyl methane (MSM). Although glucosamine inhibits inflammation and stimulates cartilage cell growth, chondroitin provides cartilage with strength and resilience. Two recent studies showed that glucosamine and chondroitin could help in reducing the pain of arthritic joints. The Glucosamine/ Chondroitin Arthritis Intervention Trial (GAIT) was a multicenter clinical trial in the United States that tested the effects of glucosamine and chondroitin for treatment of knee osteoarthritis (see Chapter 4). The study concluded that the combination of glucosamine and CS is effective in treating moderate-to-severe knee pain caused by osteoarthritis.97 A diet supplement composed of red ginseng (43.5%), glucosamine (25.0%), shark cartilage (25.0%), ascorbic acid (5.0%), and manganese chloride (1.5%) has been developed for relieving arthritic symptoms. When tested individually, glucosamine and shark cartilage showed inhibition of vascular permeability by 29.6% and 32.9%, respectively. Red ginseng (500 mg/kg) and shark cartilage showed inhibition of carrageenan-induced paw edema.100 Elations™, a new clear fruit juice containing glucosamine, chondroitin, boron, vitamin C, and calcium has been developed.101 Shark cartilage has been found to possess several therapeutic effects against diseases such as arthritis and tumor. Administration of CS/glucosamine is one of the five ways to treat arthritis naturally. (The other ways are exercise, diet, heat and cold therapy, and acupuncture.) CS has antiarthritis and antijoint inflammation properties besides having anticancer properties. Administration of CS along with glucosamine (obtained from hydrolysis of chitin, from crustacean shell waste) is useful for the treatment of osteoarthritis and rheumatoid arthritis. Glucosamine inhibits inflammation and stimulates cartilage cell growth, whereas chondroitin provides the cartilage with strength and resilience.97 The results of the largest clinical trial, GAIT conducted by the U.S. National Institutes of Health, showed a combination of glucosamine and CS had significant effect over placebo on osteoarthritis of the knee with moderate to severe pain.102,103 Shark cartilage is an ideal source of angiogenic and tumor growth inhibitors. The oral administration of cartilage extract was efficacious in reducing angiogenesis. Furthermore, shark cartilage has been reported to have therapeutic efficacy in the treatment of progressive systemic sclerosis and neurovascular glaucoma.103–105 Purified antiangiogenic factors from shark cartilage, such as U-995 and neovastat (AE-941), also showed antiangiogenic and antitumor activity. Squalamine from certain species of shark (see Chapter 12) is a low-molecular-weight aminosterol that shows strong antitumor activity when combined with chemotherapeutic materials.

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The angiogenic tissue inhibitor of metalloprotease 3 (TIMP-3) and tumor suppressor protein (snm23) genes from shark cartilage were cloned and characterized.106 Cartilage deficiency from pathology, trauma, or congenital defects is currently treated with prostheses, cartilage grafts, and scaffolds. A scaffold should be biocompatible and should allow cells’ adherence and migration with subsequent differentiation and proliferation. A porous scaffold will provide the necessary environment. Engineered cartilage includes agarose, alginate, gelatin, collagen derivatives, and a cellular cartilage matrix. Hybrid scaffolds with alginate with synthetic polymers offer more promise in clinical applications. Alginate gel sheets have recently been evaluated as a potential scaffold for cell proliferation since alginate can propagate chondrocyte growth and cartilaginous matrix production. Alternately, agarose gels can support chondrocyte growth. Agarose is a solid at body temperature, but requires higher temperature to melt. Agar at 1–3% (w/w) gives optimal nutrient and waste exchange; however, permeability is affected at higher agar concentrations. Recent studies showed that after 18 months, 47% of grafts incorporating agarose gels developed a morphologically stable hyaline cartilage. Collagens I and II have been a well-described scaffold for cartilage regeneration. Collagen can be fabricated as a gel, sponge, or foam and is subject to enzymatic degradation. In recent years, crosslinked collagen has been popularized because of its improved mechanical properties and slow degradation rates. A period of 6 weeks is required for a stable natural graft. Chitin and chitosan when combined with CS maintained the chondrocytic phenotype and support PG production.107,108 Tissue-engineered skin substitutes provide opportunity to overcome the shortage of skin autograft by culturing keratinocytes and dermal fibroblasts in vitro. A bilayer gelatin–chondroitin sulfate–hyaluronic acid (gelatin–CS–HA) biomatrices has been developed. This bilayer skin substitute not only has a positive effect in promoting wound healing, but also has high rate of graft take. It has the potential for application to extensively and deeply burned patients suffering from severe skin loss.109

6.8 COMMERCIAL PRODUCTS The global chitin production potential has been estimated at 118,000 t. However, based on the assumption of the centralized large-scale processing facilities, which are almost totally export oriented in many developing countries, a global chitin production potential of about 76,000 t has been estimated.7 The varied applications of chitosan have encouraged many companies to initiate production on a commercial scale. Japan is the major producer of chitin and chitosan from the shells of crabs and shrimp.14 Recent years have witnessed a marked growth in the dietary chitosan preparations with therapeutic and health attributes, which are commercially available. Most of these preparations are in the tablet or capsule form, with a few preparations in the powder form. Chitosan is available in a wide variety of commercial products with different deacetylation grades and molecular weights or viscosities, and hence have different functional properties.19 However, neither chitosan nor chitin has yet been cleared by the U.S. FDA for human consumption.110 In the European market, chitosan is sold in the form of dietary capsules to assist weight loss, and in some countries such as Japan it is added to foods such as noodles,

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potato crisps, and biscuits. The Global Industry report analyzes the worldwide markets for chitin and chitosan. The chitosan end-use segments analyzed are water treatment, cosmetics, food and beverages, healthcare, agrochemicals, biotechnology, etc., and profiles 53 companies that are involved in the business.111 The current market for shark cartilage in the United States is worth $170 m. However, since consumers find CS—a bioactive component of shark cartilage—therapeutically more effective, the sale of shark cartilage has stabilized.111 Some of the commercial chitosan products include “Fat Absorb,” a U.S. product containing 250 mg of chitosan per capsule; “Seaborne” range of products such as “EssentialSea” and “EssentialSeaPlus,” containing chitosan combined with other nutrients such as lecithin, vitamins C and E, garlic, and β-carotene (mostly available in Japan); “Minfat,” a fat trimmer, marketed in Malaysia, which claims to absorb 21 times its weight of fat; and chitosan-fortified fruit juices and chocolates, which were marketed in the United States.7 In addition to the preceding compounds, there is promising opportunities in harnessing seafood waste as a source of several other nutraceuticals also. Scientists from the Institute of Water and Atmospheric Research, New Zealand, have recently isolated some potent skincare products from fish processing discards. Some of these compounds include antiwrinkle cream from male stungeon gonads that can be incorporated in different skincare lines for their ultraviolet (UV) protection action and antiaging effects. Body lotions and makeup using cod sperm as a water binder has also been isolated. The proteins of cod sperm stimulate the immune system. Furthermore, chitosan and chondroitin sulfate can also be used in a wide range of skincare products, in shampoos and toothpaste as a gelling agent, and as a moisturizer.112 In conclusion, the crustacean waste product chitin and its deacetylated derivative chitosan offer unique characteristics, namely, biocompatibility, biodegradability to harmless products, nontoxicity, physiological inertness, antibacterial properties, gelforming properties, hydrophilicity, and remarkable affinity to proteins and metals. Because of these advantages, chitin and particularly chitosan are finding applications in several fields including agriculture, biotechnology, food, medicine, and also in several industrial processes. The potential of chitin and chitosan as sources of bioactive compounds useful for human has been understood. Chitin- and chitosanbased materials, as yet underutilized, are predicted to be widely exploited in the near future in a range of applications suggesting the scope of converting shellfish waste to economic advantages. Glucosamine, the hydrolyzed product of chitosan, along with CS from shark cartilage are highly effective in the treatment of arthritis and osteoporosis. There is a good scope for commercial production of these compounds from marine fishery waste.

REFERENCES 1. FAO, The State of World Fisheries and Aquaculture, Food and Agriculture Organization of the United Nations, Rome, 2006. 2. Venugopal, V. and Shahidi, F., Traditional methods to process underutilized fish for human consumption, Food Rev. Int., 14, 35, 1998. 3. de Holanda, H. D. and Netto, F. M., Recovery of components from shrimp (Xiphopenaeus kroyeri) processing waste by enzymatic hydrolysis, J. Food Sci., 71, C298, 2006.

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4. Sultanbawa, Y. and Aksnes, A., Tuna process waste—an unexploited resource, Infofish Int., 3, 37, 2006. 5. Pigott, G. M., Enzyme hydrolysis of fish waste for animal feed and fertilizer, in Seafood Safety, Processing and Biotechnology, Shahidi, F., Jones, Y., and Kitts, D. D., Eds., Technomic, Lancaster, PA, 1997, p. 249. 6. Shahidi, F., Shellfish discard utilization, in Seafood Safety, Processing and Biotechnology, Shahidi, F., Jones, Y., and Kitts, D. D., Eds., Technomic, Lancaster, PA, 1997, p. 131. 7. Subasinghe, S., Chitin from shellfish waste—health benefits over shadowing industrial uses, Infofish Int., 3, 58, 1999. 8. Shahidi, F. and Synowiecki, J., Isolation and characterization of nutrients and value added products from snow crab (Chinocetes opilio) and shrimp (Pandalus borealis) processing discards, J. Agr. Food Chem., 39, 1527, 1991. 9. Mandeville, S., Yaylayan, V., and Simpson, B. K., Proximate analysis, isolation and identification of amino acids and sugars from raw and cooked commercial shrimp waste, Food Biotechnol., 6, 51, 1992. 10. Synowiecki, J. and Al-Khateeb, N. A., Production, properties, and some new applications of chitin and its derivatives, Crit. Rev. Food Sci. Nutr., 43, 145, 2003. 11. Pan, B. S., Recovery of shrimp waste for flavor, in Advances in Fishery Technology and Biotechnology for Increased Profitability, Voigt, M. N. and Botta, J. R., Eds., Technomic, Lancester, PA, 1990, p. 437. 12. No, H. K. and Meyers, S. P., Preparation and characterization of chitin and chitosan—a review, J. Aquat. Food Prod. Technol., 4, 27, 1999. 13. Tharanathan, R. N. and Kuttur, F. S., Chitin—the undisputed biomolecule of great potential, Crit. Rev. Food Sci. Nutr., 43, 61, 2003. 14. Muffler, K. and Ulber, R., Downstream processing in marine biotechnology, Marine Biotechnol., 2, 86, 2005. 15. Alasalvar, C. and Taylor, T., Seafoods—Quality, Technology and Nutraceutical Applications, Springer, Heidelberg, 2002, p. 123. 16. Marquardt, F. H. and Carreno, R. R., The production of colorless chitin from Antarctic krill (Euphausia superba) shell waste, Arch. Fischereiwisenshaft, 41, 159, 1992. 17. Muzzarelli, R., Clinical and biochemical evaluation of chitosan for hypercholesterolemia and overweight control, in Chitin and Chitinases, Jolle`s, P. and Muzzarelli, R. A. A., Eds., Birkhauaser, Switzerland, 1999, p. 293. 18. Gooday, G. W., Chitinases, in Enzymes in Biomass Conversion, ACS Symposium Series, American Chemical Society, Washington, DC, 1991, p. 478. 19. Sen, D. P., Selected by-products from sea, in Advances in Fish Processing Technology, Allied Publisher, New Delhi, 2005, p. 616. 20. Ramachandran Nair, K. G. et al., Chitin preparation from prawn shell, Ind. J. Poultry Sci., 22, 40–43, 1987. 21. Krajewska, B., Application of chitin- and chitosan-based materials for enzyme immobilizations: a review, Enzyme Microb. Technol., 35, 126, 2004. 22. Han, X.-Q. and Shahidi, F., Extraction of harp seal gastric proteases and their immobilization on chitin, Food Chem., 52, 71, 1995. 23. Knorr, D., Uses of chitinous polymers in foods—a challenge for food research and development, Food Technol., 38(1), 85, 1984. 24. Win, N. N. and Stevens, W. F., Shrimp chitin as substrate for fungal chitin deacetylase, Appl. Microbiol. Biotechnol., 57, 334, 2001. 25. Neugebauer, W. A., Neugebauer, E., and Brezinski, R., Determination of the degree of acetylation of chitin–chitosan with picric acid, Carbohydr. Res., 189, 363, 1989. 26. Shahidi, F. and Abuzaytoon, R., Chitin, chitosan and co-products: chemistry, production, applications and health effects, Adv. Food Nutr. Res., 49, 94, 2005.

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27. Jeon, Y. J., Shahidi, F., and Kim, S. K., Preparation of chitin and chitosan oligomers and their applications in physiological functional foods, Food Rev. Int., 16, 159, 2000. 28. Natarajah, N. et al., Sorption behaviour of crawfish chitosan films as affected by chitosan extraction process and solvent types, J. Food Sci., 71, 33, 2006. 29. Radhakrishnan, A. G. and Prabhu, P. V., Chitosan preparation from prawn waste, Res. & Ind. (India), 16, 265, 1971. 30. Madhavan, P. and Ramachandran Nair, K. G., Chitosan from squilla, Fish. Technol., 12, 81, 1975. 31. Strusczyk, H. and Kivekas, O., Recent developments in microcrystalline chitosan applications, biological materials for wound healing, in Advances in Chitin and Chitosan, Brine, C. J. and Sandford, P. A., Eds., Elsevier Allied Science, London, 1992, p. 549. 32. Chung, Y. C. et al., Preparation and important functional properties of water-soluble chitosan produced through Maillard reaction, Bioresource Technol., 96, 1473, 2005. 33. Prashanth, K. V. H. and Tharanathan, R. N., Chitin/chitosan: modifications and their unlimited application potential—an overview, Trends Food Sci. Technol., 18, 117, 2007. 34. Goyal, G. K., Therapeutic benefits of pro- and pre-biotics: a review, Ind. Food Ind., 26, 41, 2007. 35. No, H. K. et al., Applications of chitosan for improvement of quality and shelflife of foods: a review, J. Food Sci., 72, R87, 2007. 36. Rodrý´guez, M. S., Albertengo, L. A., and Agullo, E., Emulsification capacity of chitosan, Carbohydr. Polym., 48, 271, 2002. 37. Benjakul, S. et al., Effect of chitin and chitosan ongelling properties of surimi from barred garfish (Hemiramphus far), J. Sci. Food Agr., 81, 102, 2001. 38. Benjakul, S. et al., Chitosan affects transglutaminase-induced surimi gelation, J. Food Biochem., 27, 53, 2003. 39. Kataoka, J., Ishizaki, S., and Tanaka, M., Effects of chitosan on gelling properties of low quality surimi, J. Muscle Foods, 9, 209, 1998. 40. Jo, C. et al., Quality properties of pork sausage prepared with water-soluble chitosan oligomer, Meat Sci., 59, 369, 2001. 41. Lin, K. W. and Chao, J. Y., Quality characteristics of reduced fat Chinese-style sausage as related to chitosan’s molecular weight, Meat Sci., 59, 343, 2001. 42. Sagoo, S., Board, R., and Roller, S., Chitosan inhibits growth of spoilage microorganisms in chilled pork products, Food Microbiol., 19, 175, 2002. 43. Butler, B. L. et al., Mechanical and barrier properties of edible chitosan films as affected by composition and storage, J. Food Sci., 61, 953, 1996. 44. Helander, I. M. et al., Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria, Int. J. Food Microbiol., 71, 235, 2001. 45. No, H. K. et al., Antibacterial activity of chitosans and chitosan oligomers with different molecular weights, Int. J. Food Microbiol., 74, 65, 2002. 46. Faug, S. W. et al., Antifungal activity of chitosan and its use, J. Food Prot., 56, 134, 1994. 47. Lopez-Caballero, M. E. et al., A functional chitosan-enriched fish sausage treated by high pressure, J. Food Sci., 70, M168, 2005. 48. Labuza, T. P. and Breene, W. M., Applications of active packaging for improvement of shelf life and nutritional quality of fresh and extended shelf life foods, J. Food Proc. Preserv., 13, 1, 1988. 49. Rwan, J. H. et al., Continuous production and microbial inhibition of hydrolyzed chitosans, J. Chinese Agr. Chem. Soc., 35, 596, 1997. 50. Kamil, J., Jeon, Y. J., and Shahidi, F., Antioxidative activity of chitosans of different viscosity in cooked comminuted flesh of herring (Clupea harengus), Food Chem., 79, 69, 2002.

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51. Jeon, Y. J., Kamil, J., and Shahidi, F., Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod, J. Agr. Food Chem., 50, 5167, 2002. 52. Matsugo, S. et al., Synthesis and antioxidant activity of watersoluble chitosan derivatives, Biochem. Mol. Biol. Int., 44, 939, 1998. 53. Xue, C. et al., Antioxidative activities of several marine polysaccharides evaluated in a phosphatidylcholine–liposomal suspension and organic solvents, Biosci. Biotechnol. Biochem., 62, 206, 1998. 54. Krochta, J. M. and Mulder-Johnston, D. C., Edible and biodegradable polymer films: challenges and opportunities, Food Technol., 51, 61, 1997. 55. Kanatt, S., Chander, R., and Sharma, A., Effect of irradiated chitosan on the rancidity of radiation-processed lamb meat, Int. J. Food Sci. Technol., 39, 997, 2004. 56. Rao, M. S., Chander, R., and Sharma, A., Development of shelf stable intermediate moisture meat products using active edible chitosan coating and irradiation, J. Food Sci., 70, M325, 2005. 57. Ouattara, B. et al., Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan, Int. J. Food Microbiol., 62, 139, 2000. 58. Koide, S. S., Chitin-chitosan: properties, benefits and risks, Nutr. Res., 18, 1091, 1998. 59. Mathew, P. T. and Nair, K. G. R., Hypocholesterolemic effect of chitin and its hydrolysed products in albino rats, Fish. Technol. (India), 35, 46, 1998. 60. Ishihara, M. et al., Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process, Biomaterials, 23, 833, 2002. 61. Benny, K. J. et al., Matrix diffusion controlled transdermal delivery of atenolol from chitosan film, in Nutrients and Bioactive Substances in Aquatic Organisms, Devadasan, K., et al., Eds., Society of Fishery Technologists, Kochi, 1994, p. 76. 62. Evvyernie, D., Conversion of chitinous wastes to hydrogen gas by Clostridium paraputrificum M-21, J. BioSci. BioEng., 91, 339, 2001. 63. Hirotsu, T. et al., Synthesis of dihydroxamic acid chelating polymers, J. Polym. Sci. Part A, 24, 1953, 1986. 64. Prabhu, P. V., Radhakrishnan, A. G., and Iyer, T. S. G., Chitosan as a water clarifying agent, Fish. Technol. (India), 13, 69, 1976. 65. Hoffman, A. S., Hydrogel for biomedical applications, Adv. Drug Delivery Rev., 54, 3, 2002. 66. Zhang, L. et al., Influence of the degree of deacetylation and molecular weights on the surface properties of chitosan, # COLL 554, National Meeting, American Chemical Society, Chicago, IL, March 25–29, 2007. 67. Ligler, F. S. et al., Development of uniform chitosan thin-film layers on silicon chips, Langmuir, 17, 5082, 2001. 68. Gopal, T. K. S. et al., Use of chitosan adhesive for the manufacture of corrogted fiberboard, Fish. Technol. (India), 28, 154, 1991. 69. Hashema, A. A. S. and Husein, S. S., Utilization of chitosan citrate as crease-resistant and antimicrobial finishing agent for cotton fabrics, Ind. J. Fibre Text. Res., 29, 218, 2004. 70. Adams, A., Chitosan for application as an organic light-emitting diode, # CHED 788, ACS National Meeting, Chicago, IL, March 25–29, 2007. 71. Kittur, F. S., Kumar, A. B. V., and Tharanathan, R. N., Low molecular weight chitosans—preparation by depolymerization with Aspergillus niger pectinase, and characterization, Carbohydr. Res., 338, 1283, 2003. 72. Chang, Ke.-L. B., Tai, M. C. and Cheng, H., Kinetics and products of the degradation of chitosan by hydrogen peroxide, J. Agr. Food Chem., 49, 4845, 2001. 73. Yoshiharu, K. et al., New techniques and functions of chitosan products, Chitin Chitosan Res., 12, 96, 2006.

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74. Saishiwa, H. et al., Production of N-acetyl-d-glucosamine from α-chitin by crude enzymes from Aeromonas hydrophila, Carbohydr. Res., 337, 761, 2002. 75. Somashekar, D. and Joseph, R., Partial purification and properties of a novel chitosanase secreted by Rhodotorula gracilis, Lett. Appl. Microbiol., 14, 1, 1992. 76. Bhushan, B., Production and characterization of a thermostable chitinase from a new alkalophilic Bacillus sp. BG-11, J. Appl. Microbiol., 88, 800, 2000. 77. Mitsutomi, M., Hata, T., and Kuwahara, T., Purification and characterization of novel chitinases from Streptomyces griseus HUT 6037, J. Ferment. Bioeng., 80, 153, 1995. 78. Lee, J. S. et al., Purification and characterization of extracellular chitinases produced by marine bacterium, Bacillus spp. LJ-25, J. Microbiol. Biotechnol., 10, 307, 2000. 79. Boucher, I. et al., Purification and characterization of a chitosanase from Streptomyces N174, Appl. Microbiol. Biotechnol., 38, 188, 1992. 80. Kurakake, M. et al., Properties of chitosanases from Bacillus cereus S1, Curr. Microbiol., 40, 6, 2000. 81. Kimoto, H. et al., Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain, J. Biol. Chem., 277, 14695, 2002. 82. Tsigos, E. et al., Chitin deacetylases, new versatile tools in biotechnology, Trends Biotechnol., 18, 305, 2000. 83. Ravikumar, M. N. V., A review of chitin and chitosan applications, React. Funct. Polym., 46, 1, 2000. 84. Chen, M. C., Yeh, G. H. C., and Chiang, B. H., Antimicrobial and physicochemical properties of methylcellulose and chitosan, Trends Food Sci. Technol., 12, 1, 2005. 85. Rustad, T. and Falch, E., Making the most of fish catches, Food Sci. Technol., 16, 36, 2002. 86. Shu, C.-K., Degradation products formed from glucosamine in water, J. Agr. Food Chem., 46, 1129, 1998. 87. Wu, T. et al., Physicochemical properties and bioactivity of fungal chitin and chitosan, J. Agr. Food Chem., 53, 3888, 2005. 88. Zheng, Z. and Shetty, K., Solid-state production of beneficial fungi on apple processing wastes using glucosamine as the indicator of growth, J. Agr. Food Chem., 46, 783, 1998. 89. Chen, W. and Chiou, R. Y. Y., A modified chemical procedure for rapid determination of glucosamine and its application for estimation of mold growth in peanut kernels and koji, J. Agr. Food Chem., 47, 19929, 2004. 90. Zhu, X. et al., Determination of glucosamine in impure chitin samples by highperformance liquid chromatography, Carbohydr. Res., 340, 1732, 2005. 91. Zhou, Z., Waszkuc, T., and Mohammed, F., Determination of glucosamine in raw materials and dietary supplements containing glucosamine sulfate and/or glucosamine hydrochloride by HPLC with FMOC-Su derivatization: collaborative study, J. AOAC Int., 88, 1048, 2005. 92. Lanham-New, S. A. and Gannon, R. H. T., Is dietary alkali supplementation the way forward for preventing osteoporosis? IFIS Publishing, http://www.foodsciencecentral. com/fsc/ixid14489, September 2006. 93. Jo, J. H. et al. Optimization of skate (Raja avirostris) cartilage hydrolysis for the preparation of chondroitin sulfate, Food Sci. Biotechnol., 13, 622, 2004. 94. Sim, J.-S., et al., Evaluation of chondroitin sulfate in shark cartilage powder as a dietary supplement: raw materials and finished products, Food Chem., 101, 532, 2007. 95. Anonymous, HealthBeat, Harvard Medical School, February 14, 2007, www.health. harvard.edu. 96. Herrero-Beaumont, G. et al., Effects of glucosamine sulfate on 6-month control of knee osteoarthritis symptoms vs. placebo and acetaminophen: results from the Glucosamine Unum In Die Efficacy (GUIDE) trial, Presentation #1203, American College of Rheomatology Conference, Washington, DC, November 15, 2006.

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97. Ohr, L. M., Joint health, Food Technol., www.ift.org, January 2006, Vol. 60, p. 57. 98. Runestad, T., Stability and taste test limits of glucosamine in beverages, Funct. Foods Nutra., 22, 24, 2004. 99. Kajimoto, O., Suguro, S., and Takahashi, T., Clinical effects of glucosamine hydrochloride diet for dry skin, J. Jap. Soc. Food Sci. Technol., 48, 335, 2001. 100. Sim, J. S. et al., Anti-arthritic effect of a new diet supplement containing red ginseng extract and glucosamine complex, Kor. J. Pharmacog., 34, 327, 2003. 101. Kim, S. K. and Mendis, E., Bioactive compounds from marine processing byproducts—a review, Food Res. Int., 39, 383, 2006. 102. Sculti, L., Arthritis benefits from shark cartilage therapy, Alternat. Complement. Ther., 35, 37, 1994. 103. McAlindon, T. E. et al., Glucosamine and chondroitin for treatment of osteoarthritis: a systematic quality assessment and meta-analysis, JAMA, 283, 1469, 2000. 104. Lee, A. and Langer, R., Shark cartilage contains inhibitors of tumor angiogenesis, Science, 221(4616), 1185, 1983. 105. Brouns, F. and Vermeer, C., Functional food ingredients for reducing the risks of osteoporosis, Trends Food Sci. Technol., 11, 22, 2000. 106. Cho, T. and Kim, Y., Sharks: a potential source of antiangiogenic factors and tumor treatments, Mar. Biotechnol., 4, 521, 2002. 107. Raghunath, J. et al., Biomaterials and scaffold design: key to tissue-engineering cartilage, Biotechnol. Appl. Biochem., 46, 73, 2007. 108. Chen, J. S. et al., Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells, J. Cell. Biochem., 78, 417, 2000. 109. Wang, T.-W. et al., The effect of gelatin–chondroitin sulfate–hyaluronic acid skin substitute on wound healing in SCID mice, Biomaterials, 27, 5689, 2006. 110. Brody, A. L., Strupinsky, E. R., and Kline, L. R., Active Packaging and Food Applications, Technomic, Lancaster, PA, 2001. 111. Anonymous, Global Industry Analysts Report, http://www.marketresearch.com, February 1, 2007, p. 215. 112. Profitt, F., A surprising use of fish waste, Infofish Int., 6, 44, 2007.

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7 Carotenoids 7.1 INTRODUCTION Carotenoids are a family of fat-soluble compounds that provide red and orange colors to plants, algae, and cyanobacteria. Whereas several vegetables such as tomatoes are rich sources of carotenoids, animal sources of carotenoids include salmonid fish, crustacean shellfish, calf liver, and eggs. Carotenoids participate in light harvesting and are essential for photo-protection in photosynthetic plant tissues. Carotenoids are synthesized by photosynthetic organisms including plants, and also by some nonphotosynthetic bacteria and fungi. Animals, including humans, do not synthesize carotenoids de novo and rely upon the diet as the source of these compounds. Over 600 carotenoids have been identified till date. The color of carotenoids is due to a chromophore consisting of a chain of conjugated double bonds. Diversity in carotenoids is essentially due to these numerous conjugated double bonds and the cyclic end groups in their elongated structures, which allow a variety of stereoisomers with different chemical and physical properties. Whereas detailed chemistry of carotenoids can be found elsewhere, this chapter essentially discusses their availability from marine organisms and biological functions, which make them potential nutraceutical molecules.

7.2 GENERAL PROPERTIES The carotenoids, found in nature, can be classified into two: hydrocarbons, such as β-carotene, and xanthophylls, the oxygenated derivatives of carotenes such as astacene, astaxanthin, canthaxanthin, cryptoxanthin, echionine, lutein, neoxanthin, violaxanthin, and zeaxanthin. β-carotene (C40H56, molecular weight 536.9) is one of the most abundant carotenoids. It is a polyunsaturated hydrocarbon and is made up of two molecules of retinal and possesses maximal provitamin A activity, whereas approximately 60 carotenoids possess varying levels of provitamin A activity. β-carotene functions in green leaves in photo energy transfer and as a photo protectant in chloroplasts. Xanthophylls are the typical yellow pigments of leaves. The most important forms commonly found among carotenoids are geometric (E/ Z) isomers (a double bond links the two residual parts of the molecule either in an E-configuration with both on opposite sites of the plane, or Z-configuration with both on the same side of the plane). Geometrical isomers of this type are interconvertible in solution. Astaxanthin is 3,3′dihydroxy-β, β′-carotene-4,4′dione and canthaxanthin, β, β′-carotene-4,4′dione. The isomers differ not only in their melting points, solubility, and stability, but also in respect to absorption affinity, color, and color intensity.1 Carotenoids are soluble in most organic solvents, but not in water. They are sensitive to oxidation, isomerization, and polymerization when dissolved in dilute solution under light and in the presence of oxygen. They can be stored in 221

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β-Carotene

Lycopene OH

HO

Lutein OH

HO

Zeaxanthin O

O

Canthaxanthin O OH

HO O

Astaxanthin

FIGURE 7.1 Chemical structures of some important carotenoids.

a cool place, in a tight, light-resistant container under inert gas without significant changes.2 Figure 7.1 gives chemical structures of some important carotenoids.

7.3 UNITS AND REQUIREMENTS In 1965, an expert committee decided to abandon the international unit (IU) of measurement for vitamin A in favor of retinal equivalents (RE), a purely dietary concept, defined as the amount of retinal plus the equivalent amount of retinal that can be obtained from the provitamin A carotenoids. The provitamin A carotenoid, β-carotene is not an essential nutrient. However, being precursor of vitamin A, dietary intakes are expressed in terms of vitamin A, as RE. One RE is equivalent

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to 1 µg retinal or 6 µg β-carotene and 1 IU is equivalent to 0.3 µg retinal or 2 µg β-carotene. There is no separate recommended daily allowance for carotenoids.3

7.4 MARINE SOURCES OF CAROTENOIDS 7.4.1

ALGAL SOURCES

One quarter of all vegetation on the planet Earth (both land and sea) consists of marine phytoplankton (see Chapter 12). Most of these algae possess various colored plastids with chlorophylls, carotenoids, and phycobiliprotein. Carotenoids help marine macroalgae survive in shallow coastal waters by offering efficient photosynthesis for acclimatization to fast changes in their environment. Xanthophyll carotenoids and nonphotochemical quenching (NPQ) are central constituents of such protection mechanisms. Red algae as a group do not have a universal carotenoid composition. Interconversions of xanthophylls have been observed in these algae. These algal species included the tropical species Gracilaria domingensis and Kappaphycus alvarezii with antheraxanthin and lutein as major xanthophylls, respectively. Interconversions of xanthophylls (violaxanthin, zeaxanthin, β-cryptoxanthin, and one unidentified carotenoid) can occur in G. domingensis.4 The microalgae, Dunaliella spp., particularly, Dunaliella salina produces significant amounts of β-carotene5 (see also Chapter 11).

7.4.2

MARINE FISHERY SOURCES

Aquatic animals contain significant amounts of carotenoid, particularly, the red-orange colored pigment, astaxanthin. The primary dietary source of carotenoids for the fish and shellfish is the algae. Secondary sources are marine animals, which have accumulated the pigments from their consumption of phytoplankton, and are subsequently consumed by other marine animals that are higher up on the food chain. The most commercially harvested crustacean species, viz., crab, shrimp, prawn, and also Antarctic krill and crayfish, contain significant amounts of carotenoids in their head and shell portions. The nature and concentrations of carotenoids in wild fish and shellfish tissues vary depending on the geographical and environmental conditions. Astaxanthin and its mono- and diesters and oxidation products of β-carotene are the major carotenoids present in the head and shell of crustaceans and remain bound to lipids.6–10

7.5 ISOLATION AND CHARACTERIZATION 7.5.1

ALGAL SOURCES

Currently, microalga are the major sources of carotenoids, which can be produced by cultivating certain high-yielding algae such as Haemotococcus spp., Dunaliella spp., and Chlorella spp.11–15 (see also Chapter 11). However, the growth phase of the algae influences yield. Sporulation of the alga results in reduction in the astaxanthin content, the spore containing only 1–2% of the pigment on dry-weight basis. Dunaliella spp. D. salina is the most halophilic eukaryotic microalga, with an optimum salinity of 22% (w/v) for growth (7–8 times seawater) that produces β-carotene, suggesting

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potential for open-air culture of the alga to produce the pigment. The pigment is natural and hence commands high price and better biological activity than synthetic products.4 However, the disadvantage is the very low biomass in the growth ponds, rarely exceeding 1 g/L, which makes harvesting an expensive process. Strain improvement for increasing growth rate is a challenge faced by the farmers.4 During processing, isolation, and concentration such as spray-drying losses and changes such as isomerization of carotenoids have been noted. Such changes, however, are minimum in the case of β-carotene isolated from Dunaliella spp. However, the carotene in dried powders of Dunaliella proved to be unstable during storage in the presence of light and air. The 9-cis isomer of β-carotene was significantly unstable than the all-trans form. A dominant photodegradative mechanism was responsible for the loss of the 9-cis isomer of β-carotene. Addition of the antioxidant, tert-butylhydroquinone (TBHQ) before drying minimized degradation of the carotene.12 The yeast, Phaffia rhodozyma is a source of astaxanthin, although it has lower astaxanthin content than algae.14 Lutein is produced by the microalga, Chlorella, which could be isolated and purified up to 98%. The pigment is commercially derived from the corn meal and is used in feed formulations.13,15 Phycocyanin has been produced by cultivation of the microalga, spirulina.16 Production and biological activities of algal components including carotenoids have been discussed in Chapter 12.

7.5.2

FISHERY SOURCES

Salmonid fishes and crustacean contain significant amounts of astaxanthin and canthaxanthin. The major marine fishery sources of carotenoids are the inexpensive and voluminous crustacean wastes, generated by the seafood industry including aquaculture of shellfish.17–20 In India, for example, the estimated potential of shrimp waste in the country is 150,000–175,000 t.21 Carotenoids can be extracted and concentrated from the crustacean wastes without losing much of the functional properties.10,19,20 Traditionally, the dried shell discards are subjected to processes that include pulverization, enzyme treatments, extraction with organic solvents, and supercritical extraction using carbon dioxide. The disadvantages of the process are high-energy requirement for solvent removal, heat sensitivity of the pigments, and presence of solvent residues in the product. Table 7.1 shows the composition of carotenoids in crustacean offal.22 Use of proteolytic enzymes could enhance extractability of carotenoids, by detaching the bound proteins. Astaxanthin in the form of carotenoprotein was extracted with a yield of 49% by treating the shrimp waste with proteolytic enzymes such as trypsin in the presence of ethylenediamine tetra acetic acid (EDTA) at a pH of 7.7 and at 4°C. At higher temperatures, extractability increased when EDTA was not required. Employing the enzymes pepsin and papain the extractability of carotenoprotein from brown shrimp was 55%. In another study, the shell waste was treated with trypsin isolated from cod viscera, which resulted in extraction of 64% of astaxanthin from the waste. Instead of trypsin, other enzymes such as alcalase and pancreatin have also been used. Alcalase was more efficient than pancreatin, increasing the recovery of astaxanthin from 4.7 to 5.7 mg/100 g of dry waste, at a degree of hydrolysis (DH) of 12%.6,18 However, in general, trypsin recovered highest amount of carotenoids from all types of head wastes. Carotenoids, being sensitive to

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TABLE 7.1 Composition of Carotenoids in Crustacean Offal Carotenoids

Shrimp

Astaxanthin Astaxanthin monoester Astaxanthin diester Asatacene Lutein Zexanthin

3.9 10.7 74.3 — 0.6

Crab 21.2 5.1 56.8 3.3 8.2 4.6

Note: The values are expressed as percent of total. Source: Adapted from Shahidi, F., in Seafood: Chemistry, Processing Technology and Quality, Chapman & Hall, Glasgow, UK, 1994, 320.

TABLE 7.2 Concentration of Astaxanthin Extracted from Soy Oil and Solvent from Shrimp Waste Astaxanthin (mg/100 g Dry Residue) Enzyme No enzyme Alcalase Alcalase Pancreatin Pancreatin

Sample Control Insoluble fraction Soluble fraction Insoluble fraction Soluble fraction

Oil 4.9 ± 2.0 5.15 ± 0.6 1.71 ± 0.3 4.71 ± 01.0 1.56 ± 0.9

Solvent 4.9 ± 2.0 11.39 ± 0.1 — 10.8 ± 1.6 —

Note: Degree of hydrolysis, 12.0; —, not determined. Source: Reprinted from de Holanda, H. D. and Netto, F. M., J. Food Sci., 71, C298, 2006. With permission from Blackwell.

oxidation, may require antioxidants during extraction. These compounds not only protected the pigments against oxidation, but also helped them retain the bright red orange color of the carotenoprotein complex.18–20 Apart from shrimp shell, crab waste could also be a source of carotenoids. Instead of plant or fish proteases, microbial proteases from Pseudomonas aeruginosa was employed to extract the pigment from crab. The crab shell waste was fermented with the bacterium for a period of 7 days, which yielded 55% of the carotene-protein.23 Methods for identification and quantification of astaxanthin, lycopene, and β-carotene have been discussed.9,24 Table 7.2 presents concentration of astaxanthin extracted from soy oil and solvent from Xiphopenaeus kroyeri shrimp waste after enzyme treatment with proteolytic enzymes.6 The head wastes of four commercially important shrimp species from India, namely, naturally available (wild) Penaeus monodon, P. indicus, Metapenaeus monocerous,

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and the aquacultured P. monodon, were extracted for their carotenoids as caroteneprotein complexes using trypsin or pepsin. These carotenoprotein were extracted by continuous stirring of homogenized shrimp head waste in citrate phosphate buffer at pH 4, and 7.6 with trypsin or pepsin at optimum temperature 45–55°C. After 2 h of continuous stirring, the sample was filtered using filter cloth. Then the filtrate was boiled at 100°C and kept overnight at an ambient temperature (28°C). Carotenoprotein precipitate was filtered under vacuum and was stored at −18°C till it was used for extraction of carotenoids using chloroform and methanol. It was observed that enzymatic extraction of carotenoids gave maximum yield over the traditional solvent extraction process and supercritical carbon dioxide extraction. Trypsin recovered the highest amount of carotenoids from all types of head wastes. The percent of recovery varied with the raw materials and the trend was P. indicus > P. monodon (cultured) > M. monocerous > P. monodon (wild). The heads of these shrimp weighed 67.4, 76.4, 65.1, and 53.3%, of their whole body weights, respectively. Some loss of carotenoids during processing of carotenoprotein cakes (CPC) was noticed. Astaxanthin was the main stable pigment and its proportion in total carotenoids increased in freeze-dried products but loss of β-carotene and their derivatives was noticed due to their sensitivity to oxygen, light, and intermediary hydroperoxide radicals of lipid peroxidation. The results showed that all the head wastes contained significant quantities of astaxanthin, although other carotenoids, such as β-carotene, canthxanthin, lutein, zeaxanthin, and crustacyanin were present, although in minor quantities. The CPC isolates could be freeze-dried for long-term storage. Freeze-drying did not affect the stability of the carotenoids. The percentage of astxanthin, β-carotene, canthxanthin, lutein, zeaxanthin, and cryptaxanthin in freeze-dried carotene-proteins from P. monodon (wild) is 79.2, 1.5, 8.1, 1.2, 1.4, and 7.9, respectively. Other shrimp species also retained these carotenoids in their freeze-dried carotene-protein complexes. The predominance of astaxanthin in the carotenoids indicated that both the frozen and freeze-dried carotene-protein

TABLE 7.3 Contents of Major Carotenoids Enzymatically Extracted from Head Wastes of Four Commercially Important Shrimps from India P. monodon (Wild)

P. indicus (Wild)

M. monocerous (Wild)

Carotenoids

Trypsin

Pepsin

Trypsin

Pepsin

Trypsin

Astaxanthin β-carotene Canthaxanthin Lutein Zeaxanthin Crustacyanin

75.3 1.5 7.1 2.0 1.4 7.9

84.5 1.5 4.2 1.9 1.5 5.9

76.4 2.8 Nil 1.7 1.9 Nil

74.5 2.3 Nil 4.0 2.4 Nil

93.9 2.3 Nil 0.4 1.9 2.5

Pepsin 83.6 2.6 Nil 1.6 1.5 6.5

P. monodon (Aquacultured) Trypsin 76.8 4.6 2.4 2.6 4.2 4.6

Pepsin 77.3 1.2 1.5 1.8 6.6 6.5

Source: Adapted from Babu M. et al., Lebensm. Wiss. Technol., 41, 227, 2008. With permission from Elsevier.

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complexes were good sources of natural carotenoids.25 Table 7.3 presents compositions of major carotenoids in enzyme extracts of different shrimp head wastes.25 Carotenes as crude carotene-protein complexes were extracted using hot vegetable or marine oil, generally at a ratio of 1:2 (v/w) at 60°C for 30 min. The extracted oil may be used in the formulation of feed for salmonid fish species, since these species are unable to synthesize the carotenoids de novo.26,27 All these studies showed that the major carotene present in shrimp is astaxanthin, although other carotenoids were also present in minor quantities.

7.6

BIOAVAILABILITY OF CAROTENOIDS

Carotenoids, in general, are bound noncovalently to protein, fiber, or dissolved in oil or in crystalline form such as in carrots, making their optimal absorption difficult. Carotenoids present in animal tissues such as crustacea exist as carotene-protein complexes, as mentioned earlier, whereas in plants they are partially concentrated in chromoplasts. The five principal carotenoids found in human plasma as a result of ingestion of plants include α-carotene, β-carotene, cryptoxanthin, lutein, and lycopene. The release of carotenoids from plant foods occurs only when the cells in the food matrix are disrupted, as is usually the case during food preparation, processing, and mastication. Following release from the food matrix, the major limiting factor governing the extent of absorption of carotenoids is their solubilization in the food digest. Since carotenoids are hydrophobic, their absorption depends not only on the release from the food matrix but also on the subsequent solubilization by bile acids and digestive enzymes culminating in their incorporation into micelles. For this reason, dietary lipids have been considered to be important cofactors for carotenoid biovailability. The major factors limiting the bioavailability of xanthophylls including lutein, zeaxanthin, capsanthin, canthaxanthin, astaxanthin, echionine, and α-cryptoxanthin are their molecular structure, interactions with other nutrients (mainly dietary fat), and the physical disposition of these pigments in the food matrix. For optimal absorption, the xanthophylls must be released from their food matrix and then transferred to lipid micelles in the small intestine. For this reason, dietary lipids have been considered to be important for carotenoid biovailability. In general, the bioavailability of carotenoids has been estimated to be about 10% in raw, uncooked vegetables. Cooking can increase the extractability of carotenoids from the plant matrix thereby improving its bioavailability.28–32 The influence of native structure and cooking on bioavailability is indicated by the example of lycopene, a carotenoid responsible for the distinctive red color of ripe tomatoes, which is usually located within cell membranes. Lycopene is 2.5 times more bioavailable to humans when present in tomato paste than in fresh tomatoes. Cooking tomato products provides better bioavailability of lycopene than raw tomato products. Cooking enhances cis-isomerization in free lycopene with the advantage that cis-isomers are more soluble in bile acid micelles.33 In the case of fishery products such as salmonid fishes and shellfish, cooking and also the action of digestive enzymes in the stomach release carotenoids from the muscle making them bioavailable.28–30,34

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7.7 FUNCTIONAL ROLES OF CAROTENOIDS Carotenoids are known to participate in different cellular activities. The major functions are activities as antioxidants (Section 8.7.1) and vitamin A (Section 8.7.2). These functions provide protection to living organisms against various diseases. Dietary carotenoids are considered to be beneficial in the prevention of diseases including certain cancers and eye disorders. They serve as precursors of vitamin A, and have a role in protecting the immune system and cellular health in the body. The scientific literature during the period 2001–2005 on these aspects, particularly pertaining to the carotenoid, astaxanthin have been reviewed.35–38 Some dietary carotenoids may have more protective roles than others, and certainly they have different antioxidant capacities in vitro. For example, zeaxanthin and lutein are the essential components of the macular pigment in the eye. These two xanthophylls showed the strongest association between dietary intake and reduced risk of macular degeneration. However, lycopene, which is a potent antioxidant, prevents cardiovascular diseases and prostate cancer. However, a combination of carotenoids and other antioxidants in food products appear to be more effective than dietary supplements of single component.39,40 Nevertheless, it has been suggested that since these compounds are not essential nutrients, there is no need to use the term as “carotenoid deficiency.”41 The major biological activities of carotenoids are discussed in Sections 7.7.1 and 7.7.2.

7.7.1

ANTIOXIDANT ACTIVITY

The energy needed by a living cell is generated in the mitochondria via multiple oxidative chain reactions, which are accompanied by the production of a large amount of reactive oxygen species (ROS) such as superoxide anion (O2•−), hydroxyl radical (HO•), alkoxy (RO•), and hydroperoxy (HOO•) radicals peroxynitrite anion (ONOO −). ROS can damage deoxyribonucleic acid (DNA) and promote diseases such as cancer, and hence need to be neutralized to maintain the proper functions of cellular components and to protect the cell from degradation and ageing. Carotenoids such as astaxanthin and lutein provide health benefits by neutralizing ROS. In exerting the antioxidant activities, the carotenoids get oxidized. A number of apocarotenals have been identified recently as a result of oxidation of β-carotene. These were also detected in processed food products such as mango juice and dried apricot.42 When carotenoids are used in combination, their antioxidant activities may vary. Antioxidant activity of mixtures containing different proportions of α-tocopherol, β-carotene, and lycopene was evaluated by the extent of inhibition of spontaneous autoxidation. It was observed that there was no synergism that occurred among the three.43 The antioxidant activity, ability to protect UV-light, antiinflammatory and other properties of astaxanthin and its possible role in human health problems have been examined. The carotenoid was found to protect the body tissues from oxidative damage with daily ingestion of natural astaxanthin, suggesting a practical and beneficial strategy in health management.44 Astaxanthin has stronger antioxidant activity, about 10 times higher than β-carotene and more than 500 times than α-tocopherol. The antioxidant activity has been demonstrated in a number of biological membranes. It has preventive effects against aflatoxin, and has been used to enhance the

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immune response of fish and shrimp for maximum survival and growth. Also, natural microalgal astaxanthin has proved to be superior in its bioactivity as compared with synthetic form. An amount of 25–100 ppm of carotenoids in the final feed has been considered to give desired pigmentation in various feeds for salmonid fishes.45 7.7.1.1

Assay of Antioxidant Activity

Validated and specific assays are needed to study antioxidant activities of various compounds. Assays of antioxidant activities can roughly be classified into two, depending upon the reactions involved, namely, assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein (LDL) autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample’s antioxidant concentrations. ET-based assays include the total phenols assay, trolox equivalence antioxidant capacity (TEAC), ferric ion-reducing antioxidant power (FRAP), and total antioxidant potential assay using a Cu(II) complex as an oxidant and 1,1-diphenyl-2-picrylhydrazyl (DPPH). In addition, other assays include scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical. On the basis of these analyses, it was suggested that the total phenols assay be used to quantify an antioxidant’s reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity.46 7.7.1.2

Antioxidant Activities of Carotenoids Containing Marine Products

Since several microalgae are rich sources of carotenoids, they also exhibit potent antioxidant activities. This has been shown with the microalga, D. salina. Extraction of antioxidants from the algae was optimized recently by combining pressurized liquid extraction with ethanol or hexane. Optimum extraction was obtained with ethanol at 160°C, although hexane extracts provided ideal antioxidant activity. Results pointed out that the extracts contained, besides all-trans-β-carotene and isomers, several different minor carotenoids that seemed to make a contribution to the antioxidant activity of the extracts.47 The antioxidant properties of acetone extracts of green microalga, Botryococcus braunii were evaluated using in vitro model systems such as DPPH, hydroxy radical scavenging, and lipid peroxidation in human LDL and rat tissues. Acetone extracts of B. braunii (equivalent to 10 ppm total carotenoid) exhibited 71 and 67% antioxidant activities in DPPH and hydroxyl radical scavenging model systems, respectively. Similarly, the extract also showed 72, 71, and 70% antioxidant activities in the liver, brain, and kidney of rats, respectively. LDL oxidation induced by Cu2+ ions was also protected (22, 38, and 51%) by the algal extract in a dose-dependent manner (4, 6, and 8 ppm levels of total carotenoid). Thiobarbituric acid (TBA) reactive substance concentration in the blood, liver, and kidney of rats were also significantly decreased in B. braunii-treated samples compared with those

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of control. It was suggested that carotenoids (violaxanthin, astaxanthin, lutein, zeaxanthin, and β-carotene; 75% of the carotenoids being lutein) and also chlorophylls a and b, and α in the B. braunii acetone extract exhibited antioxidant activities suggesting that the microalga can be effective in protecting biological systems against various oxidative stresses in vitro.48 In view of the excellent antioxidant activities, microalgae could be incorporated in diets as antioxidants.47 Shellfish has been shown to possess antioxidant activity. Free radical scavenging properties of hepatopancreas extracts of the shrimp, Pleoticus muelleri were evaluated by electron paramagnetic spin resonance (EPR) spectrometry against the DPPH radical. Feeding trials were carried out on juveniles (1 g initial weight) held in aquaria. Each diet, with different concentrations of vitamins A and E were fed for 25 days. The control groups were fed with fresh squid mantle and with a vitamin-free diet. For all of the diets, the extracts exhibited strong DPPH radical scavenging activity, suggesting that the tissue is a powerful natural antioxidant. Individuals fed with different concentrations of vitamin E showed the strongest effect on the DPPH radicals, reducing the DPPH radicals to 50%, after an incubation period of 3 min. In contrast, the extracts of control animals, fed with squid mantle, had the weakest antioxidant activity (4%). These data indicated that the presence of vitamin E in the diet could provide immediate protection against free radicals.49 The antioxidant activities of muscles of lean and fatty fish have been measured using model systems. The model system uses known antioxidants such as δ-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), and propyl gallate in a muscle-oil emulsion system. Oxidation was measured using thiobarbituric acid–reactive substances (TBARS) and sensory analysis. Using the method, it was observed that in both the lean muscle–canola oil model system and in herring muscle, the hydrophilic antioxidants, propyl gallate and TBHQ, were more effective in providing oxidative stability than the lipophilic antioxidants, δ-tocopherol and BHT.50

7.7.2

VITAMIN A ACTIVITY

Carotenoids, in general, serve as precursors of vitamin A, and therefore, are termed as provitamin, which is converted by the body to vitamin A. The most potent dietary precursor of vitamin A is β-carotene (see also Chapter 9). Based on the functional role as precursors of vitamin A, the carotenoids are categorized as follows: (1) vitamin A precursors that do not pigment, such as α-carotene, (2) pigments with partial vitamin A activity, such as cryptoxanthin, (3) nonvitamin A precursors that do not pigment or pigment poorly, such as violaxanthin and neoxanthin, and (4) nonvitamin A precursors that pigment, such as lutein, zeaxanthin, and canthaxanthin. Provitamin A carotenoids are synthesized exclusively by higher plants and photosynthetic microorganisms.41 Carotenoids received from the diet act as precursors for the production of retinoids. Retinoids such as retinol (vitamin A), retinal (the main visual pigment), and retinoic acid (which controls morphogenesis) play important functions as visual pigments and signaling molecules. The human body converts β-carotene to vitamin A via body tissues as opposed to the liver. Its deficiency, reported as the common dietary problem, affecting children worldwide, leads to xerophthalmia and blindness. Controlling such deficiency in developing countries may require not only vitamin A supplementation but also the introduction of new plant-derived

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foods with increased provitamin A (β-carotene) levels. However, canthaxanthin, a red carotenoid pigment, chemically related to β-carotene, has no vitamin A activity. The xanthophylls lutein and zeaxanthin, found in the macular region of the retina, have been implicated in helping to protect the eye against oxidative damage and cataracts. Lutein helps shield the eye from damaging sunrays. With advancing age, lutein declines in the eye.3,51–54

7.8 BENEFITS OF DIETARY CAROTENOIDS The functional benefits of carotenoids have been attributed to their capacity to quench singlet oxygen and act as free radical scavengers in vivo. For example, the benefits associated with astaxanthin intake include cardiovascular disease prevention, anticancer activity, boosting of immune system, bioactivity against Helycobacter pylori, and cataract prevention. An inverse relationship between the dietary intake of carotenoid-rich foods (shellfish, fruits, and vegetables) and the incidence of some cancers (lung, breast, colon, and prostate), UV-induced skin damage (erythema or sunburn), coronary heart disease, cataracts, and macular degeneration has been reported.53,54 Table 7.4 gives the effects of β-carotene on various cells and cellular systems.35,45

7.8.1

HYPERCHOLESTEROLEMIC ACTIVITY

The protective role of antioxidant carotenoids could be an important function in controlling the oxidative modification of LDL, a key early step in the pathogenesis of arthrosclerosis. A high level of LDL cholesterol is a primary risk factor for the disease. When LDL is oxidized (by endothelial cells, smooth muscle cells, and macrophages) numerous chemical changes occur. The content of polyunsaturated fatty acids (PUFA) is reduced with increase in lipid peroxides and aldehydes. The products of LDL oxidation are cytotoxic, leading to induction of endothelial cell dysfunction. Oxidized LDL also affects secretion of various growth factors and cell signals that can affect atherosclerosis. Antioxidants nutrients, α-tocopherol, ascorbic acid, and β-carotene, have been shown to decrease the susceptibility of LDL to

TABLE 7.4 Some Effects of β-Carotene on Various Cells and Cellular Systems Cells Mammary and mouse Human lymphocytes Human polymorpho mononuclear leukocytes Human peripheral blood mononuclear cells Melanoma T cells

Activity Inhibits alveolar lesions Inhibits proliferation Prevents decrease in antigen expression Prevents decrease in antigen expression Increases differentiation, reduces adenylate cyclase Suppress activation

Source: Adapted from Dufosse, L. et al., Trends Food Sci. Technol., 16, 389, 2005; Hussein, G. et al., J. Natural Prod., 69, 443, 2005.

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oxidation in vitro. In addition, they can also increase the resistance of LDL to oxidation when given to animals and humans. Because levels of these nutrients in plasma can be increased by dietary supplementation with minimal side effects, they may be effective in the prevention of coronary artery disease.55 Carotenoids including astaxanthin, can have hypercholesterolemic activities. Astaxanthin can also act in modulating blood fluidity, ameliorating nitric oxides, and improving vascular elastin and coronary wall thickness in hypertension.36 Lycopene and β-carotene are the major carotenoids along with other nutrients in tomatoes such as folate, potassium, vitamin C, and vitamin E that are associated with reduced risk of cardiovascular disease. The cardioprotective functions provided by the carotenoids and other nutrients may include the reduction of LDL, cholesterol, homocysteine, platelet aggregation, and blood pressure.33,55–59

7.8.2

ANTICANCER AND OTHER ACTIVITIES

Carotenoids such as astaxanthin provide protections against several forms of cancer. Astaxanthin has markedly attenuated the promotion of hepatic metastasis induced by restraint stress in mice. This effect was suggested to be through inhibition of the stress-related peroxidation. Recent studies on human and animal cells have demonstrated that a connexin 43 (Cx43) protein, the most widely expressed connexin in tissues, is upregulated by chemo-preventive retinoids and carotenoids leading to a decreased neoplasia. Reports have also described enhanced cell-mediated immune response by carotenoids. Astaxanthin has been found to suppress T-cell activation comparably to two commonly used antihistamines, cetirizine and azelastine. In addition, astaxanthin has a profound role in combating inflammatory responses.33,36 Oxidative stress induced by hyperglycemia may possibly cause dysfunction of pancreatic β-cells and various forms of tissue damage in patients with diabetes mellitus. Animal studies showed that astaxanthin ameliorated the progression and acceleration of diabetic nephropathy. It also preserved the β-cell function of insulin secretion and decreased the higher level of blood glucose in the diabetic animal.36,57,59

7.8.3

FUNCTIONS OF CAROTENOIDS IN AQUACULTURE

The growth of the aquaculture industry is placing heavy demand on carotenoids for several functions, which include pigmentation, antioxidant functions, as dietary source of provitamin A, cellular protection from photodynamic damage, enhancement of growth and reproductive potential. Astaxanthin is widely used in salmonid and crustacean aquaculture to provide the pink color characteristic of that species. This application has been well documented for over two decades and is currently the major market driver for the pigment. Synthetic astaxanthin dominates the world market but recent interest in natural sources of the pigment has increased substantially. Evidence suggests that these pigments may perform vital roles in growth and reproductive success in crustaceans. Feeding trials have shown that broken spores of the alga Haematococcus pluvialis can be used in the feed to pigment the muscle of trout and salmon.4 Dietary carotenoids are the sole biological precursors of retinoids in crustaceans, which in turn play a prominent role in many developmental processes including

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embryonic development and differentiation of various cell types. The presence of receptors of retinoic acid in crustaceans and presence of retinoids in the neuroendocrine complex and in reproductive tissue suggests an important role of these metabolites in shrimp physiology.60 In addition, the effect of carotenoid sources on the skin coloration of fish has been reported. Red porgy (Pagrus pagrus) were fed a diet supplemented with commercial astaxanthin, β-carotene, or lycopene. The carotenoids were added to the level of 100 ppm in each diet, whereas a noncarotenoidsupplemented diet served as control. Astaxanthin increased total carotenoid content in the dorsal skin area whereas β-carotene and lycopene seemed to have had no significant effect. Astaxanthin was the only carotenoid source that had a significant effect on skin hue, promoting a reddish coloration to the dorsal skin area and a ventral hue similar to wild red porgy.61 The fast-growing tropical lobster Panulirus ornatus is a good aquaculture candidate. A 12-week experiment, assessing growth, survival, and tissue carotenoid levels of juvenile P. ornatus was conducted. Lobsters were fed either pelleted feeds supplemented with astaxanthin and containing 30–120 mg total carotenoid per kg or one of two fresh mussel reference feeds—blue Mytilus edulis and green-lipped Perna canaliculus. Whole lobster carotenoid increased with increasing dietary astaxanthin. Although dietary astaxanthin, over the investigated range, did not affect growth rate or survival, there was a dose–response increase in tissue carotenoid contents and darkening of the exoskeleton pigmentation.62 Canthaxanthin is an intermediate in the metabolism of β-carotene to astaxanthin. Canthaxanthin is used as feed additive for coloring salmonids to enhance the reddish color of flesh of fresh and processed fish. Edible shrimps and prawns (e.g., Penaeus japonicus), salmon, and sprat contain a certain amount of canthaxanthin. Wild trout contains canthaxanthin in a ratio approximately 1:5 to astaxanthin. The European Union as per EU Council Directive 70/524/EEC as E-161g has authorized canthazanthin for use in animal feed as a coloring agent in poultry feeds, fish feeds, and also for coloring Strasborg sausage and medical products.1 The pigment is currently authorized for use as a coloring agent up to a level of 80 mg/kg in feeds for salmonids; and when combined with astaxanthin, a maximum level of 100 mg/kg feed is permitted. For salmonids, for example, trout, this would result in a concentration of about 4 mg/kg flesh.1 The efficiency of utilization of dietary astaxanthin using microalgae for flesh pigmentation of Atlantic salmon and rainbow trout has been demonstrated. For salmon, astaxanthin is even considered as a vitamin essential for the proper development and survival of juveniles. However, the predominant source of carotenoids for salmonids has been synthetic astaxanthin, which has been used for pigmentation for the last several years, with United States Food and Drug Administration (U.S. FDA) approval in 1996.45

7.8.4

POULTRY FEED

Canthaxanthin alone or together with astaxanthin is also used as a source of pigment for broilers to modify the color of egg yolk and also to obtain a yellow hue of the skin. The deposition of canthaxanthin in hen eggs and broiler skin/fat is related to the concentration of the pigment in the animal diet; however, this relationship is not linear. Many nutritional, physiological and environmental factors are involved in the process of canthaxanthin deposition that explains the great variability of the results

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obtained in practice, especially for fish. The Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) Joint Expert Committee on Food Additives (JECFA) (1996) assessed intake of canthaxanthin supplied through animal feeds and found an acceptable daily intake (ADI) of 0.03 mg/kg body weight. Canthaxanthin has a very low toxicity. No carcinogenic effects were noted in rats or mice fed the carotenoid at 1000 mg/kg body weight/day for 2 years. Long-term ingestion caused only deposits of the pigment in retina; such deposits are reversible.63 The algal xanthophylls, peridinin, fucoxanthin, and others have also uses as yolkpigmenting agent for the poultry.4

7.8.5

BIOTECHNOLOGY

Interests in the dietary functions of carotenoids has led to studies on their biosynthesis in plants and microorganisms, the pathways for which have been elucidated in recent years together with identification of genes for their biosynthesis. Further, some of the genes have also been successfully engineered to overproduce carotenoids of interest in plants. However, despite spectacular achievements in the metabolic engineering of plant carotenogenesis, much work is still ahead to better understand the regulation of carotenoid biosynthesis and accumulation in plant cells. New genetic and genomic approaches are now in progress to identify regulatory factors that might significantly contribute to improve the nutritional value of plant-derived foods by increasing their carotenoid levels.64

7.9

COMMERCIAL STATUS

The worldwide carotenoid demand was around U.S.$935 million in 2005, and is growing at an estimated average annual rate of 2.9%.44,65 Currently, the algal carotenoids, β-carotene and astaxanthin command high price. β-carotene from the alga D. salina was the first commercialized high-value algal product and there are now major producers of the product in Australia, United States and Israel. The β-carotene is sold mainly as an extract or suspension in vegetable oil, or as β-carotene-rich algal powder for use in the food-coloring industry, the health food, and pharmaceutical industry. Other common sources of natural astaxanthin are the green algae H. pluvialis, and the red yeast, Phaffia rhodozyma. In food and pharma industries, β-carotene is in demand as a natural colorant, cancer preventive, and free radical trapper. Their antioxidant activity has caused a surge in the nutraceutical market for the encapsulated product.4 A commercial form of astaxanthin, Zanthin, was approved by the U.S. FDA for use as a dietary supplement. Zanthin is a supercritical carbon dioxide extract of H. pluvialis.66 Astaxanthin is approved by U.S. FDA. Formulations containing astaxanthin are soft gelatin capsules containing 100 mg equivalent of total carotenoids, skin care cream containing astaxanthin as one of the ingredients, food and feed formulations for shrimp and fish. In conclusion, carotenoids are recognized antioxidants and, therefore, with increasing awareness of the dietary functions of antioxidants, the demand for carotenoids is increasing. Carotenoids are also highly sought after as natural food colors. The current production source is microalgae, which requires expensive and cumbersome cultivation techniques. Alternately, the voluminous marine shellfish wastes

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could offer a viable alternative of carotenoids, particularly astaxanthin. With increasing recognition of their health benefits, carotenoids from marine sources have potential both as coloring agents and bioactive compounds to capture markets, including for the rising aquaculture industry.

REFERENCES 1. European Commission, Opinion of the Scientific Committee on Animal Nutrition on the use of canthaxanthin in feeding stuffs for salmon and trout, laying hens, and other poultry, Adopted on April 17, 2002, http://www.eu.nl/food/fs/sc/scan/out81_en.pdf# search=%22canthaxanthin%20EU%22, accessed on April, 2006. 2. Britton, G. et al., Carotenoproteins, in Carotenoid Chemistry and Biochemistry, Britton, G. and Goodwin, T. W., Eds., Pergamon, Oxford, 1981, p. 237. 3. Swanson, M. A. and Evenson, P., Nutritional additives, in Food Additives, 2nd ed., Branen, A. L. et al., Eds., Marcel & Dekker, NY, 2002, p. 225. 4. Anderson, M. et al., Different patterns of carotenoids composition and photosynthesis acclimation in two tropical red algae, Mar. Biol., 149, 653, 2006. 5. Borowitz, M. A., Products from microalgae, Infofish Int., 5, 21, 1993. 6. de Holanda, H. D. and Netto, F. M., Recovery of components from shrimp (Xiphopenaeus kroyeri) processing waste by enzymatic hydrolysis, J. Food Sci., 71, C298, 2006. 7. Sachindra, N.M., Bhaskar, N., and Mahendrakar, N.S., Carotenoids in solonocera indica and Aristeus alcocki, Deep-sea shrimp from Indian waters, J. Aquatic Food Prod. Technol., 15, 5, 2006. 8. Shahidi, F., Metusalach, P., and Brown, J. A., Carotenoid pigments in seafoods and aquaculture, Crit. Rev. Food Sci. Nutr., 38, 1, 1998. 9. Breithaupt, D. E., Identification and quantification of astaxanthin esters in shrimp (Pandalus borealis) and in a microalga (Haematococcus pluvialis) by liquid chromatography-mass spectrometry using negative ion atmospheric pressure chemical ionization, J. Agric. Food Chem., 52, 3870, 2004. 10. Sachindra, N. M., Bhaskar, N., and Mahendrakar, N. S., Carotenoids in different body components of Indian shrimps, J. Sci. Food Agri., 85, 67, 2005. 11. Ip, P.-F. and Chen, F., Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark, Proc. Biochem., 40, 733, 2005. 12. Orset, S. et al., Spray-drying of the microalga Dunaliella salina: effects on β-carotene content and isomer composition, J. Agric. Food Chem., 47, 4782, 1999. 13. Shi, X.-M., Jiang, Y., and Chen, F., High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture, Biotechnol. Progr., 18, 723, 2002. 14. Valderrama, J. O., Perrut, M., and Majewski, W., Extraction of astaxantine and phycocyanine from microalgae with supercritical carbon dioxide, J. Chem. Eng. Data, 48, 827, 2003. 15. Li, H.-B., Jiang, Y., and Chen, F., Isolation and purification of lutein from the microalga Chlorella vulgaris by extraction after saponification, J. Agric. Food Chem., 50, 1070, 2002. 16. Sarada, P., Pillai, M. G., and Ravisankar, G. A., Phycocyanin from Spirulina sp: Influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin, Proc. Biochem., 34, 795, 1999. 17. Sachindra, N.M. and Mahendrakar, N.S., Process optimization for extraction of carotenoids from shrimp waste with vegetable oils, Bioresource Technol., 96, 1195, 2005. 18. Chakrabarti, R., Carotenoprotein from troical brown shrimp shell waste by enzymatic process, Food Biotechnol., 16, 81, 2002.

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19. Sachindra, N.M., et al., Recovery of carotenoids from ensilaged shrimp waste, Bioresource Technol., 98, 1642, 2007. 20. Cano-Lopez, A., Simpson, B. K., and Haard, N. F., Extraction of carotenoprotein from shrimp processing waste with the aid of trypsin from Atlantic cod, J. Food Sci., 52, 503, 1987. 21. MPEDA, Marine Products Export Development Authority, Cochin, India, Annual Report, 2006. 22. Shahidi, F., Seafood processing by-products, in Seafood: Chemistry, Processing Technology and Quality, Shahidi, F. and Botta, J. R., Eds., Chapman & Hall, Glasgow, UK, 1994, p. 320. 23. Sachindra, N.M., Bhaskar, N., and Mahendrakar, N.S., Carotenoids in crabs from marine and fresh waters of India, Lebensm. Wiss. U. Technol., 38, 221, 2005. 24. Kanchan, N. and Patrick, L. H., Isolation of lycopene and β-carotene, http://cas. bellarmine.edu/chem117a/lab/lycopene.htm, 1999. 25. Babu, M. et al., Effect on carotenoid pigments during enzymatic extraction and freezedrying of carotenoprotein from shrimp head waste, Lebensm. Wiss. Technol., 41, 227, 2008. 26. Shahidi, F. and Synowiecki, J., Isolation and characterization of nutrients and valueadded products from snow crab (Chinocecetes opilia) and shrimp (Pandalus borealisa) processing discards, J. Agric. Food Chem., 39, 1527, 1991. 27. Chen, H. M. et al., Color stability of astaxanthin pigmented rainbow trout under various packaging conditions, J. Food Sci., 49, 1337, 1985. 28. Faulks, R. M. and Southon, S., Carotenoids, metabolism and disease, in Handbook of Nutraceuticals and Functional Foods, Wildman, R. E. C., Ed., CRC Press, New York, 2001, p. 147. 29. Faulks, R. M. and Southon, S., Challenges to understanding and measuring carotenoid bioavailability, Biochim. Biophys. Acta, 1740, 95, 2005. 30. Yeum, K. and Russell, R. M., Carotenoid bioavailability and bioconversion, Annu. Rev. Nutr., 22, 483, 2002. 31. Parada, J. and Aguilera, J. M., Food microstructure affects the bioavailability of several nutrients, concise review, J. Food Sci., 72, R21, 2007. 32. van het Hof, K. H. et al., Dietary factors that affect the bioavailability of carotenoids, J. Nutr., 130, 03, 2000. 33. Omoni, A. O. and Aluko, S., The anti-carcinogenic and anti-atherogenic effects of lycopene: a review, Trends Food Sci. Tech., 16, 344, 2005. 34. Zaripheh, S. and Erdman, J. W., Factors that influence the bioavailability of xanthophylls, J. Nutr., 132, 531S, 2002. 35. Hussein, G. et al., Astaxanthin, a carotenoid with potential in human health and nutrition, J. Nat. Prod., 69, 443, 2005. 36. Higuera-Ciapara, L., Valenzuela, F., and Goycoolea, F. M., Astaxanthin: a review of its chemistry and applications, Crit. Rev. Food Sci. Nutr., 46, 85, 2006. 37. Wildman, R. E. C., Handbook of Nutraceuticals and Functional Foods, CRC Press, Boca Raton, FL, 2001, p. 143. 38. Krinsky, N. I. and Johnson, E. J., Carotenoid actions and their relation to health and disease, Mol. Aspects Med., 26, 459, 2005. 39. Krinsky, N. J., Cellular aspects of carotenoid actions, in Handbook of Antioxidants, Cadenas, E. and Packer, L., Eds., Marcel Dekker, New York, 1996, p. 315. 40. Wildman, R. E. C., Handbook of Nutraceuticals and Functional Foods, CRC Press, Boca Raton, FL, 2001, p. 542. 41. Olson, J. A., Vitamin A, retinoids and carotenoids, in Modern Nutrition in Health and Disease, 8th ed., Shils, M. E., Olson, J. A., and Shiki, M., Eds., Williams & Wilkins, Baltimore, 1994, p. 287.

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42. Rodrigueza, E. B. and Rodriguez-Amaya, D. R., Formation of apocarotenals and epoxycarotenoids from β-carotene by chemical reactions and by autoxidation in model systems and processed foods, Food Chem., 101, 563, 2007. 43. Castro, L. A. et al., Optimization of the antioxidant capacity of a mixture of carotenoids and α-tocopherol in the development of a nutritional supplement, Food Res. Int., 38, 861, 2005. 44. Guerin, M. et al., Haematococcus astaxanthin: applications for human health and nutrition, Trends Biotechnol., 21, 210, 2003. 45. Dufosse, L. et al., Microorganisms and microalgae as sources of pigments for food use: a scientific oddity or an industrial reality? Trends Food Sci. Technol., 16, 389, 2005. 46. Huang, D. D., Ou, R., and Prior, R. L., The chemistry behind antioxidant capacity assays, J. Agric. Food Chem., 53, 1841, 2005. 47. Miguel, H. et al., Optimization of the extraction of antioxidants from Dunaliella salina microalga by pressurized liquids, J. Agric. Food Chem., 54, 5597, 2006. 48. Rap, A. R. et al., Antioxidant activity of Botryococcus braunii extract elucidated in vitro models, Agric. Food Chem., 54, 4593, 2006. 49. Diaz, A. C. et al., Antioxidant activity in hepatopancreas of the shrimp (Pleoticus muelleri) by electron paramagnetic spin resonance spectrometry, J. Agric. Food Chem., 52, 3189, 2004. 50. Raghavan, S. and Hultin, H. O., Model System for testing the efficacy of antioxidants in muscle foods, J. Agric. Food Chem., 53, 4572, 2005. 51. Mahan, L. K. and Escott-Stump, S., Krause’s Food, Nutrition & Diet Therapy, 9th ed., Saunders, W. B., PA, 2000. 52. Handelman, G. J., The evolving role of carotenoids in human biochemistry, Nutrition 17, 818, 2001. 53. Fraser, P. D. and Bramley, P. M., The biosynthesis and nutritional uses of carotenoids, Prog. Lipid Res., 43, 228, 2004. 54. Rudge, K., Foods with Protective Benefits, IFIS Publ., http://www.foodsciencecentral. com/fsc/ixid3738, 2001. 55. Jiala, I. and Fuller, C. J., Oxidativity modified LDL and atheroscelorosis: an evolving plausible scenario, Crit. Rev. Food Sci. Nutr., 36, 341, 1986. 56. Willcox, J. K. et al., Tomatoes and cardiovascular health, Crit. Rev. Food Sci. Nutr., 43, 1, 2003. 57. Willcox, J. K., Ash, S. L., and Catignani, G. L., Antioxidants and prevention of chronic disease, Crit. Rev. Food Sci. Nutr., 44, 275, 2004. 58. Johnson, E. J., The role of carotenoids in human health, Nutr. Clin. Care, 5, 56, 2002. 59. Stahl, W. and Sies, H., Bioactivity and protective effects of natural carotenoids, Biochim. Biophys. Acta, 1740, 101, 2005. 60. Cabello-Linan, M. A. et al., Bioactive roles of carotenoids and retinoids in crustaceans, Aquaculture Nutr., 8, 299, 2002. 61. Chatzifoti, S. et al., The effect of different carotenoid sources on skin coloration of cultured red porgy (Pagrus pagrus), Aquacult. Nutr., 36, 15, 2001. 62. Barclay, M. C. et al., Comparison of diets for the tropical spiny lobster Panulirus ornatus: astaxanthin-supplemented feeds and mussel flesh, Aquaculture, 12, 117, 2006. 63. Anonymous, Safety assessment and potential health benefits of food components based on selected scientific data, Crit. Rev. Food Sci. Nutr., 39, 203, 1999. 64. Botella-Pavý´, A. P. and Rodrý´guez-Concepcio, M., Carotenoid biotechnology in plants for nutritionally improved foods, Physiol. Plantarum 126, 369, 2006. 65. www.bccresearch.com, accessed on September 2007. 66. Ohr, L. M., Riding the nutraceutical wave, Food Technol., 95, August 2005, www.ift.org.

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Sources of 8 Marine Vitamins and Minerals 8.1

INTRODUCTION

Vitamins and minerals are essential for life since their deficiencies lead to various health disorders. Marine sources are rich in several vitamins and minerals. Marine fatty fish species contain significant amounts of fat-soluble vitamins A and D. Seafood items are rich in important minerals compared to red meat. Seaweed species contain minerals such as calcium and phosphorus and also some vitamins, such as vitamin E, in good amounts, which make them nutritionally important for specific applications such as animal and agriculture feeds.1 The vitamins and minerals from these sources are also significantly bioavailable. Products derived from bones and other calciferous tissues of seafood processing wastes have good uses because of their high mineral content. An introductory note on the general aspects of vitamins and minerals is provided before the discussion on marine sources of vitamins and minerals and their nutraceutical properties.

8.2

VITAMINS

Vitamin A is available in fat, in which it remains dissolved. The human body converts β-carotene in the diet into vitamin A (see Chapter 7). Vitamin A has a wide variety of functions including specific roles in vision, embryogenesis, cellular differentiation, growth and reproduction, immune status, and taste sensation. Vitamin A deficiency is a public health problem in several countries, according to the World Health Organization. The deficiency causes night blindness in hundreds of thousands of children every year. In addition, its deficiency also causes skin disorders. Vitamin A deficiency has been treated worldwide either by single intramuscular injection of large amounts of the vitamin, ranging from 1,00,000 to 2,00,000 international units (IU), or injection at intervals of 6 months to 1 year. Clinical deficiency can also be treated with β-carotene. In the United Kingdom, the recommended nutritional intake (RNI) is 700 μg and 600 μg retinal equivalents (RE) per day for males and females, respectively (1 RE is equivalent to 1 µg of retinal). The U.S. recommended dietary allowance (RDA) is 1000 μg RE/day for men and 800 μg RE/day for women. The recommended dietary intake (RDI) values according to the World Health Organization are 600 μg and 500 μg RE/day for men and women, respectively.2 Thiamin (vitamin B1) is relatively labile to heat, ionizing radiation, and acid. The RNI for the vitamin is 0.4 mg/1000 kcal. Assuming the food intake to be equivalent to 2000 kcal/day and 20% loss due to cooking, an amount of 1.4 mg/day is recommended for an adult. In the United Kingdom, there is a mandatory fortification of white and brown flour with thiamin to about 0.24 mg/100 g of flour. Riboflavin is a 239

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water-soluble vitamin. Milk, egg, liver, and lean meat are some of the sources of this vitamin. The RNI for vitamin B1 is 1.3 mg/day for male, 1.1 mg/day for female, and 1.45 mg/day for pregnant women.2 Folate and folic acid (various derivatives of pteroyl glutamic acid) are necessary for the production and maintenance of new cells, especially important during periods of rapid cell division and growth—such as throughout infancy and pregnancy. Folic acid may act as effective antioxidants in vivo, although natural folates cannot be really considered as food antioxidants. Folate deficiency in early pregnancy may lead to increased risk of neural tube defects (NTDs) in infants. Inadequate folate intake has also been associated with esophageal, gastric, and pancreatic cancers, and brain disorders such as depression. The vitamin is also necessary for proper metabolism of amino acids and fatty acids, normal nerve function, formation of red blood cells, and also to keep skin healthy. Deficiency of the vitamin in adults results in inflammation of skin along with redness, numbness of hands and feet, and cracks and fissures appear at the corners of the mouth. Fish is a major dietary source of the vitamin in addition to French beans, cabbage, milk, poultry, and eggs. Vitamin B6 tends to get destroyed with prolonged cooking, which is why the food sources should not be overcooked.3 In nature, folates are covalently bound to macromolecules. Some folate-binding proteins (especially pteroyl monoglutamic acid [PGA]) in fortified milk products decrease the bioavailability of folate.4 Folic acid and its physiological reduced forms, 7,8-dihydrofolate, 5,6,7,8-tetrahydrofolate, and 5-methyltetrahydrofolate have approximately 3.5–7.5-fold, 12–16-fold, and 44–71fold higher antioxidant activities, respectively, than folic acid. Their antioxidant activities are comparable to those of vitamins C and E, which are commonly accepted as the effective water- and lipid-soluble antioxidants. Their activities may become important in view of nutritional supplementation and fortification of food with the vitamin.5 The RNI for adult and children is 0.2 mg/day and 0.4 mg/day for pregnant women. A supplementary dose of 1 mg/day would not cause adverse effects.2 Vitamin D is known as the sunshine vitamin and may be viewed as an important functional nutrient. Vitamin D is generally obtained from exposure to sunshine, although some foods contain this vitamin. The deficiency of this vitamin is associated with rickets, osteoporosis, muscle weakness, and decreased immune function. The highest natural level of vitamin D obtained from sunshine is 225 nmol/L. A diet rich in vitamin D appears to protect people from developing potentially cancerous growths in the colon. Furthermore, this vitamin reduces the risk of pancreatic cancer. Vitamin D also reduces colorectal cancer risk.6 The vitamin D allowance is expressed as IU per day per individual. One IU of vitamin D is defined as the activity of 0.025 µg of cholecalciferol. The recommended intake for vitamin D for most people is 400 IU/day. The U.S. adequate intakes of vitamin D for people aged 51–70 and over 70 are 400 IU and 600 IU, respectively, and the safe tolerable upper intake level based on the U.S. Nutrition Board methodologies has been revised from 2000 to >10,000 IU/day. Elevated doses of the vitamin did not result in any significant alteration in serum calcium or phosphorus levels in healthy public. Supplementation of the vitamin in the diets of the elderly to a level up to 800 IU/day possibly reduces fractures, particularly in those on a marginal calcium and vitamin D intakes.6–8

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Vitamin E (α-tocopherol) is a natural antioxidant. It exists in eight different forms, all of which have been recognized as potent antioxidants in vitro. Deficiency of vitamin E is found in debilitated patients who have had severe and prolonged period of fat malabsorption. The deficiency can be avoided by the consumption of vitamin-rich foods such as oils, green leafy vegetables, or vitamin E supplements. The RDA levels for vitamin E are 22.5 IU or 15 mg for men and women. Men and women taking vitamin E supplements for years have a lower risk of heart disease. The combined effect of vitamins and phytochemicals seems to have much greater power than one nutrient taken alone.9–11 Apprehensions have been raised on the efficacy of vitamin E to function as an antioxidant in the body, when supplied as a supplement. The presence of vitamin E in the diet can offer muscle tissue protection against free radicals. This has been confirmed in hepatopancreas extracts of shrimp Pleoticus muelleri. Feeding trials were carried out on juveniles (1 g initial weight) held in aquaria. Each diet, with different concentrations of vitamins A and E, was tested for a period of 25 days. The control groups were fed with fresh squid mantle and a vitamin-free diet. For all of the diets, the extracts exhibited strong antioxidant activity, suggesting that the tissue is a powerful natural antioxidant. Individuals fed with different concentrations of vitamin E showed the strongest effect on the free radicals. In contrast, the extracts of control animals fed with squid mantle showed the weakest antioxidant activity.12 The RNI for vitamin B12 is 1.5 μg/day in the United Kingdom.2 Vitamin K is a group of homologous fat-soluble compounds derived from 2-methyl-1,4-naphthoquinone. RDI has not been established for the vitamin. However, an intake of 0.2 mg/kg body weight per day is probably adequate.2 Table 8.1 gives the recommended intake and utilization of vitamins.

8.2.1

VITAMIN CONTENTS OF SEAFOOD

The vitamin contents of seafood items have been discussed recently.13,14 Marine fish oils are rich sources of vitamins A, D, and E. Vitamin A is concentrated mostly in fish liver oils. Halibut and cod liver oils are rich sources of vitamins A and D. Sardine contains up to 4500 IU of vitamin A and up to 500 IU of vitamin D per 100 g of meat, with an average of 125 μg/g of oil. The vitamin A found in small fish species is particularly bioavailable.13 The popular fish salmon contains up to 25% and 12% protein and lipids, respectively. A 3.5 oz portion provides 90% of the daily need of vitamin D. Herring, mackerel, salmon, and lake trout contain varying amounts of vitamin D in their tissues. Patients who consumed the amount of vitamin D and calcium contained in daily servings of dairy products and fish were 40% less likely to develop polyps than those who got little or no vitamin D.14 Seafood provides moderate amounts of thiamin. However, much of thiamin is destroyed by heat and oxygen, or is lost in cooking water or when exposed to ionizing radiation. The average content of thiamin in 155 fish species is between 6 and 434 mg/100 g of meat. Fish also contain modest amounts of biotin, folic acid, niacin, and pantothenic acid. The best sources of pyridoxine (vitamin B6) are salmon and tuna, and to some extent shellfish. Modest amounts of riboflavin are present particularly in the dark flesh of some species such as canned herring, mackerel,

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TABLE 8.1 Recommended Intake and Utilization of Vitamins Vitamin

Name

A

Retinol

D E Coenzyme Q K

Calciferol Tocopherol Ubiquinone Menaquinone

C — B1 — B2 B6 — B12 Panthothenic acid a b

Ascorbic acid Biotin Thiamine Nicotinic acid Riboflavin Pyridoxal, pyridoxine, and pyridoxamine Folic acid Cyanocobalamin

Recommended Intake Per Day 700 μg and 600 μg REa per day for males and females, respectively 400 IUb 15 mg Not known No RDI established 0.2 ng/kg body weight per day is suggested 40 mg 0.01–0.02 mg 1.4 mg 10–20 mg 1.1–1.3 mg About 1.25 mg 0.2 mg 1.5 μg 5–10 mg

One RE is equivalent to 1 µg of retinal. One IU of vitamin D is the activity of 0.025 µg of cholecalciferol.

Source: Adapted from Food Standards Agency, UK, http://www.food.gov.uk/, accessed September 2007; Rivlin, R. S., in Handbook of Food and Nutrition, CRC Press, Boca Raton, FL, 2002, 1313.

and pilchard. Pyridoxine is present in fish and shellfish in reasonable amounts, with tuna and salmon being rich in this vitamin. Vitamin B12 (cyanocobalamin) is present in animals as adenosylcobalamin and methylcobalamin. It is required for normal neurological function. Intestinal microflora also provide the vitamin to some extent. In general, seafood contains 0.89–42 μg of vitamin B12 per serving (3 oz). Clams containing 35 μg of vitamin B12 per serving are the richest source of the vitamin followed by crustaceans and flesh fish sources, which contain 3–5 μg of vitamin B12. Shellfish has about 20 μg of the vitamin. Clams, oysters, crab, salmon, and rainbow trout provide 2–14 times the RDA of the vitamin; therefore, adding seafood to the diet is the best way to get the vitamin.15 Fish cannot synthesize vitamin E, and hence the concentration of this vitamin, mainly α-tocopherol, is related to feed. The vitamin E contents of 100 g haddock fillet, shrimp, and scallop is about 0.6 mg.16 Being an antioxidant, vitamin E could be used in food product development. For example, animal feed could be enriched with polyunsaturated fatty acids (PUFAs).

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However, there is a possibility of enhanced lipid oxidation in calf meat, which were fed the feed before slaughter. The oxidation could be controlled by fortification of PUFA-enriched feed with vitamin E. The vitamin was given to the animal in the feed at a concentration of 4 g/day for 90 days before slaughter. This diet increased the α-tocopherol concentration in muscle membrane from 2.6–2.8 to 6.5–7.0 µg/g of fresh weight. The undesirable reactions during storage including high concentrations of oxymyoglobin were controlled efficiently by the presence of a critically high concentration of α-tocopherol in the muscle tissues, suggesting that PUFA-rich meat could be produced without the problem of oxidation using vitamin E.17

8.2.2

VITAMINS IN SEAWEEDS

Seaweeds are rich in vitamins (see also Chapter 9). They are also considered as good sources of vitamin B12.18,19 The vitamin contents in the frond and stem of the edible seaweed Durvillaea antarctica and dried Ulva lactuca were determined. The fronds of D. antarctica contained γ-tocotrienol, δ-tocopherol, and α-tocopherol at the levels, 652 mg/kg, 246 mg/kg, and 179.4 mg/kg, respectively, whereas 258 mg/kg of α-tocopherol was present in the stem. U. lactuca showed a high γ-tocopherol level (963.5 mg/kg).19,20 Feeding vitamin B12-deficient rats with dried purple laver (nori) significantly improved the vitamin B12 level.20

8.2.3

INFLUENCE OF PROCESSING ON VITAMINS

Vitamins are considered to be the most susceptible to heat treatment, but the magnitude of loss depends on the specific vitamins and the conditions employed. The loss may be due to leaching of water-soluble vitamins into the cooking medium, and destruction of unstable vitamins under certain treatment conditions. A combination of oxygen, light, and heat causes a greater loss of nutrients than any one of these factors individually. Folate and vitamin B6 are susceptible to destruction due to oxidation. Riboflavin is reasonably stable during cooking, but is sensitive to light as it decomposes on exposure to ultraviolet rays. The fat-soluble vitamin A and carotenes are relatively stable at normal cooking temperatures, but the high temperatures used in frying can produce oxidative losses and isomerization of the carotenes, with significant losses in biological activity. Vitamin E is slowly destroyed during frying and is decomposed by light.21 Although influence of processing on nutrients is important, current interests are more centered with respect to bioavailability of nutrients in foods essential for the physiological functions of the body.3,14

8.3

MINERALS

Minerals are inorganic elements that retain their chemical identity in a food. Minerals can be divided into two groups: major and trace. Major minerals are those present in amounts greater than 5 g in the human body and include calcium, phosphorus, potassium, sulfur, sodium, chloride, and magnesium. There are more than a dozen trace minerals in the human body. Minerals as micronutrients have immense potential as therapeutic components in the diet. Mineral additives are commercially available in the elemental form. The choice of minerals depends on bioavailability of the

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mineral in a particular salt form, their solubility, and effects on final products.22,23 The major minerals are discussed below. The topics include their availability from marine products, development of some functionally active mineral products from bones, and their applications. Calcium is one of the most important minerals essential for the human body to perform many important physiological functions. Calcium is needed to form bones and keep them strong. Deficiency in calcium will give rise to hypocalcia symptom or even more serious calcium-malnutrition disease. If calcium needs are not met through dietary intake, the body will use calcium from bones, leading to weak, porous bones eventually leading to the bone disease osteoporosis. The RDA of calcium for humans is 1300 mg for ages 9–18, 1000 mg for ages 19–50, and 1200 mg for those above 50 years.24 Research on calcium requirement in children has indicated that their average daily intake of the mineral was declining and that no gender or age group over 9 years met the recommended daily intake of 1300 mg. Calcium is commercially available as calcium phosphate monobasic and anhydrous, calcium phosphate tribasic, calcium acid pyrophosphate, calcium carbonate, calcium glycerophosphate, calcium phosphate dibasic, calcium sulfate anhydrous, etc. Cereal is an appropriate vehicle to fortify calcium, in the form of calcium carbonate. Bioavailability of calcium in cereal is equivalent to that in milk.25 Phosphorus is the second major component of bone and teeth. It is important as a major regulator of energy metabolism. Phosphorus is required by about 300 body enzymes and also plays a structural role in the formation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Deficiency of phosphorus results in loss of appetite, bone loss, and pain. With excessive intake of the element, the level of calcium in blood may be reduced. The RDA of phosphorus for male and female (9–18 years) is 1250 mg and 700 mg/day for those above 19 years. Phosphorus is available as salts of calcium, magnesium, and sodium. Magnesium is found in bones followed by muscles, soft tissues, and body fluids. The RDA for males between 19 and 30 years is 400 mg and for males 31 years and above is 420 mg. For females between 19 and 30 years the RDA is 310 mg. Commercial forms include magnesium gluconate anhydrous and dehydrate, and magnesium pyrophosphate di- or tribasic.22 The three minerals, potassium, sodium, and chloride, are known as electrolytes because of their ability to dissociate into positively and negatively charged ions when dissolved in water. Potassium is the major cation of intracellular fluid. Along with sodium, it maintains normal water balance, osmotic equilibrium, and acid–base balance. Potassium promotes cellular growth and helps maintain normal blood pressure. Sources of the element are potassium salts such as gluconate, glycerophosphate, and iodide. There is no RDA either for sodium or chloride. An amount of 500 mg/day is considered safe. An average American consumes 3.375 g of salt, mainly as sodium chloride. The daily sodium limit recommended by the U.S. Food and Drug Administration (FDA) is 2.3 g.22 Iron is a crucial component of oxygen-carrying proteins, hemoglobin, of red blood cells. Iron is also involved in energy metabolism catalyzed by the action of many enzymes. Anemia is a result of inadequate iron. When the body does not get sufficient iron, it loses many of the red blood cells that circulate oxygen, reducing physical and mental work capacity. Other deficiency signs are reduced immunity, susceptibility to infection, and inability to

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maintain body temperature in a cold environment. Iron deficiency anemia (IDA) is a significant challenge in developing countries. Iron-deficient pregnant women are at a higher risk of abortions, low birth weight, and preterm babies. In children, IDA retards growth, impairs cognitive performance, and reduces physical activity. It also accelerates the mortality and morbidity rate in women. Iron is widely available from a variety of foods. In meat products, heme and nonheme irons are found in a ratio of 2:3. Heme iron found in meat is more absorbable than nonheme iron found in plants and vegetables. Hindrance to nonheme iron absorption can be caused by oxalic acid found in spinach, phytic acid in wheat bran and legumes, tannins in tea, and polyphenol in coffee. Nonheme irons can be made absorbable by eating foods rich in vitamin C such as citrus fruits, tomatoes, leafy greens, fortified cereals, and juices. Iron and vitamin C-rich food include meat, dried fruits, legumes, dark green vegetables and vegetables such as cauliflower, cabbage, and carrot, leafy greens such asspinach and broccoli, and whole grains.26 The RDA for males between 16 and 18 years is 12 mg, and for those above 19 years is 10 mg. The value for females between 11 and 50 years is 15 mg, and for those above 50 years is 10 mg. Pregnancy raises the RDA level to 30 mg. Commercial sources are ferric ammonium citrate, ferric phosphate, pyrophosphate, gluconate, ferrous lactate, and reduced iron, among others.22 Table 8.2 shows the contents of iron in the human body. Zinc promotes cell division, growth and repair of tissue, and is also important in wound healing. Zinc requirement is high in pregnant and lactating women. Zinc is present over 70 enzymes. Zinc is required to activate kymalin, a hormone that induces the transformation of white blood cells into lymphocytes, which perform specific functions. It plays an important role in specific immune defenses such as humoral and cell-mediated. Zinc deficiency causes retarded growth, skin changes, and loss of appetite. A strong correlation exists between zinc and iron deficiency and cognition—a neurological disorder affecting the ability to perceive, think, and remember. Zinc is primarily found in meat, fish, poultry, milk, and milk products.

TABLE 8.2 The Body Content of Iron Types of Iron Essential iron Hemoglobin Myoglobin, Cytochrome, and enzymes Storage and transport iron Ferritin and hemosiderin Transferrin Total iron

Male (70 kg)

Female (60 kg)

3.1 2.7 0.4

2.1 1.8 0.3

0.9 0.89 0.003 4.0

0.5 0.41 0.003 2.6

Note: The values are given in grams. Source: Adapted from Berdanier, C. D., Ed., in Handbook of Food and Nutrition, CRC Press, Boca Raton, FL, 2002, 141. With permission from Taylor & Francis Ltd. (www.informaworld.com).

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Commercial forms are gluconate and oxide or sulfate salts of zinc. Higher calcium diets have been found to significantly reduce zinc absorption and zinc balance in postmenopausal women. Zinc toxicity can cause deficiency in copper and can harm the immune system. Zinc supplementation was found to improve cell-mediated immune response in older populations. The RDA for males is 11 mg/day and for females it is 8 mg/day.9 In the United Kingdom, the RNI for zinc is 5.5–9.5 mg/day.22,27 Copper is necessary for the maintenance of blood vessels, tendons, and bone and for the activity of many enzymes. Copper is required to carry oxygen in red blood cells. The element is required for infant growth, host defense, bone strength, iron transport, and cholesterol and glucose metabolism. Deficiency leads to anemia and bone abnormalities. Sources of the mineral are seafood, organ meat, oysters, nuts, and seeds. An average serving of fish or marine invertebrates such as oysters and clams provide between 45 and 60% of the daily requirement of the mineral. No RDAs for copper and manganese have been indicated. Established safe and adequate daily dietary intake (ESADDI) is 1.5–3 mg/day. Commercial forms are gluconate and sulfate salts. Manganese is a part of several enzymes. Sources are manganese chloride, gluconate, glycerophosphate, sulfate, or citrate. Manganese is available from crustaceans in sufficient quantities, especially lobsters, and can satisfy the total human requirement of essential microelements.28 Selenium plays an important role in the functioning of the immune system, thyroid hormone metabolism, and reproduction. It is also a part of the body’s antioxidant defense system, preventing damage to cells and tissues. Selenium is an important component of the enzyme glutathione peroxidase, which functions as an antioxidant. By increasing the antioxidant levels, selenium may minimize the risk of certain types of cancers and coronary heart diseases. Besides, selenium provides protection against mercury and cadmium toxicity. A supplement consisting of selenium and also vitamins A, C, and E has been reported to prevent hearing loss. Recommended dose of selenium is 0.075 mg/day for men and 0.06 mg/day for women.2,29 Iodine is a part of the thyroid hormone present in the thyroid gland. Functional and developmental abnormalities can occur in the absence of a healthy thyroid gland. The recommended allowance of iodine for adults is 150 µg/day. During pregnancy and lactation, an additional dose of 25 µg/day and 50 µg/day is recommended, respectively. However, iodine deficiency is prevalent throughout the world, making it responsible for the worldwide phenomenon of brain damage and mental retardation. During pregnancy, infancy, or early childhood, deficiency may lead to endemic and irreversible cretinism in infants or children. However, adequate iodine intake can reverse goiters, commonly known as iodine deficiency symptom. However, it may be noted that excessive iodine can be toxic. Graves’ disease is the most common form of hyperthyroidism. Calcium and vitamin D losses may occur in cases of hyperthyroidism, and supplementation with a multivitamin is recommended. Iodized salt is the main source of iodine. Other sources of the element include potatoes, spinach, and almond.22 Iodine forms a part of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). These hormones are involved in the maintenance of metabolic rate, cellular metabolism, and integrity of connective tissue. Iodized salt is the main source of this element. Table 8.3 gives the recommended intake and utilization of minerals.

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TABLE 8.3 Recommended Intake of Minerals Nutrient Iron Calcium Phosphate Iodine Magnesium Zinc Selenium Copper Manganese Molybdenum Chloride

Recommended Intake Per Day 1.8 mg 1000 mg 1000 mg 0.14 mg 400 μg 5.5–9.5 mg 6.0–7.5 μg 1.2 mg 2 mg 75 μg 2400 mg

Source: Adapted from Food Standards Agency, UK, http://www. food.gov.uk/, accessed September 2007. With permission.

8.3.1

MINERAL CONTENTS OF SEAFOOD

The total contents of minerals such as sodium, potassium, calcium, magnesium, and phosphorus and microelements such as selenium, fluorine, iodine, cobalt, manganese, and molybdenum in raw marine fish muscle and invertebrates are roughly in the range 0.6–1.5% wet weight. Zinc, iron, and selenium are rich in products of animal origin including seafood. Shellfish contain nearly twice the amount of minerals compared to finfish. Oysters are especially rich in zinc, iron, and copper. Oysters, clams, and shrimp contain more calcium than other fish and meat. Most fresh marine fish may be considered as moderately low sodium foods delivering approximately 140 mg sodium per serving. However, the sodium content of most processed fish and seafood products (frozen, canned, smoked, and cured) is substantially high, ranging from 300 to 900 mg/100 g. The higher contents are the result of conventional onboard handling and processing treatments such as brining and storage in refrigerated seawater. Battered and frozen seafood contain an average of 400 mg sodium per 100 g. The sodium content of fresh fish fillets ranges from 39 to 90 mg/100 g.30 Seafood is a source of calcium, its contents varying from 6 to 120 mg/100 g depending on the species. The calcium content may be as low as 15 mg in the case of mackerel; 15–50 mg in catfish, haddock, and oysters; and above 100 mg in pollock, salmon, and trout. It is well documented that consumption of whole small fish is nutritionally beneficial providing a rich source of calcium.30,31 Fresh fish are a good source of potassium containing 250–320 mg of the element per 100 g. Shellfish (clams, oysters, and scallops) and fish having dark colored flesh such as bluefish, herring, mackerel, sardines, and smelt are reasonably good sources of iron, supplying 1–2 mg/100 g of muscle. The iron content in 100 g meat may vary as 0.9 mg in cod, flounder, and pollock; 0.9–2.0 mg in carp, catfish, salmon, and trout; and above 2 mg

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in clams, oysters, and shrimp. The calcium and iron found in small fish species are particularly bioavailable.13 Consumption of sardines, whether raw or fried in olive oil enhances dietary iron availability. The bioavailability of iron from a fish diet has been examined.32 Mollusks and crustaceans are good sources of zinc and copper. Copper, which is necessary for the maintenance of blood vessels, tendons, and bone, is available in sufficient quantities in crustaceans, especially lobsters. Oysters and clams provide between 45 and 60% of the daily requirement of the mineral. Against a daily recommended value of 1000 mg of phosphorus, 300 mg of the mineral could be obtained from 100 g meat of salmon or sardine. Its content is less than 200 mg in clams, flounder, and oyster. Catfish, cod, and pollock contain 200–300 mg of the mineral.30,31,33 Mineral contents and proximate composition of selected seafood such as Parapenaeopsis atlanta, Panulirus regius, Penaeus durarum, Parapenaeopsis spp., and Penaeus kerathurus were good sources of essential minerals such as P, Fe, Mg, K, Na, and Ca, whereas Cu, Zn, and Mn contents were low.34 Antarctic krill is a good source of fluoride, which is concentrated in carapace and exoskeleton. Fluoride in doses of 50–70 mg as sodium fluoride per day can increase calcium balance, bone mineralization, and in vitro collagen production.35 Table 8.4 shows the fluoride contents in different parts of krill compared to that of deep-sea prawn (Pandalus borealis).36 Fish, particularly tuna, is a good source of selenium. However, in general, shellfish tend to be richer sources than finfish.28 Marine fish and shellfish are rich sources of iodine, oysters being the richest, followed by clams, lobster, shrimp, crawfish, and ocean fish. An average serving of fish or marine invertebrate can satisfy the total human requirement for essential microelements.28,33 Soluble protein powders from processing by-products of Alaska pollock (Theragra chalcogramma), namely, viscera, liver, heads, trimmings, and frame are rich in potassium, sodium, phosphorus, sulfur, magnesium, and calcium.37 Table 8.5 gives the mineral contents of protein powders from Alaska pollock processing by-products. Trace metal contents of nine fish species harvested from the Black and Aegean seas were determined by microwave digestion and atomic absorption spectroscopy and were found to be: 0.73–1.83 μg/g copper;

TABLE 8.4 Fluoride (mg%) in Fat-Free Dry Matter of Raw Krill and Deep-Sea Prawn Body Parts Muscle Exoskeleton Carapace Cephalothorax Whole body

Antarctic Krill (Euphausia Superba)

Deep-Sea Prawn (Pandalus Borealis)

240 333 426 369 240

1.8–9.1 — 3.6–17 3.2–15 1.8–9.1

Source: Adapted from Soevik, T. and Braekkan, O. R., J. Fish. Res. Board Can., 36, 1414, 1979. With permission.

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TABLE 8.5 Mineral Contents (%) of Protein Powders from Alaska Pollock Processing By-Products Mineral Content Phosphorus Potassium Calcium Magnesium Copper (ppm) Zinc (ppm) Ferrous (ppm)

Frame 1.06 3.48 0.04 0.11 <0.1 13 11

Head 0.92 2.06 0.03 0.05 12.5 10.0 <1.0

Trimmings 1.68 4.40 0.07 0.36 <0.1 3.0 104

Source: Adapted from Sathivel, S. and Bechtel, P. J., Int. J. Food Sci. Technol., 41, 520, 2006. With permission from Blackwell.

0.45–0.90 μg/g cadmium; 0.33–0.93 μg/g lead; 35.4–106 μg/g zinc; 1.28–7.40 μg/g manganese; 68.6–163 μg/g iron; 0.95–1.98 μg/g chromium; and 1.92–5.68 μg/g nickel. The levels of lead and cadmium in fish samples were higher than the recommended legal limits for human consumption.38 8.3.1.1

Fish Bone as a Source of Minerals

Bone comprises a significant part of seafood processing wastes. Filleting of fish generates a large amount of fish frames. For example, backbone wastes from processing of Atlantic cod (Gadus morhua) account for approximately 15% of the wet weight of the fish.39 Chemical characteristics of fish bones (from cod, Alaska pollock, yellowfin sole, hoki, conger eel, and mackerel) have been reported. The crude protein (40.7%, dry weight basis) and collagen contents (5.86%, dry weight basis) and amino acid composition of hoki bone were higher than those of the other fish bones, but were lower than those of terrestrial animal bone. The crude lipid contents and total eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) composition of yellowfin sole, conger eel, and mackerel bones were in the range 22.8–43.9% and 15.6–23.8%, on a dry weight basis, respectively. It was concluded that hoki bones may be effectively utilized as a processing material for collagen or gelatin.40 Inorganic minerals constitute approximately 65% of bones, the extracellular organic matrix making the remaining portion.39,40 The inorganic constituents of bone ashes from 15 fish species including those belonging to Teleostomi and Elasmobranchs were examined by both x-ray diffraction analysis and elemental analysis. All the samples were classified into three groups composed mainly of either hydroxyapatite (HAP; Ca10(PO4)6(OH)2), tricalcium phosphate (TCP), or a mixture of HAP and TCP. Sea bream, horse mackerel, carp, and shark had HAP-type phosphate as in the case of cattle, swine, and fowl, whereas Japanese anchovy had TCP-type phosphate. HAP–TCP types were found in the bone ashes of sardine, mackerel, tilefish, croaker, triggerfish, lizard fish, Spanish mackerel, flying fish, conger eel, and flatfish. Molar ratios of calcium with or without either magnesium

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or sodium to phosphate of the examined bone ashes coincided with the theoretical values of HAP and TCP.41 The major ash components of fish bones from cod, Alaska pollock, yellowfin sole, hoki, conger eel, and mackerel were calcium and phosphorus, and contents of these minerals in 100 g crude ash were 37.1–38.6% and 18.0–18.5%, respectively. The Ca and P contents in 100 g crude ash of cod and Alaska pollock bones were greater than those of animal bones, suggesting the use of these fish bones as a calcium source.40 In another study, proximate, mineral and amino acid from fish bone collected from Alaska pollock and Pacific cod (Gadus macrocephalus) were analyzed. The samples showed a mineral content of about 55% (dry weight basis). Fish bone meal containing 90% solids is a good source of calcium and phosphorus for mammals and poultry, with 15.3% calcium and 7.7% phosphorus. Concentrations per gram of other minerals of nutritional interest in fish bone meal include magnesium (0.23%), potassium (0.47%), sodium (1.21%), iron (50 mg), manganese (34 mg), and zinc (142 mg). The fish bone was used to prepare different grades of meal having a nitrogen-phosphorus-potassium (NPK) values of 6-9-0.5, 6-12-0.5, and 6-13-0.5. The use of fish bone meal has shown potential for use as a fertilizer capable of functioning as a slow release phosphorus fertilizer, particularly in acidic streams.42 8.3.1.2

Calcium from Fish Bone

Fish bone is considered as a potential source of calcium. Fish bone material derived from processing of large fish is a useful calcium source. To use fish bone as a calcium fortificant, the bone should be converted into an edible form by softening its structure. This can be achieved by utilizing different methods including hot water treatment and addition of hot acetic acid solutions. Superheated steam has been used to reduce the loss of soluble components from fish tissue, which enabled better recovery of bone within a shorter period. Furthermore, the treated bones need to be subjected to saponification, degreasing, and degumming. Biological utilization and clinical trial showed that the bone preparation is a good source of bioavailable calcium, much better than common calcium powders, suggesting the potential of fish bone as a source of dietary calcium.43,44 The cooking of fish bone in superheated steam was studied to utilize mackerel fish bone as a calcium source in human diet. The softening rate of mackerel spine and the moisture loss of mackerel meat were determined for samples cooked at 120 and 130°C under pressures in the range from 1.2 to 2.7 kg/cm 2. The softening reaction conformed to an apparent first-order reaction and was dependent on the temperature of the superheated steam. There was a small loss of moisture in the range 2.5–7.9% during cooking.43 Ca-based powder was derived from Alaska pollock bones by autoclaving in water. Autoclaving for an optimum period of 40 min resulted in a powder having appreciable soluble calcium content. Autoclaving for longer periods did not affect the mineral yield, and soluble calcium ratio. It was concluded that the Ca-based powder from Alaska pollock autoclaved in water for 40 min was superior to Ca-based powders derived using other methods. Ca-active powder could be solubilized by agitation at pH 2 and 37°C for 1 h. Instead, agitation at pH 7 and 37°C for 3 h also resulted in dissolution of Ca up to 59%.40,45 Instead of steaming,

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treatment with different acids has been examined to extract calcium from the Alaska pollock frame. Hydrochloric acid gave a value of 60% extraction of calcium, lactic acid gave a value of 30%, whereas acetic acid had the weakest effect. A preparation of calcium powder obtained from the fish bone had 38% Ca and 18% P, with a ratio of 2:1. There were traces of Cu, Mn, Zn, Se, and Fe also.46 Functionally active mineral compounds from seafood waste have been isolated by enzyme treatment and characterized. The skeleton discarded from industrial processing of hoki (Johnius belengerii) was digested by a heterogeneous enzyme extracted from the intestine of bluefin tuna (Thunnus thynnus) to utilize the bone as a source of calcium. The tuna intestine crude enzyme (TICE) could effectively biodegrade the hoki bone matrices composed of collagen, noncollagenous proteins, carbohydrates, and minerals. A fish bone phosphopeptide (FBP) containing 23.6% of phosphorus was isolated from the hoki bone hydrolyzates degraded by TICE using affinity chromatography and gel permeation chromatography. The FBP, having a molecular weight of 3.5 kDa, interacted with calcium without the formation of insoluble calcium phosphate. The results provided evidence that the carnivorous fish intestine enzyme (TICE) could degrade the teleost (J. belengerii) bone, and the fish bone oligophosphopeptide prepared by the enzymatic degradation of the bone could be utilized as a nutraceutical with a potential calciumbinding activity.47 Peak bone stone is the bony structure situated near the vertebral column of dorsal fin base obtained from fishes such as ghol (Protonibea diacanthus), and koth (Otolithes biauratusa). About 900 g of peak bone stone is obtained from a fish of an average weight of 12 kg, with a yield of 6–7%. From large-sized fishes, up to 13 peak bone stones can be obtained (Figure 8.1). There is potential to use the product as a raw material for calcium powder (Devadasan, K., Courtesy of Central Institute of Fisheries Technology, Cochin, personal communication).

8.3.2

MINERALS FROM SEAWEEDS

Seaweeds have exceptionally high ash content (>20%), and therefore are rich sources of minerals. However, there could be seasonal variations in the concentrations of minerals such as zinc, cadmium, copper, manganese, iron, cobalt, nickel, and molybdenum. Zinc, cadmium, copper, iron, nickel, and cobalt concentrations could be the highest in the spring and lowest in the autumn, probably reflecting levels of metabolic activity and climatic factors. Furthermore, these minerals could reach the highest concentrations in specimens habituated in areas where rivers draining mineralized areas enter the sea. Species of brown (Fucus vesiculosus, Laminaria digitata, and Undaria pinnatifida) and red (Chondus crispus and Porphyra tenera) seaweeds are edible, which contain a high proportion of ash (21.0–39.3%). In brown algae, the ash content (30.1–39.3%) was higher than that in red algae (20.6–21.2%). Atomic absorption spectrophotometry of the ashes indicated that these seaweeds contained macroelements (8–18 g/100 g) consisting of sodium, potassium, calcium, and magnesium and trace elements (5.1–15.2 mg/100 g) consisting of iron, zinc, manganese, and copper, which are higher than those reported for edible land-based plants. Because of high mineral contents, edible brown and red seaweed could be used as a food supplement to meet the recommended daily intake of essential minerals.48

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FIGURE 8.1 Peak bone from ghol fish. (Courtesy of Central Institute of Fisheries Technology, Veraval Research Center, India).

The ash contents of edible seaweed that had been canned (Saccorhiza polyschides and Himanthalia elongata) or dried (H. elongata, Laminaria ochroleuca, U. pinnatifi da, Palmaria spp., and Porphyra spp.) ranged from 19 to 34 g/100 g dry weight.49 Analysis of the chemical composition of 11 species of temperate seaweeds has been reported. Corallina officinalis had high ash and high calcium (182 ppm) contents. In contrast, Porphyra spp. had low ash and low calcium (19.9 ppm) contents. The other nine species had values within those reported for the two species mentioned earlier.50 The edible seaweed Porphyra vietnamensis growing along seven different localities of the central west coast of India was analyzed for mineral composition (Na, K, Ca, Mg, B, Pb, Cr, Co, Fe, Zn, Mn, Hg, Cu, As, Ni, Cd, and Mo) by a method called inductively coupled plasma atomic emission spectroscopy (ICP-AES). The concentration ranges (expressed in milligrams per 100 gram dry weight) of minerals found for each sample were as follows: Na, 24.5–65.6; K, 1.76– 3.19; Ca, 1.40–6.12; Mg, 4.0–5.90; Pb, 0.01–0.15; Cr, 0.13–0.22; Co, 0.06–0.20; Fe, 33.0–298; Zn, 0.93–3.27; Mn, 4.22–10.00; Hg, 0.01–0.04; Cu, 0.54–1.05; As, 1.24–1.83; Ni, 0.02–0.25; Cd, 0.14–0.55; Mo, 0.02–0.03; and B, 0.02–0.07. It could

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therefore be used as a food supplement to improve the nutritive value of the omnivorous diet.51

8.3.3

BIOAVAILABILITY OF MINERALS

Bioavailability of minerals is an important parameter in utilization of fish bone as animal feed or fertilizer.52 The bioavailability of minerals from a feed depends on the concentration of the feed used. The apparent availabilities (intake) of protein, phosphorus, and selected minerals from rainbow trout by-products were determined. The deboned fish meal had relatively high concentrations of protein, low concentrations of phosphorus, and many minerals. When the fish were fed diets containing incremental concentrations of fish bones, the apparent availabilities of phosphorus, calcium, magnesium, and iron decreased as the fish bone content in the basal diet increased. Reducing the bone fraction of high-ash (high-phosphorus) by-product meals is therefore an essential approach to use such ingredients in lowpollution fish feeds.53 The availabilities of phosphorus to juvenile (5.6 g weight). Atlantic salmon (Salmo salar) from primary and secondary calcium phosphate, primary sodium phosphate, and fish bone meal were determined by measuring the retention of phosphorus in a 12 week feeding trial. Semipurified casein-based diets were fed keeping dietary phosphate concentrations slightly below the requirement. An unsupplemented basal diet (4.6 g phosphate per kilogram) and a presumed phosphate-sufficient diet (primary calcium phosphate, 11 g phosphate per gram) were also fed. Whole body phosphate concentrations declined in fish fed with the deficient diets indicating that the fish utilized all the available phosphate. Retention of the phosphate sources was basal diet, 72%; primary calcium phosphate, 86%; secondary calcium phosphate, 91%; primary sodium phosphate, 131%; and fish bone meal, 51%. The results indicated that the inorganic salts had higher availability than phosphate from fish bone meal.54 The bioavailability of minerals from seaweed-enriched food products is appreciable. This was examined in pakoda, a traditional snack food in India, which was supplemented with Enteromorpha compressa (Linnaeus)—a seaweed rich in minerals and dietary fiber. With increase in the seaweed level, pakoda samples showed increases in ash, protein, and total dietary fiber contents. There was a nearly fivefold increase in iron content (26.4–126 mg%) and fourfold increase in calcium content (30.1–124 mg%). Bioavailability of iron in Enteromorpha, and pakoda containing 7.5% of the seaweed was dependent on pH. In the alkaline condition of intestine, having a pH of 7.5, there was no significant bioavailable iron. But in acidic conditions (pH 1.35, gastric condition), the bioavailability of iron in pakoda containing seaweed was found to be higher (27.1%) than that in the free seaweed. Reducing power (155–222 μg/g) increased as the seaweed level increased from 0 to 15%. But seaweed addition was found to decrease the free radical-scavenging activity and the total phenol content. Also, incorporation of Enteromorpha at 7.5% in the pakoda did not affect sensory quality of the preparation.55 These studies suggest that most seaweeds can be rich sources of bioavailable iron and other minerals. The iodine content of Indian seaweeds has attracted special attention. Of the 20 species of marine algae of the Saurashtra coast of India, Asparagopsis spp. contained the maximum

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bioavailable iodine. Rare elements such as rubidium and arsenic were reported in marine algae. However, some seaweeds such as Enteromorpho, Caulerpa, and Gracilaria spp. were reported to harbor radioactivity.56

8.4 BONE HEALTH IN HUMAN The process of bone formation requires adequate and constant supply of nutrients such as calcium, protein, magnesium, vitamin D, vitamin K, and fluoride. However, there are several other vitamins and minerals needed for metabolic processes related to bone including manganese, copper, boron, iron, zinc, vitamin A, vitamin K, vitamin C, and the B vitamins. Magnesium is involved in a number of activities favoring bone strength, preservation, and remodeling. Fluorine and strontium have bone-forming effects. However, high amounts of these elements may reduce bone strength. Boron is especially effective in the case of vitamin D, magnesium, and potassium deficiencies. Vitamin K is essential for the activation of osteocalcin. The RDA for the vitamin is 80 μg/day.57,58 As discussed in Chapter 4, osteoporosis (also referred to as porous bone disease) is a skeletal disorder, affecting mostly elderly people, characterized by low bone mass and its microarchitectural deterioration. The disease leads to bone fracture due to an imbalance between bone resorption and formation. With the rapid rise in ageing population world over, bone health will be a determining factor of public health. Current estimates of osteoporosis on a global scale suggest 1 in 3 women and 1 in 12 men above 55 years will suffer from osteoporosis in their lifetime. In the United Kingdom, approximately 3 million people suffer from osteoporosis. The projected rise of osteoporotic hip fracture–related economic loss worldwide is U.S.$6.26 million in the year 2050 (compared to 1.66 million in 1990) suggesting the phenomenal future economic impact of osteoporosis.59 The risk of developing osteoporosis increases with an insufficient supply of calcium and vitamin D, the vitamin being necessary for the absorption of the mineral. Therefore, nutritional intervention might be a first choice for early inhibition of postmenopausal- and age-related bone loss. Calcium and vitamin D are basic components in most preventive strategies, and hence supplements containing these nutrients can be an alternative to drugs to treat osteoporosis. Magnesium is involved in a number of activities of supporting bone strength, prevention, and remodeling. Fluorine and strontium have bone-forming effects. Vitamin K is essential for the activation of osteocalcin. An intake of 1200 mg of calcium per day as recommended by the U.S. RDA (1989), U.S. DRI (1997), or assumed normal intake of food components may lead to considerable reduction or even prevention of bone loss, especially in the individuals mentioned earlier. Several calcium-fortified products are in the market and the demand for these products is growing continuously. Increasing the recommended amounts or adequate intake of food components may lead to a considerable reduction or even prevention of bone loss. Complete foods or supplements are preferred over the supplementation of a single nutrient because of the complexity of natural bone loss, the diversity in dietary habits, and nutrient–nutrient interactions. Prevention or treatment with nutrients is most effective in late postmenopausal women and the elderly.35,60,61

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8.4.1

255

FUNCTIONAL ROLE OF FISH BONE COMPONENTS IN BONE HEALTH

Adequate seafood consumption can also positively influence the bone mineral density (BMD) in people of all ages. Harvard researchers, who tested more than 10,000 individuals whose age ranged from 25 to 64, found that the high consumption of seafood is significantly associated with higher BMD in women, especially those who take more than 250 g of seafood per week.62 Fish bones contain a proper balance of calcium and phosphorus and hence can be used as a calcium food supplement. Bones from small fish is a good source of calcium. Osteoporosis could also be treated using a hormone called calcitonin from salmon (see Chapter 4). The hormones help to regulate calcium and decrease bone loss.57 Fish bone powder is a potential value-added by-product of the tuna processing industry.63 The calcium containing powder from fish bone could be used for the fortification of fish products such as surimi. Calciumfortified mackerel surimi gel and its quality and stability during storage at 5°C were investigated. The powder from Alaska pollock bone was added at a concentration of 0.9%. Soluble calcium content of the fortified surimi gel was 105 mg% compared to a value of 2.9 mg% for unfortified sample. Cold storage did not affect moisture contents, amino acid compositions, and soluble calcium and phosphorus contents of the fortified surimi, although slight increase in pH, contents of volatile basic nitrogen and histamine, peroxide values, and brown pigment formation were noted. In addition to enhanced calcium, the surimi also had superior lysine and higher EPA and DHA contents. Results suggested that the calcium-fortified surimi gel was a nutritive, functional, and safe food.45,63–65 Currently, fish bone material has been used for application in animal feed. In addition to its mineral contents, fish bone material can also become an important source for biomedical applications. Recently, HAP has been introduced as a bone graft material in a range of medical and dental applications due to their similar chemical composition. Generally, bone substitution materials such as autografts, allografts, and xenografts are used to solve problems related to bone fractures and damages. But, none of these materials provide a perfect bone healing due to mechanical instability and incompatibility. Currently, calcium phosphate bioceramics such as tetracalcium phosphate, amorphous calcium phosphate, TCP, and HAP are identified as suitable bone substitution materials.41

8.5

COMMERCIAL PRODUCTS

According to the U.S. National Institutes of Health (NIH), more than half of the U.S. adults take some form of vitamin or mineral supplement at a total cost of U.S.$23 billion a year. Vitamins E and C are taken as popular antioxidants. Such supplements also include two broad classes of plant chemicals called polyphenols and carotenoids (including β-carotene and lycopene). Most supplements considered as antioxidants contain at least one of these, often as a pure chemical and sometimes as a concentrated extract. The total market value of vitamins is rising at an average annual growth rate of less than 1% and will reach $2272 million by 2007.66 In India, the nutrition segment has already crossed U.S.$750 million in the first half of 2007. Within the nutrition market, almost all categories that include anemics, protein supplements, and minerals are showing significant growth rates, minerals

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showing the highest growth rate of 14%. Vitamins as multivitamin capsules form the second largest therapeutic segment in the pharmaceutical industry after antibiotics. The vitamin market with 650 odd brands is valued at U.S.$370 million, annually growing at a rate of 9% (Times of India, June 21, 2007). High potency “one a day” formulations of essential nutrients including vitamins, minerals, and herbs specifically designed to meet the special needs of growing teenagers are becoming popular. These are claimed to assist in keeping the skin healthy and free from pimples and acne. Extra B vitamins help to fight fatigue and enhance energy levels.66,67 Tuna bones were in use as a source of calcium in Japan in the mid-1980s in institutional feeding programs for the elderly and in school meal programs. A fish processing company in Tokyo has successfully processed scales of sardine into an easily absorbable, edible food supplement containing calcium and collagen.65 A commercial marine minerals preparation, which contains all the minerals from the ocean water, and also seaweed is available in the United States. It also contains sardine bones. The product is rich in calcium, iodine, zinc, copper, and sodium.68 To summarize, marine products are good sources of vitamins and, particularly, minerals. Regular consumption of these products could help provide the body adequate amount of vitamins and minerals. There is a potential for production of mineral supplements such as calcium from fish bone waste, which can be used for fortification of diverse food products. Furthermore, minerals from these sources are also bioavailable. There is also the possibility of preparation of oligophosphopeptide employing enzymatic degradation of fish bone, which could be utilized as a nutraceutical with a potential calcium-binding activity. Regular consumption of either seafood or intake of seafood-based mineral supplements can be effective in keeping bones healthy by delaying osteoporosis.

REFERENCES 1. IFT Scientific Status Summary, Use of vitamins as additives in processed foods, Food Technol., 41(9), 163, 1987. 2. Food Standards Agency, UK, http://www.food.gov.uk/, accessed September 2007. 3. Parada, J. and Aguilera, J. M., Food microstructure affects the bioavailability of several nutrients, J. Food Sci., 72, R21, 2007. 4. Verwei, M. et al., Effect of folate-binding proteins on bioavailability of folate from milk products, Trends Food Sci. Technol., 16, 307, 2005. 5. Gliszczyn´ska-S´wigło, A., Folate as antioxidants, Food Chem., 4, 1480, 2007. 6. Anonymous, HEALTHbeat extra: the virtues of Vitamin D, Harvard Medical School Publications, www.health.harvard.edu, March 27, 2008. 7. Parrish, D. B., Determination of vitamin D in foods: a review, Crit. Rev. Food Sci. Nutr., 12, 29, 1979. 8. Geller, J. and Pressman, P., Vitamin D—the sunshine hormone, Food Technol., 61, 21, 2007, www.ift.org. 9. Rivlin, R. S., Vitamin deficiencies, in Handbook of Food and Nutrition, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 1313. 10. Griffith, H. W., Vitamins, Herbs, Minerals & Supplements: The Complete Guide, Fisher Books, New York, 1998. 11. Anonymous, The antioxidant myth: a medical fairy tale—health, New Scientist, 2563, 40, 2006.

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12. Diaz, A. C. et al., Antioxidant activity in hepatopancreas of the shrimp (Pleoticus muelleri) by electron paramagnetic spin resonance spectrometry, J. Agr. Food Chem., 52, 3189, 2004. 13. Roos, N. et al., Understanding the link between agriculture and health: fish and health, 2020, International Food Policy Research Institute, Washington, DC, www.ifpri.org, accessed November 2007. 14. Venugopal, V., Seafood Processing: Adding Value through Quick Freezing, Retortable Packaging and Cook Chilling, CRC Press, Boca Raton, FL, 2006, Ch. 15. 15. Bailey, L. and von Castel Roberts, K., Potential benefits associated with vitamin B12, Proc. Seafood Science and Technology Symposium of the Americas, 2006, http://sst. ifas.ufl.edu. 16. Batternfeind, J. C., The tocopherol contents of food and influencing factors, Crit. Rev. Food Sci. Nutr., 9, 337, 1977. 17. Granit, R. et al., Effects of vitamin E supplementation on lipid peroxidation and color retention of salted calf muscle from a diet rich in polyunsaturated fatty acids, J. Agr. Food Chem., 49, 5951, 2001. 18. Bender, A. E., Dictionary of Nutrition and Food Technology, Butterworths, London, 1980. 19. Ortiz, J. et al., Dietary fiber, amino acid, fatty acid and tocopherol contents of the edible seaweeds, Ulva lactuca and Durvillaea antarctica, Food Chem., 99, 98, 2006. 20. Takenaka, S. et al., Feeding dried purple laver (nori) to vitamin B12-deficient rats significantly improves vitamin B12 status, Brit. J. Nutr., 85, 699, 2001. 21. March, B. E., Effect of processing on nutritive value of food: fish, in Handbook of Nutritive Value of Processed Food, Vol. 1, Food for Human Use, Rechcigl, M., Ed., CRC Press, Boca Raton, FL, 2001. 22. Swanson, M. A., Nutritional additives, in Food Additives, 2nd ed., Bransen, A. L. et al., Eds., Marcel Dekker, New York, 2002, p. 225. 23. Gregor, J. L., Effect of dietary protein and minerals on calcium and zinc utilization, Crit. Rev. Food Sci. Nutr., 28, 249, 1989. 24. Food and Nutrition Board, Recommended Dietary Allowances, 12th ed., National Academy Press, Washington, DC, 2006. 25. Tobelmann, R., Implementing calcium fortification: an industry case study, J. Food Comp. Anal., 14, 241, 2001. 26. Trivedi, M. B., Big Bazaar (India), February 10, 2007, p. 20. 27. Lall, S. P., Macro and trace elements in fish and shellfish, in Fish and Fishery Products—Composition, Nutritive Properties and Stability, Ruiter, A., Ed., CAB International, Oxfordshire, UK, http://www.cabi.org/, 1995, p. 187. 28. Sidwell, V. D., Chemical and nutritional composition of finfishes, whales, crustaceans, mollusks and their products, NOAA technical memorandum, NMFS/Sec-II, U.S. Department of Commerce, Washington, DC. 29. Finley, J. W., Increased intakes of selenium-enriched foods may benefit human health, J. Sci. Food Agr., 87, 1620, 2007. 30. March, B. E., Effect of processing on nutritive value of food: ish, in Handbook of Nutritive Value of Processed Food, Vol. 1, Food for Human Use, Rechcigl, M., Jr., Ed., CRC Press, Boca Raton, FL, 1982, p. 363. 31. Silva, J. L. and Chamul, R. S., Composition of marine and freshwater finfish and shellfish species and their products, in Marine and Freshwater Products Handbook, Martin, R. E., et al., Eds., Technomic, Lancaster, PA, 2000, p. 31. 32. Seiquer, I. et al., Consumption of raw and fried sardine (Clupea pilchardus) as a protein source of diets: effects of iron metabolism in rats, J. Sci. Food Agr., 82, 1497, 2003. 33. Kinsella, J. E. et al., Components of seafood, in Seafood: Effects of Technology on Nutrition, Marcel Dekker, New York, 1990.

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34. Ogunlade, I., Olaofe, O., and Fadare, T., Chemical composition, amino acids and functional properties of selected seafoods, J. Food Agr. Environ., 3, 130–133, 2005. 35. Schaafsma, A., de Vries, P. J. F., and Saris, W. H. M., Delay of natural bone loss of higher intakes of specific minerals and vitamins, Crit. Rev. Food Sci. Nutr., 41, 225, 2001. 36. Soevik, T. and Braekkan, O. R., Fluoride in Antarctic krill (E. superba) and Atlantic krill (M. norvegica), J. Fish. Res. Board Can., 36, 1414, 1979. 37. Sathivel, S. and Bechtel, P. J., Properties of soluble protein powders from Alaska pollock (Theragra chalcogramma), Int. J. Food Sci. Technol., 41, 520, 2006. 38. Uluozlu, O. D. et al., Trace metal content in nine species of fish from the Black and Aegean Seas, Turkey, Food Chem., 104, 835, 2007. 39. Gildberg, A., Arnesen, J. A., and Carlehog, M., Utilisation of cod backbone by biochemical fractionation, Proc. Biochem., 38, 475, 2002. 40. Kim, J. S., Choi, J. D., and Koo, J. G., Component characteristics of fish bone as a food source, Agr. Chem. Biotechnol., 41, 67, 1998. 41. Hamada, M. et al., Inorganic constituents of bone of fish, Fish. Sci. (Tokyo), 61, 517, 1995. 42. Johnson, R. B., Nicklason, P. M., and Barnett, H. J., Macro- and micronutrient composition of fish bone derived from Alaskan fish meal processing: exploring possible uses for fish bone meal, Proc. Conf., Advances in Seafood Byproducts, University of Alaska Fairbanks, Alaska, 2003, p. 201. 43. Ishikawa, M. et al., Effect of vapor pressure on the rate of softening of fish bone by super-heated steam cooking, Nippon Suisan Gakkaishi, 56, 1687, 1990. 44. Shungan, X., Calcium powder of freshwater fish bone, J. Shanghai Fish. Univ., 5, 246, 1996. 45. Kim, J. S., Choi, D. S., and Kim, D. S., Preparation of calcium-based powder from fish bone and its characteristics, Hanguk-Nongwhahak-Hoechi., 41, 147, 1998. 46. Changh, X. et al., Studies on the preparation of active calcium from pollack frame, J. Ocean Univ. Qingdao, 25, 173, 1995. 47. Jung, W. K. et al., Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous intestine crude proteinase, Food Chem., 91, 333, 2005. 48. Ruperez, P., Mineral content of edible marine seaweeds, Food Chem., 79, 23, 2002. 49. Sánchez-Machado, D. I. et al., Fatty acids, total lipid, protein and ash contents of processed edible seaweeds, Food Chem., 85, 439, 2004. 50. Marsham, S. et al., Comparison of nutrition chemistry of a range of temperate seaweeds, Food Chem., 100, 1331, 2007. 51. Subba Rao, P. V., Mantri, V. A., and Ganesan, K., Mineral composition of edible seaweed, Food Chem., 102, 215, 2006. 52. Das, P., Raguramulu, N., and Rao, K., Determination of bioavailable zinc from plant foods using in vitro techniques, J. Food Sci. Technol. (Mysore), March/April 2006. 53. Sugiura, S. et al., Utilization of fish and animal by-product meals in low-pollution feeds for rainbow trout Oncorhynchus mykiss (Walbaum), Aquacul. Res., 31, 585, 2000. 54. Nordrum, S. et al., Availability of phosphorus in fish bone meal and inorganic salts to Atlantic salmon (Salmo salar) as determined by retention, Aquaculture, 157, 51, 1997. 55. Mamatha, B. S. et al., Studies on use of Enteromorpha in snack food, Food Chem., 101, 1707, 2007. 56. Krishnamurthy, V., Seaweed research and utilization in India, Proc. Seaweeds-2004, Seaweed Res. Utilization Association and Central Marine Fisheries Research Institute, Cochin, 2004, pp. 7–13. 57. Palacios, C., The role of nutrients in bone health from A to Z, Crit. Rev. Food Sci. Nutr., 46, 621, 2006.

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58. Berdanier, C. D., Ed., Handbook of Food and Nutrition, CRC Press, Boca Raton, FL, 2002, p. 141. 59. Lanham-New, S. A. and Gannon, R. H. T., Is dietary alkali supplementation the way forward for preventing osteoporosis? IFIS, http://www.foodsciencecentral.com/fsc/ ixid14489, September 2006. 60. Gogg, R. et al., Nutraceutical therapies for degenerative joint diseases, Crit. Rev. Food Sci. Nutr., 45, 145, 2005. 61. Brouns, F. and Vermeer, C., Functional food ingredients for reducing the risks of osteoporosis, Trends Food Sci. Tech., 11, 22, 2000. 62. Anonymous, Benefits and risks of vitamins and minerals, Harvard Medical School Newsletter, September 27, 2007, [email protected]. 63. Sultanbawa, Y. and Aksnes, A., Tuna process waste—an unexploited resource, Infofish Int., 3, 37, 2006. 64. Kim, J. S. et al., Improvement of the functional properties of surimi gel using fish bone, Hanguk-Nongwhahak-Hoechi, 41, 175, 1998. 65. Anonymous, Seafood consumption for stronger bones, Infofish Int., 3, 74, 2007. 66. Anonymous, Seatone, a high potency maximum strength mussel extract from Newzeland, http://www.healtheries.co.nz/page.php?id=25&alpha=S. 67. BCC Research Report ID: FOD010C MA 02481, October 2003, 190, www.bccresearch. com. 68. http://www.seagateproducts.com/marine-minerals.html.

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Nutritional 9 Seaweed: Value, Bioactive Properties, and Uses 9.1

INTRODUCTION

The term “seaweed” (also known as kelp) is the collective name for marine macroalgae, which are the plants of the sea (see Chapter 2). The major pigments that give color to the algae include chlorophyll a, b, and c; β-carotene; phycocyanin; xanthophylls; and phycoerythrin. All these pigments are of great potential for applications in food, pharmaceuticals, and cosmetics. Brown seaweed is usually large, ranging from the giant kelp that can often be as long as 2–4 m, whereas red seaweed is usually smaller, generally ranging from a few centimeters to about a meter in length. Of the large number of species of seaweed found all over the world, a few species of red algae have been in use for at least a few centuries. The earliest record of use of seaweed dates back to 2700 BC in the compilation of “Chinese Herbs” by Emperor Shen Nung.1 Seaweed has been an important dietary component since, at least, fourth century in Japan and sixth century in China.2 In Korea also consumption of seaweed is a common practice. In these countries, red and brown seaweed powders are traditionally consumed as condiment agents in a variety of foods. Average algal consumption per person in Asia and Africa has been very low between 3 and 13 g.3 Hydroclathrus, Caulerpa, Eucheuma, Gracilaria, and Acanthohora spp. are used as green salad vegetable, whereas the coarser Gracilaria and Eucheuma spp. are pickled.4,5 In Asian countries, seaweed are directly used for several culinary purposes, whereas, in the west, such direct uses are limited; they are almost exclusively used for the phycocolloid industry for extraction of important food hydrocolloids including carrageenan, alginic acid, and agar.5–7 From time immemorial seaweed have also been used as manure in the coastal areas. This chapter discusses composition, nutritional value, and some functional properties of seaweed. Detailed functional properties of individual seaweed hydrocolloids will be covered in the subsequent chapter. Aspects such as ecological, morphological, and genetic aspects are not coming within the purview of these discussions.

9.2 PROCESSING OF SEAWEED Fresh seaweed collected from the sea are usually washed and dried before they are used directly as food or subjected to extraction of hydrocolloids. If dried properly, seaweed can be stored for a number of years without appreciable loss of gel-forming ability of the hydrocolloids.8 High-temperature drying and cooking may cause significant loss of vitamin C in brown seaweed. The three common drying methods employed are sun drying, oven drying, and freeze drying.9 261

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9.3 IDENTIFICATION OF SEAWEED It is important to identify seaweed species for maximizing their commercial use. A method has been developed for identification of some commercially important seaweed species based on the electrophoretic pattern of proteins present in the algae. The sodium dodecyl sulfate-polyacrylamide gel electrophorsis (SDS-PAGE) patterns of Palmaria palmata (Dulse), Chondrus crispus (Pioca), Porphyra umbilicalis (nori), Gracilaria verrucosa (Ogo-nori) showed variation in protein patterns with respect to season of harvest, for all species, some protein bands were characteristic of the plants. The reference pattern of P. palmata was composed of six protein bands with apparent molecular weights between 59.6 and 15.2 kDa. The G. verrucosa pattern was constituted of eight permanent bands. Two pattern bands, with apparent molecular weights of 49.1 and 45.9 kDa, differentiated the G. verrucosa profile from other patterns. C. crispus could be identified by a reference pattern composed of seven bands; three, with close molecular weights (49.3, 46.2, and 43.2 kDa), which were characteristic of this species. The P. umbilicalis pattern showed seven bands with molecular weights between 73.1 and 15.9 kDa, whereas a band with a molecular weight above 70 kDa appeared to be specific to the Porphyra.10

9.4

PROXIMATE COMPOSITION

Seaweed, in general, can be valuable food sources since they contain protein, lipids, vitamins, and minerals. However, because of traditional dietary habits, globally very few of the world’s available seaweed species are used as commercial food sources. Seaweed contain vitamins, minerals, proteins, and fibers, and therefore are nutritionally important. Determination of proximate composition helps to assess their nutritional and industrial importance. Generally, most seaweed contain high ash (indicating appreciable amounts of diverse minerals), high fiber, low proteins, and moderate amounts of fatty acids. In general, seaweed are good sources of minerals and functionally active hydrocolloids. A number of reports are available on the composition of seaweed. Eleven species of macroalgae (including four species from commercially important genera) were analyzed for moisture, ash, fat, protein, crude fiber, calorific value, and calcium contents. Corallina officinalis had low protein (6.9 ± 0.1%), low calorific value (2.7 MJ/kg), and high ash (77.8%) and calcium contents (182 ppm). The exploited Porphyra spp. had high calorific value (18.3 MJ/kg−1), low ash content (9.3%), high protein (44.0%), and low calcium content (19.9 ppm) on dry weight basis. The other species examined had intermediate values, but tended to be more similar to Porphyra than to Corallina spp.11 Compositions of the red seaweed species, Hypnea charoides and H. japonica and that of Ulva lactuca were compared. The total ash contents ranged between 21.3 and 22.8% on dry weight basis, while crude lipids were in the range of 1.42–1.64%. Enteromorpha spp. is a seaweed present almost year round in Mexico. It often causes unpleasant appearance and foul odor from decomposition by microorganisms, causing heavy expenses in cleaning beaches. The alga was examined to understand its food value. Chemical analysis indicated that Enteromorpha spp. had 9–14% protein, 2–3.6% ether extract, and 32–36% ash. It also contained 10.4 and 10.9 g n-3 (omega-3) and n-6 (omega-6) fatty acids per 100 g of total fatty acids, respectively. The protein had a high digestibility of 98%. The alga

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was found to be acceptable in terms of microbiological quality. Salmonella spp. were not found and mesophyllic aerobic and fecal coliform were within acceptable limits. Therefore, Enteromorpha spp. was recommended for human consumption in view of its proximate composition including high content of minerals.12 The compositions of the edible seaweed Durvillaea antarctica (frond and stem) and dried U. lactuca were determined, including the contents of soluble dietary fiber (SDF), insoluble dietary fiber (IDF), and total dietary fiber (TDF) amino acid and fatty acid profiles along with tocopherols (vitamin E) and tocotrienols (pro-vitamin E). Results showed that U. lactuca contained 60.5% TDF, whereas D. antarctica frond and stem contain 71.4 and 56.4% of TDF, respectively. Levels for the different amino acids per 100 g proteins varied significantly, ranging from 508 to 2020 mg.13 The contents of dietary fibers were comparable between red and brown algae classes (29.1–62.8% semidried weight), with the lowest and highest concentrations of dietary fiber in Laminaria spp. and Hizikia fusiforme, respectively. Other algae, Sargassium spp. have been examined for its proximate composition.8,14,15 These studies indicate that nutritional values of seaweed vary greatly. Table 9.1 gives nutritional composition of some species of seaweed. Influence of processing on the composition of seaweed has been examined. Canning or drying did not significantly affect their nutritional value of seaweed. The total lipid, protein, ash, and individual fatty acid contents of canned edible seaweed, Saccorhiza polyschides and Himanthalia elongate, or dried H. elongata, Laminaria ochroleuca, Undaria pinnatifida, Palmaria spp., and Porphyra spp. were determined. Total lipid content ranged from 0.70 to 1.80 % on dry weight basis. The four most abundant fatty acids were C16:0, C18:1ω9, C20:4ω6, and C20:5ω3. Unsaturated fatty acids predominated in all the brown seaweed studied, and saturated fatty acids in the red seaweed, but both groups are balanced sources of omega-3 and omega-6 acids.

TABLE 9.1 Nutritional Composition of Some Species of Macroalgae Species Cladophora rupestris Ceromium spp. Polysiphonia spp. U. lactuca Porphyra spp. Dumontia contorta Mastocarpus stellatus Osmondea pinnatifida Laminaria digitata Corallina officinalis

Moisture

Ash

Protein

Fat

CF

NDF

Calcium

68.5 87.4 77.2 79.6 77.1 87.7 64.9 86.4 86.1 31.5

16.8 27.1 19.2 17.8 9.3 17.8 15.6 32.3 23.9 77.8

29.8 31.2 31.8 29.0 44.0 31.7 25.4 27.3 15.9 6.9

1.0 0.6 0.05 0.5 0.7 0.12 3.0 4.3 0.5 0.3

45.7 5.1 4.3 2.8 1.1 2.0 1.8 6.5 7.7 8.3

15.9 33.7 52.8 32.9 33.5 34.3 16.6 25.6 13.0 9.4

49 95.1 104 53.7 19.9 51.6 38.7 89.1 73.4 182

Note: CF, crude fiber; NDF, non-digestible fiber. The values are given as percent of total. Calcium content is given in ppm. Source: Adapted from Marsham, S. et al., Food Chem., 100, 1331, 2007. With permission from Elsevier.

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Ash contents ranged from 19.07 to 34% and protein contents from 5.46 to 24.11%, on dry weight basis.16 Eucheuma seaweed flour samples contained relatively low levels of lipid-soluble material and 23–31% ash. Water-soluble components of seaweed flours comprised of 70–90%. Acid-insoluble material in seaweed flours was high with values ranging between 4.8 and 11.3%. Insoluble solids residues of seaweed flours averaged 10–30%, with the major sugar component being glucose, followed by mannose and galactose.17 The major nutritionally important compounds of seaweed have been examined in detail, as discussed later in this chapter.

9.4.1

PROTEINS AND AMINO ACIDS

In general, red and brown algae species demonstrate large differences in their protein contents. The protein contents of products varied widely from 26.6 ± 6.3% in red algae to 12.9 ± 6.2% in brown algae varieties. All essential amino acids were detected in the seaweed species tested and red algae species featured uniquely high concentrations of taurine when compared to brown algae varieties.18 The protein content depended upon season and environmental growth conditions. For example, protein content of brown algae species (Laminaria japonica, H. fusiforme, or U. pinnatifida), is relatively low with 7–16%, on dry weight basis. In contrast, red algae, P. palmata (Dulse) and Porphyra tenera, contain 21–47%. In a recent report, the crude protein content of red seaweed varieties was found to be 31% on semidried weight, whereas that of brown algae was only 14%, as shown in Table 9.1. Comparable protein contents for these macroalgae classes were described in other studies. Seaweed proteins contain all the essential amino acids, the levels of which are sufficient to meet requirements.18 Therefore, red algae varieties represent an important source of protein. The amino acid score and the essential amino acid index were higher in red algae and U. pinnatifida, whereas Laminaria spp. and H. fusiforme have a low nutritive value protein. Enteromorpha spp. has 9–14% protein, which has a high digestibility of 98%.12 With respect to their high protein level and their amino acid composition, Porphyra spp. and Undaria spp. appeared to be an interesting source of food proteins.19 In general, seaweed have a lower protein efficiency ratio than that of casein.20 Table 9.2 gives protein quality of some algae.21,22 Major amino acids of seaweed are glycine, arginine, alanine, and glutamic acid. Among the essential amino acids, lysine with a chemical score of 53% appeared to be the most limiting when compared with the essential amino acid pattern of egg protein.23 Although the crude protein contents of the red seaweed, H. charoides and H. japonica were significantly higher than that of the green seaweed, U. lactuca, the proteins from the three seaweed species contained all the essential amino acids, the levels of which were comparable to those of the FAO/WHO requirement.24 The total essential amino acids of H. charoides, H. japonica, and U. lactua were 425, 424, and 376, respectively. In addition, the swelling capacity, WHC, and oil-holding capacity showed positive correlation with total protein contents and also fiber contents.25 The nutritional values of seaweed protein concentrates isolated from two red seaweed (H. charoides and H. japonica) and one green seaweed (U. lactuca) were evaluated by determining their in vitro protein digestibility and amino acid profiles. Both protein extractability and in vitro protein digestibility (88.7–88.9%) of the red

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TABLE 9.2 Protein Quality of Some Algae Algal Species Porphyra spp.b Porphyra spp.c Undaria pinnatificki Laminaria spp. Hizikia fusiforme a

b c

Amino Acid Score

Essential Amino Acid Index

Lys-Met-Cys-TrpThr-Scorea

61.7 64.0 61.3 31.4 40.0

89.6 91.2 91.2 65.9 80.9

1.0 1.0 1.0 0.7 0.7

Data taken from Gersovitz, M., Munro, H., and Scrimshaw, N., FAO/WHO/UNU Technical Report Series 724, World Health Organization, Geneva, 1985; FAO, FAO Fisheries Technical Paper #288, Food and Agriculture Organization of the United Nations, Rome, 1987. Porphyra spp. from Japan and Korea. Porphyra spp. from China.

Source: Adapted from Dawczynski, C. et al., Food Chem., 103, 891, 2007. With permission from Elsevier.

seaweed were significantly higher than those of green seaweed (85.7%). The total amounts of essential amino acids in the three seaweed were high (36.2–40.2%). All three seaweed were rich in leucine, valine, and threonine but lacked cystine. However, except for sulfur-containing amino acids and lysine, the levels of all essential amino acids were higher than those of the FAO/WHO requirement pattern.25 The proximate composition, amino acid profile, and also some physicochemical properties of two subtropical red seaweed (H. charoides and H. japonica) and one green seaweed (U. lactuca) were investigated. The total dietary fiber and ash ranged from 50.3 to 55.4% and 21.3 to 22.8%, respectively, on dry weight basis. These were the two most abundant components in these seaweed, whereas their crude lipid contents were very low in the range of 0.42–1.64%. Although the crude protein content of the red seaweed (H. charoides and H. japonica) was significantly higher than that of the green (U. lactuca), the three seaweed proteins contained all essential amino acids, the levels of which were comparable to those of the FAO/WHO requirement.24 Among the three seaweed, two red seaweed exhibited significantly better physicochemical properties, which were similar to some commercial fiber-rich food ingredients.25 The application of proteomics in alga research is still in its infancy. A recent report describes the establishment of the proteome of a red alga of economic importance, Gracilaria changii. Four protein extraction methods were compared for their suitability to generate G. changii proteins for two-dimensional gel electrophoresis. The phenol/chloroform protein extraction method gave the best resolution of the proteins. Using these gels and mass spectrometry, several proteins including pigment proteins, metabolic enzymes, and ion transporters were identified. These findings highlight the potential of using proteomic approaches for the investigation of G. changii protein.26

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LIPIDS

In general, marine macroalgae varieties contained low amounts of lipid in the range of 2.3 ± 1.6%, based on semidry sample weight.18 Seaweed are sources of polyunsaturated fatty acids. In the three seaweed products, namely, D. antarctica (frond and stem) and dried U. lactuca the most abundant fatty acid was C18:1, which in U. lactuca accounted for 27.42 ± 2.60%; in D. antarctica, 25.36 ± 3.10% and 25.83 ± 2.52% in leaves and stem, respectively.13 Interestingly, the fatty acids distribution of seaweed products showed high levels of omega-3 fatty acids and demonstrated a nutritionally ideal omega-6/omega-3 free fatty acid ratio. The predominant free fatty acids in various seaweed products was eicosapentaenoic acid (C20:5, n–3) with concentrations as high as 50% of total fatty acid content.18 G. changi contained a higher composition of unsaturated fatty acids (74%), mainly the omega fatty acids and 26% of saturated fatty acids, mainly palmitic acid (together with relatively high levels of calcium and iron).23 The lipid concentrations of the 34 seaweed varieties analyzed were low (2 g/100 g semidried weight). The macroalgae varieties tested contained high concentrations of PUFA. Eicosa pentaenoic acid (EPA) concentrations were high (>50% of total fatty acid methylester) in H. fusiforme and some red algae varieties. In Porphyra spp., from Japan and Korea, U. pinnatifida, and H. fusiforme omega-3 PUFAs were the main representatives of this group, suggesting these seaweed varieties as a rich source of omega-3 PUFA (17.9–52.3%).25 Supercritical carbon dioxide (SC-CO2) extraction has been examined for isolation of lipids from seaweed. Lipids from the subtropical red seaweed (H. charoides) were extracted by this method within the temperature and pressure ranges of 40–50°C and 24.1–37.9 MPa, respectively. In general, the extraction rates of algal lipids increased with pressure and temperature. The combined effect of pressure and temperature on the solubility of individual omega-3 fatty acids in the supercritical CO2 varied with its carbon chain length. The concentrations of C18, C20, and C22 n–3 fatty acids, extracted under different pressure and temperature conditions, were significantly different. Proportions of PUFA increased significantly while that of saturated fatty acids decreased with increasing pressure, advocating feasibility of the method.27 The major flavor compound common in all seaweed groups (red, brown, and green) is DMS. Green seaweed flavor is mostly due to DMS and a group of unsaturated fatty aldehydes (8Z, 11Z, 14Z)-heptadecatrienal. Both these compounds are formed enzymatically. Brown seaweed flavor is not due as much to DMS and aldehydes as in the green seaweed, which are due to β-ionone and cubenol. Red seaweed flavor is least influenced by aldehydes. Flavor of the popular seaweed, Nori, is mainly due to DMS, carotenoid derivatives, and aldehydes. Bromophenol found in the red seaweed and iodine in the brown and red seaweed are their characteristic compounds. The former has a disinfectant-like odor and the latter gives odor of sea air. Kelp contains abundant amounts of iodine (70–2300 ppm) in the fresh fronds. Iodine is used as an additive in table salt to prevent thyroid gland disease.28

9.4.3

VITAMINS AND MINERALS

The vitamin and mineral contents of seaweeds have been discussed in Chapter 9. In addition, G. changi, an edible seaweed contains several nutrients including

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vitamin C, β-carotene, free fatty acid and amino acid contents. G. changgi showed vitamin A activity of 865 μg retinol equivalents per 100 g sample.23 In D. antarctica, γ-tocotrienol (651.7 mg/kg), δ-tocopherol (245.9 mg/kg), and α-tocopherol (179.4 mg/kg) were present in fronds; α-tocopherol (258 mg/kg) was present in stem. U. lactuca, showed a high γ-tocopherol level (963.5 mg/kg).13 The distribution of iodine in Sargassum kjellmanianum was studied using neutron activation analysis combined with chemical and biochemical separation techniques. The results indicate that iodine is mainly bound with protein, followed by pigment and polyphenol, and little with polysaccharides, such as algin, fucoidan, and cellulose. The observation is significant for the mechanism of enriching iodine of algae and utilization of alga iodine.29

9.4.4

POLYSACCHARIDES

Seaweed are essentially valued for their high contents of polysaccharides, which include agar, alginates, and carrageenans, collectively named as phycocolloids, hydrocolloids, or gums, which have important function as food fibers (see Section 10.5). The major sugar components of seaweed polysaccharides are galactose, mannose, and glucose. Galactose and 3,6-anhydro-d-galactose are the major constituents of agar, carrageenan, and furcellaran, whereas d-xylose and 6-O-methyl-d-galactose are present in lower proportions (up to 2%) in several Japanese seaweed agar. Because of their high water solubility, gel forming and other rheological functions, these polysaccharides are also called hydrocolloids. The crude fiber contents of the red seaweed, H. charoides and H. japonica, and green seaweed, U. lactuca, are in the range of 46–55% on dry weight basis.13,18 The yield, spectral characteristics, contents of 3,6-anhydro-d-galactose and sulfate contents are some of the factors determining the quality of commercial seaweed in terms of its hydrocolloid contents. For example, the largest proportion of structural units of polysaccharide from the red alga, Callophyllis hombroniana consisted of alternating 3-linked β-d-galactopyranosyl 2-sulfate and 4-linked 6,6-anhydo-α-d-galactopyranosyl 2-sulfate units, indicating that the polysaccharide was a θ-carrageenan.30 Several seaweed species have been analyzed for their contents of polysaccharides, particularly that of carrageenan (see also Chapter 7). Analysis of Brazilian seaweed species, Hypnea musciformis and Gigartina spp., showed presence of κ-carrageenan, an important type of carrageenan. However, many other species yielded another carrageenan, namely, the ι-type. The types of carrageenans can have important influence in the commercial applicability of the seaweed. The lowest carrageenan yield was found in Cryptonemia crenulata (5%), and the highest in Gigartina spp. (72%). No significant variation was observed in seaweed from different collection sites.31 New methodologies and instruments have provided insight into the relationships between the chemical structure and the gelling characteristics of these polysaccharides. In recent years, developments in multi- and low-angle laser-light diffusion detectors coupled to high-performance size exclusion chromatography have rendered easy determinations of molecular weights and distributions of these galactans.32 A rapid enzyme-linked lectin assays (ELLA) and enzyme-linked immunosorbent assay (ELISA) for determination of food-grade gums and thickeners has been described. The assay can also be used for determination of specific gums and thickeners in fruit jelly desserts, coating films, and pet foods.33

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9.5 DIETARY FIBER One of the major uses of seaweed is as dietary fiber, because of their high contents of polysaccharides. Therefore, this aspect is discussed in detail.

9.5.1

DEFINITION

Dietary fiber is a complex material consisting of plant cell walls, structurally complex and chemically diverse polysaccharides and other associated substances. In 2001, the American Association of Cereal Chemists adopted the definition as “that fraction of the edible part of plants or their extracts, or synthetic analogues that are resistant to the digestion and absorption in the human small intestine, usually with complete or partial fermentation in the large intestine.”34,35 Functional fiber consists of isolated, nondigestible carbohydrates and lignin that have beneficial physiological effects in human. Total fiber is the sum of dietary fiber and functional fiber.34 According to the definition adopted by the Food and Nutrition Board of the National Academy of Sciences, United States, in 2002, “dietary fiber consists of no-digestible carbohydrates and lignin that are intrinsic and intact in plants.” The European Union has not adopted any definition for fiber. However, in several European countries inclusion of separately measured resistant oligosaccharides as dietary fiber is accepted for labeling purpose.36 The preceding definitions require that the components included are not only indigestible in the small intestine, but have beneficial physiological effect typical for the dietary fiber. Of late, the definition has broadened to include not only nonedible parts of vegetables but also fibers of animal origin such as chitosans, which are derived from the exoskeletons of crustaceans and squid pens.37 The fiber in a diet may be in soluble and insoluble forms. Foods containing soluble fiber include whole grain foods such as breakfast cereals and multigrain bread, vegetables such as carrot and celery, oatmeal, nuts, legumes, and pears. Soluble fiber such as gums and pectin is abundant in whole grain barley and oats, as well as in fruits such as ripe strawberries and bananas. Seaweed carrageenan is considered a soluble fiber. Conventionally, oat products have attracted as a major source of fiber, which are rich source of β-glucans. Whole grain barley and dry milled barley products such as flakes, guts, flour, and pearled barley, which provide at least 0.75 g soluble fiber per serving are good sources of dietary fiber. Insoluble fiber helps make stools bulkier relieving constipation. Unlike other nutrients, fiber is not attacked by the enzymes of the stomach and small intestine and therefore reaches the colon undegraded.38

9.5.2

HEALTH BENEFITS

The consumption of a diet rich in fiber is generally considered beneficial for human health. Increased fiber intake leads to a decreased food transit time and increased stool bulk. Consumption of insoluble fibers such as cellulose and hemicellulose, as found in bran, leafy vegetables, or fruit skins serve as roughage and help to reduce the caloric value of diets, which is important in obese and diabetic conditions. The soluble fiber forms a viscous indigestible mass in the gut and helps trap digestive enzymes, cholesterol, starch, glucose, and toxins, which are then expelled through the feces. The soluble fraction of fibers has a hypocholesterolemic effect, possibly

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TABLE 9.3 Binding of Bile Acids by Dietary Fibers from Various Sources Including Seaweed Fiber Low-viscosity alginate High-viscosity alginate Agar Carrageenan Wheat bran Lignin Pectin

Cholate 26 113 57 36 119 138 73

Chenodeoxycholate 56 35 86 90 61 159 —

Deoxycholate 168 150 183 184 238 — 999

Note: The studies used bile acids conjugated with taurine rather than basic salts. All tests were carried out at appropriate neutral pH for various lengths of time ranging from 10 min to 2 h. Source: Reprinted from Brownlee, I. A. et al., Critical Rev. Food Sci. Nutr., 45, 497, 2005. With permission from Taylor & Francis Ltd. (www.informaworld.com).

due to augmented gastrointestinal content interfering with micelle formation and lipid absorption, or an increase and excretion of neutral sterols and biliary acids.39 By reducing absorption of food including high-fat items, soluble fiber can help obese people reduce the amount of starch digestion and glucose uptake from their food and also help diabetics control the disease. Excess of bile acids in the digestive system are also excreted as a result of their interaction with β-glucans in the fiber.40 Table 9.3 gives bile acid-binding properties of dietary fibers from various sources including seaweed.41 The fact that fiber can bind a large amount of water makes it highly useful from a physiological point of view, since it enlarges the volume of the aqueous phase of the food pellet and slows down the absorption of nutrients in the intestine. This also leads to modification of the function of the intestine by shortening the time in transit, diluting the contents, and supplying substrates that will ferment in the bowel. This and the effects of fiber on glucose absorption indicate that, in general, consumption of fiber induces a lower risk of diseases. The main benefits are reduction of intestinal absorption rate, reduction of colonic luminal toxicity and systemic effects, alteration of colonic microflora, and direct action on colonic mucosa. Other benefits include reduction of cholesterolemia, reduction of the glycemic load on the body, and intestinal functions leading to changes in pattern of absorption of nutrients.42–44 Epidemiological studies show a correlation between high-fiber diets and a lower incidence of some chronic disorders such as cardiovascular disease and colon cancer. In addition, recent reviews have pointed out a number of dietary fibers and prebiotics are immunostimulatory, essentially attributed to alteration in the colonic microflora. Dietary fiber has been implicated in modulating the colonic mucus barrier that can function as a barrier to reduce the chance of colonic infection and disease.45–47 Table 9.4 shows advantages of fiber in foods. Despite these benefits, it has been, however, cautioned that the association between risk of disease and diet is multifactorial

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TABLE 9.4 Advantages of Fiber in Foods Increase bulk quantity Reduce calorific value of diets Soluble fibers such as gums forms a viscous indigestible mass Reduces absorption of cholesterol and hence coronary heart disease Reduces glucose absorption and controls diabetics Reduces absorption of any toxins from the food Control obesity

TABLE 9.5 Technological Functionality of Dietary Fibers Functional Property

Advantage

Water holding/binding capacity

Soluble fibers such as algal fibers, pectin, gums, and glucans have higher WHC than cellulosic fibers. Algae fibers, depending on type, can bind water 20 times their own weight Depends on porosity of the fiber rather than molecular affinity. Watersoaked fibers have more fat-binding capacity Soluble fibers from algae form highly viscous solutions, a property which also make them useful as thickners in foods Fibers such as carrageenans, chitosan, and pectin form gel network which imbibe water and solutes in the network. Network formation depends on factors such as temperature, concentration, ions and pH Many types of fibers possess the capacity of binding minerals favoring reduced metal-induced functions such as lipid oxidation

Fat-binding capacity Viscosity Gel-forming capacity

Chelating capacity

and that with the present state of knowledge, fiber cannot be isolated as the sole factor. Furthermore, some negative effects of fiber cannot be ruled out. For instance, fiber can slow down the digestion of proteins and absorption of nutrients. Fibers can also cause reduction in availability of nutrients including vitamins and minerals.48 Table 9.5 gives technological functionality and Table 9.6 indicates physiological functionality of dietary fibers including alginate and carrageenan from seaweed. Despite the recognized benefits of dietary fiber, the intake of fiber around the world is far from adequate. In the United States, the current trends in the food hydrocolloids sector focus on development of commercial hydrocolloid products to meet consumer demand for high-fiber (and low-carbohydrate foods).50 The United States Food and Drug Administration (U.S. FDA) observed that food containing barley reduces risk of coronary heart disease. According to the American Dietetic Association, the current recommended fiber intakes for adults range from 25 to 30 g/day or 10 to 13 g/1000 kcal, and the insoluble/soluble ratio should be 3:1. In Europe, the fiber intake currently recommended is 20 g/person/day, but in developing countries the range is 60–120 g/day. FDA adopts soluble fiber health claim.51

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TABLE 9.6 Physiological Functionality of Dietary Fiber Function Carcinogenesis Reduction of cholesterolaemia Modification of glucaemic response

Changes in intestinal function Reduction of nutrient availability Antioxidant activity

9.5.3

Description

Reference

Modification Fibers reduce cholesterol uptake from food by interfering with lipid absorption and increased excretion of neutral steroids and bile acids Reduce calorific value of diets through viscositydependent lowering of contact of food with the absorbent intestinal epithelium and also retarded emptying of gastric tract through increased release of cholecystokinine Increase bulk quantity soluble fibers form viscous indigestible mass with food. Viscosity-dependent shortening food transit time Some fibers can inhibit pancreatic enzymes and interfere with the absorption of nutrients including vitamins and minerals Leads to reduced formation of oxidized lipids reducing their adverse effects

38 39, 48

48

48

38

49

FIBER FROM SEAWEED

Since seaweed contain a significant amount of soluble polysaccharides, they have potential function as dietary fiber. The seaweed polysaccharides possess a higher WHC than cellulosic fibers. There is interest in seaweed hydrocolloids for human nutrition as they can act as dietary fiber, their physiological effects being closely related to their physicochemical properties such as solubility, viscosity, hydration, and ion-exchange capacities in the digestive tract.32,52,53 A low level of seaweed (1% w/v) completely inhibited amylase activity, whereas extracted algal fibers (including alginates) had no effect. This suggests that seaweed may contain a specific inhibitor of amylase that may increase a reduction in glycemic response.54 The physicochemical properties of the red seaweed were similar to some commercially available fiber-rich food.25 An antiobesity diet has been developed recently based on seaweed or seaweed hydrocolloids. For overweight people and diabetics, a diet food formula consists of natural ingredients including agar, carrageenans, alginate, and the microalgae chlorella, and spirulina. The formula supplies the nutrients a body requires and cannot be metabolized after ingestion.55 Since apart from the use of dietary fiber, seaweed hydrocolloids are also used as food additives (see Chapter 11), the digestive fates of these soluble polysaccharides have received attention. These include the colonic fermentation; algal food additives (alginates, carrageenans, and agars); association between carrageenans and ulcerative colitis; toxicity; breakdown of carrageenans in the gastrointestinal tract; and the digestive fate of agars and other polysaccharides obtained from algae (laminarins, fucans, xylans).56 Three seaweed substrates (a partly soluble fiber (carrageenan)

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extracted from Eucheuma cottonii, a poorly viscous soluble fiber (xylan) extracted from P. palmata, and a highly viscous soluble fiber (alginate) extracted from L. digitata) were fed to pigs to examine their effects on the physicochemical properties of pig digesta, and evaluate changes in algal fibers in relation to the digestive conditions prevailing in different compartments of the gut. Results showed that carrageenan was partly insoluble in the gut and increased the intestinal bulk. Owing to its water retention capacity and poor fermentation in the colon, it showed potential to regulate fecal excretion. Gel formation by alginate in the stomach was partly reversible and the resultant high viscosity of the intestinal content was involved in reducing glycemic and insulinaemic responses. It was concluded that the physicochemcial characteristics of the digest depended on the intrinsic qualities of the ingested fiber, their concentration in the different digestive compartments, and the pH and ionic conditions prevailing in the gut.57 The capacity of carrageenan and agar, and also, xanthan and other gums to bind Ca2+, Zn2+, and Fe3+ in vitro was investigated at neutral and acidic pH, with a view to understand the influence of the hydrocolloids on bioavailability of cations. For divalent cations, no binding was detected under acidic conditions, whereas at neutral pH, correlation was observed between extent of binding and the cation-exchange capacity of the polysaccharides. Results indicated that the interactions are electrostatic and attributable to carboxyl and sulfate groups in the polyanionic polysaccharides.49,58,59 Since seaweed species also possess antioxidant and other functional activities, as will be discussed, there is an additional advantage of using seaweed as dietary fiber.

9.5.4

ENRICHMENT OF FIBER IN FOODS WITH SEAWEED

Seaweed can be used to enrich fiber contents of foods that are generally low in this component. For example, fishery products, which otherwise possess high nutritional properties, are poor in fiber contents. These products can be enriched with seaweed to improve functional properties such as water-binding and gelling. There is good potential for use of fiber particularly in restructured fishery products, to improve their viscosity and hence texture. The modification of emulsifying capacity by the addition of fibers is important for the sausage and fish-processing industries. The additives used to enhance gelling of fish muscle have been essentially carrageenans, which also prevented syneresis in fish gels during freezing/thawing. Alginate, an algal polysaccharide, is widely used in the food industry as a stabilizer, or as a thickening or emulsifying agent. As an indigestible polysaccharide, alginate may also be viewed as a source of dietary fiber. The function of aliginate as fiber has been recognized, particularly its effects on intestinal absorption and the colon, indicating its potential use as a dietary supplement for the maintenance of normal health, or the alleviation of certain cardiovascular or gastrointestinal diseases.60

9.5.5

SEAWEED AS DIETARY SUPPLEMENTS

The favorable chemical composition make many seaweed species important dietary supplements, since they are potentially good sources of polysaccharides, minerals, and certain vitamins, which make them nutritionally more valuable than many land vegetables.6,34 For example, it was suggested that in comparison with many common vegetables, high levels of fiber, minerals, and omega-3 fatty acids, and moderate

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concentrations of lipids and proteins could make G. changii suitable for health food application.23 A comparative study of the influence of two edible seaweeds, Nori (P. tenera) and Wakame (U. pinnatifida), on dietary nutritive utilization was performed. Male adult Wistar rats were fed diets containing Nori, Wakame, or cellulose as source of dietary fiber for a period of 2 weeks. All the diets contained similar amounts of dietary fiber (5%), protein (14%), and ash (5%). Intake, body weight gain, food efficiency (weight gain/food intake), apparent digestibility, and retention coefficients for protein, fat, and minerals (calcium, iron, magnesium, zinc, sodium, and potassium) were evaluated. The addition of Nori or Wakame did not affect body weight of rats or food efficiency. Fresh and dry stool weights were higher in seaweed-fed rats than in the control group. Seaweed-fed animals showed significantly lower apparent digestibility of protein and fat, but more effective use of nitrogen was absorbed. Apparent digestibility and retention coefficients for calcium, magnesium, zinc, iron, sodium, and potassium were lower for seaweed-fed rats, and showed lower values for Wakame than Nori. The seaweed could be a good source of dietary fiber in diet (see Section 9.5), but they may modify digestibility of dietary protein and minerals.35

9.5.6

SEAWEED AS SOURCES OF BIOACTIVE COMPOUNDS

Apart from hydrocolloids that can function as fiber, seaweed are sources of biologically active phytochemicals, which include carotenoids, phycobilins, fatty acids, polysaccharides, vitamins, sterols, tocopherol, phycocyanins among others.61,62 Many of these compounds have been recognized to possess biological activity and hence beneficial for use in human and animal healthcare. Some of the potential benefits included control of hyperlipidemia, thrombosis, tumor, and obesity. Some seaweed have been recognized to possess antioxidant activity as well as bacteriostatic effects against microorganisms including pathogens.61,63 Recently, much attention has been paid on the antitumor, anticholesterolemic, and antioxidant activities of seaweed constituents. PUFAs from seaweed have been found to have profound cytotoxic effects against human cancer cell lines. Interestingly, the occurrence of conjugated polyenes had no relation with the geographical location as two other red seaweed species belonging to the genus Gracilaria did not show any presence of arachidonic acid (AA).64 Recently, a botanic salt, designated as “Saloni-K,” a mixture of 30% potassium chloride and 60% sodium chloride has been isolated from the seaweed, Kappaphycus alvarezii and from the plant, Salicornia brachiata. The salt has the potential to reduce blood pressure. The potassium chloride content in the salt helps in the slackening of muscles thereby reducing the effect of sodium chloride, which causes high blood pressure. The salt is being commercially produced for Indian markets (Ghosh, P. K., Personal communication, May 21, 2007). Table 9.7 indicates availability of pigments and some other bioactive compounds from algal species. The major biological activities of seaweed components are discussed later in this chapter. 9.5.6.1

Antioxidant Activity

The problems caused by the reactive oxygen species (superoxide anion [O−2], hydroxyl radical [HO•], peroxy [ROO•], alkoxy [RO•], and hydro peroxy [HOO•] radicals) are alleviated by the presence of antioxidants (see Chapters 4 and 5). Antioxidants have

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TABLE 9.7 Pigments and Some Other Compounds from Algal Species Product Tocopherol Phycocyanin Fucoidan Fucoxanthin β-carotene Astaxanthin Canthaxanthin Phlorotannins/phloroglucinols Sesquiterpenes Furanones

Algal Sources Various seaweed Spirulina, Porphyridium, Rhodelia, etc. F. vesiculosus Brown seaweed, Pheophyceae Dunaliella salina Haemotococcus pluvialis (fresh water alga) Dunaliella salina Eisenia bicyclis, Ecklonia kurome L. obtuse, L. rigida Red algae such as D. pulchra

Source: Adapted from Borowitzka, M. A., Infofish Int., 5, 21, 1993. With permission from Infofish.

been shown to reduce blood pressure and lower cardiovascular diseases.65 Butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), and tertiarybutyl hydroquinone (TBHQ) are synthetic antioxidants used in food and pharmaceutical industries. But these antioxidants have been suspected to cause toxicity.66 Therefore, interest is focused toward finding natural antioxidants as additives in food systems. Being from natural sources, these antioxidants have the advantages of consumer confidence and acceptance and may not warrant any safety tests from the regulatory authorities.67,68 The interest in natural antioxidants is, perhaps, more in the pharmaceutical industry due to an urgent need for development of antiageing factors. Seaweed extracts have been found to exhibit antioxidant activities. Potential antioxidant activities of enzymatic extracts from seven species of brown seaweed were evaluated using four different assays for the scavenging action of reactive oxygen species (ROS), which included measurement of 1,1-diphenyl-2-pricrylhydrazyl (DPPH) free radical, superoxide anion, hydroxyl radical, and hydrogen peroxide. The brown seaweed were enzymatically hydrolyzed to prepare water-soluble extracts by using two enzyme sets of carbohydrases and proteases, each set containing five enzymes from commercial sources. The enzymatic extracts exhibited more prominent effects in hydrogen peroxide scavenging activity (approximately 90%) compared to other scavenging activities. The activities of these extracts, in general, were comparable or higher than those of the commercial antioxidants, BHA and BHT. The antioxidant activities, particularly of two extracts, were dose-dependent and also thermally stable. These two enzymatic extracts were also found to reduce oxidation-dependent DNA damage by almost half. The activities indicated a marked correlation with phenol contents of the extracts. The results suggested the use of enzymatic extracts of the brown seaweed as a rich source of antioxidants.69 Table 9.8 gives scavenging activities of reactive oxygen species and total phenol contents of brown seaweed extracts prepared by enzymatic digestion using a commercial carbohydrase. Dried Scytosiphon lomentaria, a brown alga, is a traditional food in

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TABLE 9.8 Scavenging Activities of Reactive Oxygen Species and Total Phenolic Contents of Brown Seaweed Extracts Seaweed

DPNH

Ecklonia cava Ishige okamurae Sargassum fullvelum Sargassum horneri Sargassum coreanum Sargassum thunbergii Scytosipon lomentaria α-Tocopherol BHA BHT

62.90 23.03 19.91 11.18 37.15 16.78 16.00 89.64 87.38 56.05

O2−

HO

H2O2

67.14 37.14 50.00 58.57 5.71 47.14 47.14 41.14 34.84 24.74

16.33 25.33 23.96 24.08 13.20 26.80 26.09 78.89 56.36 46.87

72.97 18.01 61.14 90.88 68.48 30.03 10.79 64.11 67.37 50.32

Total Phenols 1162 213 240 272 945 261 115 — — —

Source: Adapted from Heo, S.-J. et al., Bioresource Technol., 96, 1613, 2005. With permission from Elsevier.

the Noto area in Japan. A water extract of the seaweed contained total phenols at about 5.5 mg catechin equivalents showed strong antioxidant activities. However, the antioxidant activities of an ethanol extract were not detected or were very low compared with the water extract. The antioxidant activity was observed not only with the ferrous chelating (binding) assay, but also in the superoxide anion radical scavenging assay.63,70 Fucus vesiculosus was extracted with water at 22°C, dilute acid, or alkali. The extracts were composed of neutral sugars, uronic acids, sulfate, small amounts of protein, and polyphenols. The main neutral sugars were fucose, glucose, galactose, and xylose. The dilute acid extract, containing fucose showed the highest antioxidant activity. Similarly, crude methanol extracts of Sargassum siliquastrum exhibited high antioxidant activity in terms of red blood cell hemolysis and lipid peroxidation assays. A fraction isolated from the methanol extract, and which contained phenolic compounds, was found to be most effective in protecting rood blood cells.71 In another comparable study, composition and the antioxidant activities of lipophilic extracts of 16 seaweed samples were analyzed by gas chromatography and gas chromatography-mass spectrometry. The diethyl ether soluble extracts of all selected seaweed exhibited various degrees of antioxidant activities, the high activities being exhibited by Rhodomela confervoides and Symphyocladia latiuscula, which were comparable with that of the well-known antioxidant, butylated hydroxytoluene. It was concluded that seaweed can be considered as a potential source for the extraction of lipophilic antioxidants.72 The antioxidant activity of seaweed could be attributed to sulfated polysaccharide, fucoidan. The activity was dependent upon the extent of sulfation of the compound, since oversulfation of fucoidan enhanced its antiangiogenic and antitumor activities.73 In addition to fucoidan, a carotenoid, fucoxanthin has also been bestowed with antioxidant activity.74 These data, in general, suggested that edible seaweed could be used as natural antioxidants by the food industry.75

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9.5.6.2

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Antibacterial and Antiviral Activities

Seaweed products could also exhibit antibacterial activity. The antibacterial activities of dietary polysaccharides (κ-carrageenan, ι-carrageenan, and λ-carrageenan, and also other gums including pectin) against eight food-borne pathogenic bacteria were examined. The bacterial strains used were Salmonella enteritidis E930-448 (phage type 4), S. typhimurium 1030-96, Vibrio mimicus 753, Aeromonas hydrophila 088, enterotoxigenic Escherichia coli K88, Listeria monocytogenes, and Staphylococcus aureus SA110, S-6, SA106, and 494. Among the polysaccharides, the carrageenans showed the most pronounced inhibitory effect. The growth of all the bacterial strains except L. monocytogenes was significantly inhibited by them, particularly by ι-carrageenan. A growth inhibition experiment using S. enteritidis showed that the inhibitory effect of the carrageenans was not bactericidal but bacteriostatic. Removal of sulfate residues eliminated the bacteriostatic effect of ι-carrageenan, suggesting that the sulfate residue(s) in carrageenan played an essential role in this effect. The results could be extrapolated to make use of the dietary polysaccharides, particularly carrageenans, as effective preservatives in various types of processed food.76 Seven seaweed were collected during spring tide from Rocky Bay on the east coast of South Africa and tested for antifungal, antibacterial, and acetyl cholinesterase (AChE) inhibitory activities. Dictyota humifusa extracts showed highest antifungal and (AChE) activities. The seaweed extracts inhibited the growth of the gram-positive bacteria, with Bacillus subtilis being more sensitive than S. aureus. D. humifusa was the only seaweed able to inhibit the gram-negative E. coli. Although seasonal variations in antifungal activity was not detected, such variations in antibacterial activity were observed, with the extracts generally having no activity in summer and having antibacterial activity in late winter (July) and early spring (September and November). D. humifusa was the most effective seaweed species, having antibacterial activity throughout the year. All the extracts tested had AChE inhibitory activity, with no seasonal variation in the levels of activity.77 The halogenated terpenes, lembyne A and lembyne B isolated from the red alga, Laurenci majuscule and L. marinensis, showed interesting antibacterial activities against several marine and terrestrial bacteria. Monoterpenes with antitubercular activity have been isolated from the marine alga Plocamium hamatum. A bromoditerpene, bromosphaerone, isolated from Sphaerococcus coronopifolius showed promising activity against the bacterium, S. aureus. Almazole D, an antibacterial oxazole dipeptide was isolated from a delesseriacean marine alga.78 A number of reports on the antibacterial importance of seaweeds belonging to Phaeophyceae, Rhodophyceae, and Chlorophyceae have been summarized.79 Several species of Halimeda produce terpenic di- or trialdehydes, which possess ichthyotoxic activity.80 The viral activity of green algae has been attributed to sphinganine amide and caulerpicin. Several diterpenoids have been reported from the brown algae, Dictyota spp. Dictyodiol, an antimicrobial diterpene was reported from D. spinulosa. Other bioactive diterpenoids are dictyochromenol and its analog, which exhibit ichthyotoxicity and cytotoxicity. Marine red algae are rich sources of a vast array of halogenated lipids. Elatone, which is a powerful inhibitor of cell division, has been isolated from Laurencia elata. The presence of prostaglandins in nonanimal sources was detected in Gracilaria spp. Prostaglandin PGE2 and PGF29 have been isolated from G. pichenoids. A pharmacologically active nucleoside

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5-iodo-5′-deoxytubercidin was isolated from Hypnea valentiae, which causes relaxation of muscles and hypothermia in mice. These observations lead to the conclusion that marine algae are potential sources of bioactive substances.81 There is a wide scope for development of these compounds for control of human pathogens. The antimicrobial activities of seaweed extracts were recently screened against ecologically relevant marine microorganisms including the pathogenic fungus Lindra thalassiae, the saprophytic fungus Dendryphiella salina, the saprophytic stramenopiles, Halophytophthora spinosa, Schizochytrium aggregatum, and the pathogenic bacterium Pseudoaltermonas bacteriolytica. In addition, the same assay microorganisms were used to examine the antimicrobial effects of lipophilic and hydrophilic extracts from 54 species of marine algae and two species of sea grasses collected from Indo-Pacific reef habitats. Overall, 95% of all species surveyed were active against one or more organisms. Extracts from the green alga Bryopsis pennata and the red alga Portieria hornemannii inhibited the growth of all assay microorganisms. These results provide evidence that antimicrobial chemical defenses are widespread among Indo-Pacific marine plants. Further, the activity profiles of the extracts suggest that antimicrobial secondary metabolites can have pathogen-selective or broadspectrum effects. To confirm these results, chemical studies will be needed to isolate and characterize the compounds responsible for the observed antimicrobial activities.82 9.5.6.3 Platelet Aggregation In a recent study, the influence of feeding brown or red seaweed on lipid levels in serum as well as platelet aggregation in rats was examined. The brown seaweed, namely, Eisenia bicyclis (Arame), Hizikia fusiformis (Hijiki), and U. pinnatifida sporophylls (Mekabu), and a red seaweed Porphyra yezoensis (Susabinori) were powdered and mixed in a ratio of 45:30:20:5 (w/w). When rats were fed a cholesterol-rich diet containing this mixture of seaweed at a level of 9–10% (w/w) for 28 days, serum total cholesterol, LDL-cholesterol, free cholesterol, and triglyceride levels declined significantly to 49.7, 48.1, 49, and 74.8%, respectively. Serum HDL-cholesterol, however, was unchanged. Though activated partial thromboplatin time, prothrombin time, antithrombin III activity, and fibrinogen levels in plasma were unchanged, the maximal ADP- and collagen-induced platelet aggregation decreased significantly to 89 and 85.5% as compared with rat fed with control diet. These results indicated that this mixture of E. bicyclis, H. fusiformis, U. pinnatifida sporophylls, and P. yezoensis, is useful for the prevention of hyperlipidemia and thrombosis in rats.83 Hem agglutinating activity of red alga Gracilaria chorda has been reported.84 9.5.6.4

Antitumor Activity

The bioactivity of seaweed products on tumor development has been a topic of great interest. The influence of the seaweed, U. pinnatifida (popularly known as Wakame in Japan) on 7,12-dimethylbenze-anthracene (DMBA)-induced rat mammary tumor was investigated. DMBA was administered to 8-week-old female Sprague–Dawley rats that developed mammary tumors. The rats were assigned randomly to three groups. The control rats were fed commercial feed while the experimental groups were given feed mixtures, which contained commercial rat feed blended with Wakame at

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1 and 5%, by weight. Changes in mammary tumor size were compared in rats for a period of 8 weeks. At the end of the experiment, mammary tumors and thyroid glands were resected to compare their weights. Serum total iodine and thyroxin levels were measured. Immunohistochemical studies for bromodeoxyuridine (BrdU) labeling, transforming growth factor-β, and apoptosis were carried out in the resected tumor. Significant suppression of tumor growth was observed in groups fed with Wakame compared with those fed-control diet. In the seaweed-fed groups the weights of resected mammary tumors were significantly lower and serum total iodine concentration was significantly higher than in the control. BrdU indices were significantly lower in seaweed fed rats. Transforming growth factor-β and apoptotic index were inversely related to BrdU. These results suggested that iodine is transported from the serum into mammary tissues and induced apoptosis through the expression of TGF-β. In conclusion, Wakame suppressed the proliferation of DMBA-induced mammary tumors.85 Fucoxanthin is commonly isolated from the wakame seaweed. Fucoxanthin remarkably reduced the viability of human colon cancer cell lines, Caco-2, HT-29, and DLD-1 and also growth of human leukemia cells. The inhibitory effect on the cancer cell lines was stronger than that of β-carotene and lycopene, the other two carotenoids whose anticancer effects are well documented. The treatment with fucoxanthin-induced DNA fragmentation, indicating apoptosis. Moreover, combined treatment with 3.8 μM fucoxanthin and 10 μM troglitazone (a specific ligand for peroxisome proliferator-activated receptor [PPAR] gamma), effectively decreased the viability of Caco-2 cells. However, separate treatments with these same concentrations of fucoxanthin nor troglitazone did not affect cell viability. These findings indicate that fucoxanthin may act as a chemo preventive or chemotherapeutic carotenoid in colon cancer cells by modulating cell viability in combination with troglitazone.65,86 In a recent work, it was shown that fucoxanthin significantly suppressed human umbilical vein endothelial cells (HUVEC) proliferation and tube formation. Fucoxanthin at 10–20 μM, effectively suppressed formation of blood vessel–like structures from CD31-positive cells, suggesting that it could suppress differentiation of endothelial progenitor cells into endothelial cells involving new blood vessel formation. The carotenoid and also fucoxanthinol suppressed microvessel outgrowth in an ex vivo angiogenesis assay using a rat aortic ring, in a dose-dependent manner. These results imply that fucoxanthin having antiangiogenic activity might be useful in preventing angiogenesis-related diseases.87 9.5.6.5

Hyperoxaluria

Hyperoxaluria is considered as one of the major risk factors for idiopathic calcium oxalate stones. Oxalate is an inert end product of carbon assimilation, mainly excreted by the kidney. Abnormalities in oxalate metabolism have been suggested as a cause for the pathogenesis of stone disease, as an excessive excretion of oxalate leads to calcium oxalate crystalloid. Application of exogenous glycosaminoglycans (GAGs) to prevent stone formation and recurrence is considered as a promising prophylactic approach. It is reported that synthetic polysaccharides such as low-molecular-weight heparin (LMWH) have reno-protective effects. Fucoidans, the sulfated polysaccharides from brown algae, are reported to have blood anticoagulant, antitumor, antimutagenic, anticomplementary, immunomodulating, hypoglycemic, antiviral, hypolipidemic,

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and anti-in ammatory activities. In addition, these sulfated polysaccharides bear similarity with heparin, possessing anticoagulant activities.88 Heparinoid-active sulphated polysaccharides from marine algae as potential blood anticoagulant agents. The potential of fucoidan from the edible seaweed F. vesiculosus in ameliorating the abnormal biochemical changes in experimental hyperoxaluria was examined. Two groups of male albino rats of Wistar strain received 0.75% ethylene glycol for 28 days to induce hyperoxaluria, and one of them received, fucoidan from F. vesiculosus, at 5 mg/kg body weight, commencing from the 8th day of the experimental period. Incongruity in the renal tissue enzymes (ALP, β-Glu, and γ-GT) were observed during hyperoxaluria along with an increased activity of oxalate metabolizing enzymes such as LDH, GAO, and XO. These changes were reverted to near normalcy with administration of fucoidan. Alterations were observed in the activities and levels of tissue enzymes, namely, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase and antioxidants such as reduced glutathione, ascorbate, and α-tocopherol antioxidants, along with high malondialdehyde levels in the hyperoxaluric group. However, normalized lipid peroxidation status and antioxidant defenses were noticed with the administration of fucoidan. Biochemical discrepancies observed in hyperoxaluria disrupt membrane integrity, favoring a milieu for crystal retention. Advocation of sulfated polysaccharides enhanced the antioxidant status, thereby preventing membrane injury and alleviating the microenvironment favorable for stone formation.89 9.5.6.6

HIV Inhibition

Regular consumption of dietary algae might help prevent HIV infection and suppress viral load among those infected. The incidence of HIV is about 1 in 10,000 adults in Japan and Korea, compared to Africa, where one in every ten adults is HIV positive. In these countries, consumption of seaweed is a habit. It has been postulated that regular consumption of dietary algae might help prevent HIV infection and suppress viral load among those who are infected. Carraguard has been shown to be effective against HIV in vitro and against herpes simplex 2 virus in animals. Testing has advanced to the stage where the international research organization, the Population Council, is supervising large-scale HIV trials of Carraguard, involving 6000 women over 4 years.90 An antiviral activity in the extract prepared from Indian seaweed has been patented.3,91 9.5.6.7

Enzyme Inhibition

Hyaluronidase (EC 3.2.1.35) is an enzyme that depolymerizes the polysaccharide hyaluronic acid, in the extracellular matrix of connective tissue. The enzyme is found both in organs (testis, spleen, skin, liver, kidney, uterus, and plancenta) and in body fluids (tears, blood, and sperm). The enzyme is known to be involved in allergic effects, migration of cancer, inflammation, and other reactions. Therefore, inhibitors of the enzyme can have anticancer and antiallergic effects and could lead to development of drugs. Polyphenols are one of the most common types of secondary metabolites in terrestrial and marine plants. Although terrestrial polyphenols are polymers based on flavonoids or gallic acids, marine algal polyphenols, phlorotannins, which are only

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known in brown algae, are restricted to polymers of phloroglucinol (1,3,5-trohydroxybenzene). The inhibitory effect of brown algal phlorotannins against hyaluronidase was evaluated by an in vitro assay. Crude phlorotannins from the brown algae E. bicyclis and Ecklonia kurome had a stronger inhibitory effect than well-known inhibitors, catechins and sodium cromoglycate. Concentration required for 50% inhibition (IC50) of the enzyme by the following six phlorotannins, namely, phloroglucinol, an unknown tetramer, eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol, and 8,8’-bieckol (hexamers), were 280, 650, >800, 140, 120, and 40 μM, respectively. In comparison, the IC50 values of catechin, epigallocatechin gallate, and sodium cromoglycate were 620, 190, and 270 μM, respectively. The results showed strongest HAase inhibitory activity of phlorotannin, which acted as a competitive inhibitor with an inhibition constant of 35 μM, suggesting scope for potential development of drug using the brown algae.92 There is ample scope for isolation of these bioactive principles from seaweeds for practical applications. Table 9.9 summarizes some bioactive compounds from seaweed and their functions.

TABLE 9.9 Some Bioactive Compounds from Seaweed and Their Functions Seaweed Sargassum vulgare Himanthalia elongate Undaria pinnatifida

Ingredient Alginic acid, xylofucans PUFAs, α-tocopherol, sterols, fiber PUFAs, α-tocopherol, sterols, fiber, folate, fucodanthin

Chondrus crispus

PUFAs, α-tocopherol, sterols, fiber, folate, fucodanthin

Ulva spp.

Sterol

Various seaweed including Sargassum lomentaria, S. latiuscula, S. ringgoldianum, Rhodomela confervoides, Wakame Fucus vesiculosus

κ-carrageenan, ι-carrageenan λ-carrageenan, fucoxanthin, fucoidan

E. bicyclis, H. fusiformis, U. pinnatifida sporophylls, and P. yezoensis Eisenia bicyclis and Ecklonia kurom phlorotannins

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Inhibition of hyaluronidase

Potential Health Effects Antiviral activity Reduction of total and LDL cholesterol Reduction of total and LDL cholesterol, certain types of cancer, antiviral activity Reduction of total and LDL cholesterol, reduction of cardiovascular disease Reduction of total and LDL cholesterol Obesity control, apoptosis of cancer cells, induction of docosa hexanoic acid (DHA), antitumor activity Sulfated polysaccharides are potential blood anticoagulant agents Prevention of hyperlipidemia and thrombosis Inhibitors of the enzyme can have anticancer and antiallergic effects

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9.6 INDUSTRIAL USES OF SEAWEED The main genera under the brown seaweed are Laminaria, Undaria, and Hizikia, which are mainly used directly as food and known as edible seaweeds, whereas Sargassum is mainly used as fertilizer. The most popular farmed red seaweed are Phorpyra, Gelidium, Gracilaria, and Kappaphycus/Eucheuma spp., which are used to extract hydrocolloids. Phorpyra is also dried and processed into nori or laver sheet.4 Seaweed have been used as sources of a wide range of chemicals for a few centuries. From the seventeenth to the nineteenth century, the brown macroalgae Fucus, Ascophyllum, and Laminaria were the major sources of soda ash (sodium carbonate) and potash (potassium carbonate), whereas in the nineteenth and early twentieth century the brown algae were used to produce iodine, ammonia, and acetone.61 As discussed earlier, seaweed are good sources of valuable nutrients, particularly, minerals, fiber, and also drugs.14,16,23,25,93–97 Therefore, they are useful as dietary components for humans, and also as fertilizer and feed for plants and animals including fish. Many macroalgal species such as Laminaria, Undaria, Chondrus, Porphyra, and Cauleprpa are being used as human food because of their nutritional quality as well as potential health benefits and have established themselves as a dietary supplements for the last several centuries in a number of Asian countries.2 Seaweed are also being used as fertilizers, soil conditioner, and source of salts. Realizing the commercial potentials of these macroalgae, research and development thrusts have been geared toward developing and improving its product applications. Several Western countries such as the United States, South America, Ireland, Iceland, Canada, and France have significantly increased the consumption, production, and marketing of seaweed.2 These countries are also commercially making use of seaweed as a source of food hydrocolloids. The functional properties of these hydrocolloids offer scope for their valuable applications as additives for water-thickening, emulsifying, and gelling agents in industries ranging from foods, pharmaceuticals, biotechnology, cosmetics, paper, textiles, and petroleum.17,38,50 The voluminous information on their physiological as well as functional properties and current applications will be discussed in the subsequent chapter (see Chapter 7). Important direct uses of seaweed are briefly mentioned in the following section.

9.6.1

AGRICULTURE

Seaweed are used in agriculture as farm yard manure and liquid seaweed fertilizers. Since seaweed contain 15–20% ash with as many as 60 minerals, as discussed earlier, they functions as good mineral supplements. These minerals and trace elements are present in water-soluble form in seaweed, and hence are readily absorbed when the manure is applied, which help better nitrification than conventional farmyard manure. In addition, bioavailability of adequate amounts of potassium, nitrogen, growthpromoting hormones, and micronutrients make seaweed an excellent fertilizer. Another reason that makes seaweed superior to chemical fertilizer is the high level of organic matter in the algae that aids in retaining moisture and minerals in the upper soil level available to the roots. Furthermore, the high fiber content of seaweed acts as soil conditioner and assists moisture retention. Seaweed species such as Spathoglossum asperum, U. fasciata, Sargassum wightii, Caulerpa chemnitzia, and Enteromorpha

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intestinalis have been used as fertilizer for gram, maize, and groundnut. Procedures adopted were soaking seeds in the seaweed extracts before planting and later use of the algae extracts as leaf spray. The seeds soaked with aqueous extract of seaweed performed better when compared to the water-soaked controls. Diluted extracts of seaweed when sprayed on plants show beneficial results in terms of health of plant, increase in growth, resistance to pest, and 25–30% higher yield.98 A concentration of 20% of aqueous extracts of Sargassum wightii and C. chemnitzia promoted the seedling growth in terms of the parameters, namely, lengths of shoot and root, fresh and dry weights, chlorophyll, carotenoids, protein and amino acid, reducing sugar contents, and amylase activities of shoot and root. Among the two seaweed tested, Sargassum wightii exhibited better responses.99 Liquid seaweed fertilizer has gained importance as foliar spray for several crops because the extracts contains growth promoting hormones, cytokinins, carotenes, folic acid, minerals (Fe, Cu, Zn, Co, Mo, Mn, Ni), vitamins (including vitamins E and B12), and amino acids. Aqueous extract of Sargassum wightii when applied as a foliar spray on Zizyphus mauritiana showed an increased yield and quality of fruits.100 Commercial seaweed extracts are now available under different trade names.101

9.6.2

ANIMAL FEED

Because of their significant nutritive value, seaweed have also been used as a component of animal feeds in many countries including Norway, France, Finland, and United States. In Norway, Rhodymenia palmate, a red seaweed is used as horse meal and Ascophyllum as pigmeal. Approximately 50000 t of wet seaweed are harvested annually to make 10000 t of seaweed meal.89 Some of the species which are currently used as cattle feed include Enteromorpha, Fucus, Gracilaria, Hypnea, Sargassum, Pandina, Laminaria, and Dictyota spp. Use of seaweed as cattle feed helps in increasing fertility and birth rates of animals. Both milk production and yield of butterfat have been found to increase by using seaweed as part of the diet. In addition, seaweed also afforded better disease resistance. Cattle fed on Laminaria spp. based diet showed better natural resistance to disease such as foot and mouth disease. This also reduced the incidence of mastitis.102 The digestibility of Saragassum spp. by bovine cattles is 55%. Experiments using 3500 sheep showed that an addition of 35 g/day of seaweed meal gave a 3.3% increase in winter wool, TABLE 9.10 Challenges in Utilization of Seaweed as Animal Feed Identification of suitable seaweed species Compositional analyses in terms of amino acids, vitamins, and minerals Collection/cultivation of suitable seaweed and development of animal feed Development of a database for information regarding sources, availability, nutritional value, and optimal incorporation levels in feeds for animals and poultry Source: Adapted from Ahuja, A. K. and Saijpaul, S. S., Proc. Symp., Nutritional Technologies for Commercialization of Animal Production System, Animal Nutrition Society of India, ICAR, New Delhi, 2004.

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which was increased further to 17% even if the sheep had no mineral supplement. Seaweed-incorporated poultry feed containing fish meal improved the color of egg yolk, presumably due to the presence of fucoxanthin. Further, hardness of the eggshells was also increased.103 Table 9.10 indicates some challenges in utilization of seaweed as animal feed.

9.6.3

FEED FOR AQUACULTURE

Seaweed can be a dietary supplement for farming fish and shellfish species facilitating their higher growth and survival rates. The seaweed Gracilariopsis bailinae was fed to hatchery-bred donkey’s ear abalone. Equal numbers of female and male abalone were stocked in 24 units, with 8 replicates per dietary treatment. Reproductive performance, which included number of spawnings, instantaneous fecundity, and egg-hatching rates, was monitored over 270 days. The diet helped better survival rates of broodstock at 88% as compared with abalone fed with mixed diets containing seaweed and artificial diets, which showed a survival rate of 75. The mean number of spawnings was not significantly different among treatments. However, diets containing seaweed with artificial diets gave higher fecundity and hatching rates. Fatty acid analysis showed that the omega-3/omega-6 fatty acid ratios of abalone hepatopancreas reflected those of their diets. The ratio of the fatty acids is over 1.3 in mature abalone ovary.104 A method for preparing wetfeed for Atlantic salmon, Salmo salar L., using a binder produced from seaweed, Ascophyllum nodosum L. has been developed. The feed was formulated with offal from the fish, haddock, saithe, pollack, cod, mackerel, and argentine. The extruded feed contained fish meal, wheat, capelin oil, vitamin, and mineral premix, astaxanthin, and seaweed binder. The feed was gellified in a solution of calcium chloride and was compared with a control commercial dry feed. On a dry matter basis, the two feeds were comparable with respect to protein, fat, and carbohydrate. Growth rates, appetite, and feed conversion were similar for the two experimental groups. Chemical and sensory analyses of the fish showed no difference between the two groups. There was a significantly higher level of lysozyme in the wet feed group than in the dry feed group, implying an immunostimulating effect of alginate. The study suggested that incorporation of seaweed could reduce feed production costs without affecting the yield.105 Seaweed and fish can be polycultured. An integrated polyculture system for seaweed (G. bailina) and milkfish (Chanos chanos) has been reported. Two stocking density combinations of 30 fingerlings per 100 m2 pond area containing 1–2 kg of the seaweed per 4 m2 net cage in brackish water ponds over four culture periods were used. Growth, net production, and survival rates were evaluated for milkfish, while specific growth and production rates of the seaweed were determined. Significantly, higher mean specific growth and mean net production rates of red seaweed were obtained. Growth rates of milkfish were positively correlated with temperature and salinity, whereas net production rates were positively correlated with temperature and total rainfall, but was inversely correlated with dissolved oxygen. The production of milkfish and red seaweed was higher during the dry season. G. bailinae growth and net production rates were positively correlated with water temperature and salinity. Results showed that milkfish can be polycultured with G. bailinae grown under the conditions mentioned earlier.106 Regional hatcheries in California use the algae

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Macrocystis pyrifera species as a main source of natural food. AquaMats® are a type of artificial seaweed designed to provide structure in ponds used for fish culture and as a substrate for the growth of aquatic plants and invertebrates which in turn are a source of nutrition to cultured species.107 In Europe, America, and Asia, certain species of seaweed namely wrack (Fucus), oarweeds (Lamina, Alaria, etc.) are used as fodder for animals. In many coastal regions, seaweed are used as fertilizer either as such or after composting. The effluents from rapidly expanding aquaculture farms could be used for cultivation of preferred seaweed species. One of the main environmental issues concerning aquaculture of shrimp and finfish is the direct discharge of significant amounts of effluents from the aquaculture ponds into coastal waters, causing pollution. There is potential for integrated cultivation of shrimp since seaweed act as scrubbers in reducing nutrient load and cleaning the environment.1 By integrating aquaculture practices, which involve utilization of nutrient-rich effluents for seaweed cultivation and use of the cultivated seaweed as feed, a balanced ecosystem approach can be developed. This ensures nutrient bioremediation capability, economic diversification by producing value-added marine crops, and increased profitability per cultivation unit for the aquaculture industry. The integrated polytrophic practice could help the aquaculture industry find increasing environmental, economic, and social acceptability.

9.6.4

ANTIFOULING AGENTS

Biofouling is the undesirable accumulation of bacteria, algae, hydroids, barnacles, mussels, etc., on the surfaces submerged in seawater, such as ship’s hull, seaside piers, jetties, and pipelines. Biofouling on ships reduces their speed causing increased fuel and maintenance costs. On static structures such as pipelines, biofouling can enhance the corrosion of metal by seawater. It has been estimated that biofouling costs shipping and other marine industries annually U.S.$6.5 billion. The most effective formulations to control the problem is currently tributyltin and other organic compounds. However, these antifouling agents could contaminate the waters. Some of the secondary metabolites produced by seaweed can defend ageist the colonization of organisms onto the surface. Early investigations on seaweed-mediated defense focused mainly on phlorotannins or phloroglucinol, which are present in most of the brown algae. Many benthic invertebrates in the field experienced tissue necrosis when they are in contact with brown algae. The red algae, Laurencia spp. are rich in brominated secondary metabolites. The major metabolites in L. obtuse are sesquiterpenes, palisadin A and B, and aplysistatin, whereas another red algae, L. rigida contain elatol and deschlorelatol. These metabolites showed strong antifouling activity against invertebrate larvae. Other compounds having antifouling activities such as halogenated furanones, detected in certain other species of red algae such as D. pulchra, have been found active at concentrations as low as 100–200 ng against ecologically relevant marine organisms including marine bacteria, the bryozoan B. neretina, and the cosmopolitan alga Ulva spp. In addition, several marine bacteria isolated from red, brown, and green algae inhibit settlement of invertebrate larvae. These dark pigmented bacteria essentially belonged to a new species, Pseudoalteromonas. They also inhibit growth of bacteria, fungi, and diatoms.108,109

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285

BIOSORPTION OF HEAVY METALS

Marine macroalgae can be used as an excellent biosorbent material. There is good scope for water treatment to remove toxic pollutants such as heavy metals because of recognized high metal sorption capacity of seaweed. Because of their low cost and ready abundance, some seaweed species have been used as biofilters to treat industrial effluent to produce potable water. Seaweed powder has been used for separation of coloring matter, inorganic matter, and small quantity of lignin from industrial effluents. Brown seaweed are interesting materials that can be used as biosorbents for heavy metals due to their high binding ability and low cost. The processes for treating industrial effluents by seaweed have been developed and patented in India. These include processes for treatment of black liquor waste from paper mills, and spent wash from distillery waste to prepare potable water.108 Some of the brown seaweed species successfully examined for water treatment were Ecklonia maxima, Lessonia flavicans, and Durvillea potatorum. The abilities of these biomasses to sequester the heavy metal ions such as copper, nickel, lead, zinc, and cadmium from solution under constant agitation are comparable. Further, the treatment is also cost-effective.110–112 The metal-binding ability is attributed to algal polysaccharides, because of their functional carboxyl groups. In metal-binding capacity, algal polysaccharides are ranked as sodium alginate > carrageenans > agar for metal binding, suggesting alginate have highest lead-binding capacity.112 Five different brown seaweed, Bifurcaria bifurcata, S. polyschides, A. nodosum, L. ochroleuca, and Pelvetia caniculata were studied for their ability to remove cadmium from aqueous solution. Kinetics of cadmium adsorption by all the algae were relatively fast, with 90% of total adsorption occurring in less than 1 h. The rate of adsorption followed a pseudo-second-order rate equation, with values between 1.66 × 10−3 and 9.92 × 10−3 g/(mg min) for the sorption rate constant. The maximum adsorptions were between 64 and 95 mg/g. Solution pH is an important parameter affecting biosorption of cadmium by algae. Although sorption was minimal at pH 2.0, it reached maximum at a pH value of 4.0. This behavior suggested that pH change can be used regenerate macroalgae columns.111 Cation binding equilibrium of dead biomass from the seaweed Sargassum muticum, Cystoseira baccata, and Saccorhiza polyschides was examined in the pH range between 2 and 8. The maximum proton binding capacities obtained ranged between 2.4 and 2.9 mol per kg.113

9.6.6

OTHER MISCELLANEOUS APPLICATIONS

An enzyme, hexose oxidase (EC 1.1.3.5) from seaweed has found application in detergent. This enzyme can convert lactose from varied sources such as agrofood wastes to lactobionic acid, which has applications in pharmaceuticals and detergents. The enzyme has been purified from the red seaweed C. crispus. Before extraction of the enzyme, carrageenans and proteins were removed by chromatography on DEAE-50 columns. The enzyme was then extracted from finely ground, freeze dried cells by ammonium sulfate precipitation and perfusion chromatography. The enzyme was subsequently purified to 100 fold by HPLC followed by Sephacryl chromatography to a final specific activity of 69 U/ml, and with a yield of approx. 10%.114

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TABLE 9.11 Future Priorities in Seaweed Research Taxonomic studies, both classical and molecular for cataloging as well as improvement Diversified uses of seaweed as feed, fodder, fertilizer, etc. Creation of a biodiversity database Refinement of processes of preparation of agar, alginate, and other products and quality control Ecological and enzyme immunoassay studies pertaining to introduction of exotic species Improvement of strains through biotechnological intervention such as tissue culture, genetic transformation by protoplast fusion and hybrid strain production, and gene and molecular mapping Source: Adapted from NAAS Policy Paper No. 22, National Academy of Agricultural Sciences, India, 2003. With permission.

Seaweed F. vesiculosus, F. serratus, A. nodosum, and Cladophora glomerata have proven to be excellent bioindicators for radioactive corrosion products released from a nuclear power plant into the marine environment. These bio-indicators have been used to map the spatial and temporal distribution of the released radioactivity. The activity has been followed up to 150 km from the power plant. The variation with time of activity concentration reflects the amount of activity discharged from the power plant, with surprisingly good resolution in time.115 Table 9.11 gives future priorities in seaweed research.

9.7 FARMING OF SEAWEED Because of their commercial importance, many seaweed species are now extensively farmed in countries such as China, Indonesia, Korea, the Philippines, Taiwan, Japan, Chile, the United States, France, Russia, and Italy. The world production of commercial seaweed has grown by 119% since 1984 and presently 221 species are utilized commercially, including 145 species for food and 110 species for phycocolloid production.116 According to a recent FAO report, aquatic plants including seaweed form the second most cultivated products, comprising 23.4% in volume and 6.2% in value.117 In 2003, it was estimated that 16 mt of wet seaweed was harvested in 40 countries as a source of food; as sources of agar, alginate, and carrageenan; as a fertilizer; as fuel; and for use in cosmetics, annually.2 The top five cultivated seaweed in the world are Laminaria, Porphyra, Undaria, Eucheuma, and Gracilari spp., which together account for approximately 6 mt of production. The most valuable aquaculture product in the world is nori, P. umbilicalis, which is cultivated in the coastal waters of Japan at a value of over U.S.$1.8 billion. In the Western world, seaweed cultivation has not advanced much beyond the experimental phase, most of which has occurred during the last 25 years.2,116 In India the Central Marine Fisheries Research Institute and Central Salt and Marine Chemicals Research Institute have done intensive work on cultivation of Gracilaria edulis and Gelidiella acerosa. Optimum yield of agar with high gel strength could be obtained from plants harvested after three months of culturing, giving an agar yield up to 40% on dry weight basis.118 India, having a coastline of more than 8000 km harbors more than 800 species of

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seaweed, suggesting rich biodiversity. Ecological studies have been undertaken for the cultivation of K. alvarezii, which was introduced to Indian coastal waters more than 10 years ago.101 The National Institute of Oceanography, Goa, India, has established a seaweed tissue culture laboratory to take up tissue cultures of economically important seaweed.91 All indigenous species of macroalgae are considered as ecologically safe for mass cultivation. Common techniques for the cultivation of seaweeds include pond culture, on-bottom culture, net culture and raft or rope culture.4 There are two methods for cultivation of seaweed, one by spore method and the other by vegetative propagation method. The former is expensive and takes more time, and therefore, for practical purposes, the latter method is being employed.119 Knowledge of biology and reproduction of seaweed is fundamental to successful cultivation. The six major factors governing seaweed cultivation are (i) selection of a suitable environment for growth, (ii) engineering of the shore for farming, (iii) protection of the crop from physical and biotic effects, (iv) probable pets and parasites, (v) proper transplanting of young germlings to the field of cultivation, and (vi) harvesting. Seaweed could be kept growing in filtered seawater to which nutrients in the form of potassium nitrate and potassium phosphate were added from time to time. Mostly, an enriched seawater medium is used. Seaweed could be cultivated in lagoons or ponds, open ocean using artificial upwelling, onshore ponds or shallow trays with a sprinkler system of irrigating with enriched seawater. The lagoon culture method perhaps is most costeffective, which uses natural areas such as estuaries and bays, which are flushed with tidal waters. Annual production rates up to 22 t (dry weight basis) per hectare have been estimated for natural populations. The vegetative propagation method involves introducing fragments of the seed material tied to bamboo poles fixed in the coastal waters for further growth. The process of cultivation of seaweed in Malaysia involves driving stakes deep into the shallow waters, which are tied with nylon monofilament lines. Fragments of seaweed weighing around 150 g are tied to the nylon lines. The plants are allowed to grow to 2–2.5 kg or more in weight in a period of 60–75 days, giving average production around 10–12 t per acre. The seaweed industry provides gainful employment and livelihood to coastal communities in southeast Asian countries, where seaweed farming provides an alternate source of livelihood for artisan fishermen.119,120 Most seaweed species are characterized by high growth rate. Photosynthetic ratio up to 1466 μmol CO2/g dry weight/h has been measured and maximum specific growth rates ranged as high as 38% per day. Each macroalgal species is unique in terms of optimal cultivation conditions. Integrated aquaculture consisting of seaweed along with fish and mollusks, as discussed earlier, is practiced in the coastal bays of China. In the Philippines, seaweed are grown as a supplementary or alternate source of income. The rapid growth of the carrageenan industry in China with its high demand for Eucheuma is fuelling further expansion in seaweed farming in the sub-region. Outside Asia, the United Republic of Tanzania provides an example of successful diversification of aquaculture into seaweed. The alga can be cultured under laboratory conditions and commercialization of this practice is well advanced.120 C. crispus (Irish moss) is abundant and commercially important red seaweed of the North Atlantic and has a long history of utilization, presently being the valuable

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source of carrageenan. The alga can be cultured under laboratory conditions, and many studies have taken advantage of this potential. The most productive giant kelp (M. prefer) population in California had an average harvestable yield of 2–8 dry t/ha. Nitrogen and organic phosphate manures can increase yield up to 20 dry t/ha.121 The geophytes Gaillardia spp., Pericardia capillaceous, Hype musciformis, and H. cornuta were cultured under field conditions. Specific growth rates appeared to be related to temperature and light intensity. Attached seaweed had higher annual yields than free-floating samples. The specific growth rates of all four seaweed were positively related to phycocolloid contents. Further, gel strength of the agar coincided with high 3,6 anhydrogalactose and sulfate contents.122 Seaweed may be susceptible to diseases. Most of seaweed diseases are fungal infections such as “raisin disease” in the seaweed, Sargassum spp. “Thalassia disease” in Thalassia testudinum, which are caused by Lindra thalassiae, and “aspergillosis” in Caribbean sea fans, which is caused by Aspergillus sydowii. Some fungi (Stramenopiles) are responsible for widespread epidemics such as “eelgrass wasting disease.” Known bacterial infections include “coralline lethal orange disease” (CLOD) in coralline algae, and “redspot disease” in the kelp Laminaria japonica, caused by bacterial pathogens. Although the etiology of new marine epidemics has been addressed, few studies have examined the foundation of disease resistance in healthy marine organisms.123,124

9.8

COMMERCIAL PRODUCTS

As mentioned in this chapter, seaweed species are cultivated in several countries because of their commercial importance. Out of the total farmed production of 16 mt in 2005, 60–70% was used for human consumption, 10–15% for extraction of hydrocolloids, whereas the remainder was used as fertilizer. About 300,000 t of seaweeds valued at U.S.$590 million was exported globally in 2005.4 Laminaria spp. known as kombu is a popular product in Japan. Dried Scytosiphon lomentaria, a brown alga, is a traditional food in the Noto area in Japan. Another seaweed product, dried Nori, which in the form of a paper is prepared from Porphyra spp. and is used in Japan in bakery products. The brown seaweed belonging to Sargassum, S. hemiphyllium has been used both as food and medicine in China and Japan.125 Dried and powdered laminaria seaweed known as kombu in Japan is a commercially important food flavoring agent. Nori, which is in the form of a paper thin yet flexible dried sheet prepared from several species of Porphyra spp. is another product used by Japanese as much as the western world. The aquatic plants used either as food or for the production of commercial products have been listed. In India, with a long coastal line, several species of seaweed are harvested, which are commercially used as animal feed, although to a limited scale.101 In Malaysia, at least 20 species of seaweed belonging to Gracilaria are available for commercial use including as a feed for aquaculture.25 Recently, China, Japan, and other countries, such as the Republic of Korea, the United States of America, South America, Ireland, Iceland, Canada, and France have significantly increased production, consumption, and marketing of seaweed.2 Many species of seaweed form the raw material for the manufacture of gums which can form characteristic gels and thickening agents. Major producers of brown

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seaweed in 1992 were China (2.9 mt), Korean Republic (407,000 t), Japan (346,000 t), Norway (189,000 t), Philippines (350000 t), and Indonesia (188,000 t).126 During the last 10 years, the overall production of seaweeds and other aquatic products reached over 16 mt with an annual growth rate of around 9.8%. As high as 92% of total production came from farming of brown seaweeds contributing over 52%, whereas about 32% was from farmed red seaweed. Philippines and Indonesia annually produce around 90,000 and 65,000 t of dried seaweed (E. cottonii), respectively.4

9.9

REGULATORY STATUS

In the European Union, food additives legislation is harmonized within itself. The legislation controlling the use of seaweed-based thickeners (e.g., carrageenan) is according to European Parliament and Council Directive 95/2/EC (as amended), this legislation lays down the foodstuffs to which the additives may be used and also any other conditions of use (such maximum levels). All permitted additives must also comply with the specific purity criteria, for the additives authorized by Directive 95/2/EC, the specifications are mentioned in Commission Directive 96/77/EC (amended).127 As regards FAO/WHO, the Codex website gives information which contains links to the Codex food additive standards and the JECFA specifications.128 In conclusion, many seaweed species are rich in nutrients and bioactive compounds that have significant therapeutic values. Their nutritional value make them valuable food supplements to enrich nutrients such as minerals and fiber.129–131 The nutraceuticals from seaweed could be used to fortify processed foods. Recognition of seaweed as sources of diverse bioactive principles are of recent origin and therapeutically important new compounds are being identified. There is good potential for use of these compounds for pharmaceutical applications. In addition, seaweed products could be used for food fortification, enrichment or encapsulation, for multipurpose applications, as discussed in other chapters of this book.

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102. Ahuja, A. K. and Saijpaul, S. S., Feed quality: nutritional implications and regulatory aspects, Proc. Symp Nutritional Technologies for Commercialization of Animal Production system, Animal Nutrition Society of India, Rai, S. N. and Siliga, J. P., Eds., ICAR, New Delhi, 2004, p. 8. 103. Kaliaperumal, N., Seaweed resources in India—status, problems and management strategies, Proc. Seminar on Marine and Coastal Ecosystems: Coral and Mangrove— Problems and Management Strategies, Vol. 2, Edward, J. K. P. et al., Eds., SDMRI Research Publication, New Delhi, 2002, p. 139. 104. Bautista-Teruel, M. N. et al., Reproductive performance of hatchery-bred donkey’s ear abalone, Haliotis asinina, Linne, fed natural and artificial diets, Aquacult. Res., 32, 249, 1991. 105. Gabrielsen, R. O. and Austreng, E., Growth, product quality and immune status of Atlantic salmon fed wet feed with alginate, Aquacult. Res., 29, 397, 1998. 106. Guanzon, N. et al., Polyculture of milkfish Chanos chanos (Forsskal) and the red seaweed Gracilariopsis bailinae (Zhang et Xia) in brackish water earthen ponds, Aquacult. Res., 34, 593, 2003. 107. Arndt, R. E. et al., The use of AquaMats® to enhance growth and improve fin condition among raceway cultured rainbow trout, Aquacut. Res., 33, 359, 2002. 108. Padmakumar, K., Marine algae as source of antifouling natural products, Seaweed research and utilization in India, Proc. Seaweed-2004, Seaweed Research Utilization Association and Central Marine Fisheries Research Institute, Cochin, India, 2004, p. 47. 109. de Nys, R. et al., Broad-spectrum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays, Biofouling, 8, 259, 1995. 110. Davis, T. A., Volesky, B., and Mucci, A., A review of the biochemistry of heavy metal absorption by brown algae, Water Res., 37, 4311, 2003. 111. Lodeiro, P. et al., Biosorption of cadmium by biomass of brown marine macroalgae, Bioresource Technol., 96, 1796, 2005. 112. Gueven, K. C., Akyuez, K., and Yurdun, T., Selectivity of heavy metal binding by algal polysaccharides, Toxicol. Environ. Chem., 47, 65, 1995. 113. Rey-Castro, C. et al., Acid-base properties of brown seaweed biomass considered as a donnan gel, Environ. Sci. Technol., 37, 5159, 2003. 114. Savary, B. J., et al., Hexose oxidase from Chondrus crispus: improved purification using perfusion chromatography, Enz. Microbiol. Biotechnol., 29, 42, 2001. 115. Nilsson, M., Mattsson, S., and Holm, E., Radioecological studies of activation products released from a nuclear power plant into the marine environment, Mar. Env. Res., 12, 225, 1984. 116. McHugh, D. J., Prospects for Seaweed Production in Developing Countries, FAO Fisheries Circular No. 968 FIIU/C968, Food and Agriculture Organization of the United Nations Publication, Rome, 2002, p. 2. 117. Anonymous, Seaweeds, Infofish Internat., 1, 73, 2008. 118. Thomas, P. C. and Krishnamurthy, V., Agar from cultured Gracilaria edulis (Gmel) Silva, Botanica Marina, 19, 115, 1985. 119. Chennumbhotla, V. S. K., Seaweed culture in India—An appraisal, Proc. Seaweed2004, Jayasankar, R. et al., Eds., Seaweed Res. Util. Assoc, and Central Marine Fisheries Res. Inst., Cochin, India, 2004, p. 23. 120. Dey, V. K., Seaweed—helping coastal communities make a living, Infofish Int., 6, 58, 2006. 121. Gerard, V. A., Seaweed, in Biomass Handbook, Kitani, O. and Hall, C. W., Eds., Gorden & Breach, New York, 1989, p. 205. 122. Friedlander, M. and Zelikovitch, N., Growth rates, phycocolloid yield and quality of the red seaweed, Gracilaria spp., Pterocladia capillacea, Hypnea musciformis, and H. cornut in field studies in Israel, Aquaculture, 40, 57, 2006.

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123. Kohlmeyer, J. and Kohlmeyer, E., Marine Mycology: The Higher Fungi. Academic Press, New York, 1979. 124. Richardson, L., Coral diseases: what is really known? Trends Ecol. Evol., 13, 438, 1998. 125. Kuda, T., Antioxidant properties of dried ‘kayamo-nori’, a brown alga Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae), Food Chem., 89, 617, 2005. 126. Infofish, Uses and markets for seaweed products—Malaysia and Thailand. Infofish Int., 4, 22, 1996. 127. Rigat, P., Information & Communication, Health and Consumer Protection DirectorateGeneral European Commission http://ec.europa.eu/comm/food/food/chemicalsafety/ additives/index_en.htm. 128. Codex Alimentarius Commission, http://www.codexalimentarius.net/gsfaonline/index. html?lang=en. 129. Darcyvrillon, B., Nutritional aspects of developing uses of marine macroalgae for the human food industry, Int. J. Food Sci. Nutr., 44, S23, 1993. 130. Urbani, M. G. and Goni, I., Bioavailability of nutrients in rats fed on edible seaweed Nori (Porphyra tenera) and Wakame (Undaria pinnatifida), as a source of dietary fibre, Food Chem., 76, 281, 2002. 131. Prosky, L., What is dietary fiber? A new look at the definition, in Advanced Dietary Fiber Technology, McCleary, B. V. and Prosky, L., Eds., Blackwell Science, Oxford, 2001, p. 63.

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10 Seaweed Hydrocolloids 10.1 INTRODUCTION As indicated in Chapter 9, the cell walls of seaweed contain polysaccharides, which include agar, alginates, carrageenans, and also minor compounds such as fucoidan and laminarin. These compounds can play varied functional roles by their ability to hold significant amounts of water, capacity to form gel, metal chelating, and other actions. These diverse functions have led to their applications in food technology, pharmaceutical sciences, biotechnology, etc. In addition, seaweeds are also sources of antioxidants, antimicrobials, and other bioactive agents. Their popularity also arises from their low cost in production and nontoxic nature.

10.2

GENERAL FUNCTIONAL PROPERTIES OF SEAWEED HYDROCOLLOIDS

Polysaccharides from seaweed have three functional roles, namely (1) biological, (2) physiological, and (3) technological functionalities. These properties vary widely depending on the type of the compounds. The biological functions are as components of the seaweed cell wall architecture and involve in cell–cell recognition, stimulation of host defense, and hydration of intracellular fluid. The important feature that makes them function in both physiological and technological roles is their ability to bind water. In the seaweed structure, the polysaccharides bind several times their weight of water (binding water 20 times their own volume); this capacity being related to the length and thickness of the fiber particle. In this respect, their waterholding capacity (WHC) is much higher than that of cellulosic fibers. The high affinity of water qualifies the polysaccharides to be known as hydrocolloids, and bestows them with technologically important functions such as texturizers, stabilizers, emulsifiers, fat reducers, film formers, shelf life extenders, and viscosity modifiers. (They are also sometimes referred to as phycocolloid, indicating their source.) Seaweed hydrocolloids are used in foods for inhibition of syneresis, decrease in dryness and toughness, increasing yield and viscosity, creation of a gel network in the presence of salts to get appropriate textures, replacement of fat to get acceptable mouth feel, and as a thickener. Seaweed hydrocolloids are commercially used in a variety of food products including bakery, confectionery, dairy, and muscle foods. (Other gums that are being used as food additives include cellulose gums, gum arabic, gum acacia, guar gum, pectin, and carboxymethyl cellulose.) The seaweed polysaccharides can also interact with other macromolecules in the food system, such as proteins, starch, and other components, to offer novel food products. The hydrocolloids, apart from improving functionality at low usage levels, help reduce product costs besides 297

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TABLE 10.1 Hydrocolloids from Some Seaweed Sources Hydrocolloid Agar, agarose Carrageenans Fucoidan Laminarin Furcellaran Alginic acid (alginate)

Source Rhodophyta (Gracilaria, Gelidium, Pterocladia) Rhodophyta (Eucheuma, Chondrus, Hypnea, Gigartina) Laminaria religiosa and other brown algae Brown seaweed Laminaria japonica Furcellaria lumbricalis Phaeophyta (Macrocystis, Laminaria, Ascophyllum)

TABLE 10.2 Major Functional Properties of Seaweed Polysaccharides Seaweed structure Cell wall architecture Cell–cell recognition Hydration of intracellular fluids Stimulation of host defense Food applications Gelling agent Stabilizer Texture modification WHC modifier Film formation Inhibits syneresis Increases yield Nutraceuticals Antioxidants Antithrombin activity Antitumor activity Cell recognition and cell adhesion or regulation of receptor functions As sources of functional oligosaccharides Dietary fiber

qualifying for other requirements such as kosher-certified ingredients.1,2 Table 10.1 shows some seaweeds as sources of hydrocolloids. The seaweed polysaccharides are also important bioactive compounds. Sulfated polysaccharides from marine algae, differing from those of land plants, are known to exhibit many physiological activities such as antioxidants, antithrombin activity, antitumor, cell recognition, and cell adhesion or regulation of receptor functions. These compounds are potent and selective inhibitors of various viruses, including herpes simplex virus (HSV), vesicular stromatitis virus, and human immunodeficiency virus (HIV). In addition, recent research has revealed voluminous information on their functional role in health management. They have also been recognized to play an important role as a dietary fiber. Table 10.2 denotes the major functional properties of

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seaweed polysaccharides. This chapter examines the major seaweed polysaccharides, their extraction, structure, analytical methods for their detection, biological as well as physiological functions, and their practical applications in various fields.

10.3 AGAR 10.3.1

SOURCE

Agar is synonymous with agar-agar, the Japanese gelatin, Japanese isinglass, vegetable gelatin, and angel’s hair. Red seaweeds (Rhodophyceae, consisting of more than 700 genera and 6000 species) are the commercial sources of agar. Agar is extracted from red algae such as Gracilaria, Gelidium, and Gelidiella spp. The genus Gracilaria (Gracilariales) is an important alga belonging to Rhodophyceae, which is the major source of agar in Japan, the United States, Mexico, Africa, and India. In the United States, agar is extracted from Gelidium cartilagineum and G. confervoides. Agar yield, quality, and biochemical characteristics not only depend on the seaweed species, but are also closely related to season, environmental parameters, growth, reproductive cycle, and the plant part studied. For example, in two agarophytes, namely, Gracilaria cervicomis and Hypnea cornea from France, the peak yields in biomass were observed during the summer with a maximum yield of 390 g/m2, whereas in the rainy season it was only 129 g/m2, suggesting a significant seasonal variation. The peak in biomass for H. cornea was recorded in April (383g/m2) and was much different from the other months. The agar yield for G. cervicornis varied from 11 to 20%, with generally higher values recorded during the dry season. Agar yield from H. cornea ranged from 29 to 41%, with a peak recorded in June.3,4

10.3.2

EXTRACTION

Agar is extracted from washed seaweed using hot water. The seaweed is dried and washed to remove adhering salt, sand, and other impurities, and then placed in retorts and subjected to extraction with water at a minimum temperature of 90°C. Subsequently it is transferred to a sedimentation tank where impurities settle down. The liquor is then filtered and poured into trays where it congeals into sheets of firm darkcolored agar gel. The gel is then frozen and held for several days. Agar being insoluble in ice water remains in the gel as a separate entity, whereas many impurities remain in water. The frozen agar is thawed when the impurities are removed in the melting ice. The freeze-thawing process helps in the removal of dissolved impurities and reduction of ash and nitrogen contents. Purified agar is washed, bleached, dried in hot (100°C) air, and then packaged. Commercial agar is produced in the form of chopped shreds, sheets, flakes, granules, or powder. Refinements on the procedure mentioned earlier have been introduced for improvement of the quality of agar by processes such as alkali treatment before extraction. For example, Gracilaria crassissima gave a good quality agar after alkali treatment, which removed alkali-labile sulfate and increased the gel strength. However, alkali treatment was not effective in G. cervicornis and Gracilaria blodgettii. As further refinement, the alkali-extracted agar was treated with 0.5% aqueous solution of sodium pyrophosphate at 95°C to

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improve the final quality of the product. The treatment reduced the sulfate content of the agar, thereby improving the gel strength of the final product. Alternately, agar extract may be treated with ethanol when agar is precipitated.5

10.3.3 COMPOSITION Agar is composed of two polysaccharides, agarose and agaropectin. Agarose consists of alternating 1,4-linked 3,6-anhydro-α-l-galactopyranose and 1,3-linked β-d-galactopyranose. Agaropectin is more complicated in structure, and contains residues of sulfonic, pyruvic, and uronic acids, in addition to d-galactose and 3,6anhydro-l-galactose. Agar contains 2–5% sulfate residues.6 G. cervicornis produced agar polymers with an occurrence of methoxylation and sulfation at the C-6 of the β-d-galactose residues, with an extra methylation due to the presence of the 4-O-methyl-α-l-galactose residue. The presence of these residues was found to be responsible for the extremely poor gelling property of the agar. However, agar produced from G. blodgettii showed the typical pattern of low degree of methylation on both galactose residues.5

10.3.4

PROPERTIES

Agar is a hydrophilic colloid. Commercial agar is quite stable and the product should not contain more than 20% moisture, 6.5% ash, and 1% insoluble matter. It is insoluble in cold water, but soluble in boiling water. A hot 1.5% solution is clear and congeals at 32–39°C to a firm resilient gel, which becomes liquid only above 80°C. The difference in gelling and melting temperatures, known as “hysteresis,” makes agar useful for applications in microbiological, pharmaceutical, and food industries. Agar sols are thermostable and generally lose about 5% of its strength if autoclaved for 1 h at 120°C and at a pH of 6.5–7.5. It is also rapidly degraded at either very high or very low pH.3 The most important attribute of agar is its gelling property and the wide range of temperatures under which it retains this property. Agarose has higher gelling ability than agaropectin. Agarose has a double-helical structure. The double helix aggregates to form a three-dimensional framework, which holds the water molecules within the interstices of the framework to produce thermoreversible gels.3,6 Sodium alginate and starch decrease the strength of agar gels, whereas dextrins and sucrose increase the gel strength of certain agars. Locust bean gum (LBG) has a marked synergistic effect on the strength of agar gels. Incorporation of LBG at 0.15% could increase the rupture strength of an agar gel by 50–200%.6

10.3.5

USES

The gelling property of agar makes it a bacteriological medium and its ideal melting and congealing temperatures, resistance to most enzymes and microorganisms, and ability to remain in solution at 40°C, which allows uniform distribution of microorganisms during their culturing.7 Agar is used for gel electrophoresis, chromatography, immunology, and immobilization of enzymes. Other uses include its use as a thickener, gelling agent, stabilizer, lubricant, emulsifier, and absorbent. Because of these properties, agar is also used in silk and paper industries, as a substitute for

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isinglass, for dying fabrics, for making beer, as a lubricant for wire drawing, as a sizing for fabric, and for photographic films and plates. Because of its nondigestible nature, colloidal and gelling properties agar has found extensive applications in food products such as icings, toppings, and meringues. In medical fields, agar is used as a laxative and for the treatment of constipation. Its action in the intestinal tract is comparable to that of cellulose of vegetable foods in aiding bowel movement. Agar is also used to make dental casts in dentistry, and as a surgical lubricant. The capacity of agar to block streptococcal adhesion to biosurface has made it a potential component in mouthwashes and spray wash for foods.6

10.4 ALGINATE 10.4.1

SOURCE

The term “algin” or “alginate” is used as a generic name for the salts of alginic acid such as sodium, potassium, ammonium, calcium, and propylene glycol alginates (PGAs). Although these compounds were first described in the United Kingdom in the 1880s, their commercial production started in 1929 in California. Algin occurs in all brown seaweeds as a structural component of the cell wall in the form of insoluble mixed salts of mainly calcium, with lesser amounts of magnesium, sodium, and potassium and is concentrated in the intracellular space. The most important sources are Macrocystis pyrifera, Laminaria, and Ascophyllum nodosum.7

10.4.2

EXTRACTION

The giant kelp M. pyrifera (which grows along the west coast of the North American continent) is mostly used for commercial extraction of algin. The seaweed has an alginic acid content of 13–14% on a dry weight basis. In Canada, algin is extracted from the rockweed A. nodosum whereas in Europe, the sources include Laminaria hyperborea and L. digitata. Important sources of algin also include Ecklonia maxima, E. cava, Eisenia bicyclis, and Lessonia nigrescens. In India, alginate is extracted from the brown seaweeds Sargassum wightii and Turbinaria conoides. Alginic acid contents of 17 species of brown algae that commonly occur in India vary from 5.3 to 16.6% on a dry weight basis. Alginic acid content is highest in rachid, which is the thickest part of a plant, whereas other parts (vesicles, leaves, etc.) have fair amounts of the phycocolloid. Japan and North Korea are other countries with a significant number of algin industries.7 Two widely practiced processes for the production of alginate are those of Green and Le Gloahec–Herter processes.3,7 In the Green’s process, fresh alga is first leached with 0.3% aqueous HCl to reduce its salt content. The alga is then chopped and shredded. It is treated with an aqueous 8–2% solution of soda ash (pH 10–11). The treatment is repeated a second time before the solids are ground in a hammer mill. It is then diluted with water and allowed to settle. The liquid part is mixed with diatomaceous earth as the filter aid, heated to 50°C, and filtered in a plate and frame filter press. The filtrate is mixed with 10–12% aqueous CaCl2, when the insoluble calcium alginate formed rises to the surface. The lower liquid layer, which contains soluble salts, organic matter, and other material is drained off and discarded.

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Calcium alginate is bleached with a 10% aqueous sodium hypochlorite, drained, and mixed with 5% HCl. The precipitated alginic acid is thoroughly washed with water to remove the calcium completely. The purified alginic acid is generally converted into the desired salt (e.g., sodium alginate) by treatment with the appropriate carbonate, oxide, or hydroxide. The salt is then dried, grounded, and packed. In the Le Gloahec–Herter process, initial leaching is done with 0.8–1.0% aqueous CaCl2 to eliminate salts and other impurities without damaging the algin. After washing with water, the material is soaked in a 5% aqueous HCl and again washed. The alga is digested with a 4% solution of soda ash at 40°C, ground well for 2–3 h, and the paste obtained is then diluted with water, bleached with hydrogen peroxide or ozone, and then centrifuged. The bleached liquor is treated with adsorbent materials (hydrated alumina, gelatinous silica) followed by precipitation with hydrochloric acid. The separated alginic acid is first washed in water and then in ethyl alcohol to remove impurities followed by drying to get alginic acid, which may be converted into appropriate salts by treating with carbonates, oxides, or hydroxides. Alginic acid may also be converted into PGA by treatment with propylene oxide under controlled conditions.3

10.4.3

COMPOSITION AND STRUCTURE

Alginates have a molecular formula of (C6H6O6)n, where the value of “n” varies from 80 to 83. Alginic acid contains three kinds of polymer units consisting of d-mannuronic acid (M), l-glucoronic acid (G), and alternates of the M and G units. These residues can combine to form segments rich in either M, G, or a mixture of M and G units. In the third type, common in bacterial alginates, the M and G units form a linear polymer—the glycuronoglycan consisting mainly of β(1–4)-linked M and G units. Other uronic acids such as galacturonic acid have also been found in alginic acid in some species of brown algae.8,9 G blocks are believed to be important to the alginate structure as a function of their Ca++ and H+ binding capability, which allows alginates to form gels in the presence of these ions. Biochemical and biophysical properties of alginate are dependent on the molecular weights and M:G ratios. MG blocks allow flexibility to the polysaccharide chain. The ratio of M and G in alginic acids are usually in the range 1.45–1.85, except in that from L. hyperborea in which it is as low as 0.45. The alginic acids from M. pyrifera and A. nodosum are similar in structure, containing approximately equal amounts of polymannuronic acid and polyguluronic acid segments. The ratio between the two forms depends on the conditions of extraction and production. In commercial products, the molecular weights generally range between 30,000 and 200,000 Da, primarily because of hydration and polymerization characteristics.7 Table 10.3 indicates the contents of alginic acid and d-mannuronic and l-glucoronic acids in some commercial seaweed and Table 10.4 shows the types of glycosidic linkages in carbohydrate polymers and their occurrence.10–12

10.4.4 PROPERTIES Alginic acid is essentially insoluble in water. Alginate requires the addition of calcium ions to form gels, without any heating or cooling. Calcium salt is insoluble in water, whereas magnesium salt is water-soluble. The solutions of soluble alginates are

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TABLE 10.3 Contents of Alginic Acid and D-Mannuronic and L-Glucoronic Acids in Some Commercial Seaweed Species M. pyrifera A. nodosum Laminaria digitata L. hyperborean E. cava E. bicyclis E. maxima a

Total Alginic Acid Contenta 13–14 20–30 14–24

29–38

D-Mannuronic

Acid

61 65 59 31 62 62 —

L-Glucoronic

Acid

39 35 41 69 38 38 —

Dry weight basis.

Source: Adapted from Grayson, M. and Eckroth, D., in Encyclopedia of Chemical Technology, Wiley Interscience, New York, 1980, Vol. 12, p. 45.

TABLE 10.4 Type of Glycosidic Linkages in Carbohydrate Polymers and Their Occurrence Class of Compounds

Glucosidic Linkage

Carbohydrate Polymer

Occurrence

α-d glucans

(1–4) Linear (1–4) (1–6) Branched (1–4) (1–6) Branched (1–4) Linear (1–4)n G(1–3) (1–3) Linear (1–3) (1–6) Branched β(1–4) Linear (1–3) (1–4)

Amylose Amylopectin Glycogen Cellulose Cereal gum Laminarin — Alginic acid Carrageenan

Plant Plant Mammals Plant cell wall Endosperm Seaweed Fungi, mushroom Seaweed Seaweed

β-d glucans

β-d glucans Alternating α-d and β-d glucans

Source: Adapted from Misaki, A., in Food Hydrocolloids: Structure, Properties and Functions, Plenum Press, New York, 1993; Shimizu, Y. and Kamiya, H., in Marine Natural Products, Academic Press, New York, 1985, 403.

transparent, colorless, noncoagulable on heating, and have a wide range of viscosity. The viscosity decreases with increasing temperature. Viscosity is not affected by pH in the range 5–10, but below pH 4.5, viscosity increases until the pH reaches 3.0 when insoluble alginic acid precipitates. At a very high pH, sodium alginate forms a gel. Algin forms agel without heating or cooling and is also independent of pH and sugar content. Because of the high affinity for water, a product having algin has a lesser tendency to leak. The viscosity of alginate can be controlled by the addition of calcium ions to sodium alginate solution. The formation of gel in the presence of calcium is due to the binding of ions on the chains forming what is often described

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Poly-G region

Poly-M region

Poly-G region

Calcium ions

“Egg-box” gel

FIGURE 10.1 “Egg-box” binding of Ca2+ in the gelation of alginate. (Reprinted from Rastall, R., Tailor-Made Food Ingredients: Enzymatic Modulation of Nutritional and Functional Properties, IFIS Publishing, Reading; UK, 2001, http://www.foodsciencecentral. com/fsc/ixid3729. With permission from IFIS Publishing, Reading, UK.)

as the “egg-box” dimmer structure. Algin-based gels are heat stable and hence find varied applications. The most useful product is the ester of algin with PGA, which varies in the degree of esterification. PGA is soluble and stable at pH 2–3, but not above 6.5.10 The carboxyl group of alginic acid readily reacts with the bases of sodium and ammonium to form water-soluble salts. Alginates are produced in many forms varying in molecular weights, calcium contents, particle size, particle form (granular or fibrous), and contents of d-mannuronic and l-glucoronic acids. Figure 10.1 shows the “egg-box” binding of Ca2+ in the gelation of alginate.13 Algin can be degraded by bacterial alginate lyase. The activity could be determined by a slab gel electrophoretic method. The method is based on renaturation of the enzyme after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent staining with cetylpyridinium chloride, and quantification of the spot by densitometric scanning. The molecular weight of alginate lyase can be determined from its position in the gel.14 Table 10.5 shows the typical properties of alginic acid.10

10.4.5

USES OF ALGINATES IN FOOD, MEDICINE, AND BIOTECHNOLOGY

In the food industry, alginates are used in the manufacture of dairy, bakery, meat, and other products, where the advantages of their thickening, gelling, and stabilizing properties are utilized. Alginate finds use as a stabilizing agent to give a smooth texture to frozen deserts, and in ice cream substitutes, chocolate milk suspensions, and various drinks. PGA, because of its solubility at low pH, is used as a thickener and

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TABLE 10.5 Typical Properties of Alginic Acid Property Moisture Ash Specific gravity Bulk density (kg/m3) Browning temperature (°C) Charring temperature (°C)

Value 13% 23% 1.5 87.4 150 340, 460

Source: Adapted from Grayson, M. and Eckroth, D., in Encyclopedia of Chemical Technology, Wiley Interscience, New York, 1980, Vol. 12, p. 45.

emulsifier in products with a low pH such as sauces, syrups, and sherbets. Various alginates also find uses in icing and glazes. Alginate-based jelly is used as a carrier for some antiseptics and drugs. Alginate products are used to control disintegration and to improve the binding of ingredients in tablets. It is also used as a bodying agent for weight-control drinks, puddings, and as a packaging film. It is used in fish feed as a binder. Films of alginate gel have been shown to prevent a certain extent of moisture loss and lipid oxidation in silver salmon and rockfish. The solutions of soluble alginates are transparent, colorless, noncoagulable on heating, and have a wide range of viscosity. The strong affinity of alginate for water is used to control moisture in food products. Viscosity can be controlled by the addition of calcium ions to sodium alginate solution.9 The important function of alginate in food is as a dietary fiber (see Chapter 10). Alginates, like other fiber types, increase wet and dry stool weight. They bind bile acids more strongly than cellulose. Alginates could also bind a wide range of toxic compounds from the gastrointestinal (GI) lumen, thereby lowering colonic and systemic exposure to these moieties.9 Animal studies have shown that inclusion of alginate at 1–3% in the diet may also reduce hypertension. In addition, it can also lower the blood glucose level. A 5 g supplement of sodium alginate to test meals containing similar levels of digestible carbohydrates, fats, and proteins was given to a cohort of diabetes type II patients. It was observed that glucose absorption rates were reduced in the presence of alginate. The low level of dietary alginate caused a reduction in blood peak glucose and plasma insulin rise (by 31% and 42%, respectively). These effects of alginates on glucose/cholesterol uptake would suggest that the inclusion of the fiber in the diet could reduce the onset of diabetes in type II patients and also obesity, possibly cardiovascular disease, as well as systemic risk factors in patients with these diseases. This effect of alginate in lowering plasma glucose/cholesterol has been attributed to its reduction in intestinal absorption and its prolongation of gastric emptying (also resulting in increased satiety). Alginate may be clinically more useful in reducing blood cholesterol and postprandial glycemia than other viscous fiber types. But the effect was less than that of carrageenan, but much larger than that of agar. In addition to functions, which include elevation of

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TABLE 10.6 Potential Applications of Alginate as Dietary Fiber Stimulates immune system Reduces intestinal absorption Increases satiety Reduces glycemic index value Modulates colonic microflora Elevates colonal barrier function

colonic barrier function and reduction of damaging potential of GI luminal contents, alginates have an effect on the colonic microflora, in terms of populations of species and the quantities of short-chain fatty acids produced. Similar effects, however, have also been noted for other dietary fibers. The function of alginates as fiber has been elegantly discussed recently.9,15,16 Table 10.6 gives the potential applications of alginate as a dietary fiber. Alginate either alone or in combination with other hydrocolloids could be used to replace fat without affecting textural characteristics of some food products. Low-fat, precooked, ground beef patties containing alginate/carrageenan combinations were comparable to regular beef patties (20% fat control) regarding yields and textural properties. Patties with an alginate/carrageenan combination had higher yields and moisture, but lower shear force values than those of alginate or carrageenan treatments within the same fat level. Patties with 10% fat were generally lower in shear value, cooking yield, and percentage of free water released as compared to their 5% fat counterparts with the same added ingredient.17 The storage stability and textural, physicochemical, and sensory quality of low-fat ground pork patties are enhanced with carrageenan as fat replacer.17 Alginate in combination with high pressure has been examined to modify the characteristics of fish meat gel. Alginate incorporation gave a gel that was harder, more adhesive, less cohesive, and more yellow than pressure-induced samples. Pressure-induced gels also differed according to the pressure applied (200 or 375 MPa), lower pressure treatment showing significantly higher values for penetration values and cohesiveness, and lower values for elasticity and lightness compared to higher pressure-treated samples.18 Table 10.7 gives the common uses of alginates in food products.9 In medicine, alginates are used in wound healing, to stimulate the immune system, and offers potential for weight reduction and also reduction of glycemic index through reduced intestinal absorption and also increased satiety. Alginate also reduces mucosal aggregation and also offers modulation of colonic microflora. Alginate gel sheets have recently been evaluated as a potential scaffold for cell proliferation since the polysaccharide can propagate chondrocyte growth and cartilaginous matrix production. Cartilage deficiency from pathology, trauma, or congenital defects is currently treated with prostheses, cartilage grafts, and scaffolds. A scaffold should be biocompatible and should allow cell adherence and migration with subsequent differentiation and proliferation. A porous scaffold will provide the necessary environment. A period of 6 weeks is required for a stable natural graft.19 Preparation and characterization of a three-dimensional porous sponge made from alginate

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TABLE 10.7 Common Uses of Alginates in Food Products Applications

Percentage of Total Applications

Foam stabilizer in beer Texturized foods

21.2

Other uses

18.9

Bakery products

14.9

19.6

Fruit preserves

6.5

Ice cream

3.8

Others

15.1

Remark PGA provides better head retention and prevents foam-negative contaminants Endows food products with thermostability and desired consistency PGA is acid stable and resists loss of viscosity. Has unique suspension and foaming properties. Hence used in soft drinks, milk drinks, sorbets, ice creams, noodles, pasta, etc Provides freeze-thaw stability and reduced syneresis to some products Commonly used as thickening, gelling, and stabilizing agents in jams, marmalades, and fruit sauces. Alginate–pectin gels are heat-reversible and gives better gel strength than individual components Gives ideal viscosity, prevents crystallization and shrinkage, helps homogenous melting without whey separation. Used in combination with other gums Desserts, emulsions (e.g., low-fat mayonnaise) and sauces, extruded foods (noodles and pasta)

Source: Adapted from Brownlee, I. A. et al., Crit. Rev. Food Sci. Nutr., 45, 497, 2005. With permission from Taylor & Francis Ltd. (www.informaworld.com).

for cell transplantation to replace damaged organs or tissues has been reported. The sponge is prepared by a three-step procedure: first, gelation of the alginate with bivalent cations, followed by freezing of the hydrogel, and finally, lyophilization to produce a porous sponge. The pattern and the extent of sponge porosity as well as its mechanical properties were influenced by the concentration and type of alginate (G:M ratio and viscosity), the type and concentration of the cross-linkers, and the freezing regime. By controlling these variables, macroporous sponges (pore size of 70–300 μm) that are suitable for cell culture were achieved. Fibroblasts seeded within the sponges preferred the pores, where they maintained a spherical shape. The alginate sponges conserved their initial volume for at least 3 months.20 Alginates also have applications in biotechnology, particularly in cell immobilization. For instance, Aspergillus niger cells were immobilized in alginate gel beads. An enzyme yield of 4230 units of activity/L was achieved using cells immobilized in 3% alginate after a curing time of 60 min. The addition of 1% olive oil stimulated lipase production. In repeated batch fermentations, the alginate-immobilized cells were stable for up to three fermentation cycles. It was concluded that alginate was a good matrix for cell immobilization.21 Apart from the uses mentioned earlier, algin finds uses in other areas as well. It is used as a plasticizer in cement, textile paints, patching plasters, crack fillers, and acoustical plaster. In these products, algin reduces water penetration and improves

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mixing and suspension of pigments and clays. The main use of alginate in the paper industry is for surface sizing of paper. It is also used in the welding industry to cast welding rods, and in textile printing where it functions as a thickening agent. Algin is also used as an emulsifying agent for vegetable and mineral oils and shampoo foam stabilizers.22

10.5 CARRAGEENAN “Carrageenan” is a generic term for a complex family of anionic polysaccharides. It is extracted from a number of red seaweeds, the principal ones being Chondrus crispus (known as Irish moss), Gigartina stellata, and those belonging to Eucheuma spp. These seaweeds are abundant in the Atlantic coast of Europe and North America, particularly Canada. C. crispus is an ecologically important and commercially valuable species of red seaweed of the North Atlantic. Eucheuma spp. is found to occur in the Philippines, Indonesia, and East Africa. E. cottonii, a red seaweed harvested mainly in the Philippines and Indonesia, is a good source of carrageenan. Other sources include Furcellaria fartiligita and Hpnea spp. There are three major types of carrageenans, namely, kappa (κ), iota (ι), and lambda (λ). E. cottonii, E. spinosom, and C. crispus are the primary sources of these carrageenans. Depending on the life cycle the carrageenan types vary. For example, in the red seaweed Gigartina pistillata, the gametophytic alga produces heterogeneous κ–ι-type carrageenan containing small amounts of nu-carrabiose, whereas the tetrasporophytic alga synthesizes a complex sulfated galactan composed of λ-, xi- and pi-carrabioses, and sulfated carrabioses containing 3-linked galactopyranose 2,6-disulfate.22,23 The contents of the hydrocolloid in Calliblepharis ciliata, C. jubata, Cystoclonium purpureum, and Gymnogongrus crenulatus vary with season, being maximum at the end of spring and minimum in autumn, and was positively correlated with growth of these algae.24,25

10.5.1

EXTRACTION AND CHARACTERIZATION

Conventionally, the seaweed is washed with water to remove salts, sands, and other extraneous matters. It is then subjected to extraction with hot water made alkaline with calcium or sodium hydroxide. The duration of extraction depends on the quality and condition of the raw material and other processing variables. The residue remaining after extraction is removed by settling. Because of its viscosity and presence of fine suspended particles the liquor requires a filter aid. Filtered liquor is concentrated by single or multiple-stage evaporation. Carrageenan is then precipitated from the aqueous solution with isopropyl alcohol. Separated carrageenan is dried under vacuum and the alcohol is recovered. The product is finally ground and packed.3,22 The hydrocolloid from Hypnea musciformis was extracted with water at pH 7 after an initial short pretreatment with cold, dilute HCl. Carrageenans were isolated by alcohol precipitation after an amylase treatment and a filtration of the extracts. The yields at 25°C and 90°C were 25% and 75% (w/w) of the dry alga, with molecular weights of 194,000 and 245,000 Da, respectively. The predominant product was κ-carrageenan (KCG).26 Carrageenan has been extracted from Sarconema filiforme

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and H. valentiae from Tamil Nadu, India. S. fi liforme had a higher content of carrageenan than H. valentiae. The physicochemical and infrared spectroscopic analyses confirmed that the phycocolloid extracted from S. filiforme and H. valentiae were ι- and κ-carrageenans, respectively.27 An alkali process has been reported for the extraction of carrageenan from Eucheuma and Gigartina spp. The direct treatment of E. cottonii with hot KOH led to a solid residue, predominantly containing carrageenan and cellulose. The presence of nu-carrageenan, the precursor of ι-carrageenan, which induces kinking in the ι-carrageenan is removed from crude preparations by alkali treatment. Extraction in hot water gave nu-/κ-carrageenan that was readily converted into κ-type with hot alkali. NaOH or KOH treatment did not seriously affect the gelling behavior of the polysaccharide. The Eucheuma carrageenan has an ash content of 20%.28 κ-Carrageenan was extracted from the red seaweed Ibaranori (Hypnea charoides Lamoroux) and purified by gelation with KOH. The total carbohydrate, ash, and water content of the polysaccharide were 70.2%, 20.4%, and 3.2%, respectively. The content of total sulfate in the ash was 19.2% and 12.9%, respectively. The polysaccharide, which gelled at 0.2% concentration, was composed of d-galactose, 3,6anhydro-d-galactose, and ester sulfate in a molar ratio of 1.2:0.9:1.2. Galactans of a hot water extract, obtained from the red seaweed Κappaphycus alvarezii were found to be composed of approximately 74% KCGs and the remaining mostly of sulfated aragans. The galactans exhibited a marked tendency to retain Ca2+ and Mg2+ cations.29 Seaweeds Furcellaria lumbricalis and Cocotylus truncatus from the Baltic Sea in Estonia contained carrageenans; the former yielded the κ-type, whereas the latter yielded ι-carrageenans.30 Κ. alvarezii, Eucheuma denticulatum, and the Κphycus spp. are the carrageenan-containing red seaweeds currently farmed in the Philippines. The Κphycus spp. (referred to as “sacol”) is of particular interest in the Philippine seaweed industry because of its improved resistance to “ice-ice” disease and its fast-growing characteristics. The three Κphycus spp. predominantly contained KCG with low levels of ι-carrageenan, methylated carrageenan, and precursor residues, whereas E. denticulatum predominantly contained ι-carrageenan.31 A λ-like carrageenan was produced from Halymenia durvillaei, a red seaweed that grows widely in almost all parts ofthe Philippines. Maximum extraction was achieved employing seaweed to hot water ratio of 1:40 (w/v). An average yield of 29% was obtained using two extractions followed by the precipitation of the carrageenan with isopropyl alcohol.32 An enzymatic method has also been described for the manufacture of carrageenans, especially κ- and ι-carrageenans.33 Recently, a single-mode microwave heating system for the extraction of carrageenans from seaweed was developed, which reduces the extraction time and consumption of organic solvents. Extraction of E. spinosum and E. cottonii using the developed system generated carrageenans having spectral characteristics comparable to the reference samples.34 Instead of conventional drying, carrageenan extracts from red algae could be dried by using microwaves (at preferably a frequency of 2450 MHz) at temperatures not exceeding 100°C. Optionally, the carrageenan solution may be preconcentrated to about 70% before drying. This process gives better dispersibility of the dried KCG, or a blend of κ-, ι-, and λ-carrageenans, than that obtained in the conventional process.35

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10.5.2

STRUCTURE

Carrageenan is a mixture of galactans that carry varying half-ester sulfate groups linked to one or more of the hydroxyl groups of the galactose units. The galactans consist of alternating units of 1,3-linked β-d-galactose and 1,4-linked α-l-galactose. Compositions of κ-, ι-, and λ-carrageenans depend on the type of seaweed. The structure of ι-carrageenan consists of an alternating disaccharide repeating unit of (1-3)-linked β-d-galactopyranosyl 1-4-sulfate and (1-4)-linked-3,6-anhydro-α-d-galactopyranosyl 1,2-sulfate residues. In ι-carrageenan, the anhydrogalactose residue carries a sulfate group, whereas it is absent in the κ-type. Commercial carrageenan has a molecular weight in the range 100,000–1000,000 Da. Various aspects of the structure of different carrageenans and sulfated galactans that are isolated from red seaweeds have been reviewed in relation to their functionality.36–38 Figure 10.2 gives chemical structures of different carrageenans.

κ-Carrageenan

−O SO 3 CH2OH

CH2 O

O

O

OH

O

O OH

ι-Carrageenan

−O SO 3 CH2OH

CH2 O

O

O

OH

O

O OSO3−

λ-Carrageenan

HO CH2OH

O

O

−O SO 3

O

CH2OSO3−

HO

FIGURE 10.2

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O

−O SO 3

Chemical structures of different carrageenans.

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10.5.3

311

PROPERTIES

The major types of carrageenans, namely, κ, ι, and λ, vary in their thickening ability and gel strength. κ- and λ-carrageenans are soluble in hot (70°C) sucrose solutions up to 65% concentrations, whereas ι-carrageenan is not easily soluble in sucrose solution at any temperature. ι-Carrageenan solution alone will tolerate high concentrations of electrolytes such as NaCl up to 20–25%, whereas KCG will be salted out. In theory, λ-carrageenan is also soluble in concentrated salt solutions, but in practice λ-carrageenan always contains some κ-compound, which makes them less saltcompatible. Sodium salts of κ- and ι-carrageenans are soluble in cold water, whereas K+ and Ca2+ of κ- and ι-carrageenans are not soluble. Ca2+ salt gives a thixotropic dispersion. A 2.5% mucilage will emulsify an equal volume of oil such as cod liver oil. Very stable emulsions can be made by mechanical methods.25 The carrageenan obtained from E. serra (Togekirinsai) in Japan is a colorless, fibrous powder with an yield of 38.3% (w/w) based on dried seaweed and 4.6% from wet seaweed. The total carbohydrate, ash, moisture, and sulfate contents of the polysaccharide were 71.4%, 21.2%, 7.1%, and 23.8%, respectively. The polysaccharide, having a molecular weight of 2.8 × 105 Da, was composed of d-galactose, 3,6-anhydro-d-galactose, and ester sulfate at a molar ratio of 1.2:1.0:1.5. It contained d-galactopyranosyl-4-sulfate and 3,6-anhydro-d-galactopyranosyl-2-sulfate and an ι-carrageenan.38 Carrageenan from six species of red algae (Callophycus spp.) from Australia polysaccharide resembled α-carrageenan, and contained 4’,6’-O-(1-carboxyethylidene) carrabiose 2-sulfate as the major repeating disaccharide.39 The viscosity of carrageenan depends on the concentration, temperature, the presence of other solutes, and the type of carrageenan and its molecular weight. A 3% aqueous solution of carrageenan produces a gel on cooling.25,40 The alkali extract had a viscosity of 2778 cP; gelling point, 37.8°C; melting point, 57.1°C; sulfate ion, 19 ppm; 3.6-anhydro-d-galactose, 30.3%; and ash content, 17.6%.41 The viscosity increases almost exponentially with the concentration. For the gelling type of carrageenan, the viscosity measurement is carried out at a high temperature (e.g., 75°C) to avoid the effects of gelation; usually 1.5% of concentration being used, whereas for the cold water–soluble (nongelling) carrageenans, viscosities are measured at 25°C at 1.0% concentration. Viscosity is usually measured with the easily operated rotational viscometers such as Brookfield’s. Salts lower the viscosity of carrageenan solutions by reducing the electrostatic repulsion among the sulfate groups. Commercial carrageenans are generally available in viscosities ranging from 5 to 800 cP (1.5% at 75°C). The solutions of carrageenans having viscosities <100 cP display the Newtonian flow, varying degrees of pseudoplasticity for sodium- and λ-carrageenan, and thixotropic characteristic in the case of calcium ι-carrageenan. The latter is typified by a decrease in viscosity with increasing shear or agitation and return to normal viscosity with lowering of shear.

10.5.4

ANALYSIS

In terms of purity, carrageenan is classified as refined carrageenan and semirefined carrageenan (SRC). SRC possesses all the properties of refined carrageenan, except the ability to form clear solutions. SRC can be used in products where clarity is not of

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particular importance such as toothpaste and industrial suspension. Since the purity and composition of commercial carrageenans vary widely, there is a need to assess its quality in various products. Various approaches available for quality assessment include colorimetric staining (total carrageenan determination), light microscopy, immunological detection, electrophoresis, nuclear magnetic resonance (NMR), as well as chromatographic methods coupled in general to chemical or enzymic depolymerization procedures (such as gas liquid chromatography [GLC] and highperformance liquid chromatography [HPLC] of the methylglycosides released by methanolysis) or high-performance anion exchange chromatography of the liberated monosaccharides by acid hydrolysis.25 A sensitive resorcinol reagent is known for several decades for the colorimetric determination of fructose, and of 3,6-anhydrogalactose in agar, carrageenan, and other algal polysaccharides.42 A dye-binding method using methylene blue has been developed. The dye binds to these polysaccharides including carrageenan, which results in a color change from blue to purple. Anionic sites in the polysaccharides bind the dye; binding is reversible, electrostatic, and the ratio between anionic sites and bound dye molecules is 1:1. Carrageenans could be determined at concentration of 0.02–0.2%.43 Binding with the dye, alcan blue, is a method for rapid determination of carrageenan in foods such as jellies and salad dressings. The precipitate complex was dissolved in monoethanolamine and determined colorimetrically at 615 nm.44 Another assay is based on the binding of the cytochemical stain ruthenium red to algal polysaccharides. The dye binds strongly at low ionic strength, but is released at increasing salt concentration. Both cell-bound and soluble extracellular fractions could be determined in microbial cultures from the same sample, which could be removed from cultures without affecting the growth rate.2 It has been suggested that several of these methods must be used in combination to accurately determine the carrageenan type and amount because of the overall high molecular mass, the high polydispersity in that mass, and the lack of sensitive chromophores or other functionalities. A 1H and 13C high-resolution NMR spectroscopy, antibody- and lectin-based assays, and Fourier transform infrared spectroscopy are some of the newer methods, which could also differentiate κ-, ι- and λ-type carrageenans.45–47 A method for the determination of ι- and κ-carrageenans in foods has been developed. Samples are homogenized and freeze-dried before the release of 3,6anhydrogalactose dimethylacetal, which is characteristic of gelling carrageenans, by mild methanolysis. The 3,6-anhydrogalactose dimethylacetal content is subsequently quantified by the reversed-phase HPLC analysis. As a consequence, this method is not applicable for the analysis of λ-carrageenans as they are devoid of 3,6-anhydrogalactose dimethylacetal. The method does not require delipidation, deproteination, or extraction procedures. Specificity of the method for determination of carrageenans, whether alone or mixed with other hydrocolloids such as pectins, alginates, xanthan gums, or galactomannans, was demonstrated.48 Development of rapid enzyme-linked lectin assay (ELLA) and enzyme-linked immunosorbent assay (ELISA) for the determination of food-grade gums and thickeners (e.g., alginates, carrageenans, xanthan, gum arabic, and guar LBGs) is described. Formats and performance of the assays are considered and case studies are presented on the use of

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ELLA and ELISA for the determination of specific gums and thickeners in fruit jelly desserts, coating films, and pet foods.49 The techniques for the determination of carrageenans in foods have been reviewed.50

10.5.5 GELATION OF CARRAGEENAN Carrageenan gels such as agar are thermally reversible in that they remelt on heating and gel again on cooling. At high temperatures, carrageenans exist in solution in a disordered fluctuating chain conformation, but on cooling a rigid ordered double-helical structure is adopted that can be melted out again on reheating. Although interchain association through double helices is the primary event in carrageenan gelation, it does not in itself lead to a cohesive network but only to small domains of about 10 chains, which further associate in the presence of cations such as potassium ions to helix–helix aggregation.51 Thermal gelation is a valuable property of carrageenans that determines their diverse applications including foods and pharmaceuticals. Carrageenans at concentrations as low as 0.3% can form thermally reversible water gels in the presence of cations. The extraction conditions influence the gel strength of the carrageenan. Optimal gel firmness (157 g/cm2) was obtained when E. cottonii was subjected to extraction for 120 min at 100°C and pH 7.40 There is a difference in the gelation behavior among different types of carrageenans. In ι-carrageenan, the transition from single to double helices was the major gelation process. Crosslinking domain in ι-carrageenan comprised rodlike structures. Gels of ι-carrageenan show thermoreversible setting and melting behavior. Thermal gelation of KCG is due to a conformational transition from single chain to double helix and subsequent association of these helices. The gelling temperature of KCG ranges between 35 and 65°C, whereas the melting temperature ranges between 55 and 85°C. The extent of hysteresis is dependent on the type of carrageenan: for KCG it is 10–15°C and for ι-carrageenans it is about 5°C. The double helix played a major role in crosslinking. Setting of KCG gels occurred at the helix-coil transition temperature. Gel strength of carrageenans is determined with a gel tester (same as for agar) using 1.5% concentration of gel at 75°C. λ-Carrageenan is poor in gel formation, which shows normal behavior of a polyelectrolyte in solution.52,53 Figure 10.3 shows the gelation of carrageenan. 10.5.5.1 Rheological Properties Rheology is the study of deformation and flow of matter (see Chapter 3). Information on rheological properties of hydrocolloids is important to optimize efficiency of thermal processes and in product design. Rheological measurements have been highly helpful to understand gelation of carrageenans. Rheological properties of gel made of ι-carrageenan isolated from E. serra were examined. The flow curves of a calcium salt of the carrageenan showed plastic behavior in the presence of 0.1–0.3% CaCl2 with a yield value in the range 0.4–7.7 Pa. The dynamic modulus increased with the increase in concentration, and gelation occurred at 0.3% concentration at room temperature. The calcium salt showed larger values than sodium or potassium salts of ι-carrageenan. The gel formation was attributed to intra- and intermolecular

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Cool Heat Aggregating cations

Sol

(Nonaggregating cations) Heat

Cool

Gel

Add aggregating cations

Soluble domains

FIGURE 10.3

Gelation of carrageenan.

associations, contributed by sulfate groups of adjacent d-galactose and 3,6-anhydrod-galactose residues through calcium bridges with ionic bonding and attractive electrostatic forces within and between the molecules.53 Effects of ions on the gelation properties of carrageenans have been investigated. Potassium and calcium ions may distinctly raise the gelling temperature of the κ- and ι-carrageenans, respectively. During gelation, ι-carrageenan chains formed well-defined double-stranded helices in the presence of Li+, Na+, K+, Rb+, or Cs+. However, gelation of KCG was accompanied by multiple aggregation of double helices in the presence of all counterions except Li+ and Na+.52,54 Influence of Ca2+ was investigated on the physicochemical properties of KCG gels. The gel (5 or 10 g/L) was completely dissolved in water, and Ca2+ ions were added to achieve Ca2+ to gel ratios ranging from <1:1 to 4:1. Gels were analyzed for thermal stability, rheological properties, turbidity, and microstructural changes. The optimal molar Ca2+ to gel ratio was found to be 1:1, resulting in gels with a fine network structure, high optical clarity, and a high elastic modulus. At higher Ca2+ levels, the network consisted of coarser strands and larger pore sizes; the gels became more turbid and the elastic modulus decreased.55 It was suggested that the gel formation of the carrageenan might be essentially attributed to intra- and intermolecular associations, contributed to sulfate groups of adjacent d-galactose and 3,6-anhydro-d-galactose residues through Ca2+ bridges with ionic bonding and attractive electrostatic forces within and between molecules.53 Apart from ions, molecular weights of the hydrocolloid also influenced gelation as shown using KCG from E. cottonii having different molecular weights. Aggregation rate was shown to decrease with the decreasing size of KCG.56 Treatment of 2% aqueous λ-carrageenan solution at ultrasound intensity, 114.7 w/cm2 for 10 min

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reduced the molecular weight from 250,000 to 67,000 Da. Lower molecular weights of λ-carrageenan showed higher solubility, the lower alcohol precipitation ratio, and lower emulsifying capacity.57 Ultrasonic technique is an interesting relatively new technique to study differences in gelation behavior of different carrageenans in aqueous solutions. Gelation of κ-, κ/ι-hybrid-carrageenan, as well as a mixture of κ- and ι-carrageenans causes an increase in ultrasonic attenuation and a decrease in ultrasonic velocity at 7.8 MHz. However, gelation of ι-carrageenan did not cause detectable changes in the ultrasonic measurement. Different gelation behaviors of a κ/ι-hybrid-carrageenan and a mixture of κ- and ι-carrageenans were observed.58,59 The studies mentioned earlier suggest that functionality of carrageenan can be modified by alteration of its structure. In addition to physical methods, enzymes can be used as potential biocatalysts to modify carrageenan structure and its functionality.36 Viscosity and linear viscoelastic behavior of κ- and ι-carrageenans in aqueous 0.2 M NaI were investigated using oscillatory and creep measurements, which showed gelation of KCG at two critical concentrations. Viscoelastic behavior differed between κ- and ι-carrageenans. KCG showed behavior indicating that it was “solutionlike” at a concentration up to 1.5%, whereas ι-carrageenan displayed properties of a typical viscoelastic gel, its storage modulus (G′) having only weak frequency dependence.60 Influence of temperatures ranging from 20 to 80°C on flow behavior of carrageenan solutions showed that the shear rate of samples increased from 0 to 300/s in 3 min, held at the highest rate for 10 min and then decreased linearly back to 0 over 3 min. Shear thinning behavior was observed in all samples for the up- and downward curves of rheograms. Yield stresses were observed in carrageenan at 20 and 40°C. Consistency coefficient and flow behavior index were both sensitive to changes in temperature and concentration.61 The flow behavior of carrageenan could be described by the Herschel–Bulkley model. Flow curves of a solution of Ca-salt of ι-carrageenan from the red seaweed (E. serra) showed plastic behavior, and yield values were estimated to be 0.4 Pa, 1.7 Pa, and 7.7 Pa at 0.1%, 0.2%, and 0.3% (w/v) concentration, respectively. Dynamic modulus of the Ca-salt of ι-carrageenan increased with the increasing concentration and gelation occurred at a concentration of 0.3% (w/v) at room temperature. The Ca-salt showed larger values than the Na- and K-salts of ι-carrageenan with respect to dynamic viscoelasticity. Na- and K-salts had very large dynamic modulus values in the presence of CaCl2, which remained constant up to 40°C. Above this temperature, the dynamic modulus decreased rapidly.53 Rheological studies can also be used to understand the interactions among hydrocolloids. Such interactions are of interest to the food industry because of their combined roles as texturizers, thickeners, and gelling agents, which could be starches, sugars, gums, proteins, or their mixtures.62 Fish gelatin can be used as a substitute for animal gelatin (see Chapter 3). However, it has relatively low gelling and melting temperature and gel strength. Effects of addition of KCG (1% w/v) to fish gelatin preparations were investigated on their physical properties. Turbidity and gel strength were studied as a function of fish gelatin concentration (0–10% w/v), pH (5–12), ionic strength (0–500 mM KCl and NaCl), the nature of added salt, and temperature (4 or 22°C). In the presence of 1% KCG and 20 mM KCl, maximum gel strength occurred at a fish gelatin concentration of approximately 2%; the

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Young’s modulus for this system decreased considerably at pH <6, but increases in pH above the isoelectric point (approximately 8.7) did not have a major effect on the gel strength.63 In another study, characteristics of gelatin and ι-carrageenan gels were examined individually for comparison. Storage and loss moduli for mixtures were higher than the sum of those for gelatin and ι-carrageenan alone, suggesting that the two components reinforce each other at three specific temperatures.64 Influence on sweeteners such as sucrose and aspartame on KCG gelation was investigated. Addition of 10% sucrose increased the G′ and G″ values for soft KCG gels slightly. No marked changes in the viscoelastic properties were observed on the addition of aspartame.65 Hydrocolloids have the ability to interact with proteins, and thus, may be able to inhibit their coagulation, possibly due to electrostatic interactions.66 Neutral and charged ι-carrageenan enhanced denaturation temperatures of proteins (lysozyme, bovine serum albumin, and whey protein isolate), presumably due to the formation of additional hydrogen bonds and blockage of hydrophobic binding sites of proteins, preventing aggregation.67 It was suggested that complexation with anionic polysaccharides protects globular protein against loss of functional properties due to –S–S– bridge formation during or after high-pressure treatment.68 Mixing carrageenan with the proteins variously increased the gelation temperature and storage moduli of gels compared with values for carrageenan alone. Protein types affected the outcome, for example, denatured soy protein had a more profound effect on the melting temperature, but a lower effect on thermal hysteresis than native soy protein. Similarly, native soy protein increased aggregation rate and maximum viscosity over those of carrageenan-β-lactoglobulin gels. Protein addition increased the melting temperature, hardness, cohesiveness, gumminess, and springiness of carrageenan gels and reduced their syneresis. It was suggested that these effects, which may be due to the thermodynamic incompatibility of carrageenan and globular proteins, could enhance polysaccharide gelling properties and may be applicable in food technology.69 Phase separation of casein micelles occurs when gums are added to milkbased fluid systems as a consequence of biopolymer incompatibility. Bulk phase separation in these systems can be prevented by the use of KCG. Scanning electron microscopic and dynamic light-scattering studies suggested that KCG (0.03%) adheres to the surface of the casein micelle with an increase in micelle concentration at 3.65% casein.70 These studies clearly established the interactions of carrageenan with other macromolecules.

10.5.6

APPLICATIONS OF CARRAGEENANS IN FOOD PRODUCT DEVELOPMENT

Carrageenan is widely used as an ingredient in a variety of foods to modify their rheological properties because of its characteristic prop erties of gel formation and its ability to interact with other food components under a variety of conditions. The interactions provide a number of additional advantages, such as improving the WHC, reduction of fat, possibility to replace animal-derived gelatin in dairy products such as yoghurts, antimicrobial activity, and opportunities for the development of vegetable-based texturized products. Since the properties of carrageenan gels vary with the concentrations of the hydrocolloid, manufacturers often use a mixture of carrageenans to obtain the desired result.71,72

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Modification of Textural Properties

Carrageenan allows textural modification of diverse food products through changes in water binding, emulsifying, and foaming properties. Process variables such as temperature, pH, ionic strength, cations, additives such as proteins, and other hydrocolloids could further optimize the texture.73 Carrageenan can increase surface hydrophobicity values of protein–hydrocolloid mixtures. The presence of hydrophobic areas on the protein surface can aid in its ability to adsorb at an oil–water or air–water interface.74 The ι- and κ-carrageenans at the 0.5% level increased cooking yield, hardness, and binding strength of 1%-salt sausage, but had little effect on the 2.5%-salt sausage. An increase in pH from 5.2 to 6.2 sharply enhanced the binding strength. Both the instrumental and sensory panel results suggested that α- and κ-carrageenans were the only acceptable gums for use in low- or highsalt beef sausage products.75 Model systems based on cooked ham were prepared containing carrageenan, NaCl, KCl, and salt-soluble meat protein isolate at 6.5–11.5% at pH 6.2. Although the properties of the meat gels were mainly governed by the concentration of the meat extract present, addition of 0.2% carrageenan led to an increase in the WHC, gel strength, and hardness. However, carrageenan did not interact with the meat proteins to participate in network formation, but could be present in the interstitial spaces of the protein network binding water and forming gel on cooling.76,77 The ability of carrageenan and also turkey collagen and soy protein to increase the usability of pepsin-solubilized elastin (PSE)-like turkey meat in a chunked and formed turkey breast roll was investigated. The meat products were evaluated for cooking and chilling losses, expressible moisture, purge loss, protein bind, cooked color parameters, and sensory properties. Addition of carrageenan, collagen, or soy protein markedly decreased purge loss and improved the texture of turkey breast rolls.78 Complexes of carrageenans and proteins such as casein and soy protein are utilized in food technology. The hydrocolloid is used with the proteins in ratios varying from 1:5 to 5:1 to alter functional properties. The thermal stability of these complexes was higher than that of KCG.79 Method of preparations of food compositions containing carrageenans has been described in a patent. The compositions comprise, approximately 55–85% by weight nutritive carbohydrate sweeteners, sufficient amounts of a gelling system to provide gel strength of 1–8 kg/cm2, and 10–20% moisture. The gelling system, which contains high methoxyl pectin and KCG, allowed a high solids level but a low viscosity when maintained above 55°C. The method of production involved forming a hot fluid gellable slurry, shaping it into pieces by starch molding, and curing it to form a gelled product.80 Surimi is a concentrate of fish myofibrillar proteins, which is used to develop restructured products having acceptable texture (see Chapter 3). The texture of surimi products could be modified by the incorporation of carrageenan at suitable concentrations in the presence of other additives. Gelation of washed blue whiting (Micromesistius poutassou) mince in the presence of 0.5% carrageenans (ι- or κ-type) or sodium alginate and cations was evaluated. Mixtures were heat set (37°C for 30 min and 90°C for 50 min), cooled, and held for 24 h at 4°C before characterization of the gel in terms of folding resistance, puncture properties, texture and stress relaxation, color, and WHC. The presence of salts influenced the texture of surimi.

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In gels containing KCG, CaCl2 affected elasticity and redness and lightness values. In alginate-containing gels, Ca2+ had an effect on cohesiveness, elasticity, folding test value, hardness, and WHC of gels, whereas the major impact of NaCl addition was on gel adhesiveness. In gels with ι-carrageenan, an interaction between KCl and CaCl2 was observed.81 Carrageenan was identified as having a significant positive effect on the gel strength of Atlantic pollock (Pollachius virens) surimi. All the three types (κ, ι, and λ) of carrageenans, especially the ι-type, improved water-holding ability of the cooked surimi gels over three freeze-thaw cycles.82 High pressure is known to affect the gelation of fish muscle proteins. Gelation with ι-carrageenan at atmospheric pressure produced gels that were more adhesive, less cohesive, lighter, and more yellowish than pressure-induced gels. For gels with KCG, the heat-induced sample was particularly hard and adhesive, with low cohesiveness, more yellowness, and lower WHC than pressure-induced gels.18 The presence of carrageenan at 0.25% stabilized the foam of sunflower proteins, which was subjected to hydrolysis using Alcalase 2.4 L. Foam overrun and stability against liquid drainage and collapse were improved by hydrolysis to a degree of 1.5%, but higher degree of hydrolysis did not further enhance the foaming properties. Comparable effects of carrageenan on other vegetable proteins have also been reported.83 Commercial carrageenans possess the ability to gel or thicken milk system and to react with milk proteins. λ-Carrageenan has the greatest ability to disperse in milk at 5–10°C and thicken it without any salts. λ-Carrageenan is insensitive to K+ and Ca2+, whereas κ- and ι-carrageenans, due to the higher content of 3,6-anhydrogalactose and the lower content of ester sulfate, are more sensitive to K+ and Ca2+, which are constituents of the milk. Although κ- and ι-carrageenans are practically insoluble in cold milk, they may be used effectively for thickening and gelling if tetrasodium pyrophosphate (TSPP) is added. The hydrocolloid is therefore used in chocolate/milk drinks or shakes, ice creams, and desserts as a stabilizer and emulsifier. In these products, carrageenans are usually blended with dextrose for uniform performance.84 The hydrocolloid can also be added to soymilk to improve viscosity and shear stress. Overall, flavored soymilks, particularly those containing chocolate flavoring and ι-carrageenan, had improved sensory attributes over plain soymilk, and all were stable for 1 month under refrigerated conditions.85 Addition of carrageenans to commercial bread doughs resulted in a higher water absorption value compared to doughs containing wheat bran only. A 10% replacement of wheat bran by carrageenan in breads enhanced loaf volume, water absorption, and improved crumb grain score compared to breads with comparable quantities of wheat bran only. Structural analysis of the bread revealed that perforations of the gluten and gelatinized starch matrix in the wheat bran breads containing carrageenan may be more uniform and smaller in size.86 Table 10.8 gives some applications of carrageenan and other seaweed hydrocolloids in food product development. 10.5.6.2

Reduction of Fat

Carrageenan can function as a fat replacer in food items including meat products, without significantly affecting their rheological properties.84,88 Carrageenan at 0.5%, either alone or in combination with cellulose, functions as a gel-forming fat substitute.

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TABLE 10.8 Some Applications of Carrageenan and Other Seaweed Hydrocolloids in Food Product Development Products Fishery products such as surimi and other fish meat gel and novel fish products, for example, fish burgers and sausages Red meat products such as turkey, restructured beef products, low-fat meat balls, and beef burgers. Bakery products Dairy products

Flavored soy milk Fruit juices, for example, apple juice Novel food product developments involving carrageenan–food component interactions Clarification of wine, colloidal stabilization of beer Reduction of sodium in foods

Polysaccharide and Action Carrageenan and alginate enhance cooking yield, hardness and bind strength, texture, and fiber content

Carrageenan increases yield, improved visual appearance, sliceability and rigidity, and decreased expressible juice. Enhances storage stability Carrageenan enhanced loaf volume and water absorption and improved crumb grain score Carrageenans, agar, and alginates act as stabilizing, thickening, and gelling agents. KCG–casein interactions stabilize ice cream. Carrageenan improves viscosity of goat milk ι-Carrageenan increased viscosity and sensory values ι-, κ-, or λ-Carrageenan, alone or with citric acid, inhibit browning Effected through KCG-induced increase in surface hydrophobicity increasing oil binding properties of proteins. KCG– ovalbumin complexes have applications in food technology Carrageenan, alginic acid Carrageenans maintain texture when sodium is replaced by potassium

References 15, 72, 87

88

86 71, 72, 84, 89

85 90 68, 71

62 87

The hydrocolloid at 0.25–0.75% helped develop a pork product having less than 10% total fat. The moisture content of raw and cooked low-fat patties was significantly higher than control patties having 20% fat. Cooking yield, fat, and moisture retention also improved significantly in low-fat patties, whereas the dimensions of the low-fat patties were maintained better than those of the control product during cooking. The sensory attributes of low-fat patties with carrageenan were similar to those of the high-fat control. The lipid profile revealed as much as 47.7% and 44.1% decrease in total lipids and cholesterol contents, respectively, with a reduction of calorie in 31% as compared to the controls. The low-fat ground pork patties had good storage stability for 35 days at 4°C.91 The hydrocolloid can function as a fat substitute in emulsified meat balls. High acceptability scores were observed for low-fat (<10%) meat balls containing salt, polyphosphates, and KCG at levels of approximately 2.7%, 0.17%, and 2%, respectively.92 Effects of carrageenan (0.3–0.7%) alone or in

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combination with a 20% pectin gel gave ideal physicochemical and textural properties to low-fat beef frankfurters. Replacement of fat with carrageenan and pectin reduced cholesterol levels significantly from 93.3 to 40.9% in high-fat (20%) frankfurter.89 At 2.5% NaCl, κ-, ι-, and λ-carrageenans affected the stability of proteins in chicken mince. Owing to the apparent dependence of carrageenan-induced changes on muscle types, it was deemed advisable to use different processing conditions for the manufacture of low-fat, water-added dark versus white poultry meat products in which polysaccharide gums are used as water-binding agents.93 Carrageenan and also other gums such as alginates, guar, or xanthan gum are used in the development of coated products to improve batter adhesion to the product through thermal gelation. The hydrocolloids can also be used to reduce fat uptake during deep fat frying of coated products including those from fish and shellfish.87,94,95 10.5.6.3 Reduction of Salt Because of adverse effects of high-dietary sodium on health, there is interest in low-salt food products. NaCl (and also fat) of a sausage formulation was reduced by KCG. KCl was added at 0.5% to all of the treatments and CaCl2 at 0, 0.5, and 0.1%. Cooking yield was increased by all the treatments, but expressible moisture levels were not significantly different, indicating that water is not chemically entrapped by carrageenan under the ionic strength conditions employed. However, since no detrimental cooking losses or fat release were detected, myofibrillar proteins appeared to maintain good functionality under these conditions, together with KCG. Results suggest that NaCl (and also fat content) can be significantly reduced without detrimental effects on texture and sensory properties.87 10.5.6.4

Flavor Perception

Carrageenan could influence the flavor of processed products. Effects of λcarrageenan concentration (0.1–0.5%) were investigated on the release of aroma compounds, namely, aldehydes, esters, ketones, and alcohols in thickened viscous solutions containing 10% of sucrose. Air/liquid partition coefficients of 43 aroma compounds were determined in pure water and in the λ-carrageenan solutions employing dynamic headspace gas chromatography. The partition coefficient increased as the carbon chain length of aroma compounds increased within each homologous series. Esters exhibited the highest volatility, followed by aldehydes, ketones, and alcohols. A suppressing effect of λ-carrageenan on the release rates of aroma compounds was noted; the extent of decrease in release rates was dependent on the physicochemical characteristics of the aroma compounds, with the largest effect occurring for the most volatile compounds. The suppression effects were attributable to the thickener and not the physical properties of the increasingly viscous systems.96,97 10.5.6.5 Fortification with Fiber As discussed in Chapter 9, there is good scope for fortification of food products with fiber to improve its nutritional as well as sensory properties. Most of the vegetable fibers used come from cereals, but lupine, rice, pea, and fruit fibers have also been used. Their essential uses in technological terms are to replace the oiliness

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produced by fats, to bind water to reduce cooking losses, and to maintain the shape of the product after cooking. Carrageenan at a concentration of 0.5–0.7% can be used to increase fiber in dietetic foods. Fishery products are known to be poor in fiber contents. Carrageenans can be used for fish product developments to enable raw fish meat to be processed like meat and poultry. Addition of carrageenan and other fibers to restructured fishery products such as sausages and patties can help to considerably improve the viscosity and texture; with KCG offering better WHC than ι-carrageenan preventing syneresis in fish gels during freezing/thawing. The possibility provides the industry with opportunities to manufacture a wide range of new products such as fresh salmon roll, low-fat fish pates, fish burgers (usually tuna), and improved-quality fish fingers and other breaded products.98 10.5.6.6 Control of Browning A combination of 0.1% of any of the carrageenans (ι-, κ-, or λ) and 0.5% citric acid was able to inhibit browning of unpasteurized apple juice containing 0.1% sodium benzoate for up to 3 months at 3°C. The observation may have practical application in the prevention of enzymic browning in fresh, raw apple juice or diced apples.90 10.5.6.7

Cryoprotective Effect

Denaturation of proteins in muscle foods during frozen storage is a serious problem (Chapter 3) that affects the texture of frozen products. Sodium tripolyphosphate (STPP) is conventionally used as a cryoprotectant to control the problem, whereas alginate or ι-carrageenan can be effective. This was shown on physicochemical and sensory properties of red hake (Urophycis chuss) mince stored at −20°C for 17 weeks. Addition of 0.4% alginate, 4% sorbitol, and 0.3% STPP protected the mince from hardening and improved its dispersibility during mixing. Alginate appeared to be responsible for preventing muscle fiber interaction through electrostatic repulsion and chelating Ca2+, thus improving dispersibility.99 LBG and λ-carrageenan at 0.1% concentration were investigated in whipped dairy cream during freezing and thawing. Although freezing caused the collapse of the foam structure of whipped cream, the addition of gums reduced changes in elastic properties, particularly in samples containing carrageenan, suggesting a cryoprotective effect of the hydrocolloid in whipped cream.100 10.5.6.8 Miscellaneous Applications Carrageenan and also alginic acid have greater wine stabilization capacity than agar. Protein flocculation and precipitation capacities of carrageenan and alginic acid were two times greater than that of agar. Alginic acid absorbed protein at a maximum concentration of <50 mg/L, whereas the maximum adsorption and precipitation capacity for carrageenan was observed at a protein concentration of >400 mg/L.101 In a trial on the application of carrageenan in brewing of wine, carrageenan was added to malt syrup at 0–40 ppm and the mixture was heated to boiling for 10 min and then rested for 1–2 h. The lowest turbidity of the product was noted using carrageenan at 30 ppm, whereas the control sample without carrageenan was most turbid. The

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clarification was due to the precipitation of proteins by the hydrocolloid.102 Use of hydrocolloids for improving shelf life and handling properties of tortillas has been pointed out.103 Use of carrageenan to save energy during extrusion cooking has been pointed out. Fourteen hydrocolloids including alginate, carrageenan, and gelatin were dry blended with corn grits at 0.1–1.0% level and at 20% moisture and was extruded in a Brabender-modified Pl-V500 laboratory extruder at 50–150°C with a 1:1 screw operating at 100 rpm. Alginate significantly lowered the torque at 50°C, but not significantly at higher temperatures. Carrageenan did not significantly reduce the torque. The ability to reduce torque by some hydrocolloids suggests their potential to save energy during extrusion cooking.104 The hydrocolloid can also improve the binding of ingredients in steamed or nonsteamed prawn feeds.105

10.5.7

BIOLOGICAL ACTIVITIES OF CARRAGEENAN

A number of biological activities of carrageenans have been reported, as discussed in the following sections. 10.5.7.1

Antimicrobial Properties

The natural antimicrobials approved by the U.S. Food and Drug Administration (FDA) include acetic acid, acetates, benzoic acid, dimethyl dicarbonate, lactic acid, lactoferrin, lysozyme, natamycin, nisin, parabens, propionic acid and its salts, sorbic acids, sorbates, and sulfites.106 Carrageenans and other hydrocolloids such as pectins, xanthan gums, acacia gums, or agars are anionic hydrocolloids. These compounds, when interacting with cationic preservatives such as lauric acid, form compounds that are stable and can be stored under ambient temperatures and humidity conditions for prolonged periods. These compounds have good antimicrobial properties.107 Another interesting application is for the control of pathogens in poultry and meat products. Salmonella typhimurium is a pathogen usually contaminating poultry. Similarly, Escherichia coli O157:H7 strain is known to be a potent pathogenic organism, which attaches with collagen I of meat and poultry. KCGs were found to almost completely prevent contamination of the poultry carcasses by the pathogen. This was evaluated using a biosensor containing S. typhimurium immobilized on the sensor chip. The observation could help in the development of new strategies to either detach or prevent attachment of pathogens to animal carcasses.108 10.5.7.2 As Growth Factor Antagonist Carrageenans are selective growth factor antagonists, which have potential for the treatment of disorders associated with the overproduction of certain growth factors. Carrageenans inhibited the binding of basic fibroblast growth factor (BFGF), transforming growth factor beta 1 (TGF-β1), and platelet-derived growth factor (PDGF) to cells. ι-Carrageenan was the most potent BFGF antagonist, whereas KCG was the most potent PDGF antagonist and λ-carrageenan the most potent TGF-β1 antagonist. None of the carrageenans, at less than 200 μg/mL, inhibited the binding of insulinlike growth factor 1 or TGF-α.109

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10.5.7.3 Antioxidant Activity The importance of antioxidant activity in the biological system has been discussed (see Chapter 5). At least some carrageenan derivatives have been reported to possess antioxidant activity. Oligosaccharides from KCG were prepared through mild HCl hydrolysis of the polysaccharide, and oversulfated, acetylated, and phosphorylated derivatives were generated. Significant antioxidant activities were exhibited by oversulfated and acetylated derivatives against superoxide radicals, phosphorylated and acetylated derivatives against hydroxyl radicals, and phosphorylated derivatives against DPPH radicals. Antioxidant activity of the carrageenans was not found to be affected by their molecular weight.110,111 10.5.7.4

Suppression of Immune Response

Carrageenans (κ, λ, and ι) can markedly suppress immune responses both in vivo and in vitro. Impairment of complement activity and humoral responses to T-dependent antigens, depression of cell-mediated immunity, prolongation of graft survival, and potentiation of tumor growth by carrageenans has been reported. The mechanism responsible for carrageenan-induced immune suppression is believed to be its selective cytopathic effect on macrophages. Systemic administration of carrageenan may, however, induce disseminated intravascular coagulation and inflict damage on both the liver and kidney. This is an important consideration in the interpretation of the effects of carrageenan in vivo.112 Effects of carrageenans (ι, κ, and λ) on host defense mechanisms of macrophages against Salmonella enteritidis infection were examined in vitro using macrophagelike J774.1 cells. λ-Carrageenan had no effect on the macrophage function, whereas ι-carrageenan reduced the Salmonella-binding and phagocytotic activities of J774.1 cells, but increased the bactericidal activity of the cells. KCG increased the cell-binding activity, but reduced the bactericidal activity. It was concluded that these compounds could affect immune function by modulation of macrophage functions.113 The antiviral effects of polysaccharides from marine algae toward mumps virus and influenza B viruses have been reported. Polysaccharides from the extracts of red algae were found to inhibit HSV and other viruses. The HIV is the retrovirus that causes acquired immunodeficiency syndrome (AIDS). Extracts from the California red algae (Schizynenia pacifica) that contained a sulfated polysaccharide belonging to carrageenans, selectively inhibited HIV reverse transcriptase. Recently, Israeli researchers have developed a microalgal ointment for treating herpes infection. The preparation contained special polysaccharides obtained from algae belonging to Dumontiaceae. The natural product was found to be effective against various herpes infections and also reduced pain. Carrageenan, fucoidan, dextrin sulfate, pentosan polysulfate, and heparin were examined for their efficacy as a vaginal microbicide. Although these compounds were found to possess anticoagulant activities, antiviral activities were also detected at very low concentrations. The aqueous extract of the brown seaweed Fucus vesiculosus showed very good anti-HIV activity, presumably due to the presence of polyphenols and both sulfated and nonsulfated polysaccharides including fucoidan.114,115 Certain sexually transmitted human papillomavirus (HPV) types are causally associated with the development of cervical cancer. Recent

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developments in virology have made it possible to perform high-throughput in vitro screens to identify HPV infection inhibitors. Comparison of a variety of compounds revealed that carrageenan could be a potent infection inhibitor for a broad range of sexually transmitted HPVs. Carrageenan acts primarily by preventing the binding of HPV virions to cells.116 10.5.7.5 Anticancer Activity One of the ways to fight cancer is to stimulate macrophage production and activity. Interferon is a natural secretion of the body that is thought to be a stimulator of the macrophage and tumor nercosis factor (TNF). KCG was depolymerized using carrageenase and the oligosaccharide was examined for antitumor activity using sarcoma 180 tumor in mouse. A carrageenan oligosaccharide with a molecular weight of 1726 Da when administered orally at a dose of 100 mg/kg mouse markedly inhibited tumor formation. The preparations influenced immunological regulation, especially the phagocytosis ratio and phagocytosis index of macrophage, which might be beneficial for the antitumor activity.117 10.5.7.6 Inactivation of Paralytic Shellfish Poison The action of KCG gel to sequester paralytic shellfish poison (PSP) (see Chapter 15) was tested and characterized. When an extract from a seaweed strain Pyrodinium bahamense var. compressum was used as a PSP solution, the PSP-sequestering property of KCG gel was found to be dependent on the gel surface area, interaction time, and concentrations of polysaccharide and monovalent cations. The characteristics of KCG as a PSP-sequestering agent point to cation exchange as the mechanism of action. The polysaccharide gel could be utilized as an agent to alleviate PSP intoxication.118 However, treatment of the intoxicated patient with the gel needs to be worked out. 10.5.7.7 Elicitor of Plant Defense Carrageenans efficiently induced signaling and defense gene expression in tobacco leaves. Defense genes encoding sesquiterpene cyclase, chitinase, and proteinase inhibitor were induced locally, and the signaling pathways mediated by ethylene, jasmonic acid, and salicylic acid were triggered. The results showed that λ-carrageenan could elicit an array of plant defense responses.119

10.5.8

BIOTECHNOLOGY

The varied applications of carrageenan in biotechnology include cell immobilization as an affinity ligand in enzyme purification, and control of pathogen contamination in meat and poultry and as antagonists of growth factors. Furthermore, application of biotechnology into carrageenan processing helps in obtaining tailor-made new, specific seaweed polymers to form a new generation of high-quality functional ingredients for foods, cosmetics, and pharmaceuticals.36

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10.5.8.1 Immobilization of Enzymes Carrageenans have been used for the immobilization of microbial cells.119 Saccharomyces cerevisiae was immobilized on 0.5–2.0 mm KCG gel, where the yeast cells were individually and uniformly distributed. Performance of the immobilized yeast was assessed in batch fermentation and in a gaslift bioreactor during a 6-month continuous fermentation. Yeast viability was >90% after batch fermentation for 2 days when the yeast count increased by a factor of 10. Immobilized yeast cell viability decreased to less than 50% after 6 months of continuous fermentation. The yeast cell count in the gel beads increased to 1.4 × 109/mL after 6 months of continuous fermentation and gave a fully fermented beer even after 6 months of continuous fermentation.120 Whole cells of phytopathogenic Erwinia chrysanthemi strains were immobilized in KCG beads and grown in a medium containing sodium polypectate as the carbon source and were used for the production of pectate lyase and proteinases. All the strains of the organism used survived immobilization into KCG beads and displayed higher pectolytic and proteinase activities than free cells in liquid suspension. It was suggested that carrageenan immobilization could provide a system to mimic conditions of E. chrysanthemi cells in infected plant tissue.121,122 10.5.8.2

Enzyme Purification

Egg white is a rich source of lysozyme, which has several applications including food preservation. KCG has potential for use in simple and efficient separation of lysozyme from salted or fresh duck and hen egg whites. KCG interacted and formed precipitates with lysozyme. Using 0.7% KCG, lysozyme was recovered from a fivefold diluted salted duck and hen egg whites, at a rate of 60–65% and 78–81%, respectively. Recovered lysozyme from the salted duck egg whites was stable during storage at 4°C for 35 days.123,124 Use of KCG as a ligand for affinity precipitation purification of pullulanase (α-dextrin endo-1,6-α-glucosidase) has been reported. KCG selectively bound Bacillus acidopullulyticus pullulanase. For affinity precipitation of pullulanase, the enzyme was added to 0.3% KCG and precipitation was initiated by the addition of KCl. The precipitate was dissolved in maltose solution and the polymer was precipitated by the addition of KCl and centrifugation. This technique gave a 50-fold purification of the enzyme.125

10.5.9

TOXICOLOGY OF CARRAGEENAN

Carrageenan is not degraded to any extent in the GI tract and is not absorbed from it. In long-term bioassays, carrageenan has not been found to be carcinogenic, and there is no credible evidence supporting a carcinogenic or a tumor-promoting effect on the colon in rodents. Toxicological studies on carrageenans and processed Eucheuma seaweed have been reviewed with particular reference to carcinogenicity studies. Available data on long-term bioassays do not provide evidence of carcinogenic, genotoxic, or tumor-promoting activity by carrageenan.126,127 A variety of carrageenans from E. spinosum were given to guinea pigs, monkeys, and rats through the diet. Fecal and liver samples were examined by gel electrophoresis to determine the integrity of

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administered carrageenans. Quantitative measurements of carrageenans were also carried out on samples of liver and urine. The studies showed that there was little or no absorption of high-molecular-weight carrageenans by guinea pigs or rats. In contrast, substantial amounts of low and intermediate molecular weight (40,000 and 150,000 Da) carrageenans were found in the livers of these animals. Urinary excretion of carrageenan was limited to low-molecular-weight material, having a molecular weight of 20,000 Da or lower. Qualitative and quantitative evidence indicated that there was an upper limit to the size of carrageenan molecules absorbed.128,129 Nevertheless, some adverse GI and immunological consequences related to the consumption of food products containing carrageenan have been reported.130 Carrageenans appear on the U.S. FDA’s Generally Recognized as Safe (GRAS) list for food additives. However, in contrast to undegraded carrageenans, poligeenans (degradation products of carrageenan, having molecular weights of 20–30 kDa) exhibit toxicological properties and hence they are not food additives. Foods containing high-molecular-weight carrageenans, however, generally do not contain poligeenan. The Joint Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA) has recommended an acceptable daily intake (ADI) of 0–75 mg carrageenan per kilogram body weight. The Scientific Committee on Food (SCF) of the European Union, which recently evaluated research data on biological effects of carrageenan, observed that the preceding ADI level could be continued. The SCF observed that it was inadvisable to use carrageenan in formula for infants that are fed from birth. However, the SCF had no objection in its use for older infants such as follow-on milk and weaning foods. For them, carrageenan may be added up to 0.3 g/L milk.131 The JECFA is of the view that based on the information available, it is inadvisable to use carrageenan or processed Eucheuma seaweed in infant formulae.132 Food-regulatory authorities have specifically defined species for use as raw materials for the extraction of the commercial seaweed gums. The U.S. FDA requires specific seaweeds used for extraction of the hydrocolloids for legal sanctity. Some seaweeds used as food and sources of commercial gums are given in Table 10.9.133

TABLE 10.9 Some Seaweeds Used as Food and Sources of Gums Danish agar (Furcellaria fastigiata) Eucheuman (Eucheuma spp.) Furcellaran agar (F. fastigiata) Hypnean (Hypnea spp.) Irish moss (Chondrus spp.) Wakame (Undaria spp.) Kombu (Laminaria spp.) Nori (Porphyra spp.) Source: Adapted from FAO, State of World Fisheries and Aquaculture, Food and Agriculture Organization of the United Nations, Rome, Italy, 2004, http://www.fao.org//docrep/ 007/y5600e/y5600e07.htm#P10_2114, accessed July 2008. With permission.

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DEGRADATION OF CARRAGEENAN

Carrageenans are susceptible to degradation by carrageenases. These enzymes are generally specific for the carrageenan types. Marine environment may contain microorganisms that can degrade carrageenan. Twelve bacterial strains, belonging to the genus Cytophaga, which decompose carrageenan were isolated from seawater. They were gram negative, facultative anaerobic, nonflagellate, long rods, formed spreading colonies, contained carotenoid pigments, and had phosphatase activities. The bacteria were also able to decompose agar, casein, gelatin, and starch.134 A KCG (EC 3.2.1.83)-producing Vibrio spp. was isolated from the surface of a marine alga. The enzyme specifically hydrolyzed KCG to neocarrabiose and neocarratetrose sulfates. No activity was observed on neocarrabiose sulfate, λ- and ι-carrageenans, or agar. The extracellular enzyme, which was purified to homogeneity, had a molecular weight of 35 kDa and maximum activity at pH 8 and 40°C. The values of pI and Km of the enzyme were 9.2 mg/mL and 3.3 mg/mL, respectively. The enzyme activity was inhibited completely by heavy metal.135 Another bacterial strain, able to degrade various sulfated galactans (carrageenans and agar) was isolated from the marine red alga Delesseria sanguinea. This extracellular enzyme had a molecular weight of 40 kDa and optimal activity at pH 7.2.136 Seven marine bacteria, which degraded a hybrid ι- and κ-carrageenan from E. spinosum were isolated.137 The degradation products of carrageenan, now referred to as poligeenan,126 formed by the action of carrageenase could be separated and characterized by a rapid size-exclusion chromatography method.126,138,139 Various nonenzymatic processes can also degrade carrageenans. These procedures include autoclaving, microwave treatment, and ultrasonication in the presence of acetate, citrate, lactate, malate, and succinate. Autoclaving in the presence of citrate or malate at 110–120°C for 120 min gave a maximum yield of oligosaccharides. Maximum depolymerization rate due to autoclaving was approximately 23% and treatment at 120°C led to the production of five to seven types of oligosaccharides.140

10.6 FUCOIDAN Fucoidan is a sulfated polysaccharide having an average molecular weight of 20,000 Da, found mainly in various species of brown seaweed. The polysaccharide was first isolated from marine brown algae in 1913 and named fucoidan. Fucoidan exists essentially in two distinct forms of glycosaminoglycans (GAGs): F-fucoidan, which is more than 95% of the fucoidans in seaweeds, is composed of sulfated esters of l-fucose and U-fucoidan, which is approximately 20% glucoronic acid. Depending on the source of algae, the sulfated polysaccharide consists mainly of l-fucose units, but can also contain other sugars such as galactose, mannose, xylose, or uronic acid and sometimes proteins. Sulfated fucan is the characteristic feature of the fucoidans. These compounds in total comprise less than 10% in composition. (Variant forms of fucoidan have also been found in animal species, including the sea cucumber.) F-fucoidan can easily be extracted from algae cell walls by treatment with hot mild acid solution. The crude fucoidan extracted with hot water (60–70°C) is further purified by hydrophobic chromatography. The precipitate is digested with water, filtered, dialyzed, and freeze-dried. Further purification is effected by fractional precipitation with cetyl trimethyl ammonium hydroxide or cetylpyridinium

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chloride. This is followed by fractional solubilization with acetic acid to release the polysaccharide from its precipitated salt, and subsequent precipitation with ethanol or KCl. A protease treatment can accelerate the release of the polysaccharide from bound proteins. Fucoidans and sulfated fucans can be subjected to enzymatic or acid hydrolysis to obtain low-molecular-weight poly- and oligomers for biological applications.141 The structure of fucoidans differs when they are extracted from different algal species; the structure of sulfated fucans of marine invertebrate origin is simpler compared to those of algae. They commonly possess a clearly regular structure and each species has its own sulfated fucan. Fucoidan was isolated from Nemacystus deciphens, which is commercially cultured at the rate of 2000 t/year in Japan. The yield of fucoidan extracted by 0.1 N HCl was 0.5% based on the wet weight of the alga. The total carbohydrate, sulfuric acid, ash, and moisture contents in fucoidan were 0.5%, 30.8%, 22.3%, and 3.8%, respectively. Fucose and galactose were the carbohydrates. The molecular weight of fucoidan was 2.4 × 105.141–143

10.6.1

BIOLOGICAL ACTIVITIES

Sulfated fucans exhibit a wide range of biological activities. The antiinflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans have been well recognized. Furthermore, fucoidans are reported to have antitumor, antimutagenic, anticomplementary, immunomodulating, hypoglycemic, antiviral, hypolipidemic, and antiinflammatory activities.144 Fucoidans obtained from nine species of brown algae inhibited leukocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate, nor the other structural features of their polysaccharide backbones significantly affected the activity. All fucoidans, except that from Cladosiphon okamuranus exhibited anticoagulant activity, whereas only fucoidans from Laminaria saccharina, L. digitata, Fucus serratus, F. distichus, and F. evanescens displayed strong antithrombin activities in a platelet aggregation test. Fucoidans from these algae also strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect that could have critical implications in tumor metastasis. The data presented provided a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.6,12,145–148 Owing to interference with molecular mechanisms of cell-to-cell recognition, fucoidans can be used to block cell invasion by different retroviruses such as HIV, HSV, cytomegalovirus, and African swine fever virus. Furthermore, these macromolecules can act as antiangiogenic agents and inhibitors of sperm binding to oviductal epithelium and sperm–egg binding in many species. In addition, algalsulfated fucoidans have antiproliferative and antitumoral properties.141,149 Fucoidan has anticoagulative properties, but is also able to bind and block the function of the selectins.143 In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that polysaccharides from L. saccharina, L. digitata, F. evanescens, F. serratus, F. distichus, F. spiralis, and A. nodosum could serve as P-selectin inhibitors. Intravenous administration of fucoidan significantly prolonged the time required for complete occlusion in arterioles and venules by almost seven- and ninefold, respectively, suggesting its potent in vivo anticoagulative effect.150 Therefore, fucoidans can be potential replacers for conventionally used anticoagulant, heparin, prepared from mammalian mucosa. Sixteen species of

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Indian marine green algae were screened for blood anticoagulant activity. Species of Caulerpa exhibited the highest activity, which is comparable to heparin.151 Fucoidan is capable of binding antithrombin, the inhibitor of blood coagulation, in a 1:1 stoichiometry. An affinity chromatography method has been developed to determine the binding of fucoidan with the protein.152 These polysaccharides are potent thrombin and factor Xa inhibitors mediated by antithrombin or heparin cofactor II. Such heparinoid-active sulfated polysaccharides have also been isolated and characterized recently from green seaweeds.153 A fucoidan having antitumor activity has been isolated from the edible seaweed L. religiosa.154 In addition to fucoidan, the antitumor activity was also attributed to the polysaccharides, carrageenans, and porphyrans. The antitumor activities of lipid fractions from Sargassum ringgoldianum against Meth A fibrosarcoma were found to be 36.1–47.1%, and those of glycolipid and phospholipid fractions from Laminaria angustata were as high as 45.9 and 58.0%. The phospholipid fraction from Porphyra yezoensis showed a very high activity.155 Recently, the potential of seaweeds to ameliorate chronic renal failure in rats has been reported. The formation of calcium oxalate, referred to as kidney stones, is a major disease affecting the elderly. Abnormalities in oxalate metabolism have been suggested as a cause for the pathogenesis of stone disease since an excessive excretion of oxalate leads to calcium oxalate crystal urea. Although the basic mechanism of stone formation is still largely shrouded in uncertainty, application of exogenous GAGs such as sodium pentosan polysulfate (SPP) to prevent stone formation and recurrence is considered as a promising approach. Synthetic polysaccharides, such as low-molecular-weight heparin (LMWH), have been reported to have renoprotective effects. Fucoidans isolated from marine organisms bear similarity to heparin, as mentioned earlier. Hence, the nephroprotective action of heparin derivatives such as LMWH and SPP could also be extended to sulfated polysaccharides, as shown in a recent study.156 The studies mentioned earlier clearly established the bioactive functions of seaweed fucoidans. Some potential biological activities of fucoidans are summarized in Table 10.10.141,144,156

TABLE 10.10 Some Biological Activities of Fucoidans Anticoagulant Inhibits microvascular thrombus formation Antitumor Immunomodulating Hypoglycemic Hypolipidemic Antiinflammatory Inhibition of cell invasion by retroviruses such as HIV, HSV, and African swine fever virus Ameliorates chronic renal failure Source: Adapted from Muffler, K. and Ulber, R., Adv. Biochem. Eng./Biotechnol., 96, 85, 2005; Shanmugam, M. and Mody, K. H., Curr. Sci., 79, 1672, 2000; Veena, C. K. et al., Food Chem., 101, 1552, 2007.

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10.7 LAMINARIN Laminarin is a polysaccharide, which is present in L. japonica, a marine brown alga. The compound can be isolated by hot water extraction, ultrafiltration, and gel chromatography. The molecular weights of laminarins range between 5 and 10 kDa. Hydrolysis of laminarin with endo-β-(1–>3)-glucanase from Bacillus circulans gives di- and penta-oligosaccharides (laminarin oligosaccharides [LO]). Treatment of mouse thymocytes with laminarin or LO suppressed apoptotic death and extended survival of cells. A mouse complementary deoxyribonucleic acid (cDNA) microarray showing the genes coding for immune response proteins were induced and apoptotic cell death proteins were reduced significantly by LO. The results suggest that LO and polysaccharides can be utilized to develop new immunopotentiating substances and functional alternative medicines.156

10.8 COMMERCIAL STATUS Currently, most agar is produced principally from two types of red seaweed: Gelidium and Gracilaria, the former Gelidium species giving the higher-quality product. Alginate production is by extraction from brown seaweeds, most of which are harvested from the wild. Most raw materials for carrageenan production, which were originally dependent on wild seaweeds, especially C. crispus, comes from two species cultivated in the Philippines, namely, Kappaphycus alvarezii and E. denticulatum. The current seaweed industry provides a wide variety of products that have an estimated total annual production value of U.S.$6 billion. Food and pharmaceutical products are worth about U.S.$5 billion. The global industry uses 7.5–8 million tons of wet seaweed annually, harvested either from wild or cultivated seaweeds. After nori and other edible seaweeds, the most significant commercial use of seaweeds is for the extraction of agars, alginates, and carrageenans. These polysaccharides, which are being increasingly used in foods, pharmaceuticals, and biotechnology and also as coatings, adhesives, and feedstocks have a global market value of ca. $500 million. Carrageenan is the most important hydrocolloid, the market for which has grown exponentially at 5%/year for the past 25 years, from 5,500 t in 1970 to over 20,000 t in 1995. Current global annual production of carrageenan is around 50,000 t consisting almost equal amounts of refined and semirefined carrageenans, whereas production of alginate is around 39,000 t. The United States is the main importer of seaweed hydrocolloid products, importing worth U.S.$188 million in 2006, whereas the European Union import carrageenan around 27,000 t. The market size of nori, hizikia, and wakame seaweeds in Japan is around 200,000 t.25 Large firms manufacturing carrageenan exist in Europe and the United States, but the manufacturers are depending on the Philippines and Chile, where red seaweeds are available in abundance.157 A recent survey of supermarkets, health food stores, and food manufacturers identified the availability of 198 carrageenan-containing food products. A questionnaire was developed and used to collect demographic and food frequency data. These data revealed that most consumers were over 30-year-olds, collegeeducated, female subjects.130 Fucoidans are now being marketed as a nutraceutical, a supposed “miracle drug,” and a food supplement because of its significant biological functions.

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In conclusion, the data presented in this chapter suggests potential for expansion of the seaweed hydrocolloid industry for food as well as other purposes. Progress in this field can undoubtedly come through the integration of marine sciences with biotechnology and other upcoming technologies and can lead to generation of new adhesives, pharmaceuticals, drag reducers, and foods from the sea.158 Recent research into the biomedical potential of seaweeds has opened a new vista of opportunities to remodel the treatment regime in various pathologies, many of which have been discussed in this chapter.

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66. Zhao, M. M. et al., Mechanisms of interaction in a protein–carrageenan system, Food Ferm. Ind., 23, 1, 1997. 67. Ibanoglu, E., Effect of hydrocolloids on the thermal denaturation of proteins, Food Chem., 90, 621, 2005. 68. Galazka, V. B. et al., Complexes of bovine serum albumin with sulphated polysaccharides: effects of pH, ionic strength and high pressure treatment, Food Chem., 64, 303, 1999. 69. Baeza, R. I. et al., κ-Carrageenan-protein interactions: effect of proteins on polysaccharide gelling and textural properties, Lebensm. Wiss. Technol., 35, 741, 2002. 70. Spagnuolo, P. A. et al., κ-Carrageenan interactions in systems containing casein micelles and polysaccharide stabilizers, Food Hydrocoll., 19, 371, 2005. 71. White, R., Reducing sugars/carbohydrates in food applications, Food Ingred. Anal. Int., 26, 6, 2004. 72. Tilly, G., Aubree, E., and Houdoux, A., Hydrocolloids to replace gelatine, Food Ingred. Anal. Int., 24, 10, 2002. 73. Totosaus, A., Guerrero, I., and Montejano, J. G., Effect of added salt on textural properties of heat-induced gels made from gum–protein mixtures, J. Texture Stud., 36, 78, 2005. 74. Uruakpa, F. O. and Arntfield, S. D., Surface hydrophobicity of commercial canola proteins mixed with κ-carrageenan or guar gum, Food Chem., 95, 255, 2006. 75. Xiong, Y. L., Noel, D. C., and Moody, W. G., Textural and sensory properties of low-fat beef sausages with added water and polysaccharides as affected by pH and salt, J. Food Sci., 64, 550, 1999. 76. Verbeken, D., et al., Influence of κ-carrageenan on the thermal gelation of salt-soluble meat proteins, Meat Sci., 70, 161, 2005. 77. Balber, F., Cooked ham manufacturing: from past to present, Asia Pac. Food Ind., 16, 46, 2004. 78. Daigle, S. P. et al., PSE-like turkey breast enhancement through adjunct incorporation in a chunked and formed deli roll, Meat Sci., 69, 319, 2005. 79. Lii, C.-yi. et al., Electrosynthesis of κ-carrageenan-ovalbumin complexes, Int. J. Food Sci. Technol., 38, 787, 2003. 80. Soumya, R. and Ryan, A. L., Method of preparing food products with carrageenan, US Patent 6663910B2, 2003. 81. Ntero, P. and Perez-Mateos, M., Effects of Na+, K+ and Ca2+ on gels formed from fish mince containing a carrageenan or alginate, Food Hydrocoll., 16, 375, 2002. 82. Llanto, M. G. et al., Effects of carrageenan on gelling potential of surimi prepared from Atlantic pollock, in Atlantic Fisheries Technological Conference, Voigt, M. N. and Botta, J. R., Eds., St. John’s, Newfoundland, Aug 27–Sep 1, 1989. 83. Carp, D. J. et al., Impact of proteins–κ-carrageenan interactions on foam properties, LWT Food Sci. Technol., 37, 573, 2004. 84. Philips, G. O. and Williams, P. A., Eds., Handbook of Hydrocolloids, Woodward Publishing, Cambridge, 2000. 85. Wang, B., Xiong, Y. L., and Wang, C., Physicochemical and sensory characteristics of flavoured soymilk during refrigeration storage, J. Food Qual., 24, 513, 2001. 86. Ward, F. M. and Andon, S. A., Hydrocolloids as film formers, adhesives, and gelling agents for bakery and cereal products, Cereal Food World, 47, 52, 2002. 87. Totosaus, A. et al., Fat and sodium chloride reduction in sausages using κ-carrageenan and other salts, Int. J. Food Sci. Nutr., 55, 371, 2004. 88. Trius, A. and Sebranek, J. G., Carrageenans and their use in meat product, Crit. Rev. Food Sci. Nutr., 36, 69, 1996. 89. Candogan, K. and Kolsarici, N., The effects of carrageenan and pectin on some quality characteristics of low-fat beef frankfurters, Meat Sci., 64, 199, 2003.

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90. Tong, C. B. S. and Hicks, K. B., Sulfated polysaccharides inhibit browning of apple juice and diced apples, J. Agr. Food Chem., 39, 1719, 1991. 91. Sharma, B. D., The storage stability and textural, physico-chemical and sensory quality of low-fat ground pork patties with carrageenan as fat replacer, Int. J. Food Sci. Technol., 39, 31, 2004. 92. Ulu, H., Effects of carrageenan and guar gum on the cooking and textual properties of low fat meat balls, Food Chem., 95, 600, 2006. 93. Amako, D. E. N. and Xiong, Y. L., Effects of carrageenan on thermal stability of proteins from chicken thigh and breast muscles, Food Res. Int., 34, 247, 2001. 94. Venugopal, V., Seafood Processing: Adding Value through Quick Freezing, Retortable Packaging and Cook-chilling, CRC Press, Boca Raton, FL, 2006, Ch. 10. 95. Annapure, U. S., Singhal, R. S., and Kulkarni, P. R., Screening of hydrocolloids for reduction in oil uptake of a model deep fat fried product, Fett-Lipid (Germany), 101, 217, 1999. 96. Bylaite, E. et al., Influence of λ-carrageenan on the release of systematic series of volatile flavor compounds from viscous food model systems, J. Agr. Food Chem., 52, 3542, 2004. 97. Juteau, A. et al., Flavour release from polysaccharide gels: different approaches for the determination of kinetic parameters, Trends Food Sci. Technol., 15, 394, 2004. 98. Borderý´as, A. L., Sa´nchez-Alonso, I., and Pe´rez-Mateos, M., New applications of fibres in foods: addition to fishery products, Trends Food Sci. Technol., 16, 458, 2006. 99. Lian, P. Z., Lee, C. M., and Hufnagel, L., Physicochemical properties of frozen red hake (Urophycis chuss) mince as affected by cryoprotective ingredients, J. Food Sci., 65, 1117, 2000. 100. Camacho, M. M., Martinez, N. N., and Chiralt, A., Stability of whipped dairy creams containing locust bean gum/λ-carrageenan mixtures during freezing-thawing processes, Food Res. Int., 34, 887, 2001. 101. Cabello-Pasini, A. et al., Clarification of wines using polysaccharides extracted from seaweeds, Am. J. Enol. Viticuli., 56, 52, 2005. 102. Wang, J. C., Application of carrageenan to wine brewing, Food Sci. China, 20, 37, 1999. 103. Gurkin, S., Hydrocolloids—ingredients that add flexibility to tortilla processing, Cereal Food World, 47, 41, 2003. 104. Maga, J. A. and Kapojuwo, G. A., Extrusion cooking of corn grits containing various levels of hydrocolloids, J. Food Technol., 21, 61, 1986. 105. Pascual, F. P. and Sumalangcay, A., Gum arabic, carrageenan and various types and sago palm starch as binders in prawn diets, South East Asian Dev. Center, Quart. Res. Rep., 5, 11, 1981. 106. CFR 2001, Code of Federal Regulations, National Archives and Records Administration, US Government Printing Office, Washington, DC, 2001. 107. Seguer-Bonaventura, J. et al., New preservatives and protective systems, Int. Patent Appl. No. WO03/094638A1, Spain, 2003. 108. Medina, M. B., Binding interaction studies of the immobilized Salmonella typhimurium with extracellular matrix and muscle proteins, and polysaccharides, Int. J. Food Microbiol., 93, 63, 2004. 109. Hoffman, R., Carrageenans inhibit growth-factor binding, Biochem. J., 289, 331, 1993. 110. Humano, Y. et al., Preparation and in vitro antioxidant activity of κ-carrageenan oligosaccharides and their oversulfated, acetylated, and phosphorylated derivatives, Carbohyd. Res., 340, 685, 2005. 111. Xue, C. et al., Antioxidative activities of several marine polysaccharides evaluated in a phosphatidylcholine–liposomal suspension and organic solvents, Biosci. Biotechnol. Biochem., 62, 206, 1998.

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112. Thomson, A. W. and Fowler, E. F., Carrageenan: a review of its effects on the immune system, Agents Actions, 11, 265, 1995. 113. Konishi, S. et al., Effects of carrageenans on the binding, phagocytotic, and killing abilities of macrophages to Salmonella, Biosci. Biotechnol. Biochem., 67, 1425, 2003. 114. Rao, D. V. and Rao, B., Drugs from marine algae-current status, in Proc. Symp. Seaweeds—2004, Seaweed Research and Utilization Association and Central Marine Fisheries Research Institute, Cochin, 2004, p. 54. 115. Baba, M. et al., Sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stromatitis virus, and human immunodeficiency virus, Antimicrob. Agents Chemother., 32, 1742, 1988. 116. Buck, C. B. et al., Carrageenan Is a Potent Inhibitor of Papillomavirus Infection, PLoS Pathog 2(7): e69 doi:10.1371/journal.ppat.0020069 (wikipedia), accessed September 2007. 117. Haijin, M., Xiaolu, J., and Huashi, G., A kappa-carrageenan derived oligosaccharide prepared by enzymatic degradation containing anti-tumor activity, J. Appl. Phycol., 15, 297, 2003. 118. Canete, S. J. P. and Montano, M. N. E., κ-Carrageenan gel as agent to sequester paralytic shellfish poison, Mar. Biotechnol., 4, 565, 2002. 119. Mercier, L. et al., The algal polysaccharide carrageenans can act as an elicitor of plant defence, New Phytol., 149, 43, 2001. 120. Pilkington, H. et al., κ-Carrageenan gel immobilization of lager brewing yeast, J. Inst. Brewing, 105, 398, 1999. 121. Guiseley, K. B., Chemical and physical properties of algal polysaccharides used for cell immobilization, Environ. Microbiol. Technol., 11, 706, 1989. 122. Muyima, N. Y. O., Zamxaka, M., and Mazomba, N. T., Comparative evaluation of pectolytic and proteolytic enzyme production by free and immobilized cells of some strains of the phytopathogenic Erwinia chrysanthemi, J. Ind. Microbiol. Biotechnol., 27, 215, 2001. 123. Yang, C. C., Chen, C. C., and Chang, H. M., Separation of egg white lysozyme by anionic polysaccharides, J. Food Sci., 63, 962, 1998. 124. Koketsu, M., Clarification of egg yolk suspension for the production of n-acetylneuraminic acid, J. Food Eng., 22, 359, 1999. 125. Roy, I. and Gupta, M. N., κ-Carrageenan as a new smart macroaffinity ligand for the purification of pullulanase, J. Chromatogr. A, 998, 103, 2003. 126. Cohen, S. M. and Ito, N., A critical review of the toxicological effects of carrageenan and processed Eucheuma seaweed on the gastrointestinal tract, Crit. Rev. Toxicol., 32, 413, 2002. 127. Tobacman, J. K., Review of harmful gastrointestinal effects of carrageenan in animal experiments, Environ. Health Perspec., 109, 983, 2001. 128. Michel, C. and Farlane, G. T., Digestive fates of soluble polysaccharides from marine macroalgae: involvement of the colonic microflora and physiological consequences for the host, J. Appl. Bacteriol., 80, 349, 1996. 129. Pittman, K. A., Golberg, L., and Coulston, F., Carrageenan: the effect of molecular weight and polymer type on its uptake, excretion and degradation in animals, Food Cosmet. Toxicol., 14, 85, 1976. 130. Shah, Z. C. and Huffman, F. G., Current availability of consumption of carrageenan containing foods, Ecol. Food Nutr., 42, 357, 2003. 131. Scientific Committee, Food, European Commission, SCF/CS/ADD/EMU/100/Final, February 21, 2003. 132. Sixty-Eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), Geneva, June 19–28, 2007.

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133. FAO, State of World Fisheries and Aquaculture, Food and Agriculture Organization of the United Nations, Rome, Italy, 2004, http://www.fao.org//docrep/007/y5600e/ y5600e07.htm#P10_2114, accessed July 2008. 134. Sarwar, G., Sakata, T., and Kakimoto, D., Isolation and characterization of carrageenandecomposing bacteria, J. Gen. Appl. Microbiol., 29, 145, 1983. 135. Araki, T., Higashimoto, Y., and Morishita, T., Purification and charaterization of κ-carrageenase from a marine bacterium, Vibrio sp. CA-1004, Fish. Sci., 65, 937, 1999. 136. Potin, P. et al., Purification and characterization of a new κ-carrageenase from a marine Cytophaga-like bacterium, Eur. J. Biochem., 201, 247, 1991. 137. Bellion, C., Hamer, G. K., and Yaphe, W., The degradation of Eucheuma spinosum and Eucheuma cottonii carrageenans by ί-carrageenases and κ-carrageenases from marine bacteria, Can. J. Microbiol., 28, 874, 1982. 138. Kuntsen, S. H. et al., A rapid method for the separation and analysis of carrageenan oligosaccharides released by ι- and κ-carrageenase, Carbohyd. Res., 33, 101, 2001. 139. Antonopoulos, A. et al., Characterisation of ι-carrageenans oligosaccharides with highperformance liquid chromatography coupled with evaporative light scattering detection, J. Chromatogr., 1059, 83, 2004. 140. Joo, D. S. and Cho, Y. S., Preparation of carrageenan hydrolysates from carrageenan with organic acid, J. Korean Soc. Food Sci. Nutr., 32, 42, 2003. 141. Muffler, K. and Ulber, R., Downstream processing in marine biotechnology, Adv. Biochem. Eng./Biotechnol., 96, 85, 2005. 142. Bilan, M. I. et al., Structure of a fucoidan from the brown seaweed Fucus serratus L., Carbohydr. Res., 341, 238, 2006. 143. Takedo, M., Nakada, T., and Hongo, F., Chemical characterization of fucoidan from commercially cultured Nemacystus deciphens, Biosci. Biotechnol. Biochem., 63, 1813, 1999. 144. Shanmugam, M. and Mody, K. H., Heparinoid-active sulphated polysaccharides from marine algae as potential blood anti-coagulants, Curr. Sci., 79, 1672, 2000. 145. Colliec, S. et al., Anticoagulant properties of a fucoidan fraction, Thromb. Res., 64, 143, 2001. 146. Ponce, N. M. A. et al., Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies, Carbohyd. Res., 338, 153, 2003. 147. Religa, P. et al., Fucoidan inhibits smooth muscle cell proliferation and reduces mitogenactivated protein kinase activity, Eur. J. Vasc. Endovasc. Surg., 20, 419, 2000. 148. Graufel, V. et al., New natural polysaccharides with potent antithrombic activity: fucans from brown algae, Biomaterials, 10, 363, 1989. 149. Aisa, Y. et al., Fucoidan induces apoptosis of human HS-sultan cells accompanied by activation of caspase-3 and down-regulation of ERK pathways. Am. J. Hematol., 78, 7, 2005. 150. Thorlacius, V. et al., The polysaccharide fucoidan inhibits microvascular thrombus formation independently from P- and L-selectin function in vivo, Eur. J. Clin. Invest., 30, 804, 2000. 151. Shanmugham, M. et al., Distribution of heparinoid-active sulfated polysaccharides in some Indian marine green algae, Ind. J. Mar. Sci., 30, 222, 2001. 152. Varenne, A. et al., Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin, Anal. Biochem., 315, 152, 2003. 153. Mao, W., Heparinoid-active sulfated polysaccharides from marine green algae, Paper presented No. MEDI, 391, 233rd ACS National Meeting, Chicago, IL, March 25–29, 2007. 154. Maruyama, H. and Yamamoto, I., An antitumour fucioidan from an edible seaweed, Laminaria religiosa, Hydrobiologia, 116, 534, 1984.

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155. Noda, H. et al., 1989. Antitumour activity of polysaccharides and lipids from marine algae, Nippon Suisan Gakkaishi, 55, 1265, 1989. 156. Veena, C. K. et al., Antioxidant activity of sulfated polysaccharide: beneficial role of sulfated polysaccharides from edible seaweed Fucus vesiculosus in experimental hyperoxaluria, Food Chem., 101, 1552, 2007. 157. Kim, K. H. et al., Anti-apoptotic activity of laminarin polysaccharides and their enzymatically hydrolyzed oligosaccharides from Laminaria japonica, Biotechnol. Lett., 28, 439, 2006. 158. Ronald, M. W. et al., Applications of biotechnology to the production, recovery and use of marine polysaccharides, Nature Biotechnol., 3, 899, 1985.

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Microalgae, 11 Marine Other Microorganisms, and Corals 11.1

INTRODUCTION

Basic features of marine microalgae and other marine microorganisms as well as corals in the marine environment have been discussed in Chapter 2. In view of the symbiotic associations among many of these organisms, it is apt to discuss marine microalgae, bacteria, and corals in one chapter. The importance of these organisms with respect to their functions as food and bioactive compounds is discussed in this chapter. Although corals will be covered in this chapter, their potential role as sources of drugs and other pharmaceutical products will be highlighted in Chapter 12.

11.2

MARINE MICROALGAE

Marine microalgae play an important role in protecting the environment by absorbing carbon dioxide through their photosynthetic activity, which is estimated to produce about 50–90% of global oxygen through photosynthesis. The sea takes up nearly half of the CO2 that is being emitted by fossil fuels since the beginning of the industrial revolution. The gas dissolves into surface waters and is used by the photoplankton for photosynthesis. Microalgal photosynthesis is efficient in fixing CO2 from both atmosphere and gases discharged from industries, and is a possible future alternative for CO2 reduction. When the plankton dies and eventually sinks to the ocean floor, it results in storage of huge amounts of carbon for future. According to a new survey, the biological activity in the twilight zone of the ocean determines whether captured carbon is stored for millennia or quickly recycled to the surface.1 Nevertheless, the photosynthetic activity exerts important influence on climate, removing nearly as much greenhouse gas, CO2 from the atmosphere like all terrestrial plants, and supplying about half of the O2 required for the human population. This, in turn, helps to maintain a balanced ecosystem, essential for all living systems and a healthy planet. If the amount of CO2 produced (due to vehicular pollution, burning of fossils, and related industrial activities) is more than the sea can absorb, it may result in global warming. Recent satellite observations and extensive oceanographic research have revealed the potential of these organisms to resist changes in global temperatures, ocean circulation, and nutrient availability. The fundamental characteristics of marine microalgae and their biotechnological roles in influencing worldwide issues have been discussed recently.2,3

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NUTRITIONAL COMPOSITION

From a nutritional point of view, they are significantly rich in many nutrients, which are also bioavailable, as compared to many vegetable and muscle foods that are absorbed in lesser quantities by the body. The unique metabolic capabilities of these nutrients offer potential for novel and promising natural products, which can fight various diseases providing support to human life. Some of the important microalgae, which have been well studied in this respect, include species of Chlorella, Spirulina, and Dunaliella, whereas other species are also being examined of late. Chlorella is a genus of single-celled green algae, belonging to the phylum Chlorophyta. It is spherical, about 2–10 μm in diameter. The alga contains green photosynthetic pigments, chlorophyll a and b, in its chloroplasts. It multiplies rapidly through photosynthesis, requiring only CO2, water, sunlight, and minerals for growth. Chlorella could serve as a potential source of food and energy because of its high photosynthetic activity. It is also an attractive food source because of its high content of proteins and other essential nutrients. Spirulina is a unicellular, photosynthetic blue-green algae (also known as cyanobacteria). It is a thallophyte (meaning having no clear distinction into leaf, stem, and root). Spirulina has no cellulose in its cell walls, which are composed of soft mucopolysaccharides. The alga is microscopic in nature, and occurs naturally in warm, alkaline, salty, brackish lakes. Its color is derived from the green pigment, namely, chlorophyll and from the blue color of a protein called phycocyanin. There are two commonly occurring Spirulina spp., which are used as human and animal food supplements, namely, Spirulina maxima and S. platensis. The genus Dunaliella includes halotolerant, unicellular, motile, green algae with exceptional morphological and physiological properties belonging to the family Chlorophyceae. It grows in high salt concentration (1.5 M NaCl). The alga is devoid of rigid cell wall and contains a single, large cup-shaped chloroplast. Marine microalgae are being hailed as the new “super food.”4 They are useful from a nutritional point of view, as compared to most vegetable and muscle foods, which makes them occupy a central position within marine food webs.5 They are rich in proteins, lipids, and essential minerals, and hence form the food to oceanic creatures helping them lead an active and reproductive life. Because of their importance as food, the nutritional compositions of marine microalgae have been examined. Comparison of data of protein content in algae is, however, difficult, primarily due to differences in the analytical methods employed. Recently, methodologies for protein extractions from different micro and macroalgae have been standardized with a view to evolve a common accurate method for determining their protein quality.6 In a detailed study, the marine microalga, Nannochloropsis spp. was analyzed for proximate composition including moisture, ash, crude protein, available carbohydrates, fiber, lipids, and energy, nitrate, nucleic acid, mineral element (Na, K, Ca, Mg, Fe, Cu, Zn, Mn, Pb, Cd, Cr, Ni, Co, and S), fatty acid, and pigment (carotenoids and chlorophyll) concentrations. On an average, the biomass contained 37.6% (w/w) available carbohydrates, 28.8% crude protein, and 18.4% total lipids. Mineral contents in 100 g of dry biomass were as follows: Ca (972 mg), K (533 mg), Na (659 mg), Mg (316 mg), Zn (103 mg), Fe (136 mg), Mn (3.4 mg), Cu (35.0 mg), Ni (0.22 mg), and Co (<0.1 mg). Toxic heavy metal contents (Cd and Pb) were negligible. The fatty acid composition was as follows: fatty acid of type, 14:0, 0.6%; 16:0, 5.0%; 16:1ω7,

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4.7%; 18:1ω9, 3.8%; 18:2ω6, 0.4%; 20:4ω6, 0.7%, and 20:5ω3, 2.2%. Under cultivation conditions, nutrient composition of the biomass was highly influenced by residence time in the photo bioreactor. The biomass harvested for short residence times was richer in protein and eicosapentaenoic acid (EPA) than biomass harvested for high residence time.7 The amino acid and gross compositions of three benthic diatoms and three chain-forming diatoms were determined during late-logarithmic growth phase. Four of the six species were rich in protein, ranging from 31 to 38%, and contained 4.9–6.5% carbohydrate, and 9.4–18% ash, on a dry weight basis. However, the other two species contained only 16% protein but had nearly twice the total carbohydrate (11–12%) and two to three times the ash (29–35%) of the other species. The protein quality based on total amino acid composition was high. All species contained 18–20% lipids. Microalgae are rich sources of lipids. The fatty acid compositions of microalgae have been shown to change in response to salinity. High levels of docosahexaenoic acid (DHA) have been detected in the lipids of dinoflagellates, together with significant amounts of EPA and other polyunsaturated fatty acids (PUFA). Dinoflagellates are capable of synthesizing DHA through the chain shortening of C24:6 (n-3). The lipids of dinoflagellates isolated from corals from a fringing reef in Japan have been studied. DHA up to 18% was found as major PUFA in polar lipids.8 An artificial medium for diatom has been developed, which is capable of maintaining long-term cultures of Haslea ostrearia and several other planktonic microalgae, and to allow physiological studies related to metal metabolism. The medium provided sufficient nutrients to allow H. ostrearia to grow as efficiently as in the enriched seawater medium, without negative impact on metabolism. The medium was capable of supporting growth of another 19 microalgae.9 Relative importance of benthic microalgae, phytoplankton, and mangroves as sources of nutrition for penaeid prawns and other coastal invertebrates has been studied. Stable isotope analyses suggested that benthic microalgae constitute major dietary component for prawns living in tidal creeks. Carbon, sulfur, and nitrogen stable isotope ratio techniques were used to evaluate the relative importance of algae in the nutrition of two penaeid prawn species on the west coast of Peninsular Malaysia. Mangrove detritus was found to contribute to the nutrition of juvenile prawns belonging to Penaeus merguiensis living within tidal creeks, but not to adult P. merguiensis, and juvenile and adult Parapenaeopsis sculptilis captured offshore. Results from radiotracer feeding studies with mangrove lignocelluloses as the food source indicated that juvenile P. merguiensis from tidal creeks assimilated mangrove carbon with an efficiency of 13.4%, which was significantly higher than that of adult P. merguiensis, which showed an efficiency of 2.1%.5

11.3 11.3.1

MAJOR COMPONENTS FROM MICROALGAE LIPIDS

Next to fatty fish such as cod, herring, and tuna (see Chapter 5), microalgae are the alternate source of PUFA. Fish derive much of the lipids including omega-3 fatty acids from planktonic organisms, where these algae serve as food. However, the limited availability and the increasing demand for fish have resulted in recognizing

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the importance of microalgae as raw material for isolation of PUFA. In comparison with fish oils, lipids from microalgae show a less complex fatty acid profile and have no malodor. Several microalgae have been screened for fatty acid profiles, with an aim to cultivate them for large-scale production of these lipids. Total lipid and total fatty acid composition were studied in 12 marine microalgae, belonging to the families Eustigmatophyceae, Chlorophyceae, Haptophyceae, and Prasinophyceae, cultured under comparable conditions. Species of the same class showed a particular fatty acid composition, indicating specific fatty acid bioconversion modes. Nannochloropsis spp. belonging to Eustigmatophyceae had the highest total lipid (10.3–16.1% of dry weight), 20:4n–6 (1.9–3.9% by weight of total fatty acids) and 20:5n–3 contents (12.1–17.8%, as wt% of total fatty acids). The abundance of highly unsaturated fatty acids (HUFA) is of interest to larval nutrition in marine aquaculture. All other species analyzed, either lacked or had low contents of long-chain HUFA, except Isochrysis galbana (Haptophyceae), which was high in 22:6n–3 (6.9%). Nannochloris and Chlorella species (Chlorophyceae) showed high proportions of short-chain PUFA, with 16:2n–6 (3.5–4.9%), 16:3n–3 (8.6–15.2%), 18:2n–6 (10.5–25.6%), and 18:3n–3 (15.3–24.3%) as major fatty acids, but low levels of HUFA such as 20:5n–3 or 22:6n–3.10 Most diatoms have a high content of EPA. The Pinguiphyceae consists of five marine unicellular algal species, which have an unusually high content of PUFA, especially EPA, ranging from 23 to 56% of the total fatty acids. They also possess other fatty acids such as DHA and docosa tetraenoic acid, C22:4,(n−3) up to 10% of total fatty acids. The high oil content together with their lack of cell wall makes them good candidates for feed. Diatoms are also used for the production of PUFA. Recent advances in heterotrophic production of EPA by microalgae have been reviewed.11 Certain species of thraustochytrids are explored as potential producer of PUFA for nutritional enrichment of food products, and used as feed additives in aquaculture. The fatty acid composition and squalene content were determined in the thraustochytrid, Schizochytrium mangrovei that was newly isolated from decaying Kandelia candel leaves in Hong Kong mangrove habitat. The major fatty acid constituents identified in all the three Schizochytrium mangrovei strains were tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), docosapentaenoic acid (2 DPA; C22:5,n−6), and DHA (C22:6,n−3). DHA was the most predominant PUFA, and the percentage of DHA (of total fatty acids) in all these strains varied from 32.3 to 39.1%. Only slight changes were observed in fatty acid composition of the Schizochytrium mangrovei strains harvested at their early (day 3) and late stationary (day 5) phases. In contrast, the cellular squalene content was affected significantly by the culture time; a significant decrease of squalene content from 0.162 to 0.035 mg/g was found in Schizochytrium mangrovei as the culture aged.12 Some of the new compounds identified in dinoflagellates include C28 PUFA.13 C37–C40 unsaturated ketones (alkenones) have been found in species of haptophytes consisting of the widely distributed coccolithophores, Emiliania huxleyi and Gephyrocapsa oceanica and noncoccolith-bearing Isochrysis spp. These compounds are straight chain in nature and contain 2–4 double bonds.14 These studies have indicated that microalgae contain novel fatty acids, making them well suited for pharmaceutical applications.15 Aspects of cultivation of microalgae for PUFA have been discussed in Section 12.5.

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CAROTENOIDS AND OTHER PIGMENTS

Many species of microalgae are excellent sources of chlorophylls, carotenoids, and phycobiliproteins. The phycobiliproteins are water-soluble pigments consisting of red-colored phycoerythrin and blue-colored phycocyanin and allophycocyanin. Studies extending over two decades have created an extensive database of the characteristic pigment compositions of different algal classes.16 Most cryophytes possess various colored plastids with chlorophylls, carotenoids, and phycobiliprotein. Alloxanthin is a xanthophyll that is unique to cryptomonads. Cells of chlorophytes are green due to chlorophyll a and b, the same predominant photosynthetic pigments as those of land plants. Some have yellowish green and red-green color due to β-carotene, astaxanthin, and others. Most of the plastid-containing phototrophic dinoflagellates contain chlorophylls a and c2, and carotenoids such as β-carotene and peridinin, the unique accessory xanthophylls in this phylum.17 Dunaliella salina, a marine species belonging to the family Chlorophyceae, is a good source of carotenoids. The algal extract obtained using combined pressurized liquid extraction with hexane contained all-trans-β-carotene and isomers and other minor carotenoids.18 Dunaliella spp. have been cultivated commercially for food supplements and carotenoid production. The advantages of carotenoid production by Dunaliella are given in Table 11.1. Botryococcus braunii is a green colonial microalga, which is used mainly for the production of carotenoids, hydrocarbons, and polysaccharides. The diatom H. ostrearia that lives in oyster ponds has the distinctive feature of synthesizing “marennine,” a blue-green pigment whose chemical nature remains unknown. This pigment is responsible for the greening of oyster gills. The intra and extracellular pigments have been extracted and characterized. There is a scope for scaling up the process to a larger production system for industrial applications.19 Commercial aspects of production of β-carotene from Dunaliella, astaxanthin from Haematococcus and lutein from chlorophycean strains have been discussed recently.20 The algal pigments have the potential to be used as natural colorants in food, cosmetics, and pharmaceuticals, particularly as substitutes for synthetic dyes.

11.3.3 STEROLS AND HYDROCARBONS Sterols such as 4a-methyl sterols and 5a-stanols are present in dinoflagellates. A different type of highly branched isoprenoid alkene, designated as botryococcene,

TABLE 11.1 Advantages of Carotenoid Production by Dunaliella Easy cultivation High pigment production (3–5% [w/w] on dry weight basis) Have both cis and trans isomers of carotenoids The algal protein is nutritive and can be used as protein supplement The algal biomass can be used as poultry feed

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occurs in green algae of the genus Botryococcus. Similar branched isoprenoid alkenes also occur in the two diatoms H. ostrearia and Rhizosolenia setigera. A new botryococcene incorporating unprecedented ketone functionality was identified in the lipid extract. In more recent research, microalgae from the genus Pavlova (Haptophyta) have been shown to contain novel 3,4-dihydroxy-4a-methyl sterols termed pavlovols. The identification of 4a-methyl sterols and 5a-stanols in the same microalgae has confirmed that such unusual sterols are not unique to dinoflagellates. An extensive database on the diverse sterol compositions of marine microalgae has been established over the past two decades.14,21

11.3.4

POLYSACCHARIDES

The polysaccharide of unicellular rhodophyte, Porphyridium spp. has the potential to be used as a thickening agent and food additive because of its high viscosity over a wide range of pH, temperature, and salinity.16 Recently, significant biological activities have been attributed to the polysaccharide from Gymnodinium spp. Another organism belonging to these genera produces a sulfated polysaccharide that shows antiviral activity against encephalomyocarditis virus.8

11.3.5

VITAMINS

A number of vitamins are present in appreciable concentrations in microalgae compared with conventional foods. Ingestion of relatively small quantities of microalgae can cover the requirements of some vitamins in animal and human nutrition, while supplementing others. Marine microalgae can thus be considered to represent a nonconventional source of vitamins or a vitamin supplement for animal or human nutrition, and therefore, can be used as food supplements or food ingredients.22

11.3.6

SINGLE CELL PROTEINS

The marine microalgae, Tetraselmis suecica, I. galbana, Dunaliella tertiolecta, and C. stigmatophora are useful as single cell protein (SCP), because of their high protein contents, which account for 39–54% of the dry matter, D. tertiolecta has the highest protein content. Lysine values were between 3 and 4.5% of protein, and were higher than those of freshwater species. The total nucleic acid content was <7% of the dry matter; this value being lower than that for yeasts or bacteria, commonly used as SCP sources. Amino acid profiles of the four species were very similar and comparable to the FAO reference protein, but with low contents of methionine and cystine and a high content of lysine.23

11.4

BIOACTIVE COMPOUNDS FROM MICROALGAE

Microalgae constitute a reservoir of natural substances of bioactive functions. Their unique metabolic capabilities offer potentials for novel and promising natural products, which can fight against various diseases and provide support to the human life. Research on microalgae is being carried out not only on physiological aspects,

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but also to isolate useful bioactive compounds. These compounds from microalgae encompass various roles as antioxidants, antimicrobials, antitumor drugs, bioadhesives, antifouling compounds, immunostimulants, and many others.4 The production of these biochemicals from microalgae is based on the exploitation of their relatively efficient photosynthetic machinery.24 Potentials of cyanobacteria as untapped sources of nutraceuticals have been recognized only recently. Furthermore, potentials for genetic modification of microalgae including cyanobacteria and feasibility of mass cultivation under controlled conditions offer molecular tools to enhance yield of bioactive compounds in substantial amounts. The biochemically active compounds from marine cyanobacteria include enzymes, polysaccharides, enzyme inhibitors, herbicides, antimyotics, multidrug resistance reverses, and antimalarial and immunosuppressive agents. Some of the novel compounds isolated from cyanobacteria include plant growth regulators that promote redifferentiation, germination and plantlet formation, tyrosinase inhibitors, UV-absorbing compounds, sulfated polysaccharides showing antihuman immunodeficiency virus (HIV) activity, and novel antibiotics with light-regulated activity. Interest in this field has resulted in cyanobacterial collections for the production of cyanobacteria-derived compounds. The origin of cyanobacterial dolastatins, potent cytotoxic compounds, originally isolated from the Indian Ocean sea hare, Dolabella auricularia has been pointed out. Cyanobacterial metabolites have proven to be invaluable tools in the dissection of signal transduction pathways in mammalian cells, and some are currently under clinical evaluation as drug candidates. It is also now realized that cyanobacteria are the true biosynthetic origin of many bioactive molecules isolated from marine invertebrates, since marine invertebrates may sequester cyanobacteria through diet or symbiosis.25 A plasmid from the marine cyanobacterium, Synechococcus spp., whose copy number is dependent on salinity, has been characterized. This plasmid is being used to develop a stable and controllable gene expression system.26 The recent reviews summarizing the developments in the field indicate interests in the field.3,27–32 The major bioactive compounds from various microalgae are briefly summarized in the following sections.

11.4.1

ANTIVIRAL COMPOUNDS

Anti-HIV activity of extracts and compounds isolated from micro and macroalgae from marine and freshwater sources has been reported. The HIV is a retrovirus that causes the acquired immune deficiency disease syndrome (AIDS). There is an increasing interest in recent years to determine the role of algal biomolecules such as sulfoglycolipids, fucoidan, sesquiterpene, and hydroquinones in anti-HIV activity. Studies using nonsulfated and sulfated homo and heteropolysaccharides isolated from algae have revealed the mechanism of binding of drugs to the virion and viral binding to host cells. Compounds and extracts with anti-HIV activity are also active against other retroviruses such as herpes simplex virus (HSV), but the amount of antiviral activity varies with the compound and the virus.33 Factors active against HSV and other viruses have been isolated from Dunaliella primolecta. The chemical structures of the active substances were identified as pheophorbide-like compounds.34 A potent antifungal, antiviral, and cytotoxic cyclic depsipeptide, Kahalalide F is a promising

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lead compound isolated from Bryopsis spp. An antiviral sphingosine derivative, erythro-sphinga-4,8-dien-N-palmitate was obtained from Ulva fasciata.17

11.4.2

ANTICANCER COMPOUNDS

Marine Cyanophyceae, and to a lesser extent other marine microalgae, is a promising source of anticancer-type natural products. For isolation of these compounds, microscopic forms that do not form mats or tufts in nature are cultured to isolate sufficient samples. Field collections of microalgae in intertidal and shallow subtidal tropical environments utilize hand collection and manipulation techniques into small-volume wide-mouth jars. Acclimation times in the laboratory environment are important for the cultivation of new cultures. Manipulation on agar plates has given the best success rate for obtaining pure cultures. The extracts of the organisms are evaluated in a series of mechanism-based anticancer screens, including protein kinase C, protein tryosine kinase, and inosine monophosphate dehydrogenase. More than 501 extracts were screened, which yielded 23 isolates active against the above enzymes. The extract of a cultured microalga, namely, Poteriochromonas malhamensis has yielded a novel chlorosulfolipid.35 Researchers at the University of Hawaii have isolated novel drugs called cryptophycins from blue-green algae for cancer treatment. The anticarcinogenic activity was examined in mice implanted with cells that cause prostate and breast cancer. The compounds appear to affect the internal structure of cancer cells helping to control the spread of the disease.36

11.4.3

ANTIOXIDANT COMPOUNDS

The all-trans-β-carotene and isomers and other minor carotenoids isolated from D. salina have been recognized as good antioxidants.18 The antioxidant properties of acetone extracts of the microalga, Botryococcus braunii, has been elucidated recently using in vitro model systems involving 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxy radical scavenging, and lipid peroxidation in human low-density lipoprotein (LDL) and rat tissues. The extracts (equivalent to 10 ppm of total carotenoid) exhibited 71 and 67% antioxidant activity in DPPH and hydroxyl radical scavenging model systems, respectively. The extract also showed 72, 71, and 70% antioxidant activity in the liver, brain, and kidney of rats. LDL oxidation induced by Cu2+ ions was prevented by the algal extract in a dose-dependent manner. The concentration of thiobarbituric acid–reactive substances in the blood, liver, and kidney of rats was significantly decreased in B. braunii-treated samples compared with those of control. The B. braunii acetone extract contained the carotenoids, violaxanthin, astaxanthin, lutein, zeaxanthin, and α, β-carotene, lutein representing more than 75% of the total carotenoids. It was suggested that the carotenoids could be exhibiting antioxidant activities.37 Antioxidant activities may vary depending on the season. The presence of symbiotic diatoms in Antarctic sponge Haliclona dancoi is influenced by marked seasonal variations during summer. The role of these diatoms as prooxidant stressors in porifera has been investigated. Both pigments and frustules were absent in sponge tissues sampled in early November at the beginning of the summer and increased from the mid of December with slightly shifted temporal trends.

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The efficiency of antioxidant defenses in the sponge showed a marked response to symbionts with clearly enhanced values corresponding to the peak of diatoms.38 Peroxidase activity was detected in cell-free extracts of strains of three species of the marine microalgae Porphyridium purpureum, Phaeodactylum tricornutum, and D. tertiolecta. Characterization of the thermally labile enzyme suggested that it is a heme-containing peroxidase with a molecular weight of approximately 36,000.39

11.4.4 ANTIMICROBIAL COMPOUNDS The antimicrobial activities of two microalgae species, Tetraselmis chuii and Chlorella minutissima, have been reported. The activities were measured in terms of growth of bacteria associated with the microalgae. A total of 17 and 30 bacterial strains were isolated from C. minutissima and T. chuii, respectively. No presumptive Vibrio strains were observed in any of the samples, as measured by the growth on TCBS agar. A high percentage of gram-positive strains were detected among the bacterial strains isolated. The isolated bacteria were screened in vitro for inhibition against two pathogenic strains. Nine of the 34 strains tested (26%) showed inhibition in vitro against either Photobacterium or Vibrio spp. Incubation of enriched Artemia in cultures of the two microalgae for 30 min resulted in a significant decrease of the bacterial load including Vibrio spp. in Artemia.40

11.4.5

ANTIHYPERTENSIVE PEPTIDES

Chlorella vulgaris and S. platensis contained peptides that inhibited angiotensin I-converting enzyme (ACE) (see Chapter 4). The peptides were separated from the proteolytic digests of two microalgae by ion exchange chromatography and gel filtration. Oral administration of fractions of peptic digest into spontaneously hypertensive rats at 200 mg/kg of the body weight resulted in marked antihypertensive effects. Further separation of the fractions by high performance liquid chromatography furnished the following active peptides: Ile-Val-Val-Glu, Ala-Phe-Leu, Phe-Ala-Leu, Ala-Glu-Leu, and Val-Val-Pro-Pro-Ala from C. vulgaris; Ile-Ala-Glu, Phe-Ala-Leu, Ala-Glu-Leu, Ile-Ala-Pro-Gly, and Val-Ala-Phe from S. platensis. These peptides had varying levels of antihypertensive activities.41

11.4.6

OTHER BIOACTIVE COMPOUNDS

The cyanobacterium, Tychonema spp. produces cyclic hexapeptides, brunsvicamide A, B, and C. Brunsvicamide B and C selectively inhibit the Mycobacterium tuberculosis protein tyrosine phosphatase B, a potential drug target for tuberculosis therapy. Brunsvicamide C contains an N-methylated N′-formylkynurenine moiety, a unique structural motif in cyclic peptides. The new peptides were related to the spongederived mozamides.42 Cyanobacteria of the genus Lyngbua are a rich source of bioactive secondary metabolites including fatty acid amides. A series of biologically active malingamides has been identified from marine cyanobacteria. Malingamide G showed immunosuppressive activity.8 A commercial carbohydrate extract from the green microalga, Chlorella pyrenoidosa, was shown to boost the immune system’s response in the flu vaccine in people aged 50–55 years.43 Chlorella spp. have been

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reported to contain anti-inflammatory and immunosuppressive properties besides having significant amounts of linolenic acid.3 About 60 dinoflagellate species are known to produce cytolytic, hepatotoxic, or neurotoxic compounds, although some marine species are responsible for harmful red tides (see Chapter 15). Chemically interesting and biologically significant secondary metabolites from Amphidinium spp., a genus of symbiotic marine dinoflagellates, were separated from the inner cells of Okinawan marine flatworms. These compounds include a series of cytotoxic macrolides, designated as amphidinolides, and

TABLE 11.2 Some Bioactive Compounds Produced by Microalgae Microalgae Cyanobacteria

Rhodophyta

Chlorophyta

Cryptophyta Heterokontophyta

Dinophyta

Haptophyta Euglenophyta

Bioactive Compounds Enzyme inhibitors such as tyrosinase inhibitor Plant growth regulators UV-absorbing compounds Sulfated polysaccharides having anti-HIV activity Novel antibiotics Herbicides antimalarial compounds Immunosuppressive agents Compounds having cytotoxic activity PUFAs Polysaccharide of rhodophyte Porphyridium spp. used as a thickening agent Polysaccharides have hypocholesterolemic agent (animals) and antiviral activity against animal viruses including Herpes simplex types 1 and 2 and also retroviruses Anticancer compounds Anti-inflammatory activity Immunosuppressive activity A Chlorella spp. contains 10% γ-linolenic (C18:3) acid Significant content of PUFAs and hence used as aquaculture feeds Microalgae belonging to this phylum are good raw materials for production of PUFAs, EPA, and DHA Diatoms as potential antioxidants and domoic aid Used as feed in aquaculture Culturing is difficult and hence difficulties in isolation of bioactive compounds However, DHA is produced by heterotrophically grown Crypthecodinum cohnii Sulfated polysaccharide Gymnodinium spp. Have antiviral activity Used as feed DHA production? Culturing is difficult for most organisms E. gracilis Z. when cultured in photoheterotrophic or photoautotrophic conditions, produces antioxidant vitamins such as β-carotene, vitamin C and E

Source: Adapted from Matsunaga, T. et al., Adv. Biochem. Engn./Biotechnol., 96, 165, 2005; Regoli, F. et al., Mar. Env. Res., 58, 637, 2004; Henkel, J., FDA Consumer magazine, January–February, 1998, U.S. FDA, Washington.

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long-chain polyketides isolated from Amphidinium spp. Their isolation, structure elucidation, synthesis, biosynthesis, and bioactivity have been discussed in a recent review.44 Table 11.2 summarizes major bioactive compounds produced by microalgae.

11.5

CULTIVATION OF MICROALGAE

Commercial cultivation of microalgae offers opportunity to isolate a vast array of products including SCPs, fuels, source of fine chemicals, and bioactive compounds. The latter include carotenoids, vitamins, fatty acids including DHA, EPA, and γ-linolenic acid (GLA), and other compounds. Growing microalgae also facilitates production of hydrogen (through biophotolysis), methane (through anaerobic digestion), ethanol (through yeast or alcohol fermentation), triglycerides (through extraction of lipids), methyl ester fuels (through transesterification of lipids), and liquid hydrocarbons. Selection of species for cultivation depends on required applications. Algae are usually cultivated in open ponds having large amounts of surface area, to be economically viable. In addition, the ponds must be maintained at low organism densities to allow the light to penetrate into the system. However, this creates processing problems since large volumes of water must be processed to recover the small volumes of biomass. Nutrients and light were needed to assist in the growth of the organism through photosynthesis. The open culture systems offer advantages in low construction cost and ease of operation. Some of the open systems include shallow ponds, which may be unstirred or paddle-wheeled, slopping cascade, tubular reactors (helix, plane, or double layered), or laminar reactors. The growth rate and maximum biomass yield of microalgal strains are influenced by cultivation parameters (light, temperature, and pH) and nutritional status (CO2, nitrogen, and phosphate concentrations). Stringent nitrogen limitation conditions are required to stimulate the organisms to produce required compounds such as lipids. However, increasing the density of cultures decreases photon availability to individual cells, because the penetration of light into microalgal cultures is poor, especially at high cell densities, adversely affecting specific growth rates. The development of efficient photobioreactors significantly extends the number of species that can be cultivated under controlled conditions and the range of extractable products, for example, β-carotene, phycocyanin, phycoerythrin, and glycerol.45 Microalgae such as Chlorella, Scenedesmus, Dunaliella, Spirulina, Porphyridium, and Haematococcus spp. have been commercially cultured. D. salina is cultured in large (up to ∼250 ha) shallow open-air ponds, with no artificial mixing. Similarly, Chlorella is grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. More recently, a helical tubular photo bioreactor system has been developed which allows these algae to be grown reliably outdoors at high cell densities in semicontinuous culture. Other closed photo bioreactors such as flat panels are also being developed. The main problem facing commercialization of new microalgae and microalgae products is the need for commercially viable closed culture systems. The high cost of microalgae culture systems is due to the requirement of light and the relatively slow growth rate of the algae.45 The rhodophyte microalgae have not been produced commercially as yet, although their polysaccharides are considered to have commercial potential. The phylum Heterokontophyta is one of the most diverse algal groups with huge

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commercial and biotechnological potentials. They range in size from microscopic single cells to giant kelp averaging several meters. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors, in 20–40 L carboys or in large plastic bags of up to 1000 L. The Haptophyta biomass is used as feed for bivalve mollusks, crustacean larvae, and zooplankton which in turn, is used as feed for fish and crustacean larvae (Botryococcus braunii).3,45–47

11.5.1

CULTIVATION OF MICROALGAE FOR LIPIDS

Several microalgae are rich sources of lipids, particularly PUFA, and are therefore used for commercial cultivation for isolation of lipids. These lipids provide alternate sources since fatty fish species such as cods, which are conventional sources of PUFA face depletion (see Chapter 5). Algal fatty acids are biosynthesized in the chloroplasts comprising the thylakoid membranes, and are chiefly esterified to glycolipids. Carbon is usually stored in these algae as glycolipids except during the exponential growth phase, such as during algal bloom, when carbon fixation through photosynthesis is directed toward growth and cell division. As a result, the proportion of omega-3 PUFA can reach 50% of total lipids. By using conventional stirred-tank fermenters, economically viable quantities of certain microorganisms that are rich in PUFA can be produced. Twelve species of microalgae, isolated from north Australian marine, freshwater and hypersaline environments, were grown under controlled conditions of temperature, pH, photon flux density, and salinity, and analyzed for ash, total protein, water-soluble carbohydrates, total lipids, and fatty acids. Highest levels of the PUFA and EPA were found in the marine diatoms Nitzschia frustulu and Nitzschia closterium (23.1 and 15.2% of total fatty acids, respectively). None of the species studied had levels of DHA greater than 1.1% of total fatty acids. Whereas, the highest total fatty acid concentration of all species in the study was found in the freshwater chlorophyte species, Scenedesmus dimorphus (105 mg/g dry weight), the hypersaline species D. salina had the highest total lipid content of 28.1% on dry weight basis, followed by N. closterium, N. frustulum, and Navicula spp., which had lipid contents in the range of 24.2–27.8% dry weight (Chlamydomonas spp. had the highest protein content of 66.9%, dry weight). N. frustulum was highlighted as a possible useful source of lipids and PUFA in mixed microalgal diets for maricultured organisms used in tropical aquaculture.48 When the algae are cultivated in a photobioreactor the nutritive value of microalgae is influenced by residence time in a photobioreactor. The biomass harvested for short residence times was richer in protein and EPA than biomass harvested for high residence time.7 Three marine microalgal species P. tricornutum, I. galbana, and Porphyridium cruentum were cultured semicontinuously. The percentage of EPA in total fatty acids increased with increasing renewal rates in nitrogen-limited cultures, but for P. tricornutum and I. galbana, a plateau around 20–25% of total fatty acids was reached with renewal rates that were not nitrogenlimiting. The studies indicated that different culture strategies should be adopted for the production of a particular PUFA depending on the microalgal species being used.49 Recently, biotechnological approaches have been made to isolate PUFA from marine microorganisms. The chief advantages of microbial production of PUFA by biotechnological means are that microorganisms produce only a single PUFA rather than the complex mixture yielded from fish or algal oils and hence better consistency

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and purity of the final fatty acid product. Furthermore, there is a scope for applying genetics to microorganisms for improved yield and hence, there is ample scope for reduction of cost of PUFA production.8 Biotechnological applications for dinoflagellates have not been intensively performed, probably because most dinoflagellates cannot be easily cultured. The only exception is the DHA produced by heterotrophically grown Crypthecodinium cohnii. The DHA produced by these cells is used as food supplements such as infant formula. Apart from the microalgae, the marine fungi (Thraustochytridae) are considered to hold particular potential as viable supplier of vegetative PUFA-rich biomass and oils, particularly omega-3 fatty acids. In a recent work, 68 isolates of thraustochytrid from 19 different Atlantic Canadian locations were screened. Some of the isolates had up to 320 mg EPA and DHA per g of the biomass. The strain Thraustochytrium striatum T91-6 produced a maximum of 28 g/L biomass, 31.4% EPA, and 4.6 g/L DHA (w/w of biomass) under optimal fermentation conditions. Furthermore, the strain was found to produce carotenoids such as β-carotene, and xanthophylls such as astaxanthin, zeathanin, and echinenone, suggesting potential of the organism for commercial production of these compounds. In another study, fatty acid composition was determined in three strains of the thraustochytrid, Schizochytrium mangrovei. The major fatty acid constituents identified in all three Schizochytrium mangrovei strains were EPA and DHA in addition to tetradecanoic acid (C14:0), hexadecanoic acid (C16:0). DHA was the most predominant PUFA, the contents of which in all the strains varied from 32 to 39% of total fat. The microalgae were cultured for production of DHA. The culture time had no significant influence on the PUFA, whereas it had influence on the cellular squalene contents.12,50–52 In a recent patented work, new strains of fungi were isolated which are capable of producing high concentrations of PUFA. These fungi were grown in fermentation medium containing nonchloride sodium salts, in particular sodium sulfate. The biomass is useful for incorporation in aquaculture feeds. A food product including either of the fungi as well as a component selected from flax seeds, rapeseeds, and soybean has also been described. The food includes a balance of long and shortchain omega-3 fatty acids.53 In addition to microorganisms, transgenic plants could also be capable of producing polyunsaturated acids.54 Production of PUFA from these organisms involves its cultivation, followed by separation by filtration or centrifugation, followed by ultrasonication, freezing, and grinding or disruption in bead mills for disruption of the cells. The lipids are directly extracted with volatile water-immiscible organic solvents in the wet state. In the following steps, the crude extract is purified using selective methods, such as chromatography and precipitation. It is important that purification steps for PUFA should take into account its high sensitivity to oxidation and therefore protection must be afforded during purification steps and storage of the PUFA (see Chapter 5).

11.5.2

CULTIVATION FOR CAROTENOIDS

In a recent exhaustive review, the most relevant features of microalgal biotechnology related to outdoor cultivation of microalgae for different carotenoids have been discussed. The carotenoids examined are β-carotene from Dunaliella, astaxanthin from Haematococcus, and lutein from chlorophycean strains. The current state of

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production technologies are based on either open-pond systems or closed photo bioreactors, and the potential of scientific and technological advances for improvements in yield and reduction in production costs for carotenoids from microalgae are being identified.20 The heterotrophic production potential of astaxanthin by the green microalga, Chlorella zofingiensis has been reported. The alga showed excellent growth on glucose-supplemented media in batch culture. The maximum specific growth rate and astaxanthin yield were 0.03/h and 10.3 mg/L, respectively, obtained at glucose concentrations of 20 g/L and 50 g/L, respectively. In the absence of light, formation of secondary carotenoids was mostly dependent on the initial carbon and nitrogen balance in the medium. Enhanced biosynthesis of astaxanthin was found in the medium with a high C/N ratio of 180. The light-independent astaxanthin-producing ability of C. zofingiensis suggested that the alga might be potentially employed for commercial production of astaxanthin on a large scale.55 The influence of culture conditions on yield and carotenoid production has been shown in the case of unicellular microalga, D. salina, which maintains physiological balance in the production of the carotenoid. Under stress conditions, such as formation of excessive free radicals, cell division inhibition, nitrogen starvation, high salinity, and temperature, this balance is disturbed and the cells generate additional amounts of the carotene. With nitrogen starvation, β-carotene production was enhanced from 1.65 to 7.05 pg per cell. Maximum carotene (8.28 pg per cell) was obtained when the cells were exposed to high light irradiance (6000 lux) and high temperature (35°C).56 The green microalga Chlorella protothecoides has been shown to produce enhanced quantity of lutein under a combination of nitrogen limitation and high-temperature stress. C. protothecoides was grown heterotrophically in batch mode in a 3.7-L fermenter containing 40 g/L glucose and 3.6 g/L urea, followed by a relatively reduced supply of nitrogen source to establish a nitrogen-limited culture. This resulted in an enhanced lutein production without significantly lowering biomass production. The cellular lutein content was 0.27 mg/g higher than that obtained in the nitrogen-sufficient culture. The improvements were also reflected by maximum lutein yield, lutein productivity, and lutein yield coefficient on glucose. This nitrogen-limited fed-batch culture was scaled up from 3.7 to 30 L, and a three-step cultivation process was developed for the high-yield production of lutein. The maximum cell dry weight concentration (45.8 g/L) achieved in the large fermenter (30 L) was comparable to that in the smaller one (3.7 L). The maintenance of the culture at 32°C, for 84 h resulted in a 19.9% increase in lutein content, but a 13.6% decrease in cell dry weight concentration as compared with the fed-batch culture (30 L).57 The process of isolation of lutein consists of extraction of the crude pigment with dichloromethane from the microalga after saponification, which gave the carotenoid with a purity of 90–98%, and yield of 85–91%.58,59

11.6 11.6.1

SOME SPECIFIC EXAMPLES OF ALGAE CHLORELLA

As mentioned earlier, Chlorella is a single-celled green algae, belonging to the phylum Chlorophyta. A few decades ago, Chlorella had attracted attention as a source of nutraceuticals. Mass production of Chlorella in large artificial circular ponds is being practiced. The nutrient profiles of Chlorella vulgaris has been

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reported.60 Dried Chlorella has about 45% protein, 20% fat, 20% carbohydrate, 5% fiber, and 10% minerals and vitamins.45,60,61 Chlorella is an important source of PUFAs.60 Chlorella is not classified as “generally recognized as safe” (GRAS) by the United States Food and Drug Administration (U.S. FDA). Although theoretically creative and promising, cultivation of Chlorella has faced some problems such as economic inviability and competition from cereals such as soybean, and hence did not capture the benefits of photosynthesis and sunlight as predicted.45,61 A functional food oil, rich in fatty acids and antioxidants, colored with carotenoids extracted from C. vulgaris has been produced recently. The alga was subjected to supercritical CO2 extraction at a pressure of 300 bar, or using acetone containing vegetable oil at room and high temperatures. The recovery of carotenoids was 100% with oil at room temperature for 17 h, 70% with oil at 100°C for 30 min, 69% with supercritical CO2 at 40°C and 300 bar. In supercritical extraction, the degree of crushing strongly influenced the extraction recovery; higher pigment recoveries were obtained with well-crushed biomass.59,60 Biological activities of Chlorella including antimicrobial, antihypertensive, and immunosuppessive activities have been mentioned earlier. In addition, the alga stimulates the activity of T cells and macrophages by increasing the interferon levels, thus enhancing the immune system against bacteria, viruses, chemicals, or foreign proteins. Chlorella has been found to exhibit antitumor properties when fed to mice. Another study found enhanced vascular function in hypertensive rats that were given oral doses of the alga.45

11.6.2

SPIRULINA

S. maxima, S. platensis, and other Spirulina spp. have been now grouped under the genus Arthrospira. S. maxima and S. platensis are therefore named as Arthrospira maxima, and A. platensis, respectively.59,61 Nutritional composition was determined for S. platensis (and also I. galbana). Data include the proximate composition, energy value, mineral elements, and fatty acid composition. Total PUFA, saturated fatty acid (SFA) contents, n-3/n-6 ratios, and EPA/DHA ratios were obtained. Protein content was high in Spirulina samples, whereas Isochrysis had the highest ash content. Spirulina is a rich source of GLA and vitamin K, and Isochrysis, a good source of Ca and Mg. Selinium content of Isochrysis is higher comparable to the other microalgae.60 Phycocyanin, the pigment that gives green color to Spirulina has been produced by cultivation of the microalga. Fresh biomass is suitable for phycocyanin extraction, since drying of the alga by either cross-flow, spray or oven drying, results in ∼50% loss of the pigment. Extraction of fresh Spirulina by freezing and thawing of cells, homogenization using a mortar and pestle in the presence of abrasive material, and homogenization using a blender at 10,000 rpm yielded 19.4 mg phycocyanin per 100 mg dry weight of the alga. Phycocyanin was stable over a pH range of 5–7.5 at 9°C, whereas temperatures beyond 40°C lead to instability. Another pigment, phycocyanobilin was separated from the phycocyanin.62 Spirulina can be produced by several methods, starting from cultivation in lakes, ponds, or earth ditches to sophisticated bioreactors and fermenters. Outdoor cultivation may take place either in open or closed system. Open system shows wide variation in productivity, which is dependent on the type of water, environmental

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temperature, and sunlight radiation. Problems of propagation of mixed culture populations and frequent contaminations by bacteria, fungi, protozoa, etc., hamper the production and affect the quality of the microalgae. In India, large-scale cultivation of S. platensis was initiated by Indo-German cooperation, with the aim of utilizing the microalga as animal feed and nutritional supplement.63 The alga was cultured in large shallow open-air ponds with no artificial mixing, or using paddlewheel-mixed ponds or circular ponds. Cultivation was carried out in a high salt and bicarbonate/carbonate (alkaline pH of ∼10) medium under light irradiation and aeration to help optimal photosynthesis by the alga. Batch tests performed at 25°C in open tanks suggested that inorganic carbon is preferentially assimilated in the form of bicarbonate and that its utilization efficiency depended on pH. For the rapid growth of cells, CO2 is often injected into the water. The efficiency of photosynthesis and utilization of CO2 reach maximum values of 6 and 38%, after 4 and 7 days, respectively. Fed-batch tests performed on CO2 showed production of biomass at 1.5 g/L under psuedo-steady-state conditions at a feeding rate of 0.25 L/day. Instead of mineral medium, the possibility of growing the alga on biogas slurry, swine waste water, cattle waste, or water from aquaculture ponds with proper supplementation of nutrients including CO2 and minerals, suggests possibility of recycling waste water for economic benefits. However, Spirulina grown in water contaminated with heavy metals can concentrate these toxic substances. In addition, infectious organisms may also be present and contaminate harvested algae. Therefore, the microalgae used for human and animal consumption should be from good quality water. Spirulina grows relatively slowly, with an output of about 5 g dry mass per m2 per day, in a medium of solid cattle waste extract in water containing NaCl at 10 g/L. The growth rate and output rate were substantially increased when the wastebased medium was fortified with carbon, nitrogen, and phosphorus. Aeration of the algal cultures considerably improved the output rate of algal biomass. After cultivation, Spirulina can be harvested by simple filtration process using cloth. They are dewatered by centrifugation and dried to a powder. The biomass is usually dehydrated by sun-, drum-, or spray drying. Spirulina are available in powder, flake, capsule, and tablet form.64 More recently, a helical tubular photobioreactor system has been developed which allow these algae to be grown reliably in outdoors at high cell densities in semicontinuous culture. Advances in development of photobioreactors allow successful cultivation of different species of microalgae under controlled conditions for a range of extractable products including β-carotene, phycocyanin, phycoerythrin, and glycerol.45,65,66 11.6.2.1

Nutritional Benefits

Spirulina contains about 65% protein, 7% crude fat, 9% minerals, 5.8% crude fiber, and 16% carbohydrates, and many pigments including chlorophyll a, xanthophyll, β-carotene, echinenone, zeaxanthin, canthaxanthin, diatoxanthin, β-cryptoxanthin, oscillaxanthin, and the phycobiliproteins such as phycocyanin and allophycocyanin. The proteins are complete, in that they contain all essential amino acids, and hence are similar to animal proteins. Besides, Spirulina proteins are highly digestible. Spirulina is a good source of protein and nutrients,

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particularly β-carotene. In addition, the alga contains many nutrients including B-vitamins, β-carotene, GLA, iron, calcium, magnesium, manganese, potassium, selenium, zinc, and flavanoids. The presence of vitamin B12 in Spirulina is of particular value to vegetarians, since absence of red meat in their diets make them depend on other sources for this vitamin. GLA is present in significant amounts in some Spirulina species. This essential fatty acid can be used in the body to form products that are anti-inflammatory and is potentially useful for individuals with rheumatoid arthritis and diabetes. Spirulina, particularly, S. maxima and S. platensis are used as a nutritional supplement, since >95% of them are digestible and, therefore, particularly beneficial for elderly people who are unable to absorb, assimilate, and utilize nutrients efficiently. The recommended dose is 3–5 g/day, but the amount used may depend on the product, the individual using it, and the indication for which it is being taken. Spirulina is a rich source of natural antioxidants, and is a good supplement for those who are fasting or dieting. Consumption of Spirulina at a dose of 1 g/day for a year has shown to decrease a type of blindness. Spirulina fits into practically any dietary plan since it is an extremely digestible, high-energy, low-calorie, low-fat natural food containing an incredibly wide range of important nutrients.67 Table 11.3 shows the composition of S. maxima on dry weight basis. 11.6.2.2

Biological Functions

Regular consumption of small amounts of Spirulina may lower cholesterol, serum lipids, and LDL cholesterol. The microalga is also thought to be helpful in the treatment of oral leukoplakia, a precancerous condition that is manifested as white patches in the mouth. However, some side effects of Spirulina such as diarrhea, nausea, and vomiting have been reported.68,69 Total phenol content of Spirulina is almost five times greater than that of Chlorella (6.86 ± 0.58 and 1.44 ± 0.04 mg tannic acid equivalent/g of algae powder, respectively). Spirulina has the highest concentration of evercetin, a potent antioxidant and anti-inflammatory compound that can be used to alleviate the symptoms of sinusitis and asthma. Phycocyanin, the pigment that gives spirulina its characteristic color, improves body immunity and has also been shown to relieve inflammation associated with arthritis and various allergies. Spirulina is effective for the clinical improvement of melanosis and keratosis due to chronic arsenic poisoning. Liver fibrosis is a chronic liver disease that will further advance to cirrhosis if severe damage continues further. A potential treatment for liver fibrosis is to inhibit activated hepatic stellate cell (HSC) proliferation and, subsequently, to induce HSC apoptosis. It has been reported that antioxidants are able to inhibit the proliferation of HSCs. The growth inhibitory effects of aqueous Spirulina and Chlorella extract on human liver cancer cells, HepG2, were studied. Results indicated that the aqueous extracts of these two algae showed antiproliferative effects on both HSC and HepG2, but Spirulina was a stronger inhibitor than Chlorella. Annexin-V staining showed that aqueous extract of Spirulina induced apoptosis of HSC, after 12 h of treatment. The antioxidant activity of Spirulina is higher than that of Chlorella. Spirulina extract inhibits HIV replication in human T cells, peripheral blood mononuclear cells, and Langerhans cells. It improves

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TABLE 11.3 Composition of S. maxima on Dry Weight Basis Protein (g/100 g) Amino Acids Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Vitamins (mg/100 g) Thiamine Riboflavin Pyridoxine Nicotinic acid Panthothenic acid Folic acid Cyanocobalamin (Vitamin B12) Γ-tocopherol

48–61% 5.0–6.0% 4.5–9.3% 6.0–15.2% 0.6–2.2% 8.2–21.8% 3.2–4.0% 0.9–1.6% 3.7–4.5% 5.6–7.7% 3.0–4.5% 1.6–2.2% 2.8–4.0% 2.7–3.2% 3.2–4.3% 3.2–4.5% 0.8–1.2% 3.9% 4.2–6.0% 5.5% 4.0% 0.3% 11.8% 1.1% 0.05% 0.02% 19.0%

Source: Reprinted from Sen, D.C. and Sarkar, S., Beverage Food World (India) 31, 45, 2004. With permission from The Amalgam Press, Mumbai, India.

weight-gain and corrects anemia in both HIV-infected and HIV-negative undernourished children; and protects against hay fever.70,71 Animal research has shown that Spirulina helps prevent heart damage caused by chemotherapy using Doxorubicin, without interfering with its antitumor activity. Spirulina reduces the severity of strokes, and improves recovery of movement after a stroke; reverses age-related neurological degenerative changes and treats hay fever.72–74 Spirulina was also shown to potentially limit brain damage from strokes and other neurological disorders. It was found that rats fed diets preventively enriched with Spirulina (or blueberries or spinach) experienced less brain cell loss and improved recovery of movement following a stroke.73 A commercial form of Spirulina was recently shown to have antiallergy effects in allergic rhinitis patients. The clinical study demonstrated that daily ingesting 2 g of Spirulina provided a significant

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reduction in the level of interleukin-4 (IL-4) in the body, which is responsible for the production of the antibody, immunoglobulin E (IgE), which mediates symptoms of allergic rhintis.75

11.6.3

DUNALIELLA

The alga can yield three major valuable products, namely, glycerol, β-carotene, and protein. In recent years, it is mainly cultivated for carotenoids. Production of β-carotene by the algae is very high, and depends on light intensity. The natural “β-carotene” extracted from the algae is a mixture of numerous carotenoids and essential nutrients that are not present in synthetic β-carotene. On average, D. salina under ideal conditions can yield 400 mg of β-carotene/m2 of cultivation area. Dunaliella spp. also contain several compounds that exhibit various biological activities such as antihypertensive, bronchodilator, analgesic, and muscle relaxant activities.76 Growth of Dunaliella spp. is comparable with that of Spirulina and requires bicarbonate as a source of carbon and other nutrients such as nitrate, sulfate, and phosphate. Initial vegetative growth phase requires 12–14 days of incubation in nitrate rich medium, under 25–30k lux light intensity. For optimal carotene synthesis, the light intensity should be reduced to 15k lux, besides, nitrate depletion and maintenance of the initial salinity. Harvesting is done by flocculation, followed by filtration; the biomass can be directly utilized for food formulation, as feed or for extraction of pigments as Dunaliella biomass is considered safe. Most of the pharmaceutical formulations are made by extracting the alga with olive or soybean oil.76 Although not from marine habitat, Haematococcus pluvialis is a green alga that can grow both under autotrophic and heterotrophic conditions. It is known for its ability to synthesize astaxanthin, up to 2.0% on dry weight basis and hence, is one of the potential organisms for commercial production of astaxanthin. Several European countries had approved its marketing as dietary supplement for human consumption. Production of the carotenoid by the freshwater alga, H. pluvialis is very attractive, but has fewer advantages than Dunaliella. The challenging task with this organism is its outdoor cultivation, which involves curtailment of contamination and control of environmental conditions such as light and temperature. The organism grows at 20–28°C, below 15k lux light intensity and at pH 6.8–7.4. Since it grows at neutral pH, contamination by bacteria, fungi, and protozoa is the main problem. The cell growth is significantly affected by the intensity of light. Because of the problems associated with open-pond cultivation, the alga is cultivated in closed tubular photobioreactors. Astaxanthin from H. pluvialis is obtained by crushing the algae and supercritical carbon dioxide extraction. The maximum total recovery of astaxanthin exceeded 97%. The red microalga genus Porphyridium is a source of biochemicals possessing nutritional and therapeutic value. These biochemicals include polysaccharides (having anti-inflammatory and antiviral properties), long-chain PUFAs, carotenoids such as zeaxanthin and fluorescent phycolipoproteins. The phycolipoproteins are accessory photosynthetic pigments, aggregated in the cell as phycobilisomes, which are attached to the thylakoid membrane of the chloroplast. The red phycolipoproteins, phycoerythrin and the blue phycobiliproteins and phycocyanin, are insoluble in water and can serve as natural colorants in

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foods, cosmetics, and pharmaceuticals. Chemically, the phycobiliproteins are built up of chromophores, the bilins, which are open-chain tetrapyrroles, covalently linked through thio—ether bonds to an apoprotein. Another source of the pigments is Phaffia rhodoxyma (Xanthophyllomyces dendrorhous) requires large amounts of feed for sufficient pigmentation leading to higher ash contents.76

11.7 MICROALGAE AS FEED FOR AQUACULTURE Because of favorable nutritional composition, several microalgae are utilized in aquaculture systems as food for larval and juvenile stages of fish, crustaceans, and mollusks. The usefulness of microalgae as feed is determined by growth rates, gross composition, and acceptability to the animal. The optimum dietary level of these algae could be ∼0.5% of diet. The microalga, N. closterium has the fastest growth rate of the benthic mat-forming diatoms. It is also rich in protein, and has been suggested as a good candidate for abalone culture. The two Skeletonema spp. having high growth rates among the chain-forming diatoms are already widely used for prawn larval culture in Australia.77 A few strains of the microalgae, belonging to Cryptophytes, such as Rhodomonas minuta and Cryoptomonas spp. have also been used for aquaculture feeds since they contain significant amounts of PUFAs.67 The phylum Haptophyta is a group of unicellular flagellates, having brownish or yellowish green color due to chlorophylls a and c1/c2 and carotenoids such as β-carotene, fucoxanthin, and others. The cells are commonly covered with scales made mainly of carbohydrates or calcium bicarbonate, and hence many species produce calcified scales. About 70 genera and 300 species have been isolated to date, most being tropical marine species, forming food for aquatic communities. The biomass is used as feed for bivalve mollusks, crustacean larvae and zooplankton, which in turn, is used as feed for fish and crustacean larvae.3,45–47 Spirulina has been examined as a feed in the aquaculture of fish and shellfish. The effect of feeding S. platensis on the growth, carcass composition, organoleptic quality, digestive enzyme activity, and digestibility of common carp was studied using an experimental diet in which the fish meal protein was replaced by Spirulina at concentrations ranging from 25 to 100%. It was observed that the diet with Spirulina as the sole source of protein resulted in better net protein utilization (NPU) in common carp although the final weight-gain, specific growth rate, food conversion ratio (FCR), and protein efficiency ratio (PER) were not affected by Spirulina supplementation. There was no significant difference in carcass moisture, protein content, and sensory quality of the fish fed Spirulina diets as compared to fishmeal-based control diet.78 Algal supplementation on the functional roles such as survival, growth, and ability to withstand low dissolved oxygen stress in kuruma prawn was examined. Natural carotenoids from astaxanthin containing alga H. pluvialis and a nonastaxanthin carotenoid-containing S. pacifica, and a synthetic astaxanthin were supplemented in formulated diets at 50 and 100 mg/kg concentrations. Formulated diet without carotenoid supplementation served as a control. The different diets were fed to juvenile kuruma prawn Marsupenaeus japonicus for 9 weeks. Dietary carotenoid effects on survival, growth, and pigmentation were compared by the treatment individually or collectively. After 9 weeks of rearing,

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Food Nutraceutical, functional food, food additive, (emulsifier and thickener), sweets

Commercial products

Colorants

Hydrocarbons (fuel), adsorbents, enzymes, and other research materials

Food (ice creams, jellies, confectionaries, and juices)

Micro algae

Cosmetics (lipsticks, creams and lotions) Pharmaceutical products

Pharmaceutical Antibiotic, antibacterial, bulking agent, binder, hard and soft capsule shell, thickener, and diagnostic agents

FIGURE 11.1 Microalgae applications in various fields. (Reprinted from Dufosse, L. et al., Trends Food Sci. Technol., 16, 389, 2005. With permission from Elsevier.)

control diet-fed prawn had significantly lower survival rate than the pigmented diets-fed prawns. No difference in weight-gain was found among all prawns. Control diet-fed prawn had 66.4% less flesh astaxanthin and 75.5% less shell astaxanthin than the pigmented diets-fed prawns. When subjected to low dissolved oxygen stress, control diet-fed prawn had higher oxygen consumption rate and shorter survival time than the prawns fed the pigmented diets.79 Figure 11.1 summarizes applications of microalgae in various fields.

11.8

MARINE BACTERIA

Marine heterotrophic bacteria are abundant in ocean sediments (see Chapter 2). Extensive chemical investigations of extracts from marine organisms have led to the discovery of a variety of secondary metabolites with antimicrobial activities against human pathogens.80 A survey of culturable heterotrophic bacteria associated with the marine ark shell Anadara broughtoni inhabiting the Sea of Japan, and their antimicrobial, hemolytic, and surface activities were undertaken. A total of 149 strains were isolated and identified phenotypically, whereas a total of 27 strains were selected to be investigated phylogenetically by 16S rRNA gene sequence analysis. The isolates capable of hemolysis were numerically abundant in the genera

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Pseudo-alteromonas, Aeromonas, and Bacillus spp. The six gram-positive isolates belonging to the genera Bacillus, Paenibacillus, and Saccharothrix and two gramnegative strains related to Pseudomonas and Sphingomonas, possessed antimicrobial activity against indicator strains and to each other. Substances with hemolytic and surface activities were isolated from a strain of Bacillus pumilus and characterized as cyclic depsipeptides with molecular weights ranging from 1021 to 1077 Da. The recovery of strains producing antimicrobial and surface-active substances suggested that microorganisms associated with the marine bivalve are potential source of bioactive metabolites.81 In another study, the extract of the sponge-associated bacterial strain Micrococcus luteus was found to exhibit potent antimicrobial activity. The major metabolite isolated was a new acyl-1-(acyl-6’-mannobiosyl)-3-glycerol. Quinolones and a phosphatidylglyceride were isolated from the sponge-associated bacterial strain Pseudomonas spp.82 Several bacterial strains isolated from green and brown marine algae displayed antibiotic activity. All epiphytic bacteria with antibiotic activity were assigned to the Alteromonas and Pseudomonas spp. Antagonism assays among the isolates demonstrated that each producer strain inhibited the growth of the other producers. Likewise, an auto-inhibitory effect was observed in all antibiotic-producing strains. Antibacterial spectra of all the strains include activity against Staphylococcus, Alcaligenes, Pseudomonas, Vibrio, Pasteurella, and Achromobacter spp. The antibiotic substances produced by these epiphytic bacteria were low-molecular-weight compounds, thermolabile, and anionic.83 The actinobacteria or actinomycetes are a group of gram-positive bacteria, which can be from the soil or marine habitats. The soil actinomycets play an important role in decomposition of organic materials, such as chitin and cellulose and, thereby, play a vital part in organic matter turnover. Actinobacteria are wellknown secondary metabolite producers and hence of high pharmacological and commercial interest. In 1940, S. Waksman discovered that soil actinomycetes produced actinomycin. Since then hundreds of naturally occurring antibiotics and other compounds have been discovered from these organisms from both terrestrial (especially from the genus, Streptomyces) and marine sources (see also Chapter 12).80

11.8.1

MARINE BACTERIA AS SOURCES OF PUFA

Bacterial fatty acids are commonly saturated and monounsaturated, ranging from C10 to C20, whereas bacterial PUFA are quite rare. Therefore, at present marine bacteria seem to be of limited use as a source of oils rich in n-3 PUFA.8 The occurrence of bacteria with the ability to produce PUFA is limited essentially to five wellknown marine genera, which all fall within distinct domains, namely, marine genera of the Proteobacteria (Shewanella, Colwella, Photobacterium, Psychromonas, and Moritella). These organisms are mostly psychrophilic, hemophilic, and piezophilic. It has been suggested that PUFA producing capacity helps these bacteria in environmental adaptation for countering the effects of elevated pressure and low temperature.84 High prevalence of PUFA producing bacteria in arctic invertebrates has been observed. More than 100 bacterial strains including Pseudomonasa, Vibrio spp., capable of producing DHA and EPA, were isolated from arctic and subarctic invertebrates and also from some fish species. A large number of bacterial species were

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detected in two species of bivalves (Chlamys islandica and Astarte spp.) and in the amphipod (Gammarus wilkitzkii). Standard taxonomic tests, supplemented with fatty acid profile analysis, showed that the PUFA producing strains belonged to marine Pseudomonas and Vibrio spp.85 Shewanella putrefaciens strain ACAM 342 was found to produce the PUFA, C18:2ω3, C18:3ω3, and C20:5ω3 under aerobic and anaerobic conditions at 15 and 25°C.86 Pathway that does not require desaturation and elongation of saturated fatty acids has been reported for synthesis of PUFA in both eukaryotes and prokaryotes.87 In addition to bacteria, fungi could also provide PUFA. A process for growing fungi, Schizochytrium and Thraustochytrium spp. and for production of omega-3 fatty acids has been patented. These fungi were grown in fermentation medium containing nonchloride-containing sodium salts, in particular, sodium sulfate. The process preferably produced microflora with a cell aggregate size that was useful for the production of feeds for use in aquaculture.53 The lipid classes of cultured Pavlova lutheri were studied. Neutral lipids and glycolipids were the major constituents and accounted for ∼57 and 24% of the total fatty acid residues, respectively. Phospholipids accounted for ∼10% of total fatty acids. The nonpolar fraction was mainly composed of triacylglycerol, whereas the polar fraction was mainly composed of monogalactosylacylglycerols (MGDG). EPA and DHA were seen distributed at varying concentrations in these fractions.88

11.8.2

MICROBIAL BIOTECHNOLOGY

Most products obtained from microbial growth are formed as secondary metabolites. Therefore, optimization of culture conditions is essential for maximum recovery of these compounds. Standardization of fermentation conditions has the potential to increase production of target compounds dramatically. Recent research over the past two decades has provided genomics, postgenomic cloning, protein expression, and gene knock-out techniques with technology to produce these metabolites. The genome sequencing of an organism gives the blueprint of its life. This blueprint establishes the basis for a comprehensive view of the cellular physiology. Knowing the sequence of all genes not only allows the identification of protein functions but also makes it possible to explore the complexity of the cellular organization of an organism. Elucidation of the structural organization of sequenced genomes has led to new insights into the physiological capacity of these organisms. This opens up new possibilities for the exploration of genes that are involved in pathways responsible for the synthesis of metabolites of biotechnological interest. Understanding these functions will be a major challenge for the next decades. Heterogeneous expression techniques are commonly deployed when the original microbe is unable to have a sufficient growth or compound production rate. In this case, the genes responsible for the production of target compound are isolated and inserted into a more favorable host, one having either a faster growth rate or more highly developed expression/ production system. Microalgal biotechnology has the potential to produce a vast array of products from virtually untapped sources.89 Only few marine genome projects have been taken up till now, such as those of methanogenic bacterium Methanococcus jannaschii, and that of the cyanobacterium Synechocystis spp. The function of the majority of genes within the sequenced

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marine genomes is not well understood. Furthermore, even if the complete set of genes of a microbial cell is available, it is mostly not known how these genes are regulated or how the proteins interact to express their functions. In order to assign potential functions to the genes of a genome, functional genome analysis techniques are used. These techniques include the expression profiling of the whole set of genes by using genomic DNA arrays or proteomics. These techniques are not only suitable for exploration of the functions of proteins, but also help to find new potential drug targets. Proteome and transcriptome analysis techniques have led to a shift from direct antimicrobial screening programs toward rational target-based strategies. The proteome technique is mainly based on two-dimensional protein gel electrophoresis, used for protein separation, and mass spectrometry, used for protein identification. Complete genome sequence of the organism of interest is mandatory for the proteome analysis. The proteome of only few marine microorganisms has been investigated so far. Most of these proteome studies explore how marine bacteria adapt to alterations in their environmental conditions. A specific example of application of genetic engineering is the production of EPA by marine cyanobacteria, which do not have the biosynthetic pathway to produce the fatty acid. The EPA synthesis gene cluster (ca. 38 kbp) isolated from a marine bacterium S. putrefaciens SCRC-2738 was cloned to the marine cyanobacterium using a broad-host cosmid vector. The cyanobacterial transcojugants grown at 29°C produced EPA, 0.12 mg/g dry cell, whereas those grown at 23°C produced EPA, 0.56 mg/g dry cell. The content of EPA grown at 23°C increased to 0.64 mg/g dry cell after 24 h incubation at 17°C. Furthermore, EPA production was improved by partial deletion of the EPA gene cluster to stabilize its expression and maintenance in host cyanobacterial cells.3,26,29 Another example is the engineering of new pathways for the production of diverse biomolecules, such as the recombinant production of carotenoids in noncarotenogenic microorganisms. Genes responsible for the formation of carotenoid backbone, the acyclic xanthophylls and several cyclic carotenoids such as astaxanthin and co-enzyme Q10 in Escherichia coli have been cloned. Fermentation of the E. coli results in the better production of the carotenoids. Similar techniques offer better scope for production of nutraceuticals using microbial biotechnology.49–51 The complete genes of three strains of the abundant cyanobacteria, Prochlorococcus have been sequenced and analyzed. The genomic database for cyanobacteria is available (http://www.kazusa.or.jp/cyano/cyano.html).

11.9

CORAL REEFS AND CORALS

Corals are the major organisms that form the basic reef structure (see Chapter 2). Corals include such diverse forms as jellyfish, hydroids, the freshwater Hydra, and sea anemones. They exist in symbiotic association among themselves and also with other marine organisms including bacteria. The role played by antioxidants as defense in symbiotic relationship is known. The metabolic reactions associated with photosynthetic organisms lead to generation of prooxidants, termed as reactive oxygen species (ROS) such as O2−, H2O2, and •OH (see Chapter 5). In response to these ROS, the symbiotic corals establish antioxidant defenses to inactivate the algae-induced prooxidant (ROS) compounds. However, above a certain threshold

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level of ROS, the antioxidants become ineffective and the oxidative damage will occur.90,91 The failure of symbiosis characterized with oxidative stress has been observed in coral bleaching, although a general enhancement of antioxidant defenses has been observed in some organisms such as demosponge Petrosia ficiformis which uses it as an adaptive response to the more elevated levels of oxygen photosynthetically produced by the cyanobacterium, Aphanocapsa feldmanni.38 When algal production of ROS is exacerbated (e.g., by elevated temperature), corals will remove the main source of oxidative damage by expelling their symbiotic algae.90–92 For example, in the star coral Montastera annularis, accumulation of oxidative damage products, antioxidants, and cellular stress capacity were correlated with increases in temperature and bleaching intensity confirming that high temperatures may contribute to triggering oxidative stress and bleaching in coral reef systems.90 A study on the content of antioxidants (superoxide dismutase, catalase, glutathione S-transferases, glutathione reductase, and glutathione peroxidases) in Mediterranean sponges showed significant seasonal changes in antioxidant efficiency, with more marked variations in tissues directly exposed to photosynthetically produced ROS. The greatest variations were observed during the summer months. The results suggested greater production of H2O2 in the symbioses during summer, supporting the hypothesis that seawater temperature can significantly modulate the prooxidant challenge.38

11.9.1

BIOLOGICAL ACTIVITY

Marine sponges contain glycolipids and phospholipids in their membranes, and are rich sources of unusual fatty acids. DHA and EPA rarely occur, whereas sponges contain very long-chain (C23–C34) fatty acids, called as demospongic acids, which constitute up to 80% of the total fatty acids. Fatty acids having more than 15 carbon atoms with characteristic double bonds at ninth and fifth carbon atoms (sometimes with additional double bonds at other positions) from the methyl group are also common. For example, the ∆-5,9, 23-triacontatrienoic methyl ester, isolated as a natural compound from the marine sponge, Chondrilla nucula, is an elastase inhibitor with the potential to be a therapeutic agent in some diseases such as chronic bronchitis. Such ∆-5,9 fatty acids have also been observed in sea anemones gorgonians and zoanthids. In addition, brominated fatty acids and branched fatty acids have also been detected in some corals including sponges. PUFA represents the most important class, accounting for about 50%, in tunicates, some popular edible ascidians, in Japan and Korea.8 The lipid content and fatty acid composition of three healthy coral species (Pavona frondifera, Acropora pulchra, and Goniastrea aspera) and of partially bleached and completely bleached colonies of P. frondifera were examined recently. The fatty acid composition did not differ among healthy corals, but differed significantly among healthy, partially bleached, and completely bleached specimens of P. frondifera. Completely bleached corals contained significantly lower lipid and total fatty acid content, as well as lower relative amounts of PUFAs and higher relative amounts of SFA, than healthy and partially bleached corals.93 Sponges and bryozoa have been found to contain such metabolites that prevent settlement or survival of fouling species. In many cases, associated bacteria were found to be the actual producing organisms of these compounds.94

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The coral reefs are highly beneficial for people with certain kinds of bone injuries. Hydroxyapatite (HA) made from the rigid exoskeletons of marine coral can fill voids caused by fractures or other trauma in the upper, flared-out portions of long bones. The material is similar to human bone in structure. When HA is implanted into a bone void, its web-like structure allows surrounding bone and fibrous tissue to infiltrate the implant and make it biological part of the body. The implants, which are blocks in precut sizes or granules used to fill in the voids, must be used with reinforcement devices such as steel rods to ensure that the fracture remains stable until it heals to avoid crack while walking or lifting weights. Growth of bone provides sufficient strength to support the weight. One of the real advantages of using coral-based implants is that they avoid a second surgery that would be necessary if a donor site is used. The U.S. FDA has approved coral-derived implants for applications such as bone loss around the root of a tooth and in certain areas of the skull. Marine carbonate as highly interconnected microporous materials is receiving attention mainly in medical applications. Three-dimensional microporous skeletons are found in certain species of coral. The size of pores is uniform and range from 15 to 500 μm, depending on the species. The carbonate framework may be used as template for the deposition of metals, ceramics, or polymers, which after removal of the carbonate by mid-acid treatment, provides an interconnected porous composite structure for varied applications.95 Besides, Caribbean sea whip, a type of coral that resembles shrubbery on the sea floor, contains the compounds called pseudopterosins. The compounds appear to have anti-inflammatory properties and have potential for treatment for skin irritations resulting from injury or infection.36

11.10

COMMERCIAL STATUS

Microalgae have industrial uses as carotenoids, vitamin, and fatty acid supplements in health food products and as feed additives for poultry, livestock, fish, and crustaceans. Despite the growing demand, worldwide, for carotenoids, which is projected to reach worth U.S.$ 1000 m by the end of the decade, microalgal pigments have not sufficiently penetrated the market. Dunaliella natural β-carotene is widely distributed today in markets under different categories, under the names, β-carotene extracts, Dunaliella powder for human use, and dried Dunaliella as animal feed. Extracted β-carotene is sold mostly in vegetable oil ranging in concentrations from 1 to 20% to color various food products and for personal use in soft gels, which contain about 5 mg of the carotenoid. The purified natural β-carotene is generally accompanied by the other carotenoids of Dunaliella, predominantly lutein, neoxanthin, zeaxanthin, violaxanthin, cryptoxanthin, and α-carotene, which form approximately 15% of the carotene concentration is marketed under the title “carotenoid mix.” Some companies were interested in these products and therefore their production, process optimization, toxicological studies, and regulatory issues were carried out. Along with this, the biotechnology of microalgae gained considerable progress and relevance, with carotenoids production representing one of its most successful domains. Nowadays some food grade pigments produced by fermentation are on the market: Monascus pigments, astaxanthin from Xanthophyllomyces dendrorhous, Arpink Red from Penicillium oxalicum, riboflavin from Ashbya

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gossypii, β-carotene from Blakeslea trispora. The successful marketing of pigments derived from algae or extracted from plants, both as a food color and a nutritional supplement, reflects the presence and importance of niche markets in which consumers are willing to pay a premium for all natural ingredients. Future trends involve combinatorial engineering and production of niche pigments not found in plants.76 Some current commercial products include a U.S. FDA-approved dietary supplement containing astaxanthin, by name “Zanthin,” extracted from the microalga, H. pluvialis. “Martek DHATM” is a commercial oil product from microalga that contains significant amount of DHA. A carbohydrate extract from the green microalga, Chlorella pyrenoidosa is claimed to boost response of the immune system to the flu vaccine.96 In conclusion, marine microalgae can act as good sources of lipids and carotenoids, whereas microalgae and other microorganisms as well as corals can be sources of large varieties of other bioactive compounds (hormones, antibiotics, and pharmaceuticals). In recent times, genetic engineering is being examined to augment yield of carotenoids from marine sources. Further research in this area can derive additional therapeutic benefits from these organisms. It may be noted that with their significant photosynthetic activity, microalgae play an important role in addressing the problem of global warming. It is important to take necessary steps to protect the marine diversity, particularly with respect to microalgae and corals to avoid any ecological damage to the environment.

REFERENCES 1. Biello, D., Picture if you will an oceanic ‘twilight zone’ of microscopic creatures hindering carbon sequestration, Sci. Am., April 26, 2007. 2. Matsunaga, T. et al., Marine microalgae, Adv. Biochem. Eng./Biotechnol., 96, 965, 2005. 3. Kurano, N. and Miyachi, S., Microalgal studies for the 21st century, Hydrobiologia, 512, 27, 2004. 4. Falkowski, P. G., The ocean’s invisible forest, Sci. Am., 287, 54, 2002. 5. Newell, R. I. E. et al., Relative importance of benthic microalgae, phytoplankton, and mangroves as sources of nutrition for penaeid prawns and other coastal invertebrates from Malaysia, Mar. Biol., 123, 595, 1995. 6. Barbarino, E. and Lourenco, S. O., An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae, J. Appl. Phycol., 17, 447, 2005. 7. Rebolloso-Fuentes, M. M. et al., Biomass nutrient profiles of the Microalga Nannochloropsis, J. Agric. Food Chem., 49, 2966, 2001. 8. Berge, J.-P. and Barnathan, G., Fatty acids from lipids of marine organisms: molecular diversity, role as biomarkers, biologically active compounds and economical aspects, Adv. Biochem. Engn./Biotechnol., 96, 49, 2005. 9. Moreaux, S. G. et al., Diatom artificial medium (DAM): a new artificial medium for the diatom Haslea ostrearia and other marine microalgae, J. Appl. Phycol., 19, 549, 2007. 10. Mourente, G. et al., Total fatty acid composition as a taxonomic index of some marine microalgae used as food in marine aquaculture, Hydrobiologia, 203, 147, 1990. 11. Wen, Z. and Chen, F., Heterotrophic production of eicosapentaenoic acid by microalgae, Biotechnol. Adv., 21, 273, 2003.

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12. Jiang, Y. et al., Fatty acid composition and squalene content of the marine microalga Schizochytrium mangrovei, J. Agric. Food Chem., 52, 1196, 2004. 13. Mansour, M. P. et al., Very long-chain (C28) highly unsaturated fatty acids in marine dinoflagellates, Phytochemistry, 50, 541, 1999. 14. Volkman, J. K., Australasian research on marine natural products: chemistry, bioactivity and ecology, Mar. Freshwater Res., 50, 761, 1999. 15. Muffler, K. and Ulber, R., Downstream processing in marine biotechnology, Adv. Biochem. Engn./Biotechnol., 96, 85, 2005. 16. Jeffrey, S. W., Vesk, M. and Mantoura, R. F. C., Phytoplankton pigments: windows into the pastures of the sea, Nature Res., 33, 14, 1997. 17. Rao, D. V. and Rao, B., Drugs from marine algae-current status, in Proc. Symp. Seaweeds—2004, Seaweed Research and Utilization Association and Central Marine Fisheries Research Institute, Cochin, India, 2004, p. 54. 18. Miguel, H. et al., Optimization of the extraction of antioxidants from Dunaliella salina microalga by pressurized liquids, J. Agric. Food Chem., 54, 5597, 2006. 19. Pouvreau, P. et al., Purification of the blue-green pigment “marennine” from the marine tychopelagic diatom Haslea ostrearia (Gaillon/Bory) Simonsen, J. Appl. Phycol., 18, 769, 2006. 20. Del Campo, J. A., Garcia-Gonzalez, M., and Guerrero, M. G., Outdoor cultivation of microalgae for carotenoid production: current state and perspectives, Appl. Microbiol. Biotechnol., 74, 1163, 2007. 21. Barrett, S. M. et al., Sterols of 14 species of marine diatoms (Bacillariophyta). J. Phycol., 31, 360, 1995. 22. Fabregas, J. and Herrero, C., Vitamin content of four marine microalgae. Potential use as source of vitamins in nutrition, J. Ind. Microbiol. Biotechnol., 5, 259, 1990. 23. Fabregas, J. and Herrero, C., Marine microalgae as a potential source of single cell protein (SCP), Biotechnology, 23, 110, 1985. 24. Arad, S. M. and Yaron, A., Natural pigments from red microalgae for use in foods and cosmetics, Trends Food Sci. Technol., 3, 92, 1992. 25. Garrigan, G. G. and Goetz, G., Symbiotic and dietary marine microalgae as a source of bioactive molecules—experience from natural products research, J. Appl. Phycol., 14, 103, 2002. 26. Matsunaga, T. and Takayama, H., Genetic engineering in marine cyanobacteria, J. Appl. Phycol., 7, 77, 1995. 27. Burja, A. and Radianingtyas, A., Marine microbial-derived nutraceuticals biotechnology: an update, Food Sci. Technol., 19, 14, 2005. 28. Takeyama, H. and Matsunaga, T., Production of Useful Materials from Marine Microalgae, Oxford & IBH Publishing, New Delhi, 1998. 29. Matsunaga, T. et al., Appl. Microbiol. Biotechnol., 45, 24, 1996. 30. Shah, V. et al., Curr. Microbiol., 40, 274, 2000. 31. Otero, A. and Vincenzini, M., Extracellular polysaccharide synthesis by Nostoc strains as affected by N source and light intensity, J. Biotechnol., 102, 143, 2003. 32. Proksch, P., Edrada, R., and Ebel, R., Current status and microbiological implifications, Appl. Microbiol. Biotechnol., 59, 125, 2002. 33. Schaeffer, D. J. and Krylov, V. S., Anti-HIV activity of extracts and compounds from algae and cyanobacteria, Ecotoxicol. Environ. Saf., 45, 208, 2000. 34. Ohta, S. et al., Anti-Herpes simplex virus substances produced by the marine green alga, Dunaliella primolecta, J. Appl. Phycol., 10, 349, 1998. 35. Gerwick, W. H. et al., J. Appl. Phycol., 6, 143, 1994. 36. Henkel, J., Drugs of the deep, FDA Consumer magazine, January–February 1998, U.S. FDA, Washington.

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37. Ranga Rao, A. et al., Antioxidant activity of Botryococcus braunii extract elucidated in vitro models, J. Agric. Food Chem., 54, 4593, 2006. 38. Regoli, F. et al., Variations of antioxidant efficiency and presence of endosymbiotic diatoms in the Antarctic porifera Haliclona dancoi, Mar. Env. Res., 58, 637, 2004. 39. Murphy, C., Moore, R. M., and White, R. L., Peroxidases from marine microalgae, J. App/.Phycol., 12, 507, 2000. 40. Makridis, P. et al., Microbial conditions and antimicrobial activity in cultures of two microalgae species, Tetraselmis chuii and Chlorella minutissima, Aquaculture, 255, 76, 2006. 41. Suetsuna, K. and Chen, J.-R., Identification of antihypertensive peptides from peptic digest of two microalgae, Chlorella vulgaris and Spirulina platensis, Mar. Biotechnol., 3, 305, 2001. 42. Muller, D. et al., Brunsvicamides A-C: sponge-related cyanobacterial peptides with Mycobacterium tuberculosis protein tyrosine phosphatase inhibitory activity, J. Med. Chem., 49, 4871, 2006. 43. Halperin, S. A. et al., Safety and immunoenhancing effect of a chlorella-derived dietary supplement in healthy adults undergoing influenza vaccination: randomized double-blind, placebo-controlled trial, Can. Med. Assoc. J., 169, 111, 2003. 44. Kobayashi, J. and Kuboto, T., Bioactive macrolides and polyketides from marine dinoflagellates of the genus Amphidinium, J. Nat. Prod., 70, 451, 2007. 45. Borowitzka, M. A., Commercial production of microalgae: ponds, tanks, tubes and fermenters, J. Biotechnol., 50, 313, 1999. 46. Jimmy, R. A. et al., The effect of diet type and quantity on the development of common sea urchin larvae, Aquaculture, 220, 261, 2003. 47. Lorenz, R. T. and Cysewski, G. R., Commercial potential for Haematococcus microalgae as a natural source of astaxanthin, Trends Biotechnol., 18, 160, 2000. 48. Renaud, S. M. et al., Microalgae for use in tropical aquaculture I: gross chemical and fatty acid composition of twelve species of microalgae from the Northern Territory, Australia, J. Appl. Phycol., 6, 337, 1994. 49. Otero, A., Garcia, D., and Fabregas, J., Factors controlling eicosapentaenoic acid production in semicontinuous cultures of marine microalgae, J. Appl. Phycol., 9, 465, 1997. 50. Burja, A. M. and Radianingtyas, H., Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: screening of strains and optimization of omega-3 production, Appl. Microbiol. Biotechnol., 72, 1161, 2005. 51. Burja, A. M. and Radianingtyas, H., Marine-microbial derived nutraceutical biotechnology: an update, Food Sci. Technol., 19, 14, 2005. 52. Aki, T. et al., Thraustochytrids as a potential source of carotenoids, J. Am. Oil. Chem. Soc., 80, 789, 2003. 53. Barday, W. R., Schizochytrium and Thraustochytrium strains for producing high concentrations of ω-3 fatty acids, US Patent No. 7 022 512 BZ, 2006. 54. Graham, I. A. et al., The use of very long chain polyunsaturated fatty acids to ameliorate metabolic syndrome: transgenic plants as an alternative sustainable source to fish oils, Nutr. Bull., 29, 228, 2004. 55. Ip, P.-F. and Chen, F., Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark, Proc. Biochem., 40, 733, 2005. 56. Pisal, D. S. and Lele, S. S., Carotenoid production from microalga, Dunaliella salina, Ind. J. Biotechnol., 4, 476, 2005. 57. Shi, X.-M., Jiang, Y., and Chen, F., High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture, Biotechnol. Progr., 18, 723, 2002.

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58. Li, H.-B., Jiang, Y. and Chen, F., Isolation and purification of lutein from the microalga Chlorella vulgaris by extraction after saponification, J. Agric. Food Chem., 50, 1070, 2002. 59. Gouveia, L. et al., Functional food oil colored by pigments extracted from microalgae with supercritical CO2, Food Chem., 101, 717, 2007. 60. Tokusoglu, O. and Unal, M. K., Biomass nutrient profi les of three microalgae: Spirulina platensis, Chlorella vulgaris, and Isochrysis galbana, J. Food Sci., 68, 4, 2003. 61. Adams, M., Superfoods for optimal health: Chlorella and Spirulina, in Spirulina platensis (Arthrospira): Physiology, Cell biology and Biotechnology. Tucson, A. Z. and Vonshak, A., Eds., Taylor & Francis, London, 1997. 62. Sarada, P., Pillai, M. G., and Ravisankar, G. A., Phycocyanin from Spirulina sp: influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin, Proc. Biochem., 34, 795, 1999. 63. Becker, E. W. and Venkataraman, L. V., Production and utilization of the browngreen alga, spirulina, in India, Biomass, 4, 105, 1984. 64. Mitchell, S. A. and Richmond, A., Optimization of a growth medium for spirulina based on cattle waste, Biol. Wastes, 25, 41, 1988. 65. Binaghi, L. et al., Batch and fed-batch uptake of carbon dioxide by Spirulina platensis, Process Biochem., 38, 1341, 2003. 66. Luis, D. A. et al., Continuous and pulse feedings of urea as a nitrogen source in fedbatch cultivation of Spirulina platensis, Aquacult. Eng., 31, 237, 2004. 67. Zhou, B. et al., Application of Spirulina mixed feed in the breeding of Bay scallop, Bioresource Technol., 38, 229, 1991. 68. Gopalan, C. et al., Nutritive value of Indian Foods, National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, 1983, p. 63. 69. Sen, D. C. and Sarkar, S., Spirulina: a classical health food, Beverage Food World (India), 31, 45, 2004. 70. Ayehunie, S. et al., Inhibition of HIV-1 replication by an aqueous extract of spirulina platensis (Arthrospira platensis), J. Acquir. Immune Defic. Syndr. Hum. Retrovirol., 18, 7, 1998. 71. Simpore, J. et al., Nutrition rehabilitation of HIV-Infected and HIV-Negative undernourished children utilizing spirulina, Ann. Nutr. Metabol., 49, 373, 2005. 72. Khan, M. et al., Protective effect of Spirulina against doxorubicin-induced cardiotoxicity, Phytother. Res., 19, 1030, 2005. 73. Wang, Y. et al., Dietary supplementation with blueberries, spinach, or spirulina reduces ischemic brain damage, Exp. Neurol., 193, 75, 2005. 74. Gemma, C. et al., Diet enriched in foods with high antioxidant activity reverse age-induced decreases in cerebellar beta-adrenergic function and increases in proinflammatory cytokines, Exp. Neurol., 22, 6114, 2002. 75. Mao, T. K., Van de Water, J., and Gershwin, M. E., Effects of a spirulina-based dietary supplement on cytokine production from allergic rhinitis patients, J. Med. Food, 8, 27, 2003. 76. Dufosse, L. et al., Microorganisms and microalgae as sources of pigments for food use: a scientific oddity or an industrial reality? Trends Food Sci. Technol., 16, 389, 2005. 77. Brown, M. R. and Jeffrey, S. W., The amino acid and gross composition of marine diatoms potentially useful for mariculture, J. Appl. Phycol., 7, 521, 1995. 78. Nandeesha, M. C., Effect of feeding Spirulina platensis on the growth, proximate composition and organoleptic quality of common carp, Cyprinus carpio L, Aquacult. Res., 29, 305, 1998.

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79. Chieu, Y.-H. and Shiau, W. C., The effects of dietary supplementation of algae and synthetic astaxanthin on body astaxanthin, survival, growth, and low dissolved oxygen stress resistance of kuruma prawn, Marsupenaeus japonicus Bate, J. Exp. Mar. Biol. Ecol., 318, 201, 2005. 80. Jensen, P. R. and Fenical, W., New natural products diversity from marine actinomycetes, in Natural Products: Drug Discovery and Therapeutic medicines, Zhang, L. and Demain, A. L., Eds., Humana Press, New Jersey, 2005, p. 315. 81. Romanenko, L. L., Isolation, phylogenetic analysis and screening of marine molluscassociated bacteria for antimicrobial, hemolytic and surface activities, Microbiol. Res., 2007 (in press). 82. Bultel-Ponce, V., Debitus, C., and Guyot, M., Metabolites from marine bacteria, Proc. Symp. Marine Lipids, Brest, France, November 19–20, 1998, Baudimant, G. et al., Eds., 2000, p. 193. 83. Lemos, M. L., Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds, Earth Env. Sci., 11, 149, 1985. 84. Yokochi, T. et al., Appl. Microbiol. Biotechnol., 49, 72, 1998. 85. Jostenseri, J.-P. and Landfaid, B., High prevalence of polyunsaturated fatty acid producing bacteria in arctic invertebrates, FEMS Microbiol. Lett., 151, 395, 1997. 86. Nichol, D. S. et al., Anaerobic production of PUFA by Shewanella putrefaciens strain ACAM 342, and Shewanella spp. SCRC-2738, FEMS Microbiol. Lett., 98, 117, 1992. 87. Metz, J. G. et al., Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes, Science, 293, 290, 2001. 88. Meireles, L. A., J. Agric. Food Chem., 51, 2237, 2003. 89. Olaizola, M., Commercial development of microalgal biotechnology: from the test tube to the marketplace, Biomol. Eng., 20, 459, 2003. 90. Lesser, M. P., Oxidative stress causes coral bleaching during exposure to elevated temperatures, Coral Reefs, 8, 187, 1997. 91. Douglas, A. E., Coral bleaching—how and why? Rev. Mar. Pollut. Bull., 46, 385, 2003. 92. Wilkinson, C. R., Executive summary, in Status of Coral Reef of the World, Wilkinson, C. R., Ed., Aust. Inst. Marine Sci., Townsville, Australia, 2000, p. 7. 93. Bachok, Z. et al., Characterization of fatty acid composition in healthy and bleached corals from Okinawa, Japan, Coral Reefs, 25, 545, 2006. 94. Kon Ya, K., et al., Fish. Sci., 60, 773, 1994. 95. White, R. A. and White, E. W., Biomedical applications of marine natural products, in Marine Biotechnology in the 21st Century: Problems, Promise and Products, National Academy Press, Washington, 2003, p. 79. 96. Ohr, L. M., Riding the nutraceutical wave, Food Technol., August 2005, 59, p. 95, www.ift.org.

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and 12 Drugs Pharmaceuticals from Marine Sources 12.1 INTRODUCTION The ocean, which hosts the richest diversity of living organisms, is a relatively unexplored source of compounds that can find uses as new pharmaceuticals and as tools in molecular and pharmacological studies. Marine pharmacology deals with the search for models of marine bioactive compounds, which, in combination with chemical synthesis and analysis of structure–activity relationships, generate new drugs against human ailments. Pharmacology of marine compounds had its beginning in 1969; since then it has increasingly looked toward the ocean for new medical advances and treatments. The science of marine pharmacognosy encompasses a broad range of aspects concerning bioactive compounds biosynthesized in organisms found in the sea. The initial work in the field done before three decades has been elegantly summarized by Baslow.1 During the past four decades, over 10,000 compounds have been isolated from marine organisms, many from sponges and other invertebrates found on coral reefs. The increasing interest in the field is shown by the fact that between 1969 and 1999, 200 patents were issued worldwide for marine-derived biochemicals with potential therapeutic activities, whereas between 1996 and 1999, about 100 new compounds were patented.2 The interest and research progresses in marine natural products as pharmaceutical compounds are also indicated by several recent reviews on the topic.3–16 This chapter discusses various compounds present in marine organisms that have potential to function as promising drugs for some human diseases.

12.2 PROSPECTS OF FINDING DRUGS FROM MARINE ORGANISMS The prospects of finding drugs from marine organisms stem from the fact that these creatures have in them several techniques to survive in the hostile oceanic environments consisting of high salinity, fluctuating temperatures, and stringent competition from diverse species. The marine organisms have met these challenges essentially by a threefold program. 1. Most marine organisms produce several secondary metabolites, which, although, are not directly involved in central physiological functions, yet contribute to their fitness and survival. They have also evolved to produce certain unique compounds that are extremely toxic, particularly to mammalian systems; the marine toxins being much more potent in toxicity compared to many terrestrial poisons. 371

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2. Cell components such as sugars, fats, proteins, nucleic acids, adenosine triphosphate (ATP), and hemoglobin of these organisms serve life-sustaining physiological functions such as growth, respiration, energy storage, and genetic information transfer similar to terrestrial animals. However, in addition, many cell compounds of marine organisms also possess certain characteristic bioactivities. For example, sulfated polysaccharides (carrageenans) possess antifungal, antibacterial, ichthyotoxic, hypotensive, and helminthic activities (see Chapter 10). Other bioactive cell components include chitin, chondroitin, fucoidan, fucoxanthin, laminarin, and several others as discussed earlier in the book. 3. Several marine organisms have adapted themselves through symbiotic association among themselves that help them survive under harsh environments (see Chapter 2).

12.2.1 MARINE SECONDARY METABOLITES AND THEIR FUNCTIONS Secondary metabolites (also called natural products) are organic compounds that are not directly involved in the normal growth, development, or reproduction of organisms. Unlike primary metabolites, absence of secondary metabolites does not result in immediate death of these organisms, but in long-term impairment of their survivability/fecundity. The function of these compounds is usually of an ecological nature, as they are used as defenses against predators, parasites, and diseases for interspecies competition and to facilitate reproductive processes (attracting by color, smell, etc.). There is a vast diversity in the secondary metabolites produced by marine organisms consisting of alkaloids, terpenoids, sulfated polysaccharides, peptides, and novel chemical compounds. Especially striking is the frequent occurrence of sesterterpenes in marine organisms, which are produced mostly by sponges. The bioactivities of secondary metabolites (and also the cell components) help combat adversities of diverse living creatures of the sea. In many cases, these compounds are less lethal, but capable of causing great pain to the attacker and thereby warning them not to consider coming back for another attack. In contrast, offensive compounds (poisons) tend to have some very different characteristics, such as fast action and degradative functions (e.g., enzymes) that help to predigest the victim. The involvement of secondary metabolites in defense is illustrated by the fact that grazers and predators less readily attack certain seaweeds and invertebrates containing such compounds in their tissues. In some cases, it has been further demonstrated that an initial grazer attack can actually promote increased production of these defensive chemical compounds, resulting in decreased attacks over time. In other cases, grazers have been able to protect against certain attaching organisms, sometimes even sequestering the defensive metabolite in their own tissues for their own protection. An example of this strategy of stolen defenses is seen in the Caribbean tiger flatworm, Maritigrella crozierae, an animal that sequesters the secondary metabolites produced by its food source—the ascidian (tunicate) Ectinascidia turbinata for its own defenses. In fact, one of the natural products produced by E. turbinata and pilfered by the predatory flatworm, the alkaloid ecteinascidin-743, is a potential drug for treating a variety of cancers (see Section 12.3.1).2 Corals including invertebrates

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such as sponges, sea slugs, and others have attracted particular attention because they possess specific chemical defenses against predators, and hence serve as potential sources of bioactive compounds for practical applications (see Section 12.5.1). The role of secondary metabolites in providing antimicrobial chemical defenses among marine organisms has also been established.5 Studies on their disease resistance could throw light on the antimicrobial activities of a variety of secondary metabolites against human pathogens. Thus, a number of antibacterial compounds have been isolated from seaweed.6,7 The marine environments have had millions of years to evolve these compounds, whereas when we bring these compounds “ashore” our systems have not had that evolutionary process to combat these compounds, facilitating their therapeutic functions. It is to be kept in mind that activities of these compounds are measured invariably on terrestrial systems and not on marine systems. The major producers of biologically active compounds are invertebrates, which comprises more than 150,000 species in the aquatic environment. These invertebrates include the tunicates and lancelets of the phylum Chordata, as well as all animal phyla other than Chordata including members of phyla Porifera (sponges), Cnidaria (coelenterates), Ctenophora, Platyhelminthes (flatworms), Nematoda (roundworms), Annelida (segmented worms), Arthropoda, Molluska, Echinodermata, Endo- and Ectoprocta, and protochordates.8 Because of their characteristic biological activities, several antibacterial and antiviral and anti-inflammatory or analgesic compounds and drugs for cancer, malaria, osteoporosis, and other diseases have been identified from marine sources. The potential uses of these compounds as medicine belong to the domain of pharmaceutical industry. The industry, by a process termed as “biomining,” screens different organisms including those from marine sources and identifies their natural products for development of drugs. During the past few years, significant attempts have been made to isolate several drugs from marine sources with an aim to use them as drugs and pharmaceuticals for human healthcare. Furthermore, understanding chemical structures of these bioactive compounds also help in the development of models for the synthesis of newer bioactive compounds.2,9 First, this chapter briefly discusses some drugs from marine sources for various diseases such as cancer, malaria, arthritis, and tuberculosis. Next, it discusses different marine sources that can offer bioactive compounds, which have potential to be developed into drugs. Finally, current problems and prospects with respect to the development of marine drugs have been pointed out.

12.3 SOME MAJOR MARINE DRUGS Several species belonging to the marine flora have been screened for natural products that can have significant biological activities and potential for drug development, as shown in Table 12.1. These efforts have yielded the isolation of a number of drugs from these sources. Table 12.2 indicates bioactive compounds produced by some marine organisms.38–40 These compounds include analgesic, anti-Alzheimer, antiangiogenic, antiasthma agents, anticancer drugs, antifungal agents, anti-inflammatory compounds, antitumor (anticancer) agents, and immunostimulatory and immunosuppressive compounds, among others (Table 12.3).41 Some major categories of the drugs has been discussed in this chapter.

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TABLE 12.1 List of Some Marine Flora Screened for General Biological Activities Class Chlorophyceae

Phaeophyceae

Rhodophyceae

Species

Nature of Biological Activity

Caulerpa toxifolia C. racemosa Codium elongatum Ag. Enteromorpha intestinalis (L.) Halimeda gracilis H. opunitia Ulva fasciata U. lactuca Dictyota atomaria Hauck Padina tetrastomatica P. commersonii Sargassum plagiophyllum Acantophora spinifera Centroceras clavulatum Ag. Cryptonemia undulate Galaxaura oblongata Lamour (Jag) Kylin Galerucella rugosa Lamon Gracilaria edulis (Gmel.) Silva Hypnea musciformis

Diuretic Hypotensive Hypotensive Antiamoebic, diuretic, and hypotensive Hypotensive, diuretic Diuretic Antiviral Anti-implantation Hypotensive Spasmolytic Hypoglycemic, diuretic Anti-implantation and diuretic Antiviral Hypotensive Hypoglycemic Hypotensive, diuretic Antiviral Diuretic Antiviral

Source: Adapted from Garg, H.S., in Nutrients and Bioactive Substances in Aquatic Organisms, Society of Fishery Technologists, Cochin, 1994, 1. With permission.

TABLE 12.2 Bioactive Compounds Produced by Some Marine Organisms Products Fish, shrimp

Sea cucumber

Jellyfish Bivalves (mollusks, clams, scallop)

Secondary Metabolites and Their Bioactivities Peptides (blood pressure control), fish bone phosphopeptide (FBP; Ca-binding activity), salmon calcitonin (Ca-regulating hormone), squalamine (antiangiogenic activity to control tumor), antioxidants Triterpene glycoside, patagonicoside A, polysaccharides and other antibacterial, antifungal, antioxidant, and anticoagulant activities Green fluorescent protein (GFP) Hypotensive agents, cardioactive substances, muscle relaxants, antibiotics, antiviral and antitumor agents, ziconotide (analgesic activity), human immunodeficiency virus (HIV)-inhibiting compound, cross-linking protein adhesive with or without chitosan, conotoxins (depsipeptides), cytotoxicity factor, designated as ES-285, from clam

References 17–20

19–22

23, 24 25, 26–28

(continued)

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TABLE 12.2 (Continued) Bryozoa Tunicates Sponges (Porifera)

Microorganisms (actinomycetes) Seaweeds

Bryostatin for cancer treatment Ecteinascidin, aplidine (also called dehydrodidemnin), didemnin B (cancer treatment) Pseudopterosins, photoactivatable fluorescent proteins (PAFPs), diterpenes, helioxenicins A–C, sponge-derived anticancer drugs such as bengamide, giroline, and manoalide Anticancer agents, fungal metabolites, and other compounds Laminarin, fucoidan, halogenated polysaccharides and lipids, and terpenephenols

4, 13 13, 29 7, 13, 30

16, 31–35 25, 36, 37

Source: Adapted from Matsunaga, T. et al., Adv. Biochem. Eng./Biotechnol., 96, 165, 2005; Bhadury, P. and Wright, P., Planta, 219, 564, 2004; Bhakuni, D.S. and Rawat, D.S., Bioactive Marine Natural Products, Springer, Netherlands, 2005, Vol. XV, p. 400; Henkel, J., FDA Consumer Magazine, 32, January–February, 1998.

TABLE 12.3 Bioactive Compounds from Marine Sources As Cure for Various Diseases Analgesic agents Anti-Alzheimer agents Antiangiogenic agents Antiasthma agent Protein synthesis inhibition Anticancer agents Antifungal agents Anti-inflammatory Antimalarials Antimicrobials Antitumor agents Antiviral agents Apoptosis induction compounds Cytotoxins Immunostimulatory agents Immunosuppressive agents Osteoarthritis Source: Reprinted from Sen, D.C. and Sarkar, S., Beverage Food World (India), 31, 45, 2004. With permission from The Amalgam Press, Mumbai, India.

12.3.1 ANTICANCER AGENTS Cancer being one of the major diseases, yet to be conquered, the various bioactive compounds from marine sources are increasingly being looked into as potential drugs.42,43 These efforts have resulted in the identification of a number of marine compounds that can treat at least certain types of cancers. Some of these marine

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anticancer compounds are currently in clinical trials. Bryostatin, produced by the bryozoan Bugula neritina, a filter-feeding invertebrate, is an exciting new form of chemotherapy. This marine drug selectively kills cancer cells without harming normal, healthy ones. Although all the existing forms of chemotherapy inhibit red blood cell production and patients often require blood transfusions, bryostatin simulates this process. This unique set of properties has resulted in promising numerous human trials of the compound.3 Other secondary metabolites from the bryozoan Biflustra perfragilis from Bass Strait have also been studied in detail. This species are particularly malodorous because of the presence of dimethyl disulfide, dimethyl sulfide, and methanethiol, and two novel sulfur-containing isoquinoline alkaloids. Novel brominated quinone methides, named euthyroideones, have been isolated from the New Zealand bryozoan Euthyroides episcopalism.43,44 Neovastat (AE-941), derived from shark, is an antiangiogenic (angiogenesis is the formation and differentiation of blood vessels) and antitumor compound derived from shark cartilage, with a molecular weight of about 500 kDa. AE-941 inhibits the binding of vascular endothelial growth factor (VEGF) to its receptors. Normally, when VEGF is secreted by tumors it binds to target endothelial receptors and directs the profusion of new capillaries to supply the tumor with nourishment. By blocking the receptor sites, AE-941 prevents the formation of the new blood vessels required by the growing tumor to sustain itself. AE-941 also inhibits the metastatic cellular machinery the tumor normally uses to disrupt the extracellular matrix of the surrounding host tissue. Additionally, the drug appears capable of inducing endothelial cell-specific apoptosis (programmed cell death). It also induces an increase production of compounds by endothelial cells that may lead to the disintegration of blood vessels present in the tumor. The antiangiogenic and antitumor activity of AE-941 was first reported in 1997. It is now in phase III trials (see Section 12.7) in several countries for renal cell carcinoma and lung cancer. The compound is efficient in stabilizing tumor progression and relieving pain in metastatic prostate cancer patients. Recently, AE-941 has also received attention as a plausible treatment against metastatic breast cancer. The antiangiogenic bioactivity of the drug further suggests it could be a valuable agent for use in patients suffering from multiple myeloma and other hematological (blood) diseases. One of the characteristics of multiple myeloma is bone marrow angiogenesis, and treatment with an antiangiogenic agent such as neovastat shows potential.3 Squalamine is an aminosterol isolated from the stomach and liver of the spiny dogfish Squalus acanthus—a common coastal shark species that is found on both the coasts of the United States and also in European waters. Squalamine appears similar to the shark-derived product, neovastat, in its ability to inhibit tumor production of VEGF and other such growth factor signals. Although the bioactivity of squalamine lies in its ability in preventing proliferation of blood vessels in tumor cells, the compound appears to be capable of inducing endothelial cell inactivation and apoptosis through the inhibition of integrin (specialized receptor protein) expression and the disruption of cytoskeletal formation. Development of squalamine as a drug has progressed to phase II clinical trials as part of a combination treatment for nonresponding solid tumors, and as a primary treatment against ovarian cancer. There is data suggesting its efficacy against prostate and brain cancers. Its potency for the treatment of vision problems relating to age-related macular

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degeneration has also been indicated.4,45 A cytotoxicity factor, designated as ES-285 has been recently isolated from the clam Mactromeris polynyma, which triggers an atypical cell death program when compared to other sphingosine-dependent apoptosis pathways.46 The anticancer agent Ara-C and antiviral drugs Ara-A and AZT developed from the extracts of sponges found on the Caribbean reef were among the earliest modern medicines obtained from coral reefs.47 Ageladine A, a promising anticancer alkaloid with potent antiangiogenic activity, was fi rst isolated from the marine sponge Agelas nakamurai. A short simple method for the synthesis of ageladine A and its analogs has been developed recently. The analogues were screened for anticancer, antiangiogenic, and kinase activities to understand the structure–activity relationship.48 Glycosphingolipids, chiefly isolated from sponges, show interesting biological activities such as immunomodulation and antitumoral activity. Arising from this class of lipids, the compound KRN-7000 is being considered as a potent anticancer agent.49 Anticancer compounds have also been isolated from marine bacteria, which are mostly in symbiotic association with sponges. A metabolite, salinosporamide A, from a marine bacterium of the genus Salinospora—a group of obligate marine actinomycetes widely distributed in marine sediments—has been isolated. The antitumor activity has been attributed to its ability to inhibit the 20S proteasomes, which are important in cellular physiology. Other examples of potential antitumor drugs from marine microorganisms are the alkaloid alteramide from an Alteromonas spp. isolated from the sponge Halichondria okadai; the diketopiperazine dimmer asperazine from a strain of Aspergillus niger obtained from the sponge Hyrtios spp.; the macrolide halichomycin from Streptomyces hygroscopicus isolated from the gastrointestinal tract of the marine fish Hallchores bleekeri; the leptosins diketopiperazine dimmers from the obligate marine fungus Leptosphaeria spp.; and the neomangicols, partly halogenated sesterpenes isolated from the mycelial extract of a wood-inhabiting marine fungus. The IC50 values of these compounds against various cell lines range from 0.1 to 10 μg/mL. More significantly, leptosins A and C displayed potent in vivo activity in sarcoma 180 ascites tumor model in mice.50 Kahalalide F (molecular weight 1477.87 g/mol; molecular formula C75H124N14O16) isolated from the Hawaiian sacoglossan Elysia rufescens appears capable of disrupting lysosome membranes within certain target cells, thereby initiating apoptosis. The drug also appears to inhibit the expression of certain specific genes that are involved in deoxyribonucleic acid (DNA) replication and cell proliferation, thereby inhibiting tumor spreading and growth.51 Other promising anticancer compounds from marine sources include spisulosine (ES-285), aplidine, ecteinascidin, diazonamide A, and vitilevuamide.4 Didemnins, especially didemnin B from the Caribbean tunicate Trididemnum solidum provoked interest in the 1980s due to their potent antitumour activity. Dehydrodidemnin (also called aplidine) isolated from the Mediterranean tunicate Aplidium albicans could prove to be a favorable substitute for didemnin B. Aplidine appears to be less toxic and even more effective than didemnin B with a broad-spectrum activity against various types of cancer.29,52 Ecteinascidin is a new anticancer agent isolated from the “mangrove tunicate” found in the Florida Keys as well as other areas of the Caribbean.4

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Scientists from CalBioMarine Technologies in Carlsbad, California, have developed a method of culturing the animal, going so far as creating an artificial version of the mangrove roots it settles on in the wild. The drug is in human trials and is one of the most promising new treatments under development.53 Cytotoxic metabolites from cyanobacteria are crytophycin from a Nostoc spp. and curacin A from a specimen of Lyngbya majuscule, which inhibits microtubule assembly by binding at the colchicin site. The potent cytotoxin dolastatin 10, primarily found in the sea hare Dolabella auricularia, has been isolated from a cyanobacterium (Symplaca spp.).50 Thiocoraline, which belongs to the chemical family known as the thiodepsipeptides, was first isolated from Micromonospora marina—an actinomycete bacterium collected from southeast African coast. The compound has been reported to show activity against cancers of breast, colon, kidney, and melanoma. Target cells appear to be inhibited through inhibition of the DNA polymerase enzyme. Recent published literature suggests that thiocoraline is still undergoing advanced preclinical evaluation. Other products such as dolastatin 10, isolated from a sea hare found in the Indian Ocean, are under clinical trials for use in the treatment of breast and liver cancers, tumors, and leukemia.4,47 Cancer chemotherapy relies to a great extent on the use of drugs targeted at topoisomerases. These enzymes that resolve DNA topological constraints in cells are the primary targets for a large diversity of natural products from different sources including marine microorganisms.7,54–56 The recent discovery of novel secondary metabolites from taxonomically unique populations of marine actinomycetes suggests that these bacteria add an important new dimension to microbial natural product research.31 The Fenical Lab at the Scripps Institution of Oceanography has screened more than 500 microbial extracts over the past years against a series of cancer chemoprevention assays. These studies have resulted in the isolation and characterization of a few anticancer agents from a marine actinomycete strain CNS-177, Streptomyces spp.32,57 Collaborative work among scientists in Japan, Switzerland, and Queensland led to the discovery of three new diterpenes, helioxenicins A–C, in the blue coral Heliopora coerulea. Sinularia flexibilis is a dominant soft coral on many Indo-Pacific coral reefs, which has been found to release toxic compounds (diterpenes) that cause tissue necrosis and death in nearby scleractinian. The ability of the diterpenes derived from soft corals to inhibit cell division suggested that they could have potential applications in cancer chemotherapy.43 The U.S. National Cancer Institute is sponsoring clinical trials of substances derived from marine invertebrates such as sea hares and bryozoans that may have use in the future as cancer treatments. A type of protein linked to cancer prevention in humans may also play a role in ageing. The internationally funded research, carried out at the Buck Institute, the United States and the Manchester University, the United Kingdom, found that proteins that prevent cancer in humans by stopping damaged cells from dividing also determine the life span of microscopic worms. The findings raise the question of whether genetic variations in specific proteins in humans may protect some people from age-associated diseases, whereas placing others at high risk of cancer. In the study, researchers genetically removed checkpoint proteins in the nematode worm Caenorhabditis elegans, which resulted in a 15–30% increase in the life span of the worms.58

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12.3.2 TUBERCULOSIS More than 9 million people across the world suffer from tuberculosis and each year 2 million people die from it. About a decade ago, the first reports concerning potent antituberculosis molecules of marine origin appeared with the isolation of two cyclic depsipeptides, massetolide A and viscosin-9, from the cultures of two Pseudomonas spp. isolated from a marine alga and a marine tube worm, respectively. Both these compounds exhibited in vitro antimicrobial activity against the tubercle bacilli. Later, antitubercle bacilli active kahalalide A and kahalalide F were isolated from sacoglossan mollusk E. rufescens and its algal diet. Kahalalide A was more active against the bacteria and less toxic. Antituberculosis compound has also been isolated from a cyanobacterium and a coral.50

12.3.3 MALARIA With an average of 2 million deaths per year, and the appearance of chloroquineresistant Plasmodium strains, malaria represents another major medical challenge. Among the marine organisms, sponges provide plausible source of potential candidates for antimalarial drugs. A diterpene analog, kalihinol A, isolated from an unidentified sponge of the genus Acanthella, displayed high antimalarial activity, with an IC50 (concentration required for 50% inhibition) of 400 ng/mL. Manzamine A and 8-hydroxymanzamine A are new bioactive compounds isolated recently from sponges. Recently, endoperoxides, especially three nonsesterterpenes, having antimalarial activities have been isolated from Diacarnus erhthraeanus, collected from the Red Sea. The phylum Cnidaria has also yielded antimalarial diterpenes with moderate in vitro activities. Lyophilized extracts of two Cyanobacteria strains of the genus Calothrix yielded two compounds, namely, calthrixins A and B, which are active in vitro against the chloroquine-resistant strains of Plasmodium FAF6, with an IC50 value of 58 nM and 180 nM, respectively. Five compounds were isolated from the culture medium of the marine fungus Halorosellinia oceanica found in Thailand. Two compounds, 5-carboxymellein and halorosellinic acid, displayed noticeable antimalarial activity against a multiresistant strain of plasmodium (P. falciparum) with IC50 values of 4 μg and 13 μg, respectively.4

12.3.4 OSTEOPOROSIS Osteoporosis, a crippling disease marked by loss of bone mass, causing about 1.5 million fractures of hip, wrist, and spine in people above 50 years (see Chapters 4, 6, and 7). Salmon, which like humans, produces a hormone called calcitonin that helps regulate calcium and decreases bone loss. The hormone inhibits the activity of specialized bone cells called osteoclasts that absorb bone tissue, enabling bone to retain mass. The salmon calcitonin is about 30 times more potent than that secreted by the human thyroid gland. Calcitonin is now also being synthesized.17 The zoanthamine family represents a group of polycyclic alkaloids that could be potent candidates for preventing osteoporosis. Zoanthamine was isolated in 1984 by Faulkner’s group from a Zoanthus spp. (phylum Cnidaria, class Anthozoa, order

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Zoanthides), collected off the Visakhapatnam coast of India. So far, 17 analogs have been isolated from different zoanthids from Indian, Arabian, or Japanese seas. Some of these compounds also possessed anti-inflammatory, cytotoxic, and analgesic activities.7 Potential of these compounds from dinoflagellates has also been indicated recently.47

12.3.5 ARTHRITIS Arthritis indicates inflammation of the joints and is a disease affecting mostly the aged population. Preventing inflammation with its associated pain and reduced mobility symptoms is a primary requirement in arthritis treatment. The principal anti-inflammatory activity is due to the carbohydrate complex that inhibits the emigration of neutrophils from the blood vessels when an inflammatory stimulus is present. Anti-inflammatory agents inhibit enzymes involved in the inflammatory process (cycloxygenases [COX], lipoxygenases, and some proteases) and in the production and release of cytokines in blood coagulation. There are two forms of the COX enzymes. COX 1 is a constitutive and natural component of the normal tissue, which performs important functions in the body. The other form, COX 2, is induced when an inflammatory state such as arthritis is present and it produces proinflammatory prostaglandins. It is therefore desirable to selectively inhibit the COX 2 enzymic activity. Generally, anti-inflammatory drugs tend to inhibit both COX 1 and COX 2 activity thus inhibiting the production of the prostaglandins. An extract, called the green lipped mussel extract (GLME), from the mussel Perna canaliculus found off the coast of New Zeland contains the therapeutic agents needed to treat arthritis. The product commercially known as “Seatone” has been shown in laboratory and clinical trials, involving both human and animal subjects, to be effective in treating both the rheumatoid and osteo forms of arthritis. GLME performs two distinct anti-inflammatory activities. The anti-inflammatory activity is due to the effect of natural content of glycosaminoglycans and long-chain fatty acids. Although the glycosaminoglycans provide proteoglycans, the latter selectively inhibits COX 2 activity. Seatone has been shown in laboratory studies to inhibit the cytokine activity that would lead to the production of cells responsible for this process. According to the manufacturers, the product also protects the stomach linings from the damaging effect of some painkillers should a person need to be taking these at the same time.59 The Caribbean coral Pseudopterogorgia elisabethae has anti-inflammatory activity and are also used as an ingredient for cosmetic skin care products. The activity of the extract is due to unusual diterpene glycosides, called pseudopterosins, which inhibit phosholipase A2.60 Other anti-inflammatory agents isolated from marine sources are salinamides A and depsipeptides from actinomycetes isolated from a jellyfish and thiotropocin, which is a sulfur-containing macrolide, among others.50,61

12.3.6 ANTIMICROBIAL AND ANTIVIRAL COMPOUNDS Bacterial biofilms are groups of bacterial cells, and the extracellular material they produce assemble on surfaces. It is believed that two-thirds of all human bacterial

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infections are caused by biofilms. This is mainly because the abundant sugars and starches (polysaccharides) that form the matrix holding biofilms together create a barrier that protects biofilms against antibiotics as well as antibodies and white blood cells.4 In the marine environment, biofilms are of particular interest to research scientists because biofilms behave differently compared to free living organisms. Any living or nonliving surface immersed in seawater rapidly acquires a bacterial biofilm. Biofilm formation can result in the death of the animal, and thus there is strong evolutionary pressure for marine eukaryotes to evolve mechanisms that inhibit or control the development of biofilms on their surfaces. Some marine eukaryotes control biofouling through the production of inhibitory chemicals that act at or near the surface of the organism. One of the fascinating aspects of benthic marine communities, particularly those associated with coral reefs, is that many of the animals are able to prevent overgrowth by fouling organisms by using chemical deterrence. Natural inhibitors can be toxic to fouling organisms such as bacteria and microalgae. One type of seaweed—a marine red alga called Delisea pulchra—that grows in Botany Bay, Australia, is almost completely resistant to the surface fouling of biofilms. The effect is due to a novel class of chemical compounds called furanones produced by seaweed. Over 40 furanones have been isolated so far. There is an exciting possibility to use furanones or similar signal blockers to block biofilm formation that can also enhance the sensitivity of bacteria to antibiotic treatment. Effective treatments for disrupting the formation of biofilms or allowing elimination of established biofilms could therefore be useful against a host of bacterial diseases including a range of skin disorders, cholera, and various infections related to surgical procedures, among many other applications. Furanone-based biomedical product applications under development include the production of antibacterial contact lenses and furanone-impregnated implants and catheters. Environmental applications include the development of marine antifouling paints and furanone-incorporated industrial products. Success in this area will greatly depend if furanones are proved nontoxic to human.62 Examples for new antibacterial agents from marine microorganisms include the massetolides A–H, cyclic depsipeptides from two Pseudomonas spp. obtained from a marine alga, marinone and debromarinone from a marine sediment– derived actinomycete, and microsphaeropsisin from a fungus isolated from a sponge, among others.50 Antiviral agents from marine microorganisms have been isolated. These include macrolactins A–F, the macrolides from a gram-positive deep-sea bacterium from the California coast, and caprolactins A and B from a marine Pseudomonas spp. These compounds inhibit herpes simplex viruses. In addition, macrolactin A is effective against HIV.50 Extracts prepared from marine bivalves such as green mussel (Perna viridis), estuarine oyster (Crassostrea madrasensis), giant oyster (Crassostrea gryphoides), estuarine clam (Meretrix casta), black clam (Villorita cyprinoides), and mud clam (Polymesoda erosa) were found to possess significant antiviral activity when tested against influenza virus strains type A and type B. Maximum inhibitory activity was found in P. viridis. The green mussel extract also possessed anti-HIV activity.25,62 A new depsipeptide from the sacoglossan mollusk Elysia ornata has also been isolated.63

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12.3.7 ANALGESIC AND HYPOTENSIVE DRUGS Ziconotide is a 25-amino acid linear peptide that is present in the predatory IndoPacific marine mollusk Conus magus. The drug has been approved by the U.S. Food and Drug Administration (FDA) in 2004. These are fish-hunting mollusks that use their venom to paralyze their prey. The remarkable analgesic activity of ziconotide, which is about 1000 times more potent than morphine, is due to its efficiency in blockage of calcium channels. Ziconotide appears to suppress pain by targeting and blocking specific neuron presynaptic ion channels (called N-type calcium channels) to short-circuit neurotransmitter release in nerves that transmit pain signals. The compound effectively blocks pain while still allowing the rest of the nervous system to function properly. This represents an important advantage over that of currently available opiates with more systematically suppressive effects.36 A neurotoxin obtained from a seagoing snail common in the Pacific has also been found to be a potent painkiller. Early clinical trials have shown that the substance relieves chronic pain and could be an alternative to morphine.17 Another compound is methopterosind from a soft coral.47 Some brown algae and their extracts have been used for centuries as hypotensive drugs in oriental medicine. Laminine, an active hypotensive agent, is isolated from Lonicera angustata. The potential pharmaceutical, medicinal, and research applications of these compounds have been pointed out.64 Table 12.4 presents the drugs derived from sponges and Table 12.5 indicates the drugs from marine sources, other than sponges and their mechanisms of action. Figure 12.1 shows the structure of some drugs of marine origin. The potentials of various marine products as sources of bioactive compounds has been discussed in the following section.

12.4

MARINE PRODUCTS HAVING POTENTIAL BIOACTIVE COMPOUNDS

Although the preceding section discussed potent drugs from various marine sources, this section discusses potentials of different marine products as sources of drugs and recent studies in the field. It is hoped that the information would encourage efforts to examine these groups of organisms for drug development.

12.4.1 CORALS Coral reefs organisms are known to possess several biologically active chemicals that can be of immense therapeutic applications. Therefore, corals and coral reefs are being targeted for studies across the world for their bioactive principles. Recent interest in the area is depicted by the study conducted by the University of Miami researchers, who using advanced sonar techniques discovered new deepwater reef sites 2000–2900 ft deep in the Straits of Florida between Miami and Bimini. The expedition was aimed at searching and testing the organisms from the reefs for the presence of new chemical compounds with the potential to treat human diseases. The efforts have led to the collection of thousands of marine organism samples and the identification of a number of promising potential drugs, which are now in various

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TABLE 12.4 Drugs Derived from Sponges Compound, Formula, Molecular Weight, and Source Bengamide A and B, C32H58N2O8, 598.812 g/mol Fijian sponges, Jaspis cf. coriacea Contignasterol (IZP-94005, IPL-576,092), C29H48O7 (08.687 g/mol) The sponge Petrosia contignata Debromohymenialdisine (DBH) (alkaloid), C11H11N5O2, 245.238 g/mol The Palauan sponge Stylotella aurantium Discodermolide, C33H55NO8, 593.792 g/mol Discodermia dissoluta (Caribbean deep sea) Giroline (girodazole), C6H11ClN4O, 190.631 g/mol Pseudaxinyssa cantharella Halichondrins (halichondrin B), C60H86O19, 1111.31 g/mol H. okadai from Japan KRN-7000, C50H99NO8, 842.323 g/mol Agelas mauritianus Manoalide, C25H38O5, 418.566 g/mol Indo-Pacific sponge Luffariella variabilis Topsentins (topsentin B1), C20H14N4O2, 342.351 g/mol Topsentia genitrix, Hexadella spp., and Spongosorites ruetzleri Dictyostatin Unidentified Jamaican sponge of family Corallistidae

Lasonolides Gulf of Mexico deep-sea sponge (Forcepia spp.)

Manazamine A, C36H44N4O, 548.761 g/mol The sponge Haliclona spp. Peloruside A The New Zealand sponge Mycale hentscheli Salicylhalamides (salicylihalamide A), C26H33NO5, 439.544 g/mol Haliclona spp.

Activity/Mechanism of Action Tumor growth inhibitor. Has antibiotic and anthelmintic activity (against the nematode Nippostrongylus braziliensis) Antiasthma agent. One contignasterol derivative, named IPL-576,092, shows promise as an oral asthma medication Anti-Alzheimer agent. Treatment against osteoarthritis Tubule-interactive agent. Apart from anticancer properties, possesses immunosuppressive and cytotoxic activity Inhibits protein synthesis in eukaryotic target cells Tubulin-interactive agent

Antitumor, immunostimulatory. Exhibits cytotoxic and antitubulin activity similar to dolastatins. Mitotic inhibition occurs by binding to tubulin Anti-inflamatory, antianalgesic. Inhibits phospholipase A2 (PLA2). Hold potentials against rheumatoid arthritis Anti-inflammatory agent. Shows promise in the treatment of pancreatic cancer. The exact mode of action is not yet fully understood, and is an area of active research Dictyostatin inhibits the growth of human cancer cells and are active against certain taxol-resistant tumors through prevention of the breakdown of tubulin during mitosis similar to the cancer drug taxol Anticancer activity. Latrunculins A and B have been shown to be cytotoxic. Alters cell shape, cytokinesis, and processes such as fertilization and early development Demonstrated activity against malaria, tuberculosis, and HIV Tubulin-interactive agent Vo-ATPase inhibitor. In addition has potential to treat tumor and osteoporosis

Source: Adapted from National Seagrant Program, Harbor Branch, California, www.marinebiotech.org, accessed December 2007.

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TABLE 12.5 Drugs from Marine Sources, Other Than Sponges Compound, Formula, and Molecular Weight

Source

Sarcodictyins, C28H36N2O6, 321 g/mol Dolastatin, C42H68N6O6S, 785.092 g/mol

The octocorals Eleutherobia spp. (Cnidaria) Corals Sarcodictyon roseum, Eleutherobia aurea Indian Ocean sea hare D. auricularia (Molluska)

Kahalalide F, C75H124N14O16, 1477.87 g/mol

Hawaiian sacoglossan mollusk (E. rufescens)

Spisulosine (ES-285), C18H40ClNO, 321 g/mol Ziconotide (PRIALT®, SNX111), C10H172N36O32S7, 2639.16 g/mol Bryostatin 1, C47H68O17, 905.033 g/mol Aplidine (Aplidin®, dehydrodidemnin B) Dideminin B, C57H89N7O15, 1112.35 g/mol Ecteinascidin-743, C39H43N30O11S, 761.84 g/mol Diazonamide A, C40H34Cl2N6O6, 765.64 g/mol Vitilevuamide

The Spisula (M. polynyma) (Molluska) Derivative of a conotoxide of cone snails Conus geographicus, C. magus (Mollusk) The bryozoan B. neritina (Ectoprocta) The tunicate A. albicans (Chordata) The tunicate T. solidum (Chordata) The tunicate E. turbinata (Chordata) The tunicate Diazona angulata (Chordata)

Eleuthrobin

Curacin, C23H35NOS, 373.596 g/mol Thiocoraline

Activity/Mechanism of Action A microtubule-binding agent similar to the cancer drug taxol Tubulin-interactive agents Mitotic inhibitor. Interferes with tubulin formation and thereby disrupts cell division by mitosis Disrupts lysosome membranes, initiates apoptosis, and inhibits tumor growth Antitumor agent Suppress pain by targeting and blocking specific neuron presynaptic ion channels Anticancer, antitumor, and immunostimulant activities Anticancer agent via apoptosis induction Anticancer agent via protein synthesis inhibition Anticancer agent via apoptosis induction Tubulin-interactive agent

The ascidians Didemnum cuculiferum, Polysyncraton lithostrotum (Chordata) L. majuscule (Cyanophyta)

Tubulin-interactive agent

M. marina, an actinomycete bacterium from Southeast Africa

Shows activity against breast, colon, and renal cancers and melanoma

Tubulin-interactive compound

Source: Adapted from National Seagrant Program, Harbor Branch, California, www.marinebiotech.org, accessed December 2007.

stages of development for treating cancer, Alzheimer disease, malaria, acquired immune deficiency disease syndrome (AIDS), and other ailments.65 Eight new noradrosinane sesquiterpenoids, laevinols A–H; a new neolemnane sesquiterpenoid, levinone A; and also other two previously known compounds were isolated from the soft coral Lemalia laevis. Their structures were elucidated and their cytotoxicity

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Drugs and Pharmaceuticals from Marine Sources O Galp 1α O

O

HN

Galp 1α O

C24H49 OH

OH

HN

HO

C22H45

O

O O

1. KRN-7000

2. Agelasphin 9a

H H

HO O

H

H

O

O

H

H

H

O

H

O

OH CO2Me

OMe O

H

H O

O

OAc

H

HO H H N

O

O O

H

O

O

O

O O

HOH2C

O

1

HO

20

3. Bryostatin 1

O

H

7

O

C13H27

HO

OAc

MeO2C

OH

C14H29

HO

385

O

N O

O

O

4. Isohomohalichondrine B

H OH O S

O MeO

NH

−

OSO3

HO +

Na

6. Squalamine H2N

5. Ecteinascidin 743

CKGKGAKCSRLMYDCCTGSCRSGKC H

N H

N H

H

OH

7. Ziconotide

FIGURE 12.1 Chemical structures of some drugs of marine origin. (Adapted from National Seagrant Program, Harbor Branch, California, www.marinebiotech.org; Matsunaga, T. et al., Adv. Biochem. Eng./Biotechnol., 96, 165, 2005. With permission from Elsevier.)

against selected cancer cells were measured in vitro.64–66 Polyaromatic alkaloids, known as lamellarins, have been isolated from the marine ascidian Didemnum spp. and mollusk Lamellaria spp. These compounds have been suggested to be of microbial origin. Samples of colonial ascidians, Aplidium spp., yielded three novel iodinated l-tyrosine alkaloids. Extracts of the colonial ascidian Synoicum castellatum yielded four compounds derived from prenylated hydroquinones. New cytotoxic cyclic heptapeptide, mollamide and patellins (cyclic peptides), have also been isolated from ascidians Didemnum molle and Lissoclinum spp. collected on the Great Barrier Reef.18,67 A compound termed as “38-bromotyramine” along with the previously reported sponge metabolite 1,3-dimethylisoguanine have been isolated. In addition, a new purine, 1,3-dimethyl-guanine, from the New Zealand ascidian Botrylloides leachi has also been isolated. Four new bromotyrosine derivatives (botryllamides A–D) from the styelid ascidian Botryllus spp. from the Philippines, and from Botryllus schlosseri from the Great Barrier Reef have also been identified.43 Corals have a narrow range of temperature tolerance, generally in the range 28–30°C (see Chapters 2 and 11). Intense sunlight and associated water temperatures can leave corals vulnerable to damage and death. The situation is further

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complicated by the release of photosynthetic oxygen by algal symbionts residing within their tissues, which, in combination with high light intensities, is a potential cause of hyperoxic toxicity and photooxidative stress. To be protected from sunlight, corals have developed sunscreens based on naturally occurring compounds in the marine organisms, with which the coral animals are able to withstand long-term exposure to physiologically damaging wavelengths of ultraviolet (UV) radiation. This resistance proved to be due to the presence of certain compounds (mycosporinelike amino acids) having nonaromatic chromophore with a high efficiency to absorb UV light. Such compounds are found in many marine organisms including sponges and sea anemones. The unique physical and chemical properties of these compounds as natural sunscreens prompted an investigation of their use in healthcare applications and in the formulation of cosmetic products. A novel UVabsorbing compound, a methylamine-substituted mycosporine, was isolated from methanol extracts of two hermatypic reef-building corals Pocillopora damicornis and Stylophora pistillata.43,68,69 Another compound dendra, a PAFP, has also been recently isolated, as mentioned earlier.69 Most sponges have siliceous spicules that serve as skeletal elements. Sponges employ protein matrices to precipitate biogenic opal in a highly ordered fashion. Thus, isolation, characterization, and eventual encoding of these proteins (cathepsins, silicateins, etc.) might lead to commercially interesting compounds.70 A library of extracts from different sponges from various locations has been prepared and kept freeze-dried as potential sources of bioactive compounds. Extensive studies using isolated human trial mycocytes exposure showed the potential of at least a few sponge extracts in protecting against cardiovascular disease.71 Researchers have discovered unique chemical compounds from a sponge, Plakinistrella, found in the Indian Ocean that could lead to new treatment for fungal infections that threaten the lives of AIDS and cancer patients. The compounds are members of a completely different class of antifungal agents called cyclic peroxide acids.47 Dragmacidins, a newly discovered class of bis(indole) alkaloids, have been isolated from a variety of marine sponges having antiviral and anticancer activities. A Spongosorites spp. collected off the southern coast of Australia gave the new alkaloid dragmacidin E. This compound and its cometabolite dragmacidin D can function as potent inhibitors of serine–threonine protein phosphatases.72 The compounds have also been detected in other sponges such as Coscinoderm lanuga and Aplysina fulva.73 Recent Australian research has shown that the tropical marine sponge Haliclona spp. from Heron Island in the Great Barrier Reef contains symbiotic dinoflagellates and nematocysts. The cytotoxic alkaloids associated with Haliclona spp., haliclonacylamines A and B, were within the sponge cells rather than within the dinoflagellates. The ability to synthesize bioactive compounds such as the haliclonacyclamines may help Haliclona spp. to preserve its remarkable ecological niche. Arenochalina mirabilis collected from the Great Australian Bight contains six tricyclic alkaloids (mirabilins A–F) isolated and identified as their N-acetyl derivatives. A new sesterterpene tetronic acid exhibiting antimicrobial activity has been isolated from another Australian marine sponge Psammocinia spp. Mycalone, a new steroid, has been isolated from the southern Australian marine sponge Mycale spp. The observed

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cytotoxicity of the New Zealand sponge Hymeniacidon hauraki has been attributed to a furan derivative of hexanoic acid. New chlorinated metabolites have been identified in the sponge Dysidea herbacea. Two new sesquiterpenes, an isonakafuran and its autoxidized derivative, were also isolated from the sponge. Both metabolites inhibited the growth of the P388 murine leukemia cell line. The sponge Dictyodendrilla spp. from Port Phillip Bay, Victoria, yielded three new oxygenated sesquiterpenes (dictyodendrillins A–C), together with the known sesquiterpene dendrolasin.43 A compound 11-epi-sinulariolide from the soft coral S. flexibilis exhibited strong algicidal properties against the common fouling alga Ceramium codii. Field experiments with treated settlement tiles confirmed that the compound was active against several fouling algae. A new sesquiterpene as an antifouling agent has been isolated.43,74 Some of the other bioactive compounds isolated from sponges include calafianin and two known compounds, aerothionin and 3,5-dibromo-2-hydroxy-4-methoxyphenylacetic acid;75 phorbasin A, a diterpene;76 callyspongamide A, a new cytotoxic polyacetylenic amide;77 epidioxy sterols as antifouling substance having repellent activity;74 antibacterial pyrroloiminoquinone;78 a new triene aldehyde having moderate growth-inhibitory activity toward HeLa S3 cells;79 stellettazole alkaloid, having antibacterial activity; two antimicrobial compounds, namely, lysoplasmanylinositols and C14 acetylenic acid;80 microxine, a new cdc2 kinase inhibitor;81 an antiangiogenic matrix metalloproteinase inhibitor;82 two new dibromotyrosine-derived metabolites;83 miraziridine A, a cysteine protease inhibitor;84 a novel diterpene, having mild cytotoxicity against acute promyelocytic leukemia, HL-60 cells;85 mycapolyols A–F, new cytotoxic compounds;85 the antineoplastic agents, scalarane-type pentacyclic sesterterpene, sesterstatin 6, having significant anticancer activities against murine P388 lymphocytic leukemia and a series of human tumor cell lines86 and cheilanthane sesterterpenes, having protein kinase inhibitor activities.33 It is to be mentioned that because of intense symbiotic association among corals and microalgae as well bacteria, it is often very difficult to separate the compounds that are being produced by the algae and bacteria from those from sponge/coral posing problems for biochemists and chemists. Discoveries of new compounds from sponges and other corals are regularly being reported in journals in the areas of organic, marine, and natural products chemistry.

12.4.2 MARINE MICROORGANISMS Microorganisms in the marine ecosystems usually produce bioactive substances that are different from known compounds from terrestrial organisms. Marine microorganisms such as bacteria, cyanobacteria, dinoflagellates, and others are generally associated with marine plants and animals. As mentioned earlier, because of their symbiotic associations, natural products isolated from higher marine organisms such as marine invertebrates are frequently of bacterial origin. It is also justifiable to assume that because marine animals and plants are continuously exposed to a large diversity of harmful microorganisms, potential hosts might produce bioactive compounds to deter microbial attack.87,88 Despite the limitations in determining the origin, several novel antimalarial antibiotics,

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antitumor polysaccharides, glucan-degrading enzymes, and aminoglycoside antibiotics have been isolated from marine microorganisms.5,9,50,89,90 Many of the microbial compounds function as signal transducers and thus regulate complex processes within marine living societies. Salinosporamide A is a potent proteasome inhibitor used as an anticancer agent that recently entered phase I human clinical trial for the treatment of multiple myeloma. The novel natural product is produced by the recently described obligate marine bacterium Salinospora tropica, which is found in ocean sediment. Salinosporamide A belongs to a family of compounds possessing a dense functionalized γ-lactam-β-lactone bicycle.34 Neomarinone, a novel metabolite possessing a new sesquiterpene- and polyketide-derived carbon skeleton, and several derivatives were isolated from the fermentation broth of a taxonomically novel marine actinomycete. Neomarinone and several of the marinone derivatives were shown to be moderately cytotoxic toward human cancer cells in in vitro testing.35 A growing number of marine fungi are the sources of novel and potentially lifesaving bioactive secondary metabolites.91 Filamentous fungi have long been used by the fermentation industry for the production of metabolites including antibiotics and enzymes. With developments in genetic engineering and molecular biology, filamentous fungi have received increased attention as hosts for recombinant DNA. Along with the developments in molecular genetics, the research on bioprocessing technologies may have a competitive advantage for production of enzymes and healthcare products including generic biopharmaceuticals from fungi.92,93 Employing variable carbon and nitrogen sources in the medium, fungi can be cultivated for the isolation of a number of metabolites such asglucoamlyase, amylase, α-glucosidase, and proteases. The current status of natural products from marine fungi and their potential as novel antibacterial, antiviral, and antiprotozoal compounds together with their possible roles in disease eradication have been discussed recently.94

12.4.3 MARINE PLANTS Marine plants consist of all photosynthezing organisms including algae, seaweeds, and grasses. Marine macroalgae have long been used as food and source of food additives (see also Chapters 9 and 10). During the past three decades, marine algae have attracted attention of both chemists and biologists all over the world as a source of bioactive compounds. Recent studies have provided substantial information on a wide spectrum of biological properties of algal components, which include antifungal, antibacterial, antiviral, ichthyotoxic, hypotensive, helminthic, and other activities. Most of these substances isolated from marine algae may be classified chemically as brominated aromatics, nitrogen heterocycles, nitrogen–sulfur heterocycles, sterols, terpenes, dibutenoloids, proteins, peptides, and sulfated polysaccharides (carrageenans). Several metabolites with unusual structures having biological activities have also been isolated from these organisms. The prominent biological activity of marine terpenes is evident in their ecological role in the marine environment, which makes them potential drugs. Several terpenoid compounds, for example, eleutherobin, sarcodictyin, and contignasterol

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derivatives, are in preclinical or clinical development. A new sesquiterpenoid metabolite and related known metabolites deoxyprepacifenol and pacifenol in the red alga Laurencia marianensis have been reported. Others include a reverse isoprenylated phenol and the related compounds from the marine brown alga Perithalia, along with a new sesquiterpenephenol, and cis-dihydroxytetrahydrofuran from the brown alga Notheia anomala. Tetrahydrofurans from N. anomala are potent and selective inhibitors of the larval development of parasitic nematodes.39 These compounds possess antifungal, antibacterial, antiviral, ichthyotoxic, hypotensive, or helminthic activities. The red alga D. pulchra produces halogenated furanones that inhibit bacterial settlement, attachment, and growth. The alga can transfer halogenated furanones to the sea hare Aplysia parvula, which is accumulated up to 13.3% of the dry weight, particularly in the digestive gland, suggesting that the sea hares differentially bioaccumulate algal metabolites. Furanones interfered with the expression of bioluminescence, swarming (surface) motility, and exoenzyme synthesis in different bacterial species. Furthermore, adhesion and swarming in a range of marine bacteria were inhibited by furanones at concentrations that did not affect growth. Other substances such as macroalgal lectins, kainoids (a neurotoxin isolated from the red seaweed Digenea simplex), and aplysiatoxins are routinely used in biomedical research and a multitude of other substances have known biological activities.95 Table 12.6 gives biological activities of some major seaweed components that have potential in drug development. Lipophilic and hydrophilic extracts of three sea grasses (and also 49 marine algae) were screened for antimicrobial activities against five ecologically relevant marine pathogens including two fungi and bacteria. Overall, 90% of the extracts screened were active against one or more organisms, whereas 77% of the extracts were active against at least two organisms. Extracts of the green algae Halimeda copiosa and Penicillus capitatus (Chlorophyta) were active against all the organisms. It was concluded that antimicrobial activities are prevalent among extracts from marine algae and sea grasses, suggesting antimicrobial defenses exist among marine plants.88 Seventeen species of marine plants were screened for analgesic activities. Among these, three mangrove species, Acanthus ilicifolius, Avicennia marina, and Excoecaria agallocha, showed dose-dependent analgesic activity at 160 mg/kg. The root of A. ilicifolius produced a maximum activity of 89.7%, whereas leaf and flower showed 57% and 38% activities, respectively. Similarly, the E. agallocha exhibited 87% activity, whereas A. marina had an activity of 27%. However, all plants were less effective when compared to morphine, having an ED50 value of 0.25 mg/kg.37 Despite the intense research efforts by academic and corporate institutions in the area of biological activities, very few seaweed products with real potential have been commercialized. Substances that currently receive most attention from pharmaceutical companies for use in drug development or from researchers in the field include sulfated polysaccharides (fucoidans) as antiviral substances and antifouling compounds. Several authors have discussed the secondary metabolites and bioactive compounds produced by marine organisms and their potentials as drugs.19,30,96,97

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TABLE 12.6 Biological Activities of Some Major Seaweed Components Relevant in Drug Development Components Fucoidans

Carrageenans and carrageenan—oligosaccharides

Fucoxanthin and laminarin (carotenoids)

Others

Biological Activities Antibacterial Antioxidant Antithrombin Antiviral (herpes, simplex, cytomegalovirus) Anticoagulant Anti-inflammatory Antiadhesive Antiangiogenic Inhibits smooth muscle cell proliferation Reduces protein kinase activity Antitumor activity Sequesters paralytic shellfish poison Elicits plant defense Immune response Anticancer activity (inhibits human leukemia cells and induces apoptosis) Antiangiogenic Antiapoptotic activity Chemopreventive and chemotherapeutic activity Brominated aromatics, nitrogen heterocycles, nitrogen–sulfur heterocycles, sterols, terpenes, dibutenoloids, proteins, and peptides

12.4.4 MARINE TOXINS AS DRUGS Some marine organisms such as microalgae elicit toxins (see Chapter 15). Some of these toxins can also function as potential drugs. Cone-shells, highly prized by collectors for their attractive patterns, have recently attracted the attention of researchers because venoms of Conus spp. provide chemical templates for the design of new drugs of value. Of the 500 species of Conus, worldwide, over 80 are found on the Great Barrier Reef in Australia. The neurotoxins isolated from cone-shell venoms are a diverse group of small, disulfide-rich peptides called conotoxins. Although the venoms of several species have caused human fatalities, toxins of certain other species show potential as novel drugs for use in pain management, epilepsy, and the prevention of stroke. The α-toxins from different species of Conus exhibit selectivity toward either muscle or brain receptors.60,20 Most of the active peptides isolated to date from the venoms have high specificities for ion channel and receptor types and subtypes, which make them valuable as diagnostic tools in the characterization of neural pathways, as therapeutic agents in medicine, and potentially as biodegradable toxic agents in agro-veterinary applications. Vibrantly colored creatures from the depths of the South Pacific Ocean harbor toxins that can act as powerful anticancer

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drugs, according to University of Madison biochemists and their Italian colleagues. These scientists have identified the structure of the toxins and provided a basic understanding that can be used to synthesize pharmaceuticals. The toxins, which are produced naturally by organisms that exist symbiotically on deep-sea sponges, work by disrupting the activity of actin, a muscle protein (see Chapter 3). Because cancer cells grow faster than other cells in the body, actin provides an easy target for drugs that could inhibit such rapid growth.20 Toxins of marine snails are also of interest, especially conotoxins in signal transmission research and due to their analgesic properties.3 Conotoxin has been produced in significant amounts.3

12.4.5 FISH AND SHELLFISH Marine fishery products including the abundant catch of underutilized fish and processing wastes are good sources of certain bioactive compounds.98 Bioactivities of certain compounds including proteins, peptides, lipids, chitin, chondroitin sulfate, and minerals from marine fishery sources have been pointed out elsewhere (see Chapters 3 through 7). Some of the fish-based compounds have been recently developed for bone diseases. For instance, an oligophosphopeptide isolated from hoki (Johnius belengerii) bone could be utilized as a nutraceutical with a potential calcium-binding activity. The compound was isolated from the fish bone waste after digestion with an enzyme extracted from the intestine of bluefin tuna (Thunnus thynnus), also a by-product from industrial processing of tuna. The hoki FBP containing 23.6% of phosphorus from the hoki had a molecular weight of 3.5 kDa, which interacted with calcium binding 41.1 mg/L of soluble calcium at a pH of 7.8, without the formation of insoluble calcium phosphate.99 Although not from any fishery item, another seawater-dependent remedy for bone disease has been identified recently. Scientists from the U.S. Department of Energy’s Lawrence Berkeley National Laboratory have harnessed the way seawater freezes to develop a porous, scaffoldinglike material that is four times stronger than material currently used in synthetic bone. These developments suggest that marine products could be used for bone therapy in the future.100 High blood pressure is a significant risk factor for heart disease and related medical conditions. Moderately elevated blood pressure is a very attractive target for the functional food/natural health product and nutraceutical industries as it is one of the relatively small group of health conditions where the consumer can readily assess alternative therapies. Researchers have been aware of the potential of bioactive peptides from food proteins for more than 20 years. Some peptides from fishery products (see also Chapter 4) could be applied for controlling blood pressure.101 Several of these products have been demonstrated to produce a therapeutically significant reduction in blood pressure in animal models and in human clinical trials. Business opportunities and challenges for this new class of compounds have also been discussed.99 A phenolic antioxidant, with an amino group and having a molecular weight of 164, has been purified from shrimp shell. The antioxidant compound was identified as 1,2-diamino-1-(o-hydroxyphenyl) propene.100 Reef fishery could be a source of biomolecules. However, the potential of fishery (at least in tropical waters) including reef fishery to sustain the amount fish that would have to be taken for practical production

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of either a nutraceutical or a pharmaceutical is doubtful because of the large amount of raw material required to isolate the negligible amount of biomolecules present. 12.4.5.1

Sea Cucumber

Sea cucumbers are found in shallow water areas of the sea to deep ocean floors and are deposit feeders—living on organic matter and associated microorganisms (see Chapter 2). Several species of sea cucumbers have been shown to possess antibacterial, antifungal, antioxidant, and anticoagulant activities. (These creatures, however, also contain toxic compounds, such as holothurin.) Antibacterial activity was detected in extracts from different body parts and eggs of the sea cucumber Cucumaria frondosa. The animal also exhibits high antioxidant activity, which is present in the digestive tract, gonads, muscles, and respiratory organ of the organism. The antioxidant activity is related to the phenol content, which varies from 22.5 to 236 mg gallic acid equivalents per 100 g dry weight, and flavonoids from 2.9 to 59.8 mg of rutin equivalents per 100 g. The oxygen radical activating capacity (ORAC) values range from 140 to 800 μmol of Trolox equivalents per gram. The activity was highest in the acetone extracts of the digestive tract, followed by muscles, gonads, and respiratory apparatus. A significant correlation was observed between ORAC values and total phenol content in extracts and fractions of gonads and muscles, but not in similar extracts of digestive tract and respiratory apparatus. The results suggested that C. frondosa tissues could be useful sources of antioxidants for human uses.102 A triterpene glycoside, leucospilotaside A, isolated from the sea cucumber Holothuria leucospilota, has been characterized.103 Relatively high antibacterial activity was present in the gastrointestinal organs and eggs from the starfish Asterias rubens and lysozymelike activity in several tissues of the starfish. Hemolytic activity could be detected in all the species tested, especially in the body wall extracts. These activities were also detected in the green sea urchin Strongylocentrotus droebachiensis, suggesting that marine echinoderms could be potential sources of novel antibiotics.21,22 A new triterpene glycoside, patagonicoside A, has been isolated from the sea cucumber Psolus patagonicus. The compound is a disulfated tetrasaccharide with a new aglycon moiety, and exhibited considerable antifungal activity against the pathogenic fungus Cladosporium cucumerinum.104 Antifungal activity of another sea cucumber, Actinopyga lecanora, has also been reported.105 The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose-rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber’s body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue.106 The cucumber cell wall polysaccharide was comparable in backbone structure with the mammalian chondroitin sulfate, but some of the glucuronic acid residues displayed sulfated fucose branches. The specific spatial array of the sulfated fucose branches in the fucosylated chondroitin sulfate not only conferred high anticoagulant activity to the polysaccharide, but also determines differences in the way it inhibits thrombin.107 Sulfated glycans exhibit a wide range of biological activities, which include anticoagulant activity, venous antithrombic activity, and recombinant HIV reverse transcriptase activity.

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The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans have been well recognized. Furthermore, fucoidans are reported to have antitumor, antimutagenic, anticomplementary, immunomodulating, hypoglycaemic, antiviral, hypolipidemic, and anti-inflammatory activities.108,109 12.4.5.2

Jellyfish

GFP is a novel compound that was first isolated from the jellyfish Aequrea in 1962. Since then numerous applications of GFP have been identified. These include its function as a reporter gene, cell biology and functional roles as researcher’s aids in drug development, and related activities. It is an invaluable tool for the biotechnologists for bioprocess monitoring to in vivo studies on protein interactions, screening of drugs, assay of protein activity, and even resolution of protein structures.23,24 A related compound has also been identified from a sea coral. A glow emitted by a variety of sea coral helped Russian scientists harness a protein that generates the light to create a tiny fluorescent tag that responds to visible light. The two-color tags are expected to help researchers follow individual proteins as they are transported inside living cells. Under a microscope, the two-color tag—called dendra (because it is derived from the sea coral Dendronephthya)—first shows up as a green glow, highlighting the otherwise invisible protein to which it is attached. The green turns to red when the tags are zapped with an intense pulse of visible blue light. Dendra is the latest addition to the growing family of PAFPs, comparable to the GFP isolated from jellyfish.70 12.4.5.3

Bivalves

Mollusks are another important species that have a wide range of uses in pharmacology. More than 400 compounds having pharmacological value have been described in the literature. Some of these can function as hypotensive agents; cardioactive substances; muscle relaxants; and antibiotics, antiviral, and antitumor agents.3,26 The marine environment contains many examples of interesting robust adhesive strategies that can inspire design of novel synthetic adhesives. Marine organisms such as mussels, barnacles, and reef-building worms employ specialized protein glues, collectively referred to as mussel adhesive proteins (MAPs), which adhere well to surfaces despite the presence of water. Studies have shown that the catecholic amino acid, l-3,4-dihydroxyphenylalanine (DOPA), which is abundantly found in MAPs plays a key role in establishing chemical interactions between MAPs and various metal, metal oxide, and polymer surfaces. The optimal curing of MAPs occurs in the presence of an oxidizing metal ion (e.g., MnO4− and Fe3+). The polymer mimics are being designed for medical and nonmedical applications. The polymers may be used as injectable liquids for surgical tissue adhesion or as components of mucoadhesive drug delivery systems.27,28 Using analogies from nature, the possibility of tyrosinase-catalyzed reactions of 3,4-dihydroxyphenethylamine (dopamine) on conferring water-resistant adhesive properties to dilute solutions of chitosan was investigated. Rheological measurements showed that the tyrosinase-catalyzed, and subsequent uncatalyzed, reactions lead to substantial increases in the viscosity of the chitosan solutions. Adhesive shear

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strengths of over 400 kPa were observed for these modified chitosan samples, whereas control chitosan solutions conferred no adhesive strength (i.e., the glass slides separated in the absence of measurable forces). High viscosities and water-resistant adhesive strengths were also observed when semidilute chitosan solutions were treated with the known cross-linking agent glutaraldehyde. These results demonstrate that the renewable biopolymer chitosan can be converted into a water-resistant adhesive. These modified adhesives could have medical applications as scaffolds for rebuilding of bones.110 The ink of squid and cuttlefish has found to possess some therapeutic activity. It is being studied as a potent homeopathic medicine (Kasim, S. A., Personal communication, 2007).

12.5 MARINE BIOTECHNOLOGY Marine biotechnology encompasses those efforts that help harnessing marine resources of the world. Recent developments in this field, besides isolation of drugs, include the development of marine ingredients in food and other industries, seaweed farming, and monitoring ocean pollution. A synthesis uniting marine natural products, ecology, aquaculture, and bioremediation research under the heading of marine biotechnology was initiated only in the 1980s. Genomic tools are now being applied to optimize the production of drugs from marine sources. In the case of marine microorganisms, attempts are focused to develop their cultures under commercial conditions. Bioprocessing strategies in microbial cultivation include solid-state or submerged-state fermentation, the latter being popular for fungi in the industry. Growth and production are affected by wide range of parameters, including cultivation medium, inoculum, pH, temperature, aeration agitation, and shear stress. Immobilization technology has also been employed for growth of microorganisms for metabolite production. Immobilization has several advantages over dispersed cells, such as easy separation, continuous culture, and downstream processes. Various immobilization methods and materials including adsorption, entrapment, crosslinking, and covalent bonding have been employed. Compared to many unicellular microbes, fermentation of filamentous fungi presents special challenges in optimization and scale-up because of the varying fungal morphological forms.93,111 Governments and intergovernmental agencies are committed to funding and expanding oceanic research. However, sustainable efforts are required to discover and study the ocean’s vast, unplumbed resources.112

12.6

DEVELOPMENT OF MARINE DRUGS

Development of a new drug starting from a marine (or terrestrial model) requires usually 12–15 years of work and an expense of US$700–800 million. The work includes fundamental research related to in vitro screening of compounds, characterization of a lead compound, and in vivo bioassays on laboratory animals followed by three phases of clinical trials in human (phase I, tolerance; phase II, therapeutic interest; and phase III, large-scale bioassays). After successful clinical trials, the drug is approved and marketed. The success rate of finding a new active chemical is about 500 times higher in marine organisms than from terrestrial sources.13

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Until now traditional screening methods have mostly been used. In recent years, the improvement of screening technologies has yielded a considerable number of potential new drug candidates and other metabolites from microorganisms of marine ecosystems. Improved screening methods and development of technology will allow immense progress in the drug development process. In recent years, the progress of screening technologies has simplified the detection of potential new drug candidates and other metabolites from microorganisms of marine ecosystems. Nuclear magnetic resonance (NMR) is one of the most essential tools needed by biotechnology facilities involved in drug discovery research because it enables to unravel the chemical structure of newly discovered compounds having disease-fighting potential. Developments in genomics and proteomics have boosted success in this field. The sequencing of marine genomes and the techniques of functional genomics (including transcriptome, proteome, and metabolome analyses) open up new possibilities for the screening of new metabolites of biotechnological interest. Although the sequencing of microbial marine genomes has been somewhat limited to date, selected genome sequences of marine bacteria and algae have already been published. Application of the techniques of functional genomics, such as transcriptome proteome, and metabolome analysis in combination with high-resolution two-dimensional polyacrylamide gel electrophoresis and mass spectrometry will lead to a better understanding of disease mechanisms and the identification of new drug targets. Techniques on the target analysis of antimicrobial compounds by proteome or transcriptome analysis of bacterial model systems are also available. An understanding of the physiology of uncultivable microorganisms by environmental genomics can lead to the discovery of new biotechnologically relevant enzymes and genes that code for bioactive metabolites. The revolutionary development in the field of functional genomics during the past 10 years supports this assumption.46 Besides, with advances in organic chemistry, synthesis of novel compounds using natural products as models and templates is also likely to make rapid progress.55,112 Current organic synthesis, however, is a challenging task as the complex molecular structure of marine metabolites. Enzymatic methods are also being coupled in synthetic processes. Enzymes can be carefully extracted from a sponge or coral and used as a tool to manufacture a chemical product. The method is to clone the enzyme responsible for the production of the drug into a host such as Escherichia coli. As yet, it has not been achieved for any marine drug, but is likely to be accomplished in the near future. The benefit of inserting the genetic material from the sponge into a bacterium is that bacteria can be fermented in large reactors and thus produce much more drug than would be available in nature. Recently, many of these compounds were discovered making use of advances in genetics and applying new screening technologies. In many instances, the discovery of a novel natural product serves as a tool to better understand targets and pathways in the disease process. The recent progress in drug discovery from natural sources including several examples of compounds has been reviewed.113 Despite these current limitations, it is felt that new discoveries will certainly be increasing in the rising future as marine biomedical research develops to meet future needs. At present, processing by-products of seafood offer an economically viable alternative for bioactive compounds such as calcitonine, chondroitin, and chitosin-derived products (see Chapter 7).

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12.6.1 PROBLEMS IN MARINE DRUG DEVELOPMENT At present, only a few compounds found in marine sources have been produced commercially, although the prospects of finding newer compounds are high. Marine invertebrates, which theoretically offer immense potentials, have so far given a few commercially viable products, whereas a few others are in different phases of clinical trials, as indicted in Table 12.7. The limited success in developing drugs from marine sources is attributed to problems such as lack of adequate and consistent supply of raw material and paucity of cost-effective techniques for their isolation and identification. Part of the problem is that most of the searches for unique compounds tend to be concentrated in tropical waters, especially around reefs. Although there is a very large and great variety of species, the biomass (the actual amount of each species) available for recovery of copious amounts of the compound is very low since the compounds are generally present in the organisms in minute quantities. Therefore, a large amount of raw material is required for the isolation of individual drugs, the average annual demands for which are likely to be in the range 1–5 kg. For example, to obtain 1 g of an anticancer agent found in tunicate, about 1 t of the organism has to be harvested. Harvesting such large amounts of rare or endangered species may be against nature and not acceptable to society as a whole. In addition, unequal distribution of the resources also hinders the availability of the raw material. Destruction of marine environments through overfishing, pollution, and also global warming are showing adverse impacts on the sustainability of many valuable organisms. As a result, concerns have been raised that opportunity to harvest marine organisms as source of these compounds for commercial purposes may become limited.3,13 The estimated taxonomical diversity of marine microorganisms in general indicates the powerful potential of novel bioactive substances produced in aquatic ecosystems. Successful cultivation is usually the prerequisite for isolation, screening, and final application of natural compounds from marine microorganisms. However, many of these organisms, which are in a symbiotic association with higher organisms, cannot be usually cultivated alone in a pure culture since their growth depends directly on the activity of their hosts. Therefore, only a minority of free-living marine microorganisms has been identified and cultivated so far. The focus on the physiology and the potential of bioactive substances of noncultivable marine microorganisms is an important challenge at present and for the future. Furthermore, the taxonomical identification of marine microorganisms in general is still in its infancy.13 Nowadays, many drug firms look to the marine environment as sources of model chemical compounds which are then synthesized in the laboratory and used for testing. If the useful compound is found, it is then scaled-up on an industrial scale for production. It is unlikely that marine resources will be used for large harvesting in the drug industry. Pricing is another problem in using marine raw material. During World War II, the drug industry used cod and salmon livers as a source of vitamin A. Prices for the liver (stated in barrels) could swing from a few dollars per barrel to over $200–$500. The enormous and erratic pricing scheme made costing of the finished product very difficult. In addition, the wild sources also had highly variable vitamin A content, also making the processing and quality assurance difficult (Wekell, J., Personal communication, 2007).

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Mollusk Nemertina Sponge (A. mauritianus) Tunicate Sponge (Luffariella variabilis) Jellyfish (Aequora victoria) Mussel Perna canaliculus

Phase II Phase II Phase I Phase II On market On market On market

GFP GLME

Reporter gene Arthritis

Antimitotic Alzheimer’s Cancer Cancer Phospholipase A inhibition

Anthelmintic Antileukemic Antibiotics Antiviral Anticancer (bladder) Antiangiogenic Antipain Inflammation/wound Anticancer Anticancer (melanoma) Anticancer

Activity

Protein Glycosaminoglycans and long-chain fatty acids

Peptide Alkaloid Glycosphingolipid Peptide —

Alkaloid Macrolide Macrocyclic depsipeptide

Cyclic amino acid Nucleoside (arabinose) β-Lactams Nucleoside Peptide Sulfated aminosterol Peptide (ώ-conotoxine)

Chemical Class

Source: Adapted from Bourguet-Kondracki, M.-L. and Kornprobst, J.-M., Adv. Biochem. Eng./Biotechnol., 97, 106, 2005; Liebezeit, G., Adv. Biotechnol. Eng. Biotechnol., 97, 1, 2005; Proksch, P., Edrada, R. A., and Ebel, R., Appl. Microbiol. Biotechnol., 59, 125, 2002.

Rhodophyceae Sponge Fungus Sponge Mollusk Shark (Squalus acanthias) Cone snail (Conus magnus) Soft coral Ascidian Bryozoan Ascidian, tunicate

Origin

On market On market On market On market On market On market Phase III Phase I Phase II/III Phase II Phase II

Status

Kainic acid Cytarabine (Ara-C) Cephalosporins Spongoadenoside (Ara-A) Keyhole limpet hemocvanin Squalamine (Squalamax) Ziconotide (SNX-111) Methopterosin Ecteinascidin-743 Bryostatin 1 Dehydrodidemnine B (Aplidin) Dolastatine-10 GTS-21 KRN-7000 Didemnin Manoalide

Name

TABLE 12.7 Drugs of Marine Origin Marketed or in Different Phases of Clinical Trials

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The problems faced with regard to raw materials could be, at least, partially addressed by aquaculture (mariculture). Aquaculture of marine invertebrates under controlled or natural conditions has been attempted at least on a limited scale. However, culturing of some marine organisms is rather difficult because of their symbiotic association, as mentioned earlier. The prerequisites for culturing organisms are larval production, development of ideal composition of the medium, and feeding and harvesting. The ascidian Ecteinascidia turbinate, which is the source of promising anticancer compound ecteinascidin-743, is currently produced by aquaculture.114 Another important issue to be considered in the fermentation of microorganisms for a bioactive compound will be variability of contents of the required compound from batch to batch. Therefore, optimization of fermentation conditions is a prerequisite.

12.7

GLOBAL INTERESTS AND COMMERCIAL STATUS

Interests in marine natural products for healthcare are worldwide, as also indicated by specific examples mentioned in this chapter. In the United States, the National Sea Grant College Program, under the National Oceanic and Atmospheric Administration, is undertaking active research in the area. Sea Grant is a network of about 30 university-based programs in coastal and Great Lakes areas that involves more than 300 institutions. The Sea Grant program has compiled information on marinebased natural products of biomedical significance. Information provided for each product entry includes compound source, bioactivity, and clinical status, as well as structural and chemical attributes and commercial development highlights.4 The University of California, San Diego (La Jolla), has an extensive program on looking at reef fishes and other species (plankton, corals, and sponges) as potential sources of the interesting novel bioactive compounds under the leadership of Dr William Fenical (http://fenical.ucsd.edu/index.htm). In the South American coast, ample potential for marine drugs has been indicated. The 8000 km of the Brazilian coast line has a great biodiversity, constituting one of the most important unexplored sources of biologically active compounds. Recently, emphasis has been laid on a multidisciplinary collaborative program for the isolation, structure elucidation, and evaluation of biological activities from sponges, ascidians, octocorals, mollusks, and fungi from the Brazilian coast.114,115 Natural products research is being carried out in both Australia and New Zealand, encompassing the great diversity of organisms in Australasian waters and the large range of compounds present. Several marinederived drugs including those from microalgae, macroalgae, microorganisms ascidians, bryozans, corals, and sponges are in advanced stages of trial, and some are likely to reach market.43 In India, the government has taken up a national coordinated program on the development of drugs from ocean to harness potential bioactive substances, which is being implemented by the Central Drug Research Institute, Lucknow. Over 4000 samples collected from 800 different species of marine flora and fauna were examined for bioactive compounds, which gave about 500 isolates exhibiting various biological activities and identification of two compounds, one for antidiabetic and another for antihyperlipidemic activity. The phase I clinical trial of the former has been completed for the single dose, and the multidose trial is underway. A 90-day chronic toxicity study on the antidyslipidemic preparation has also been completed successfully in monkeys.46

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There are a few marine-derived drugs in the late stages of development particularly in the anticancer, antiviral, and anti-inflammatory areas. Some algal metabolites are being used clinically such as kainic acid as a vermifuge and cephalosporin as an antibiotic. The extract of the red seaweed Digenia simplex, an efficient vermifuse, is commercially available in some parts of Asia as “Helminal.” Kainic acid and allokainic acid are the active principles isolated from it. Chondriamaide C, an indole alkaloid, was isolated from the extract of Chondria atropurpurea that showed good anthelmintic activity. A α-1-glyceryl-d-mannoside-4-ammonium salt was found in Cologlossa leprieuri, which is a traditional Chinese drug used as an anthelmintic. An Indonesian specimen of the red alga Ceratodictyon spongiosum contained the anti-inflammatory cyclic heptapeptide ceratospongamide. The protein phosphatase 2A inhibitor, thyrsiferyl 23-acetate, was isolated from Laurencia obtusa. A lipid peroxidation inhibitor martegragin A was isolated from Martensia fragilis. Brominated diphenyl ether, which inactivates a glucosidase enzyme, was reported from Odonthalia carynbifera. Marine adenine cytokinins (MACs) is a new commercial seaweed product made from Fucus serratus L. Cytokinins were isolated from this extract by cation exchange and paper and high-performance liquid chromatography, and were detected by bioassay-guided fractionation using the soybean callus assay. Trans-zeatin, dihydrozeatin, and isopentenyladenosine were identified by gas chromatography-mass spectrometry (GC-MS) as being the dominant cytokinins in the seaweed. The allokainic acids that show mild action against worms have been found to show neurophysiological activity when given intravenously.97,116,117 Table 12.7 depicts the molecules of marine origin already marketed as drugs or currently in different phases of clinical trials. In addition to drugs, marine biotechnology offers other products such as those for agriculture (plant growth regulators, fertilizers, fungicides, pesticides and feeds, and aquaculture), industry (chemicals, coatings, detergents, paper, and textiles), foods and beverages (nutraceuticals), and environmental remediation and pollution control (bioindicators, water/waste water treatments). The global market for marine biotechnology products and processes was estimated at US$2.1 billion in 2002, a 9.4% increase from 2001. The non-U.S. market for marine biotechnology applications in 2002 was at more than $1.6 billion and is projected to grow annually at 6.4.118 In conclusion, marine organisms provide ample scope for the development of drugs that can address various human ailments. A few drugs of marine origin are already being used in the treatment of some cancers and other ailments. There is a great scope for the search for novel compounds from the marine environment that can serve as models for drug development. With the current technologies in bioengineering and synthetic chemistry, pharmaceutical industry can achieve success in the development of drugs from marine resources.

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97. Sarma, A. S., Daum, T., and Muller, W. E. G., Secondary Metabolites from Marine Sponges, Ullstein-Mosby, Verlag, Berlin, 1993. 98. Kim, S. K. and Mendis, E., Bioactive compounds from marine processing by-products— a review, Food Res. Int., 39, 383, 2006. 99. Jung, W. K. et al., Preparation of hoki (Johnius belengerii) bone oligophosphopeptide with a high affinity to calcium by carnivorous intestine crude proteinase, Food Chem., 91, 333, 2005. 100. BLR, Berkeley Lab Research, Secrets of the Sea Yield Stronger Artificial Bone, Press Release, January 31, 2006, http://www.lbl.gov/Science-Articles/Archive/MSDartificial-bone.html. 101. Muir, A. D., Natural peptides in blood pressure control. A review, Agro Food Ind. HiTech., 16, 15, 2005. 102. Seymour, T. A., Jing Li, S., and Morrissey, M. T., Characterization of a natural antioxidant from shrimp shell waste, J. Agr. Food Chem., 44, 682, 1996. 103. Mamelona, J. et al., Quantification of phenolic contents and antioxidant capacity of Atlantic sea cucumber Cucumaria frondosa, Food Chem., 104, 1040, 2007. 104. Murray, A. P. et al., Patagonicoside A: a novel antifungal disulfated triterpene glycoside from the sea cucumber Psolus patagonicus, Tetrahedron, 57, 9563, 2001. 105. Kumar, R., et al., Antifungal activity in triterpene glycosides from the sea cucumber, Bioorg. Med. Chem., 2007. 106. Paulo, A. S., Mourao, S., and Bastos, G., Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea, Eur. J. Biochem., 166, 639, 1987. 107. Mouraso, P. A. S. et al., Inactivation of thrombin by a fucosylated chondroitin sulfate from Echinoderm, Thrombosis Res., 102, 167, 2001. 108. Mao, W., Heparinoid-active sulfated polysaccharides from marine green algae, # MEDI, 391, 233rd American Chemical Society National Meeting, Chicago, IL, March 25–29, 2007. 109. Shanmugham, M. et al., Distribution of heparinoid-active sulfated polysaccharides in some Indian marine green algae, Ind. J. Mar. Sci., 30, 222, 2001. 110. Yamada, K. et al., Chitosan based water-resistant adhesive. Analogy to mussel glue, Biomacromolecules, 1, 252, 2000. 111. Punt, P. J., Filamentous fungi as cell factories for heterologus protein production, Trends Biotechnol., 20, 200, 2002. 112. Colewell, R. R., Fulfilling the promise of biotechnology, Biotechnol. Adv., 20, 215, 2002. 113. Gullo, V. P. et al., Drug discovery from natural products, J. Ind. Microbiol. Biotechnol., 33, 523, 2006. 114. Bourguet-Kondracki, M.-L. and Kornprobst, J.-M., Adv. Biochem. Eng./Biotechnol., 97, 106, 2005. 115. Berlinck, R. G. S. et al., Challenges and rewards of research in marine natural products chemistry in Brazil, J. Nat. Prod., 67, 510, 2004. 116. Rao, D. V. and Rao, B., Drugs from marine algae-current status, in Proc. Symp. Seaweeds—2004, Seaweed Research and Utilization Association and Central Marine Fisheries Research Institute, Cochin, 2004, pp. 54–57. 117. Stirk, W. A. et al., Seasonal variation in antifungal, antibacterial and acetylcholinesterase activity in seven South African seaweed, J. Appl. Phycol., Online, www. Springerlink.com, January 20, 2007. 118. BCC Research, Evolving Neutraceutical Business. Report ID: FOD013B, p. 135, 2003, www.bccresearch.com.

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Nutraceuticals 13 Marine for Food Fortification and Enrichment 13.1 INTRODUCTION It is well known that optimal health could be realized only through adequate intakes of nutrients. Developments in the area of human nutrition in the twentieth century led to identification, isolation, and purification of many nutrients. Most foods may be deficient in one or more nutrients. Sometimes nutrients, which are present in original food material, may be partially or fully lost as a result of processing, storage, packaging, and handling. The modern food processing technology is aware of the possibility of loss of nutrients at various stages of processing and therefore takes necessary steps to minimize such losses. In view of consumer interests in nutrition, a major area of modern food technology is related to formulation and designing of foods to achieve specific nutritional quality objectives.1,2

13.2

DIETARY GUIDELINES

Regulatory agencies the world over provide appropriate guidelines on intake of nutrients for the purpose of maintenance of public health. Food-based dietary guidelines (FBDGs) are advisory notes from regulatory agencies for healthcare professionals and consumers on requirements of adequate nutrient contents in diet, with a view to meet the nutritional needs. These guidelines can address the relevant public health concerns regarding whether they are related to dietary insufficiency or are in excess. The establishment of FBDGs is a key strategy to reach the nutritional goals of a population and an important tool in national food and nutrition policy development. These guidelines aim to promote general nutritional well-being as well as to prevent and control both ends of the spectrum of malnutrition: under and overnutrition.2,3 In 1997, the Food and Nutrition Board of the U.S. National Academy of Sciences issued new guidelines for nutrients now known as dietary reference intakes (DRIs). These new values were designed to address both serious vitamin and mineral deficiencies and daily intakes that promote good health.4,5 The recommended daily allowances (RDAs) were developed by the Food and Nutrition Board to serve as the benchmark of nutritional adequacy in the United States as the minimum values needed to avoid serious disease. The RDA for protein for U.S. men aged 19–24 is 58 g and for women of the same age group is 49 g. The recommendation for fat is 30% or less of total energy required. Over the years, the RDAs have undergone modification as the safe upper levels of micronutrients so that consumers can ingest these substances 405

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at levels that prevent deficiency and the pathogenesis of chronic disease but avoid intakes that could have problematic effects. Scientific knowledge regarding the roles of nutrients in addressing classical nutritional deficiency diseases (such as rickets) and to the reduction of risks of chronic diseases such as osteoporosis, cancer, and cardiovascular disease expanded dramatically since the inception of the RDAs.6,7 On November 9, 2004, the United States Food and Drug Administration (U.S. FDA) published a draft guideline for the food industry, entitled “Substantiation for Dietary Supplement Claims Made under Section 403(r)(6),” which is intended to describe the amount, type, and quality of evidence a manufacturer has to substantiate a claim under the Section of the Act (see Appendix).

13.3

SUPPLEMENTATION

Supplementation refers to periodic administrations of pharmacological preparations of nutrients as capsules or tablets or by injection when substantial or immediate benefits are necessary for the group at risk. Nutritional supplementation should be restricted to vulnerable groups, which cannot meet their nutrient needs through food (such as women of childbearing age, infants and young children, and elderly people).8,9 Some examples are supplementation with iron, which is recognized as the only option to control or prevent iron deficiency anemia in pregnant women and that with folic acid for women of childbearing age who have had a child with neural tube defect to prevent recurrence. Eating more of foods rich in antioxidants, such as the vegetables and citrus fruits has been shown to be protective. When this is not possible due to certain health conditions, nutritional supplements could be used.5,10 Vitamin E and β-carotene supplementations are popular approaches to address cardiovascular disease and lung cancer. The concept of “dietary supplement” (DS) was initiated in 1994 when the U.S. Congress passed the Dietary Supplement Health and Education Act (DSHEA). The “DS” went beyond vitamin, pills, and tablets, allowing the addition of specific metabolites that had some demonstrated relationship to a disease/health condition. The DSHEA also allowed the product to bear a health-based structure–function claim with specific rules as to what could be said.11 In addition, a DS has to have a supplement facts panel instead of nutrition facts panel. The act did not define functional foods or nutraceuticals, but these terms began to be used simultaneously with DS at that time. Under the DSHEA, the DS manufacturer is responsible for ensuring that a DS is safe before it is marketed. In general, no claim should be made for a food that represents the food that is intended to cure, mitigate, treat, or prevent any disease, since such a claim can cause a food to become subject to regulation as a drug. FDA regulations allow “health claims” that a substance in the diet on a regular basis “may help reduce the risk” of a named disease. Recently, however, apprehensions have been raised in using the approach of supplementation to address these diseases. It has been now recognized that the most promising outcome with respect to nutrition and positive health is through dietary patterns, and not nutrient supplements. It was suggested that the relative presence of some foods and the absence of certain other foods was more important than the level of individual nutrients consumed. Notwithstanding justification for nutrient

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supplements to certain segments of the population (e.g., the elderly), there were insufficient data to justify an alteration in public health policy from one that emphasizes food and diet to one that emphasizes nutrient supplements.12,13 Supplements may not be necessary if a variety of foods are being eaten, which can ensure good supply of daily nutrients, since a balanced diet provides all the nutrients required by the body. While supplements provide some minerals and vitamins, they do not provide all of the food components needed for good health, since there are at least 42 nutrients needed each day. Further, physiological stress conditions like surgery, trauma, and burns do increase the need for nutrients. In addition, there are some apprehensions on the harmlessness of supplements. Earlier unrecognized risks caused by nutrient toxicity and nutrient interactions have surfaced during intervention studies. Very high doses of many vitamins like A, B6, C, and D as also certain minerals, if taken regularly may cause grave health problems. Excess of one nutrient causes either a nutritional imbalance or increase in the requirement of other nutrients. Apprehensions have also been raised on the efficacy of certain antioxidants such as vitamin E to function in the body, when supplied as supplement.14,15 Because of these reasons, it is preferable to depend on nutritive foods rather than supplements.16

13.4

FOOD FORTIFICATION AND ENRICHMENT

One of the techniques to compensate nutritional deficiencies in food is by a process known as fortification, that is, external addition of the nutrient to the food. The added nutrients are called “fortificants” or “additives,” which are added to a commonly consumed food, referred to as the “vehicle.” It is possible for a single nutrient or group of micronutrients to be added to the vehicle. Such externally added nutrients include vitamins, minerals, proteins, amino acids, and fatty acids, which enhance the nutritional value of the food. In addition to improving the nutritive value, inclusion of certain additives also help improve other qualities of the food. Thus, vitamins C and E and β-carotene may function as antioxidants in foods, β-carotene may enhance color, while proteins and specific fatty acids modify the texture and therapeutic values of foods.15 The technique, probably, is the easiest way to reach the population segments at risk of chronic diseases due to such deficiencies. For example, iodine, vitamin A, and iron deficiencies are important public health problems in developing countries and often coexist in vulnerable groups, such as pregnant women and young children. These deficiencies can be corrected by fortification of the respective nutrients. Addition of iodine to common salt is the earliest example of fortification to address the disease of goiter, which was recommended by the French chemist Boussingault in 1833. The technique was commercialized in 1924 in the United States. In later years, with the advent of nutritional labeling and increased public interest in nutritional properties of food, fortification of foods became a major tool for the food industry for marketing of processed foods. During recent years, several foods have been targeted to enhance their nutritional value by appropriate inclusion of additives. Most vitamins cannot be synthesized by the body and must be supplied by the diet. Examples include fortification of milk with vitamin D, margarine with vitamin A, and breakfast drink substitutes with vitamin C. Protein level of breads can be increased up to 12% by addition of protein-rich fortifiers

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such as milk. Fortifications of wheat flour with iron and vitamin B1 and salt with iodine are other successful examples. Apart from its well-conceived aim of prevention of diseases, in modern times, fortification is increasingly being used not only to protect against deficiencies but also to receive maximum health benefits.9,16 The term “additive” referred in fortification technology is defined differently by different regulatory agencies. Under the U.S. law, components described as additives may have a nutritional element. However, according to the European Union, an additive is defined as a compound that provides a technological rather than nutritional function to the food. It is also important that the product should be acceptable to individuals who may have a reduced appetite or interest in food. In a workshop held by the International Life Science Institute (ILSI), in Lisbon in 1997, experts from key European countries concluded that addition of nutrients can provide an effective and safe strategy to improve actual micronutrient intake and status.10 Successful instances of fortification-induced eradication of diseases include the conquest of pellagra in the United States by niacin fortifiction of white flour, the virtual elimination of goiter by iodination of salt and the reduction of neural tube defects by folate fortification of cereal grains.9–11

13.4.1 REQUIREMENTS FOR FORTIFICATION There are at least three essential requirements or conditions that must be met in any fortification program: The fortificant should be effective, bioavailable, acceptable, and affordable; the selected food vehicle should be easily accessible and a specified amount of it should be regularly consumed in the diet. Further, detailed production instructions and monitoring procedures should be in place and enforced by law. The development of a fortification policy must be based on rigorous criteria that embrace both positive and adverse consequences.11–13,16 Information on commercial aspects of techniques, nutrient databases, and formulation worksheets for food product development have been provided.13,16 The important factors need to be considered when formulating a food product with added nutrients are given in Table 13.1.13 There are important scientific and policy issues that must be resolved before programs for fortification can be considered. The data needs, constraints, and limitations for fortification programs have been identified recently, which include five general areas, namely, (1) human requirements and nutritional status, (2) bioavailability, (3) interactions among nutrients, (4) interactions of fortification nutrients with carrier foods, and (5) the safe upper limits (or upper reference levels) for these nutrients. While all these aspects need to be discussed in developing a fortificaton strategy, the domain of greatest controversy is the establishment of upper reference levels or nutrient toxicity.10 It is to be noted that the older population generally differs from younger adults not only by age but also by health status. The elderly group is not a homogenous population and their different health and social problems influence their nutritional status. Attention must be paid to environmental, phychological, and physical parameters in evaluating their nutritional status and interactions.11–13,17 Sometimes, fortification may have adverse impact on acceptability of the product. Unappealing flavor of fortified foods is often a hurdle. The problem can be addressed

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TABLE 13.1 Factors Need to Be Considered When Formulating Food Products with Added Nutrients Overall product composition

Ingredients interactions

Processing considerations

Shelf life and packaging

Nutrigenomics

The physical and chemical properties of a food such as pH, water activity, oil, or water content influence nutrient stability. Macroingredients such as protein and fiber may affect stability and bioavailability of nutrients. Fortification can cause change in sensory characteristics. Essential minerals such as iron can cause adverse reactions in the color and flavor of foods, while ascorbic acid can lower pH and impart tartness. Need corrective steps to combat these changes Interaction among the nutrients and with other food components is a key factor in viable added nutrients present in food products. For example, vitamin C may improve the absorption of iron, which in turn can accelerate degradation of vitamins Most vitamins are unstable at high temperatures; while most nutrients are not adversely affected by heat. Freezing is generally beneficial for nutrients; however, blanching and washing can cause loss of water-soluble vitamins Package selection is affected by intended use and shelflife considerations. For example, vitamin C and βcarotene must be protected from oxygen. Shelf-life loss can be overcome by adding appropriate nutrient overages Nutrigenomics is how nutrients affect genes and enable foods to be developed that can be used to prevent and treat diseases. The application of nutrigenomics may allow food product developers to better target condition-specific foods

Source: Adapted from www.fortitech.com.

by addition of sweetener, food acids, chemical derivatization of nutrients, microencapsulation, and addition of bitterness inhibitors, as shown in Table 13.2.

13.5

SOME EXAMPLES OF FOOD FORTIFICATION

13.5.1 IODINE Deficiency of iodine is because of its sparse distribution in the earth’s surface resulting in its low contents in plant foods. Although foods of marine origin are naturally rich sources of iodine (see Chapter 9), their consumption is not enough throughout the world to take care of the problem. The situation has made iodine deficiency disorders exceedingly common among populations. Common salt is a recognized vehicle for fortification of iodine and the fortified salt is probably the ideal way to virtually

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TABLE 13.2 Methods to Make Fortified Foods More Palatable Methods Addition of sweeteners Food acids

Chemical derivatization/ substitution

Microencapsulation Addition of bitterness inhibitors

Example/Remark Addition of sweeteners such as aspartame (l-aspartyll-phenylalanine) and saccharin to reduce the intensity of off-flavor Food acids such as dl-malic or citric acid reduce intensity of off-flavors and bitterness by chelation of metals. Acidity of the product can be varied to reduce intensity of bitterness Protection of sulfur groups of cysteine and methionine by derivatization such as acetylation of methionine to N-acetyl-methionine to improve taste. Replacement of cysteine with cystine gives a neutral taste. Solubility of the product is an important criterion for acceptability Coating of functional food additive helps its slow release into the food matrix. Bitter peptides could be coated with a hardened edible fat or wax Cyclodextrins, maltols, etc., plastein reaction in the case of protein hydrolyzates

eliminate iodine deficiency disorders. The safe level of iodine fortification usually lies between 25 and 50 mg/kg salt. The actual amount should be specified according to the level of salt intake and magnitude of deficit at the country level.18–21

13.5.2 VITAMINS Addition of vitamins to foods is often necessary to enhance the nutritive value of food with a view to provide health protection, without causing any health risks to any consumer group. International expert groups have aimed at establishing tolerable upper intake levels for vitamins (and also minerals), although lack of conclusive data on their safety is a major obstacle to this work. Assuming 95% intake of vitamins and minerals from food together with a daily multivitamin mineral pill, the calculation of total dietary intake levels of all vitamins and minerals can be calculated to develop a fortification strategy.20,22 Folic acid is a typical candidate for fortification. An adequate intake of folic acid is very important for women of childbearing age for proper birth weight and reduction of neural tube defects. A higher intake of folic acid may lower homo-cysteine levels in adults since elevated plasma homo-cysteine levels are considered an independent risk factor for heart disease. In addition, folate may improve the mental condition of the elderly population. Most population groups may not easily reach the required level of folic acid; therefore, folic acid fortification has been recommended. The United States initiated mandatory folic acid fortification of cereal-grain products in January 1998, with an approved level of 140 mg/100 g product, which will increase the average woman’s intake by only 100 mg/day. Dairies generally enrich milk by introduction of vitamin A and vitamin D, riboflavin, niacin, and thiamine hydrochloride as dry mix. Vitamin C is destroyed by the heat treatment of milk, so it is added into milk in the oxidized form or as dehydro-ascorbic acid. New data continue to emerge regarding the health benefits of

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vitamin D beyond its role in bone. The intakes associated with those benefits suggest a need for levels of supplementation, food fortification, or both that are higher than current levels. The current tolerable upper level of vitamin D established by the U.S. Food and Nutrition Board is 50 µg, or 2000 IU. A group of U.S. researchers has found that this upper intake level could be raised fivefold. A human clinical trial data published support a significantly higher upper level. A prevailing concern, however, exists regarding the potential for toxicity related to excessive vitamin D intakes. Collectively, the absence of toxicity in trials conducted in healthy adults that used vitamin D dose ≥250 µg/day (10,000 IU vitamin D3) supports selection of this value as the upper limit.23,24 Inclusion of vitamins (and minerals) may, however, contribute to the problems by adding a persistent metallic note; such flavor interactions may be additive or complementary to each other.

13.5.3 MINERALS Deficiencies of calcium and iron may be common among population that do not take adequate amounts of milk and meat.15,18 The prevalence of iron deficiency and anemia in vegetarians and in populations of the developing world is significantly higher than in omnivore populations (see also Chapter 8). Food fortification with iron is recommended when dietary iron is insufficient or the dietary iron is not bioavailable. Absorption of heme iron is high (20–30%) and its bioavailability is relatively unaffected by dietary factors. The traditionally eaten staple foods represent an excellent vehicle for iron fortification. Examples of foods, which have been fortified, are wheat flour, corn (maize) flour, rice, salt, sugar, cookies, curry powder, fish sauce, and soy sauce. Incorporation of vitamin C and other enhancers promote absorption of iron. A rapid colorimetric method for determination of iron in fortified and unfortified foods has been reported. The method consists of hot acid extraction of iron in the presence of hydroxylamine, and measuring the color after mixing the extract with a chromogen reagent, namely, bathophenanthroline disulfonic acid.25. Increased intakes of selenium-enriched foods may benefit human health. Selenium supplementation of populations with low or deficient in the mineral may improve measures of health and reduce the risk of cancer, especially prostrate cancer in men. Foods may contain different amounts and chemical forms of selenium, and therefore the benefits of the mineral may depend on the particular type of the mineral consumed.26 A multicomponent fortification of salt involving iodine, vitamin A, and iron has been attempted. Potassium iodate, retinyl palmitate, and ferric pyrophosphate were microencapsulated in hydrogenated palm fat by spray cooling. The microcapsules were added to common salt. During storage for 6 months, color change in the triplefortified salt was negligible, and iodine losses were only about 20%. Losses of retinyl palmitate were 12% during the period, suggesting encapsulation by spray cooling an ideal way for fortification of salt with the nutrients.21 Fortification of cereal staple foods is a potentially attractive intervention for zinc deficiency, which could target the vulnerable population groups of children and pregnant women. Zinc fortification would perhaps decrease the prevalence of stunning in many developing countries having low-zinc diets.2 Calcium has been high on the lists of nutrients recommended

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for fortification, as a result of widespread deficiency of the mineral, especially among the elderly. Fortification has been suggested to enhance the intake of calcium among the U.S. population, which, despite more than 20 years of awareness of the importance of calcium to health, remains suboptimal. As a result, since the beginning of 1999, calcium-fortified foods have appeared in large numbers in the United States and most of them have met with commercial success. Over 1000 calcium-fortified products were introduced in the last 5-year period, more than two-thirds in the beverage and snack categories. The different calcium salts such as gluconates, oxides, and sulfates, most commonly used as supplements or fortificants exhibit comparable bioavailabilities. Choice of salt will depend mainly upon cost, compatibility with the manufacturing process, and consumer acceptability. However, interaction with food, tablet, or beverage matrices can degrade intrinsic absorbability substantially. As a consequence, each product must be explicitly tested to establish the degree to which its calcium is available to consumers.6,7,27 A survey of consumer preferences for minerals revealed that 67% of the general public believed that they were deficient in calcium, magnesium, iron, zinc, and potassium. They prefer beverages as good source of calcium and ∼23% of the population consume such beverages once per day, and 24%, one to six times per week.28

13.5.4 CAROTENOIDS The overall intake of several natural antioxidants including carotenoids present in foods has been associated with lower incidence of various ageing diseases. Besides these, carotenoids have other functions including its usage as food colorants (see Chapter 8). Carotenoids have been therefore used to fortify foods. A functional food, oil, rich in fatty acids and antioxidants, colored with carotenoids extracted from the microalga Chlorella vulgaris, has been developed. For extraction of the carotenoid, the microalga was crushed in the presence of vegetable oil and ethanol or acetone and subjected to supercritical CO2 fluid extraction (SFE) at a pressure of 300 bar and varying temperatures. The recovery of carotenoids was 100% with oil at room temperature for 17 h, 70% with oil at 100°C for 30 min, 69% with supercritical CO2 at 40°C and 300 bar.29 The behavior of multicarotenoids systems in a functional food has been studied. The interactions between α-tocopherol, β-carotene, and lycopene in the formulation of a nutritional supplement using a simplex-centroid design and response surface methodology (RSM) were examined. The antioxidant activity (AOA) of the product was evaluated by determining the inhibition of spontaneous autoxidation. The results showed that no synergism occurred between the three compounds when rat brain homogenate was used as the oxidizable substrate, and also suggested that RSM can be applied to estimate the behavior of mixed ingredients in nutritional supplements.30

13.5.5 PROTEINS AND AMINO ACIDS Populations who largely consume cereal-based diets need to get adequate proteins. Cereal products such as breads are ideal candidates for protein fortification.

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Conventional breads are low in protein (8.5–9.5%) but quite high in carbohydrates (45–52%). Protein level of breads is increased by the addition of fortifiers such as milk solids, extra gluten, soybean flour, and cottonseed flour. In the process, some of the starches of flour are replaced by the fortifiers, leading to increase in the level of proteins up to 12%. Uses of protein supplements are quite common among athletes and physically active adults. Instead of whole protein, protein hydrolyzates can be used for the purpose. Protein hydrolyzates consist of a small fraction of free amino acids and short peptides, a broader spectrum of medium-sized peptides, and substantial amount of high-molecular-weight materials (see Chapters 3 and 4). Nutritionally complete products intended to address metabolic disorders usually have their protein components, partially or wholly provided by amino acids or by protein hydrolyzates. Protein hydrolyzates, being often bitter in taste, may require certain additives to mask their bitterness (see Chapter 3). When amino acids are used as fortifiers, they added in the product well above their threshold level. Since most amino acid products have unappealing taste, these products are flavored using sweet fruity notes to enhance their acceptability. The majority of such clinical nutrition products are in the form of either a readymade drink or as a powder to be dissolved in water. Therefore, while preparing the product, the insolubility of several amino acids such as tyrosine, cystine, and histidine needs to be addressed. For example, tyrosine has a poor solubility of 45 mg% in water at 25°C. The solubility could be improved by using amino acid derivatives such as hydrochlorides, converting them to ester or by the use of soluble peptide containing these amino acids. The relationships between physicochemical and functional properties of protein hydrolyzates in nutritional products are shown in Table 13.3.

TABLE 13.3 Relationship between Physicochemical and Functional Properties of Protein Hydrolysates in Nutritional Products Chemical and Physicochemical Properties Molecular size

Surface activity (hydrophobicity) Interactions with carbohydrates and lipids

Interactions with minerals

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Functional Properties Solubility Viscosity Gelation Emulsifying capacity Flavor Osmolarity Emulsifying capacity Foaming Browning (Maillard reaction) Flavor formation Solubility Thermal stability Solubility

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13.5.6 PROBIOTICS Since Metchnikoff’s first observation that the unique longevity of the Bulgarians might be associated with yogurt in their diet, there has been considerable interest in the health benefits of foods containing “therapeutic microorganisms.” The animal or human gut is inhabited with a heavy load of bacteria belonging to diverse nature; at least one-third of these bacteria are useful in the digestion of food. It is believed that if these positive therapeutic microorganisms are maintained, the unwanted bacteria are less able to cause disease and irritation. Probiotics (meaning “pro-life”) are foods containing live beneficial bacteria. Probiotics are defined as “live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance.”31 Two of the most common probiotic strains are lactobacilli and bifidobacteria. Lactobacilli are beneficial microorganisms of particular interest because of their long history of use for processing foodstuffs and for preserving food by inhibiting invasion by other microorganisms that cause food-borne illness or food spoilage (see also Chapter 3). The largest category of foods containing lactic acid bacteria is fermented or cultured dairy products. It is advisable to get probiotics from foods than from supplements, as there is a synergistic effect between the components of food and probiotic cultures. Foods such as yogurt and buttermilk are the most recognized foods providing probiotics for the gut. These dairy products provide a number of high-quality nutrients including calcium, protein, bioactive peptides, and conjugated linoleic acid, which strengthen the body’s immunity and gastrointestinal functioning. Table 13.4 shows health benefits of probiotics. In contrast to probiotics, prebiotics are the foods that help the probiotics grow and multiply. Wheat, onions, garlic, banana, tomatoes, etc., are examples of prebiotics. Biomedical research currently offers new prospects of application of lactobacilli for applications and this may have far-reaching results in the future. Many fermented products containing lactobacilli have been recently released on the market. Some of them contain new Lactobacillus spp. of nondairy origin, such as L. rhamnosus GG, isolated from the healthy human intestinal flora. In view of current interests in the field, the European Food Safety Authority (EFSA) has adopted a generic approach to

TABLE 13.4 The Health Benefits of Probiotics Synthesis of vitamins, primarily B vitamins Increased availability of nutrients Decreased lactose intolerance Boost of immune response Control of pathogenic bacteria by antimicrobial substances such as cytokines, butyric acid, and bacteriocins Stimulation of the growth of lactic acid-producing bacteria, favoring digestion, especially milk and milk products Strengthening of the gastrointestinal membrane, for protection against infection Prevention of the growth and activity of unwanted bacteria, including the bacterium, Helicobacter pylori, known to cause peptic ulcers

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the safety assessment of microorganisms used in food/feed and the production of food/feed additives. New specific guidelines, granting a “long-standing presumption of safety” status to Lactobacillus genus based on its long history of safe use has been proposed.31,32

13.6 MARINE INGREDIENTS FOR FOOD FORTIFICATION AND SUPPLEMENTATION Marine products offer a number of ingredients for a variety of fortification programs. These include polyunsaturated fatty acids (PUFA) (omega-3 fatty acids), proteins, minerals, glucosamine, and chondroitin sulfate. These will be discussed in the following sections.

13.6.1 OMEGA-3 FATTY ACIDS The PUFA (omega-3 fatty acids) particularly, eicosa pentaenoic acid (EPA), and docosahexaenoic acid (DHA) have been recognized as important therapeutic agents, as discussed in Chapter 5. One of the major sources for the requirement of PUFA is through regular consumption of marine products. They can also be derived from other PUFA-rich products such as algal oil or on the availability of foods containing linoleic and linolenic acid, which are elongated and desaturated for conversion into PUFA. It has been, however, recognized that during the past few decades, intake of omega-3 fatty acids by general public is rather poor, resulting in an undesirable increase in the ratio of omega-6 to omega-3 fatty acids. This discrepancy has been mainly attributed to increase in consumption of seed oils (corn, soybean, palm, etc.), which are rich in omega-6 fatty acids. The strategy to get adequate quantity of PUFA includes keeping omega-6 PUFA in the diet as low as possible, consumption of omega-3 PUFA-rich diet and intake of omega-3 PUFA supplements including microencapsulated fish oil (FO). EPA and DHA, which can serve as important fortificants for the development of nutritional products that can address several biological functions.33,34 Marine fishes are good sources of omega-3 fatty acids, the isolation of which has been discussed in detail. Several marine oils are available for DS and functional food manufacture, providing sources of EPA and DHA, and in some cases vitamins A and D. Oils ranging from cod liver to sardine are offered by several manufacturers.34,35 Table 13.5 indicates some commercial sources of PUFA.16,35 13.6.1.1

Marine Oil-Fortified Products

Marine oil has been used to fortify food products such as mayonnaise, margarine, bread spreads, and bakery products. Recently, increased incorporation of DHA into margarines and baby foods has been promoted to enhance brain-memory development. Bread spreads containing 40% fat incorporating refined, deodorized, unhydrogenated FO in vegetable oil and butter oil has been developed. The product contained wheat powder and gelatin as emulsifying agents and potassium sorbate as preservative and antioxidants. Other products include salad dressing and bakery products.34 Supplementation with EPA and DHA has been recognized to be more effective than use of their precursors such as linoleic acid in the food, since the bioconversion

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TABLE 13.5 Commercial Sources of Essential Fatty Acids Fatty Acid

Product

EPA

Seafood

DHA

Seafood

Animal Single cell oils

Omega-6 LA

Vegetable oil

ARA

Single-cell oil Animal

Individual Source Alaska pollock Cod Menhaden Salmon Tuna Antarctic krill Sardine Alaska pollock Cod Menhaden Salmon Tuna Atlantic krill Egg yolk Crypthecodinium Schizochytrium Corn Soy Canola Mortierella Egg yolk

Levels (%) 12.5–14 10 10 12 6 Varies Varies 6–7 10 13 4 17 — 2 47 25 59 50 30 8 4

Source: Adapted from Ohr, L.M., Food Technol., August 2005, 95; Swanson, M.A. and Evenson, P., Food Additives, 2nd ed., Marcel Dekker, New York, 2002. With permission from Taylor & Francis Ltd. (www.informaworld.com).

efficiency is less. Further, such fortified products can have improved bioavailability of omega-3 PUFA.36 The current thinking among scientists is that it is better to consume both the EPA and DHA rather than DHA alone since both are found in FO at a ratio of around 1:5.33 Fish could be cultured under specific conditions such as diet, to have appreciable levels of PUFA in their muscle. Development of PUFA-enriched Atlantic salmon was reported using aquaculture. Four diets containing either 100% of Peruvian FO, capelin oil, soybean oil, or rapeseed oil as supplemental oil were fed to triplicate groups of salmon for 135 days. After slaughter, half of the fish were smoked, while the rest were analyzed as raw (without smoke treatment). For smoked and raw fish, the left fillet was analyzed as fresh fillet while the right fillet was frozen and stored at −20°C for 2 or 4 months before analyses. Fish-fed diets enriched with FO had firmer texture than fish-fed rapeseed oil diet. Water-holding capacity (WHC) and color characteristics were influenced by dietary oil source. Fish-fed FO-diets had significantly higher color than fish-fed vegetable oil–diets. Frozen storage decreased the firmness of raw fillets and the WHC of raw and smoked fillets. Color was affected by

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frozen storage, whereas muscle carotenoids concentrations slightly decreased. Lipid oxidation was more pronounced in fish-fed high levels of omega-3 fatty acids, which increased with frozen storage.37 In another comparable study, poultry feed was supplemented with FO at a concentration of 2–4%. The FO-fed chicken was later used to make chicken frankfurters. These frankfurters had higher contents of EPA and DHA and a lower content of omega-6 fatty acids. No significant differences were found in pH, cooking yield and moisture, fat, protein, ash, and cholesterol contents, and sensory quality.38 13.6.1.2

Process Optimization

The principal technological hurdle in amending the diet with FO, however, is the high degree of unsaturation and associated sensitivity to oxidation. The resulting compounds adversely influence flavor apart from having negative consequences on the biological system. The problem could be addressed by certain control measures. These include storing the products at refrigerated temperatures and excluding oxygen from the packages. Another alternative is addition of precursors of PUFAs, such as linolenic acid. However, due to poor bioconversion these have to be added in extra measures to give sufficient DHA as required in the product.39 Partial hydrogenation is another method to control oxidation of PUFA. Incorporation of partially hydrogenated oils in baked goods did not show oxidation as judged by sensory analysis. Mayonnaise-containing specially deodorized unhydrogenated menhaden oil was found to have a shelf life of 14 weeks under nitrogen, which is an acceptable shelf life for a refrigerated product. FO in the hydrogenated form has also been used as a component of margarines. The objective was to enable people to consume 1–2 g of EPA and DHA per day by means other than eating fish or resorting to DSs. The product was commercially acceptable with shelf life of 10 weeks, and quality comparable to the all-vegetable control product. Hydrogenated and partially hydrogenated menhaden oil, which generally contain 13% EPA and 8% DHA, has been given generally recognized as safe (GRAS) status by the U.S. Food and Drug Administration (see Chapter 5). Menhaden oil mixed with defatted soy flour extract demonstrated the greatest stability by producing the lowest TBA reactive oxidation products and retaining the highest concentrations of DHA and EPA after heating at 150°C for 30 min. A range of 62.8–71.5% of DHA and 67.7–75.9% of EPA remained in the FO with defatted soy flour extract, while only 29.9% of DHA and 37.2% of EPA were retained in the FO with no addition. The effect was due to the presence of the highest level of total phenolic content (11.3 μg catechin equivalent/g) in the defatted flour extract, with 55 mg/g isoflavones in the defatted soy flour extract The order of free radical scavenging capability measured was also for the defatted soy flour extract.40 Polyols have been incorporated into FO emulsions as a means for the inhibition of lipid oxidation and also for suppression of fishy flavor. Selected polyols were evaluated for their performance as antioxidants and modifiers of oxidation pathways in a model system. Oil/water (O/W) emulsions were prepared with freshly steamdeodorized menhaden oil. A layer of emulsion in aluminum pans held at 5°C was exposed to 2550 lx fluorescent lights for 24 h before peroxide values and volatile flavor compounds were analyzed. AOA was observed for fructose, sucrose, raffinose,

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sorbitol, or mannitol when incorporated at 16% of the aqueous phase into model FO-in-water emulsions. Peroxide values were suppressed 10–18% in treated samples compared to control samples. The data supported a hypothesis that either or both free radical scavenging and transition state metal chelation activities were provided by polyols in FO emulsions. Also, polyols retarded the water-requiring retroaldol decomposition of (E,Z)-2,6-nonadienal to (Z)-4-heptenal in the model systems and the reaction may be involved in some suppression of fishy flavors in emulsions.41 Flavonoids obtained from agrofood products are natural antioxidants in control of oxidation of PUFA. Besides increasing the shelf life of seafood, addition of these compounds was recognized beneficial due to the induction of apoptosis of cancer cells. Thus, fish muscle supplement with bioactive antioxidants appears to give a stable functional food offering the combined action of omega-3 fatty acids and natural polyphenols.42 FO with 33% omega-3 fatty acids was microencapsulated through spray-drying in a matrix of n-octenylsuccinate-derivatized starch and either glucose syrup or trehalose. Samples showed no difference in physicochemical properties as determined by measurement of particle size, oil droplet size, true density, and Braunauer, Emmett and Teller (BET) surface adsorption parameters. Upon storage at low relative humidity, lipid oxidation was decreased in trehalose-containing samples indicating that in the amorphous state trehalose is a more suitable wall material for microencapsulation than glucose syrup. The retarded oxidation of trehalose-containing samples may be attributed to the unique binding properties of trehalose to dienes. At 54% relative humidity, a rapid oxidation of the microencapsulated oil was observed upon crystallization of trehalose, which limits the range of applications to products to be stored at low humidity.43 The influence of fortification of selected instant foods using two types of encapsulated FO powders on their sensory qualities was determined. Since FO had some adverse sensory effects on the product, incorporation of flavorings for masking of undesirable odors allowed higher levels of FO addition to instant foods. The decrease of sensory quality of flavored and nonflavored samples stored in the air-permeable conditions was detected after 1 and 3 weeks, respectively. However, vacuum-packed samples showed no changes in sensory quality. The results showed that it was possible to fortify instant foods with microencapsulated FO at limited levels, especially when spray-dried powders were used. It was also observed that one portion of fortified instant foods might provide up to 16% of the minimal recommended daily intake of long chain omega-3 PUFA.44 13.6.1.3

Therapeutic Benefits of PUFA-Fortified Products

The therapeutic benefits of omega-3 PUFA have been discussed in Chapter 5. Such benefits accrued from consumption of fortified products are discussed in this section. Influence of feeding commercial diets fortified with omega-3 and omega-6 PUFA on arachidonic acid (ARA) content, PUFA equivalent (PE) (calculated as 0.15 × linolenic acid [LA] + [EPA] + [DHA]), and ratio of omega-6 to omega-3 PUFA acids were determined in different meat portions including breast, thigh, and fillets in chickens, turkeys, common carp, and rainbow trout. Arachidonic acid (AA) content was in the range 20 mg/100 g in fillets of rainbow trout fed the diet with linseed oil (LO) to 138 mg/100 g of thigh meat of chickens fed the diet based on maize, for a period of 90 days. AA content in breast meat of turkeys fed the diet with LO or FO did not

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differ from that of rainbow trout fillet. Apart from all fish samples, both breast and thigh meat of turkeys fed the diet with either LO or FO gave the recommended value of <4 for the ratio of omega-3 to omega-6 PUFA. AA content in the tissue increased significantly with increasing dietary LA in both chicken and all turkey tissues.45 Supplementing the diet with DHA and also ARA can have beneficial effects in human amnesic patients. The patients were given 240 mg of ARA and DHA or 240 mg of olive oil for 90 days. Subjects with mild cognitive dysfunction showed a significant improvement in immediate memory functions and attention. Subjects with organic brain lesions showed a significant improvement in immediate and delayed memory functions.46 Dietary supplementation of arachidonic and dosahexaenoic acids improves cognitive dysfunction. Taking high dose of omega-3 supplements daily decreased the severity of symptoms associated with ankylosing spondylitis, a chronic disease that mainly affects joints of the spine and hips. The patients were given a high dose (4.55 g) or low dose (1.95 g) daily supplement of omega-3 fatty acids. The subjects in the high dose group exhibited a notable decrease in disease. Supplements of omega-3 FO appeared to be an alternative treatment with fewer side effects to nonsteroidal antiinflammatory rugs for the treatment of nonsurgical neck or back pain. Omega-3 fatty acids had an effect comparable to ibuprofen.47,48 A fortifying agent containing the fatty acids has been successfully examined for supplementation of baby foods. The study showed that babies who were breastfed from birth to 4–6 months and then randomly weaned—either to the formula supplemented with DHA and ARA or to a formula without them. The babies fed with the supplemented formula had improved visual activity at 1 year of age, compared to the babies fed with the nonsupplemented formula. Some infant formula developers are now offering DHA and ARA fortified formulas for toddlers up to 24 months old. Food manufacturers are also taking advantage of this opportunity by adding DHA to their food products. It is likely that such fortified foods in the markets may increase due to understanding of the role of these fatty acids in brain development and also cancer prevention.49 13.6.1.4 Regulatory Status In the United States, most essential fatty acids are GRAS because of their historical presence in the diet, and are therefore exempt from the premarket approval.49,50 The U.S. FDA in 2004 announced the availability of a qualified health claim for reduced risk of CHD on conventional foods that contain EPA and DHA. This approved health claim is believed to contribute to the influence of omega-3-containing products in the markets. The FDA recommends consumption of not more than a total of 3 g of EPA and DHA per day, with no more than 2 g/day from a DS. Being able to differentiate food products that contain both EPA and DHA means that food companies can create a source of strategic competitors advantageously because of the presence of these nutraceutical compounds in their products.49,50 Table 13.6 indicates recommendations for supplementation of infant formula with DHA and ARA by expert panels.16 13.6.1.5

Marketing Campaigns

In view of the recognized nutritional advantages of EPA and DHA, marketing campaigns have been launched for many marine fish products that tend to affirm that

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TABLE 13.6 Recommendations for Supplementation of Infant Formula with DHA and ARA Recommendation Preterm ARA (% of formula fat) DHA (% of formula fat) Preterm ARA (% of formula fat) DHA (% of formula fat) EPA/DHA ratio

BNF a

ISSFALb

FAO/WHOc

0.30% 0.3%

1.0–1.5% 0.5–1.1%

1.0% 0.8%

— — —

— — >5:1

0.6% 0.4% 10:1

Note: DHA, docosahexaenoic acid; ARA, arachidonic acid; —, not given. BNF, British Nutrition Foundation, 1993. b ISSFAL, International Society for the Study of Fatty Acids and Lipids, 1994. c FAO/WHO, Food and Agriculture Organization/World Health Organization, 1993. a

Source: Adapted from Swanson, M.A. and Evenson, P., Food Additives, 2nd ed., Marcell Dekker, New York, 2002. With permission from Taylor and Francis Ltd. (www.informaworld.com).

consumption of these fish is an appropriate method of satisfying consumer’s need for a variety and nutritious, tasty, and healthy foods. These campaigns have resulted in positive changes in consumer attitude toward seafood.51 According to a recent survey, 47% consumers associated omega-3 fatty acids with heart health and 32% considered themselves deficient in the compounds. Therefore, omega-3 fatty acids– fortified products are now generally accepted. Such products include frozen desserts, muffins, breads, sauces, margarine, pasta, cheese spreads, tuna burgers, yogurts, and salad dressings. These fatty acids are also finding use as nutrient supplement to flour in countries such as Germany and Norway. Since encapsulation prevents oxidation (see Chapter 14), encapsulated products are also available in the markets.52,53 Canada, perhaps, is the first country to recommend fortification of infant formula with omega-3 fatty acids. One of the fortificants for the purpose is a new high-stability powder. MarinolTM OMEGA-3 HS Powder, a FO is one of several popular fortified products.54,55 It is important to note that while developing such omega-3 PUFAfortified foods, the total fat content, particularly the level of saturated fatty acids, must not exceed the dietary guidelines. The fortified food must be convenient, palatable, with no fishy odor/flavor and no fishy eructation following consumption. The food matrix should provide minimum or no resistance for release of omega-3 PUFA in the gastrointestinal tract to ensure maximum bioavailability. Particular attention needs to be paid to the material used for micro emulsification, as this may be an important criterion for the bioavailability of long-chain omega-3 PUFA.

13.6.2 MARINE PROTEINS Proteins from seafood by-products can have a range of dynamic properties and could preferentially be used in foods as binders, emulsifiers, and gelling agents.

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Seafoods are recognized sources of high-quality proteins with many desirable functional properties. The fish protein powders are also good sources of potassium, phosphorus, and magnesium and amino acids (see Chapter 3). Fish protein hydrolyzates are generally used for the modification of functional properties of foods and in dietetic foods as a source of small peptides and amino acids. Its high dispersibility makes it suitable as a replacer for milk proteins and as an additive to cereal foods, soups, and bread and crackers.56 Some entrepreneurs such as the Association of Danish Fish Processing Industries and Exporters have commercially produced fish-based protein powders for use in frozen products to enhance their functional properties such as water binding and frozen stability (see Chapter 3).

13.6.3 MINERALS Fish bone material derived from processing of large fish is a useful calcium source where the quantity of calcium is concerned. To use fish bone for food fortification purposes, the bone should be converted into an edible form by softening its structure. This can be achieved utilizing different methods including hot water treatment and hot acetic acid solutions addition (Chapter 8). A natural calcified mineral source is available for use in both functional foods and DSs, providing bioactive calcium, magnesium, and trace minerals for bone health and overall wellness. AquaminTM is derived from Lithothamnion spp., a sea plant. The product is harvested from the portions of the sea plant that have naturally broken down and settled to the bottom of the sea. The ingredient is available in three forms, providing opportunities for a wide variety of applications, from baked goods and beverages to confections and DSs.55

13.6.4 GLUCOSAMINE Glucosamine is a well-recognized nutraceutical for joint pain relief, which helps in the repair and maintenance of cartilage (see Chapter 6). Oral glucosamine supplementation has demonstrated effectiveness in combatting osteoarthritis. Glucosamine is absorbed easily into the human intestine because of its low molecular weight. Glucosaminoglycans and glycoproteins allow cells in tissues to hold together. In particular, N-acetyl-glucosamine is the final form, which together with glucoronic acid is polymerized to lubricant, hyaluronic acid. They are necessary for contraction and maintenance of virtually all connective tissues and lubricating fluids in the body. Daily intake of 1500 mg glucosamine sulfate may be the preferred treatment for knee osteoarthritis.57,58 A diet-supplement composed of red ginseng (43.5%), glucosamine (25%), shark cartilage (25%), ascorbic acid (5%), and manganese chloride (1.5%) has been developed for relieving arthritic symptoms. Use of N-acetyl glucosamine (produced from chitin hydrolysis) as an additive in milk products has been described in a Chinese patent.58 Enzymic production of N-acetyl-d-glucosamine (GlcNAc) from crab shell α-chitin was investigated with a view to using the GlcNAc as a functional food supplement or therapeutic agent. A crude enzyme preparation from Aeromonas hydrophila H-2330 was used for hydrolysis at 17°C, as the chitinases in the crude enzyme mixture were inactivated at higher temperatures. Yields of GlcNAc from α-chitin were 66–68% after 10 days.59 Potential of supplementing beverages with glucosamine in hydrochloride or sulfate forms has been examined

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with respect to their taste, consumer acceptability, and marketing potential.60 An orange juice supplemented with glucosamine at 750 mg per serving has also been marketed recently in the United States. According to a company press release, two 8 fl oz servings deliver 1500 mg of glucosamine. Recently, a commercial glucosamine product has been accorded GRAS status by the U.S. FDA. The GRAS status permits the product to be used in a variety of specific mainstream foods and beverages.61 Recently, N-acetyl glucosamine has been found feasible to fortify milk.62

13.6.5 CHONDROITIN SULFATE The therapeutic effects of chondroitin in the cure of joint pain have been discussed (see Chapter 6). The compound is ideally obtained from shark cartilage. Recently, the content and biochemical properties of chondroitin sulfate in shark cartilage and finished cartilage powders being used as nutraceutical supplements were evaluated by analyzing unsaturated disaccharides after treatment with the enzyme, chondroitinase, and the results were compared with the specifications on the product labels. The recovery ranged from 95 to 102%. Furthermore, the average molecular weight and the origins of chondroitin sulfate in shark cartilage and finished products were evaluated by agarose gel-electrophoresis and assessment of disaccharide compositional patterns, respectively. Quantitative and compositional analysis of disaccharides after enzymatic depolymerization showed that the amount of CS in the samples was up to 29%. In the finished products, the content of chondroitin sulfate was 21.3% and its average molecular weight in the cartilage and finished product was approximately 40 kDa.63

13.7 COMMERCIAL STATUS The U.S. National Marine Institute (NMI) observed that 65% of adults used a fortified food or beverage and 65% used a functional food in 2005. The NMI has projected a value of $35.6 billion in sales of functional and fortified foods in 2006.64 The popularity of functional and fortified foods has increased in recent years due to a combination of strong marketing campaigns and growing body of scientific evidence that have contributed to rising consumer awareness and interests. The carbonated beverage industry, which has been facing negative criticism from the general public for the adverse effects of their products on health, is diverting to new beverages fortified with nutrients such as vitamins and minerals. The current interest in fortified products is also indicated by presentation of a total of 217 papers in the functional foods and health category under the symposium grid of Agricultural and Food Chemistry organized by the American Chemical Society at San Fransisco, in September 2006. Of the various marine ingredients, omega-3 fatty acids are the most popular fortificants used by the industry. Foods fortified with omega-3 fatty acids are available, particularly in the United States, Europe, Japan, and Southeast Asia. These products include bread items and other baked goods, dairy products such as milk, cheese, yogurt, and chocolate milk, and infant formulae.58,65 Some companies are marketing specialty drink products in Japan that are enriched with DHA. It has been estimated that opportunities for omega-3 fatty acids in the global nutrition and food fortification markets could reach an annual value of $500 million.64 There are several forms

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of FO supplements, which are available in 400–2000 mg capsules. A typical 1 g soft gel capsule of FO contains 180 mg of EPA and 120 mg of DHA. Natural FO capsules containing 50% EPA and DHA in a 1:5 ratio are now available. A more concentrated form of FO is the semisynthetic ethyl ester product containing 85% EPA/DHA. One such product contains 490 mg of EPA ethyl ester and 350 mg of DHA ethyl ester per 1 g capsule. Stable FO powders (such as Marinol TM OMEGA-3 HS Powder) have been commercially developed recently.66 A commercial omega-3 powder is claiming to provide the health benefits of fish, without the taste and smell of fish. Another encapsulated omega-3 fatty acid from menhaden oil ingredient has also been developed to fortify baked goods. A commercial product, MEG-3™ containing powdered FO that delivers the health benefits of both EPA and DHA has been developed. A patented microencapsulation technology provides superior process tolerance and ease of formulation, without adding any fishy taste or smell to your product. The product provides food manufacturers with the highest concentration of bioavailable omega-3 in the marketplace.65 Recently, a new milk with omega-3 fatty acids derived from FO has been introduced. The new line of premium half-gallons, Kemps Plus Milk, is now available in grocery stores throughout Minnesota and Wisconsin.67 Recommended FO products must contain antioxidants such as tocopherol to protect against their oxidation. Further, FO products that contain high quantities of vitamin A and D, which could be toxic, should not be used. In Europe and the United States these products are presented as having important cardiovascular benefits and labeled as FO products. Such claims were generally supported by a long list of clinical studies demonstrating the advantages of FO consumption on lowering triglycerides.12 Currently, the estimated North American consumption for FO-based omega-3 fatty acids in DSs excluding infant formula is 6–10 times higher compared to the food sector. Sales of supplements in the United States were worth $230 million in 2004. Omega-3 fatty acids received a recent boost in the consumer awareness with both the FDA’s 2004 qualified heart-health claim and the revised 2005 Dietary Guidelines for Americans. The dietary guidelines recognize that “limited evidence suggests an association between consumption of fatty acids in fish and reduced risks of mortality from cardiovascular disease for the general population.”65 In addition to those mentioned earlier, several DSs of marine origin have recently entered commercial markets. Some of these include astaxanthin, extracted from the microalga, Haematococcus pluvialis, and other microalgae products such as spirulina and chlorella, a carbohydrate extract from chlorella, claimed to boost the immune system against influenza, chitosan as a weight loss supplement, among others.35 Table 13.7 gives examples of some commercially available microencapsulated omega-3 fatty acid powder products and Table 13.8 provides examples of some commercial foods fortified with omega-3 PUFA.49,64,68–70 In conclusion, several marine ingredients, because of their recognized biological activities have entered the nutraceutical market as food supplements. While the prominent ones are the omega-3 fatty acids, particularly DHA, other promising ingredients of seafood origin include calcium supplements, glucosamine, chondroitin sulfate, and proteins. Of these, glucosamine and chondroitin sulfate have already shown commercial promises. There are good prospects to avail therapeutic benefits of marine ingredients through fortification technology.

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TABLE 13.7 Examples of Some Commercially Available Omega-3 Fatty Acid Powder Products Product

Ingredients

Microencapsulated FO rich in EPA and DHA, light yellow Microencapsulated FO high in EPA and DHA, light yellow Encapsulated tuna oil, high in DHA, less fishy odor

Natural FO concentrate powder, typical taste

Gelatin and sucrose matrix, coated in starch, sodium ascorbate, ascorbate, tocopherol, and tricalcium phosphate Caseinate and sucrose matrix, coated in starch, ascorbyl palmitate and sodium ascorbate Sodium cseinate, dextrose, monohydrate, dried glucose syrup, sodium ascorbate, mixed natural tocopherol, lecithin, dialpha tocopherol, and ascorbyl palmitate Carbohydrate, antioxidants, and free-flowing agent

TABLE 13.8 Examples of Some Commercial FO Powder Products and their Major Ingredients Food Products Beverage Bakery products

Bar FO capsules Spread Various foods

Various foods Infant formulaea Whole wheat breadb Margarine Milk

Trade Name/Brand

DHA/EPA and Dose

Great Circles, USA Ultrabalance, USA Irish Pride, Ireland British Bakeries, U.K. Allied Foods, NZ Wegman’s Food Markets, USA Coles high Top bread, Australia AP Foods omega-3 bread, Australia Great Circles, USA Most brands, Australia MEG-3TM Ocean Nutrition, USA MD Foods, U.K. Golden Vale, Ireland NovomegaTM National Starch Association (USA) and Omega Protein (USA) Kellog Co. and Martek, USA Mead Johnson Nutritionals, USA Arnold Food, Co., USA

DHA/EPA

DHA from microalgae DHA, ARA, and omega-6 fatty acids DHA/EPA

Blue Band Idee! Uniliver, Netherlands St Ivel’s Advance milk, U.K. Farmer’s bestmilk, Australia Brown’s (Heart Plus) milk, Australia

DHA, ALA, and B vitamins Omega-3 fatty acids 21.2 mg/250 ml 150 mg/250 ml

FO (EPA/DHA) FO (EPA/DHA) FO (EPA/DHA) OMEGA-3 fatty acids 37 mg/2 slices 200 mg/35g serve FO (EPA/DHA) 300 mg/capsule omega-3 encapsulated powder FO (EPA/DHA) FO (EPA/DHA) EPA/DHA

(continued)

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(Continued)

Partially skimmed milk Yogurtc

Neilson Dairy Oh! Partially Skimmed Milk, Canada Woodstock Water Buffalo Co., VT

Orange juice

Oh Mega J, Canada Shwartz Sparky Ornage, UK

a b

c

425

From cows that are fed diet rich in DHA 100 mg EPA and DHA in 170 g serving Omega-3 fatty acids

Data taken from Ohr, L.M., Food Technol., 59(4) 63, 2005. Data taken from Sloan, A.E., Food Technol., April 2006, p. 23; Garg et al., 2006, Newsletter, U.S. Institute of Food Technologists, Washington, June 13, 2007. Data taken from Ohr, L.M., Food Technol., March 2006, p. 81.

Source: Adapted from Swansson, M.A. and Evenson, P., Food Additives, Marcel Dekker, New York, 2002.

REFERENCES 1. Padgaonkar, S. V., Designed foods, Newsletter, Association of Food Scientists and Technologists (India), Mumbai Chapter, July 2006. 2. WHO, Diet, nutrition and prevention of chronic diseases, Report of a Joint FAO/WHO Expert Consultation, Technical Report Ser. 916, World Health Organization, Geneva, 2003. 3. Weever, C. and Schneeman, B., Revised dietary guidelines promote healthy life style, Food Technol., 59(3), 51, 2005. 4. Kurtzweil, P., An FDA guide to dietary supplements, in Nutrition, 11th ed., Cook-Fuller, C. C., Ed., Pushkin/Mcgraw Hill, Guilford, CT, 1999, p. 27. 5. Read, M., The health-promoting diet through life: adults, in Handbook of Nutrition and Food, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 299. 6. Kennedy, G., Nantel, G., and Shetty, A. P., The Scourge of ‘hidden hinger’: Global Dimensions of Micronutrient Deficiencies, Food and Nutrition and Agriculture, Food and Agriculture Organization of the United Nations, Rome, 2002, p. 3. 7. Kennedy, E., Dietary guidelines, food guidance and dietary quality, in Handbook of Nutrition and Food, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 339. 8. Rafferty, K., Walters, G., and Heaney, R. P., Calcium fortificants: overview and strategies for improving calcium nutriture of the U.S. Population, J. Food Sci., 72, R152, 2007. 9. Rowan, C., Fighting through the functional maze, Food Eng. Ingr., October 14, 2001, p. 5. 10. Miller, S. A. et al., Calcium and vitamin D deficiencies: a world issue, FAO Food, Nutr. Agric., 20, 27, 1997. 11. FAO, Food fortification technology and quality control, FAO Technical Meeting, Rome, 1996, p. 60. 12. Uzzan, M., Nechrebeki, J., and Labuza, T. P., Thermal and storage stability of nutraceuticals in a milk beverage dietary supplement, J. Food Sci., 72, E109, 2007. 13. Fortitech Inc., New York, www.fortitech.com, accessed on September 2007. 14. Lichtenstein, A. H., Robert, M., and Russell, R. M., Essential nutrients: food or supplements? Where should the emphasis be? J. Am. Med. Assoc., 294, 351, 2005. 15. Anonymous, The antioxidant myth: a medical fairy tale—health, New Scientist, 2563, 40, 2006. 16. Swanson, M. A. and Evenson, P., Nutritional Additives, in Food Additives, 2nd ed., Bransen, A. L., Ed., Marcel Dekker, New York, 2002.

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17. Perelson, A. M. and Ellenbogen, L., Rationale for use of vitamin and mineral supplements, in Handbook of Nutrition and Food, Berdanier, C. D., Ed., CRC Press, Boca Raton, FL, 2002, p. 1333. 18. Huma, N. et al., Food fortification strategy—preventing iron deficiency anemia: a review, Crit. Rev. Food Sci. Nutr., 47, 259, 2007. 19. Rastall, R., Tailor-made food ingredients: enzymatic modulation of nutritional and functional properties, IFIS, http://www.foodsciencecentral.com/fsc/ixid3729, December 2001. 20. Ramussen, S. E. et al., A safe strategy for addition of vitamins and minerals to foods, Eur. J. Nutr., 45, 123, 2006. 21. Wegmuller, R., Development, stability, and sensory testing of microcapsules containing iron, iodine, and Vitamin A for use in food fortification, J. Food Sci., 71, S181, 2006. 22. IFT Scientific Status Summary, Use of vitamins as additives in processed foods, Food Technol., 41(9), 163, 1987. 23. Hathcock, J. N. et al., Risk assessment for vitamin D, Am. J. Clin. Nutr., 85, 6, 2007. 24. WHO, Human vitamin and mineral requirements, Report of a joint FAO. WHO Expert Consultation, Thailand, World Health Organization, Geneva, 2002. 25. Kosse, J. et al., A rapid method for iron determination in fortified foods, Food Chem., 75, 1371, 2001. 26. Finley, J. W., Increased intakes of selenium-enriched foods may benefit human health, J. Sci. Food Agri., 87, 1620, 2007. 27. Tobelmann, R., Implementing calcium fortification: an industry case study, J. Food Cmp. Anal., 14, 241, 2001. 28. Rokesh, S., New product trends: consumer research: Prepared Foods, www.preparedfoods.com. 29. Gouveia, L. et al., Functional food oil colored by pigments extracted from microalgae with supercritical CO2, Food Chem., 101, 717, 2007. 30. Castro, L. A. et al., Optimization of the antioxidant capacity of a mixture of carotenoids and α-tocopherol in the development of a nutritional supplement, Food Res. Int., 38, 861, 2005. 31. Fuller, R., A review of probiotics in man and animals, J. Appl. Bacteriol., 66, 365, 1989. 32. Bernardeau, M., Guguen, M., and Vermoux, J. P., Beneficial lactobacilli in food and feed: long term use, biodiversity and proposal for realistic safety assessment, FEMS Microbiol. Rev., 30, 487, 2006. 33. Garg, M. L. et al., Means of delivery recommended levels of long chain omega-3 polyunsaturated fatty acids in human diets, J. Food Sci., 71, R66, 2006. 34. Bimbo, A. P., Fish oils: past and present food uses, J. Am. Oil Chem. Soc., 66, 1717–1726, 1989. 35. Ohr, L. M., Riding the nutraceutical wave, Food Technol., August 2005, 95, www. ift.org. 36. Kyle, D. J., Essential fatty acids as food additives, in Food Additives, 2nd ed., Chapter 11, Branen, A. L., Davidson, O. M., Salminin, S., and Thorngate, J. H., Eds., Marcel Dekker, NewYork, 2002, p. 277. 37. Regost, C., Jacobsen, J. V., and Rora, A. M. B., Flesh quality of raw and smoked fillets of Atlantic salmon as influenced by dietary oil sources and frozen storage, Food Res. Int., 37, 259, 2004. 38. Jeun-Horng, L., Yuan-Hui, L., and Chun-Chin, K., Effect of dietary fish oil on fatty acid composition, lipid oxidation and sensory property of chicken frankfurters during storage, Meat Sci., 60, 161, 2002.

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39. Chapman, K. W. and Regenstein, M., Use of fish oil in food products, in Seafood Safety, Processing and Biotechnology, Shahidi, F., Jones, Y., and Kitts, D. D., Eds., Technomic, Lancaster, PA, 1997, p. 151. 40. Bhale, S. D., Oregano and rosemary extracts inhibit oxidation of long-chain n-3 fatty acids in menhaden oil, J. Food Sci., 72, C504, 2008. 41. Faraji, H. H., and Lindsay, R. C., Characterization of the antioxidant activity of sugars and polyhydric alcohols in fish oil emulsions, Agric. Food Chem., 52, 7164, 2004. 42. Medina, I. et al., Natural bioactive antioxidants for the enrichment of seafood, Abstract No. AGFD 169, American Chemical Society 232nd Symposium, San Francisco, September 10–14, 2006. 43. Drusch, S. et al., Physicochemical characterization and oxidative stability of fish oil encapsulated in an amorphous matrix containing trehalose, Food Res. Int., 39, 807, 2006. 44. Kolanowski, W. and Jaworska, D., Evaluation of sensory quality of instant foods fortified with omega-3 PUFA by addition of fish oil powder, Eur. Food Res. Technol., 225, 715, 2006. 45. Komprda, J. et al., Arachidonic acid and long-chain omega-3 polyunsaturated fatty acid contents in meat of selected poultry and fish species in relation to dietary fat sources, J. Agric. Food Chem., 53, 6804, 2005. 46. Kotani, S. et al., Dietary supplementation of arachidonic and dosahexaenoic acids improves cognitive dysfunction, Neurosci. Res., 56, 159, 2006. 47. Sundstrom, K. et al., Supplementation of patients with ankylosing spondylitis, Scandinav. J. Rheumatol., 35, 359, 2006. 48. Ohr, L. M., Revisiting Omega-3, Food Technol., March 2007, www.ift.org, p. 57. 49. Ohr, L. M., Nutraceuticals and functional foods—functional fatty acids, Food Technol., 59(4), 63, 2005. 50. Camire, M. E. and Kontor, M. A., Dietary supplements: nutritional and legal considerations, Food Technol., 53, 87–96, 1999. 51. Trsondsen, T. et al., Consumption of seafood – the influence of overweight and health beliefs, Food Qual. Pref., 15, 361, 2004. 52. Pszczola, D. E., Encapsulated ingredients, Food Technol., 52(12), 70, 1998. 53. Katz, F., Research priorities move towards health and safe, Food Technol., 54(12), 58, 2000. 54. Anononymous, Fish oil revisited: an old favourite with fresh appeal. NutraceuticalsNow, Spring, 2005, 28. 55. Sloan, A. E., The heart of the matter, Funct. Foods Nutraceut., April 2004, 16. 56. Frokjaer, S., Use of hydrolysates for protein supplementation, Food Technol., 48(10), 86, 1994. 57. Herrero-Beaumont, G. et al., Effects of glucosamine sulfate on 6-month control of knee osteoarthritis symptoms vs. placebo and acetaminophen: results from the Glucosamine Unum In Die Efficacy (GUIDE) trial, Presentation 1203 at American College of Rheomatology Conference, November 15, 2006. 58. Ohr, L. M., Joint health, Food Technol., www.ift.org, January 2006, 57. 59. Saishiwa, H. et al., Production of N-acetyl-D-glucosamine from α-chitin by crude enzymes from Aeromonas hydrophila H-2330, Carbohy. Res., 337, 761, 2002. 60. Runestad, T., Stability and taste test limits of glucosamine in beverages, Funct. Foods Nutraceut., 22, 24, 2004. 61. Anonymous, Newsletter, Institute of Food Technologists, Washington, March 14, 2007. 62. Xu, Q., Liu, J., and Yuan, Z., The use of N-acetyl glucosamine as additive in milk products, Chinese Patent WO2004/093556A1, 2004.

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63. Sim, J. S. et al., Evaluation of chondroitin sulfate in shark cartilage powder as a dietary supplement: raw material and finished product, Food Chem., 101, 532, 2007. 64. Sloan, A. E., Top ten functional food, Food Technol., April 2006, www.ift.org. 65. Ocean Nutrition Canada, www.oceancanada.com. 66. Broek, A. and Gerritsen, J., Omega-3 fish oil: improved powder opens up new markets. Nutraceuticals-Now, Autumn, 26, 2004. 67. Institute of Food Technologists, Weekly newsletter, Washington, January 16, 2008. 68. Ohr, L. M., Nutraceutical ingredients for good digestion, Food Technol., 61, July 2007. 69. Garg, et al. 2006, Newsletter, U.S. Institute of Food Technologists, Washington, June 13, 2007. 70. Swansson, M. A. and Evenson, P., Nutritional additives, in Food Additives, Brown, A. L., Ed., Marcel Dekker, New York, 2002, p. 225.

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Macromolecules 14 Marine as Nutraceutical Carriers and Biofilms 14.1

INTRODUCTION

In nature, the food matrix plays an important role in the maintenance of physiological value of nutrients. The “natural” food structures may be one of the four broad categories: (1) fibrous structures assembled from macromolecules into tissues for specific functionality, such as muscle; (2) fleshy materials from plants that are hierarchal composites of hydrated cells that are bonded together at the cell walls, such as tubers, fruits, and vegetables; (3) encapsulated embryos of plants that contain a dispersion of starch, protein, and lipids assembled into discrete packets such as grains and pulses; and (4) a unique complex fluid called milk, containing several nutrients in a state of dispersion. Thermal and physical processing, mastication, and to a limited extent digestion breaks down the matrix releasing the nutrients and rendering them available for absorption and function in the living organism. It is generally agreed that various nutraceuticals when isolated from their natural biological systems may not exhibit full bioactive functions that are associated with them had they been in the natural environments, that is, the food microstructure or the food matrix. Generally, only a small proportion of molecules retain their functions after oral administration. Furthermore, retention of physiological functionality of nutraceuticals is a challenge. A number of reasons have been held responsible for this situation, such as insufficient gastric residence time, low permeability and solubility within the gut, instability due to the conditions encountered during food processing (temperature, oxygen, and light) or in the gastrointestinal tract (pH, enzymes, and presence of other nutrients). These in turn, influence the potential health benefits of nutraceutical molecules. Similarly, drugs, which are formulated into various dosages and are intended to obtain the desired therapeutic responses, may exhibit undesirable actions that are related to a particular route of administration.1 Therefore, there is a need for edible delivery systems for the food, medical, and pharmaceutical industries for optimal retention and delivery of nutraceuticals at their sites of absorption and biological functions. The fact that these delivery systems must be edible puts constraints on the type of ingredients and processing operations that can be used to create them. At the outset, this chapter focuses on various types of carriers of nutraceuticals followed by a discussion on marine macromolecules-derived matrices and their potential applications as carriers and biomembranes for marine nutraceuticals and functional molecules.

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FUNCTIONS OF A DELIVERY SYSTEM

Nutraceuticals isolated from various sources including those of marine origin generally require suitable matrices to improve their stability during storage and administration till they reach their specific site of action. Incorporation of bioactive compounds such as vitamins, probiotics, bioactive peptides, and antioxidants and also drugs into carrier systems provides a simple way to retain their functionality and develop novel functional foods that may have enhanced physiological benefits. The efficiency of a nutraceutical to be made available for a specific physiological function is generally referred to as its bioavailability. Other terminologies used include bioconversion fraction, bioefficacy, bioaccessibility, and bioequivalence. In general, these imply the potential of a nutrient to make a functional impact through its bioactivity.1 It is important to create a carrier matrix similar to the food, for successful delivery of the isolated nutrients to target sites to derive its maximum bioavailability and effective physiological functions. The functions of the carrier matrices therefore include (i) maintenance of the active molecular form until the time of consumption, (ii) delivery of the active form to the physiological target within the organism, and (iii) increasing the effectiveness at the absorption site and ensure optimal dosage delivery.2

14.3 MATRIX DESIGN FOR DELIVERY OF NUTRACEUTICALS During the past few decades, food technologists and pharmacologists have developed a number of carrier matrices for this purpose. Polymer-based delivery systems that trap molecules of interest within networks have been extensively developed for the biomedical and pharmaceutical sectors to protect and transport bioactive compounds to

TABLE 14.1 Functionalities, Working Principles, and Technologies in Delivery of Active Ingredients in Foods Functionality Control of migration Control of migration of oxygen Water vapor Flavors Control of release

Reaction control Stabilization of biological materials

Principle

Technology

Amorphous carbohydrate in glassy state Lipid coating

Spray drying and extrusion Spray chilling

Amorphous carbohydrate in glassy state

Fluidized bed coating

Hydrogel, coacervates, alginate complexes, gelatin capsules, emulsion, and complex fluids High-molecular-weight starches Change of local conditions Amorphous carbohydrate in glassy state

Complex coacervation, emulsions Extrusion (starches) Liposomes and vesicles Freeze-drying, spray drying

Source: Adapted from Ubbink, J. and Kruger, J., Trends Food Sci. Technol., 17, 244, 2006. With permission from Elsevier.

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perform target functions.3 Such carrier materials are those that are capable of forming films. These molecules not only constitute efficient carriers of food ingredients, but also provide them added functionality in foods and beverages. The materials that can be used include carbohydrates, proteins, lipids, gums, and cellulose. The choice depends on factors such as the nature of the core material; the expected requirements such as control of oxidation, stability, and control release of flavor and other ingredients; the process of encapsulation; labeling concerns; and economics. While choosing a matrix, it is important to ensure that the material is biocompatible and does not pose any safety problem.3 The major steps in the delivery of active ingredients are processing, formulation, and encapsulation.4 Table 14.1 summarizes the functionalities, working principles, and technologies in the delivery of active ingredients in foods. The prominent techniques for matrix development for nutrient delivery are encapsulation and the use of biodegradable films and membranes.

14.4

ENCAPSULATION

Encapsulation or microencapsulation of bioactive substances, nutrients, and probiotics is becoming a widely used technology in the pharmaceutical and food industries. The term “encapsulation” denotes the coating of functional food additives and nutraceuticals to modify their diffusional properties and control the interactions with environments, until the time of release when it is desired. The coating material used should be of edible nature. Preferably, it should be insoluble in aqueous solution, solid at oral cavity temperatures, and degradable by stomach or intestinal enzymes. Encapsulation in food technology helps tailoring of ingredients for specific uses and solving functional problems such as instability, poor solubility, and off-flavor. The technology has been used in the food industry for more than 70 years as a way to provide barriers to functional ingredients. In effect, nearly any material that needs to be protected, isolated, or slowly released at a particular time can be encapsulated. Encapsulated systems help in controlled release of vitamins, minerals, colors, and flavors in foods and facilitate customers to perceive improved product quality. Bioactive molecules and beneficial microorganisms may be protected by encapsulation during their transit in the digestive system to the absorption sites. Encapsulated forms of ingredients such as fish oils help their use as supplements in various products.5 Table 14.2 gives the advantages of encapsulation.

TABLE 14.2 Advantages of Encapsulation Permits timed release of encapsulated ingredients Enhances stability to temperature, moisture, and light Masks undesirable flavors Reduces negative interactions with other compounds Promotes easier handling by preventing lumping and improving flowability and mixing properties Avoids extra dosages of ingredients such as vitamins to compensate storage losses

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CLASSIFICATION

Encapsulated particles are classified based on the size of the particle, namely, microencapsules, when the size range is between 0.2 and 5000 μm; macrocapsules, when the size is larger than 5000 μm; and nanocapsule, when the size is smaller than 0.2 μm. The sizes of the encapsulated systems are useful to modify the properties of encapsulated nutrients through mechanism of release, particle size, and physical forms. For example, microencapsulation process can help mask undesirable odors and flavors, such as those of fish oil, in the final product. Microencapsulation properties may also be changed according to specific ingredient applications, and also composition and cost. Application of nanotechnology to develop nanoparticles is a promising tool for drug and nutraceutical delivery, and is discussed in the subsequent sections.

14.4.2

TECHNIQUES OF ENCAPSULATION

The techniques of microencapsulation include spray drying, spray chilling, extrusion, liposomes, cocrystallization, and freeze-drying. Chemical techniques include coacervation and molecular inclusion. Several authors have discussed the technology of encapsulation.5–10 14.4.2.1

Spray Drying

Spray drying remains to be the dominant process technology for microencapsulation. The process involves the development of an aqueous carrier phase, which is fed into a hot air dryer chamber by an atomizing head where the particles are immediately heated. A film is formed at the droplet surface retarding diffusion of the functional compound, whereas the water molecules are allowed to diffuse rapidly and migrate to the surface and vaporize. The dried particles are removed so as to prevent their overheating and are immediately dehydrated. The requirements of a carrier for development through spray drying are a high degree of solubility, limited viscosity at 35–45% solubility range, emulsifying properties, good drying properties, nonhygroscopic character, bland taste, nonreactivity, and low cost. Spray chilling, also referred to as coacervation, is based on the controlled phasing out of two interactive water-soluble polymers from solution to form a mixed polymer coacervate film around a lipid droplet.3,4,7 Encapsulation by spray cooling has been used for highly stable microcapsules containing iodine, vitamin A, and iron for salt fortification. The status of iron (ferrous or ferric) and type of food matrix strongly affect its relative bioavailability in humans, as recently observed.11 Microencapsulation could be a promising method for better bioavailability of iron. Potassium iodate, retinyl palmitate, and ferric pyrophosphate were microencapsulated in hydrogenated palm fat by spray cooling. The microcapsules were added to the local salt. During storage for 6 months, color change in the fortified salt observed was within the acceptable limits, and iodine losses were ∼20% comparable to the iodized salt. There was only 12% loss of retinyl palmitate during the storage period. No sensory difference was detected, and the overall acceptability of the fortified salt was good.12 Alginate in combination with silica gel has been used for microencapsulation.13

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Liposomes

Liposomes can be used as carrier systems for both water- and oil-soluble ingredients. Preparation of liposomes involves the development of a mixture of multi- and unilamellar structures with a mean diameter of 6 μm. Use of phospholipid-rich fractions, extracted from fat globule membranes, as liposomes renders oil-soluble flavors suspendable in aqueous systems. Liposome-entrapped flavors offer a wide range of new applications since they remain entrapped even after they are in contact with water. Vitamins that are entrapped in liposomes are protected, thereby avoiding the need for overage to compensate for processing and storage losses. Liposomeentrapped nutrients appear to be potential candidates as artificial diets.5 14.4.2.3

Microemulsion

Emulsion technology is particularly suited for the design and fabrication of delivery systems for encapsulating bioactive lipids (e.g., omega-3 fatty acids, carotenoids, and phytosterols). A recent review has discussed major bioactive lipids that need to be delivered within the food industry and different emulsion-based technologies for delivery systems including conventional emulsions, multiple emulsions, multilayer emulsions, solid lipid particles, and filled hydrogel particles. Each of these delivery systems could be produced from food-grade (Generally Recognized As Safe [GRAS]) ingredients (such as lipids, proteins, polysaccharides, surfactants, and minerals) using simple processing operations (e.g., mixing, homogenization, and thermal processing). This knowledge can be used to facilitate the selection of the most appropriate emulsion-based delivery system for specific applications.14 Flavors are delicate ingredients in any food formula. They are usually volatile; preserving them is often a major concern for food manufacturers. Encapsulation can be employed to coat the flavors to impart protection against evaporation, chemical reaction, or migration in a food. Other benefits of encapsulation of flavors are conversion of a liquid flavor into an easily dispensable powder, protection of specific flavor or key flavor components from change, and providing controlled release functionality in a product application. Encapsulation of flavors has been attempted and commercialized using different methods such as spray drying, spray chilling or spray cooling, extrusion, freeze-drying, coacervation, and molecular inclusion. Encapsulated flavors can be released in the mouth at body temperature. Protected in matrices of carbohydrates and fats, these flavors have designated release temperatures so that the flavor added to the food before processing is retained unaltered after cooking.10 Microemulsion is a potential delivery system for flavor compounds and bioactive ingredients. Microemulsions, which are formed using proteins, polysaccharides, lecithin, and other low-molecular-weight emulsifiers, individually and in various combinations, are spray dried to form microcapsules. Oil-in-water emulsions allow persistence of flavor and stability of molecules. The amount of oil that can be delivered in these formats varies from 1 to 30%. During spray drying, a significant proportion of the oil can migrate into the surface of the powder particle, which readily oxidizes and can cause off-flavors in food products. To improve the oxidative stability of the microencapsulated product, antioxidants are added either to the oil or to the powder, or both. Microemulsification has added advantages such as long shelf

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life, masking of the taste and flavor of fish, and improved bioavailability of encapsulated nutrients such as omega-3 polyunsaturated fatty acid (PUFA). Most of the encapsulated omega-3 oil products are based on the formation of fish oil emulsions. The technology has allowed the fortification of frequently used nutritional supplements.15,16 Bland refined fish oils, vitamins, minerals, meat processing aids, and flavors are examples of ingredients that can be encapsulated for use in pet foods.5 Apart from flavor, carotenoids have been subjected to microencapsulation. Waterin-oil-in-water (W1/O/W2) emulsions containing carotenoids having 25 and 35% final solid contents were spray dried producing microcapsules. Morphology, encapsulation efficiency, and larger particle size were observed in capsules having higher biopolymers blend to primary emulsion ratios and solids content. However, they showed relatively higher carotenoids degradation kinetics than microcapsules made with lower biopolymers blend to primary emulsion ratios and solids content, which exhibited poorer morphology, encapsulation efficiency, and smaller particle size.17

14.5 SOME NOVEL DELIVERY SYSTEMS A recent growth is the development of a delivery system named BioSwitch consisting of charged, cross-linked biopolymers in which active compounds are bound. The system responds to an external stimulus (such as change in temperature, pH, and concentration of certain metabolites) by releasing the active compound. As a result, the released compound is active only when required. The efficacy of BioSwitch as a delivery system for nutraceuticals has been evaluated using a computer-simulated in vitro gastrointestinal model. The model accurately simulates digestion processes and mimics the body’s peristaltic movements, and also allows sampling at different stages of digestion to study the integrity of nutraceuticals during passage through the gastrointestinal tract. Several applications of BioSwitch have been suggested, which include active antimicrobial food packaging, delivery of nutraceuticals, and disinfection of contact lenses.18 A nano-sized transport system, referred to as “product micelle,” has been developed recently for efficient delivery of ingredients. In this system, hydrophobic substances are rendered water-soluble, whereas hydrophilic substances are rendered fat-soluble. The process allows several ingredients to be combined precisely in the same ratio in each product micelle, having spherical particle size of 10–30 nm in diameter. The micelles are thermodynamically stable aggregates held together by van der Waals forces. They have high thermal, mechanical, and pH stability. The technique allows improved bioavailability and development of products having efficient antioxidative and other functional properties.19 Figure 14.1 shows the diagram of a typical product micelle.

14.5.1

MARINE MACROMOLECULES AS DELIVERY SYSTEMS

Different marine macromolecules have major applications as delivery materials. These include myofibrillar proteins, chitosan, carrageenan, alginate, and fucoidan. Proteins possess unique functional properties, which allow them to be an ideal material as carriers including encapsulation of nutraceuticals.3 The gel-forming property of proteins bestows unique ability making them efficient carrier molecules (see Chapter 3). Gels of diverse mechanical and microstructural properties can be

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Marine Macromolecules as Nutraceutical Carriers and Biofilms Product micelle Shell

435

Core (active substance)

30 nm

FIGURE 14.1 An artist’s view of a product micelle. (Courtesy of Hohenheim, S. and Biesalski, H.K., AQUANOVA AG, Birkenweg 8–10, Darmstadt, Germany, www.aquanova.de.)

formed, offering the possibility of developing biocompatible carriers for sensitive nutraceuticals in a wide variety of foods. Owing to the multiple functional groups in polypeptide chains and possibility of their interactions among themselves or with nutraceuticals to form three-dimensional structures, food proteins can be excellent delivery systems for delivery of nutraceuticals including vitamins, probiotics, bioactive peptides, and antioxidants to the site of action in active forms. The food proteins matrices could be in the form of hydrogel, micro or nanoparticles, all of which can be tailored for specific applications in the development of innovative functional food products. Other advantages of such protein matrices include their high nutritional value, abundance of renewable sources, and acceptability as naturally occurring food component, and susceptibility to digestive enzymes. The ability to control the particle size of protein materials is of primary importance not only for determining food product properties such as taste, aroma, texture, and appearance, but also for determining the release rates of the carried bioactive compounds and ultimately the amount that is absorbed into the body and hence the overall efficacy of the compounds. By decreasing the matrix size from micro- to nanometers, new protein vehicles with improved delivery properties could be developed. With improvements in manufacturing technologies, new strategies for stabilization of fragile nutraceuticals and development of novel approaches to site-specific carrier targeting, food protein–based materials have been suggested to play an important role as carrier molecules.3,20 Chitin and chitosan are abundantly available polysaccharides from shellfish processing, as discussed earlier (see Chapter 6). These polysaccharides either alone or

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in combination with others may be used for encapsulation of ingredients and also living cells.21 The seaweed hydrocolloids, alginate, carrageenans, and fucoidan are potential capsule materials, and have been used as matrices for delivery of nutraceuticals such as proteins, enzymes, vitamins, and antioxidants.5 Their function as encapsulation material is derived from their ability to form gels under a variety of conditions (see Chapter 10). Entrapment within spheres of calcium alginate gels, and to a lesser extent, potassium carrageenan and agarose, stands out as the most promising and versatile technique for immobilizing living organisms such as bacteria, cyanobacteria, fungi, plant, and animal cells. The food matrix has been found relevant in the absorption of vitamins. The absorption efficiency of highly lipophilic food microconstituents including the fat-soluble vitamins (A, E, D, and K), carotenoids, and phytosterols depends on factors such as the presence of fat and the type of food matrix. The potential uses of such systems in industry, medicine, and agriculture are numerous and range from production of ethanol from yeast or monoclonal antibodies from hybridomas to mass production of artificial seeds by entrapment of plant embryos.5 Matrices of marine origin such as alginate and chitosan have been developed for encapsulation of probiotics. Among different probiotics, Lactobacilli have played a crucial role in the production of diverse fermented products. Dairy products are obvious carriers of probiotics, thus, yogurt (fermented milk) and cheeses containing probiotics are well established in the market. In general, probiotic bacteria (see Chapter 13) exhibit a low ability to survive the harsh conditions of the gastrointestinal tract and need to be protected to preserve their activity, and several food matrices and encapsulation techniques have been successfully used for this purpose. Microencapsulation with alginate improved the viability of probiotic organisms in freeze-dried yogurt stored for 6 months at 4 and 21°C. Sodium alginate along with fructooligosaccharides or isomaltooligosaccharides and a peptide was examined to be used as a coating material to encapsulate the probiotics, Lactobacillus acidophilus, L. casei, Bifidobacterium bifidum, and B. longum. The proportions of the prebiotics, peptide, and sodium alginate were optimized using response surface methodology and the survival of microencapsulated probiotics was evaluated under simulated gastric fluid test. The results indicated that 1% sodium alginate mixed with 1% peptide and 3% fructooligosaccharide as the coating material would produce the highest viable probiotic counts. Addition of prebiotics on the walls of probiotic microcapsules provided improved protection for the active organisms. The probiotic count in the microcapsules remained viable for 1 month.21–24 Suitability of carrageenan to encapsulate enzyme preparations has been recognized. A proteinase preparation (Flavourzyme) was encapsulated using κ-carrageenan for cheese making. Cheese treated with the encapsulated enzyme showed higher rates of proteolysis than the control cheese throughout the ripening process. The rate of proteolysis was greater in cheese made from milk by incorporating κ-carrageenanencapsulated proteinase than proteinase encapsulated in gullan. The moisture content of cheese with added gum capsules was higher than that of the control cheese containing no proteinase. Differences in textural and sensory quality between treated and control cheeses were consistent with the release of proteinases from the capsules.25 Marine polysaccharides have also been successfully employed for encapsulating a number of vitamins, particularly vitamin E.21

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437

ENCAPSULATION OF MARINE INGREDIENTS

Ingredients from the sea can offer a range of functional and nutritional benefits as foods, as discussed in Chapters 3–12. Stability and functionality of these ingredients can be vastly improved by appropriate encapsulation techniques, mostly using marine polysaccharides, as discussed in the following section.

14.6.1

POLYUNSATURATED FATTY ACIDS

PUFA are prone to oxidation because of their high degree of unsaturation (see Chapter 5). Encapsulation has been carried out to control the oxidation. However, encapsulation of PUFA is not a solution to completely arrest the oxidation process. For instance, PUFA encapsulated by hot air drying using the single droplet method showed a relatively rapid oxidation during the early stages of storage, and the oxidation process leveled off during prolonged storage.26 Nevertheless, the oxidation could be controlled by encapsulation into a powdery matrix in a two-step process: the first step involves the emulsification of the PUFA with a dense solution of gum arabic or maltodextrin as the matrix material, and the second step involvesthe rapid dehydration of the emulsion. The encapsulated PUFA oxidized much more slowly than the untreated one.27 In another study, fish oil with 33% omega-3 fatty acids was microencapsulated by spray drying in a matrix of n-octenylsuccinate-derivatized starch and either glucose syrup or trehalose. Samples showed no difference in physicochemical properties as determined by particle size, oil droplet size, density, and surface characteristics. On storage at low relative humidity, lipid oxidation was decreased in trehalose-containing samples indicating that the amorphous trehalose is a suitable material for microencapsulation than glucose syrup. At 54% relative humidity, a rapid oxidation of the microencapsulated oil was observed on crystallization of trehalose, suggesting a need for high humidity requirement to control oxidation of the oil.28,29 Calcium alginate capsules have been commercially used to encapsulate shark liver oil (SLO). However, the capsules could be permeable to the oil. The problem could be solved by coating the capsules with a polyelectrolyte complex membrane of chitosan and alginate. An encapsulation efficiency (expressed as the percentage of SLO entrapped) of 87% (w/w) was obtained using 6% (w/v) alginate solution. The capsules could be degraded in vitro by enzymes such as lipases. There was no release of oil from the capsules up to 4 h at pH 1.2, but the capsules became very fragile when they were immersed in the enzyme solution at pH 7.4.30 In another report, SLO was encapsulated into a composite matrix comprising linseed sodium pectate, alginate, and chitosan. The oil was loaded into a linseed pectin–alginate solution at 2% (w/v), and gel beads were formed by dropwise addition into a CaCl2 bath and further coated by chitosan. Loading efficiency was greater in the capsules having 28% pectin, which retained 73% of SLO, whereas capsules of pure alginate retained 65% of the oil.31 Tuna oil-in-water emulsions (5% tuna oil in 100 mM acetate buffer, pH 3) stabilized either by lecithin membranes (primary emulsions) or by lecithin–chitosan membranes (secondary emulsions) were produced. The secondary emulsions were

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prepared using a layer-by-layer (LbL) electrostatic deposition method that involved adsorbing cationic chitosan onto the surface of anionic lecithin-stabilized droplets. The secondary emulsions had better stability to droplet aggregation than primary emulsions exposed to thermal processing (30–90°C for 30 min), freeze-thaw cycling (−18°C for 22 h followed by holding at 30°C for 2 h), high sodium chloride contents (200 mM), and freeze-drying. The addition of corn syrup solids decreased the stability of primary emulsions, but increased the stability of secondary emulsions.32 Encapsulated forms of fish oil, in particular, holds good for fortification. However, the bioavailability of the oil is important in determining the nutritional value of the product. In a study, the bioavailability of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from salmon patties fortified with fish oil (DHA:EPA ratio, 1.8; total DHA + EPA, ∼2.2 g) was compared to that of unfortified salmon patties (DHA:EPA ratio, 1.9; total DHA + EPA, ∼1.1 g), and also fish oil capsules (DHA: EPA ratio, 1.6; total DHA + EPA, 500 mg) in healthy older adults. The studies indicated that fish oil incorporated into the salmon patties was bioavailable.33 Fish oils and dry powders with microencapsulated oils are commercially available. The microencapsulated powders are used mainly in products such as bakery and milk items. They are produced by special spray drying process. Several patents on microencapsulation technologies for the protection and encapsulation of fish oil exist. A number of companies manufacture and sell microencapsulated fish oil powders for use in food products. However, the levels of incorporation that can be achieved with the existing technologies are very low and the amounts of long-chain omega-3 PUFA required to meet recommended allowances are impractical in most cases. Further work in this area needs to concentrate on the development of convenience foods, suitable for fortifying with larger amounts of omega-3 PUFA per serve, in a palatable format.34

14.6.2

GLUCOSAMINE AND CHONDROITIN SULFATE

Glucosamine is commonly consumed in combination with chondroitin sulfate as a cure for arthritis (see Chapter 6). Usually these compounds are added to milk before consumption. However, the compounds could be very labile when processed in a milk beverage, which has a pH of ∼6.5, whereas lowering the pH to 4.5 enhanced their stability. When heated in a milk beverage at 100°C or more, glucosamine caused instantaneous destabilization of milk proteins resulting in aggregation and precipitation of milk proteins. Nevertheless, heating at 80°C or lower did not destabilize proteins and showed less than a 5% loss. Similarly, only ∼10% of chondroitin sulfate was lost when held at 100–121°C for 30 min, suggesting that pasteurization of a glucosamine or chondroitin sulfate–enriched beverage is feasible at 80°C. Thus, a milk beverage could be a convenient vehicle for delivery of glucosamine and chondroitin sulfate.35 Under the U.S. Dietary Supplement Health and Education Act (DSHEA), fluid skim milk can be used to serve as a vehicle to deliver nutraceuticals dietary ingredients. In a recent study, the efficacy of milk to deliver nutracaeuticals was evaluated. Milk beverages enriched with various nutraceutical ingredients such as glucosamine, chondroitin sulfate, and others including soy isoflavonones, creatine, and lactoferrin were thermally processed at different combinations of temperatures from 72 to 138°C for different holding times using a microthermics pilot plant thermal processing unit and were incubated at refrigeration, room, or elevated storage temperatures.

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TABLE 14.3 Coating Materials Used in Food Encapsulation Materials Gums Carbohydrates

Lipids Proteins Inorganic material Synthetic polymers Other synthetic compounds

Examples Agar, sodium alginate, carrageenan, gum acacia, and gum arabic Corn syrup, pectin, dextrose, modified starches, sucrose, modified cellulose such as acetyl and carboxymethyl celluloses, maltodextrins, carrageenans, chitosanetc Bees wax, neutral lipids, stearic acid, paraffin, etc. Albumin, casein, gelatin, collagen, fish myofibrillar proteins, soy proteins, wheat gluten, corn zein, etc. Calcium silicate, sulfate, and clay Acetonitrile and polybutadiene Ethyl vinyl acetate, polyethylene, polyvinyl alcohol, and polyvinyl acetate

Residual concentrations of the active compounds were measured. Results showed a very good stability of chondroitin sulfate in the milk environment, whereas glucosamine, lactoferrin, and creatine showed only limited stability during either processing or storage. Glucosamine destabilized the milk protein system at boiling temperature or higher, which caused drastic precipitation in the heat exchangers. Nevertheless, all the tested nutraceutical compounds can be used to design milk beverage dietary supplements. The deployment of this technique also opens a new avenue for increase in milk consumption.35 The common coating materials used for encapsulation are given in Table 14.3.

14.7

BIODEGRADABLE AND EDIBLE FILMS

This section discusses the potential role played by marine macromolecules as biodegradable and edible films. At the onset, a brief introduction on these films is provided. Packaging technology has made significant progress during the past few decades parallel to the progress in the field of food and pharmaceutical sciences. A variety of synthetic packaging materials are now available for food and pharmaceutical products to maintain their quality during storage with minimum deteriorative changes. Synthetic packaging materials, mostly derived from petroleum products, however, are recognized to cause environmental hazards because of their nondegradable nature resulting in serious waste disposal problems. In recent times, there is a growing consumer demand for packaging products that are environment friendly, safer, and nontoxic. Apart from consumer interest, other factors driving development of the biodegradable packaging include the increase in crude oil prices, recognition of the new applications of bioplastics, increased economic viability, and the potential of nanotechnology for development of novel biodegradable films with improved properties. In view of these reasons, there is an increasing interest in developing packaging materials using renewable natural biopolymers such as polysaccharides and proteins. These materials, which are nontoxic and biodegradable, do not produce environmentally harmful by-products. Films made from such materials are variedly

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designated as biodegradable, biocompatible, environment friendly, renewable, biopolymer, edible, or green. Biodegradable packaging materials are being manufactured from refuse such as sugarcane waste and other sustainable plant by-products, including corn, potatoes, and bamboo. Potatoes and corn have dominated the biodegradable food packaging patent landscape. Apart from reduction of synthetic packaging, other advantages include possibility of newer ways of utilization of biopolymers, development of biopolymer-based active packaging, and uses of biofilms as carriers in fortification technologies.21,36–40 The biopolymers used for packaging may be divided into three broad main divisions based on their origin and protection as follows: 1. Polymers directly extracted or removed from biomass. Polysaccharides such as starch, cellulose, alginate, chitosan, and carrageenans and proteins such as casein, whey, collagen, gluten, and soy. These polymers are hydrophilic in nature and function as gas barriers. 2. Polymers produced by chemical synthesis using renewable biobased monomers such as polylactic acid, which is a biopolyester formed from lactic acid monomers. 3. Polymers produced by microorganisms or genetically modified bacteria. Bacterial cellulose, polyhydroxy alkonates (PHA), and polyhydroxy butyrates (PHB) are examples. These either alone or in combination with synthetic plastics or starch produce excellent packaging films. Although biodegradable and edible films may not completely replace synthetic plastics and perhaps even unnecessary, there is a good scope for the development of environment-friendly packaging materials using these polymers, which in addition offers a simple method to address the problem of marine waste disposal. It is unlikely that one polymer will be able to provide all the required properties such as low gas permeability or high water resistance, etc. Hence, it is necessary to use multiple materials for composite, laminate, or coextruded materials.40 Table 14.4 gives the benefits and possible uses of natural biopolymer-based packaging materials. TABLE 14.4 Benefits and Possible Uses of Natural Biopolymer-Based Packaging Materials Edible Biodegradable Supplement the nutritional value of foods Enhanced sensory characteristics of food Reduced packaging volume, weight, and waste Possibility of incorporation of antimicrobials and antioxidants Control over intercomponent migration of moisture, gases, lipids, and solutes Individual packaging of small particulate foods such as nuts and raisins Microencapsulation and controlled release of active ingredients Possible use in multiplayer food packaging together with nonedible films Low-cost and abundant renewable raw material Source: Adapted from Rhim, J.W. and Ng, P.K.G., Crit. Rev. Food Sci. Nutr., 47, 411, 2007. With permission from Taylor & Francis Ltd. (www.informaworld.com).

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441

EDIBLE FILMS

Edible films are thin layers of edible materials applied to food as a coating or placed on or between food components. The application of edible, protective coatings and films to foods to prolong the storage life has been practiced for the past several centuries. For instance, enrobing foods in fat, a practice called “larding” was used in sixteenth century in England. Soy films have traditionally been employed in the Orient to wrap and shape ground meat or vegetables. In recent times, gelatin is being used as sausage casings. Several food-based macromolecules such as proteins and polysaccharides can be used for the development of edible films. These natural sources include starch and cellulose from various agroproducts, zein (corn protein), pectin (agro waste), butylhydroxy butyrate (microbial product), polylactic acid, whey (milk protein), alginate and carrageenan (seaweed), chitosan (crustacean shell waste), collagen (constituent of skin, tendon, and connective tissue), and gelatin (partial hydrolysis product of collagen).41–43 14.7.1.1

Properties

Edible films have several properties comparable to those of synthetic packaging materials. They inhibit the migration of moisture, gases, and aromas and improve the mechanical integrity or handling characteristics of food products. For many food applications, the most important functional property of an edible film is control of moisture migration from the packaged food. This is because critical level of water activity (aw) must be maintained in many foods if the product is to exhibit optimum quality. Microorganisms require certain critical aw values for proliferation. In general, bacterial, yeast, and mold growth is inhibited below aw of 0.85, 0.70, and 0.60, respectively. Deteriorative chemical and enzymatic reactions are also strongly influenced by aw.44 In addition to moisture, edible films can also serve as a barrier to oxygen, carbon dioxide, and solute transmission in food systems, besides functioning as carrier of some active substances such as antimicrobial and antioxidant agents. Edible packaging can also retard the loss of volatile flavors and aromas. Another advantage is that they can coat small pieces or portions of food, which would be advantageous to products such as fish sticks. Furthermore, their barrier properties can be optimally enhanced by complementing with other types of packaging. A number of potential and innovative uses of edible films to improve food safety, extension of shelf life, and possibility of cost reduction of packaging materials have been identified.41–43,45 Active packaging is a promising upcoming area. In active packaging, the packaging material and the environment interact during food preparation and storage, resulting either in an improved product quality and safety and an extended shelf life or in the attainment of some product characteristics that cannot be obtained by any other means. The most well-developed active packaging technology is oxygen scavenging. Reduction of oxygen in a package by this method can inhibit oxidative reactions as well as growth of microorganisms. However, a reduction in oxygen concentration to a very low level may encourage the growth of anaerobic pathogenic microorganisms such as Clostridium botulinum in the package. Antimicrobial packaging, which has become one of the most interesting and challenging

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topics in the area, overcomes this problem. Incorporation of approved antibacterial compounds such as nisin in films can suppress the growth of Listeria monocytogenes in the packaged food. Several antimicrobial packaging systems have been discussed recently.46 In addition to antimicrobial compounds, edible packaging can go a long way in the delivery of functional foods since as a matrix it helps in fortification and functions as a carrier of ingredients such as vitamins and antioxidants, among others. The conceptual approach to develop functional foods through novel packaging technology is termed as “bioactive packaging,” in which a food package or coating is given the unique role of enhancing food impact over the consumer’s health. The technologies include integration of novel technologies such as micro- and nanoencapsulation and enzyme encapsulation and immobilization. All of these technologies are found to have excellent allies in particular properties exhibited by the biopolymers.47 A typical example is the technical feasibility of fortification of rice with folic acid using coating with edible polymers. A concentrated premix of rice can be prepared in a rotating coating pan by first spraying with folic acid solution, followed by coating with a suitable ingredient such as pectin and then drying. The important advantage is that the loss of folic acid during washing could be minimized in the products. However, the rice should be cooked in minimum water to avoid loss of the vitamin since no polymer could satisfactorily retain folic acid during boiling in excess water. The premixes had a higher water uptake ratio than raw milled rice. There was no significant sensory difference observed between the qualities of cooked fortified rice and raw milled rice.48,49 Despite the potential benefits, some disadvantages of edible films have been noted. Relatively poor mechanical and water vapor barrier properties of these films cause limitations for their industrial use. Protein and polysaccharide films generally act as good barriers against oxygen at low to intermediate relative humidity, whereas are poor barriers against water vapor due to their hydrophilic nature. Current research efforts have been focused on the modification of natural biopolymer-based films to improve their mechanical and water vapor barrier properties and also employing nanotechnology (see Section 14.7.4.1).39,50

14.8

MARINE MACROMOLECULES AS FOOD COATINGS AND EDIBLE FILMS

Marine macromolecules such as fish myofibrillar proteins, collagen and gelatin, chitin and chitosan from crustacean waste, and alginate as well as carrageenans from seaweeds are good raw materials for the development of biodegradable/edible films. Their use as carriers of bioactive compounds is discussed in detail in this section.

14.8.1

MARINE PROTEINS

One of the methods of using low-cost fish, which are abundantly available, is to develop protein films that can enhance the storage quality of high-value fishery products.51 Casting of such films can make use of inherent properties of proteins, which include their ability to form networks, plasticity, and elasticity. Protein films

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are formed through the partial denaturation of polypeptide chains by the addition of a solvent, alteration of pH, addition of an electrolyte to cause cross-linking, or application of heat. Films are subsequently formed when the partially denatured peptide chains bond together primarily through hydrophilic and hydrogen bonds resulting in the formation of a matrix.52 Fish myofibrillar proteins have been examined as a raw material for biopackaging. However, the impediments in this respect arise because of the inherent characteristics of fish structural proteins such as poor water solubility and sensitivity to denaturation by heat. These problems have been solved with the development of a process to prepare aqueous dispersions of fish meat. The dispersion is made using a mild acid-induced gelation of washed fish structural proteins. The proteins in the dispersion are also heat stable. The methodology for the preparation of thermostable dispersion of fish myofibrillar proteins has been discussed in detail (see Chapter 3). Films have been prepared from dispersions of proteins from shark (Scoliodon laticaudus), threadfin bream (Nemipterus japonicus), and dhoma (Johnius dussumieri).53 Coating of fresh fish fillets with the dispersion prepared from the same fish meat could enhance the chilled shelf life of the fillets. A typical example was shown in the case of the freshwater fish rohu (Labeo rohita), which was coated with an aqueous dispersion of the fish meat itself. To make the dispersion, a few pieces of washed rohu meat was homogenized in water and its pH was lowered to a value of 3.5–4.0 by the addition of a few drops of acetic acid. The dispersion had a protein content of 3%, and apparent viscosity of 1 Pa s. Fresh rout steaks were dipped for 1 h in this dispersion. The treated steaks were packaged in polyethylene pouches and stored under ice, which resulted in extended storage life of the product. It was observed that the dispersion-coated steaks gave a shelf life of 32 days as compared to 20 days for noncoated steaks stored under the same conditions. The effect was due to the antimicrobial activity of the protein coating, essentially due to low pH and presence of acetic acid, which is an antimicrobial additive.54 The shelf life of the dispersioncoated steaks could be further enhanced by exposure to gamma radiation at 1 kGy. Although, the acidic nature of the dispersion could cause some bleaching of fish pigments in the fillets during prolonged chilled storage, this could be prevented by incorporating 0.5% butyl hydroxyanisole or ascorbic acid in the dispersion as an antioxidant.55 Comparable results were obtained with a marine fish and seer fish too. Coating of seer fish steaks with the dispersion prepared from the same fish enhanced the chilled storage life of seer steaks from 15 to 20 days.56 The fish-based edible films can also enhance the stability of frozen fishery products. The major quality losses in frozen fish are moisture loss, lipid oxidation, and discoloration. Such changes are particularly significant during frozen storage of mince of fatty fish species. The potential of edible coatings from mackerel as a coating for blocks of mackerel mince to prevent weight loss and rancidity during prolonged frozen storage has been suggested. Unglazed mackerel mince blocks lost 35% of its initial weight due to moisture sublimation when stored at −18°C for 80 days. Conventionally, glazing with water is used to prevent weight losses in frozen fishery products. However, water glazing could not completely prevent dehydration loss during frozen storage. Glazing of the mince blocks with protein dispersion prepared from the same fish was found beneficial. The coated fish mince blocks exhibited

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only 17% weight loss. Furthermore, about 50% reduction in lipid oxidation was also observed in the dispersion-glazed mackerel mince blocks.56 The fish water-soluble proteins from blue marlin (Makaira mazara), which are normally discarded during processing, can be used for the production of edible films. An aqueous solution of 3% protein containing 1.5% glycerol as plasticizer having a pH of 10 was prepared by heating at 70°C. The solution was dried at 25°C for 20 h. The transparent film formed had appreciable flexibility and lower water vapor permeability (WVP) than protein films made from soybeans, rice bran, and casein. The characteristics of protein film were influenced by its pH. Protein solubility in films was high at acidic (2–3) and alkaline (8–12) pH ranges. Films were not formed between pH 4 and 6 because of the poor protein dispersion around the isoelectric point. Effect of different plasticizers including glycerol and polyethylene glycol (PEG) on the properties of the protein film from blue marlin was also studied. As the concentration of glycerol increased, the tensile strength (TS) decreased with concomitant increase in elongation at break (EAB) and WVP. The film containing both glycerol and PEG in a ratio of 2:1 exhibited maximum elongation value, whereas it reduced the water vapor barrier properties. The TS of the films was higher when prepared at acidic and alkaline pH ranges, whereas, EAB was almost constant irrespective of pH. The pH of film-forming solutions had no effect on WVP, light transmission, film solubility, and enzymatic hydrolysis. WVP of myofibrillar protein films were slightly lower than those of other protein-based edible films and higher by one to three orders of magnitude than those of synthetic films. It was suggested that the hydrophobic interactions contributed to film formation at both alkaline and acidic pH ranges, whereas at neutral pH it was ionic bonding.57–60 Edible film was also cast from tilapia proteins containing glycerol after heating at 90°C for 30 min. The properties of the film were evaluated in terms of color, opacity, mechanical properties, viscoelasticity, and thermal properties.61 Table 14.5 shows the TS, EAB and WVP of myofibrillar protein-based films and synthetic polymer films. A thermomolding process was developed for fish myofibrillar protein-based films. Effects of moisture content of protein powder (2–32%), thermomolding process conditions and process temperature (150–250°C) on mechanical properties, and characteristics of the films were assessed using response surface methodology. At low temperature and moisture content (200°C and 2.2%, respectively), glassy translucent materials were obtained. Expansion and development of a foamed structure were accompanied by increases in temperature and initial water content. Process conditions affected expansion yield, which in turn caused changes in the mechanical properties. High temperature and low water content caused relatively high thermal degradation of materials and also brought about Maillard reactions.52 14.8.1.1 Collagen and Elastin Collagen casings have been used in the food industry for a long time, particularly in sausage making.62 Recently, use of fish collagen in the manufacture of biopolymer films has been reported. Collagens from different species of fish were extracted using acetic acid, which were used to form biodegradable films. Differences in the mechanical film properties such as, TS, Young’s modulus, elongation, and WVP were found among the films. Films formed from collagen of New Zealand hoki and New Zealand ling had

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TABLE 14.5 TS, EAB, and WVP of Myofibrillar Protein-Based Films and Synthetic Polymer Films pH of Film-Forming Solution 2 3 7 10 12 Synthetic films LDPE PE PVDC

TS (MPa)

EAB (%)

WVP (1010 g/m/s/Pa)

15.1 ± 3.1 15.2 ± 19 8.0 ± 3.3 11.2 ± 1.4 16.7 ± 2.4 16.5 ± 0.9

29.5 ± 4.2 27.6 ± 9.5 29.3 ± 21.7 30.1 ± 17.7 28.1 ± 14.0 >1000

0.75 ± 0.1 0.66 ± 0.3 0.72 ± 0.2 0.77 ± 0.03 0.78 ± 0.14 0.02 ± 0.006

81.6 ± 3.2 65.6 ± 10.8

19.0 ± 6.0 18.0 ± 5.0

0.198 ± 0.015 0.002 ± 0.0000

LDPE, low-density polyethylene; PE, polyester; PVDC, polyvinylidene chloride. Source: Adapted from Shiku, Y., Hamaguchi, P. Y., and Tanaka, M., Fish. Sci., 69, 1026, 2003. With permission from Blackwell.

greater elongation, TS, and elasticity in comparison with similar films from Irish fish species. Addition of plasticizers could improve the properties of the film.63 Biomaterials based on elastin and elastin-derived molecules are increasingly investigated for their application in tissue engineering. The interest is further fueled by the remarkable properties of this structural protein, such as elasticity, self-assembly, long-term stability, and biological activity. Elastin can be applied in biomaterials in various forms. These include insoluble elastin fibers, hydrolyzed soluble elastin, recombinant tropoelastin (fragments), repeats of synthetic peptide sequences, and as block copolymers of elastin, possibly in combination with other (bio)polymers. The properties of various elastinbased materials and their current and future applications have been evaluated.64 14.8.1.2

Gelatin

Gelatin, perhaps, was the earliest edible film used for food products. Fish skin is a good source of gelatin, as discussed earlier (see Chapter 3). Gelatin is useful during encapsulation of thermolabile pharmaceuticals and in coating of photographic paper. Researchers have further sought to develop gelatin derivatives or modified gelatins such as cold water–soluble gelatin, hydrolyzed gelatin to develop bioactive peptides, and esterified gelatin.65–67 Edible films were prepared from the skins of brown stripe red snapper (Lutjanus vitta) and bigeye snapper (Priacanthus macracanthus). Higher contents of gelatin resulted in higher thickness and mechanical properties (TS and EAB) of the film, but lower WVP than those with lower protein content. Films prepared from the bigeye snapper gelatin exhibited lower mechanical properties than those prepared from the brown stripe red snapper gelatin. Incorporation of glycerol during casting markedly decreased the transition temperature and transition enthalpy of the films. Films from the bigeye snapper

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skin gelatin had lower content of cross-links with the concomitant increase in degradation peptides, compared to films from the brown stripe red snapper gelatin.68

14.8.2

MARINE POLYSACCHARIDES

Marine polysaccharides including alginate, chitosan, carrageenan, and fucoidan are ideal raw materials for the development of edible/biodegradable films because of their ability to form gel. Formation of the gel structures involves intermolecular association or cross-linking of polymer chains to form a semirigid three-dimensional matrix, which entraps and immobilizes the solvent. The gel could be converted into films, the properties of which could be enhanced by additional techniques such as copolymerization with multicomponent systems involving other hydrocolloids and incorporation of additives. These films are environment friendly because of their susceptibility to biodegradation by enzymes such as carrageenase and chitinase from microbial and other sources. Furthermore, being nontoxic the films can be used for packaging food products for environmental protection against bacterial contamination, moisture loss, and control of oxidation. They can also function as carriers of bioactive compounds.21,47,69 These properties with respect to different polysaccharides are discussed in the following sections. 14.8.2.1

Chitosan

Chitin and chitosan are inexpensive besides being nontoxic, biodegradable, and biocompatible. Chitosan is more versatile as compared to its precursor, chitin, in film-forming properties (see also Chapter 6).21 Chitosan has the capacity to form semipermeable coatings which when used in foods, prolong their shelf life by acting as barriers against air and moisture. The use of chitosan in food applications is particularly promising because of biocompatibility and nontoxicity. The process employed and solvent types used can influence the characteristics of the chitosan film. For example, chitosan from crawfish waste dissolved in acetic or formic acid formed flexible and transparent films that are desirable in packaging applications. Chitosan acetate films maintained lower moisture contents at any relative humidity level compared to chitosan formate films. The molecular weight of chitosan significantly influenced the sorption isotherm of chitosan formate films but not chitosan acetate films. The apparent viscosities of the coatings were dependent on the extent of deacetylation of chitin, the precursor of chitosan.70–72 Barrier properties, namely, permeability of oxygen, water vapor, and ethylene and mechanical properties, namely, TS and percentage EAB were determined in fresh and stored chitosan film plasticized with glycerin. It was observed that after an initial decrease in permeability during the first 2 weeks of storage, the mean oxygen permeability (4.6 × 10−5 cc/m-day-atm) and mean ethylene permeability (2.3 × 10−4 cc/m-day-atm) remained constant, whereas the mean water permeability (2.2 × 10−1 g/m per day) decreased with respect to storage time. TS values (15–30 MPa) decreased and percentage elongation values (25–45%) increased with respect to storage.73 Cross-linking and graft copolymerization of chitin and chitosan with synthetic monomers offer additional techniques to improve the properties of the biodegradable packaging films. Chitosan-based edible coating

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TABLE 14.6 WVPs of Some Composite Films from Marine Polysaccharides Basic Film Starch:alginate:glycerin (B) in the ratio 5.2:2.1:0.63

Chitosan

Composite Film Component

WVP

L

T

RH (%)

None B + lecithin–LA (2.1) B + lecithin–PA (2.1) B + lecithin–SA None Chitosan:LA Chitosan:PA Chitosan:BA Chitosan:AM

5.1 2.2 2.9 1.3 314.6 154.3 233.3 526.7 477.3

0.11 0.13 0.10 0.12 0.03 0.03 0.03 0.03 0.03

25 25 25 25 25 25 25 25 25

50–100 50–100 50–100 50–100 0–100 0–100 0–100 0–100 0–100

Abbreviations: WVP, water vapor permeability (g/mm2/day); L, thickness (mm, corrected to two decimal); T, temperature (°C); RH, relative humidity. Component: LA, lauric acid; PA, palmitic acid; SA, stearic acid; BA, butyric acid. Source: Adapted from Wu, Y. et al., Adv. Food Nutr. Res., 44, 347, 2002; Wong, D.W.S., J. Agr. Food Chem., 40, 540, 1992. With permission.

can be used as a vehicle for incorporating functional ingredients such as antioxidants, flavors, colors, antimicrobial agents, and nutraceuticals. Several studies have reported successful incorporation of calcium, vitamin E, and potassium into chitosan film formulation to prolong the shelf life and to enhance the nutritional value of fruits.74–76 Table 14.6 gives the WVPs of some composite films from marine polysaccharides.77 Chitosan has antimicrobial activities against a wide variety of microorganisms including fungi, algae, and bacteria. Antimicrobial activity of chitosan films against microorganisms including Staphylococcus aureus, and Propionobacterium propionicum has been reported.78–80 Besides, chitosan also possesses antioxidant activity, as discussed in Chapter 6. Making use of these properties, chitosan films could be used to enhance the shelf life of refrigerated fishery products. The influence of chitosan coating on shelf stability of Atlantic cod (Gadua morhua) and herring (Clupea harengus) has been examined in terms of different microbiological and biochemical parameters. Three chitosan preparations from snow crab (Chinoecetes opillo) processing wastes having different molecular weights were used. Coating of the fish fillets with chitosan having an apparent viscosity of 360 cP resulted in a 29–40% reduction in relative moisture loss for cod sample after storage up to 12 days at 4°C. The coating also significantly reduced lipid oxidation in the products. It was also observed that the preservative effect of chitosan and viscosity of the coating were interrelated. The study concluded that coatings with chitosan having apparent viscosities in the range 57–367 could be effective in shelf life extension of the fish fillets.80,81 Melanosis or black discoloration during storage is a problem in crustaceans, including shrimp, which affects its commercial value. The problem could be controlled by chitosan coating.82

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Chitosan films have been examined as coating for other food products too. Employing its antioxidant property, chitosan extended the refrigerated shelf life of meat products.83,84 The antioxidant activity of the polysaccharide could be further enhanced by gamma irradiation.83 Chitosan films reduced lipid oxidation in precooked beef patties during storage at 4°C (also see Chapter 10). Chitosan subjected to limited demineralization and deproteinization was employed as an egg-coating material to preserve internal quality of eggs up to 2 weeks longer than the control noncoated eggs during storage at 25°C. The coating also decreased weight loss. Furthermore, consumers could not differentiate the coated eggs from the control noncoated eggs.85 Chitosan films have the potential to prevent browning of fruits and vegetables (see Chapter 10). 14.8.2.2

Carrageenan

All the different types of carrageenans (λ, κ, and ι) possess gel-forming properties (see Chapter 10), which could be utilized to produce biodegradable films. Film prepared by mixing 2% κ-carrageenan, 0.1% KCl, 0.75% PEG, and 0.75% glycerol was examined for use as a potential packaging material for mackerel mince to prevent moisture loss and lipid oxidation. The fish mince patties were vacuumpackaged with the film and stored at temperatures ranging from +20 to −15°C. Weight loss and lipid oxidation were measured during storage. Packaged or nonpackaged samples stored at 20, 10, and 0°C showed a 60% weight loss between 2 and 15 days of storage, whereas the corresponding weight loss when stored at −15°C was ∼3% after 25 days. Nonpackaged samples stored at 0°C showed a steady increase in lipid oxidation, whereas carrageenan-wrapped product exhibited lower degree of lipid oxidation.86 Carrageenan film also reduced browning in apple slices87 and lipid oxidation and moisture loss in beef patties during storage at 4°C. The film was as effective as polyvinyl chloride (PVC) film in reducing moisture loss.84 Carrageenan could replace polyethene, which is used to coat the paper used for packaging oily or greasy foods. Both carrageenan-coated papers and films were highly impermeable to lipid, κ-carrageenen-coated paper showing maximum impermeability, followed by λ- and ι-carrageenan films. Lipid impermeability increased as the thickness of the κ-carrageenan layer increased. Carrageenan-coated papers weighing between 4 and 5 kg/ream (278 m2) showed lipid resistance comparable to that of polyethylenecoated papers. The lipid impermeability of carrageenan films was approximately 10 times higher than that of carrageenan-coated paper. κ-Carrageenan-coated papers having more than 4 kg/ream were shown to have adequate lipid barrier properties to be used for packaging greasy foods.39,88 14.8.2.3

Alginate

Algin has versatile uses in food products, microencapsulation, as microsphere vectors for drug delivery, for making dental impressions, as absorbent in dressings, antireflux therapies, in the dye industry, separation of milk whey, and many others. Alginate forms gel without heating or cooling, but needs the addition of calcium ions (see Chapters 9 and 10). Films of alginate gel have been shown to prevent moisture loss and lipid oxidation to a certain extent in fishery products. The solutions of soluble alginates are transparent, colorless, noncoagulable on heating, and have a

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wide range of viscosity. Alginate has a strong affinity for water and can be used to control moisture in food products.89

14.8.3

MULTICOMPONENT FILMS

Films made of more than one food-based natural component could be developed that possess excellent barrier and mechanical properties. Multicomponent edible films and coatings consisting of blends of various polymers, polysaccharides, proteins, and lipids have been developed to have cooperative functionalities. Properties of these films may be improved by taking advantage of specific characteristics of each component of the film. Such biobased multicomponent films, which resemble synthetic packaging material, may provide new atmospheric conditions and improve the quality of specific food products during storage. Marine resources such as chitosan, carrageenan, lipids, and proteins can be highly useful in the development of multicomponent eco-friendly packaging materials. A polysaccharide imparts structural cohesion and serves as a structural matrix, whereas a protein gives rise to a tight structure by inter- or intramolecular folding, and lipid increases hydrorepulsive character.90 Most of the multicomponent films containing a lipid as a moisture barrier and a high polar polymer as a structural matrix has been well studied. These films have been formed by two basic approaches: “coating technique” and “emulsion technique.” The former involves casting or laminating a lipid onto a dried edible base film to form a bilayer or laminated film, whereas the latter involves adding a lipid to a film-forming solution before film casting, and thus creating an emulsified film.42 The limitation of chitosan as a film for food packaging is its high sensitivity to moisture. The problem could be addressed by incorporating lipid into the film. Thus, blending chitosan with poly-3-hydroxybutyric acid can reduce water sensitivity of chitosan. In the composite film, the lipid component provides enhanced barrier to water vapor, whereas the hydrocolloid component provides necessary mechanical strength. The enzyme, transglutaminase, can be used to enhance cross-linking of chitosan with protein ingredients such as whey protein or ovalbumin to improve barrier properties.91,92 Composite films based on combinations of κ-carrageenan and chitosan, having molecular weights of 5.1 × 105 Da and 1.71 × 105 Da, respectively, were compared to films of pure κ-carrageenan or chitosan in terms of TS, elongation, and WVP. All the films were prepared with the addition of ascorbic acid up to 3%. The TS was highest in pure κ-carrageenan films with 2% ascorbic acid, whereas elongation was highest in pure chitosan films with no ascorbic acid, both types of films showing comparable WVP. Composite films showed intermediate levels of TS, but showed lower values of elongation and permeability than either of the pure films.93 A collagen–chitosan composite membrane has been developed at the Central Institute of Fisheries Technology, Cochin, India. The collagen and chitosan were from fish air bladder and prawn shell, respectively. The absorbable membrane could function as an artificial skin for treatment of burns and wounds, besides having potential application in dentistry (Devadasan, K., Personal communication, 2008). Table 14.7 gives the tensile properties of chitosan-based nanocomposite films. Composite film containing carrageenan and corn zein has been developed. The process consists of immersing preformed κ-carrageenan films into 10–95% corn zein in ethyl alcohol with PEG and glycerol at 20% and 24% of zein (w/w),

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TABLE 14.7 Tensile Properties of Chitosan-Based Nanocomposite Films Film Type Chitosan Na-MMt Cloisite 30B Nanosilver Silver ion

Thickness (µm)

TS (MPa)

Eb (%)

E (MPa)

64.0 ± 6.0 70.0 ± 9.2 63.3 ± 2.3 64.7 ± 9.0 61.3 ± 5.0

32.9 ± 0.7 35.1 ± 0.9 36.8 ± 3.3 35.9 ± 1.9 38.0 ± 3.4

54.6 ± 3.0 50.3 ± 11.7 66.3 ± 5.3 46.3 ± 7.6 38.9 ± 1.4

135.6 ± 2.1 191.3 ± 8.0 184.6 ± 5.1 259.2 ± 11.1 455.4 ± 25.1

TS, tensile strength; Eb, elongation; E, Young’s modulus of elasticity; Na-MMt, Sodium montmorillonite. Source: Reprinted from Rhim, J.-W. and Ng, P.K.G., Crit. Rev. Food Sci. Nutr., 47, 411, 2007. With permission from Taylor & Francis Ltd. (www.informaworld.com).

TABLE 14.8 Properties of Film Based on Composite Film of Gelatin and Sodium Alginate Thickness TS Young’s modulus Puncture resistance Water vapor transmission rate

0.364 mm 3.595 MPa 0.069 MPa 5.367 kg 42.897 g H2O/day/100 m2

Source: Reprinted from Liu, L., Kerry, J.F., and Kerry, J.P., J. Food Agr. Environ., 3, 51, 2005. With permission from the publishers, http:// www.isfae.org/scientificjournal.php.

respectively. WVP, water solubility, TS, and swelling ratio of corn zein-coated κ-carrageenan films decreased significantly with the increase in corn zein concentration. All the corn zein-coated carrageenan films showed heat sealing properties, although their sealing strength was less than 50% of corn zein film. However, corn zein coating affected the color of κ-carrageenan films, causing an increase in yellowness.39 Carrageenan could be used as a reinforcing material for potato starch-based edible wrapping paper. A combination of 0.5% carrageenan, 3% glycerol, and 5% sorbitol increased the foldability of potato starch edible paper to 60 times. The film was reasonably transparent, with a transparency value of 76%.94 Carrageenan–starch film can be used as meat casings to provide high strength and excellent adhesion to meat. Composite film consisting of gelatin and sodium alginate has been developed.95 Some of the recent developments in the field include preparation of bariumlinked alginate membrane and wet spun chitosan–collagen composite fibers. Table 14.8 gives the properties of film based on composite film of gelatin and sodium alginate.

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451

ACTIVE PACKAGING

“Active packaging” or sometimes referred to as “smart packaging” is intended to sense internal or external environmental changes in a package and to respond to it by changing the properties or attributes of the packaging film and hence the internal environment of the package. In response to the dynamic changes in the current consumer demand and market trends, the area of active packaging is becoming increasingly significant. Principal active packaging systems include those that involve oxygen scavenging, moisture absorption and control, carbon dioxide and ethanol generation, and antimicrobial migrating and nonmigrating systems.41 The role of active packaging, in conjunction with other food processing and packaging techniques, is to enhance the preservation of contained food and beverage products. The technology has attracted much attention from the food industry because of the increase in consumer demand for minimally processed, preservative-free products. The different categories of active packaging concepts with particular regard to the development, activity of antimicrobial packaging and its effects on food products, and the current and future applications have been reviewed.49 Antimicrobial packaging is a promising form of active packaging and encompasses any packaging technique(s) used to enhance the shelf life of food products by controlling microbial growth in them. The antimicrobial substances in the packaging materials function by extending the lag period of microbes in the food. These films contain antimicrobial agents such as lysozyme and nisin. Lysozyme (1,4-β-N-acetyl muramidase, EC 3.2.1.17) is a 14,600 Da enzyme present in avian eggs, milk, tears, and fish. It is active against gram-positive bacteria. Nisin is a 34 amino acid peptide secreted by Lactococcus lactis spp. having a molecular weight of 3500, but usually existing as a dimer with molecular weight of 7000, which affects gram-positive bacteria. Care needs to be taken in developing antimicrobial films so that the process of incorporation of the compounds does not adversely affect their bioactivity. Activity of antimicrobial films is based on the diffusivity of preservatives incorporated into the films. In addition, the films could be safe for packaging of food.96,97 Antioxidants containing active packaging is another area that can be developed to control lipid oxidation in foods. Antioxidants (ascorbyl palmitate and α-tocopherol) were incorporated into 10% (w/w) whey protein isolate (WPI) coating solution containing 6.67% (w/w) glycerol. Before incorporation, the antioxidants were mixed either using powder blending or ethanol solvent-mixing. The WPI solutions, adjusted to appropriate viscosity and turbidity, were dried on a flat surface to produce WPI films. The films produced by ethanol mixing were more transparent than the films produced by powder blending. Oxygen permeability of the powder-blended sample was lower than that of the alcohol-blended product. However, both the diffusivity and solubility of oxygen were statistically the same in both the films. WPI films had very low oxygen solubility. Permeability of antioxidant-incorporated films was not enhanced compared to the control WPI films.98 14.8.4.1

Marine Polysaccharides for Active Packaging

Antimicrobial films from marine polysaccharides such as carrageenan, chitosan, and alginate have been developed recently.99,100 A diffusion cell was used to determine

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the diffusivity of antimold compounds. The release of potassium sorbate incorporated into κ-carrageenan-based antimicrobial film as a function of pH (3.8–7.0) and temperature (5–40°C) was determined. Kinetics of potassium sorbate release was found to follow Fick’s law of diffusion. As the temperature decreased from 40 to 5°C, the diffusion coefficient also decreased at pH 5.2. However, diffusion was unaffected by changes in solution pH.99 Inhibition of surface spoilage bacteria in processed meats by the application of antimicrobial films prepared from chitosan has been observed.101 Antimicrobial effect of garlic oil-incorporated chitosan edible film was compared to conventional food preservatives, namely, potassium sorbate, bacteriocin, and nisin. Incorporating garlic oil up to 100 μL/g, potassium sorbate at 100 mg/g, or nisin at 51,000 IU/g of chitosan were found to have antimicrobial activity against S. aureus, L. monocytogenes, and Bacillus cereus. At these levels, the films were physically acceptable in terms of appearance and mechanical and physical properties.102 A fish skin gelatin film containing lysozyme has been developed.103 Synergistic effects of lysozyme and nisin, in sodium alginate and κ-carrageenan-based biopolymer films toward various food spoilage bacteria and pathogens were evaluated. Films were prepared using 2% alginate and 1% carrageenan with 0.75% plasticizer containing equal proportions of PEG and glycerol together with various combinations or a single addition of 100 μg nisin per milliliter, and 0.1% lysozyme solutions were heated with stirring and then spread onto the glass plates for cooling at 25°C. Dry films were peeled off and held in polyethylene at 25°C and 50% relative humidity for 3–4 days. Antimicrobial activity of films was measured using agar diffusion tests. Sodium alginate-based films exhibited higher extent of inhibition than carrageenan-based films with the same antimicrobial compound additions. TS and EAB values were, however, significantly reduced in films with added antimicrobials.104 Heat-press and cast methods were used to prepare nisin-containing biopolymer films based on κ-carrageenan and chitosan, and the antimicrobial activity of each film was investigated. Films contained 104 IU nisin per gram. The amount of nisin released was determined by a diffusion bioassay using Micrococcus luteus ATCC 10240 as the test organism. Both heat-press and cast type of film inhibited the microorganism; cast films with a biopolymer coating exhibited higher antimicrobial activity than heat-pressed films, as the release rate of nisin from the latter was very slow. Of the cast films examined, chitosan films containing nisin appeared to exhibit the maximum inhibitory activity.105 Antimicrobial packaging can be a promising tool for protecting ready-to-eat meat products against contamination of L. monocytogenes.106 Gelatin film containing an antimicrobial compound has been developed to prolong the shelf life of fishery products.107 Table 14.9 indicates the various applications of marine macromolecules for antimicrobial packaging. Carrageenan at 0.5% concentration could be used for active packaging (see Section 14.7.3.), in combination with antibrowning agents for extending the shelf life of minimally processed apple slices. The antibrowning treatment involves dipping the slices in one or more solutions of citric, ascorbic, or oxalic acids as well as organic acid plus calcium chloride mixtures. Respiration rates, color, firmness, sensory properties, and microbiological quality were assessed during the storage of vacuum-packaged slices at 3°C for 2 weeks. Coatings and antibrowning agents

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TABLE 14.9 Some Applications of Marine Macromolecules for Antimicrobial Packaging Biopolymer

Antimicrobial Agents

Alginate

Lysozyme and nisin

Alginate

Carrageenan

Glucose oxidase Nisin — Sorbate, bacteriocin Sodium benzoate and potassium sorbate Acetic/propionic acid Nisin Chlortetracycline, oxytetracyclin, ethylenediaminetetraacetic acid (EDTA), lysozyme, nisin Lysozyme and nisin

Gelatin

Antimicrobial film

Chitosan Chitosan Chitosan Agar Carrageenan

Food Preservation/ Bacterial Control Control of various food spoilage bacteria Fish Skim milk, beef, poultry Fresh fish Various pathogens Butter Poultry Culture media M. luteus Control of various food spoilage bacteria Spoilage control of tilapia fillets

Reference 104 97 81 102 97 97, 105 97

97 107

extended the shelf life of apple slices by 2 weeks, by maintaining color and reducing counts of mesophilic and psychrotropic microorganisms. The coating reduced initial respiration rates by 5%. Mixtures of acid/calcium chloride had a synergistic effect on color. Coated apple slices maintained acceptable sensory scores for color, firmness, flavor, and overall preference, even after 14 days.87 14.8.4.2

Casting of Films

A range of methods for casting biodegradable/edible films have been reported, which include plate casting, spraying, dipping, and enrobing. Biodegradable edible packaging films are generally prepared by wet casting of the aqueous solution on a suitable base material followed by drying. Choice of the base material is important to obtain films, which can be easily removed without any tearing and wrinkling. Heat-press and cast methods were used to prepare nisin-containing biopolymer films based on κ-carrageenan and chitosan.104,105 Extrusion technology has been successfully employed within the food industry for the manufacture of a wide range of food products. Recently, a process for extrusion-based manufacture of biodegradable packaging films from food-based polymers has been developed. The parameters examined included extrusion temperature, feeding rate of the material, plasticizer (glycerol) concentration, and screw speed. The raw materials included gelatin/sodium alginate blends among other biopolymers. Sodium alginate/gelatin blends at setting temperatures ranging from 120 to 135°C at 50% glycerol concentration produced a film that had optimal TS, Young’s modulus, puncture resistance, color, turbidity, and vapor transfer rate.95

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14.9 NANOTECHNOLOGY “Nanoparticle technology” is the general term for the unique properties of submicron-sized particles. The technique allows the creation and use of structures, devices, and systems that have novel properties and functions because of their small or intermediate size in the range 1–100 nm diameter. It helps reinforcement of the packaging film by dispersion of nanometer-sized particles in the polymer matrix, thereby improving mechanical, thermal, optical, and physiochemical properties and decreasing gas permeability with very low filler loading, typically 5% or lower.39,50 With regard to biological systems, the advantages of nanotechnology are improved solubility, target ability, adhesion to tissues, and possibility for better delivery of nutraceuticals. Despite recent developments in the field, at present, the potential nanotechnology in biopackaging is rather limited due to insufficient knowledge of the physicochemical aspects of nanoparticle systems organization and of the interactions between bioactive molecules and their carrier matrices. Further advances are needed to turn the concept of nanosizing into a realistic practical technique for the next generation of molecule delivery systems.108

14.9.1

NANOTECHNOLOGY FOR MARINE POLYSACCHARIDE FILMS AND PARTICLES

Nanotechnology offers possibilities for development of novel chitosan films. Traditionally, mineral fillers such as clay and silica are incorporated in film preparation to reduce its cost and improve performance. A chitosan-based nanocomposite films using nanoclay particles such as Cloisite 30B, nanosilver, and silver ion were prepared by the solvent casting method. For this, a chitosan film solution was prepared by dissolving 2% chitosan (w/v) in 1% acetic acid solution and 2% glycerol. The clay solution was prepared by dispersing layered nanoclay particles in the same solvent as that used for the film-forming solution. The clay solution was added to the polymer solution dropwise and the resulting mixture was subjected to high shear mixing and ultrasonic treatment. The resulting solution was allowed to dry in ambient or elevated temperature conditions to make a free-standing film and then cured.39 The ability of milk proteins to interact strongly with charged polysaccharides such as chitosan opens up further possibilities for making novel hybrid nanoparticles.109 There is potential to develop marine macromolecules-based nanoparticles for cancer therapy, as shown by a recent study. Biodegradable nanotubes were fabricated through the LbL assembly technique of alternate adsorption of alginate and chitosan onto the inner pores of polycarbonate template with the subsequent removal of the template. The assembled materials exhibited good film-forming ability. Changing the assembled layers controlled the thickness of nanotube walls. Confocal microscopy images showed that the assembled alginate–chitosan (alg/chi) nanotubes can be internalized into the cancer cell readily. The cell viability experiment proves that the alg/chi nanotubes have low cytotoxicity. The final assembled nanotubes have presented good biodegradability and low cytotoxicity.110 The technique can be extended to biosensor development. A hydroxyl-containing antimony oxide bromide (AOB) nanorods were synthesized by a hydrothermal method. The AOB nanorods were combined with the biopolymer chitosan to form an organic–inorganic hybrid material, and a biocompatible, crack-free and porous chi–AOB composite film.

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The advantages of organic–inorganic hybrid materials are that they facilitate direct electron transfer and excellent bioelectrocatalytic activity. The prepared biosensor containing horseradish peroxidase displayed good sensitivity and reproducibility, wide linear range, low detection limit, fast response, and excellent long-term stability. The chi–AOB composite film could be used efficiently for the entrapment of other redox-active proteins and may find wide potential applications in biosensors, biocatalysis, biomedical devices, and bioelectronics.111 Nanofibers of chitosan membrane or mat has also been developed using electron spinning of carboxymethyl chitosan, which has potential medical benefits.112

14.10 HYDROGELS AND MEMBRANES FOR THERAPEUTIC APPLICATIONS Hydrogel is an infinite water-swollen network of hydrophilic polymers that can swell in water and hold a large amount of water while maintaining a network structure. Over the past decade, hydrogels have been studied extensively in biomedical and pharmaceutical applications, primarily due to their ability to protect drugs from hostile environments and to deliver them in response to environmental stimuli such as pH and temperature. Protein hydrogels are undoubtedly the most convenient and widely used matrix in food applications. These gels are attractive carriers for controlled release of bioactive molecules.

14.10.1 MARINE MACROMOLECULES AS HYDROGELS AND MEMBRANES FOR DRUG DELIVERY Marine polysaccharides have significant applications as biomembranes and hydrogels for delivery of drugs and other therapeutic applications. A few of these are discussed. Alginate gels have the potential to be used as the implantation material for hormone-producing cells and encapsulated Langerhans islets. The breakthrough in genetics of alginate-producing bacteria also opens up the prospect of polysaccharide engineering.113 The manufacture of highly stable and elastic alginate membranes with good cell adhesivity and adjustable permeability has been reported. Clinical-grade, ultra-high viscosity alginate is gelled by diffusion of barium ions followed by use of the “crystal gun.” Burst pressure of well-hydrated membranes is between 34 and 325 kPa depending on the manufacturing and storage conditions. NaCl-mediated membrane swelling can be prevented by partial replacement of salt with sorbitol, allowing cell culture on the membranes. Water flows induced by sorbitol and raffinose (probably diffusional) are lower than those caused by PEG. The properties of the film such as hydraulic conductivity, mechanical stability, and other characateristics have been reported.114 A novel biocompatible blended fiber was prepared by blending chitin with collagen. Chitosan (3.3%) and tropocollagen or collagen in aqueous 2% acetic acid–methanol (2:1, v/v) solutions were spun through a viscose-type spinneret in an aqueous 5% ammonia solution containing 40–43% ammonium sulfate at room temperature to afford a white fiber of chitosan–tropocollagen. The tropocollagen content (up to 50% by weight) in the blended fiber affected their tenacity and elongation values only to a minor extent. The blended fiber was chemically N-modified at the fiber state by treatment with a series of carboxylic anhydrides and aldehydes

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to afford the corresponding N-modified fiber. A transparent blended hydrogel of N-acetylchitosan (chitin) with tropocollagen was produced from the aforementioned mixed solution by treatment with acetic anhydride, and its membrane and sponge sheet were also prepared from the hydrogel.115 A composite collagen–chitosan membrane has the potential to treat periodontal defects in dentistry. A chitosan membrane has been developed at the Central Institute of Fisheries Technology, Cochin, India, that is claimed to have the potential to replace Teflon (Devadasan, K., Personal communication). Transdermal delivery is a method designed to deliver drugs continuously at a controlled rate through the skin to the systemic circulation. Chitosan can be used as a safe carrier for transdermal delivery of drugs, which can eliminate some of the problems of conventional dosage forms.116 A process to prepare mixed capsules of chitosan with other macromolecules such as soy albumin has been reported. When a 2% solution of chitosan (pH 5) was dropped into a 5% solution (pH 8) of soy globulin, a thin chitosan–globulin membrane was formed on the surface of the chitosan capsules. The diameter of the capsules obtained was approximately 3 mm.117 A novel dual cross-linked complex gel bead for oral delivery of protein drugs has been reported recently. The composite capsules composed of carboxymethyl chitosan and alginate was prepared with the dual cross-linking agents, calcium chloride and dialdehyde starch. The stability of complex beads increased compared to the gel beads prepared with the one-component cross-linking agent, which indicated that the dual-component cross-linking agents were more advantageous. The complex beads seemed to withstand the acidity of gastric fluid without liberating substantial amounts of loaded protein and retard protein release in the intestine, suggesting that the dual cross-linked complex beads could be a suitable carrier for oral protein drug delivery.118 A drug delivery system consisting of liposome–chitosan nanoparticle complexes targeted to ocular surface has also been developed recently.119 Biodegradable glucose-sensitive in situ gelling system based on chitosan for pulsatile delivery of insulin was developed. The glucose-sensitive gels responded well to varied glucose concentrations in vitro, indicating their ability to function as environment-sensitive systems. Insulin loaded onto the gels was optimized and was found to affect the rheological behavior of these gels. These gels released the entrapped insulin in a pulsatile manner in response to the glucose concentration in vitro. Furthermore, the formulations when evaluated for their in vivo efficacy in streptozotocin-induced diabetic rats at a dose of 3 IU/kg showed their ability to release insulin in response to glucose concentration and were preferred because of its better performance compared to plain insulin formulation used as the control. These results indicated that biosensitive chitosan in situ gelling systems have substantial potential as pulsatile delivery systems for insulin.120 Alginates are also being evaluated as an insulin delivery system for diabetic patients.112 The development of selective delivery systems for cancer diagnosis and chemotherapy is one of the most important goals of current anticancer research. Various self-assembled nanoparticles as candidates to shuttle radionuclide and drugs into tumors were evaluated. By combining different hydrophobic moieties and hydrophilic polymer backbones, various self-assembled nanoparticles were prepared, and their in vivo distributions in tumor-bearing mice were studied by radionuclide imaging.

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Of these, fluorescein isothiocyanate-conjugated glycol chitosan (FGC) nanoparticles exhibited highly selective tumoral localization. Scintigraphic images obtained one day after the intravenous injection of FGC nanoparticles clearly delineated the tumor against adjacent tissues. This work can provide scope for engineering of nanoparticles and for their use in cancer therapy and diagnosis, potentially facilitating the delivery of multiple therapeutic agents and imaging probes at high local concentrations.121 Another area of application is gene therapy. Gene therapy using polymers such as chitosan shows good biocompatibility. Folic acid–chitosan– deoxyribonucleic acid (DNA) nanoparticles have been synthesized and their in vitro cytotoxicity have been evaluated. The nanoparticles presented a mean size of 118 nm and 80% cellular viability. Gel electrophoresis showed intact DNA within the carriers.122

14.10.2

MARINE POLYSACCHARIDES AS SCAFFOLDS

Skin repair is an important field of tissue engineering, especially in the case of extended third-degree burns, where the current treatments are still insufficient in promoting satisfying skin regeneration. A biodegradable scaffold in tissue engineering serves as a temporary skeleton to accommodate and stimulate new tissue growth. Chitosan and other marine polysaccharides can be used for the development of hydrogels to be used in tissue engineering for skin regeneration. Bio-inspired bilayered physical hydrogels consisting of chitosan and water were processed and applied to the treatment of full-thickness burn injuries. The first layer constituted of a rigid protective gel that ensured good mechanical properties and gas exchanges. The second soft and flexible layer allowed the material to follow the geometry of the wound and ensured a good superficial contact. Veterinary experiments were performed on pigskins, and biopsies up to 293 days were carried out by histology and immunohistochemistry. The results showed that chitosan materials were well-tolerated and promoted good tissue regeneration. They induced inflammatory cells migration and angiogenetic activity, favoring a high vascularization of the neotissue. After day 22, type I and IV collagens were synthesized under the granulation tissue and the formation of the dermal– epidermal junction was observed. After 100 days, the new tissue was quite similar to a native skin, especially by its esthetic aspect and great flexibility.123 Hydrogels derived from natural proteins and polysaccharides are ideal scaffolds for drug delivery since they resemble the extracellular matrices of the tissue consisting of various amino acids and sugar-based macromolecules. The injectable polymer scaffolds are biocompatible and biodegradable. A new class of biodegradable and self-cross-linking biopolymers as injectable hydrogels derived from oxidized alginate and gelatin has been developed. The periodate-oxidized sodium alginate having appropriate molecular weight and degree of oxidation rapidly cross-links proteins such as gelatin in the presence of small concentrations of sodium tetraborate (borax) to give injectable systems for tissue engineering, drug delivery, and other medical applications. The rapid gelation in the presence of borax is attributed to the slightly alkaline pH of the medium as well as the ability of borax to complex with hydroxyl groups of polysaccharides. The gelling time decreased with the increase in concentration of alginate. The degree of cross-linking was found to increase with the increase in the degree of oxidation of alginate, whereas the swelling ratio and the

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degree of swelling decreased. The gel was found to be biocompatible and biodegradable. The potential of the system as an injectable drug delivery vehicle and as a tissue-engineering scaffold was demonstrated by using primaquine as a model drug and by encapsulation of hepatocytes inside the gel matrix, respectively.124 Potential of the alginate–gelatin hydrogel containing borax for wound dressing has also been reported. Wound dressings that can be formed in situ offer several advantages over the use of preformed dressings, such as conformability without wrinkling or fluting in the wound bed, ease of application, and improved patient compliance and comfort. The hydrogen was found to fulfill many critical requirements in a wound dressing material. The composite matrix has the hemostatic effect of gelatin, the wound healing-promoting feature of alginate, and the antiseptic property of borax to make it a potential wound dressing material. The hydrogel was found to have a fluid uptake value of 90% of its weight, which would prevent the wound bed from accumulating exudates. The hydrogel can maintain a moist environment over the wound bed in moderate to heavily exuding wound, which would enhance epithelial cell migration during the healing process. Using a rat model, it was demonstrated that within 2 weeks, the wound covered with gel was completely filled with new epithelium without any significant adverse reactions.125 Hybrid alginate peptides and peptides derived from laminin and elastin, were also developed for wound dressing. Its efficacy was examined for cell attachment and proliferation using normal human dermal fibroblasts (NHDF). The hybrid peptides promoted attachment of NHDF and strong NHDF proliferative activity. The alginate dressings linked with the hybrid peptides showed significantly greater epithelialization and a larger volume of regenerated tissue. The dressings linked with the hybrid peptides could be promising especially for wounds with impaired healing.126 Development of a biodegradable porous scaffold made from chitosan and alginate polymers having improved mechanical and biological properties has been reported. The mechanical properties were attributable to the formation of a complex structure of chitosan and alginate. Bone-forming osteoblasts readily attached to the chitosan–alginate scaffold, proliferated well and deposited calcified matrix. The in vivo study showed that the hybrid scaffold had a high degree of tissue compatibility. Calcium deposition occurred as early as the fourth week after implantation. The scaffold can be prepared from solutions of physiological pH, which may provide a favorable environment for incorporating proteins with less risk of denaturation.127 Although the total joint replacement has become commonplace in recent years, bacterial infection remains a significant complication in following this procedure. One approach to reduce the incidence of joint replacement infection is to add antimicrobial agents to the bone cement that is used to fix the implant. The use of quaternary ammonium derivatives of chitosan derivative nanoparticles (QCS NP) as bactericidal agents in poly(methyl methacrylate) (PMMA) bone cement with and without the antibacterial compound, gentamicin, was examined. The antibacterial activity was tested against S. aureus and S. epidermidis. A 103-fold reduction in the number of viable bacterial cells on contact with the surface was achieved using QCS NP at a nanoparticle/bone cement weight ratio of 15%. Growth inhibition of the microorganisms on the surface of the chitosan derivative nanoparticles (CS NP) and QCS NP-loaded bone cements was clearly

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observed. The CS NP and QCS NP also provided a significant additional bactericidal effect to gentamicin-loaded bone cement. The antibacterial effectiveness remained high even after the modified bone cements were held immersed for 3 weeks in an aqueous medium, whereas no cytotoxic effect of the CS NP- and QCS NP-loaded cements was shown. Mechanical tests indicated that the addition of the CS and QCS in nanoparticulate form allowed the retention of strength of the bone cement to a significant degree. The results indicated a new promising strategy for combating joint implant infection.128 Preparation and initial characterization of novel calcium titanium phosphate–alginate and hydroxyapatite–alginate microspheres as enzyme delivery matrices and bone regeneration templates has been reported.129 Chitosan in combination with other polymers can function as good delivery system for drugs, nutraceuticals, and enzymes. A new microsphere delivery system based on cross-linking chitosan with fucoidan, a seaweed polysaccharide, was evaluated as a drug carrier. The microspheres (also designated as fucospheres) were prepared by cross-linking the oppositely charged biopolymers. The shape and surface morphologies of the particles were evaluated by scanning electron microscopy, and the size, charge, and encapsulation capacity of the microspheres were determined. The amount of the protein, bovine serum albumin, released from the microspheres into the phosphate buffered saline (pH 7.4) was determined spectrophotometrically. The concentrations of fucoidan, chitosan, and protein were 1.5–2.5%, 0.25– 0.75%, and 0.25–0.75%, respectively. Smooth and spherical microspheres between the size range of 0.61 and 1.28 µm were obtained. Bovine serum albumin was efficiently encapsulated up to 89% into the microspheres; the highest encapsulation obtained with microspheres containing 2.5% of fucoidan.130 Multicomponent systems containing chitosan and β-lactoglobulin core shell nanoparticles were successfully prepared with the aim of developing a biocompatible carrier for the oral administration of nutraceuticals. The effects of pH and initial concentrations of native and denatured β-lactoglobulin on the properties of the nanoparticles were investigated. Uniform nanoparticles were prepared by ionic gelation with sodium tripolyphosphate. β-Lactoglobulin loading efficiency spanned a broad range (1–60%) and was highly sensitive to formulation pH. This adsorption can be mainly attributed to electrostatic, hydrophobic interactions and hydrogen bonding between β-lactoglobulin and chitosan. Brilliant blue release experiments showed that the nanoparticles prepared from native β-lactoglobulin had favorable properties to resist acid and pepsin degradation in simulated gastric conditions. When transferred to simulated intestinal conditions, the β-lactoglobulin shells of the nanoparticles were degraded by pancreatin.131 Table 14.10 gives some salient biomedical applications of chitosan.

14.11

COMMERCIAL STATUS

Currently, microencapsulated marine omega-3 PUFA powder products are commercially available, as discussed in Chapter 13. At present, only a few companies are producing biodegradable packaging materials on a large scale. Biodegradable packaging is a niche market, about 1–2% of the food packaging segment, which accounts

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TABLE 14.10 Biomedical Applications of Chitosan Applications Wound healing, burn therapy

Hemodialysis membranes

Drug delivery matrix

Removal of toxins Hemoperfusion column by chitosan and its oligomers Artificial cartilage scaffolds

Anticholesterol drug Dental bioadhesive biodegradable sutures Composite films with alginate, fucoidan, β-lactoglobulin, etc.

Salient Property Forms tough, water-absorbent, biocompatible films that promotes tissue growth. N-carboxy butyl chitosan finds use in reconstruction of cutaneous tissue due to its similarity with glycosaminoglycans Chitosan–cellulose-blended membranes using trifluoroacetic acid as a cosolvent have improved dialysis properties in artificial kidney. Improved permeability of chitosan–cellulose composite films Functions as an inexpensive carrier encapsulating nutraceuticals and drugs. Chitosan enhances dissolution properties of poorly soluble drugs. Prevents drug irritation in stomach. (Gel formation of chitosan at acidic pH provides antacid and antiulcer activities.) Helps transdermal delivery Chitosan-encapsulated activated charcoal can remove toxins and bilirubin A good adsorbent for hemoperfusion should be highly specific, selective in their binding, and blood compatible. Chitosan is used as a selective adsorbent for antigen/antibodies Chitin and chitosan when combined with chondroitin sulfate maintains the chondrocytic phenotype and supports proteoglycan production to treat cartilage deficiency Reduces lipid absorption by trapping neutral lipids and reduces cholesterol A composite collagen–chitosan membrane has potential for treatment of infra- and suprabony periodontal defects in dentistry Various processes in tissue engineering for skin recovery, scaffolds, etc.

for about 40% of the $460 billion global packaging industry. Industrial applications of the existing technologies including biopackaging have been discussed.6 Uses of marine polysaccharides for therapeutic applications on a commercial scale are in infancy, although a significant huge potential exists. Gelatin developed from cod skin at Fiskeriforskning, Norway, is being used as a soluble base for the photoresist in television tubes and for coating photographic paper. Because of its low melting point of 10°C, it is also useful for encapsulation of drugs.132 Recent research is focusing on potential applications of marine macromolecules in physiology and medicine, as indicated by the increasing number of research publications in the field. The foregoing discussion points out the valuable applications of marine polysaccharides as environment-friendly packaging material and carrier macromolecules. They can also be used in bio and active packaging, encapsulation and delivery of nutraceuticals and drugs, and tissue engineering. Use of marine macromolecules for encapsulation and delivery of nutraceuticals of marine origin, such as PUFAs, glucosamine, and bioactive peptides allow vast opportunities for total utilization of marine products for healthcare and also to address environmental problems caused by synthetic packaging materials.

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73. Butler, B. L. et al., Mechanical and barrier properties of edible chitosan films as affected by composition and storage, J. Food Sci., 61, 953, 1996. 74. Muzarelli, R. A. A., Chitosan-based dietary foods, Carbohyd. Polym., 29, 309, 1996. 75. Hirano, S., Chitin: biotechnological applications, Biotechnol. Ann. Rev., 2, 237, 1996. 76. No, H. K. et al., Applications of chitosan for Improvement of quality and shelf life of foods: a review, J. Food Sci., 72, R87, 2007. 77. Wong, D. W. S., Chitosan-lipid films: microstructure and surface energy, J. Agr. Food Chem., 40, 540, 1992. 78. Takeda, H., Antibacterial activity of chitosan sheet, Chitin Chitosan Res., 12, 192, 2006. 79. Coma, V. et al., Edible antimicrobial films based on chitosan matrix, J. Food Sci., 67, 1162, 2002. 80. Jeon, Y. J., Kamil, J. Y. V. A., and Shahidi, F., Chitosan as an edible invisible film for quality preservation of herring and Atlantic cod, J. Agr. Food Chem., 50, 5167, 2002. 81. Cakii, S. et al., Application of chitosan and its phosphatic derivatives for quality preservation of fresh fish stored at refrigerated temperature, 2nd Joint Trans-Atlantic Fisheries Technology Conference, Quebec, October 29–November 1, 2006. 82. Simpson, B. K. et al., Utilization of chitosan for preservation of raw shrimp (Pandalus borealis), Food Biotechnol., 11, 25, 1997. 83. Rao, M. S., Chander, R., and Sharma, A., Development of shelf stable intermediate moisture meat products using active edible chitosan coating and irradiation, J. Food Sci., 70, M325, 2005. 84. Wu, Y. et al., Moisture loss and lipid oxidation for precooked beef patties stored in edible coatings and films, J. Food Sci., 65, 300, 2000. 85. Kyon, H., Prinyawiwatkul, W., and Meyers, S. P., Comparison of shelf life of eggs coated with chitosans prepared under various deproteinization and demineralization times, J. Food Sci., 70, S379, 2005. 86. Hwang, K. T., Rhim, J. W., and Park, H. J., Effects of κ-carrageenan-based film packaging on moisture loss and lipid oxidation of mackerel mince, Korean J. Food Sci. Technol., 29, 390, 1997. 87. Lee, J. Y. et al., Extending shelf-life of minimally processed apples with edible coatings and antibrowning agents, LWT Food Sci. Technol., 36, 323, 2003. 88. Rhim, J. W. et al., Lipid penetration characteristics of carrageenan-based edible films, Korean J. Food Sci. Technol., 30, 379, 1998. 89. Brownlee, I. A. et al., Alginate as a source of dietary fiber, Crit. Rev. Food Sci. Nutr., 45, 497, 2005. 90. Wu, Y. et al., Development and application of multicomponent edible coatings and films: a review, Adv. Food Nutr. Res., 44, 347, 2002. 91. Di Pierro, P., Transglutaminase-catalyzed preparation of chitosan-ovalbumin films, Enz. Microbiol. Technol., 40, 437, 2007. 92. Di Pierro, P. et al., Chitosan-whey protein edible films produced in the absence and presence of transglutaminase, Biomacromolecules, 7, 744, 2006. 93. Park, H. J. et al., Mechanical and barrier properties of chitosan-based biopolymer films, Chitin Chitosan Res., 5, 19, 1999. 94. Conggui, C. et al., The application of konjac glucomannan and carrageenan in developing edible wrapping paper made from potato starch, Food Sci. China, 25, 98, 2004. 95. Liu, L., Kerry, J. F., and Kerry, J. P., Selection of optimum extrusion technology parameters in the manufacture of edible biodegradable packaging films derived from food-based polymers, J. Food Agr. Environ., 3, 51, 2005. 96. Padgett, T., Han, I. Y., and Dawson, P. L., Incorporation of food grade antimicrobial compounds in biodegradable packaging films, J. Food Qual., 61, 1330, 1998. 97. Cha, D. S. and Chinnan, M. S., Biopolymer-based antimicrobial packaging: a review, Crit. Rev. Food Sci. Nutr., 44, 223, 2004.

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98. Han, J. H. and Krochta, J. M., Physical properties of whey protein coating solutions and films containing antioxidants, J. Food Sci., 72, 308, 2007. 99. Choi, J. H. et al., Diffusivity of potassium sorbate in κ-carrageenan based antimicrobial film, Lebensm. Wiss. Technol., 38, 417, 2005. 100. Begin, A. and Van Calsteren, M. R., Antimicrobial films produced from chitosan, Int. J. Food Sci. Technol., 26, 63, 1999. 101. Tsai, G. J. and Su, W. H., Antibacterial activity of shrimp chitosan against Escherichia coli, J. Food Protect., 62, 239, 1999. 102. Pranoto, Y., Rakshit, K. K., and Salokhe, V. M., Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin, Lebensm. Wiss. Techol., 38, 859, 2005. 103. Bowler, C. K. et al., Characterization of fish skin gelatin gels and films containing the antimicrobial enzyme, lysozyme, J. Food Sci., 71, M141, 2006. 104. Cha, D. S. et al., Antimicrobial films based on Na-alginate and κ-carrageenan, Lebensm. Wiss. Technol., 35, 715, 2002. 105. Cha, D. S. et al., Release of nisin from various heat-pressed and cast films, LWT Food Sci. Technol., 36, 209, 2003. 106. James, M. E. et al., Control of Listeria monocytogenes on the surface of refrigerated ready-to-eat chicken coated with edible zein film coatings containing nisin and calcium propionate, J. Food Sci., 67, 2754, 2002. 107. Qu, C. Y. et al., Using gelatin-based antimicrobial edible coating to prolong shelf life of tilapia fillets, J. Food Quality, 25, 213, 2002. 108. Shefer, A. and Shefer, S., Novel encapsulation system provides controlled release of food ingredients, Food Technol., 57(11), 40, 2003. 109. Singh, H., Nanoencapsulation systems based on milk proteins and phospholipids, AGFD 123, National Meeting, ACS, Chicago, IL, March 25–29, 2007. 110. Yang, Y. et al., Assembled alginate/chitosan nanotubes for biological application, Biomaterials, 28, 3083, 2007. 111. Lu, X., Wen, Z., and Li, J., Hydroxyl-containing antimony oxide bromide nanorods combined with chitosan for biosensors, Biomaterials, 33, 5740, 2006. 112. Akira, S., Electrospinning of chitosan and chitosan derivatives, Chitin Chitosan Res., 12, 166, 2006. 113. Skjak-Braek, G., Alginates—a target molecule for genetic engineers and a versatile material for the biotechnologist, 3rd International Marine Biotechnology Conference, Tromsoe, Norway, August 7–12, 1994. 114. Zimmermann, F. et al., Physical and biological properties of barium cross-linked alginate membranes, Biomaterials, 28, 1327, 2007. 115. Hirano, S. et al., Wet spun chitosan–collagen fibers, their chemical N-modifications, and blood compatibility, Biomaterials, 21, 997, 2000. 116. Rao, S. B., Bai, M. V., and Naseema, K., Studies on the safety and potential of chitosan as a biomaterial, Proceedings of the Symposium on Harvest and Post Harvest Technology of Fish. Society of Fisheries Technologists (India), Devadasan, K. et al., Eds., 1994, p. 82. 117. Murakami, R. and Takashima, R., Mechanical properties of the capsules of chitosan– soy globulin polyelectrolyte complex, Food Hydrocolloids, 17, 885, 2003. 118. Zheng, H. et al., Novel dual crosslinked complex gel bead based on carboxymethyl chitosan/alginate for oral delivery of protein drugs, PMSE 377, 233rd National Meeting, ACS, Chicago, IL, March 25–29, 2007. 119. Diebold, Y. et al., Ocular drug delivery by liposome–chitosan nanoparticle complexes (LCS-NP), Biomaterials, 28, 1553, 2007. 120. Kashyap, N. et al., Design and evaluation of biodegradable, biosensitive in situ gelling system for pulsatile delivery of insulin, Biomaterials, 28, 2051, 2007.

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121. Cho, Y. W. et al., In vivo tumor targeting and radionuclide imaging with self-assembled nanoparticles: mechanisms, key factors, and their implications, Biomaterials, 28, 1236, 2007. 122. Mansouri, S. et al., Characterization of folate-chitosan-DNA nanoparticles for gene therapy, Biomaterials, 27, 2060, 2005. 123. Boucard, N. et al., The use of physical hydrogels of chitosan for skin regeneration following third-degree burns, Biomaterials, 28, 3478, 2007. 124. Balakrishnan, B. and Jayakrishnan, A., Self-cross-linking biopolymers as injectable in situ forming biodegradable scaffolds, Biomaterials, 26, 3941, 2005. 125. Balakrishnan, B. et al., Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin, Biomaterials, 26, 6335, 2005. 126. Hashimoto, T. et al., Development of alginate wound dressings linked with hybrid peptides derived from laminin and elastin, Biomaterials, 25, 1407, 2004. 127. Li, Z. et al., Chitosan–alginate hybrid scaffolds for bone tissue engineering, Biomaterials, 26, 3941, 2005. 128. Shi, Z. et al., Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles, Biomaterials, 27, 2440, 2005. 129. Ribeico, C. C. et al., Calcium phosphate–alginate microspheres as enzyme delivery matrices, Biomaterials, 25, 4363, 2004. 130. Sezer, A. D. and Akbuğa, J., Fucosphere—new microsphere carriers for peptide and protein delivery: preparation and in vitro characterization, J. Microencapsul., 23, 513, 2006. 131. Chen, L., and Subirade, M. Chitosan/b-lactoglobulin coreshell nanoparticles as nutraceutical carriers, Biomaterials, 26, 6041, 2005. 132. Anonymous, Gelatin from cod skin, Infofish Int., 5, 71, 2007.

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Hazards with 15 Safety Marine Products and Their Control 15.1 INTRODUCTION The foregoing chapters discussed various functional and nutraceutical aspects of marine resources and their importance in healthcare. Although, in general, marine food products are considered reasonably safe, concerns regarding presence of toxins, heavy metals, pathogenic microorganisms, etc., in the products are growing stronger particularly with respect to international seafood trade. Whereas marine algae, both macroalgae and microalgae, are good sources of nutraceuticals and polysaccharide food additives, as discussed in previous chapters, safety of algae, with respect to toxins, is of paramount importance in determining overall safety of marine products. Some marine organisms have also evolved to produce certain unique compounds for their defence. Some of these compounds are extremely toxic, particularly to mammalian systems; the marine toxins being more potent in toxicity compared with many terrestrial poisons (see Chapter 12). In addition, some seafood items are also known to cause allergic reactions to consumers. Therefore, this chapter discusses the safety hazards of marine products and some measures for controlling the risks.

15.2

FOOD-BORNE HAZARDS

A hazard is defined as a biological, chemical, or physical agent in food, or a condition of food with the potential to cause harm. Whereas an estimate of the probability and severity of the hazard to populations caused by consumption of foods is called risk. Diseases caused by consumption of food contaminated with pathogenic microorganisms are food infections, whereas those resulting biotoxins are referred to as food intoxications. The severity of these risks depends upon the nature of contamination, and may range from mild diarrhoea to death. Despite rapid strides in food technology, worldwide food poisoning outbreaks have increased in recent years. The World Health Organisation (WHO) has observed that in Asia-Pacific region alone, more than 700,000 people die every year from consuming contaminated food.1 The major reasons for such outbreaks are increase in population, tourism, industrialization, mass production of foods, consumer demand for “lightly processed” foods, changes in eating habits, and rise in international trade of food associated with lesser care in development of processed foods. Although modern intensive fishing practices contribute to increasing the availability of marine products, in an attempt to maximize production, many times hygienic quality of the product may be at risk 467

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TABLE 15.1 Major Classes of Food-Borne Hazards Hazard Type Naturally occurring toxins Organic pollutants

Heavy metals

Unconventional agents

Description Toxins such as biotoxins, cyanogenic glycosides, and mycotoxins such as aflatoxin and ochratoxin A Compounds that accumulate in the environment and the human body. Examples are dioxins and oxychlorinated biphenyls (PCBs). Exposure may result in a wide variety of adverse effects in humans Include lead, mercury, and cadmium. These cause neurological damages, particularly in infants and children. Exposure to cadmium can also cause kidney damage, usually in the elderly people Examples are agents responsible for bovine spongiform encephalopathy (BSE or “mad cow disease”)

of being neglected. Industrial pollution and mass discharge of untreated sewage into the sea are also the contributing factors. Besides the use of antibiotics to control microorganisms in aquaculture ponds and increase growth rate of animals has raised concern about enhancing antibiotic resistance of human pathogens. Premarket review and approval followed by continuous monitoring are necessary to ensure the safe use of pesticides, veterinary drugs, and food additives. Table 15.1 gives major classes of food-borne hazards. Food-borne illnesses are defined as diseases, usually either infectious or toxic in nature, caused by agents that enter the body through the ingestion of food. Foodborne diseases are a widespread and growing public health problem, both in developed and developing countries. A great proportion of these cases can be attributed to contamination of food and drinking water with pathogenic microorganisms. Different foods, including seafood and other commodities such as rice and vegetables have been implicated in outbreaks of cholera. The global incidence of food-borne disease is difficult to estimate. In industrialized countries, the percentage of the population suffering from food-borne diseases each year has been reported to be up to 30%. An estimated 76 million cases of food-borne illness occur each year in the United States costing between $6.5 billion and $34.9 billion in medical care and loss of productivity.2 Developing countries bear the brunt of the problem due to the presence of a wide range of food-borne diseases, including those caused by parasites. The high prevalence of diarrheal diseases in many developing countries suggests major underlying food safety problems. Diarrhea is also a major cause of malnutrition in infants and young children. It has been reported that in 2005 alone 1.8 million people died from diarrhea-related diseases.3

15.3 TYPES OF HAZARDS OF MARINE PRODUCTS With increasing interests in marine products and rising international trade of the commodity, there has been rapid rise in hazards related to marine products. Fish, shellfish, and other marine organisms are responsible for at least one in six food

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poisoning outbreaks with a known etiology in the United States.4–6 Seafood was responsible for diseases in other parts of the world also. In the 30-year period during 1971–1990, seafood caused 32% of total food poisoning outbreaks in Korea and 22% in Japan.7 The public health issues associated with seafood have been grouped as environment; process, distribution, or consumer-induced.8,9 Seafood-borne illness can be broadly divided into intoxications and infections. In the first case, the causative agent is a toxic compound that contaminates the seafood or is produced by a biological agent in the marine product. If the agent is biological, intoxication will occur even if the agent is dead as long as it has previously produced enough toxins to precipitate the symptoms of illness. In the case of infections, the causative agent (bacteria, viruses, or parasites) must be ingested alive resulting in its invasion of the intestinal mucous membrane or other organs to produce endotoxins.10 Depending on the target population segments, the various hazards associated with seafood could be broadly grouped into three (1) those which can cause illness in healthy adults (microbial pathogens such as Clostridium botulinum, C. perfringens, Salmonella typhimurium, Shigella, Vibrio cholerae, V. parahaemolyticus, hepatitis A virus, Norwalk-like viruses), and biotoxins from algae and other sources and chemical contaminants such as heavy metals, and polychlorinated biphenyls [PCBs]; (2) those not capable of causing illness in healthy adults, but are dangerous to susceptible people such as immuno-compromised individuals, children, elderly people, and pregnant women (pathogens such as Listeria monocytogenes and V. vulnificus); and (3) those microorganisms having uncertain pathogenicity (Aeromonas hydrophila and Plesiomonas shigelloides).11 Table 15.2 gives a general ranking of hazards with respect to seafood safety. Microbial hazard is the most important concern. However, possible loss in nutritional value as a result of different processing methods is a concern that is probably equally important to microbiological hazards. Concerns such as presence of toxins, environmental contamination, pollutants, and food additives are relatively of less importance.12 Table 15.3 presents seafood hazard categories in order of decreasing risks.13 The various types of hazards with respect to marine products is discussed followed by possible control measures to contain these hazards.

TABLE 15.2 Ranking of Food Safety Hazards Ranking

Hazard

Relative Risk

1 2 3 4 5

Microbial content Pollutant chemicals Natural toxins Pesticide residue Food additive

100,000 100 100 1 1

Source: Adapted from Ashwell, M., J. Royal College Phys., 24, 23, 1990.

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TABLE 15.3 Seafood Hazard Categories in Order of Decreasing Risks Category

Description

Example

1

Those consumed raw without any cooking

2

Nonheat processed raw foods often consumed with additional cooking Lightly preserved fish products (with <6% salt in water phase, pH > 5.0) Semipreserved fish (salt >6%) or pH < 5.0 with added preservatives Mildly heat-processed (pasteurized, cooked, hot smoked) fish products Heat processed (sterilized, packed in sealed containers)

Mollusks including fresh and frozen mussels, clam, oysters, and raw fish such as sushi/sushimi Fresh/frozen fish and crustacea

3

4

5

6

Salted marinated, fermented, cold smoked fish Salted, marinated fish, fermented fish, caviar Precooked, breaded fillets

Canned, retort-pouch packaged items

Source: Adapted from Huss, H.H., Reilly, A., and Ben Embarek, P.K., Food Control, 11, 149, 2000; Ashwell, M., J. Royal College Phys., 24, 23, 1990. With permission.

15.3.1 MICROBIOLOGICAL HAZARDS Fish muscle is sterile immediately after harvesting. However, contamination of both pathogenic and nonpathogenic microorganisms can take place from various sources including water, fishing vessel, processing units, handling equipment, and handling persons. Once contaminated, growth and survival of these organisms are dependent on nutrient status of food, physiological attributes, and extrinsic factors such as temperature, gas environment, and processing factors.14 Fish from cold water generally have an abundance of gram-negative organisms, whereas, mesophilic, gram-positive organisms are predominant in fish from tropical waters. Most of the organisms belonging to gram-negative genera include Pseudomonas, Alteromonas, Moraxella, Acinetobacter, Vibrio, Flavobacterium, and Cytophaga spp. Gram-positive bacteria often belong to the genera, Micrococcus and Bacillus. During ice storage, psychrotrophic gram-negative organisms comprising of Pseudomonas and Alteromonas spp. predominate, which are highly proteolytic and are involved in spoilage of fishery products during chilled storage.15,16 15.3.1.1

Bacterial Pathogens

Seafood may be a vehicle for many bacterial pathogens from various sources. Shellfish, especially the filter-feeding bivalve mollusks (oysters, scallops, mussels, clams,

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and cockles) can accumulate pathogenic bacteria in the alimentary tract. Since the alimentary tract of these bivalves forms the major edible portion for humans, these mollusks can pose potent hazards to humans, unless care is taken to thoroughly clean them before consumption. Since the seafood-borne pathogens represent a serious public health concern, international food safety agencies are working toward a more risk-based approach to targeting food safety policy. To achieve this, it is necessary to understand the relationship between incidence of food-borne illness and consumer exposure to specific food-borne pathogens in the food supply.17 Seafood-borne pathogenic bacteria may be divided into three groups, according to their ecology, and origin, namely, (i) those belonging to the aquatic environment, (ii) those belonging to the general environment, and (iii) those from the animal/human sources.11,18 The indigenous (aquatic and general) bacterial pathogens in fish are generally quite low. Pathogenic bacteria can be introduced into marine waters mainly by commercial shipping activity through fouling, oil fall, discharge of sewage, and sediment, although visible light and biotic components of seawater were the important inactivating factors of marine pathogens.19 Consumption of seafood contaminated with fecal organisms continues to pose large-scale health threat. The hazard is severe due to the fact that many of these pathogens can survive chill temperatures.20 At least 10 genera of bacterial pathogens have been implicated in seafood-borne diseases. These include Salmonella spp., Shigella spp., pathogenic Escherichia coli, and Campylobacter spp., V. cholerae, V. parahaemolyticus, Aeromonas spp., Plesiomonas spp., Yersinia enterocolitica, C. botulinum, and L. monocytogenes. The leading cause of food-borne illness during the last few years was salmonellosis followed by shigellosis, staphylococcal intoxication, and gastroenteritis.18 Salmonella spp. including S. paratyphi and S. enteritidis have been detected in shrimp and bivalves in several parts of the world. Shellfish has been a common carrier of these pathogens. Other shellfish-borne pathogens include fecal coliforms, S. aureus, and V. parahaemolyticus in addition to viruses such as Norwalk virus and Hepatitis A virus.21 Many seafood-importing countries do not permit the presence of these organisms in imported consignments, therefore, safety regulations with respect to handling, processing, and storage are required for their acceptance. The pathogens of concern associated with seafood are listed in Table 15.4, whereas Table 15.5 gives seafoodborne illness associated with these pathogens from different environments.22 Salmonella spp. are among the most important causes of gastrointestinal disease in the world. Presence of these organisms in seafood indicates contamination with sewage. There is a higher prevalence of Salmonella in tropical than in temperate waters, although seasonal variations occur. Salmonella spp. have been reported in fishponds, which are usually scavenged by birds and, therefore, have been found in the gut of cultured fish. Surveys have revealed that 21% of Japanese eel culture ponds, 5% of North American catfish ponds, and 22% of shrimp ponds in one of the major shrimp-exporting countries in Southeast Asia are contaminated with Salmonella spp.13 In the United Kingdom, S. typhi was detected in more than 1.6% of shellfish samples from open harvesting waters.23 Food infection by Salmonella spp. leads to nausea, vomiting, abdominal cramps, and fever. Outbreaks of Salmonella food infection have been associated with raw oysters, salmon, tuna shrimp, sole, and

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TABLE 15.4 Bacterial Pathogens Associated with Seafood from Different Environments Group

Bacterial Pathogens

Pathogenic bacteria indigenous to aquatic environment Pathogenic bacteria indigenous to general environment Pathogenic bacteria in the animal/human reservoir

V. cholerae, V. parahaemolyticus, V. vulnificus, C. botulinum types B, E, F., P. shigelloides, and A. hydrophila Listeria monocytogenes C. botulinum types A and B C. perfringens and Bacillus cereus Salmonella spp., Shigella spp., Pathogenic E. coli, C. jejuni, Y. enterocolitica, and Staphylococcus aureus

TABLE 15.5 Seafood-Borne Illness Associated with Bacterial Pathogens

Pathogenic Bacteria

Seafood Vector

Minimal Dose for Infection (cfu/g)a

Vibrio paramolyticus V. cholera

Crustaceans, fish Shellfish, fish

105–106 102–106

C. botulinum type E C. perfringens A. hydrophila Listeria monotycogenes Bacillus cereus Salmonella spp.

Fish, shellfish, smoked Sporadic incidences Shellfish Raw seafood, smoked, salted Seafood, squid, prawn Shrimp, mollusks, fish

0.1–1 mg toxin 105–108 105–106 >102

Shigella Y. enterocolitica E. coli

Fish, mollusks Fish/shellfish Fish/shellfish

S. aureus

Contamination from infected persons

106–109 >102 101–102 107–109 101–109, depends on strain 105–106 or 0.14–0.19 mg enterototoxin

Clinical Presentation Diarrhea, nausea, vomiting Abdominal pain, vomiting, and profuse watery diarrhea, may lead to severe dehydration and possibly death, unless fluid and salt are replaced Paralysis, diarrhea, death Diarrhea, seldom lethal Vomiting, diarrhea Diarrhea, vomiting, nausea Diarrhea, nausea, vomiting Fever, headache, nausea, vomiting, abdominal pain, and diarrhea Severe diarrhea, cramps, vomiting Diarrhea, vomiting, fever Diarrhea, fever Diarrhea, cramps, vomiting

a

Colony forming units per gram. Source: Adapted from Lalitha, K.V. and Thampuran, N., Fishery Technol. (India), 43, 118, 2006. With permission from Society of Fishery Technologists (India), Cochin.

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other fishery products. The predominant species in Europe is S. enteritidis. In the United States, nontyphoidal salmonellae have been associated with fish and shellfish. S. partyphi and S. enteritidis have been found in shrimp and bivalves. The infective dose of Salmonella is relatively high for healthy individuals and very low for the elderly or medically compromised individuals. Shigella spp., particularly, S. dysenteriae causes an illness called shigellosis, which causes mild diarrhea, fever, abdominal cramps, and severe fluid loss. These organisms are transferred to seafood through sewage pollution of the coastal environment or by contamination after harvest. Shigella spp. have also been isolated in aquacultured fishery products. The hazards from these organisms can be prevented by eliminating chances of human waste contamination of water supplies and by improved personal hygiene.17,13,23 E. coli strains that colonize the intestinal tract are generally harmless. There are at least four types of pathogenic E. coli. These are enteropathogenic, enterotoxigenic, enteroinvasive, and enterohaemorhagic strains. E. coli 0157:H7 is both enterohaemorhagic and cytoxin-producing strain, and hence, hazardous. Infection by this organism is a global public health concern.24 Most infections appear to be related to contamination of water. Where animal manure, particularly bovine manure, is used as pond fertilizer in aquaculture, there is a risk that pathogenic strain of E. coli O157:H7 may be present in pond water. Some people infected with E. coli suffer permanent kidney damage. Hazards from E. coli can be prevented by heating seafood sufficiently to kill the bacteria, holding seafood below 4.4°C and by preventing postcooking cross-contamination.22 Currently, more than 10 Vibrio spp. are known to be involved in human infections acquired by consumption of contaminated foods and water. These organisms being salt tolerant, occur in marine and estuarine waters, whereas V. cholerae and V. mimicus also occur in fresh water. All members of this group show an increase in abundance in warmer waters and an apparent reduction in numbers during cooler months. Vibrio spp. are frequently isolated from seafood. Some Vibrio spp. are both human and fish pathogens. The diseases associated with these organisms are characterized by gastroenteritic symptoms varying from mild diarrhea to cholera. Infections caused by Vibrio spp. are largely classified into two distinct groups V. cholera infection and noncholera Vibrio infections. Only two cholera serotypes, V. cholera 01 and 0139, have been shown to cause the disease. Marinated raw fish, crabs, and undercooked seafood or shellfish have been implicated as vehicles for the transmission of cholera. The hazards associated with consumption of raw fishery products, particularly farmed finfish and crustaceans harboring V. cholerae and V. parahaemolyticus have been major causes of gastroenteritis in Japan. Hazard from V. parahaemolyticus is due to the consumption of inadequately cooked or refrigerated crustaceans and fish and is responsible for certain food poisoning outbreak cases.25 In recent years, the incidence of infection by Vibrio spp. are increasing in many parts of the world, and this has been attributed to the emergence of a new serotype.26 C. botulinum is an anaerobic pathogen, responsible for food-borne botulism, a potentially lethal paralytic disease caused by ingesting preformed botulinum neurotoxin released by the bacterium. The organism is widely distributed in ocean sediments, and hence contaminates fish. C. botulinum type E is the most common clostridia in fishery products.27,28

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Listeria spp. have been isolated from tropical fish, shrimp, crab, lobster tail, and surimi-based products.20 L. monocytogenes has been identified as the cause of listeriosis in humans, and can be lethal for fetuses, pregnant women, neonates, and immunocompromised persons. L. monocytogenes is frequently reported from fish items, particularly aquaculture products from temperate regions; however, it is rarely detected in tropical waters. Severe listeriosis can cause meningitis, abortions, septicemia, and a number of other maladies. Shrimp and shellfish in the United States, New Zealand, and Australia have been implicated in listeriosis.29 Y. enterocolitica is a psychrotrophic bacterium, which causes gastrointestinal disease, characterized by appendicitis-like symptoms, fever, and abdominal pain accompanied by diarrhea and vomiting. Outbreaks have been associated with oysters and fish. Poultry guts invariably harbor Campylobacter spp. and hence use of poultry-based manure may pose hazards in inland and coastal aquaculture. Campylobacter jejuni, an emerging food-borne pathogen, is the leading cause of diarrhea worldwide and the second most common cause in the United States. In Ireland, thermolphilic Campylobacter spp. were found in 42% of 380 shellfish.30 Nevertheless, the risk associated with consumption of cultured fish infested with this bacterium is low. Plesiomonas spp. and A. hydrophila are common contaminants of estuarine waters, and have also been isolated from fishery products. The former has been implicated in the outbreaks of gastroenteritis through fish consumption. P. shigelloides is an emerging cold-tolerant pathogen, mostly associated with water, both fresh water and seawater in warm weather. P. shigelloides has been implicated as the etiologial agent, causing diarrhea after consumption of seafood in Hong Kong, the United States, and India.24,31 Pathogenic organisms including L. monocytogenes, Y. enterocolitica, and A. hydrophila, are capable of survival even at refrigerated temperatures, posing a threat to the safety of refrigerated products.19 Staphylococcus aureus does not appear as a part of the natural microflora of newly caught fish, but could be present in processed products such as cooked crustaceans and fish, possibly transferred from food handlers. S. aureus food poisoning is caused by the ingestion of food that contains one or more enterotoxins of the organism. S. aureus elaborates an enterotoxin on improperly stored seafood, especially if the fish is garnished with cream sauces or mayonnaise. The disease is characterized by nausea, vomiting, abdominal cramps, and fever. Hazards from this bacterium can be prevented by not abusing chilled storage temperature and also by proper hygiene.32 Food safety issues associated with cultured fish and shrimp differ from region to region and from habitat to habitat and vary according to the method of production, management practices, and environmental conditions.33 In addition, there is recent evidence that viable but noncultivable (VBNC) state of microbes may be formed under stressing environments such as inadequate processing conditions. This is a cause of concern because the microbial pathogens in such a state may retain the capacity to cause infection after ingestion by the consumer, despite their inability to grow under conditions employed for determining their presence in food. VBNC organisms are potentially dangerous public health problems.34 Filter-feeding organisms invariably harbor viruses such as Hepatitis-type A, Norwalk virus, and small round viruses (calicivirus, astrovirus, and parvovirus).

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The symptoms of diseases caused by these viruses include paralysis, meningitis, respiratory illness, and myocarditis among others. Consumption of raw bivalves is a major cause of viral disease as a result of filtration of water by these shellfish. Viral disease has been a major reason for the setback in shrimp aquaculture during the past few years. Hepatitis A virus gets contaminated on shrimp harvested from water-containing human sewage. The virus is more resistant to heat and drying. The incubation period for onset of symptoms varies from 15 to 45 days.2,20 A recent study used expert elicitation to attribute food-borne illness associated with each of the 11 major food-borne pathogens (nine pathogens plus Toxoplasma gondii and noroviruses) to 11 broad food categories. A total of 42 nationally recognized food safety experts contributed to the formal elicitation survey. The data were used to attribute case, hospitalization, and death incidence estimates to foods according to pathogen. The estimates indicated that 15 food pathogen pairs accounted for 90% of the illnesses, whereas 25 pairs accounted for 90% of hospitalizations and 21 pairs for 90% of deaths.35 15.3.1.2 Histamine Poisoning Histamine poisoning, also referred as scombroid poisoning, is a worldwide problem occurring in countries where consumers ingest fish containing high levels of the amino acid, histidine. The disease is linked to postharvest contamination and improper storage of fish. Histamine, responsible for scombroid poisoning, is formed through decarboxylation of histidine by microorganisms, such as Enterobactaeriacea, some Vibrio spp., and Klebsiella spp. The amino acid is present in significant amounts in fish of the scombroid family (mackerel, tuna). The principal histamineproducing bacteria, Morganella morganii, grow at neutral pH, but they can also grow in the pH range 4.7–8.1 and requires 5% NaCl for optimal growth. Histamine poisoning is a mild disease; incubation period is very short, ranging from a few minutes to a few hours. Symptoms are headache, dizziness, nausea, and vomiting. The human body will tolerate certain amount of histamine without any reaction. The ingested histamine will be detoxified in the intestinal tract by at least two enzymes, the diamine oxidase and histamine N-methyltransferase. This protective mechanism can be eliminated if intake of histamine or other biogenic amines is very high or if the enzymes are blocked by other compounds.36 Histamine level <5 mg in 100 g is generally safe for consumption. Rapid chilling of fish immediately after harvest is the most important strategy for preventing the formation of scombrotoxin, especially for fish that are exposed for warmer waters or air. This will minimize the activity of histidine decarboxylase and hence formation of histamine. As control measure of this poisoning, a maximum concentration of 20 mg histamine in 100 g flesh has been prescribed by the European Union regulations, and also as per the Codex Alimentarius Standards. The U.S. Food and Drug Administration (U.S. FDA) has permitted 50 mg per 100 g Canada, Denmark, India, and Sweden have permitted levels of 20 mg, whereas Germany has permitted 30 mg% histamine. There are rapid test kits available for detecting scombroid toxins making use of enzyme-linked immunosorbent assay (ELISA), enzyme mmunoassay, and color tests.37–40

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15.3.2

Marine Products for Healthcare

INSECTS

A number of insects such as flesh flies (Saracophagidae), beetles (Dermesates, Cornestes, and Necrobia spp.), and mites (Lardoglyphus and Lyrophagus spp.) infest fish, particularly during sun drying.41 The most destructive pest is the hide beetle, D. maculatus Deg. A case study of losses in traditionally cured fish showed infestation of blowfly as the major cause of losses in dried sardine products.42 Low dose irradiation can eliminate insect infestation problem in seafood (see Section 15.10.5.1).

15.3.3 ALGAL TOXINS Marine algae form the first stage of the food web (see Chapter 11). Algae are invariably present in both marine and freshwater, and are known to contribute to taste and odor in water supplies. If the algae are toxic, the hazard is likely to be passed on to those who feed on such algae. There is a strong chance that some marine algae may be infected with disease, which may hamper its utilization as food or as sources of nutraceuticals and bioactive compounds.43 Fortunately, although there are over 4000 species of marine algae, only 70–80 species are known to produce toxins. Nevertheless, if the concentration of these algae exceeds certain limit, it results in manifestation of their toxins. For instance, the blooming of dinoflagellates may cause a reddish or yellowish discoloration of water, when their concentrations exceed 106 cells/L. Such extreme concentrations of the algae are called “red tide” and the effect is significant formation of the algal toxin causing mass mortality in fish and invertebrates. The phenomenon occurs as a function of water temperature, light, salinity, presence of nutrients, and other environmental conditions. Red tides have devastated fish farms and fishing areas in the seas around Hong Kong, Africa, and Japan.44,45 Microalgal blooms have frequently occurred in coastal areas under certain nutrient conditions such iron limitation. Their massive growth despite physiological iron-deficiency has long been a matter of concern. Iron is an essential trace metal and a limiting factor for microalgal growth, but concentrations of bioavailable iron in seawater are low. Recently these algae, which include the raphidophyte, Heterosigma akashiwo, the dinoflagellate, Heterocapsa triquetra, and the diatom, Ditylum brightwellii were found to utilize insoluble iron such as ferric phosphate and ferric sulfide for their growth, suggesting that during red tide the algae adopt specific strategies for the utilization of various iron sources and growth.46 Pigment analysis provides an effective alternative for investigating phytoplankton dynamics during red tide and other algal blooms. The majority of toxin-producing dinoflagellates are photosynthetic, estuarine, or coastal shallow water forms that are capable of producing benthic resting cysts. Freshwater species are not known to produce toxins. Dinoflagellates were indicated by the characteristic pigment peridinin, since significant correlation was found between concentration of peridinin and dinoflagellate density. A decrease in peridinin and an increase in fucoxanthin, a major carotenoid in diatoms, marked the shift in phytoplankton composition from dinoflagellates to diatoms at the end of the red tide.47 Most marine toxins, except tetrodotoxins, are produced by microalgae. These compounds are generally considered secondary metabolites, not essential for the basic metabolism and growth of the producing species. Some of the secondary

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metabolites may manifest as toxins, as suggested in the cases of dinoflagellates, diatoms, and cyanobacteria. Dinoflagellates are responsible for the production of the majority of these toxins, although only a few dozen species out of the several thousands of dinoflagellates identified so far are known to produce toxic metabolites. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), diarrhetic shellfish poisoning (DSP), and ciguatera fish poisoning (see Section 15.6.1). Among the diatoms, only the genus Pseudonitzschia produces toxic compounds. Literature on methods for identification of toxic dinoflagellates, their toxins, and potential applications of the toxins have been reviewed recently.48,49 Toxins produced by cyanobacteria (blue–green algae) that occur in the marine environment are responsible for human diseases. Nodularia spumigena was the first cyanobacterium recognized to cause animal death. During blooms some strains of Microcystis, belonging to cyanobacteria, produce the hepatotoxin, microcystins. The toxin produced by N. spumigena, called nodularin, acts as a hepatotoxin, in that it induces massive hemorrhages in the liver of mammals and causes disruption of the liver structure. However, among the more than 80 microcystins identified to date, only a few occur frequently and in high concentrations. Microcystin-LR is among the most frequent and most toxic microcystin congeners. Microcystins usually occur within the cells; substantial amounts are released to the surrounding water only in situations of cell lysis. Frequently occurring cyanobacterial genera that contain these toxins are Microcystis, Planktothrix, and Anabaena. A concentration of 58 μg/L of water was measured in Microcystis bloom in August 2004. Microcystins have been shown to be detrimental to the health of humans, animals, and the ecosystem. To date, however, there have been no reports of human poisoning by N. spumigen. Inhalation of a sea spray aerosol containing fragments of marine dinoflagellate cells and toxins (brevetoxins) released into the surface by lysed algae can be harmful to humans, particularly children. The signs and symptoms are severe irritation of conjunctivae and mucous membranes (particularly of the nose) followed by persistent coughing, sneezing, and tingling of the lips.3 Most documented cases of human injury through cyanotoxins involved exposure through drinking water. Disease symptoms include abdominal pain, nausea, vomiting, diarrhea, sore throat, cough, headache, blistering of the mouth, atypical pneumonia, and elevated liver enzymes in the serum, as well as hay fever symptoms, dizziness, fatigue, and skin and eye irritations. In addition to the hazard caused through drinking water, marine cyanobacterial dermatitis (known as “swimmers’ itch” or “seaweed dermatitis”) is a severe skin disease that may occur after swimming in seas containing blooms of certain species of marine cyanobacteria. The symptoms are itching and burning within a few minutes to a few hours after swimming in the sea, where the cyanobacteria are suspended. These toxins are also highly inflammatory and are potent skin tumor–promoting compounds. Available data, however, indicate that the risk for human health associated with the occurrence of marine toxic algae or cyanobacteria during recreational activities is limited to a few species and geographical areas. Allergic or irritating dermal reactions of varying severity have also been reported from a number of freshwater cyanobacterial genera, after recreational exposure. Bathing suits and particularly wet suits tend to aggravate such effects by accumulating cyanobacterial material and enhancing

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disruption of cells and liberation of cell contents. The WHO has recommended a limit of 1 μg/L of alga for drinking water supplies and 20 μg/L for recreational exposure causing moderate risk.3 Interactions between toxic algae and bacteria can sometimes be of detrimental consequences.50 Some marine bacteria living in association with algae are able to produce sodium channel–blocking toxins and a role of these bacteria in the toxicity of dinoflagellates has been hypothesized. Toxicity tests on bacterial extracts were performed using in vivo (mouse assay) and in vitro (cell culture) tests and by HPLC. The algae belonging to the genus Dinophysis were detected throughout the year, whereas those belonging to Alexandrium genus were present in winter and spring. Sixteen bacteria out of 61 strains isolated from the algae were found to be producers of toxic substances that could block sodium channels in cells.51 The presence of EPA and DHA and 18:5 (n−3) fatty acids have been linked to potential toxicity in a dinoflagellate Gymnodinium. It was also suggested that the mode of action in the ichthyotoxicity of these harmful bloom-forming flagellates is correlated to oxygen radical formation.52 The diarrhetic shellfish poisoning toxins okadaic acid (OA) and dinophysistoxin1 (DTX-1) are potent phosphatase inhibitors produced by certain species of marine dinoflagellates. OA can cause hyperphosphorylation of a broad range of animal and higher plant proteins, but little is known regarding the effects of the DSP toxins on marine organisms or their biological function. A variety of microalgae, including a clone of Prorocentrum lima known to produce both OA and DTX-1, were incubated with solutions of OA and in one case DTX-1 or a combination of OA and DTX-1. The effects of DTX-1 on microalgal growth were found to be equivalent to those of OA, and the effects of both toxins in combination were simply additive.53 Therefore, production and processing of these microalgae to extract bioactive compounds requires caution. Presence of algae in substantial amounts is characterized by the earthy smelling of water, which has been attributed to the production of geosmin and 2-methyl isoborneol (MIB).54,55 Other compounds responsible for odor include n-hexanal and n-heptanalm, which are produced by many cyanobacteria and diatoms. Dead algae can also cause tastes and odors. These can be through release of the compounds during cell lysis or as a result of bacterial decay of the dead algae, when odor-bearing sulfur compounds are also produced. Other sources of odor include zoonoplankton, protozoa, and fungi. The odor can be removed by passing the water through activated charcoal.56 Accumulation of these odor-bearing compounds in fishery products is a problem, necessitating steps for their removal. Conditions that optimize MIB and geosmin reduction by protein solubilization were evaluated in channel catfish. For both MIB and geosmin spiked fish, acid and alkaline solubilization effectively reduced off-flavor. Average MIB and geosmin levels for untreated, unprocessed fish were 1.396 and 1.992 ppb, respectively. The levels for treated fish were: 0.194 ppb MIB and 0.398 ppb geosmin.57 Apart from the microalgal species, mentioned earlier, macroalgae can also be sometimes toxic. Red seaweeds have been shown to induce chronic ulceration. The algal polysaccharides elicit toxic effect by binding trace metals and rendering them unavailable. The brown algae, Alaria spp. and Rodomenia spp. produce degenerative

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neural abnormalities, resulting in disturbed coordination and demylination of the cerebrum. Laminine achoine is a hypotensive agent from brown algal species of the Laminaria, Undaria, and Eklonia. Green algae, the benthic genus Caulerpa spp., are known to be toxic during the rainy months and injury to the plant thallus causes extrusion of toxin. A toxin named caulerpin causes anesthesia in the mouth, besides respiratory distress. Halogenated monoterpenes have been isolated from the red algae, Plocamium spp. and brown alga, Cystaphora spp. The red algae Asparagopsis tixiformis contains many halo-compounds. The major algal component is bromoform, but the organism also contains the antiseptic, iodoform, halogenated acetones, butenones, and other substances.39,58,59 Several species of seaweeds from India, however, were found safe as indicated by animal feeding studies. Acute and subacute oral feeding of the seaweeds up to 12 weeks did not produce any toxic effects in rats.60 Presence of toxins in seafood has been known for a long time, essentially as a result of the phenomenon of algal blooms, as discussed earlier. Presence of marine algal toxins is a cause of seafood-associated outbreaks. Fishes that feed on toxic dinoflagellates or toxic herbivore fish are potential safety hazards for consumers. These toxins become a problem primarily because they get concentrated in shellfish and fish that are subsequently eaten by humans, causing shellfish poisoning. There are a number of different seafood poisoning syndromes associated with toxic marine algae, which include ciguatoxins, PSP, ASP, DSP, neurotoxic shellfish poisoning (NSP), and azaspiracid shellfish poisoning (AZP), as discussed later in this chapter. The fish poisoning (ichthyotoxism) can be classified into three, namely, ichthyosarcotoxin, ichthyotoxin, and ichthyohaemotoxin. Ichthyosarcotoxin is characterized by fish having toxins within their musculature, viscera, or skin. Examples are ciguatera fish poisoning, puffer fish poisoning, histamine poisoning, clupeotoxin, hallucinogenic fish poisoning, cyclostomotoxic fish poisoning, and elasmobranches poisoning. Ichthyotoxin includes fishes, which produce a toxin that is generally confined in the gonads and ichthyohaemotoxin indicates fishes that have toxins within their blood.39 15.3.3.1 Paralytic Shellfish Poisoning PSP is caused by a group of toxins (saxitoxins and derivatives) produced by dinoflagellates of the genera Alexandrium, Gymnodinium, and Pyrodinium. Shellfish such as clams and mussels harvested during occurrence of “red tide” are vectors of this toxin. Digestive glands containing PSP toxins were isolated from toxic scallops. Such poisons or toxins are not destroyed by heat or processing. However, gonyautoxin (GTX) 2 and 3, saxitoxin (STX), and neosaxitoxin (NEO) are sensitive to higher temperatures and higher pH values. Under gentle heating conditions and low pH, GTX 2 and 3 increased slightly. The increase in saxitoxin may possibly be due to the conversion of GTX 2/3 and NEO into STX. Heating at 130°C at pH 6–7 could reduce the levels of individual toxins in the homogenate. Instead of the standard mouse assay, a new biomolecular assay to screen for PSPs that uses the 3H-saxitoxin binding assay based upon a saxiphilin isoform from the centipede Ethmostigmus rubripes has been developed.61,62

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15.3.3.2

Marine Products for Healthcare

Ciguatera Poisoning

Ciguatera poisoning is a serious threat to public health and fisheries development in tropical and subtropical regions. Ciguatera poisoning, causing gastrointestinal and neurological symptoms, is associated with a number of fish species, most notably reef fish such as groupers, sea basses, and snappers. It has been estimated that the worldwide incidence may be in the order of 50,000 cases per year.63 The toxin is produced by the dinoflagellate, Gambierdiscus toxicus, which is living around coral reefs and is passed up the food chain so that the eating of contaminated fish can be a hazard to human health. Ciguatoxins arise from biotransformation in the fish of precursor toxins produced in the dinoflagellates and it causes disease when present in 1 ppb (0.1 μg/kg) in the fish flesh. Increased production of toxic dinoflagellates are seen when reefs are disturbed by hurricanes, blasting of reefs, etc. More than 400 species of fish, all found in tropical waters were reported to be affected by ciguatera. Toxins can be detected in gut, liver, and muscle tissue of fish. Apart from mouse assay, methods based on reverse-phase HPLC/MS, have been used to identify Pacific ciguatoxins in fish.39,62 NSP has been limited to the Gulf of Mexico and areas off the coast of Florida. Brevetoxins are highly lethal to fish.21 15.3.3.3 Puffer Fish Poisoning Puffer fish poisoning or tetrodotoxin (TTX) is one of the most potent nonprotein toxins known for numerous fish poisonings. TTX is mainly found in the liver, ovaries, and intestines of various species of puffer fish. The toxin is named after the order, Tetraodonitidae (common fishes are puffer fish, ballon fish, globe fish, fugu, and blow fish), since many of these fish often carry the toxin. The precise mechanism in production of this toxin is not clear, but apparently symbiotic bacteria are involved. The toxin is produced by bacteria, absorbed on or precipitated with plankton, transmitted to TTX-bearing animals such as small gastropods and starfish and then transmitted to fish and large gasropods. TTX is a potent toxin with LD50 of 2 mg for man. The minimum dose necessary to cause symptoms has been estimated to 0.2 mg. Symptoms of ingesting the toxins found in puffer fish include tingling around the lips and in the extremities followed by problems speaking, loss of balance, muscle weakness and paralysis, vomiting, and diarrhea. In extreme cases, there may be respiratory paralysis that can lead to death.64 15.3.3.4 Diarrhetic Shellfish Poisoning Until recently, little focus was given to the presence of DSP toxin esters in seafood products. Okadaic acid and its esters have been found the causative agent of diarrhetic shellfish poisoning. Samples of Danish surf clams (Spisola spp.) and blue mussels (Mytilus edulis) harvested during 1999–2004 contained okadaic acid and its esters, which ranged from 224 to 2516 μg/kg in surf clams.65 In addition to okadaic acid, dinophysistoxin-1 (DTX-1) is also potent diarrhetic shellfish poisoning toxin, which is inhibitor of the enzyme, phosphatase.53

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15.3.3.5 Amnesic Shellfish Poisoning ASP is due to domoic acid, an amino acid produced by the diatom such as Nitzschia pungens. The first reported incidence of ASP occurred in the winter of 1987/88 in eastern Canada, where over 150 people were affected and 4 deaths occurred after consumption of cultured blue mussels. The symptoms of ASP vary greatly from slight nausea and vomiting to loss of equilibrium and memory loss.17,66 15.3.3.6 Other Biotoxins Sardines and herrings belonging to the family Clupediae are reported to contain the toxin, clupeotoxin, which is derived from certain types of dinoflagellates. Outbreak of this poisoning is rather rare. The toxin appears to be concentrated in the viscera of fish, which does not destroy on cooking. The symptoms are a shark metallic taste in mouth, nausea, vomiting, diarrhea, and respiratory paralysis. Consumption of certain types of fishes such as mullets and goatfishes results in hallucinogenic poisoning. The poison is found in the flesh of fishes and appears to be concentrated in the head of fishes and occurs seasonally, usually during summer months. Symptoms are intense weakness, nightmares, constrict chest pains, and burning of the throat. The flesh, skin, and slime of some hagfish are considered to contain a toxic compound. Many species of shark such as tiger shark and gray reef shark and rays are known for their poisonous liver and musculature. The symptoms include gastrointestinal disorders and respiratory distress. Most haemotoxic compounds belong to the eel fishes, which contain toxic principles in their serum. Whole body, except blood, of the eels is edible. The symptoms are vomiting, weakness, neurological problems, and death. In most instances, cooking destroys the poison. Consumption of livers of certain mammals such as bearded seal (Erignatuas barbatus) also lead to poisoning, manifested by headache, muscle soreness, anorexia, and hair loss.39 A detailed analysis of the toxins from the hepatopancreas of mussels from northern Adriatic sea has been reported. Along with some toxins of DSP, these toxins and analogues are responsible for a variety of human seafood poisonings throughout the world. A new type of toxin, the chlorosulfolipid-1 has been isolated, which is completely different in structure from the DSP-toxins isolated so far.67 During the summer of 2001, a bloom of N. spumigena occurred in the Gippsland Lakes area of Southern Victoria, Australia, which resulted in concentration of the toxin, nodularin in mussels and in prawn viscera. Although nodularin concentrations in the flesh of finfish remained low, boiling the seafood redistributed toxin between viscera and flesh. The results were used to restrict some seafood harvesting.68 15.3.3.7

Implications of Biotoxins

The outbreaks caused by algal toxins are compounded by the fact that most toxins are heat stable and are not inactivated by cooking. Since it is not possible to visually distinguish toxic from nontoxic fish and shellfish, many countries have biotoxin monitoring programs to protect public health. Presence of biotoxins has been responsible for incidents of wide scale deaths of sea life.39 Progress in

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analytical chemistry has enabled isolation and structural identification of algal several neurotoxins from cyanobacteria.3 The increased frequency and distribution of harmful algal blooms worldwide and their influence on seafood safety have led to international collaborative studies to address the problem.62 A recent international symposium on algal toxins, discussed various topics, which inlcuded marine harmful algae, mechanism and toxicity of marine toxins, ostreopsis and related health problems, aerosol exposure, palytoxin and related toxins, mechanism of action and toxicity of okadoic acid, domoic acid, saxitoxins, palytoxin and methods for their detection, the European legislation on marine biotoxins, and validated methods for algal toxin measurements in seafood as well as risk characteristics of algal toxins.69 Table 15.6 gives shellfish poisoning and its effects on human health,39 whereas Table 15.7 shows tolerable levels of shellfish poisoning in some countries.39,70,71 TABLE 15.6 Shellfish Poisoning and Its Effects on Human Health Name of Toxin

Affected Organism

Biochemical Agent

Causative Organism

Symptoms in Human

Alexandrium spp., Pyrodinium bahamensei, Gymnodinium spp. Dinophysis acuminata, Prorocentrum lima

Neurological, due to blockage of neuronal and muscular Na+ channel Gastro-intestinal disturbances, acute diarrhea, nausea, vomiting, abdominal pain Respiratory distress, eye and nasal membrane irritation, neurological disorders Abdominal cramps, vomiting, disorientation, and memory loss (amnesia)

PSP

Mussels, clams, oysters, scallops

Saxitoxin tetra

DSP

Mussels, clams

Dinophysistoxin (DTX-1), okadaic acid

Neurotoxic shellfish poisoning

Mussels, clams, oysters

Brevetoxin

Gymnodium breve, Prychodiscus breve

Amnesic shellfish poisoning (ASP)

Blue mussels, razor clams, scallops

Phycotoxic syndrome (domoic acid)

VSP

Oyster, shortneck clam

Venerupin poisoning

Pseudo-Nitzschia multiseries, Pseudodelicatissima, Paustralis, Nitzschia pungens Prorocentrum spp.

Callistin poisoning Azaspiracid poisoning

Gain clam

Cholinergic compound Asari poisoning

Mussels

Callista spp. Protopendinim spp.

Gastrointestinal prevail, damage of liver and kidney Asthma, gastrointestinal disorders Gastrointestinal illness

Source: Reprinted from Kalidas, C. and Anand, P.S., Ind. Food Ind., 25, 52, 2006. With permission from Association of Food Scientists and Technologists (India).

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TABLE 15.7 Tolerable Levels of Shellfish Poisoning in Some Countries Country

Product

Toxin

Tolerable Level (in 100 g Product)

Australia Canada European Union Hong Kong Japan Korea

Shellfish Mollusk Bivalves Shellfish Bivalve Bivalves

India Singapore Norway Various countries

Bivalve Bivalve Bivalve Shellfish

Saxitoxin PSP, DSP PSP PSP PSP, DSP Gonyautoxin DSP PSP Saxitoxin Saxitoxin ASP

80 μg 40 μg 80 μg 400 MU 400 MU, 5MU 400 MU 5 MU; 0–60 μga 80 μg 80 μg 80 μg 20 μga

a

Data taken from Huss, H.H., FAO Fisheries Technical Paper. No. 334, FAO, Rome, 1993, p. 169.

Note: MU, mouse unit, is the minimum amount of purified toxin (μg) required to kill a 20 g mouse in 1 min when 1 mL solution of extract at pH 4.0 is injected interperitoneally. Source: Adapted from Kalidas, C. and Anand, P.S., Ind. Food Ind., 25, 52, 2006; Huss, H.H., FAO Fisheries Technical Paper No. 348. FAO, Rome, Italy, 1995. With permission.

15.3.4

PARASITES

Parasites including flatworms, roundworms, and protozoa infest the gills, viscera, and skin of marine, freshwater, as well as farm-raised fish and shellfish, and can pose health hazards to consumers.72 More than 50 species of helminth parasites from fish and shellfish are known to cause disease in man. Most are rare and involve only slight to moderate injury but some pose serious potential health risk. The most common parasites associated with fish include nematodiases, trematodiases, and cestodiases. Fish-borne nematodiases (roundworms) can be detected in human as incidental infections, whose natural definitive hosts include marine mammals, birds, and pigs. The mode of infection is ingestion of fish containing infective larvae. Anisakids (particularly A. simplex) are among the most common nematodes in marine fishes. Others include Ascaris lumbricoides, Trichuris trichura, T. spiralis, Capillaria philippinensis, and Pseudoterranova decipiens.17,68 Cod, whitefish, and salmonids can carry T. spiralis. Fish-borne trematodiases are major diseases in various parts of the world, causing morbidity and complications leading to death. The two major genera of importance for human health are clonorchis and opsthorchis. Clonorchiasis, caused by clonorchis, is endemic in some countries in East

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Asia. For liver fluke species such as Clonorchis sinensis, Opsthorchis felineus, and O. viverrini, the intermediate hosts are snails and freshwater fish, whereas dogs, cats, and humans are the final hosts where the fluke lives and develops in bile ducts of the liver. Other trematodes of importance are Nanophetus salmincola and Crytotyle lingua, the latter existing under the skin of gadoids such as herring and mackerel. Cestodiases (tapeworms), which mature in small intestine, cause cestodes infection in human. The disease is not very pathogenic. Cestode infection from fish seems to be confined to species of Diphyllobothrium and most reports are concerned with infection in North America from eating raw salmon. Nevertheless, Diphyllobothrium spp. are transmitted by various species of freshwater, marine, and anadramous fish. The smallest of the parasites are the protozoa, which are single-celled organisms. The three most important ones in food are Entameba histolytica, Giardia lamblia, and Toxoplasma gondii. A protozoan infection is caused by Chloromyscium thyristes. It occurs in a number of species of fish although its presence is difficult to detect in fresh fish. During ice storage, the flesh of badly infected fish becomes softened by proteolytic enzymes produced by the parasite. Abnormal color of shrimp may be due to infestation of a protozoan that decomposes the meat, giving it a soft and white appearance.73–76 Contamination of molluskan shellfish with parasites has been a problem for a long time. Since the late 1800s, when shellfish related illness was first reported in the United States, there have been more than 400 epidemics of food-borne diseases and more than 14,000 gastroenteritis cases related to consumption of contaminated molluskan shellfish. The parasites, Cryptosporidium parvum, C. hominis, and C. meleagridis, were found in 65% samples from commercial harvesting sites. Oysters have been found to carry the parasites more than any other seafood. Clams, mussels, cockles, and scallops are less of a public health concern because they are usually consumed cooked or steamed.22,77

15.3.5

FUNGI AND OTHERS

Contamination of fishery products with fungi such as Aspergillus flavus, A. parasiticus, A. rubur, and Penicillium veridicatum mycotoxins may also be possible. These are transmitted through agricultural components used in feed. The fungi grow at 10–40°C and pH 3–11. The effect of mycotoxin is acute hemorrhage in the gastrointestinal tract, liver damage, and death.42 Oilfish (Ruvettus pretiosus) and Escolar (Lepidocybium flavobrunneum) belonging to the family of Gempylidae are consumed in several European countries. These two species do not metabolize wax esters that occur naturally in their diet and, as a consequence, these wax esters are stored in the body of these fish. The oil content of the muscle meat of oil fish and Escolaramounts to 18–21% and the oil contains >90% wax esters. Human case reports suggest an association between the consumption of the two fish species with diarrhea and other gastrointestinal disturbances, presumably due to the wax esters. However, proper preparation practices may prevent such incidence.78

15.3.6

CHEMICAL HAZARDS

In the late 1980s, seafood attracted significant media attention as a carrier of environmental pollutants, when the tendency of some fish to absorb and concentrate

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heavy metals such as mercury and other industrial pollutants were recognized. Some species of fish may contain significant levels of chemicals, which include heavy metals such as antimony, arsenic, cadmium, lead, mercury, and selenium; organic chemicals such as PCBs, dioxins, and insecticides; and processing-related compounds such as sulfites (used in control of blackening of shrimp); nitrosamines; and contaminants such as antibiotics and hormones (sometimes used in aquaculture). Uses of chemicals in food production and processing have contributed to increase in chemical hazards. The WHO has identified 18 chemical contaminants in food, which include industrial chemicals, organochlorine pesticides, organophosphorus pesticides, and mycotoxins.79 In the sea, pollutants are potentially bioconcentrated in marine organisms and sediments, and subsequently transferred to man through food. Their levels are generally high in older, larger, predatory fish and marine mammals.80 The use of antibiotics such as chloramphenicol to control diseases has resulted in accumulation of residues of these compounds in shrimp and other products.81 However, contamination of fishery products by chemicals is generally low. Nevertheless, high contents of cadmium, lead, zinc, and copper in popular fishery products sold in some markets have been noted.82 Heavy metal pollution of the marine environment has long been recognized as serious concern. Heavy metals can be classified as potentially toxic (arsenic, cadmium, lead, and mercury), probably essential (nickel, vanadium, and cobalt), and essential (copper, zinc, iron, and manganese). Toxic elements can be very harmful even at low concentrations when ingested for a long period. Mercury is a toxic metal that can damage the developing brain. Too much mercury may affect behavior, learning, and thinking patterns of children. Methyl mercury presents greater concern for adverse health effects. Babies in the womb, nursing babies, and young children are at greatest risk for adverse health effects from mercury exposure. Studies have shown that all fish contain some mercury, with varying concentrations based on the species, location, age, and other factors. Large predatory ocean fish such as swordfish, tuna, king mackerel, tilefish, and shark can bioaccumulate methyl mercury in their edible portions. Levels may be 1000–10,000 times greater in these fish than in any other food.83 It must be noted that because methyl mercury is distributed throughout the muscle, skinning and trimming does not significantly reduce its contents in fillets. For this reason, determination of the chemical quality of aquatic organism, particularly the contents of heavy metals in fish is extremely important with respect to human health.84 The U.S. Center for Disease Control’s most recent survey, however, indicates that blood mercury level in the United States was unchanged through 1999–2000, during the period when seafood consumption escalated. Methyl mercury in seafood supply has also not changed in through the recent decades.85 Toxicity symptoms of zinc include nausea, vomiting, and diarrhea. Exposure to cadmium can cause kidney damage, usually seen in the elderly people.84 Arsenic species contents in raw and cooked edible seaweed and the bioaccessibility (maximum soluble concentration in gastrointestinal medium) of arsenosugars (glycerol ribose, phosphate ribose, sulfonate ribose, and sulfate ribose) were examined. An in vitro digestion (pepsin, pH 2; pancreatin-bile extract, pH 7) was applied to estimate arsenosugar bioaccessibility. Cooking of Undaria pinnatifida and Porphyra spp. did not alter the arsenic species present in the methanol–water extract, but it produced 2–5 times

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increase (2 and 5 times) in the content of arsenic in Hizikia fusiforme. In all of the seaweeds analyzed, arsenosugar bioaccessibility was high (>80%) and did not vary as a result of cooking. Arsenosugar degradation as a result of cooking was also not observed.86 Compared with capture fisheries, aquacultured fishery products are more exposed to hazards, both biological as well as chemical, due to possibilities of their contamination from both freshwater and coastal ecosystems. The products get contaminated from some of the chemicals when they are used to sterilize pond soils between production cycles. Nevertheless, when used as per good aquaculture practices, none of the water treatment compounds can be considered a hazard. The routine addition of antibiotics to animal feed to contain the problem of bacterial pathogens in coastal an inland aquaculture environments has given rise to antibiotic-resistant bacteria. Therefore, presence of antibiotic residues in imported aquacultured fishery products is a matter of major concern to regulatory agencies. High levels of chloramphenicol in aquacultured shrimp have adversely affected international trade in the commodity.87 Presence of PCBs in farmed salmon has created some controversy. However, the reported content of the chemicals was below the tolerance level of 2000 ppb established by the **U.S. Food and Drug Administration. The WHO has fixed an upper limit of 4.5 ng of polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in fish, other marine animals, and their products and by-products.88 The safety issues associated with aquaculture products have been addressed by international bodies. A FAO/NACA/WHO Study Group evaluated options for implementing risk management strategies to reduce or eliminate risks associated with products from aquaculture systems and recommended food safety measures to control these hazards.89

15.3.7

SEAFOOD ALLERGY

Food allergies are generally transient and are most frequent in infants and most serious in adults. For example, in infants, allergy to cows’ milk is common. Food allergy is due to antigenicity, which denotes the capacity of molecules (antigens) to interact with immunological-specific antibodies. Factors defining immunogenicity include foreignness, molecular size, chemical complexity, antigen presentation, and genetic capacity of the host. Unlike their common role as nutrients, some proteins, in particular are allergic. Glycoproteins present in a number of foods may be major food allergens. Certain proteins may not exert their allergenicity unless they are released from protein body organelles in which they are naturally present. Digestion, hydration, interactions with other proteins, and other matrix effects may contribute to the ability of a protein reach the sites of immune action in the gastrointestinal mucosa and thus exert their potential allergenicity. Crustaceans such as shrimps, lobster, and crab have known to be the most common allergic food items among seafood. Shrimp water or vapor from cooked shrimp or other crustaceans such as lobster or crab have been known to be a source of some allergens in susceptible individuals.90–92 In recent years, the molecular identification of seafood allergens, mapping of epitopes, expression of recombinant allergens, and the elucidation of immunological mechanisms of allergies have been carried out. The fish allergen, known as Allergen M (later designated as GAD m 1 or Gad c 1) isolated from Atlantic cod in 1969 is

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among the first food allergen identified. Gad m 1 consists of 113 amino acids and a glucose molecule with a molecular weight of 12 kDa. The allergen is a calciumbinding protein and is involved in muscle relaxation. The compound has since been isolated from Atlantic salmon. The molecular identity of shrimp allergen was elucidated in early 1990s and identified as tropomyosin, which has been confirmed in other crustaceans including lobster and crab. Arginine kinase, an enzyme, is also known to be an allergen among crustaceans. The commonly consumed mollusks include members of class Gastropoda (limpet and abalone), bivalves (scallop, clam, mussel, oyster), and cephalopods (cuttlefish, squid, and octopus). There have been a number of reports on allergies of mollusks including snail, abalone, limpet, cuttlefish, and squid. Among bivalves, it is known that oysters can cause hypersensitive reactions upon ingestion, as well as occupational reactions in sensitized workers. The allergens from oysters have been identified as cra g 1 and cra g 2 (tropomyosin, a 38 kDa protein) and Hal m 1 (49 kDa protein).93 Chinese scientists from the Ocean University of China revealed that treating prawns with a combination of heat and irradiation (see Section 15.4.5.1) significantly reduced the level of proteins responsible for allergic reactions. They found that levels of “Pen a 1,” one of the major allergens, decreased 20-fold after treatment.94

15.4 CONTROL OF HAZARDS The foregoing discussion summarized the various hazards that can jeopardize the safety of marine products. In view of the situation, efforts are needed to protect global consumers against these hazards. With rise in international trade, seafood is being subjected to increasingly greater regulation and control. As a result, issues related to environmentally sustainable practices and seafood safety are assuming greater significance. Various governments, under the guidance of international agencies such as FAO and WHO are working with farmers and fishermen to ensure safe supply of food including marine products at various stages of selection, processing, storage, and consumption. Algal and seafood-related health hazards have also forced the global regulatory authorities to introduce stringent quality standards in traded fishery products. This section discusses various possible measures to control different types of seafood-borne hazards.

15.4.1 CONTROL OF BIOTOXINS Marine biotoxins are responsible for a substantial number of seafood-borne diseases, as pointed out earlier. Control of marine biotoxins is difficult and disease cannot be entirely prevented. Appearance of an alga or a fish does not give any indication of presence of toxin. Further, the toxins are mostly nonprotein in nature and extremely stable, and therefore, cooking, smoking, drying, salting, etc., do not destroy them. Therefore, presence of biotoxins has raised an important issue of the safety of marine products necessitating strict monitoring of the products harvested under such conditions. The knowledge about various seafood toxins and their significance in production of food-borne intoxication is very essential for establishing consumer safety with respect to marine products. The knowledge on the toxicological status of marine products would help judicious exploitation of the resources for the betterment

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of mankind. Because adequate surveillance is difficult, few immediate management options are available. The precautionary measures include guidelines to avoid areas with visible algal concentrations or algal scum in the sea as well as on the shore and adequate public information. Medium- to long-term measures are identification of the sources of nutrient pollution (in many ecosystems, phosphorus, or nitrogen) and significant reduction of these nutrient inputs to effectively reduce proliferation of potentially harmful algae. For protection from health outcomes not due to cyanotoxin toxicity, but rather to the irritating or allergenic effects of other cyanobacteria, a guideline level of 20,000 cyanobacterial cells/mL (corresponding to 10 mg chlorophyll/L under conditions of cyanobacterial dominance) has been suggested.3,95,96 The major preventive measure is inspection and sampling from fish areas and shellfish beds, and analysis for toxins. The mouse bioassay is often used for this purpose, which is confirmed by HPLC and other chromatographic methods. Since control at the level of phytoplankton is difficult, if high levels of toxin are found, commercial harvesting is halted.97 Removal of toxin by depuration of filter-feed animals such as mussels may have some potential, but the process is time-consuming and expensive.97 In addition to the conventional mouse bioassay, efforts have been made to develop simple and reliable methods for toxin monitoring. Members of a zooplankton group called rotifers, which measure a mere 400 μ are sensitive to toxins and, therefore, may be used as a biological method to monitor the health of estuaries and streams.98 Recently an automated system has been developed for algal monitoring.99 Researchers from the Swawnsea University, Wales claim that they have made a breakthrough in the war against red tides. The technique involves growth of certain zooplankton.45 Despite concerns regarding red tide, however, at present time, only a few seafood toxins cause a significant public health problem worldwide. The Asia Pacific Economic Cooperation (APEC) task team on algal biotoxin regulations recommended the creation of a group of regulatory authorities on seafood safety with regard to harmful algal bloom (HAB). The regulatory procedures are being standardized by the APEC.96 Regulatory agencies are also giving periodical warnings against consumption of fishery products containing biotoxins. The U.S. FDA recently released industry and consumer advisories regarding safe sources of puffer fish (also known as fugu, bok, blowfish, globefish, swellfish, balloonfish, or sea squab). Puffer fish caught in the mid-Atlantic coastal waters of the United States, typically between Virginia and New York, are safe to consume.100

15.4.2

REMOVAL OF ALLERGENS

There is currently no way to treat food allergies, except avoiding certain foods for a period of 1–2 years, which can result in loss of sensitivity. Some attempts have, nevertheless, been made to treat food allergy. For instance, protein hydrolysates can be effective in treatment of some food allergies.101 The problem of allergy with respect to shrimp has been addressed recently, using heat and irradiation treatment, as mentioned earlier. Furthermore, a molecule necessary to resist food allergies has been identified recently, thereby offering a potential target for therapy. It was suggested that by delivering allergens with this molecule, allergic reactions could be controlled.94

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489

CONTROL OF PARASITES

The problem of parasite infestation of molluskan shellfish is compounded by the fact that some products such as oyster are quite often eaten raw. Control measures to reduce the public health problems due to parasite infestation of fishery products include legislation and surveillance. In principle the problem can be attacked at three levels, namely, (i) avoidance of capture of nematode-infected fish by selecting specific fishing grounds, species or age groups, (ii) removal of nematodes from fish by hand over a candling table, and (iii) application of techniques to kill nematodes in the flesh. All nematodes can be killed when heated to 55°C for 1 min. Therefore, hot-smoking, pasteurization, and sous-vide cooking and other light heat treatment can inactivate parasites. Freezing to −20°C and maintaining this temperature for at least 24 h will kill all nematodes. At present only freezing and heating are the two mentioned steps that are applied in commercial fishery.76 Other potential methods of sanitization include ozonation and high-pressure processing. Low dose irradiation can effectively address the problem of parasites in fishery products (see Section 15.5.5.1).102

15.4.4 CONTROL OF CHEMICAL HAZARDS Chemical hazards can be controlled by specifying quality of raw material and ingredients. Removing the skin and fat from the fish before cooking them can reduce the levels of PCBs. The Codex Alimentarius Commission, the U.S. FDA, and the European Union have laid down action plans to limit these chemical contaminants. The U.S. FDA has set action levels for maximum permitted concentrations of 5.0 mg DDT and its breakdown products, DDE and TDE, and lower limits for dieldrin and aldrin. It is likely that these limits may be further lowered to improve the quality of the processed products. Some examples of maximum residual chemical contaminants in fish per human consumption include dieldrin, 0.1 mg/kg (Denmark); total levels of DDT, DDE, and DDD of 2 mg/kg (Denmark); PCB, 2 mg/kg (Sweden); lead, 2 mg/kg (Denmark); and mercury, 0.5 mg/kg (EEC).17 Advisories for seafood consumption to address the hazard of heavy metal poisoning have been provided by regulatory agencies. The U.S. FDA recommends that women who are pregnant or nursing and young children should eliminate shark, swordfish, king mackerel, and tilefish (also referred to as golden bass or golden snapper) from their diets completely and limit their consumption of other fish to 12 oz/week (3–4 servings/week) to minimize exposure to methyl mercury. The FDA has stipulated that the concentration of mercury in edible portion should not exceed 0.5–1.0 ppm on wet weight basis.11 Recently, the U.S. State of Alaska has provided guidelines for fish consumption suggesting that women who are or can become pregnant, nursing mothers, and children aged 12 years and under should limit their consumption of the fish that are known to have elevated mercury levels. The guidelines have categorized fish consumption into four categories: category 1—limit consumption of sablefish, rougheye, rockfish, medium-sized halibut (9–18 kg), halibut, and medium-sized lingcod to ≤4 meals/week (or ≤16 meals/month); category 2—limit consumption of medium–large halibut to ≤3 meals/week (or ≤12 meals/month); category 3—limit consumption of large lingcod, yellow eye rockfish, and large

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halibut to ≤2 meals/week (or ≤8 meals/month); and category 4—limit consumption of salmon shark, spiny dogfish, very large lingcod, and very large halibut to ≤1 meal/week (or ≤4 meals/month). The fish consumption limitations listed earlier assumes a person eats fish from a single category listed earlier, and that an adult meal size is 6 oz. Women who are or can become pregnant, nursing mothers, and children aged 12 years and under who consume fish from the categories listed earlier during a given month may also consume unlimited quantities of fish known to be low in mercury (e.g., salmon) during that month103 (see also Chapter 2). The report might help lay to rest outgoing fears that a contamination from pollutants such as methyl mercury and microorganisms including viruses make seafood consumption unsafe.104 Monitoring of waters is an important method to reduce accumulation of metals in fishery products. Mussel, being filter-feeding mollusks, can be used as bioindicator organisms to assess heavy metal contents in coastal waters.105,106

15.4.5

CONTROL OF MICROBIOLOGICAL HAZARDS

Control of microbial quality has been traditionally based on the principle that ideally all pathogenic microorganisms should be absent in food. However, the concept of “zero tolerance” is impracticable and hence, permitting a certain level of pathogens in some raw foods is explicitly acknowledged. According to the Codex Alimentarius Commission guidelines, fish and fishery products should not contain microorganisms in any amount that may represent a hazard to public health.21 Heating seafood sufficiently, holding chilled seafood below 4°C, preventing postcooking crosscontamination, salting, and acidification of food can minimize hazards from pathogens such as E. coli and Y. enterocolitica.19 Seafood-related health hazards have forced the global regulatory authorities to introduce stringent quality standards in traded fishery products. The Department of Food Safety of the WHO together with other international organizations such as FAO and Codex Alimentarius Commission take efforts to strengthen food safety systems, promote good manufacturing practices, and educate retailers and consumers about appropriate food handling. The WHO is also promoting in-country laboratory-based surveillance of priority food-borne diseases in humans and animals, as well as the monitoring of pathogens in food.87,107 15.4.5.1 Food Irradiation Food irradiation is a cold process that has significant potential to enhance the shelf life and improve microbial safety of food products including fishery items. For irradiation of food, generally, ionizing radiations emitted by radioisotopes, 60Cobalt and 137Cesium, and also electron beams are used for food preservation. 60Co isotope (half-life, 5.27 years) emits two gamma rays of 1.17 and 1.33 million electron volts (MeV), whereas 137Cs (half-life, 30.2 years) emits gamma ray of 0.66 MeV. In recent times, irradiation using electron beams has been popular. Irrespective of the sources, the effects of irradiation with respect to food components and contaminant microorganisms are generally comparable. The quantity of energy absorbed by the food during irradiation is called “absorbed dose.” The SI unit for irradiation dose is the Gray (Gy), which is equal to the absorption of energy equivalent to 1 J/kg of absorbing material. One Gy is equivalent to 100 rad, or “radiation absorbed dose.”

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TABLE 15.8 Dose Required for 90% Inactivation (D10 Value) of Some Microorganisms in Fish/Shellfish Media Pathogen S. aureus V. cholerae V. flavialis V. mimucus V. parahaemolyticus V. parahaemolyticus V. vulnificus V. alginolyticus A. hydrophila A. hydrophila Shigella flexneri Salmonella paratyphi A S. paratyphi B S. typhimurium S. typhi Streptococcus fecalis Bacillus cereus Listeria monocytogenes Yersinia enterocoliticus Hepatitis A virus

Medium Surface of prawns Surface of prawns Shrimp paste Shrimp paste Shrimp homogenate, 1% NaCl Fish paste Shrimp paste Shrimp paste Fish homogenate Shrimp paste Shrimp paste Oyster paste Oyster paste Shrimp or fish paste Crab meat Shrimp paste Shrimp or fish paste Shrimp or fish paste Shrimp or fish paste Clams and oysters

Temperature (°C)

Atmosphere

D10 Value (kGy)

−10 ± 2 −10 ± 2 −20 −20 −20

Air Air Vacuum Vacuum Vacuum/air

0.29 0.11 0.44 0.75 0.44/0.07

Ambient −20 −20 0 0 Frozen 5 5 0–2 — — 0–2 0–2 0–2 —

Air Vacuum/air Vacuum Air Air — — — Air — — Air Air Air Air

0.03/0.06 0.30/0.35 0.19 0.14 0.10 0.22 0.75 0.85 0.10–0.15 0.87 5.0–7.5 0.2–0.3 0.15–0.25 0.10–0.15 2.02

Source: Reprinted from Venugopal, V., Doke, S.N., and Thomas, P., Crit. Rev. Food Sci. Nutr., 39, 391, 1999. With permission from Taylor & Francis Ltd. (www.informaworld.com).

Low-dose irradiation can have three major applications with respect to fishery products, which include extension of their refrigerated shelf life, hygienization of the products from pathogens, and inactivation of insects and their eggs from dried products. One of the major benefits of low-dose irradiation of seafood is inactivation of contaminant microbial pathogens such as Salmonella and Vibrio spp., which are sensitive to radiation.102 Table 15.8 gives radiation dose required for 90% inactivation (D10 value) of some microorganisms in fish/shellfish media of pathogenic microorganisms.100 The high penetration power of gamma radiation helps elimination of these microorganisms even from frozen fishery products; and the process of sanitization is called radicidation.108 Table 15.9 depicts important radiation processes for seafood, their treatment, and storage conditions. Other potential benefits of irradiation with respect to marine products are summarized in Table 15.10.100 Food irradiation plants have been installed in several countries, and the know-how for such plants has been gained by successful running of pilot plants in a number of countries including Belgium, China, India, the Netherlands, and the United States. Unconditional clearance for irradiation of fish items for shelf life extension,

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TABLE 15.9 Important Radiation Processes for Seafood, Their Treatment and Storage Conditions Treatment and Storage Temperature −1 to +3°C (postirradiation storage, under ice)

−10 to −20°C Packaged, frozen, ready-to-export fish can be treated before shipment. Frozen storage 15–30°C Ambient storage

Radiation Process

Benefits

Radurization (radiation pasteurization) Dose: 1–3 kGy Reduction of initial microbial content by 1–2 log cycles. Specific reduction of spoilage causing organisms Radicidation (radiation hygienization) Dose required: 4–6 kGy Elimination of nonspore forming pathogens such as Salmonella, Vibrio, and Listeria Radiation disinfestation Dose required <1 kGy Elimination of eggs and larvae of insects

Extends chilled shelf life of marine and freshwater fishery products two to three times Additional benefit includes reduction of nonspore forming pathogens Improvement of hygienic quality of frozen, materials for export such as frozen shrimp, cuttle fish, squid, finfish, fillets, and IQF items

Dry products free from spoilage due to insects. From dried fishery products including fish meal and feed for aquaculture Inactivation of Salmonella spp. and other pathogens

TABLE 15.10 Potential Benefits of Ionizing Radiations in Seafood Processing Extension of shelf life of fresh fish under chilled condition Elimination of pathogens in fresh and frozen seafood Hygienization of individually quick frozen fishery products Reduction of pathogens, including viruses from oysters Removal of off-odors from some fish and shellfish species Removal of allergens Reduction in fecal coliforms in live hard-shell clams Hygienization of aquafeed Hygienization of fish meal Development of shelf stable products using combination treatments Source: Adapted from Venugopal, V., Doke, S.N., and Thomas, P., Crit. Rev. Food Nutr., 39, 391, 1999.

disinfestation, and frozen (hygienization) has been accorded by a number of countries. The International Consultative Group on Food Irradiation (ICGFI) established under the aegis of FAO, IAEA, and WHO in 1984 helped to evaluate the global developments in the field of food irradiation. Till its expiry on May 8, 2004, ICGFI provided advices on the application of the technology and furnished information to

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its members. The recently created International Council on Food Irradiation (www. icfi.org) is intended to familiarize users with the benefits of irradiation to completely protect the consumers against seafood-borne health hazards.109 The unique possibility of irradiation of prepackaged, ready-to-export foods provides added advantage of prevention of postprocess contamination during handling. Integration of radiation treatment with conventional processing can enhance safety and shelf stability of fishery items to meet international quality standards.102

15.4.6

HAZARD ANALYSIS CRITICAL CONTROL POINT

Hazard analysis critical control point (HACCP) is the internationally recognized ideal method for assuring product safety by controlling food-borne safety hazards. HACCP helps to identify and monitor points where there is risk of contamination of a hazard. At present, it is the widely acknowledged, cost-effective method of controlling hazards in fishery products. Introduction of HACCP-based regulations for fish and fish products, particularly in the European Union and the United States, has triggered the need for seafood production under the HACCP system in most fish exporting countries. The underlying principle of HACCP is to make seafood processors responsible for assuring safe seafood. The seven elements of the HACCP system are (1) identification of potential hazards, (2) determination of critical control points (CCPs, a critical control point is defined as a point in the processing steps where the failure to effectively control a potential hazard may create an unacceptable risk), (3) description of the critical control point, (4) establishment of a monitoring system, (5) establishment of corrective action when CCP goes out of control, (6) establishment of procedures for verification, and (7) establishment of documentation and record keeping. HACCP, with its seven principles, form the framework for the rational consideration of actual hazards of seafood. Hazards may be caused by the fish species and its environment, or by the processing method, as discussed earlier. With HACCP, processors can use their own scientific/technological principles to establish the hazard prevention system that works for a particular processing situation. The U.S. FDA, as per notification dated December 18, 1995 (Federal Register, 21 CFR Parts 123 and 124), has made mandatory for all seafood processors exporting to the United States to adopt HACCP-based seafood quality assurance system for fish and fish products from December 18, 1997 onward. The regulations require that all seafood products must have been processed in accordance with both the HACCP principles.110,111 These requirements apply to both imported as well as domestic products. The HACCP system has been described by several authors.110–114 The HACCP system has been harmonized with the General Principles of Food Hygiene of the Codex Alimentarius Commission and the official regulations of the European Union. Practical experience generated during the last few years has showed that HACCP systems can control most of the hazards related to indigenous pathogens. Nevertheless, HACCP system cannot ensure complete safety of processed seafood and other marine products. Some of the weaknesses of the system include presence of pathogens in molluscs and fish to be consumed raw or steamed (undercooked) and growth of L. monocytogenes in some lightly preserved fish products (e.g., cold smoked fish). It is recommended that consumers should be warned about a possible

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risk in the case of the former and a limiting shelf life of the products should be recommended for the latter product. In conclusion, marine products sometimes may pose health hazards, which include presence of pathogenic bacteria, HAB toxins, and environmental pollutants such as heavy metals, drugs, and chemicals. Concerns related to these health hazards have attracted the attention of regulatory authorities worldwide. Efforts in this direction have led to a realization of proper understanding of food hazards and needs for measures for their control. These measures have moved from a paradigm of inspection to that of prevention, to contain the hazards. It was recognized that apparent absence of toxin or pathogen does not guarantee safety of the consumer. However, preventive measures must be very much in place to ensure consumer safety of the products. Worldwide implementation of the HACCP protocol is the result of such a realization. In the future, the need for eco-labelling and HACCP certification may be made mandatory for all seafood products. Given the inherent complexities associated with the safety and quality of marine products, a multidisciplinary approach, from the environmental to the food sciences, is often needed to ensure their adequate safety. Such efforts would ensure wholesome marine products as sources of food, nutraceuticals, bioactive compounds, and drugs.

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Appendix A.1

SOME INTERNATIONAL AND NATIONAL ORGANIZATIONS RELATED TO MARINE PRODUCTS

Food and Agriculture Organization, www.fao.org. Food Contaminant and Residue Information System, http://www-infocris.iaea. org/en/default.htm. World Resources Institute, www.wri.org. World Fish Center, www.worldfishcenter.org. World Trade Organization, www.wto.org. Global Aquaculture Alliance, http://www.gaalliance.org. Australia Recreational and Commercial Fish Handbook, http://www.filofish. com. Food Safety Information System, www.nal.usda.fda.gov. American Tilapia Association, http://ag.arizona.edu/azaqua/ata.html. Center for Disease Control, USA, www.cdc.gov/. Department of Agriculture, USA, www.usda.gov. Environment Protection Agency, USA, www.epa.gov/. EPA-OST (Environment Protection Agency, Office of Science and Technology), USA Fish Consumption Advisory Site, www.epa.gov/ost/fish. European Economic Commission, Food-Linked Agro-Industrial Research (FLAIR), Concerted Project on Harmonisation of Safety Criteria for Minimally Processed Foods, www.harmony.alma.ac.be. Fishery Statistics, Australia, http://www.daff.gov.au/. Fish Health, http://groups.msn.com/fishhealth. Fishbase, http://www.cgiar.org/. Center for Food Safety and Applied Nutrition, Food and Drug Administration, vm.cfsan.fda.gov/. Food Navigator, www.foodnavigator.com. USDA Food and Nutrition Information Center, www.nal.usda.gov/fnic/ foodborne/haccp/index.shtml. Health Focus International, www.healthfocus.net. Institute for Aquaculture, www.stir.ac.uk/aqua/. Institute of Food Technologists, Washington, DC, www.ift.org. Marine Science Institute, www.utmsi.zo.utexas.edu. National Fisheries Institute, USA, www.nfi.org. National Food Processors Association, USA, www.nfpa-food.org. National Marine Fisheries Service, USA, www.nmfs.gov. National Science Foundation, USA, www.nsf.gov. National Science Foundation, Seagrant Program, USA, www.mdsg.umd.edu/ NSGO/mdsg/. North Atlantic Fisheries College, www.nafc.ac.uk. 501

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Ocean Nutrition, Canada, www.ocean-nutrition.com. Pacific States Marine Fisheries Commission, www.vims.edu/. Seafood Data Search, www.seafood.com. Seafood Network Information Center, USA, http://seafood.ucdavis.edu/. Virginia Institute of Marine Center, USA, www.vms.edu. National Sea Grant Program, www.marinebiotech.org. Seafood Magazine, www.seafoodinternational.com.

A.2 DIETARY COMPONENTS AND COMPOSITION OF FOODS NIH Office of Disease Prevention, http://odp.od.nih.gov/agency.aspx#link21. The International Food Information Council (IFIC) Guidelines for Communicating the Emerging Science of Dietary Components for Health, (www.ific. org/nutrition/functional/guidelines/index.cfm), http://ific.org/. The USDA National Nutrient Database for Standard Reference, Release 19, 2006, http://www.nal.usda.gov/fnic/foodcomp/search/. European Foods, cost99/eurofoods. Inventory of European Food Composition, http://food.ethz.ch/. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids (macronutrients), 2005, Food and Nutrition Board, The National Academies Press, 500 Fifth Street NW, Lockbox 285, Washington, DC, 20055, http://www.nap.edu/catalog.php?record_id=11762. Nutrient Contents and Fortification, www.fortitech.com. Mahan, L. K. and Escott-Stump, S., Eds., Krause’s Food, Nutrition and Diet Therapy, Saunders, Philadelphia, PA, 2004 (book).

A.3 FOOD HAZARDS AND SAFETY The U.S. Food and Drug Administration’s Informational Site on Food Allergens, http://www.cfsan.fda.gov/∼dms/ffalrgn.html. Policy Statement on Food Safety. The Institute of Food Science and Technology, http://www.ifst.org/. Francis, G. A., Microbiological Safety Issues in Prepared Chilled Produce, IFIS Online, www.ifis.org. Food and Drug Administration’s Hazards and Control Guidance which consists of 21 chapters and 8 appendices on implementing HACCP, http://www. cfsan.fda.gov/~comm/haccp4.html. International Database on Insect Disinfestation and Sterilization (IDIDAS), http://www-ididas.iaea.org. Food and Agriculture Organization Training and Reference Centre for Food and Pesticide Control, www.iaea.org/trc. U.S. FDA, HACCP with respect to Environmental Chemical Contaminants and Pesticides, http://www.cfsan.fda.gov/~comm/haccp4i.html, http://www.cfsan. fda.gov/~acrobat/haccp4i.pdf, http://www.cfsan.fda.gov/~comm/haccp4j.html,

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http://www.cfsan.fda.gov/~acrobat/haccp4j.pdf, http://www.cfsan.fda.gov/ ~comm/haccp4x5.html. U.S. FDA Guide for the Control of Molluscan Shellfish, 2005, http://www. cfsan.fda.gov/~ear/nss3-toc.html. Guidelines on Fresh and Frozen Seafood: Selecting and Serving It Safely, U.S. Food and Drug Administration, Washington, DC, August 2006, http:// www.cfsan.fda.gov/. U.S. FDA, 2005 Census of Aquaculture, http://www.nass.usda.gov/aquaculture/.

A.4 TRADE RELATED Astra Information Systems Inc., www.astrainf.com. European Seafood Exposition, www.euroseafood.com. International Boston Seafood Exhibition, www.bostonseafood.com. International West Coast Seafood Show, www.westcoastseafood.com. Mediterranean Seafood Exposition, www.medseafood.it. Net.Yield TM by Lan Infosystems Inc., www.laninfo.com. ParityProTM Food Enterprise System, www.paritycorp.com. Seafood Business, www.seafoodbusiness.com. Seafood Processing Europe, www.europrocessing.com. SeaSoft, Computer Associates, www.caisoft.com. Singapore Seafood Exhibition/Seafood Processing Asia, www.singaporeseafood.com. WiseFishTM by Maritech International, www.wisefish.com. Buyers’ Guide, http://buyersguide.ift.org/cms/. U.S. Institute of Food Technologists Healthful Foods Directory, 2007, http:// buyersguide.ift.org/cms/?pid=1000237.

A.5 FISH NETWORK GLOBEFISH Fishery Industries Division FAO Viale delle Terme di Caracalla 00100 Rome, Italy Email: [email protected] Website: www.globefish.org INFOFISH 1st Floor, Wisma PKNS Jalan Raja Laut PO Box 10899 50728 Kuala Lumpur, Malaysia Email: [email protected] Website: www.infofish.org

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EUROFISH PO Box 0896 UN Centre, Midtermolen 3 DK-2100 Copenhagen, Denmark Email: [email protected] Website: www.eurofish.dk INFOYU Maizidian Street, Chaoyang District Beijing 100026, P. R. China Email: [email protected] INFOSAMAK 71, Boulevard Rahal El Meskini 16243 Casablanca, Morocco Email: [email protected] Website: www.infosamak.org INFOPECHE Tour C-19 Etage Cite Administrative, Abidjan 01 Cote D’lvoire Email: [email protected]

A.6

BOOKS

Alasalvar, C. and Taylor, T., Seafoods—Quality, Technology and Nutraceutical Application, Springer, Heidelberg, 2002. Beier, R. C., Pre-Harvest and Post-Harvest Food Safety, Blackwell Publishing, Ames, IA, 2004. Blunt, J. W. and Munro, M. H. G., Dictionary of Marine Natural Products, CRC Press, Boca Raton, FL, 2008. Barswanti, L. and Gualtieri, P., Algae: Anatomy, Biochemistry and Biotechnology, CRC Press, Boca Raton, FL, 2005. Connell, J. J., Control of Fish Quality, 4th ed., Martson Book Services Ltd., Oxford, UK, 1995. Huss, H. H., Ababouch, L., and Gram, L., Assessment and Management of Seafood Safety and Quality, Fisheries Technical Paper No. 444, FAO, Rome, Italy, 2003. Joseph, J., Mathew, P. T., Joseph, A. C., and Muralidharan, V., Eds., Product Development and Seafood Safety, Central Institute of Fisheries Technology, Cochin, 2003. Kraemer, K., Hoppe, P. P., and Packer, L., Nutraceuticals in Health and Disease, Marcel Dekker, New York, 2001, p. 318. Kurtzweil, P., An FDA guide to dietary supplements, in Nutrition, 11th ed., Cook-Fuller, C. C., Ed., Ruskin/McGraw-Hill, Guilford, CT, 1999. Lees, M., Ed., Food Authenticity and Traceability, Woodhead Publ. Co. Ltd., Cambridge, UK, 2003. Novak, J. S., Sapers, G. M., and Juneja, V. J., Microbial Safety of Minimally Processed Foods, CRC Press, Boca Raton, FL, 2002.

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505

Onwulata, C. I., Ed., Encapsulated and Powdered Foods, Taylor & Francis, Boca Raton, FL, 2005. Seaweed Recipes, Central Marine Fisheries Research Institute, Indian Council of Agricultural Research, Cochin, India, 2002. Surendran, P. K. et al., Eds., Seafood Safety, Central Institute of Fisheries Technology, Cochin, 2003. Tarver, T., ‘Just add water’—regulating and protecting the most common ingredient, IFT Status Summary, J. Food Sci., 73, R1, 2008. Wildman, R. E. C., Handbook of Nutraceuticals and Functional Foods, CRC Press, Boca Raton, FL, 2001, p. 542. Taylor, W. W., Schechter, M. G., and Wolfson, L. G., Globalization: Effects on Fisheries Resources, Cambridge University Press, Cambridge, UK, 2007, p. 550.

A.7 FISH COMPOSITION AND CONSUMPTION GUIDELINES TABLE A.1 Average Contents of Vitamins in Certain Commercially Important Seafood Seafood Flounder Halibut Herring Cod Mackerela Sardine Haddock Swordfish Pollocka Tuna Eel Oyster Shrimpb Lobster Mussel Cuttlefish a b

Average Content of Vitamins 1

2

3

4

5

6

7*

8

9

10

11

12

10 32 38 7 100 20 17 20 — 450 980 93 2 — 54 3

— 5 25 1 4 11 — — — 4.5 20 8 — — — —

360 850 1.5* 1.0* 1.3* — 390 — — — — 850 — 1.5* 750 2.6*

3 — 2.7 3 7 — 1.2

220 78 40 55 130 20 50 50 170 160 180 160 51 130 160 70

210 70 220 46 360 250 170 80 170 160 320 200 34 88 220 50

3.4 5.9 3.8 2.3 7.5 9.7 3.1 7.6 — 8.5 2.6 2.2 2.4 1.8 1.6 2.6

— 305 940 256 460 — 221 — — 660 — 320 80 2 — —

250 420 450 200 630 960 2.5 — — 460 280 220 130 1* 76 390

— 3 5 22 4 — — — — — — 10 500 5 — —

5 9 5 8 1 — 9 2 3 15 13 7 12 16 33 —

1 1 9 — 9 140 737 600 3 4 1 15 2 970 8 —

— — 40 100 — — — —

From Atlantic ocean. Brown shrimp.

Note: The values are given for 100 g edible portion. 1, vitamin A; 2, vitamin D; 3, vitamin E; 4, vitamin K; 5, vitamin B1; 6, vitamin B2; 7, nicotinamide; 8, pantothenic acid; 9, vitamin B6; 10, biotin; 11, folic acid; and, 12, vitamin B12. The values are given in microgram except otherwise indicated. *, values given in milligram; —, not reported. Source: Adapted from Souci, S. W., Fachmann, W., and Kraut, H., Food Composition and Nutrition Tables, 7th ed., Medpharm Sci. Publ., Stuttgart, Germany & CRC, 2008. With permission from Taylor & Francis, Boca Raton, FL (www.informaworld.com).

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TABLE A.2 Average Contents of Some Minerals in Certain Commercially Important Seafood Seafood

Average Content of Minerals

Flounder Halibut Herring Cod Mackerela Sardine Haddock Swordfish Pollocka Oyster Brown shrimp Lobster Mussel Cuttlefish a

1

2

3

4

5

6

7

8

9

10

160 67 117 72 80 100 116 102 100 160 146 270 296 387

278 446 160 360 380 — 301 336 336 184 230 220 280 273

21 28 21 26 30 24 24 17 17 32 67 24 30 —

48 14 14 28 12 85 38 8 8 82 92 61 24 27

180 202 250 194 268 258 176 376 376 157 224 234 200 143

297 550 1.1* 320 1.2* 2.4* 610 — 200 3.1* 605 1.0* 4.2* 800

520 495 585 396 585 — 270 —

110 — — 127 — — 35 —

22* 2.2* 1.6* 1.8* 700

120 160 210 480 —

26 27 42 229 42 32 135 — 88 58 91 100 156 —

25 — 43 28 43 58 30 — 20 25 50 130 80 65

From Atlantic ocean.

Note: The values are given for 100 g edible portion. Minerals: 1, sodium; 2, potassium; 3, magnesium; 4, calcium; 5, phosphate; 6, iron; 7, zinc; 8, fluoride; 9, iron; and 10, selenium. Unless otherwise stated, concentrations of minerals 1 to 5 are given in milligram, whereas those marked 6–10 are given in microgram. *, mg; —, not reported. Source: Adapted from Souci, S. W., Fachmann, W., and Kraut, H., Food Composition and Nutrition Tables, 7th ed., Medpharm Sci. Publ., Stuttgart, Germany & CRC, 2008. With permission from Taylor & Francis, Boca Raton, FL (www.informaworld.com).

TABLE A.3 Composition per 100 g of Edible Portion of Some Processed Fishery Products Products

A

B

C

D

E

F

G

H

I

J

K

L

M

I II III IV V VI VII VIII IX

0 0 24 0 0 0 0 17 19

60.9 56.2 75.1 65.5 65.6 70.0 70.0 58.4 74.6

199 214 98 199 188 126 107 217 99

19.6 15.1 22.8 20.4 21.5 18.8 22.6 23.7 23.5

10.3 9.0 0.8 13.0 11.3 5.4 1.8 13.6 0.6

0.9 2.8 0.2 3.7 2.3 1.1 0.4 2.8 0.2

7.5 19.3 0 0 0 0.7 0 0 0

0.1 0 0 0 0 0 0 0 0

0.3 0.7 0 0 0 0 0 0 0

80 52 55 33 28 300 150 550 0.8

0.5 0.8 0.7 1.0 1.2 2.7 1.1 2.9 1.0

100 380 120 170 150 370 1590 650 320

— 0 — 34 43 32 0 0 0

Note: Products: I = cod in batter, fried in oil; II = fish finger, grilled; III = haddock, steamed flesh; IV = herring, grilled; V = mackerel, fried; VI = pilchard, canned in tomato sauce; VII = prawn, boiled; VIII = sardine canned in oil, drained; IX = tuna, canned in brine. A = inedible waste; B = water, g; C = energy, kcal; D = protein, g; E = fat, g; F = saturated fatty acids, g; G = carbohydrates, g; H = total sugar, g; I = fiber, g; J = calcium, mg; K = iron, mg; L = sodium, mg; M = vitamin A, µg; — = not reported. Source: Adapted from Manual of Nutrition, 10th ed., Ministry of Agriculture, Fisheries and Food, London, HMSO, London, 1995. With permission.

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507

Age/gender group

12 years and below

13 years and above

Female

Male

Could become pregnant, pregnant, or lactating

Yes

May benefit from consuming seafood, especially those with relatively higher concentrations of EPA and DHA A reasonable intake would be two 3-oz servings (or for children, age-appropriate servings) but can safely consume 12 oz/week Can consume up to 6 oz (or for children, age-appropriate servings) of white (albacore) tuna per week and should avoid large predatory fish such as shark, swordfish, tilefish, or king mackerel

No

Consume seafood regularly, for example, two 3-oz servings weekly If consuming more than two servings per week, choose a variety of seafood types There may be additional benefits from including seafood comparatively high in EPA and DHA

Contaminants in seafood vary according to local conditions; consume locally caught seafood only if appropriate after checking your state advisories

FIGURE A.1 Diagram highlighting the variables that group consumers into target populations that face different benefits and risks and should receive tailored advice. (Reprinted from Committee on Nutrient Relationships in Seafood: Selections to Balance Benefits and Risks, Food and Nutrition Board, Seafood Choices: Balancing Benefits and Risks, Nesheim, M. C. and Yaktine, A. L., Eds., The National Academic Press, Washington DC, 2007. With permission.)

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A.8 MICROBIOLOGICAL STANDARDS TABLE A.4 Microbiological Criteria for Fish and Fishery Products as Required by European Countries Microorganisms

Standard

Pathogen Salmonella spp.

Absent in 25 g (n = 5, c = 0). In addition, pathogens and toxins thereof that are to be sought according to risk evaluation, must not be present in quantities such as to affect the health of consumers

Indicator of poor hygiene a. Staphylococcus aureus b. Coliform (thermotolerant, 44°C) c. Escherichia coli Mesophilic bacteria (30°C) a. Whole product b. Shelled or sucked product except crab meat c. Crab meat

a. m = 100, M = 1000, n = 5, c = 2 b. m = 10, M = 100, n = 5, c = 2 c. m = 10, M = 100, n = 5, c = 1 a. m = 0, M = 100,000, n = 5, c = 2 b. m = 50,000, M = 500,000, n = 5, c = 2 c. m = 100,000, M = 1,000,000, n = 5, c = 2

Note: m, limit below which all the results are considered satisfactory; M, acceptability limit beyond which the results are considered unsatisfactory; n, number of units comprising the sample; c, number of sampling units giving bacterial counts between m and M. The quality of a batch is considered to be satisfactory where all the values observed are three times m or less. The samples are acceptable where the values observed are between 3 m and 10 m (M) and where c/n = 2/5 or less. The quality of a batch is considered to be unsatisfactory in all the cases where values are above M, where c/n is greater than 2/5.

TABLE A.5 The Japanese Government Bacteriological Standard and the Tokyo Metropolitan Governmental Bacteriological Guidelines for Frozen Foods Product

Frozen foods for serving without cooking Frozen foods for cooked serving (to be cooked before freezing) Frozen foods for cooked serving (not cooked before freezing) Frozen fish and shellfish for raw serving Frozen cooked octopus

Type of Bacteria 1

2

<105/g <105/g

5

6

N

N

N

N

N

N

N

N

<3 × 106/g

3

4

N

<105/g

N

<105/g

N

N

7

20 20

(Continued )

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TABLE A.5

509

(Continued)

Product

Type of Bacteria 1

Frozen oyster for raw serving

<5 × 106/g

Frozen fish and shellfish for processing

<5 × 106/g

2

3

4

5

6

7

<230 MPN/ 100g N

25

Note: 1, Standard aerobic plate counts per gram; 2, Coliform per 0.01 g; 3, E. coli per 0.01 g; 4, Vibrio parahaemolyticus per 0.01 g; 5, Salmonella spp. per g; 6, S. aureus per 0.01 g; 7, volatile basic nitrogen milligram per 100 g; N, negative; MPN, most probable number. Source: From MPEDA Newsletter, 4, 16, 2001, Marine Products Export Development Authority, Cochin. With permission.

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Index A Active packaging technology, marine biodegradable and edible films, 451–453 Agar, 299–301 applications, 300–301 composition and properties, 300 extraction, 299–300 source, 299 Agriculture fungal contaminants in, 484 seaweed applications in, 281–282 Algae carotenoids in, 223–224 drugs and pharmaceuticals from, 388–390 nutritional composition, 262–267 omega-3 fatty acids in, 173–174 pigments and bioactive compounds in, 274 protein quality in, 264–265 toxins, as safety hazards, 476–483 amnesic shellfish poisoning, 481 biotoxins, 481–483 ciguatera poisoning, 480 diarrhetic shellfish poisoning, 480 paralytic shellfish poisoning, 479 puffer fish poisoning, 480 Alginates, 301–308 composition and structure, 302 food, medical, and biotechnology applications, 304–308 macromolecule carriers and biofilms, encapsulation, 437–438 macromolecule carriers and edible films, 448–449 properties, 302–304 source and extraction, 301–302 Alkoxy radicals, carotenoids, 228–231 Allergies to seafood removal of, 488 safety hazards and, 486–487 Amino acids fish and seafood, histamine poisoning, 475 fortified foods, 412–413 microalgae, 341 protein nutritive values and, 104–116 in seaweed, 264–265 soluble protein powder, 110–111 Amnesic shellfish poisoning, 481 Analgesics, marine sources of, 382

Angiotensin I-converting enzyme (ACE) inhibitory activity bioactive peptides, 122–124 microalgae, 347 Animal feed carotenoids in, 233–234 fish protein in, 81–83 seaweed in, 282–283 Ankylosing spondylitis, polyunsaturated fatty acids and, 162 Antarctic krill fish protein hydrolyzates, nutritive value, 111–114 as food source, 29–30 nutritive value and biochemical composition, 110–111 Antibacterial/antimicrobial compounds bioactive marine peptides, 120–121 carrageenan, 322 chitosan, 197–198 macromolecule biodegradable and edible films, 441–442 marine drugs and pharmaceuticals, 373, 380–381 microalgae, 347 seaweed, 276–277 Anticancer compounds carotenoid activity, 232 carotenoids and, 228–232 carrageenans, 324 marine drugs, 375–378 microalgae, 346 polyunsaturated fatty acids and, 158 seaweed bioactivity and, 277–278 Antifouling agents, seaweed in, 284–285 Antifreeze compounds marine peptides, 126–127 proteins, 130–131 Antihypertensive peptides, microalgae, 347 Antimony oxide bromide (AOB) nanotechnology, edible films, 454–455 Antioxidants active packaging technology, 451–453 bioactive marine peptides, 121–122 carotenoids, 228–231 carrageenan, 323 chitosan, 198–199 current research on, 9 fatty acids, 152–154

511

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512 Antioxidants (Contd.) health effects, 152–154 microalgae, 346–347 seaweed sources, 273–275 Antithrombin, bioactive peptides, 125 Antiviral compounds carrageenans, 323–324 hydrocolloids (seaweed), 298–299 marine drugs and pharmaceuticals, 373, 380–381 microalgae, 345–346 seaweed, 276–277 Aplidine, 377–378 Aquaculture carotenoid activity in, 232–233 fish protein, animal feed, 82–83 marine product applications, 17–19 microalgae, 349–352 applications in, 358–359 seaweed in, 283–284, 286–288 Ara-C agent, marine sources, 377 Arachidonic acid, food fortification and supplementation, 418–419 Arthritic agents, marine sources for, 380 Artificial reefs, coral reef restoration and, 42 Astaxanthins carotenoids, 221–222 chitin recovery, 186–189 dietary function of, 228–231 poultry feed and, 233–234 in seafood waste, 225 Asthma, polyunsaturated fatty acids and, 160 Autoxidation carotenoids, 229–231 fatty acids, 151–155

B Bacteria algal toxin interactions, 478–483 marine bacteria habitat and resources, 45–46 safety hazards, 470–475 probiotic bacteria, nutritive value, 114–116 Behavior, polyunsaturated fatty acids and, 160–161 Bile acids, dietary fiber and, 269 Bioactive compounds carrageenan, 322–324 coral reef and corals, 363–364 fucoidan, 328–329 marine drugs and pharmaceuticals, 374–375 bivalves, 393–394 corals, 382–387

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Index fish and shellfish, 391–392 jellyfish, 393 microorganisms, 387–388 plants, 388–390 sea cucumber, 392–393 toxins, 390–391 microalgae, 344–349 anticancer compounds, 346 antihypertensive peptides, 347 antimicrobial compounds, 347 antioxidant compounds, 346–347 antiviral compounds, 345–346 seafood peptides, 116–127 angiotensin I-converting enzyme inhibition, 122–124 antibacterial activity, 120–121 antioxidant activity, 121–122 antithrombin activity, 125 calcitonin, 125 calcium-binding activity, 120 commercial status, 133–135 functional roles, 119–120 human immunodeficiency virus-1 protease inhibition, 125 immunostimulant activity, 124 isolation, 117–119 obesity control, 120 physiological functions, 125–127 seaweed sources, 273–280 Bioavailability carotenoids, 227 microalgae, 340–341 minerals in seafood, 253–254 nutrients, 11–12 Biodegradable films hydrogel drug delivery systems, 455–457 marine macromolecules, 439–440 Biofilms, marine drugs and pharmaceuticals, 373, 380–381 Biofouling, seaweed applications in, 284–285 Bioinformatics, functional food development and, 9 Biomining, marine products, 17–19 Biopolymer-based packaging materials, marine biodegradable and edible films, 440–441 BioSwitch encapsulation system, macromolecule carrier delivery, 434–437 Biotechnology alginate applications in, 304–308 carotenoid applications, 234 carrageenans, 324–325 chitosan applications, 203 marine bacteria, 361–362 marine drugs and pharmaceuticals, 394 Biotoxins, hazard analysis and control, 481–483, 487–488

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Index Bivalves, drugs and pharmaceuticals from, 393–394 Black band disease (BBD), coral reefs and corals, 41 Bleaching, coral reefs and corals, 41 Blood pressure, polyunsaturated fatty acids and, 156 Blood protein, fish products, 75–76 Bone density and health bioactive peptides and, 120–127 in humans, marine-based minerals and, 254–255 polyunsaturated fatty acids and, 161–162 Brain function, polyunsaturated fatty acids and, 160–161 Bromodeoxyuridine (BrdU), anti-cancer activity, 278

C Calcitonin bioactive peptides, 125 bone health and, 255 Calcium alginate binding, 304 bone health and, 254–255 carrageenan, 314–316 in fish bone, 250–254 marine sources of, 244–254 Calcium-binding activity, marine peptides, 120 Canada, functional food regulations and standards, 12–14 Cancer carotenoids and, 228–232 carrageenans, 324 marine drugs and pharmaceuticals, 375–378 microalgae, 346 polyunsaturated fatty acids and, 158 seaweed bioactivity and, 277–278 Canthaxanthin, aquaculture and, 233 Carbohydrate polymers, alginates, 302–304 Cardiovascular disease, polyunsaturated fatty acids and, 156–158 Carotenoids anticancer activity, 232 antioxidant activity, 228–230 aquaculture functions, 232–233 bioavailability, 227 biotechnology, 234 commercial status, 234–235 dietary benefits, 231–234 encapsulation, 434 fortified foods, 412 functional roles, 228–231 general properties, 221–222

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513 hypercholesterolemic activity, 231–232 isolation and characterization, 223–227 marine sources, 223 microalgae, 343 antioxidants, 346–347 cultivation, 351–352 in poultry feed, 233–234 units and dietary requirements, 222–223 vitamin A activity, 230–231 Carrageenan, 308–327 analysis, 311–313 bioactivity of, 322–324 biotechnology and, 324–325 browning control, 321 cryoprotective effect, 321 degradation, 327 extraction and characterization, 308–309 fat reduction and, 318–320 fiber fortification, 320–321 flavor perception, 320 food product applications, 316–321 gelation, 313–316 macromolecule carriers and edible films, 448 active packaging technology, 452–453 properties, 311 rheological properties, 314–316 salt reduction, 320 structure, 309–310 textural modification with, 317–318 toxicology, 325–326 Casting methods, macromolecule carriers and edible films, 453 Catalysts, chitosan applications, 204 Cellular processes carotenoid activity, 231–232 marine drugs and pharmaceuticals, 372 polyunsaturated fatty acids, 156 Chemical hazards in marine products, 484–486 removal technologies, 489–490 Chemical modifications, seafood protein, 60 Chemotherapeutic agents, marine sources for, 378 Chitin applications, 191–192 edible films, 446–448 isolation of, 187–190 properties, 190–191 sources, 186–187 specifications, 196 structure, 190 Chitinases, structure and properties, 207 Chitin oligosaccharides, 204–206 Chitosan, 192–204 antimicrobial activity, 197–198 antioxidant activity, 198–199

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514 Chitosan (Contd.) applications, 194–196, 204–206 biomedical applications, 459–460 biotechnology, 203 catalytic support and packaging, 204 derivatives, 194–196 edible films, 199–200, 446–450 nanotechnology, 454–455 food, 196–197 hydrogel, 203 medical applications, 202 nutritional role, 200–202 oligosaccharides, 204–206 antibacterial/antimicrobial activity, 198 physicochemical properties, 193–194 properties, 193–194 specifications, 196 structure, 192 water treatment, 203 Chitosanases, structure and properties, 207–208 Chlorella, 352–353 Chloride, marine sources of, 244–254 Chlorophylls, microalgae, 343 Chondroitin sulfate food fortification and supplementation, 422 macromolecule carriers and biofilms, 438–439 structure and properties, 209–213 Ciguatera poisoning, toxin structure and properties, 480 Coating materials, macromolecule carriers and biofilms, 441–453 active packaging, 451–453 alginates, 448–449 antimicrobials, 451–453 carrageenan, 448 casting methods, 453 collagen and elastin, 444–445 food encapsulation, 438–439 gelatin, 445–446 multicomponent films, 449–450 polysaccharides, 446, 451–453 proteins, 442–445 Cod head, gelatin extracts, 87–88 Cold-adapted enzymes, 131–133 Collagen amino acid composition, 90 fish protein, 83–85 macromolecule carriers and biofilms, 444–445 Commercial status carotenoids, 234–235 chitin and chitosan, 213–214 coral reef and corals, 364–365 hydrocolloids, 330–331 marine-based vitamins and minerals, 255–256

CRC_52632_Index.indd 514

Index marine biodegradable and edible films, 459–460 marine drug and pharmaceuticals, 398–399 marine nutraceuticals, 422–425 seafood proteins, 93–94, 133–135 seaweed in, 286–289 squalene, 175–178 Connective tissue marine protein in, 83–91 shark cartilage and chondroitin sulfate, 209–213 Consumer preferences changing trends in, 34–39 food consumption data, 10–11 food selection preferences, 5–6 functional foods, 9–10 global consumption of seafood, 33–34, 36–37 seafood demand growth, 27–29 Consumption guidelines, seafood, 38–39 Control technology, safety hazards, 487–494 allergen removal, 488 biotoxins, 487–488 chemical hazards, 489–490 food irradiation, 490–493 hazard analysis critical control point, 493–494 microbiological hazards, 490 parasite removal, 489 Cooking, protein nutritive value, effects of, 108–109 Copper, marine sources of, 246–254 Coral reef and corals basic properties, 362–363 biological activity, 363–364 commercial status, 364–365 drugs and pharmaceuticals from, 382–387 habitat and resources, 39–42 bleaching and other problems, 41–42 fisheries and, 40 restoration efforts, 42 symbiotic associations, 40 structure and properties, 362–363 Corals, marine compounds from, 17–19 Crocin bleaching assays, carotenoids, 229–231 Crustacean waste. See also Chitin; Chitosan carotenoids in, 224–227 aquaculture and, 232–233 global availability, 185–186 Cryoprotective agents, carrageenan, 321 Cyanobacteria, 347–348

D Deacetylated chitin (DAC), structure, 192 Deep-sea fauna, as food source, 29–30 Degradation, carrageenan, 327 Dehydrodidemnin, 377–378

9/5/2008 3:14:55 PM

Index Delivery systems, macromolecule carriers and biofilms, 430 encapsulation technology, 434–436 hydrogel drug delivery systems, 455–457 Demand, consumer, seafood, 27–29 Detergents, seaweed applications in, 285 Diabetes, polyunsaturated fatty acids and, 161 Diarrhetic shellfish poisoning okadaic acid, 478–483 toxin structure and properties, 478–480 Diatoms, microalgae, 341 Diet carotenoids in, 222–223 antioxidant properties, 228–231 benefits of, 231–232 vitamin A, 230–231 fiber sources alginate, 305–308 carrageenan, 320–321 seaweed, 268–280 antibacterial/antiviral activities, 275–277 antioxidants, 273–275 antitumor activity, 277–278 bioactive compounds, 273–280 definition, 268 dietary supplements, 272–273 enzyme inhibition, 279–280 food enrichment, 272 health benefits, 268–271 HIV inhibition, 279 hyperoxaluria, 278–279 platelet aggregation, 277 health protection and, 2–4 marine nutraceuticals and, 405–406 omega-3 fatty acids current intake levels, 165–166 recommended consumption levels, 166–168 protein requirements, 103–104 seafood proteins in, 51 Dietary reference intakes (DRIs), marine nutraceuticals, 405–406 Dietary supplements marine nutraceuticals, 406–407 seafood protein, 63–74, 109–116 seaweed fiber, 272–273 Dimethylsulfoxide (DMS), in seaweed, 266 1,1-Diphenyl-2-picrylhydrazyl (DPPH) carotenoid antioxidants, 229–230 microalgae antioxidants, 346–347 seaweed bioactivity and, 274–275 Diterpenes, marine sources for, 378 Docosahexaenoic acid (DHA), microalgae, 341 Drugs and pharmaceuticals

CRC_52632_Index.indd 515

515 macromolecule carriers and biofilms, hydrogel drug delivery systems, 455–457 marine products analgesic/hypotensive drugs, 382 anticancer agents, 375–378 antimicrobial/antiviral compounds, 380–381 arthritis, 380 bioactive compounds, 374–375 biotechnology, 394 bivalves, 393–394 classification, 373–382 corals, 382, 384–387 development process, 394–398 fish and shellfish, 391–392 global market and commercial status, 398–399 jellyfish, 393 limitations of, 394–398 malaria, 379 microorganisms, 387–388 osteoporosis, 379–380 overview, 371 plants, 388–390 potential development of, 371–373 sea cucumber, 392–393 sponges, 382–387 toxins, 390–391 tuberculosis, 379 Dunaliella, 357–358

E Ecteinascidin, 377–378 Edible films chitosan, 199–200, 446–448 marine macromolecules, 441–453 active packaging, 451–453 alginates, 448–449 antimicrobials, 451–453 carrageenan, 448 casting methods, 453 collagen and elastin, 444–445 gelatin, 445–446 multicomponent films, 449–450 polysaccharides, 446, 451–453 proteins, 442–444 Eicosa pentaenoic acid (EPA), food fortification and supplementation, 415 Elastin, macromolecule carriers and biofilms, 444–445 Emulsions, seafood protein, 55–56 Encapsulation advantages of, 431–432 macromolecule carriers and biofilms, 431–434, 437–439

9/5/2008 3:14:55 PM

516 Encapsulation (Contd.) classification, 432 glucosamine and chondroitin sulfate, 438–439 liposomes, 433 marine products, 437–439 microemulsion, 433–434 polyunsaturated fatty acids, 437–438 spray drying, 432 techniques, 432 Enrichment processes marine nutraceuticals, 407–409 seaweed fiber in food, 272 Environment and ecosystems chemical hazards, 484–485 removal technologies, 489–490 marine habitat, 23–25 seaweed applications in, 284–286 Enzymes carrageenan immobilization and purification, 325 chitin/chitosan degradation, 207–209 cold-adapted, 131–133 marine enzymes, 127–130 seafood protein modification of, 60–61 seaweed inhibition of, 279–280 Estimated safe and adequate daily dietary intakes (ESADDIs), nutrients intake, 12 Extracellular gastric enzymes, marine sources, 128–130

F Fat replacement alginates, 306–308 carrageenan, 318–320 Fatty acids. See also Polyunsaturated fatty acids (PUFA) food fortification and supplementation, 415–420 marine lipids, 145–146 omega-3 fatty acid levels, 148–150 oxidation, 150–155 antioxidants, 152–154 marine fishery product lipids, 154–155 terminology and symbols, 145–146 Fermented fish products, protein content, 80–81 nutritive value, 114–116 Fiber carrageenan fortification, 320–321 seaweed as source of, 268–280 antibacterial/antiviral activities, 275–277 antioxidants, 273–275 antitumor activity, 277–278 bioactive compounds, 273–280 definition, 268

CRC_52632_Index.indd 516

Index dietary supplements, 272–273 enzyme inhibition, 279–280 food enrichment, 272 health benefits, 268–271 HIV inhibition, 279 hyperoxaluria, 278–279 platelet aggregation, 277 Films, edible, chitosan, 199–200 Fish and fishery products. See also Seafood carotenoids in, 223–225 dietary guidelines for, 16–19 drugs and pharmaceuticals from, 391–392 fish meat mince/mince-based products, 63–65 fungal contaminants, 484 habitat and resources, 25–39 Antarctic krill and deep-sea fauna, 29–30 consumption trends, 34–39 demand and concerns, 27–29 food security and, 32–33 global consumption patterns, 33–34 landing, 26–27 mariculture, 31–32 new species identification, 31 sea cucumbers, 30–31 underutilized fisheries, 29 histamine poisoning, 475 insect safety hazards, 476 lipid oxidation, 154–155 macromolecule carriers and biofilms, 442–444 microalgae as feed, 358–359 mineral content of, 247–254 muscle structural proteins, function of, 61–62 omega-3 fatty acid sources, 16–19, 168–174 extraction, 168–170 properties, 170–173 parasites in, 483–484 removal technologies, 489 per capita fish supply, 33–34 protein nutritive value in, 106–107 protein powders commercial status, 422–425 mineral content, 248–254 nutritive value, 110–111 thermostable dispersions, 71–75 raw muscle protein content, 52–54 reef-associated, 40 safety hazards, 467–487 toxins as safety hazard, 481–483 world fish production and capture, 26–27 Fish ball, protein content, 64–65 Fish bone bone health and, 255 food fortification and supplementation, 421 mineral sources in, 249–254

9/5/2008 3:14:55 PM

Index Fish bone phosphopeptide (FBP) calcium-binding activity, 120 minerals in, 251–254 Fish oil commercial status, 422–425 extraction, 168–170 fish feed applications, 176–178 food fortification and supplementation, 415–420 as functional food, 16 macromolecule carriers and biofilms, encapsulation, 437–438 properties, 170–173 vitamin content of, 241–243 Fish protein concentrate (FPC) blood proteins, 75–76 krill sources, 75 protein nutritive value, 109–116 squid, 75 structure and properties, 70–80 thermostable dispersions and powders, 71–75 Fish protein hydrolyzates (FPH) bioactive peptides, 119–127 food fortification and supplementation, 420–421 immunostimulant activity, 124 nutritive value, 111–114 structure and properties, 76–80 Flavor enhancement carrageenan, 320 fortified foods, 407–409 Fluoride, in seafood, 247–254 Foaming capacity, seafood protein, 56 Folate and folic acid, marine sources, 240–243 Food and food additives alginates, 304–308 carrageenan analysis, 311–313 applications, 316–321 browning control, 321 cryoprotective effect, 321 fiber fortification, 320–321 textural modifications, 317–318 chitosan, 196–202 consumer selection patterns, 5–6 fatty acid terms and symbols, 145–146 fiber sources, seaweed, 268–272 food-borne hazards, 467–468 functionality, 2 irradiation technology, 490–493 marine nutraceuticals carotenoids, 412 chondroitin sulfate, 422 commercial status, 422–425 dietary guidelines, 405–406 fortification and enrichment, 407–409 iodine, 409–410

CRC_52632_Index.indd 517

517 macromolecule carriers and biofilms, 430–459 marine oil-fortified products, 415–417 marketing campaigns, 419–420 minerals, 411–412, 421–422 omega-3 fatty acids, 415 probiotics, 413–414 process optimization, 417–418 proteins and amino acids, 412–413, 420–421 PUFA-fortified products, 418–419 regulatory status, 419 supplementation, 406–407 vitamins, 410–411 microalgae, 340–341 protein functions in, 58–61 seaweed as, 271–272 Food-based dietary guidelines (FBDGs), marine nutraceuticals, 405–406 Food consumption data (FCD), functional food design and, 10–11 Food flavorings, seafood products, 92 Food security, seafood production and, 32–33 Free radicals, bioactive marine peptides, 121–122 Fucoidan, 327–329 Fucoxanthin, anti-cancer activity, 278 Functional foods classification, 6–7 consumer surveys on, 9–10 defined, 3–4, 6 design criteria, data for, 10–16 food consumption data, 10–11 nutrient bioavailability, 11–12 nutrient reference standards and recommendations, 12 diet and health protection, 2–4 increased popularity, 8–9 limitations of, 8–9 marine products, 16–19 marketing and trade of, 14–16 nutraceuticals, 4–6 overview, 1–2 recent developments in, 7–9 safety and regulation, 12–14 seven-step design, development, marketing process for, 15 Fungi, as marine product safety hazard, 484

G Galactans, carrageenan, 310 Gamma irradiation, chitosan, 198–199 Gelatin agar, 299–301 alginates, 306–308 amino acid composition, 90 applications, 91

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518 Gelatin (Contd.) extraction from marine sources, 86–89 fish protein, 85–86 antioxidant peptides, 121–122 macromolecule carriers and edible films, 445–446, 450 molecular composition, 88–91 Gelation carrageenan, 313–316 seafood protein, 56–58 surimi/surimi-based products, 66–70 Global consumption patterns carotenoids, 234–235 marine drug and pharmaceuticals, 398–399 seafood, 33–34, 36–37 Global markets, functional foods, 16 Glucosamine food fortification and supplementation, 421–422 macromolecule carriers and biofilms, 438–439 structure and properties, 209 Glycomacropeptide (GMP), obesity control, 120 Glycosidic linkages, alginates, 302–304 G-protein-coupled receptors, polyunsaturated fatty acids, 163–164 Graft materials, alginates, 306–308 Green process, alginate extraction, 301–302 Growth factor antagonists, carrageenan, 322 Gums, carrageenan, 326

H Habitat and resources, marine products bacteria, 45–46 coral reef and corals, 39–42 bleaching and other problems, 41–42 fisheries and, 40 restoration efforts, 42 symbiotic associations, 40 environment and ecosystem, 23–25 fishery products, 25–39 Antarctic krill and deep-sea fauna, 29–30 consumption trends, 34–39 demand and concerns, 27–29 food security and, 32–33 global consumption patterns, 33–34 landing, 26–27 mariculture, 31–32 new species identification, 31 sea cucumbers, 30–31 underutilized fisheries, 29 Hazard analysis critical control point (HACCP), marine product safety, 493–494 Health diet and, 2–4 polyunsaturated fatty acids and, 165

CRC_52632_Index.indd 518

Index seaweed fiber and, 268–271 shellfish toxins and, 482–483 Heavy metals, seaweed biosorption of, 285–286 Herpes simplex virus (HSV), microalgae bioactivity and, 345–346 Highly unsaturated fatty acids (HUFA), microalgae, 342 Histamine poisoning, fish and seafood, 475 Human immunodeficiency virus-I (HIV) microalgae bioactivity and, 345–346 protease inhibiting activity, 125 seaweed inhibition, 279 spirulina bioavailability, 355–357 Hyaluronidase, seaweed inhibition, 279–280 Hydrocarbons carotenoids, 221–222 microalgae, 343–344 Hydrocolloids (seaweed) agar, 299–301 applications, 300–301 composition and properties, 300 extraction, 299–300 source, 299 alginates, 301–308 composition and structure, 302 food, medical, and biotechnology applications, 304–308 properties, 302–304 source and extraction, 301–302 carrageenan, 308–327 analysis, 311–313 bioactivity of, 322–324 biotechnology and, 324–325 browning control, 321 cryoprotective effect, 321 degradation, 327 extraction and characterization, 308–309 fat reduction and, 318–320 fiber fortification, 320–321 flavor perception, 320 food product applications, 316–321 gelation, 313–316 properties, 311 rheological properties, 314–316 salt reduction, 320 structure, 309–310 textural modification with, 317–318 toxicology, 325–326 commercial status, 330–331 composition, 271–272 fucoidan, 327–329 functional properties, 297–299 laminarin, 330 Hydrogels chitosan applications, 203 macromolecule carriers, 455–459 drug delivery systems, 455–457 polysaccharide scaffolds, 457–459

9/5/2008 3:14:56 PM

Index Hydrogen atom transfer (HAT) assay, carotenoids, 229–231 Hydrolyzates, fish protein, 76–80 Hydrophilicity, agar, 300 Hydroxyl radicals, carotenoids, 228–231 Hypercholesterolemic activity, carotenoids, 231–232 Hyperoxaluria, seaweed bioactivity, 278–279 Hypotensive drugs, marine sources of, 382

I Immune response, carrageenan suppression, 323–324 Immunostimulant activity, bioactive peptides, 124 Industrial applications agar, 300–301 seaweed agriculture, 281–282 animal feed, 282–283 antifouling agents, 284 aquaculture, 283–284 future research on, 285–286 heavy metal biosorption, 285 seaweed in, 281–286 Infancy, polyunsaturated fatty acids in, 158–160 Infant formula, marine nutraceutical supplementation, regulatory guidelines, 420 Insects, safety hazards of, 476 International recommendations and standards, nutrients intake, 12 Iodine food fortification, 409–410 marine sources of, 246–254 Iron, marine sources of, 245–254 Irradiation technology, food products, 490–493

J Japan, functional food regulations and standards, 12–14 Jellyfish drugs and pharmaceuticals from, 393 food products from, 92–93

K Kamaboko, protein content, 81 Kelps. See Seaweed Krill mineral content of, 247–254 protein in, 75 Kuruma prawns, fish protein hydrolyzates, nutritive value, 111–114

CRC_52632_Index.indd 519

519

L La Gloahec-Herter process, alginate extraction, 301–302 Laminarin, 330 Landing statistics, seafood capture, 26–27 Lipids, marine, 144–150 fatty acids, 145–146 fish oils, extraction, 168–170 microalgae, 341–342 cultivation, 350–352 nutritional value of, 155–168 oxidation in fishery products, 154–155 seaweed, 266 structural profile, 146–150 Liposomes, macromolecule carriers and biofilm encapsulation, 433 Low-density liproprotein (LDL) autoxidation carotenoids, 229–231 hypercholesterolemic activity, 231–232 microalgae antioxidants, 346–347 Low-molecular-weight chitosans (LMCs), 2005–206 Lycopene, antioxidant properties, 228–231

M Macromolecule carriers and biofilms, nutraceuticals active packaging, 451–453 alginate, 448–449 biodegradable and edible films, 439–442 carrageenan, 448 casting methods, 453 chitosan, 446–448 collagen and elastin, 444–445 commercial status, 459–460 delivery systems, 430 encapsulation, 431–434, 437–439 food coatings and edible films, 442–453 gelatin, 445–446 hydrogels and membranes, 455–459 marine proteins, 442–444 matrix delivery design, 430–431 multicomponent films, 449–450 nanotechnology, 454–455 novel delivery systems, 434–437 overview, 429–430 polysaccharides, 446, 451–455, 457–459 Malaria agents, marine sources for, 379 Mariculture, habitat and environment for, 31–32 Marinbeef product, protein content, 65 Marine bacteria anticancer agents, 377–378 structure and properties, 359–362 Marine flora, drugs and pharmaceuticals, 373–375

9/5/2008 3:14:56 PM

520 Marine lipids, 144–150 fatty acids, 145–146 oxidation, 154–155 seafood lipid profile, 146–150 Marine plants, drugs and pharmaceuticals from, 388–390 Marine products bioactive peptides, 119–127 carotenoids, 223 antioxidant activities of, 229–230 drugs and pharmaceuticals analgesic/hypotensive drugs, 382 anticancer agents, 375–378 antimicrobial/antiviral compounds, 380–381 arthritis, 380 bioactive compounds, 374–375 biotechnology, 394 bivalves, 393–394 classification, 373–382 corals, 382, 384–387 development process, 394–398 fish and shellfish, 391–392 global market and commercial status, 398–399 jellyfish, 393 malaria, 379 microorganisms, 387–388 osteoporosis, 379–380 overview, 371 plants, 388–390 potential development of, 371–373 sea cucumber, 392–393 sponges, 382–387 toxins, 390–391 tuberculosis, 379 as functional foods, 16–19 habitat and resources bacteria, 45–46 coral reef and corals, 39–42 bleaching and other problems, 41–42 fisheries and, 40 restoration efforts, 42 symbiotic associations, 40 environment and ecosystem, 23–25 fishery products, 25–39 Antarctic krill and deep-sea fauna, 29–30 consumption trends, 34–39 demand and concerns, 27–29 food security and, 32–33 global consumption patterns, 33–34 landing, 26–27 mariculture, 31–32 new species identification, 31 sea cucumbers, 30–31

CRC_52632_Index.indd 520

Index underutilized fisheries, 29 overview, 23 seaweed, 42–43 microalgae, 43–45 marine enzymes, 127–130 applications, 129–130 isolation, 127–129 microalgae, 339–362 minerals, 243–254 bioavailability, 253–254 fish bone, 249–251 seafood, 247–254 seaweed, 251–253 nutraceuticals, 405–425 macromolecules, 429–460 omega-3 fatty acid sources, 16–19, 168–174 processing of carotene from, 223–227 chitin, 186–192 chitin oligosaccharides, 204–206 chitosan, 192–204 commercial products, 213–214 enzyme degradation, 207–209 global availability, 185–186 glucosamine, 209 minerals in, 249–254 overview, 185 seaweed, 261 shark cartilage and chondroitin sulfate, 209–213 protein nutritive value, 109–116 physiological functions, 125–127 safety hazards algal toxins, 476–483 amnesic shellfish poisoning, 481 bacterial pathogens, 470–475 biotoxins, 481–483 chemical hazards, 484–486 ciguatera poisoning, 480 classification, 468–469 control technology, 487–494 diarrhetic shellfish poisoning, 480 food-borne hazards, 467–468 fungi, 484 histamine poisoning, 475 insects, 476 microbiological hazards, 470–475 overview, 467 paralytic shellfish poisoning, 479 parasites, 483–484 puffer fish poisoning, 480 seafood allergy, 486–487 secondary metabolites, 372–373 vitamins, 239–243 processing effects, 243 seafood, 241–243 seaweed, 243

9/5/2008 3:14:56 PM

Index Marketing and trade guidelines food fortification and supplementation, marine nutraceuticals, 419–420 functional foods, 14–16 Matrix design, nutraceuticals, 434–437 Medical applications agar, 300–301 alginates, 304–305, 306–308 chitosan, 202 marine products, 17–19 spirulina, 355–357 Membrane technology, macromolecule carriers, 455–459 drug delivery systems, 455–457 polysaccharide scaffolds, 457–459 Microalgae aquaculture applications, 358–359 bioactive compounds, 344–349 anticancer compounds, 346 antihypertensive peptides, 347 antimicrobial compounds, 347 antioxidant compounds, 346–347 antiviral compounds, 345–346 biological functions, 355–357 carotenoids and other pigments, 343 chlorella, 352–353 cultivation, 349–352 carotenoids, 351–352 lipids, 350–351 dunaliella, 357–358 habitat and resources, 43–45 lipids, 341–342 marine bacteria, 359–362 microbial biotechnology, 361–362 nutritional benefits, 354–355 nutritional composition, 340–341 overview, 339 polysaccharides, 344 polyunsaturated fatty acids, 360–361 single cell proteins, 344 spirulina, 353–357 sterols and hydrocarbons, 343–344 toxins in, 476–483 vitamins, 344 Microbiological hazards marine products, 470 removal technologies, 490 Microcystins, algal toxins, 477–483 Microemulsion, macromolecule carriers and biofilm encapsulation, 433–434 Microorganisms, drugs and pharmaceuticals from, 387–388 Mince/mince-based products, fish protein, 63–65 Minerals food fortification and supplementation, 421 fortified foods, 411–412 intake recommendations, 247

CRC_52632_Index.indd 521

521 marine sources of, 243–254 bioavailability, 253–254 fish bone, 249–251 seafood, 247–254 seaweed, 251–253 in seaweed, 266–267 Molluscs, chitin content, 186–187 Multicomponent edible films, basic properties, 449–450 Mussel-derived scavenging peptide (MRSP), 121–122 Myofibrillar protein films, basic properties, 442–445 Myosin, fish muscle structure and protein content, 52–54

N Nanotechnology, macromolecule carriers and edible films, 454–455 Neovastat, shark sources for, 376 Nonprotein nitrogen (NPN), raw muscle protein content, 52–54 Nutraceuticals consumer selection patterns, 5–6 defined, 3–4 food fortification and enrichment carotenoids, 412 chondroitin sulfate, 422 commercial status, 422–425 dietary guidelines, 405–406 fortification and enrichment, 407–409 iodine, 409–410 marine oil-fortified products, 415–417 marketing campaigns, 419–420 minerals, 411–412, 421–422 omega-3 fatty acids, 415 probiotics, 413–414 process optimization, 417–418 proteins and amino acids, 412–413, 420–421 PUFA-fortified products, 418–419 regulatory status, 419 supplementation, 406–407 vitamins, 410–411 in food products, 4–5 macromolecule carriers and biofilms active packaging, 451–453 alginate, 448–449 biodegradable and edible films, 439–442 carrageenan, 448 casting methods, 453 chitosan, 446–448 collagen and elastin, 444–445 commercial status, 459–460 delivery systems, 430 encapsulation, 431–434, 437–439

9/5/2008 3:14:56 PM

522 Nutraceuticals (Contd.) food coatings and edible films, 442–453 gelatin, 445–446 hydrogels and membranes, 455–459 marine proteins, 442–444 matrix delivery design, 430–431 multicomponent films, 449–450 nanotechnology, 454–455 novel delivery systems, 434–437 overview, 429–430 polysaccharides, 446, 451–455, 457–459 marine sources of, 16–19 omega-3 fatty acids and, 162–163 shark cartilage and chondroitin sulfate, 209–213 Nutrients bioavailability, 11–12 carotenoids, 222 antioxidant properties, 228–231 chitosan, 200–202 marine nutraceuticals, 405–407 microalgae, 340–341 reference standards, 12 seafood proteins, 104–116 fermented fishery products, 114–116 fish protein hydrolyzates, 111–114 powders, 110–111 processing effects on, 107–109 quality evaluation methods, 104–106 supplements, 109–116 in seaweed, 262–267 spirulina, 354–355 Nutrigenomics, 8–9

Index nutritional value, 155–163, 165–174 asthma, 160 blood pressure, 156 bone health, 161–162 brain function, 160–161 cancer, 158 cardiovascular disease, 156–158 cellular processes, 156 consumption recommendations, 166–168 current intake levels, 165–166 diabetes, 161 health benefits, 155–163 indication, 165 marine fish sources, 168–173 mode of action, 163–164 nonfish sources of, 173–174 nutraceutical potentials, 162–163 obesity, 160 pregnancy and infancy, 158–160 seafood content, 147–150 vitamin content of, 241–243 Osteoporosis in humans, marine-based minerals and, 254–255 marine-base medicines for, 379–380 polyunsaturated fatty acids and, 161–162 Overexploitation, fish and seafood stocks, 27–29 Oxidation, fatty acids, 150–155 antioxidants, 152–154 marine fishery product lipids, 154–155 Oxygen radical absorbance capacity (ORAC), carotenoids, 229–231

P O Obesity control bioactive peptides, 120 dietary fiber and, 269–271 polyunsaturated fatty acids and, 160 Okadaic acid (OA), diarrhetic shellfish poisoning, 478–483 Omega-3 fatty acids commercial status, 422–425 microencapsulated compounds, 459–460 food fortification and supplementation, 415–420 marketing campaigns, 419–420 processing optimization, 417–418 regulatory guidelines, 419 therapeutic benefits, 418–419 marine sources of, 16–19, 168–174 extraction, 168–170 properties, 170–173 nonfish sources, 173–174

CRC_52632_Index.indd 522

Packaging technology, marine biodegradable and edible films, 439–442 active packaging, 451–453 Paralytic shellfish poison carrageenan, 324 toxin structure and properties, 479 Paramyosin, fish muscle structure and protein content, 53–54 Parasites as marine product safety hazard, 483–484 removal technologies, 489 Peroxy radicals, fatty acids, 151–155 Pharmaceuticals. See Drugs and pharmaceuticals Phenolic compounds, seaweed bioactivity and, 274–275 Phosphorus, marine sources of, 244–254 Phycobilipoproteins, microalgae, 343 Pigments, microalgae, 343 Plant defense, carrageenans, 324 Platelet aggregation, seaweed, 277

9/5/2008 3:14:57 PM

Index Polysaccharides laminarin, 330 marine biodegradable and edible films active packaging technology, 452–453 basic properties, 446–447 classification, 440 hydrogel drug delivery systems, 457–459 nanotechnology, 454–455 microalgae, 344 in seaweed, 267–272 hyperoxaluria, 278–279 seaweed hydrocolloids, 297–299 agar, 299–301 Polyunsaturated fatty acids (PUFA). See also Fatty acids carotenoids and, 231–232 food fortification and supplementation, 415–420 marketing campaigns, 419–420 process optimization, 417–418 regulatory status, 419 therapeutic benefits, 418–419 macromolecule carriers and biofilms, 437–438 marine bacteria, 360–361 marine lipids, 144–150 fatty acids, 145–146 seafood lipid profile, 146–150 microalgae, 341 cultivation, 350–352 lipids, 341–342 nutritional value, 155–168 indication, 165 lipids, 155–163, 165–174 mechanism of action, 163–164 omega-3 fatty acids, 155–163, 165–174 asthma, 160 blood pressure, 156 bone health, 161–162 brain function, 160–161 cancer, 158 cardiovascular disease, 156–158 cellular processes, 156 consumption recommendations, 166–168 current intake levels, 165–166 diabetes, 161 health benefits, 155–163 indication, 165 marine fish sources, 168–173 mode of action, 163–164 nonfish sources of, 173–174 nutraceutical potentials, 162–163 obesity, 160 pregnancy and infancy, 158–160 overview, 143–144 oxidation, 150–155 antioxidants, 152–154 marine fishery product lipids, 154–155

CRC_52632_Index.indd 523

523 in seaweed, 266 squalene, 174–178 commercial aspects, 175–178 functionality, 174–175 vitamin E and, 242–243 Postharvest effects, seafood proteins, 62–63 Potassium carrageenan, 314–316 marine sources of, 244–254 Poultry feed, carotenoids in, 233–234 Pregnancy, polyunsaturated fatty acids and, 158–160 Probiotic bacteria fortified foods, 414–415 nutritive value, 114–116 Processing of marine products carotene from, 223–227 chitin, 186–192 chitin oligosaccharides, 204–206 chitosan, 192–204 commercial products, 213–214 enzyme degradation, 207–209 global availability, 185–186 glucosamine, 209 irradiation technology, 490–493 marine drug development, 394–398 marine nutraceuticals, food fortification and supplementation, 417–420 minerals in, 249–254 overview, 185 protein nutritive value, effects on, 107–109 seaweed, 261 shark cartilage and chondroitin sulfate, 209–213 vitamins, 243 Proteases, marine sources, 129–130 Protein efficiency ratio (PER), seafood proteins, 104–116 Protein hydrolysates, fortified foods, 412–413 Protein-protein binding, seafood protein gelation, 57–58 Proteins, seafood animal feed and, 81–83 antifreeze proteins, 130–131 bioactive peptides, 116–127 angiotensin I-converting enzyme inhibition, 122–124 antibacterial activity, 120–121 antioxidant activity, 121–122 antithrombin activity, 125 calcitonin, 125 calcium-binding activity, 120 functional roles, 119–120 human immunodeficiency virus-1 protease inhibition, 125 immunostimulant activity, 124 isolation, 117–119 obesity control, 120 physiological functions, 125–127

9/5/2008 3:14:57 PM

524 Proteins, seafood (Contd.) blood proteins, 75–76 chemical modification, 60 chitin, 186–189 coatings and edible films, 442–444 collagen, 83–85 commercial aspects, 93–94, 133–135 connective tissue proteins, 83–91 diet requirements, 51, 103–104 emulsifying capacity, 55–56 enzymatic modifications, 60–61 fermented fish products, 80–81 fish meat mince/mince-based products, 63–65 fish protein concentrate, 70 foaming capacity, 56 food flavorings, 92 food fortification and supplementation, 420–421 fortified foods, 412–413 functional properties, 54–58, 61–63 gelatin, 85–91 gelation, 56–57 hydrolyzates, 76–80 jellyfish sources, 92–93 krill as source of, 75 marine enzymes, 127–130 applications, 129–130 isolation, 127–129 microalgae, 344 aquaculture, 358–359 modification of, 59–60 nutritive value, 104–116 fermented fishery products, 114–116 fish protein hydrolyzates, 111–114 powders, 110–111 processing effects on, 107–109 quality evaluation methods, 104–106 supplements, 109–116 overview, 51 physical functions in food, 58–61 postharvest changes in, 62–63 in raw fish muscle, 52–54 rheological properties, 57–58 roe from eggs, 93 sea cucumber, 92 seaweed, 264–265 solubility, 55 squid as source of, 75 supplements from, 63–70 surimi and surimi-based products, 65–70 thermostable dispersions and powders, 71–74 Proximate composition analysis, seaweed, 262–267 lipids, 266 polysaccharides, 267 proteins and amino acids, 264–265 vitamins and minerals, 266–267

CRC_52632_Index.indd 524

Index Puffer fish poisoning, toxin structure and properties, 480 Pyridoxine, in seafood, 242–243

Q Quality controls, protein nutritional quality, 104–106

R Reactive nitrogen species (RNS), health effects, 152–154 Reactive oxygen species (ROS) carotenoids, 228–231 coral reef and corals, 362–363 fatty acids, 151–155 health effects, 152–154 seaweed bioactivity and, 273–275 Red tide, as safety hazards, 476–479 Regulatory guidelines chitins and chitosans, 208–209 food enrichment marine nutraceuticals, 408–409 omega-3 fatty acids, 419 functional foods, 12–14 seaweed production, 289 Relative protein value (RPV), fish protein, 106–107 Restocking, coral reef restoration, 42 Retinal equivalents carotenoids, 222 vitamin A, 239–240 Rheology carrageenan, 314–316 seafood protein gelation, 57–58 Riboflavin, marine sources, 239–243 Roe, marine sources of, 93 Roe herring, fish protein hydrolyzates, 77–80

S Safety hazards, marine products algal toxins, 476–483 amnesic shellfish poisoning, 481 bacterial pathogens, 470–475 biotoxins, 481–483, 487–488 chemical hazards, 484–486 ciguatera poisoning, 480 classification, 468–469 control technology, 487–494 diarrhetic shellfish poisoning, 480 food-borne hazards, 467–468 fungi, 484 histamine poisoning, 475 insects, 476

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Index microbiological hazards, 470–475 overview, 467 paralytic shellfish poisoning, 479 parasites, 483–484 puffer fish poisoning, 480 seafood allergy, 486–487 Safety regulations chitins and chitosans, 208–209 functional foods, 12–14 Salinosporamide A, 377–378 Scaffold materials alginates, 306–308 marine biodegradable and edible films, 457–459 Sea cucumbers consumer interest in, 92 drugs and pharmaceuticals from, 392–393 as food source, 30–31 Seafood allergies, 486–487 removal technologies, 488 bacterial pathogens in, 470–475 bioactive peptides, 116–127 angiotensin I-converting enzyme inhibition, 122–124 antibacterial activity, 120–121 antioxidant activity, 121–122 antithrombin activity, 125 calcitoning, 125 calcium-binding activity, 120 functional roles, 119–120 human immunodeficiency virus-1 protease inhibition, 125 immunostimulant activity, 124 isolation, 117–119 obesity control, 120 physiological functions, 125–127 chemical hazards in, 484–486 cold-adapted enzymes, 131–133 consumption guidelines, 38–39 flavor classifications for, 35–36 habitat and resources, 25–39 Antarctic krill and deep-sea fauna, 29–30 consumption trends, 34–39 demand and concerns, 27–29 food security and, 32–33 global consumption patterns, 33–34 landing, 26–27 mariculture, 31–32 new species identification, 31 sea cucumbers, 30–31 underutilized fisheries, 29 irradiation technology, 490–493 marine enzymes, 127–130 applications, 129–130 isolation, 127–129 mineral content of, 247–254 per capita fish supply, 33–34

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525 processing wastes chitin, 186–192 chitin oligosaccharides, 204–206 chitosan, 192–204 commercial products, 213–214 enzyme degradation, 207–209 global availability, 185–186 glucosamine, 209 overview, 185 shark cartilage and chondroitin sulfate, 209–213 protease treatment of, 78–79 proteins animal feed and, 81–83 antifreeze proteins, 130–131 blood proteins, 75–76 chemical modification, 60 collagen, 83–85 commercial aspects, 93–94, 133–135 connective tissue proteins, 83–91 as dietary component, 51 dietary requirements, 103–104 emulsifying capacity, 55–56 enzymatic modifications, 60–61 fermented fish products, 80–81 fish meat mince/mince-based products, 63–65 fish protein concentrate, 70 foaming capacity, 56 food flavorings, 92 functional properties, 54–58, 61–63 gelatin, 85–91 gelation, 56–57 hydrolyzates, 76–80 jellyfish sources, 92–93 krill as source of, 75 modification of, 59–60 nutritive value, 104–116 fermented fishery products, 114–116 fish protein hydrolyzates, 111–114 powders, 110–111 processing effects on, 107–109 quality evaluation methods, 104–106 supplements, 109–116 overview, 51 physical functions in food, 58–61 postharvest changes in, 62–63 in raw fish muscle, 52–54 rheological properties, 57–58 roe from eggs, 93 sea cucumber, 92 solubility, 55 squide as source of, 75 supplements from, 63–70 surimi and surimi-based products, 65–70 thermostable dispersions and powders, 71–74 vitamin content of, 241–243

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526 Seafood protein powders nutritive value, 110–111 thermostable dispersions, 71–75 Seaweed basic properties, 261 commercial status, 288–289, 330–331 as dietary fiber source, 268–280 antibacterial/antiviral activities, 275–277 antioxidants, 273–275 antitumor activity, 277–278 bioactive compounds, 273–280 definition, 268 dietary supplements, 272–273 enzyme inhibition, 279–280 food enrichment, 272 health benefits, 268–271 HIV inhibition, 279 hyperoxaluria, 278–279 platelet aggregation, 277 drugs and pharmaceuticals from, 388–390 farming of, 286–288 food fortification and supplementation, 421 habitat and resources, 42–43 hydrocolloids agar, 299–301 alginates, 301–308 carrageenan, 308–327 commercial status, 330–331 fucoidan, 327–329 functional properties, 297–299 laminarin, 330 identification, 262 industrial applications, 281–286 agriculture, 281–282 animal feed, 282–283 antifouling agents, 284 aquaculture, 283–284 future research on, 285–286 heavy metal biosorption, 285 lipids, 266 marine compounds from, 17–19 minerals in, 251–254, 266–267 nutritional composition, 263–264 polysaccharides, 267 processing, 261 proteins and amino acids, 264–265 proximate composition, 262–267 regulatory status, 289 vitamins in, 243, 266–267 Secondary metabolites, marine drugs and pharmaceuticals, 371–37 Semirefined carrageenan (SRC), properties, 311–313 Shark cartilage food fortification and supplementation, 422 structure and properties, 209–213 Shark compounds, anticancer agents, 376 Shark liver squalene, 174–175

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Index Shark oil, extraction, 169–170 Shellfish carotenoids in, 223–227 drugs and pharmaceuticals from, 391–392 parasites in, 483–484 toxins in, 479–483 amnesic shellfish poisoning, 481 ciguatera poisoning, 480 diarrhetic shellfish poisoning, 478–480 paralytic shellfish poisoning, 324, 479 puffer fish poisoning, 480 Shrimp carotenoids in, 223–227 fish protein, animal feed, 82–83 mineral content of, 247–254 Sodium, marine sources of, 244–254 Solubility alginates, 302–303 seafood protein, 55 Species identification and development, new marine products, 31 Spirulina, 353–357 biological applications, 355–357 fish feed, 358–359 nutritional benefits, 354–355 Sponges, drugs and pharmaceuticals from, 382–387 Spray drying, macromolecule carriers and biofilm encapsulation, 432 Squalene, 174–178 commercial aspects, 175–178 functionality of, 174–175 Squid, protein in, 75 Stabilizers, carrageenan, 321–322 Sterols, microalgae, 343–344 Superoxide anions, carotenoids, 228–231 Surimi/surimi-based products fish muscle structural protein gelation and, 72–73 protein content, 65–70 protein nutritive value, 108–109 Symbiotic associations coral reef and corals, 40 marine drugs and pharmaceuticals, 372

T Terpenes, drugs and pharmaceuticals from, 388–390 Textural modifications, carrageenan, 317–318 Thermostable dispersions and powders, fish proteins, 71–75 Thiamin (vitamin B1), marine sources, 239–243 Thiobarituric acid, carotenoid antioxidants, 229–230 Thiocoraline, 378

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Index Thraustochytrids cultivation, 351–352 microalgae, 342 Threadfin bream, protein composition and characteristics, 73–74 Total radical trapping antioxidant parameter (TRAP) assay, carotenoids, 229–231 Toxicology, carrageenan, 325–326 Toxins, marine algal toxins, 476–483 amnesic shellfish poisoning, 481 bacterial pathogens, 470–475 biotoxins, 481–483 chemical hazards, 484–486 ciguatera poisoning, 480 classification, 468–469 control technology, 487–494 diarrhetic shellfish poisoning, 480 drugs and pharmaceuticals from, 390–391 food-borne hazards, 467–468 fungi, 484 histamine poisoning, 475 insects, 476 microbiological hazards, 470–475 overview, 467 paralytic shellfish poisoning, 479 parasites, 483–484 puffer fish poisoning, 480 seafood allergy, 486–487 Transdermal drug delivery, marine macromolecule hydrogels, 455–457 Trolox equivalence antioxidant capacity (TEAC), carotenoid antioxidants, 229–230 Tuberculosis therapy, marine sources for, 379 Tuna intestine crude enzyme (TICE), minerals, 251–254 Tuna silage amino acid composition, 115–116 gelatin peptide composition, 119–120

527

V Value-added seafood products, consumer preferences for, 35–36k Vascular endothelial growth factor (VEGF), marine sources, 376–377 Viable but noncultivable (VBNC) bacterial pathogens, seafood safety and, 474–475 Vitamin A carotenoid activity, 230–231 marine sources, 239–243 Vitamin B12, in seafood, 242–243 Vitamin D bone health and, 254–255 marine sources, 240–243 Vitamin E, marine sources, 241–243 Vitamins fortified foods, 410–411 intake and utilization recommendations, 242–243 marine sources, 239–243 processing effects, 243 seafood, 241–243 seaweed, 243 microalgae, 344 in seaweed, 266–267

W Water treatment, chitosan applications, 203 Wound healing, alginates, 306–308

X Xanthophylls carotenoids, 221–222 dietary function of, 228–231

U Underutilized fisheries, species classified as, 29 United States, functional food regulations and standards, 12–14

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Z Zinc, marine sources of, 245–254

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