Probiotics, Prebiotics, and Synbiotics: Bioactive Foods in Health Promotion 9780128021897, 2572592592, 0128021896

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Probiotics, Prebiotics, and Synbiotics

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Probiotics, Prebiotics, and Synbiotics Bioactive Foods in Health Promotion

Edited by

Ronald Ross Watson

University of Arizona, Division of Health Promotion Sciences, Mel and Enid Zuckerman College of Public Health, and School of Medicine, Arizona Health Sciences Center, Tucson, AZ, USA

Victor R. Preedy

Department of Nutrition and Dietetics, Nutritional Sciences Division, School of Biomedical & Health Sciences, King’s College London, London, UK

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 125 London Wall, London, EC2Y 5AS, UK 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA 225 Wyman Street, Waltham, MA 02451, USA The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK © 2016 Elsevier Inc. All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including ­photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, ­methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-802189-7 For information on all Academic Press publications visit our website at http://store.elsevier.com/ Printed and bound in the United States of America Publisher: Nikki Levy Acquisition Editor: Andrea Topping Editorial Project Manager: Billie Jean Fernandez Production Project Manager: Caroline Johnson Designer: Ines Cruz

Contents Contributors xix Preface xxv Acknowledgments xxvii Biographies xxix

Part I Prebiotics in Health Promotion 1. Prebiotics and Probiotics: An Assessment of Their Safety and Health Benefits Arturo Anadón, María Rosa Martínez-Larrañaga, Irma Arés and María Aránzazu Martínez  1 Introduction 3   2 Prebiotic Concept 4   3 Use of Prebiotics 4 3.1 Use of Prebiotic as Medical Purposes 6 3.2 Prebiotic Sources 7 3.3 Prebiotics and Resistance to Gastrointestinal Infections 7   4 Evaluation of Prebiotic 8 4.1 AFCSF Product Specification/ Characteristics of the Prebiotic 9 4.2 Functionality 9 4.3 Qualifications 9 4.4 Safety 9   5 Probiotics Used in Food 10  6 Synbiotic 11   7 Safety Aspect of Probiotics 12 7.1 In�Vitro Studies 13 7.2 Animal Studies 13 7.3 Noninvasive Tests in Animal Models and Humans 13 7.4 Studies in Humans 13 7.5 Epidemiological and Postmarketing Surveillance 13   8 Prebiotic and Probiotic Efficacy Evidence 14 8.1 In�Vitro Evidence 14 8.2 Animal Models 14

8.3 Human Case Studies 8.4 Human Trials   9 Prebiotic and Probiotic Claims 9.1 European Union 9.2 The United States 10 Qualified Presumption of Safety (QPS) Concept of Microorganisms Used in Food 10.1 Taxonomic Status of Candidate Organisms for QPS Assessment 10.2 Purpose and Advantages of QPS 10.3 Requirements of QPS 11 Conclusion Acknowledgments References

15 15 15 15 17

19 19 20 20 20 21 21

2. Pre- and Probiotic Supplementation in Ruminant Livestock Production Mitchel Graham Stover, Ronald Ross Watson and Robert J. Collier 1 Introduction 25 2 The Ruminant 26 2.1 Anatomy and Physiology of the Ruminant Gastrointestinal Tract 26 2.2 The Ruminant Gut Microbiota 26 2.3 Microbial Stimulation of Regulatory Immune Mechanisms 27 2.4 Microbial Contributions to Ruminant Nutrition and Metabolism 28 3 Prebiotics 28 3.1 Prebiotic Substances 28 3.2 Prebiotics and Their Effect on Probiotic Supplementation and the Gut Microbiota 29 4 Probiotics 29 4.1 Probiotic Species and Blends 29 4.2 Probiotics: Ruminant Performance 30 4.3 Probiotics, Methanogens, and the Environment 33 5 Discussion and Conclusions 33 References 34

v

vi  Contents

3. Prebiotic Addition in Dairy Products: Processing and Health Benefits Elisa Carvalho de Morais   1   2  3   4   5   6   7   8   9 10 11

Functional Foods Prebiotic Ingredients β-Glucan Resistant Starch Inulin-Type Fructans The Role of Fructans in Plants Chemical Structure of Fructans Physicochemical Properties of Inulin Structural and Rheological Aspects Inulin as a Fat Replacer Effects of Process and Process Conditions 12 Oligofructose 13 Functional Effects of Prebiotics on the Health 14 Sensory Aspects 15 Prebiotics in Dairy Products 16 Perspectives References

37 37 38 38 38 38 39 39 39 40 40 40 41 41 42 43 43

4. Low-Lactose, Prebiotic-Enriched Milk Francisco J. Plou, Barbara Rodriguez-Colinas, Lucia Fernandez-Arrojo and Antonio O. Ballesteros 1 Human Milk Oligosaccharides 2 Galacto-Oligosaccharides (GOS) and Fructo-Oligosaccharides (FOS) in Dairy Products 3 Enzymatic Synthesis of GOS 4 In Situ Formation of GOS in Milk 5 GOS Formation in Milk with β-Galactosidase from B. circulans 6 GOS Formation in Milk with β-Galactosidase from K. lactis 7 Effect of Temperature on GOS Formation in Milk 8 Proposed Method to Obtain Low-Lactose, Milk-Enriched in GOS References

47

47 48 50 50

54 55 55

Nina Kirmiz and David A. Mills Introduction Intestinal Microbiota in Breast-Fed Infants Human Milk Composition and Complexity Antimicrobial Activities in Human Milk

61 61 62 63 65 66 66 67 67 68 68

6. Probiotics and Prebiotics for Promoting Health: Through Gut Microbiota Manoj Kumar, Ravinder Nagpal, Rajkumar Hemalatha, Hariom Yadav and Francesco Marotta  1 Introduction   2 Human Gut Microbiota: Complexities, Diversities, Functionalities   3 Gut Microbiota Balance in the Triangle of Nutrition, Health, and Disease   4 Factors Influencing the Gut Microbiota   5 Modulation of Gut Microbiota Composition   6 Probiotics: Foundation and Definition   7 Health Benefits of Probiotics   8 Probiotics’� Effects on Intestinal Microbiota and Environment  9 Prebiotics 10 Future Prospects and Expectations References

75 75 76 77 77 77 78 80 81 82 82

51

5. Intestinal Microbiota in Breast-Fed Infants: Insights into Infant-Associated Bifidobacteria and Human Milk Glycans  1   2   3   4

  5 Human Milk Glycans   6 HMO Structures and Properties   7 Structure-Function Relationships of HMOs   8 Bifidobacterial Strategies of HMO Consumption   9 Human Milk Glycoproteins and Glycolipids 10 Consumption of Human Milk Glycoconjugates by Bifidobacteria 11 Bifidobacteria and Health Benefits to the Infant 12 Infant Formula 13 Conclusions Acknowledgments References

59 59 60 60

7. Prebiotics in Human Milk and in Infant Formulas Jose M. Moreno Villares 1 Introduction 2 Development of the Immune System in Infants 3 Breast Milk and Defense Against Infections and Allergic Manifestations 4 What Are Prebiotics? 5 Human Milk Oligosaccharides 6 Prebiotics in Infant Formulas 7 Side Effects 8 Regulation of the Addition of Prebiotics to Infant Formulas

87 88 88 90 90 92 96 96

Contents   vii

9 Conclusions References

96 97

8. Prebiotics and Probiotics in Infant Nutrition Antonio Alberto Zuppa, Giovanni Alighieri, Antonio Scorrano and Piero Catenazzi  1 Introduction 101   2 Development and Physiology of the Gastrointestinal Ecosystem 101  3 Prebiotics 104 3.1 Definition 104 3.2 Characteristics 105   4 Human Milk Oligosaccharides 105   5 Nonhuman Milk Oligosaccharides 106 5.1 Oligosaccharides from Animal Milks 106 5.2 Nonmilk Oligosaccharides 106 5.3 Mechanisms of Action 107 5.4 Side Effects 108  6 Probiotics 108 6.1 Definition 108 6.2 Characteristics 109 6.3 Mechanisms of Action 110 6.4 Side Effects 111  7 Symbiotics 112   8 Use of Prebiotics in Pediatrics 112 8.1 Nonmilk Oligosaccharides and Prebiotic Effects of Breast Milk 112 8.2 Nonmilk Oligosaccharides and the Prevention of Infections and Allergies 113 8.3 Nonmilk Oligosaccharides and Other Effects 113   9 Use of Probiotics in Pediatrics 114 10 Acute Diarrhea 114 10.1 Antibiotic-Associated Diarrhea 115 10.2 Necrotizing Enterocolitis 115 10.3 Allergy 116 11 Other Pediatric Uses 117 11.1 Constipation 117 11.2 Inflammatory Bowel Disease 118 11.3 Irritable Bowel Syndrome 119 11.4 Helicobacter pylori Infection 119 11.5 Lactose Intolerance 119 11.6 Respiratory Tract Infections 120 11.7 Urinary Tract Infections 120 11.8 Obesity and Diabetes 121 11.9 Prebiotics and Probiotics in Infant Formulas 121 12 Conclusion 122 References 122

  9. Synthesis of Prebiotic GalactoOligosaccharides: Science and Technology Ali Osman 1 Introduction 135 2 Galacto-Oligosaccharides (GOS): Chemical Synthesis vs. Biocatalysis 135 3 Synthesis of GOS Using Galactosyltransferases 136 4 Synthesis of GOS Using β-Galactosidases 136 4.1 Mechanism of Catalysis by β-Galactosidases 136 4.2 Hydrolysis vs. Transgalactosylation During Lactose Hydrolysis by β-Galactosidases 136 4.3 Factors Affecting GOS Synthesis Using β-Galactosidases 138 4.4 Degree of Polymerization and Glycosidic Linkages in GOS Mixtures 141 5 Types of Biocatalysts Used in GOS Synthesis 142 5.1 Whole Cell Biocatalysts 142 5.2 Free β-Galactosidases 143 5.3 Recombinant β-Galactosidases 143 6 Improving the GOS Synthesis Process 145 6.1 Immobilization of β-Galactosidases 145 6.2 Protein Engineering 148 6.3 Reaction Medium Engineering 148 7 Future Developments 149 References 149

10. Prebiotics as Protectants of Lactic Acid Bacteria N. Romano, E. Tymczyszyn, P. Mobili and A. Gomez-Zavaglia 1 Introduction 2 Physical Chemistry of the Preservation of Lactic Acid Bacteria and Probiotics 3 Use of Prebiotics as Protectants of Starters 4 Prebiotics as Probiotic Protectants in Food Matrices 5 Prebiotics as Probiotic Protectants in the Gastrointestinal Tract 6 Conclusions References

155 156 157 159 160 160 160

11. Prebiotic Agave Fructans and Immune Aspects L. Moreno-Vilet, R.M. Camacho-Ruiz and D.P. Portales-Pérez 1 Chapter Points 2 Introduction

165 165

viii  Contents

3 Agave Plant: Origin and the Role of Fructans 4 Chemical Structures of Agave Fructans 5 Overview of the Immune System 5.1 Innate Immunity 5.2 Acquired Immunity 5.3 Gut-Associated Lymphoid Tissue 6 Mechanism of Prebiotics 7 Health Implication of Agave Fructans: In�Vivo and In�Vitro Studies 7.1 Prebiotic Effect 7.2 Obesity, Blood Lipids, and Cholesterol 7.3 Toxicological Studies 7.4 Immunological Studies 7.5 Cancer 8 Discussion of Immune Aspects of Agave Fructans 9 Conclusions Acknowledgments References

166 166 168 168 168 169 169 170 170 173 174 174 175 175 177 178 178

Rok Orel and Lea Vodušek Reberšak 181 183 184 185 186 188 189

13. Structural Characteristics and Prebiotic Effects of Lotus Seed Resistant Starch Baodong Zheng, Yi Zhang and Hongliang Zeng 1 Introduction 2 Structural Characteristics of LRS3 2.1 Particle Morphology 2.2 Structure Properties 3 Prebiotic Effects of LRS3 3.1 Proliferation Rate of Bifidobacteria 3.2 Growth Curve of Bifidobacteria 3.3 Production of Short-Chain Fatty Acids 3.4 Tolerance Tests 4 Mechanisms Underlying the Prebiotic Effects of LRS3 4.1 Effect of Particle Morphology on the Prebiotic Effects of LRS3 4.2 Effect of Crystalline Pattern on the Prebiotic Effects of LRS3

207 208 208 208 208 208 209 209

Part II Probiotics in Food 14. Probiotic Lactobacillus Strains from Traditional Iranian Cheeses Seyed Mohammad Bagher Hashemi

12. Prebiotics Use in Children 1 Introduction 2 Prebiotics and Short-Chain Fatty Acids 3 Clinical Effects in Children 3.1 Gastrointestinal Transit and Resorption of Nutrients 3.2 Protection Against Infections and Treatment of Acute Diarrhea 3.3 Prevention of Allergy References

4.3 Effect of Double Helix Structure on the Prebiotic Effects of LRS3 4.4 Biological Mechanisms 5 Future Trends 5.1 Combination of Single Mechanism and Multiple Mechanisms 5.2 Proliferation Signaling Pathways of Probiotic Bacteria of LRS3 5.3 Food Application of LRS3 6 Conclusions References

195 196 196 197 203 203 203 204 205 207 207 207

 1 Introduction   2 Isolation and Identification of Candidate Probiotic Strains from Traditional Iranian Cheeses   3 Acid and Bile Resistance   4 Autoaggregation and Coaggregation   5 Cell Surface Hydrophobicity and Epithelial Cell Adhesion   6 Antimicrobial Activity   7 Antibiotic Susceptibility   8 Cholesterol Removal and Effect on the Fatty Acid Profiles   9 Carbon Source Utilization 10 Antioxidant Activity 11 Encapsulation 12 Conclusion References

215

215 216 218 218 219 220 221 221 223 223 223 223

15. Safety of Probiotic Bacteria Mohammad Abdollahi, Amir Hossein Abdolghaffari, Maziar Gooshe and Farnaz Ghasemi-Niri 1 Introduction 227 2 Pharmacology of Probiotics 228 3 Uses of Probiotics 228 4 Pathogenicity and Infectivity of Probiotic Bacteria 228 4.1 Registering Probiotic Products: Key Initiatives in Probiotic Safety Concerns 229 4.2 Evaluation of the Safety of Probiotics 230 4.3 Human Studies 232

Contents ix

5 Conclusion References

237 237

16. Stressors and Food Environment: Toward Strategies to Improve Robustness and Stress Tolerance in Probiotics Vittorio Capozzi, Mattia Pia Arena, Pasquale Russo, Giuseppe Spano and Daniela Fiocco 1 Introduction 245 2 Food Manufacturing Process and Associated Stress 245 3 Stress Response in Probiotic Bacteria 247 3.1 Acid Stress 247 3.2 Heat Stress 248 3.3 Food-Associated Stressors 249 4 Strategies to Improve Robustness and Stress Tolerance in Probiotic 249 4.1 Encapsulation 249 4.2 Carrier Media and Protective Agents 250 4.3 Prebiotic Fibers 250 4.4 Addition of Protective Agents to Counteract Acid Challenge 250 4.5 Stress Adaptation and Cross-Protection 251 4.6 Selection of Resistant Strains 251 4.7 Recombinant DNA Technology 252 5 Concluding Remarks 253 References 253

17. Effect of Food Composition on Probiotic Bacteria Viability E. Sendra, M.E. Sayas-Barberá, J. Fernández-López and J.A. Pérez-Alvarez 1 Introduction 257 2 Effect of Food Processing on Probiotic Bacteria and Prebiotic Ingredients 259 2.1 Effect of Food Processing on Probiotic Bacteria 259 2.2 Effect of Food Processing on Prebiotics 262 3 Sensory Aspects of Probiotic, Prebiotic, and Symbiotic Foods 262 4 Food Formulation Effects on Probiotic Viability 263 4.1 Effects of Food Ingredients on Probiotic Viability 263 4.2 Effects of Food Formulation on Probiotic Activity 265 5 Conclusions and Future Prospect 266 References 266

18. Probiotics and Antibiotic Use Arthur C. Ouwehand and Julia Tennilä 1 Introduction 2 Probiotics and Microbiota Maintenance 3 Probiotics and Reduction of Antibiotic Side Effects 4 Future of Probiotics in AAD: Claiming the Effect 5 Health Economics of Probiotics in AAD 6 Conclusions References

271 272 273 275 275 275 276

19. Multistrain Probiotics: The Present Forward the Future Valentina Giacchi, Pietro Sciacca and Pasqua Betta 1 Introduction 2 Definition 3 Pharmacokinetics 4 Mechanisms of Action 4.1 Direct Effects 4.2 Indirect Effects 5 Single- and Multistrain Probiotics 6 Probiotics and Microbiota 7 Safety 8 Use of Multistrain Probiotics in Clinical Practice 8.1 Gastrointestinal Diseases 8.2 Probiotics and Urinary Tract Diseases 8.3 Probiotics and Atopic Diseases 9 Conclusions References

279 280 280 280 280 281 283 288 290 290 290 292 293 294 294

20. Production of Probiotic Cultures and Their Incorporation into Foods Edward R. Farnworth and Claude P. Champagne 1 Introduction 2 Production of Probiotic Cultures for Foods or Food Supplements 3 Ensuring Delivery of Viable Cultures in Foods and Supplements 3.1 Delivering as Food Supplements 3.2 Delivering by Processed Foods 4 Addition of Probiotics to Foods Ensuring Efficacy 4.1 Strain Selection 4.2 Effective Dose 4.3 Effect of Food Matrix 4.4 Using Encapsulation 4.5 Simulated GIT Conditions

303 303 305 305 306 309 309 309 310 310 312

x  Contents

5 Concept of Probioactive 5.1 Probioactives from the Food Matrix 5.2 Probioactives from Bacterial Metabolism 5.3 Protection of Probioactives 6 Conclusion References

312 313 313 314 314 314

21. Probiotics and Other Microbial Manipulations in Fish Feeds: Prospective Update of Health Benefits F.J. Gatesoupe 1 Introduction 2 Intestinal Microbiome in Fish 3 Probiotics in Fish 4 Prebiotics and Other Dietary Manipulations 5 Relevance of Fish as Model Species 6 Conclusion References

319 319 321 323 324 324 324

22. Current and Future Applications of Bacterial Extracellular Polysaccharides Adrian Pérez-Ramos, Montserrat Nácher-Vázquez, Sara Notararigo, Paloma López and Mª Luz Mohedano 1 Introduction 329 2 Classification of EPS 329 3 Current Applications of EPS in the Food Industry 332 3.1 Usage of EPS as Food Additives 332 3.2 In Situ Production of EPS 332 4 Bacterial EPS and Human Health 334 4.1 EPS as Prebiotics and Immunomodulators 335 4.2 Role of EPS Improving Bacterial Probiotic Properties 335 4.3 Potential Effect of EPS as Coadjuvant for Treatment of Diseases 336 5 Bacterial EPS and Animal Health 336 5.1 Animal Models to Study the Role of EPS in�Vivo 336 5.2 Unhealthy Effects of EPS in Animals 337 5.3 Beneficial Effects of EPS in Animals 337 6 Conclusions and Perspectives 338 Acknowledgments 338 References 338

23. Probiotic and Prebiotic Dairy Desserts Flávia C.A. Buriti, Raquel Bedani and Susana M.I. Saad 1 Introduction

345

2 Points to be Considered When Developing Probiotic and/or Prebiotic Dairy Desserts 346 2.1 Regulatory Requirements 346 2.2 Gel Formation and Prebiotic Gelling Properties 347 2.3 Preparation of Probiotic Strains for Incorporation into Refrigerated Dairy Desserts 348 3 Probiotic Desserts 349 3.1 General Effects of the Food Matrix on Physicochemical Characteristics and Probiotic Viability 349 3.2 Interactions Among Probiotic Microorganisms During Storage 351 3.3 Protective Effect of Food Ingredients on Probiotic Bacteria 351 4 Probiotic and Prebiotic Dairy Desserts 352 4.1 Influence of Probiotic Cultures and Prebiotic Ingredients on Flavor, Texture, and Acceptance 352 4.2 Inulin as Fat Substitute in Low-Fat Milk-Based Desserts 354 5 Concluding Remarks 356 References 356

24. Lactobacillus paracasei-Enriched Vegetables Containing Health Promoting Molecules P. Lavermicocca, M. Dekker, F. Russo, F. Valerio, D. Di Venere and A. Sisto 1 Introduction 361 1.1 Probiotic Bacteria and Beneficial Effects 361 1.2 Lactobacillus paracasei as Probiotic 361 1.3 Probiotic L. paracasei and Vegetables 362 2 L. paracasei-Enriched Cabbage as Source of Health-Promoting Phytochemicals and Carrier of Probiotic Cells 363 3 L. paracasei-Enriched Artichokes as a Symbiotic 365 3.1 The Artichoke as a Source of Bioactive Compounds 365 3.2 Probiotic Artichokes and GI Function 365 References 367

25. Probiotics from the Olive Microbiota Anthoula A. Argyri, Efstathios Z. Panagou and Chrysoula C. Tassou 1 Introduction 2 Assessment of the Probiotic Potential of Microorganisms from Olive Microbiota

371 372

Contents xi

3 Use of Probiotic Strains in the Production of Probiotic Table Olives 3.1 Fermentation—Use of Starters 3.2 Table Olives Inoculated with Non-Olive Origin Probiotic LAB 3.3 Use of Olive Origin Probiotic LAB in Table Olive Production and Storage References

378 378 379 381 385

26. Kimchi (Korean Fermented Vegetables) as a Probiotic Food Kun-Young Park and Ji-Kang Jeong 1 Introduction 391 2 Preparation and Fermentation of Kimchi 392 2.1 Kimchi Preparation and Recipe 392 2.2 Kimchi Fermentation and LAB 393 3 Various Health Benefits of Kimchi 394 3.1 Antimutagenic and Anticancer Effects 394 3.2 Antioxidative and Antiaging Effects 395 3.3 Antiobesity Effects 397 3.4 Serum Cholesterol and Lipid-Lowering Effects 398 3.5 Other Health Benefits 398 4 Salt and NO3 Content in Kimchi 399 5 Probiotic and Functional Properties of Kimchi LAB 400 5.1 Probiotic Function of Kimchi LAB 400 5.2 Antioxidative Effects 400 5.3 Antimicrobial Activity 401 5.4 Antimutagenic and Anticancer Effects 401 5.5 Immune-Regulation Effects 402 5.6 Antiiflammatory and Antiallergic Effects 402 5.7 Antiobesity, Cholesterol, and Lipid-Lowering Effects 404 6 Conclusion 404 References 404

27. Probiotics as Potential Adsorbent of Aflatoxin S. Mohd Redzwan, Rosita Jamaluddin, Farah N. Ahmad and Ying-Jye Lim 1 Probiotics 2 Binding of Probiotics to Food Carcinogens and Mutagens 3 Aflatoxin and Historical Background 4 Aflatoxin Metabolites 5 Physical Binding of Aflatoxin to the Bacterial Cell Wall 6 In�Vitro Experiments and Animal Studies of Probiotics as Potential Aflatoxin Adsorbent

409 409 410 410 410

411

7 Human Clinical Trials: A Way Forward to Study the Efficacy of�Probiotic Bacteria as Potential Adsorbent of Aflatoxin 8 Conclusion References

416 416 417

Part III Synbiotics: Production, Application, and Health Promotion 28. β-Glucans and Synbiotic Foods Mattia Pia Arena, Pasquale Russo, Daniela Fiocco, Vittorio Capozzi and Giuseppe Spano 1 β-Glucans: Chemistry and Sources 2 Beneficial Influence of β-Glucans on Human Health 3 Network of Human Health Promoting 4 Microbial β-Glucans Fermentation: A Metabolic Overview 5 β-Glucans for Synbiotic Foods Production 6 Concluding Remarks Acknowledgments References

423 425 426 426 427 430 430 430

29. Probiotics and Synbiotics in Lactating Mothers Leila Nikniaz, Reza Mahdavi, Zeinab Nikniaz and Hossein Nikniaz 1 Introduction 2 Effects of Probiotic or Synbiotic Supple­ mentation on Breast Milk Immune Factors 3 Effects of Probiotic or Synbiotic Supplementation on Total Antioxidant Capacity of Human Breast Milk 4 The Effect of Probiotic or Synbiotic Supplementation on the Breast Milk Microbiological Composition 5 The Traditional Hypothesis: "A Contamination" 6 The Revolutionary Hypothesis: "Active Migration" 7 The Effect of Probiotic or Synbiotic Supplementation on Maternal Nutritional Status and Infants’� Health References

435 435

437

439 439 440

441 442

30. Synbiotics and the Immune System Felicita Jirillo, Emilio Jirillo and Thea Magrone 1 Introduction 449 2 Interaction of Microbiota with Host Immunity 450

xii  Contents

3 Prebiotics, Probiotics, and Synbiotics 4 Synbiotics and Immune Response 5 Conclusion Acknowledgment References

451 452 455 455 455

1.1 Definition of Acute Gastroenteritis 1.2 Incidence and Disease Burden 1.3 Causes 2 Intestinal Microfloria and Mucosal Barrier 2.1 Intestinal Microflora 2.2 Mucosal Barrier 3 Treatment and Synbiotics 4 Conclusions References

459 461

35. Symbiotics, Probiotics, and Fiber Diet in Diverticular Disease

31. Synbiotics and Immunization Against H9N2 Avian Influenza Virus Seyedeh Leila Poorbaghi and Masood Sepehrimanesh 1 Avian Immune System 2 Avian Influenza 3 Association of Probiotics, Prebiotics and Synbiotics with Immunity 4 Immunity Against AIVs 5 Conclusion References

462 463 465 466

32. Probiotics, Prebiotics, Synbiotics, and Foodborne Illness Eleni Likotrafiti and Jonathan Rhoades 1 Introduction 2 Inhibitory Mechanisms of Probiotics Against Pathogenic Bacteria 3 Reduction of Human Enteric Pathogen Carriage by Food Animals 3.1 Mammals 3.2 Poultry 3.3 Aquatic Animals 3.4 Concluding Comments 4 Inhibition of Pathogens in Food Products Prior to Consumption 5 Pathogen Inactivation in the Gut 6 Concluding Remarks References

469 469 471 471 471 471 472 472 473 473 474

33. In�Vitro Screening and Evaluation of Synbiotics Maria Lena Skalkam, Maria Wiese, Dennis Sandris Nielsen and Gabriella van Zanten 1 Introduction 2 Screening of Synbiotic Combinations 3 Models of the Human Gastrointestinal Tract 4 Cell Assays 5 Future Perspectives References

477 477 479 480 482 483

488 488 489 490 494 495

Edith Lahner and Bruno Annibale 1 Introduction 2 Diverticular Disease—Definition and Epidemiology 3 Which Options to Treat Diverticular Disease? 4 Diverticular Disease and ProbioticsSymbiotics 5 Diverticular Disease and Dietary Fiber/ Prebiotics 6 Conclusion References

501 501 502 503 507 511 511

36. Gut Microbiota: Impact of Probiotics, Prebiotics, Synbiotics, Pharmabiotics, and Postbiotics on Human Health Saikiran Chaluvadi, Arland T. Hotchkiss, Jr. and Kit L. Yam 1 Introduction 515 2 Gut Microbiota 516 3 Evolving Field of Probiotics 517 3.1 What is More Important, Survival or Efficacy? Does Survival also Guarantee Efficacy? 517 3.2 Beneficial Commensals or Traditional Probiotics 520 3.3 Pharmabiotics and Postbiotics 521 4 Conclusions 521 References 522

37. Potential Benefits of Probiotics, Prebiotics, and Synbiotics on the Intestinal Microbiota of the Elderly Raquel Bedani, Susana Marta Isay Saad and Katia Sivieri

34. Synbiotics and Infantile Acute Gastroenteritis Zuhal Gundogdu 1 Introduction

487 487 488

487

1 Introduction 2 Elderly Population 3 The Gut Microbiota

525 525 526

Contents xiii

4 The Gut Microbiota and Aging 5 Probiotics, Prebiotics, and Synbiotics During Aging and Their Potential Beneficial Effects on Intestinal Microbiota 6 Concluding Remarks References

527

530 534 535

38. Synbiotics in Gastrointestinal Surgery Masahiko Yano, Masaaki Motoori, Keijiro Sugimura and Koji Tanaka 1 Prevention of Infectious Complications After GI Cancer Surgery 1.1 Upper GI Surgery 1.2 Hepatobiliary Surgery 1.3 Colorectal Cancer Surgery 2 Prevention of Colonic Carcinogenesis in Postcolectomy and Postpolypectomy 3 Prevention of Adjuvant Therapy-Related Toxicity 4 Conclusions References

539 539 541 542 545 545 546 546

39. Probiotics, Prebiotics, Synbiotics, and Other Strategies to Modulate the Gut Microbiota in Irritable Bowel Syndrome (IBS) Eamonn M.M. Quigley 1 Introduction 2 Novel Concept: Alterations in the Microbiota and Inflammation in the Pathophysiology of IBS 3 Treatment Strategies in IBS 3.1 Diet 3.2 Antibiotics 3.3 Probiotics, Prebiotics, and Synbiotics 4 Conclusion References

549

550 550 551 551 552 553 553

40. Gut Microbiota and IBS C. Bucci, A. Santonicola and P. Iovino 1 The Normal Microbiota: An Essential Factor for a Healthy Gut 2 Gut Microbiota in IBS: Harmful or Beneficial? 3 Is the Alteration of Gut Microflora Harmful? 4 Is the Alteration of Gut Microbiota Beneficial? References

557 558 558 561 563

41. Synbiotics: A New Strategy to Improve the Immune System from the Gut to Peripheral Sites Alberto Finamore, Ilaria Peluso and Mauro Serafini 1 Introduction 567 1.1 Gut Immunity, Microbiota and Cross Talk 568 1.2 Synbiotics and Allergy 568 1.3 Synbiotics and Obesity 569 1.4 Synbiotics, Gut Immunity and Inflammatory Diseases 570 1.5 Synbiotics, Mineral Absorption and Immune Function 571 2 Conclusions 572 References 572

42. Probiotics and Prebiotics for Prevention of Viral Respiratory Tract Infections Hamid Ahanchian and Seyed Ali Jafari 1 Introduction 1.1 Viral Infections and Asthma 1.2 Viral Infection in Infants 2 Mechanisms of Action 3 Clinical Trials 3.1 Children 3.2 Adults and the Elderly 4 Summary References

575 576 576 576 578 578 580 581 581

43. Synbiotics in the Intensive Care Unit Diya Mohammad and Lee E. Morrow  1 Introduction   2 The Rationale for Synbiotic Therapy in the ICU   3 Commonly Studied Synbiotic Preparations   4 Synbiotics in Severe Acute Pancreatitis   5 Positive SAP Trials   6 Negative SAP Trials   7 Synbiotics in Elective Surgery   8 Synbiotics in Liver Transplantation   9 Synbiotics in Esophageal Surgery 10 Synbiotics in Critically Ill Trauma Victims 11 Synbiotics to Prevent VAP 12 Positive VAP Trials 13 Negative VAP Studies

585 585 585 586 586 587 587 588 588 588 589 589 589

xiv  Contents

14 Synbiotics in Hepatic Disease 15 Synbiotics in Minimal Hepatic Encephalopathy 16 Synbiotics and Diarrheal Disorders 17 Conclusions References

590 590 590 590 591

44. Properties of Probiotic Bacteria: A Proteomic Approach Masood Sepehrimanesh 1 Introduction 2 Identification of Unknown Microorganisms According to Reference Proteome Map 3 Evaluation of Virulence Factors 4 Identification of Pathogenic from Nonpathogenic Microorganisms 5 Detection of Surface Proteins in Microbiota 6 Analysis of Secretome 7 Evaluation of Environmental Effects on the Growth and Functions of Microorganisms 8 Evaluation of Probiotic Bacteria 9 Conclusion References

593

595 596 597 598 598

O. Martinez-Augustin and F. Sánchez de Medina 1 Introduction 2 The Intestinal Mucosal Barrier Function 3 Innate Immune Response and Pattern Recognition Receptors as Regulators of MBF 4 Oligosaccharides in Human Milk 5 Nonprebiotic Effects of Prebiotics 5.1 NDOs and Intestinal Epithelial Cells 5.2 NDOs and Leukocytes 5.3 NDOs and Mucus Layer 5.4 NDOs and Microbial Adhesion 6 Conclusions References

619 619

623 623 624 624 627 628 628 629 630

Part IV Probiotics in Health 47. Probiotics and Physical Strength

599 600 600 603

45. Symbiotic Bacteria and Gut Epithelial Homeostasis Rheinallt M. Jones  1 Introduction   2 Sensing the Microbiota by the Intestinal Epithelium   3 Deliberate Generation of Physiological Levels of ROS Within Cells   4 ROS Signaling and Reactive Cysteines   5 Bacterial-Induced ROS Generation in the Intestinal Epithelium   6 Cell Signaling Pathways Activated by Bacterial-Induced ROS Generation   7 Bacterial-Induced Generation of ROS and Cell Motility   8 Lactobacilli-Induced ROS Generation and Epithelial Growth   9 Keap1/Nrf2/ARE Signaling and BacterialInduced Cytoprotection 10 Microbiota-Induced Cell Proliferation and Colorectal Cancer 11 Future Perspectives 12 Conclusions References

46. Nonprebiotic Actions of Prebiotics

605 606 606 607 607 608 609 609 610 611 612 613 613

Alok S. Tripathi and Shreesh J. Marathe 1 Introduction 635 2 Bone Strength 635 2.1 Bone Mass Density and Bone Mineral Content 636 2.2 Oxidative Stress and Bone Homeostasis 637 2.3 Anti-inflammatory Activity and Bone Volume Fraction 637 3 Protein 638 4 Muscle Wasting 638 References 638

48. Probiotics in Invasive Candidiasis Jacopo Colombo and Angela Arena 1 Candida 2 Pathogenesis of ICs 3 Prevention of ICs 4 Probiotics and ICs 5 Conclusion References

641 643 646 647 649 650

49. Probiotics and Usage in Bacterial Vaginosis Somayeh Ziyadi, Aziz Homayouni, Sakineh Mohammad-Alizadeh-Charandabi and Parvin Bastani 1 Bacterial Vaginosis 2 Probiotic and BV

655 655

Contents xv

2.1 Route of Administration 2.2 Administration Vehicles 2.3 Appropriate Strains for Treatment of BV 2.4 Appropriate Dose for Treatment of BV 2.5 Effect of Treatment Duration 3 Conclusion Acknowledgment References

656 656 657 657 657 657 658 658

50. Evidence and Rationale for Probiotics to Prevent Infections in the Elderly Patrick Alexander Wachholz, Paulo José Fortes Villas Boas and Vânia dos Santos Nunes 1 Background 2 Gastrointestinal Infections 3 Common Cold and Airways Infections 4 Genitourinary Infections 5 Final Considerations References

661 662 663 664 665 665

51. Probiotics Usage in Childhood Helicobacter pylori Infection Caterina Anania, Camilla Celani, Claudio Chiesa and Lucia Pacifico 1 Introduction 669 2 Mechanisms of Action of Probiotics on H. pylori 670 3 Probiotics for the Treatment of H. pylori Infection 671 3.1 Probiotics in Association with Antibiotics for the Treatment of H. pylori 671 3.2 Utilization of Probiotics Alone for Eradication or Prevention of H. pylori 674 4 Probiotics and Antibiotic-Associated Gastrointestinal Side Effects During H. pylori Eradication Therapy 676 5 Conclusions 678 References 678

52. Lipoic Acid Function and Its Safety in Multiple Sclerosis Amirreza Azimi and Mohammad Khalili  1  2  3  4   5   6   7

Introduction De Novo Synthesis of LA α-LA and Functions α-LA as a Cofactor Antioxidant Effect Antiinflammatory Effect Metal Chelating

683 683 683 684 684 684 684

  8 Plasma Pharmacokinetics and Safety of α-LA   9 Multiple Sclerosis 9.1 Definition and Pathobiology 9.2 Treatment 9.3 Disease-Modifying Treatment 10 Plasma Pharmacokinetics and Safety of α-LA in Experimental and Clinical MS 11 Lipoic Acid and Multiple Sclerosis References

685 686 686 686 686 687 687 688

53. Probiotics and Health: What Publication Rate on Probiotics, Prebiotics, and Synbiotics Implies? Behjat Shokrvash, Aziz Homayouni, Laleh Payahoo, Mohammad-Hossein Biglu, Elnaz Vaghef Mehrabany and Mohammad Asghari Jafarabadi 1 Introduction 2 Materials and Methods 3 Results 4 Discussion Acknowledgments References

691 692 692 697 698 698

54. The Cholesterol-Lowering Effects of Probiotic Bacteria on Lipid Metabolism Selcen Babaoğlu Aydaş and Belma Aslim 1 Introduction 699 2 Cholesterol Metabolism 700 2.1 Bile Acid Metabolism 701 3 Hypercholesterolemia and Atherosclerosis 701 4 The Relationship of the Composition of Intestinal Microbial Flora with Lipid Metabolism 703 5 Cholesterol-Lowering Mechanisms of Probiotics 705 5.1 Bile Salts Tolerance and Deconjugation of Bile Acids by BSH of Probiotics 706 5.2 Co-precipitation of Cholesterol with Deconjugated Bile 708 5.3 Cholesterol Binding to Cell Walls and Assimilation of Cholesterol by Probiotics 708 5.4 Reduction in the Host Absorption of Cholesterol 709 5.5 Inhibition of Hepatic Cholesterol Synthesis by SCFAs of Probiotic Origin 709 5.6 Conversion of Cholesterol into Coprostanol 709 5.7 Some Other Mechanisms Proposed for Cholesterol-Lowering Effects of Probiotics 710

xvi  Contents

6 In�Vivo Studies 6.1 Functional Food with Probiotics and Appropriate Probiotic Dosage in Cholesterol Reduction 7 Obesity and Probiotics 8 Conclusions References

710

713 714 715 716

55. The Use of Prebiotics, Probiotics, and Synbiotics in the Critically Ill

  8 Vaginal Probiotic Administration 746 8.1 Probiotic Selection 746 8.2 Drying of Probiotics 747 8.3 Dosage Forms for Vaginal Administration 747   9 Oral versus Vaginal Administration of Probiotics 749 10 Conclusion 749 Acknowledgments 749 References 750

Eva H. Clark and Jayasimha N. Murthy  1 Introduction   2 Immunology of Probiotics in the Critically Ill   3 Type of Probiotic Therapy Matters in the ICU   4 The Amount of Probiotic Supplied   5 The Method of Administration   6 Time Probiotic is Administered and Duration of Therapy   7 ICU Patients  8 Diarrhea   9 Ventilator-Associated Pneumonia 10 Severe Acute Pancreatitis 11 Abdominal Surgery Patients 12 Liver Transplantation Patients 13 Trauma Patients 14 Critically Ill Children and Probiotics References

723 724 725 726 726 727 727 728 729 731 732 732 733 733 734

56. Gynecological Health and Probiotics Sandra Borges, Joana Barbosa and Paula Teixeira  1 Introduction   2 Vaginal Microbiota   3 Urogenital Infections 3.1 Bacterial Vaginosis 3.2 Vulvovaginal Candidiasis 3.3 Urinary Tract Infection   4 Antibiotic as Therapeutic of Urogenital Infections   5 Probiotics as Alternatives or Complements to Conventional Treatments   6 Antagonistic Properties of Probiotics 6.1 Adherence 6.2 Lactic Acid 6.3 Hydrogen Peroxide 6.4 Bacteriocins 6.5 Biosurfactants   7 Probiotics and Prebiotics

741 741 742 742 742 742 743

743 744 744 745 745 745 746 746

Part V Probiotics and Chronic Diseases 57. Probiotics in Inflammatory Bowel Diseases and Cancer Prevention Jean Guy LeBlanc and Alejandra de Moreno de LeBlanc 1 Introduction 755 2 Probiotics and Inflammatory Bowel Diseases 755 2.1 Mechanisms of Action of LAB Against IBD 755 3 Probiotics in Cancer Prevention 761 3.1 Probiotics and Fermented Products in Colon Cancer Prevention and Treatment 761 3.2 Effects in the Prevention and/or Treatment of Nonintestinal Tumors 763 4 Conclusions 765 References 766

58. Resistant Starch as a Bioactive Compound in Colorectal Cancer Prevention Amir Amini, Leyla Khalili, Ata K. Keshtiban and Aziz Homayouni 1 Introduction 1.1 Colorectal Cancer 1.2 Preventive Methods 1.3 Application of Prebiotics 1.4 Treatment Methods 2 Resistant Starch 2.1 RS Types 2.2 RS Health Advantageous 3 Epidemiological Studies 4 Conclusion Acknowledgment References

773 773 774 774 775 775 776 776 777 777 778 778

Contents xvii

59. Probiotics in Cancer Prevention, Updating the Evidence

61. Probiotics Usage in Heart Disease and Psychiatry

Davood Maleki, Aziz Homayouni, Leila Khalili and Babak Golkhalkhali

Fatemeh Ranjbar, Fariborz Akbarzadeh and Aziz Homayouni

1 Introduction 781 2 Probiotics 782 3 Cancer 783 3.1 Epidemiology 783 3.2 Etiology 783 3.3 Risk Factors 783 3.4 Treatment 784 4 Probiotic and Cancer 784 4.1 Probiotics and Anticancer Drug Metabolism 784 4.2 Animal Studies 784 4.3 Human Studies 785 5 Appropriate Probiotic Strains for Use in Cancer Therapy 785 6 Effective Dosage of Probiotics for Cancer Therapy 786 7 Duration of Probiotic Therapy in Patients With Cancer 786 8 Mechanisms by Which Probiotic Bacteria May Inhibit Cancer 788 9 Conclusion 788 Acknowledgments 788 References 789

1 Introduction 2 Probiotics 3 Psychobiotics 4 Mechanisms of Action 4.1 Probiotics and CHD 4.2 Psychobiotics and Depression 5 Conclusions and Future Trends Acknowledgment References

60. Cardiovascular Health and Disease Prevention: Association with Foodborne Pathogens and Potential Benefits of Probiotics Irene Hanning, Jody Lingbeck and Steven C. Ricke 1 Introduction 793 2 Direct Affects 793 3 Indirect Affects 794 3.1 Probiotics, Cardiac Health, and Obesity 794 3.2 Probiotics, Cardiac Health, and Hypertension 795 3.3 Probiotics, Cardiac Health, and Hypercholesterolemia 796 3.4 Probiotics, Cardiac Health, and Foodborne Pathogens 796 4 Emerging Issues 801 5 Conclusions 802 Acknowledgment 802 References 802

807 807 808 808 808 809 810 810 810

62. Intestinal Microbiota and Susceptibility to Viral Infections: Role of Probiotics Vicente Monedero and Jesús Rodríguez-Díaz 1 Introduction 813 2 Gastrointestinal Viruses 813 3 Microbiota of the Gastrointestinal Tract and Virus Susceptibility 815 4 Suggested Antagonistic Mechanisms of Probiotics Against Intestinal Viral Pathogens 816 5 Rotaviruses, Noroviruses, and the Intestinal Microbiota 818 6 Efficacy of Probiotics Against Enteric Viruses in In�Vitro Models, Animal, and Clinical Trials 820 6.1 Efficacy of Probiotics: In�Vitro Data 820 6.2 Animal Models 821 6.3 Clinical Trials 823 7 Conclusions 823 References 824

63. Probiotics and Usage in Urinary Tract Infection Somayeh Ziyadi, Parvin Bastani, Aziz Homayouni, Sakineh Mohammad-AlizadehCharandabi and Fatemeh Mallah 1 Urinary Tract Infection 2 Probiotics 3 Probiotic and Urinary Tract Infection 4 Conclusion Acknowledgment References

827 828 828 829 829 829

xviii  Contents

64. Probiotics: Immunomodulatory Properties in Allergy and Eczema Lorenzo Drago and Marco Toscano 1 Introduction 2 Probiotics: An Innovative Therapeutic Strategy References

831 832 835

65. Prebiotics and Probiotics for the Prevention and Treatment of Food Allergy Shu-E Soh and Lynette Pei-Chi Shek 1 Introduction 2 Immunomodulatory Effects of Probiotics and Prebiotics 3 Effects of Probiotics and Prebiotics in Animal Models of Food Allergy 4 Prevention of Food Allergy 4.1 Probiotics and Synbiotics 4.2 Prebiotics 5 Treatment of Food Allergy 6 Implications for Future Research 7 Conclusion References

839 839 840 841 841 841 844 844 846 846

66. Probiotics and Prebiotics for the Prevention or Treatment of Allergic Asthma Marek Ruszczyński and Wojciech Feleszko 1 Introduction 2 Probiotics: Mechanisms of Action 3 Clinical Effects of Probiotics and Prebiotics in the Treatment of Allergic Asthma 3.1 Prebiotics 3.2 Probiotics 4 Role of Probiotics and Prebiotics in Preventing Allergic Asthma 4.1 Prebiotics 4.2 Probiotics 5 Final Remarks 5.1 Prebiotics 5.2 Different Strains 5.3 Adverse Effects 6 Conclusion References

849 849 851 851 852 852 858 858 858 858 862 862 862 862

67. Amelioration of Helicobacter pylori-Induced PUD by Probiotic Lactic Acid Bacteria Baljinder Kaur and Gaganjot Kaur   1 Peptic Ulcer Disease 1.1 Causes of Peptic Ulcer Disease 1.2 Symptoms of PUD

865 865 865

 2 H. pylori Infection and its Association with Gastric Mucosa 866   3 Association of H. pylori with PUD 866   4 Disease Prevalence 867 4.1 International Scenario 867 4.2 Indian Scenario 868   5 Genome Organization of H. pylori 868   6 Role of Host Cell Factors in H. pylori Infection and Progression of PUD 875   7 Role of Virulence Factors in H. pylori Pathogenesis 876 7.1 Role of cagA 876 7.2 Role of vacA 879 7.3 Role of iceA 879 7.4 Role of nikR 879 7.5 Role of rocF 879 7.6 Role of BabA 879 7.7 Role of SabA 880 7.8 Role of OipA 880 7.9 Role of AlpA/B 880 7.10 Role of DupA 880   8 Role of Host Tumor Suppressors: p53 and RUNX3 880   9 Genetic Predisposition to PUD 881 10 Mode of Transmission of H. pylori Infection 882 11 Diagnosis of H. pylori Infection 882 12 Treatment and Preventive Measures 882 13 Drug Resistance Phenotype of H. pylori 883 14 Bioactive Compounds Showing Anti-H. pylori Activity 885 15 Mechanism of H. pylori Inhibition by Probiotic Lactic Acid Bacteria 886 15.1 Competitive Exclusion 886 15.2 Alteration in Polyamine Metabolism 887 15.3 Production of Bacteriocins 887 15.4 Production of Polyphenols 887 15.5 Host Cell Immune Modulation 888 16 Conclusions 889 References 889

Index 897

Contributors Numbers in parenthesis indicate the pages on which the authors’ contributions begin.

Amir Hossein Abdolghaffari (227), Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, and Tehran University of Medical Sciences, International Campus (TUMS-IC), Tehran, Iran Mohammad Abdollahi (227), Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran Hamid Ahanchian (575), Queensland Children's Medical Research Institute, The University of Queensland, Brisbane, Queensland, Australia, and Department of Allergy and Immunology, Mashhad University of Medical Sciences, Mashhad, Iran Farah N. Ahmad (409), Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia Fariborz Akbarzadeh (807), Clinical Cardiovascular Research Center, Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran

Irma Arés (3), Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain Anthoula A. Argyri (371), Hellenic Agricultural Organisation DEMETER, Institute of Technology of Agricultural Products, Lycovrissi, Attica, Greece Belma Aslim (699), Department of Biology, Faculty of Science, Gazi University, Ankara, Turkey Selcen Babaoğlu Aydaş (699), Gazi University Vocational School of Health Services, Gölbaşı-Ankara, Turkey Amirreza Azimi (683), MS Research Center, Neuroscience Institute, Tehran University of Medical Sciences, Tehran, Iran Antonio O. Ballesteros (47), Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain Joana Barbosa (741), CBQF-Centro de Biotecnologia e Química Fina—Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/ Porto, Porto, Portugal

Giovanni Alighieri (101), Division of Neonatology, Catholic University of the Sacred Heart, Rome, Italy

Parvin Bastani (655, 827), Department of Obstetrics and Gynecology, Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Amir Amini (773), Department of Food Science and Technology, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran

Raquel Bedani (345, 525), Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil

Arturo Anadón (3), Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain

Pasqua Betta (279), NICU, AOU Policlinico-Vittorio Emanuele, Catania, Italy

Caterina Anania (669), Department of Pediatrics and Child Neuropsychiatry, Sapienza University of Rome, Rome, Italy Bruno Annibale (501), Department of Medicine, Surgery and Translational Medicine, Sant’Andrea Hospital, Sapienza University of Rome, Rome, Italy Mattia Pia Arena (245, 423), Department of Agriculture, Food and Environment Sciences, University of Foggia, Foggia, Italy Angela Arena (641), Department of Anesthesia, Intensive Care and Palliative Care, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy

Mohammad-Hossein Biglu (691), Department of Medical Information Sciences, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran Sandra Borges (741), CBQF-Centro de Biotecnologia e Química Fina—Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/ Porto, Porto, Portugal C. Bucci (557), Department of Medicine and Surgery, University of Salerno, Salerno, Italy Flávia C.A. Buriti (345), Department of Pharmacy, Center of Biological and Health Sciences, State University of Paraíba, Campina Grande, Brazil xix

xx  Contributors

R.M. Camacho-Ruiz (165), Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico Vittorio Capozzi (245, 423), Department of Agriculture, Food and Environment Sciences, University of Foggia, and Promis Biotech Srl, Foggia, Italy Piero Catenazzi (101), Division of Neonatology, Catholic University of the Sacred Heart, Rome, Italy Camilla Celani (669), Department of Pediatrics and Child Neuropsychiatry, Sapienza University of Rome, Rome, Italy Saikiran Chaluvadi (515), Harris Tea Company, Moorestown, New Jersey; U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania, and Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA Claude P. Champagne (303), Food Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Quebec, Canada

Lucia Fernandez-Arrojo (47), Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain J. Fernández-López (257), Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche, Orihuela Alicante, Spain Alberto Finamore (567), Functional Food and Metabolic Stress Prevention Laboratory, Council for Agricultural research and Economics (CRA-NUT), Rome, Italy Daniela Fiocco (245, 423), Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy F.J. Gatesoupe (319), INRA, UR 1067, Nutrition Aquaculture et Génomique, Plouzané, France Farnaz Ghasemi-Niri (227), Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran Valentina Giacchi (279), Pediatric Department, AOU Policlinico-Vittorio Emanuele, Catania, Italy

Claudio Chiesa (669), Institute of Translational Pharmacology, National Research Council, Rome, Italy

Babak Golkhalkhali (781), Department of Surgery, University of Malaya, Kulalampur, Malaysia

Eva H. Clark (723), Baylor College of Medicine, Houston, Texas, USA

A. Gomez-Zavaglia (155), Center for Research and Development in Food Cryotechnology, CIDCA, CCT-La Plata, Argentina

Robert J. Collier (25), Department of Animal Sciences, School of Animal and Comparative Biomedical Sciences, Agricultural Research Center, University of Arizona, Tucson, Arizona, USA Jacopo Colombo (641), Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy Alejandra de Moreno de LeBlanc (755), Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucumán, Argentina Elisa Carvalho de Morais (37), Department of Food and Nutrition, School of Food Engineering, University of Campinas, Campinas, Brazil M. Dekker (361), Wageningen University, Food Quality and Design Group, Wageningen, The Netherlands D. Di Venere (361), Institute of Sciences of Food Production, National Research Council of Italy, Bari, Italy Lorenzo Drago (831), Laboratory of Technical Sciences for Laboratory Medicine, Department of Biomedical Sciences for Health, University of Milan, Milan, Italy Edward R. Farnworth (303), Food Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Quebec, Canada Wojciech Feleszko (849), Department of Pediatric Pneumology and Allergy, The Medical University of Warsaw, The Medical University Children's Hospital, Warszawa, Poland

Maziar Gooshe (227), Students’ Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran Zuhal Gundogdu (487), Child Health and Diseases Department, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey Irene Hanning (793), Food Science and Technology Department, University of Tennessee, Knoxville, Tennessee, USA Seyed Mohammad Bagher Hashemi (215), Food Science and Technology Department, College of Agriculture, Fasa University, Fasa, Iran Rajkumar Hemalatha (75), Microbiology and Immunology Division, National Institute of Nutrition, Hyderabad, India Aziz Homayouni (655, 691, 773, 781, 807, 827), Department of Food Science and Technology, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Arland T. Hotchkiss, Jr. (515), U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania, USA P. Iovino (557), Department of Medicine and Surgery, University of Salerno, Salerno, Italy

Contributors   xxi

Susana Marta Isay Saad (525), Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil Mohammad Asghari Jafarabadi (691), Department of Epidemiology, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran Seyed Ali Jafari (575), Department of Pediatrics Gastroenterology, Mashhad University of Medical Science, Mashhad, Iran Rosita Jamaluddin (409), Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia Ji-Kang Jeong (391), Department of Food Science and Nutrition, Kimchi Research Institute, Pusan National University, Busan, South Korea

Jean Guy LeBlanc (755), Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucumán, Argentina Eleni Likotrafiti (469), Department of Food Technology, Laboratory of Food Microbiology, A.T.E.I. of Thessaloniki, Thessaloniki, Greece Ying-Jye Lim (409), Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia Jody Lingbeck (793), SeaStar International, Fayetteville, Arkansas, USA Paloma López (329), Centro de Investigaciones Biológicas, Madrid, Spain Mª Luz Mohedano (329), Centro de Investigaciones Biológicas, Madrid, Spain

Felicita Jirillo (449), Department of Agroenviromental and Territorial Sciences, University of Bari, Bari, Italy

Thea Magrone (449), Department of Basic Medical Sciences, Neuroscience and Sensory Organs, University of Bari, Bari, Italy

Emilio Jirillo (449), Department of Basic Medical Sciences, Neuroscience and Sensory Organs, University of Bari, Bari, Italy

Reza Mahdavi (435), Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Rheinallt M. Jones (605), Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA Baljinder Kaur (865), Department of Biotechnology, Punjabi University, Patiala, Punjab, India Gaganjot Kaur (865), Department of Biotechnology, Punjabi University, Patiala, Punjab, India Ata K. Keshtiban (773), Department of Food Science and Technology, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Mohammad Khalili (683), Neuroscience Research Center, Tabriz University of Medical Science, Tabriz, Iran Leyla Khalili (773), Department of Nutrition, Faculty of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Leila Khalili (781), Department of Nutrition, Tabriz University of Medical Sciences, Tabriz, Iran Nina Kirmiz (59), Food Science & Technology, and Foods for Health Institute, University of California, Davis, California, USA

Davood Maleki (781), Hematology and Oncology Ward, Urmia University of Medical Sciences, Urmia, Iran Fatemeh Mallah (827), Department of Obstetrics and Gynecology, Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Shreesh J. Marathe (635), Geetadevi Khandelwal College of Pharmacy, Akola, Maharashtra, India Francesco Marotta (75), ReGenera Research Group for Aging Intervention, Milano, Italy María Aránzazu Martínez (3), Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain O. Martinez-Augustin (619), Department of Biochemistry and Molecular Biology II, School of Pharmacy, Granada, Spain María Rosa Martínez-Larrañaga (3), Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain

Manoj Kumar (75), Microbiology and Immunology Division, National Institute of Nutrition, Hyderabad, India

Elnaz Vaghef Mehrabany (691), Department of Nutrition, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran

Edith Lahner (501), Department of Medicine, Surgery and Translational Medicine, Sant’Andrea Hospital, Sapienza University of Rome, Rome, Italy

David A. Mills (59), Food Science & Technology; Foods for Health Institute, and Viticulture & Enology, University of California, Davis, California, USA

P. Lavermicocca (361), Institute of Sciences of Food Production, National Research Council of Italy, Bari, Italy

Mitchel Graham Stover (25), Department of Veterinary Science, School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona, USA

xxii  Contributors

P. Mobili (155), Center for Research and Development in Food Cryotechnology, CIDCA CCT-La Plata, Argentina

Vânia dos Santos Nunes (661), Internal Medicine Department, UNESP – Universidade Estadual Paulista, Botucatu, Brazil

Diya Mohammad (585), Division of Pulmonary and Critical Care Medicine, Creighton University Medical Center, Omaha, Nebraska, USA

Rok Orel (181), Department of Gaastroenterology, Hepatology and Nutrition, Children’s Hospital, University Medical Center Ljubljana; University of Ljubljana, and Institute for Probiotics and Functional Foods, Ljubljana, Slovenia

Sakineh Mohammad-Alizadeh-Charandabi (655, 827), Department of Midwifery, Faculty of Nursing and Midwifery, Tabriz University of Medical Sciences, Tabriz, Iran S. Mohd Redzwan (409), Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia Vicente Monedero (813), Institute of Agrochemistry and Food Technology (IATA-CSIC), Paterna, Spain Jose M. Moreno Villares (87), Servicio de Pediatría, Madrid, Spain L. Moreno-Vilet (165), Biotecnología Industrial, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Guadalajara, Jalisco, Mexico Lee E. Morrow (585), Division of Pulmonary and Critical Care Medicine, Nebraska-Western Iowa Veterans' Affairs Medical Center, Creighton University Medical Center, Omaha, Nebraska, USA Masaaki Motoori (539), Department of Gastroenterological Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Jayasimha N. Murthy (723), Baylor College of Medicine, Houston, Texas, USA Montserrat Nácher-Vázquez (329), Centro Investigaciones Biológicas, Madrid, Spain

de

Ravinder Nagpal (75), Division of Laboratories for Probiotics Research, Juntendo University Graduate School of Medicine, Tokyo, Japan Dennis Sandris Nielsen (477), Department of Food Science, University of Copenhagen, Copenhagen, Denmark Hossein Nikniaz (435), Young Researchers and Elite Club, Tabriz Branch, Islamic Azad University, Tabriz, Iran Leila Nikniaz (435), Tabriz Health Services Management Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Zeinab Nikniaz (435), Liver and Gastrointestinal Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Sara Notararigo (329), Centro de Investigaciones Biológicas, Madrid, Spain

Ali Osman (135), Arla Strategic Innovation Centre (ASIC), Arla Foods amba, Aarhus, Denmark Arthur C. Ouwehand (271), DuPont Nutrition & Health, Kantvik, Finland Lucia Pacifico (669), Department of Pediatrics and Child Neuropsychiatry, Sapienza University of Rome, Rome, Italy Efstathios Z. Panagou (371), Agricultural University of Athens, Department of Food Science and Human Nutrition, Athens, Greece Kun-Young Park (391), Department of Food Science and Nutrition, Kimchi Research Institute, Pusan National University, Busan, South Korea Laleh Payahoo (691), Department of Nutrition, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran Ilaria Peluso (567), Functional Food and Metabolic Stress Prevention Laboratory, Council for Agricultural Research and Economics (CRA-NUT), Rome, Italy J.A. Pérez-Alvarez (257), Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche, Orihuela Alicante, Spain Adrian Pérez-Ramos (329), Centro de Investigaciones Biológicas, Madrid, Spain Francisco J. Plou (47), Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain Seyedeh Leila Poorbaghi (459), Department of Avian Medicine, School of Veterinary Medicine, Shiraz University, Shiraz, Iran D.P. Portales-Pérez (165), Laboratorio de Inmunología, Biología Celular y Molecular, Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico Eamonn M.M. Quigley (549), Division of Gastroenterology and Hepatology, The Lynda K. and David M. Underwood Center for Digestive Disorders, Houston Methodist Hospital and Weill Cornell Medical College, Houston, Texas, USA Fatemeh Ranjbar (807), Research Center of Psychiatry and Behavioral Sciences, Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran

Contributors xxiii

Lea Vodušek Reberšak (181), Department of Gaastroenterology, Hepatology and Nutrition, Children’s Hospital, University Medical Center Ljubljana, Ljubljana, Slovenia Jonathan Rhoades (469), Department of Food Technology, Laboratory of Food Microbiology, A.T.E.I. of Thessaloniki, Thessaloniki, Greece Steven C. Ricke (793), Center for Food Safety and Food Science Department, University of Arkansas, Fayetteville, Arkansas, USA Barbara Rodriguez-Colinas (47), Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain Jesús Rodríguez-Díaz (813), Microbiology Department, School of Medicine, University of Valencia, Valencia, Spain N. Romano (155), Center for Research and Development in Food Cryotechnology, CIDCA, CCT-La Plata, Argentina Pasquale Russo (245, 423), Department of Agriculture, Food and Environment Sciences, University of Foggia, and Promis Biotech Srl, Foggia, Italy F. Russo (361), Laboratory of Nutritional Pathophysiology, I.R.C.C.S. “Saverio de Bellis,” National Institute of Digestive Diseases, Bari, Italy Marek Ruszczyński (849), Department of Pediatrics, The Medical University of Warsaw, The Medical University Children's Hospital, Warszawa, Poland Susana M.I. Saad (345), Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil F. Sánchez de Medina (619), Department of Pharmacology, School of Pharmacy, CIBERehd, University of Granada, Granada, Spain A. Santonicola (557), Department of Medicine and Surgery, University of Salerno, Salerno, Italy M.E. Sayas-Barberá (257), Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche, Orihuela Alicante, Spain Pietro Sciacca (279), Pediatric Department, AOU Policlinico-Vittorio Emanuele, Catania, Italy Antonio Scorrano (101), Division of Pediatrics, Neonatal Intensive Care Unit, Cardinale G. Panico Hospital, Tricase (Lecce), Italy E.

Sendra (257), Departamento de Tecnología Agroalimentaria, Universidad Miguel Hernández de Elche, Orihuela Alicante, Spain

Masood Sepehrimanesh (459, 593), Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Mauro Serafini (567), Functional Food and Metabolic Stress Prevention Laboratory, Council for Agricultural Research and Economics (CRA-NUT), Rome, Italy Lynette Pei-Chi Shek (839), Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Behjat Shokrvash (691), Department of Health Education & Promotion, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran A. Sisto (361), Institute of Sciences of Food Production, National Research Council of Italy, Bari, Italy Katia Sivieri (525), Department of Food and Nutrition, School of Pharmaceutical Sciences, São Paulo State University, Araraquara, Brazil Maria Lena Skalkam (477), Department of Food Science, University of Copenhagen, Copenhagen, Denmark Shu-E Soh (839), Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Giuseppe Spano (245, 423), Department of Agriculture, Food and Environment Sciences, University of Foggia, Foggia, Italy Keijiro Sugimura (539), Department of Gastroenterological Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Koji Tanaka (539), Department of Gastroenterological Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Chrysoula C. Tassou (371), Hellenic Agricultural Organisation DEMETER, Institute of Technology of Agricultural Products, Lycovrissi, Attica, Greece Paula Teixeira (741), CBQF-Centro de Biotecnologia e Química Fina—Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/ Porto, Porto, Portugal Julia Tennilä (271), DuPont Nutrition & Health, Kantvik, Finland Marco Toscano (831), Clinical Chemistry and Microbiology Laboratory, IRCCS Galeazzi Orthopaedic Institute, Milan, Italy Alok S. Tripathi (635), P. Wadhwani College of Pharmacy, Yavatmal, Maharashtra, India E.

Tymczyszyn (155), Laboratory for Molecular Microbiology, Department of Science and Technology, National University of Quilmes, Bernal, Argentina

F. Valerio (361), Institute of Sciences of Food Production, National Research Council of Italy, Bari, Italy Gabriella van Zanten (477), Department of Food Science, University of Copenhagen, Copenhagen, Denmark

xxiv  Contributors

Paulo José Fortes Villas Boas (661), Internal Medicine Department, UNESP – Universidade Estadual Paulista, Botucatu, Brazil Patrick Alexander Wachholz (661), Internal Medicine Department, UNESP – Universidade Estadual Paulista, Botucatu, Brazil Ronald Ross Watson (25), Mel and Enid Zuckerman College of Public Health, and School of Medicine, Arizona Health Sciences Center, University of Arizona, Tucson, Arizona, USA Maria Wiese (477), Department of Food Science, University of Copenhagen, Copenhagen, Denmark Hariom Yadav (75), Diabetes, Endocrinology and Obesity Branch, Clinical Research Center, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

Kit L. Yam (515), Department of Food Science, Rutgers University, New Brunswick, New Jersey, USA Masahiko Yano (539), Department of Gastroenterological Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan Hongliang Zeng (195), College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, China Yi Zhang (195), College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, China Baodong Zheng (195), College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, China Somayeh Ziyadi (655, 827), Department of Midwifery, Faculty of Nursing and Midwifery, Tabriz University of Medical Sciences, Tabriz, Iran Antonio Alberto Zuppa (101), Division of Neonatology, Catholic University of the Sacred Heart, Rome, Italy

Preface Humanity lives in an environment filled with bacteria and other microbes. Similarly people are colonized with bacteria, which have significant influences on health, digestion, absorption of nutrients, and wellness or disease.

PREBIOTICS IN HEALTH PROMOTION This book brings together experts working on the different aspects of supplementation with foods, prebiotics, to stimulate growth of some bacteria as well as bacterial preparations for health promotion and disease prevention. Their expertise and experience provide the most current knowledge to promote future research. Dietary habits and thus intestinal bacterial content can be altered. Therefore, the conclusions and recommendations from the various chapters provide a basis for change. Expert reviews define and support the actions of bacteria modified bioflavonoids and fibrous materials, as well as other materials that are part of dietary vegetables. As such, probiotic bacteria with healthpromoting activities may have biological activity. Topics in this section include safety and health benefits, livestock production, enriched milk, feed for infants as well as children, role in human milk, immune aspects, and structural characteristics.

PROBIOTICS IN FOODS Therefore, their role in food is a major emphasis, along with discussions of which agents may be the active components. The book’s overall goal is to provide the most current, concise, scientific appraisal of the efficacy of key foods and constituent bacteria in preventing disease and improving the quality of life. Topics in this section include safety, use in cheese, dairy desserts, on vegetables, olives, and kimchi, improving robustness and stress tolerance, effects of concurrent antibiotic use on probiotics, benefits in fish food, probiotic polysaccharides, and removal of aflatoxin.

SYNBIOTICS: PRODUCTION, APPLICATION, AND HEALTH PROMOTION This book reviews and often presents new hypotheses and conclusions about the effects of different bioactive

c­ omponents of probiotics to prevent disease and improve the health of various populations. Diet and nutrition are vital keys to controlling or promoting morbidity and mortality from chronic diseases. A frequent goal of nutrition is to reduce disease caused by pathogens as bacteria are frequently viewed as causes of disease and illness. However, many intestinal inhabitants are really critical to preventing colonization by disease-causing pathogens. Symbiotics maybe more effective and certainly could have different actions beyond nutrition, especially when they are modified by intestinal flora (synergism). Gnotobiotic animals without intestinal flora do not grow normally, showing the need for some intestinal bacteria. So what is the role of bacteria? How is it that some bacteria can cause disease, but others make us healthier? How do prebiotics and probiotics interact to improve biological activity? Topics in this section include β-glucans and synbiotic foods, in lactating mothers, on the immune system, anti-viral immunization, gastroenteritis, diverticular disease and foodborne illnesses, post-intestinal surgery, gut microbiota, Irritable Bowel Syndrome, in the intensive care unit, and nonprebiotic actions of prebiotics.

PROBIOTICS IN HEALTH Can bacteria be used to promote health or prevent ­disease? If so, how and which ones? Clearly, bacteria compete with each other for the ability to grow in the intestine, which ultimately influences which will secrete protective or ­ disease-causing mediators. As ever-increasing levels and types of antibiotics are used, not only are pathogens ­affected but beneficial gut flora are also changed and killed. How does one restore gut flora in a selective manner during and after antibiotic therapy for other pathogens? Topics in this section include Probiotics in invasive candidiasis, physical strength, bacterial vaginosis, prevent infections in the elderly, childhood Helicobacter pylori infection, multiple sclerosis disease, on lipid metabolism, in the critically ill, and gynecological health.

PROBIOTICS AND CHRONIC DISEASES Probiotics are dietary supplements, now a multibillion-dollar business that is built on little research data. For example, the U.S. Food and Drug Administration is pushing this industry, xxv

xxvi  Preface

with the support of Congress, to base its claims and products on scientific research. Because common dietary bacterial preparations are over-the-counter and readily available, this book is useful to laypeople who can apply it to modify their lifestyles, as well as to the growing ­nutrition, food ­science, and natural product community. This book ­focuses on the growing body of knowledge about the role of various intestinal bacteria in reducing disease. The multitude of biomolecules in dietary fruits and vegetables, prebiotics, play crucial roles in health maintenance and bacterial growth. They may, therefore, be more effective and certainly could have different actions beyond nutrition. Topics in this section include prevention and treatment of colorectal cancer, inflammatory bowel diseases, heart disease, psychiatry, susceptibility to viral infections, urinary tract infection, immunomodulatory properties, in food, allergy, asthma, and eczema, and amelioration of Helicobacter pylori.

This is especially true when they are modified by i­ntestinal flora (synergism). Bioavailability of important constituents of fruit and vegetables plays a key role in their effectiveness. Their roles in gastrointestinal disease, heart disease, and old age are defined. Each vegetable contains thousands of different biomolecules, some with the potential to promote health or retard disease and cancer. By use of probiotics, people can dramatically expand their exposure to protective chemicals and thus readily reduce their risk of multiple diseases. Specific foods, individual fruits or vegetables, and their by-products are biomedicines with expanded understanding and use. However, which bacteria and their metabolisms of biomolecules in vegetables or fruits best prevent disease or promote health? This book’s focus is on probiotics and their role in biomodulation of natural products to produce active agents from inactive molecules in dietary fruits and vegetables.

Acknowledgments The work of Dr. Watson’s editorial assistant, Bethany L. Stevens, in communicating with authors and working on the manuscripts was critical to the successful completion of the book. It is very much appreciated. The encouragement, advice, and support of Caroline Johnson, Carrie Bolger, and Billie Jean Fernandez at Elsevier in producing the book was very helpful. Support for Ms. Stevens’ and Dr. Watson’s editing was graciously provided by the Natural

Health Research Institute (www.naturalhealthresearch.org) and Southwest Scientific Editing & Consulting, LLC. The encouragement and support of Elwood Richard and Dr. Richard Sharpee was vital. Finally, the work of the librarian at the Arizona Health Science Library, Mari Stoddard, was vital and very helpful in identifying key researchers who participated in the book.

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Biographies Ronald Ross Watson

Victor R. Preedy

University of Arizona Health Sciences Center, Tucson, USA

King’s College London, School of Biomedical and Health Sciences, London, UK

Ronald Ross Watson, Ph.D., attended the University of Idaho but graduated from Brigham Young University in Provo, Utah, with a degree in chemistry in 1966. He earned his Ph.D. in biochemistry from Michigan State University in 1971. His postdoctoral schooling in nutrition and m ­ icrobiology was completed at the Harvard School of Public Health, where he gained 2 years of postdoctoral research experience in immunology and nutrition. From 1973 to 1974, Dr. Watson was assistant professor of immunology and performed research at the University of Mississippi Medical Center in Jackson. He was assistant professor of microbiology and immunology at the Indiana University Medical School from 1974 to 1978 and associate professor at Purdue University in the Department of Food and Nutrition from 1978 to 1982. In 1982, Dr. Watson joined the faculty at the University of Arizona Health Sciences Center in the Department of Family and Community Medicine of the School of Medicine. He is currently professor of health promotion sciences in the Mel and Enid Zuckerman Arizona College of Public Health. Dr. Watson has published over 450 articles in peer-reviewed journals and has an h-index of 25.

Victor R. Preedy, BSc Ph.D. DSc FIBiol FRIPH FRSH FRCPath, is professor of Nutritional Biochemistry in the Department of Nutrition and Dietetics, King’s College London and professor of Clinical Biochemistry in the Department of Clinical Biochemistry, King’s College London. He is also Director of the Genomics Centre, Kings College London. Professor Preedy gained his Ph.D. in 1981 and in 1992 he received his Membership of the Royal College of Pathologists, based on his published works. He was elected a Fellow of the Royal College of Pathologists in 2000. In 1993, he gained a D.Sc. degree for his outstanding contribution to protein metabolism. Professor Preedy was elected as a Fellow to the Royal Society for the Promotion of Health (2004) and The Royal Institute of Public Health (2004). In 2009, he was elected as a Fellow of the Royal Society for Public Health (RSPH). The RSPH is governed by Royal Charter and Her Majesty the Queen is its Patron. Dr. Preedy has published over 550 articles, which includes over 160 peer-reviewed manuscripts based on original research and 85 reviews and 30 books. He has an h-index of 25. His interests pertain to matters concerning Public Health and how this is influenced by nutrition, addictions, and other life style factors.

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Part I

Prebiotics in Health Promotion

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Chapter 1

Prebiotics and Probiotics: An Assessment of Their Safety and Health Benefits Arturo Anadón, María Rosa Martínez-Larrañaga, Irma Arés and María Aránzazu Martínez Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain

1 INTRODUCTION The intestinal microflora or microbiota is a large bacterial community that colonizes the gut, with a metabolic activity that affects the physiology and pathology of the host’s mucosal immune system. Intestinal bacteria are useful in promoting human health, but certain components of microflora, in genetically susceptible individuals, contribute to various pathological disorders, including Crohn’s disease and ulcerative colitis, which are the two main types of inflammatory bowel disease (IBD). These diseases are characterized by persistent mucosal inflammation at different levels of the gastrointestinal tract (GIT). Clinical and experimental observations indicate an imbalance in protective and harmful microflora components in these disorders. The intestinal tract performs many different functions. The functions of microbiota include (1) nutrition (fermentation of nondigestible substrates that results in production of short-chain fatty acids, absorption of ions, and production of amino acids and vitamins); (2) protection (the barrier effect that prevents invasion by alien microbes); and (3) trophic effects on the intestinal epithelium and immune system (development and homeostasis of local and systemic immunity) (Guarner, 2007). In addition to absorption and digestion, it is also the body’s largest organ of host defense. Part of the intestinal mucosal barrier function is formed by a common mucosal immune system, which provides communication between the different mucosal surfaces of the body. Our gut microbiota can be pictured as a microbial organ placed within a host organ. It is composed of different cell lineages with a capacity to communicate with one another and the host. The gut microbiome contain more than 100 times the number of genes in our genome and endows us with functional features that we have not had to evolve ourselves (Turnbaugh et al., 2007). The GIT of a newborn baby is sterile. Soon after birth, however, the GIT is colonized by numerous types of microorganisms. Colonization is complete after approximately 1 week, but the numbers and species of bacteria fluctuate markedly during the first months of life (Rautava et al., 2012). On the other hand, there are now numerous studies demonstrating differences in the composition of the gut microbiota between allergic and nonallergic individuals, as well as between infants living in countries with a high and a low prevalence of allergy and between healthy and allergic infants. There is a range of new prebiotics and probiotics emerging and their market in food is growing rapidly. The prebiotic and probiotic must be assessed for health benefits and safety before they can be introduced in food products. The functional foods containing prebiotic compounds and probiotic bacteria have a great potential for the agro-food industry, consumers, and public health. Probiotics and prebiotics are fundamental ingredients of fermented milks and yogurts, which account for the most important fraction of the overall market for functional food. They have become the cornerstone of food innovation in the past few years. For this reason, the present review intends to express the main health benefits of interest for prebiotics and probiotics, as well as the main requirements for their studies and assessments. There are certainly safety concerns for the consumer with regard to the selection and dosage of nondigestive substances, mainly carbohydrates, and their ability to be tolerated, and the selection of nonpathogenic bacteria strains. But there is consensus on the prebiotic metabolic substrates (e.g., digestibility, composition, dosage, and specificity of metabolization) and on the selection of bacterial strains (e.g., counts, survival of gastrointestinal passage, growth conditions, nonpathogenicity, nontoxinogenicity, stability, and identity) (Przyrembel, 2001; Anadón et al., 2014). The current European Union (EU) legislation covers substances with a physiological effect, such as prebiotic compounds and probiotic bacteria. Any claims proposed for these substances must not only be based on and substantiated by the generally accepted scientific data, but also informative and comprehensible to the consumers. The EU regulations will Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00001-0 © 2016 Elsevier Inc. All rights reserved.

3

4  PART | I  Prebiotics in Health Promotion

prohibit any claims referring to the prevention, treatment, or cure of a human disease for a food in contrast to that proposed by other countries, such as Canada and the United States (Sanders et al., 2005). One of the most difficult endeavors facing those in the field of prebiotics and probiotics is the substantiation of efficacy needed to support claims of health benefits. Prebiotic and probiotic foodstuffs with identifiable functions can be rightly considered as functional following the Consensus Document of the Scientific Concepts of Functional Foods in Europe (Anonymous, 1999). This document states that a food can be regarded as “functional” if it is satisfactorily demonstrated to affect beneficially one or more target functions in the body, beyond adequate nutritional effects, in a way that is relevant to either an improved state of health and well-being and/or a reduction of risk of disease. Functional foods must remain foods and they must demonstrate their effects in amounts that can normally be expected to be consumed in the diet; they are not pills or capsules, but part of a normal food pattern.

2  PREBIOTIC CONCEPT A prebiotic was defined by Gibson and Roberfroid (1995) as “a non-digestive food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health.” These authors revised this concept and proposed a new prebiotic definition as a “selectively fermented ingredient that allows specific changes; both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health” (Gibson et al., 2004; Roberfroid, 2007). The latest definition results in an equalization of “prebiotic” and “bifidogenic” and includes in the definition the prebiotic index (i.e., it gives the absolute increase of the fecal bifidobacteria concentration per gram of daily consumed prebiotics). According to this definition, candidate prebiotics must fulfill the following criteria that are to be proven by in vitro and in vivo tests: (1) nondigestibility (resistance to low pH gastric acid, enzymatic digestion, and intestinal absorption); (2) fermentation by the intestinal microbiotica; and (3) selective stimulation of growth and activity of intestinal bacteria (De Vrese and Scherezenmeir, 2008). Also, the prebiotics have been defined as “a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota” (FAO, 2007). This definition arose from observations that particular dietary fibers bring about a specific modulation of the gut microbiota, particularly increased numbers of bifidobacteria and/or lactobacilli cell counts, or a decrease in potential harmful bacteria is a sufficient criterion for health promotion. In regular terms, prebiotics are food for bacterial species and are considered beneficial for health and well-being. It is scientifically accepted that prebiotics are valuable dietary additions for modulating the growth and activity of specific bacterial species in the colon that are considered health supporting.

3  USE OF PREBIOTICS Although prebiotics and probiotics probably share common mechanisms of action (especially modulation of the endogenous flora), they differ in their composition and metabolism. The fate of prebiotics in the GIT is better known than that of probiotics. It is generally accepted that prebiotics have a selective effect on the microbiota that results in improved health of the host; therefore, a substance is considered prebiotic when it fulfills the following aspects: resistance to digestion, fermentation by the large intestinal microbiota, and a selective effect of the microbiota. Prebiotics should have selective effects on the microbiota. Prebiotics, like other low digestible carbohydrates, exert an osmotic effect in the GIT so long as they are not fermented; when they are fermented by the endogenous flora (i.e., at the place where they exhibit their prebiotic effect), they also increase intestinal gas production (Roberfoid and Slavin, 2000). The prebiotic, or rather bifidogenic, effects depend on the type and concentration of the prebiotic, and on the bifidobacteria concentration in the intestine of the host. No simple dose-effect relationship exists. Only carbohydrates such as inulin [β(2-1-)-fructans] and oligofructose (OF) [β(2-1-)fructans], (trans-)galacto-oligosaccharides (TOS or GOS) (galactose oligomers and some glucose/lactose/­galactose units), or lactulose, synthetic, nondigestible sugar, all of which are nondigestible but can be fermented by the intestinal flora, fulfill the criteria (De Vrese and Scherezenmeir, 2008). Inulin-type fructans are the best documented oligosaccharides for their effect on intestinal bifidobacteria and are considered important prebiotic substrates. The prebiotics usually employed and the candidate ones are indicated in Table 1.1. With the exception of inulin (a mixture of fructo oligo- and polysaccharides), the known prebiotics are mixtures of undigestible oligosaccharides (i.e., chains consisting of 3-10 carbohydrate monomers). Oligosaccharides are carbohydrates of low molecular weight with a degree of polymerization values (2 and 9). They exhibit properties typical of dietary fibers and are found in several vegetables as fructans (e.g., asparagus, onions, garlic, and leeks), as stachyose in soybeans, as well as in the human breast milk and cow’s milk. Oligosaccharides are readily water soluble and exhibit some sweetness, which decreases with increasing chain length. They also have water-binding and gelling properties; therefore, the putative use as a fat substitute increases with the number of hexose molecules and reticulation (Delzenne, 2003).

Prebiotics and Probiotics  Chapter | 1  5

TABLE 1.1  Common and Emergent Prebiotics Functional Food Type of oligosaccharides Recognized prebiotics

Fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), galacto-oligosaccharides (GOS)/ transgalactosylated oligosaccharides (GOS/TOS), inulin, isomalto-oligosaccharides, lactulose, pyrodextrins, soy-oligosaccharides (SOS)

Emergent prebiotics

Genti-oligosaccharides, Gluco-oligosaccharides, isomalto-oligosaccharides (IMO), lactosucrose, levans, pectic-oligosaccharides, resistant starch, sugar alcohols, xylo-oligosaccharides (XOS)

TABLE 1.2  Mechanisms of Prebiotics ●

Increased expression or change in the composition of short-chain fatty acids to colonocytes during fermentation of prebiotics carbohydrates

●

Increased fecal weight and a mild reduction in luminal colon pH

●

A more acidic pH and modulation of the intestinal flora, especially growth stimulation of carbohydrate-fermenting bacteria

●

Decreased concentration of putrefactive, toxic, mutagenic, or genotoxic substances and bacterial metabolites, as well as of secondary bile acids and cancer-promoting enzymes

●

The bifidobacteria and lactobacilli (increased by oligosaccharides) exhibit low β-glucuronidase and nitroreductase activity

●

Decreased nitrogenous end products and reductive enzymes

●

Production of butyric acid reinforces the regenerative of the intestinal epithelium (i.e., through its pro-apoptotic potency)

●

Increased expression of the binding proteins or active carriers associated with mineral absorption

●

Enhanced immunity and modulation of mucin production

The glycosidic bonds of oligosaccharides are resistant to hydrolysis by intestinal digestive enzymes and hence are poorly degraded in the upper regions of the GIT, thus reaching the colon intact where oligosaccharides serve as a fermentable substrate. The colonic microbes ferment the nondigestible oligosaccharides to produce short-chain fatty acids (e.g., acetic, propionic, and butyric acid), lactic acid, and gases (e.g., carbon dioxide, methane, and hydrogen). Finally, it is known that the ingestion of prebiotics can elevate indigenous bifidobacterium and lactobacillus levels in the colon. Because of fermentation in the large intestine, the ingestion of higher quantities of prebiotics may lead to flatulence, abdominal disorders, and diarrhea. High levels of oligosaccharides (i.e., >10 g/day) may produce intestinal discomfort and flatulence. Oligosaccharides have recently been recognized as components of dietary fiber because of their interesting physiological effects, which are similar to those of well-known “soluble” fibers (Flamm et al., 2001). The prebiotic carbohydrates are not digested by human enzymes but fermented by the flora of the large intestine. Thus, they increase biomass, feces weights, and feces frequency, and they have a positive effect on constipation and on the health of the mucosa of the large intestine (Cherbur, 2002; Nyman, 2002). The fermentation of oligosaccharides in the cecum-colon could contribute to the protection against colon cancer (De Vrese and Scherezenmeir, 2008; Delzenne, 2003). A summary of mechanisms are expressed in Table 1.2. A number of oligosaccharides have been assessed for their prebiotic potential. The dose and duration for nutrition purposes are as follows: inulin (8-40 g/day, 15-64 days); fructo-oligosaccharides (FOS) (4-12.5 g/day, 8-12 days); GOS (7.5-15 g/day, 7-21 days), soy-oligosaccharides (SOS) (10 g/day, 21 days); and lactulose (3-20 g/day, 14-28 days) (Conway, 2001). Overall, the dosage levels for most health benefits will range from 3 g/day for short-chain FOS to 8 g/day for mixed short- and long-chain inulin, although more may be safely consumed according to individual tolerance (Marteau and Flourie, 2001; Douglas and Sanders, 2008). Rao (1999) reviewed the dose in relation to the extent of the elevation of bifidobacteria and indicated than even the dose of saccharide ranged from 8 to 40 g/day, there was no correlation with the resultant elevation of bifidobacteria. In general, 10-20 g oligofructose (OF) or inulin, regardless of whether ingested in a liquid or solid meal, is considered to be without side effects. In a trial with 80 individuals, the ingested quantity, after which at least one of the tested symptoms (headache, belching, flatulence, bowel contractions, or liquid stools) had been observed, was

6  PART | I  Prebiotics in Health Promotion

between 31 and 41 g OF, corresponding to 0.04-0.06 g/kg body weight (De Vrese and Scherezenmeir, 2008). The consumption of 80 g/day of OF in one study gave 4 of 12 test subjects diarrhea (Clausen et al., 1998). Prebiotics can be also used as supplement and special food. Supplements may provide an easy way to boost prebiotic fiber consumption, giving consumers a clear, convenient, and fool-proof way to obtain a particular type of prebiotic and dose level. Clearly labeled probiotic supplements can be sprinkled directly on food; stirred into beverages; or taken as capsules, tablets, or chewables. Because the most commonly available prebiotics are water soluble and completely clear in water, they are easily incorporated into most foods and are undetectable. Special foods—such as sports drinks, weight-loss powders, ready-to-drink protein meal replacers, and nutrition bars—provide a popular way for people to obtain prebiotic fiber. These food items often contain FOS, some form of inulin, or resistant starch for their fiber content and prebiotic advantages, although there may be no prebiotic-associated label claims (Douglas and Sanders, 2008).

3.1  Use of Prebiotic as Medical Purposes Prebiotics are used for medical purposes—and frequently used in intestinal nutrition products. They are used with adult and pediatric patients presenting with a wide range of medical conditions, including diabetes, cancer, renal failure, pressure ulcers, metabolic stress, trauma, and immunosuppression (Ross Products Division, 2005). Prebiotic enrichment of these liquid products is used as a means to provide short-chain fatty acids to colonocytes via fermentation, to normalize and maintain bowel function, to improve colon integrity, and to build colonization resistance in a hospital setting. These characteristics make prebiotics appropriate for use in patients with antibiotic-associated diarrhea; various irritable bowel conditions, including colitis; and general bowel maintenance while receiving a formulated diet for medical nutrition therapy (Seidner et al., 2005). When used in appropriate amounts, the effect of prebiotic fiber may also lead to an alteration in nitrogen excretion that is advantageous to renal patients (Younes et al., 1995, 2001). Table 1.3 highlights the use indications of prebiotics. Intestinal bacteria are useful in promotion of human health, but certain components of microflora, in genetically susceptible individuals, contribute to various pathological disorders, including IBD. For this reason, the use of prebiotics in IBD,

TABLE 1.3  Use Indications for Prebiotics Prebiotics to be used

Possible mechanism

References

Alleviation of constipation

Lactulose, fructooligosaccharides, galactooligosaccharides

Osmotic effect and modulation of indigenous microflora

Gibson et al. (2004)

Treatment of hepatic encephalopathy

Lactulose, Lactitol

Bacterial incorporation of nitrogen and acidification of the colonic environment which in turn reduces the breakdown of nitrogen-containing compounds to ammonia and other potential cerebral toxins

Delzenne (2003) and Marteau and Boutron-Ruault (2002)

Inflammatory bowel diseases (IBD)

Inulin, fructo-oligosaccharides, galacto-oligosaccharides

Regulating immune responses to commensal and pathogenic bacteria

Cherbut et al. (2003), Schultz et al. (2004), Furrie et al. (2005), and Kelly et al. (2005)

Prevention of cholesterol gallstones

Oligosaccharides (fructooligosaccharides, isomaltooligosaccharides, galactooligosaccharides, palatinose condensate, raffinose, and soybean oligosaccharides)

Stimulating the growth of bifidobacteria in vitro and in vivo

Mitsuoka et al. (1987) and Kohmoto et al. (1988)

Prevention of infections of intestinal origin

Oligosaccharides

Contributing to a greater resistance to infection. Most of Bifidobacterium species have scavenging function

Mitsuoka (1990)

Prebiotics and Probiotics  Chapter | 1  7

such as Crohn’s disease, ulcerative colitis, and pouchitis, is very important. These diseases are characterized by persistent mucosal inflammation at different levels of the GIT. In the GIT, the inflammatory capacity of commensal bacteria varies (some bacteria are proinflammatory), whereas other attenuate inflammatory responses. Wolf et al. (2005) provide an informative overview of the medical uses of FOS at the levels found in enteral products. The use of these products to provide total nutrition will deliver efficacious amounts of prebiotic fiber to hospitalized patients, generally in the range of 10-15 g/day. Patients not receiving formulated diets simply start with 1 g/day for the first week, increasing by 1 g/week until a 3 g level is attained. The maximum dose that is generally recognized as safe (GRAS) for all persons older than age 1 is 20 g, although much higher doses have also been suggested as safe (Douglas and Sanders, 2008).

3.2  Prebiotic Sources 3.2.1 Fructans Fructans are a group of naturally occurring oligosaccharides and FOS found in milligram quantities in onions, bananas, wheat, artichokes, garlic, and other whole foods (Chow, 2002). They are also extracted from chicory or are produced from sucrose for use in the food industry. Despite their similarities, the fructans remain distinct from each other in origin, structure, and fermentation characteristics (Douglas and Sanders, 2008). In vitro testing is not sufficient for prebiotic qualification or claims of efficacy because this method cannot approach the dynamic nature of colonic metabolism. There are also method limitations that involve the metabolism of the resident microflora as well as that of the host. These factors contribute to wide variations in measurable colony-forming unit counts, short-chain fatty acid and enzyme levels, and other measurements of outcome (Blaut, 2002). Many factors can confound results, including the chemical composition of the proposed prebiotic, its fermentation profile, the study design, the baseline distribution of a subject’s colonic microbiotica, the methodologies used in observing an effect in a particular subject group, and the statistical constructs used to interpret the data (Scholz-Ahrens et al., 2001).

3.2.2  Resistant Starch Non-fructan prebiotics are also under investigation for their fermentation characteristics, prebiotic effect, and health benefits. The resistant starch has been the subject of numerous studies that document a prebiotic effect, both as a single ingredient and in combination with FOS. Resistant starch is found in raw potatoes, cooked and cooled starchy products (retrograde starch), and in unripe fruits such as bananas. Appreciable amounts of resistant starch exist in many commercial food products due to processing effects on starch (Douglas and Sanders, 2008). Resistant starch is also manufactured specifically for use in the food industry. The standard dose for resistant starch is about 20 g/day, but low doses ranging from 2.5 to 5 g/day have demonstrated a prebiotic effect; the difference in dosing is due to the varying fermentation profiles of prebiotic ingredients. The bread and cereal categories are filled with products that include meaningful amounts of resistant starch or inulin for fiber content and sometimes energy reduction. It is reported that 20 g/day of resistant starch is a minimum healthy dose (Cassidy et al., 1994). Bounik et al. (2004) found that short-chain FOS, SOS, GOS, and type III resistant starch measurably raised fecal count of the Bifidobacterium species at reasonable dose ranges of 2.5-5 g/day within 7 days of administration. It is also important to validate markers that provide predictors for efficacy on human health. This difficult process requires mechanistic and epidemiological studies for validation. One large barrier to development of biomarkers relevant to the study of probiotics and prebiotics is that the composition of the human gut flora is not fully characterized and the significance of the presence, absence, or certain levels of different genera, species, or strains of bacteria is not understood.

3.3  Prebiotics and Resistance to Gastrointestinal Infections The gut microflora and the mucosa themselves may act as barriers against invasion by potential pathogens. Bifidobacteria and lactobacilli can inhibit pathogens such as Escherichia coli, Campylobacter, and Salmonella spp. The lactic microflora of the human GIT is thought to play a significant role in the improved colonization resistance (Gibson et al., 1997). These authors stated different mechanisms that can be used: 1) Metabolic end products, such as acids, excreted by these microorganisms may lower the gut pH, in a microniche, to levels below those at which pathogens are able to effectively compete; 2) Competitive effects from occupation of normal colonization sites;

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3) Direct antagonism through natural antimicrobial excretion (lactic acid bacteria produce inhibitory peptides); 4) Competition for nutrients and blocking of pathogen adhesion sites in the gut; and 5) Enhancement of the immune system. Moreover, many lactobacilli and bifidobacteria species are able to excrete natural antibiotics, which can have a broad spectrum of activity (Gibson et al., 2005). A potential correlation exists with reduced pathogen resistance, decreased numbers of bifidobacteria in the elderly, and the production of natural resistance factors. In essence, the natural gut flora may have been compromised through reduced bifidobacteria numbers and may have a diminished ability to deal with pathogens. If prebiotics are used to increase bifidobacteria or lactobacilli toward being the numerically predominant genus in the colon, an improved colonization resistance will result. Several studies have been conducted using human subjects, although the dose, substrate, duration, and volunteers vary. A general observation was the greater bifidogenic effect of substrates in subjects with a low initial bifidobacteria count (107/g feces) than in those with high initial number (109.5/g feces) (Hidaka et al., 1986). Also, a negative correlation between bifidobacteria and Clostridium perfringens was observed, suggesting that the former may inhibit growth of the latter in the intestine, supporting earlier studies (Wang and Gibson, 1993; Gibson and Wang, 1994).

4  EVALUATION OF PREBIOTIC According to the Food and Agricultural Organization of the United Nations (FAO) Technical Meeting on Prebiotics (FAO, 2007), the flowchart in Figure 1.1 shows how to evaluate and substantiate that a product is a prebiotic. The steps to be followed are explained here.

Component characterization—source, origin, purity, chemical composition, and structure

Product formulation, vehicle, concentration, and amount

Functional characterization In Vitro / animal testing

Safety assessment In Vitro and/or animal, and/or Phase 1 human study if not GRAS or equivalent

Double blind, randomized, controlled human trial (RCT) with simple size and primary outcome appropriate to determine if product is efficacious. Minimum proof of a correlation between the measurable physiological outcomes and modulation of the microbiota at specific site

Preferably second independent RCT study to confirm results

Prebiotic

FIGURE 1.1  Guidelines for the evaluation and substantiation of prebiotics.

Prebiotics and Probiotics  Chapter | 1  9

4.1  AFCSF Product Specification/Characteristics of the Prebiotic The component to which the claim of being prebiotic is attributed must be characterized for any given product. This includes source and origin, purity, chemical composition and structure, vehicle, concentration, and amount in which it is to be delivered to the host.

4.2 Functionality At a minimum, there needs to be evidence of a correlation between the measurable physiological outcomes and modulation of the microbiota at a specific site (primarily the GIT, but potentially also other sites such as vagina and skin). It is necessary to correlate a specific function at a specific site with the physiological effect and its associated time frame. Within a study, the target variable should change in a statistically significant way. The change should be biologically meaningful for the target group, and consistent with the claim to be supported. Substantiation of a claim should be based on studies with the final product type, tested in the target host. A suitably sized randomized control trial (compared to a placebo or a standard control substance) is required, preferably with a second independent study. Examples of physiological outcomes due to administration of prebiotics could be: l l l l l l l l

Satiety (measured toward carbohydrates, fats, and total energy intake); Endocrine mechanisms regulating food intake and energy usage in the body; Effects on absorption of nutrients (e.g., calcium, magnesium, trace elements, and protein); Reduced incidence or duration of infection; Blood lipid and classic endocrine parameters; Bowel movement and regularity; Markers for cancer risk; and Changes in innate and acquired immunity that are evidence of a health benefit.

4.3 Qualifications Qualifications for a prebiotic can be component (chemical substance or a food grade component), health benefit (measurable and not due to the absorption of the component or due to the component acting alone, and overriding any adverse effects), or modulation (changes in the composition or activities of the microbiota in the target host). A prebiotic can be a fiber but a fiber is not necessarily a prebiotic. It was stated that bifidogenic effects are not sufficient without demonstrated physiological health benefits. It is also recognized that determining the events that take place within compartments of the intestine are often difficult; specific site sampling or more sophisticated methods can reliably link microbiota modulation with health benefits—for example, fecal analysis.

4.4 Safety It is recommended that the following issues need to be covered in any safety assessment of a prebiotic final product formulation: l

l l

l

When the product has a history of safe use in the target host, such as GRAS or its equivalents (e.g., the qualified perception of safety (QPS) in EU, also discussed in this revision), then it is suggested that further animal and human toxicological studies may not be necessary. Safe consumption levels with minimal symptoms and side effects should be established. The product must not contain contaminants and impurities. The contaminants should be identified and measured, and the impurities should be well characterized and submitted to toxicity evaluation if needed. Based on current knowledge, the prebiotic should not alter the microbiota in such a way as to have long-term detrimental effects on the host.

For functional ingredients, animal models can be used to ascertain the target organs and effects that are produced as a result of toxicity. The extent of testing necessary for a functional ingredient is increased in response to the lack of understanding of potential for toxicity as a result of inadequately characterized products. The following criteria must be met to derive a safe level of exposure without additional toxicology testing (Kruger and Mann, 2003): 1. Active component(s) and related substances are well characterized and there is adequate understanding of the lack of potential for toxicity at the human dose levels recommended based on existing data from the literature.

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2. Impurities are well characterized and there is an adequate understanding of the lack of potential for toxicity based on existing data from the literature. 3. The manufacturing process is standardized and reproductive. When either the active component(s) or impurities are either not fully characterized or there is not enough data available to evaluate potential for toxicity, the following preclinical toxicological information is needed to assess the functional ingredient: toxicity studies in vitro and in vivo, including mutagenicity studies, reproduction and teratogenicity studies, pharmacokinetics and special pharmacology studies, as well as long-term feeding studies, following a tiered approach on a case-by-case basis. An element that must be considered in the design of animal studies for functional ingredients is the margin of safety between the no-observed-adverse-effect level (NOAEL) determined in the animal studies and the anticipated human level of intake (Anadón et al., 2014).

5  PROBIOTICS USED IN FOOD Probiotics are commonly defined as viable microorganisms (yeast and lactic acid bacteria) that exhibit a beneficial effect on the health of the host when they are ingested, although the health benefits are strain-specific and not species- or genusspecific. Many health effects attributed to probiotic microorganisms are related, among others, to the GIT, showing the ability to survive through the upper GIT, and be capable of surviving and growing in the intestine (acid and bile resistant). Also, probiotics are safe for human consumption, produce antimicrobial substances such as bacteriocins, and have the ability of adherence to human intestinal cell lines and colonize the intestine. The actions of microorganisms are useful to assist the GIT by breaking down sugars and carbohydrates to promote good digestion, boosting the immune system, maintaining proper intestinal pH, and successfully competing with pathogens. Among the expectations for probiotics, a number of strains have been shown to modulate the intestinal microflora and to prevent the duration and complaints of rotavirus-induced diarrhea. Probiotic bacteria also reinforce the intestinal wall by crowding out pathogenic microorganisms, thereby helping to prevent their attachment to the human gut, where they have been shown to be safe. The consumption of probiotics influences various aspects of the innate nonspecific immune system such as promotion of mucin production; inhibition of pathogens; decrease in gut permeability, macrophage activation, and phagocytic capacity; and exhibit natural killer cell activity. Regarding the adaptive immune system, the effects observed are an increase in the production of antibodies (IgA, IgM, and IgG), and an influence in the arrangement of both branches of the immune system by the production of cytokines and other regulatory elements. Most probiotics are marketed as foodstuffs or drugs. Lactobacillus, Leuconostoc, and Pediococcus species have been used extensively in food processing throughout human history, and ingestion of foods containing live bacteria, dead bacteria, and metabolites of these microorganisms have taken place for a long time (Mäyrä-Mäkien and Bigret, 1993). Today, the most widely used probiotics include lactobacilli, bifidobacteria, and some nonpathogenic strains, mostly of human origin, which confer a health benefit on the host and enable the prevention or improvement of some diseases when administered in adequate amounts. An important fact is probiotics must retain their viability during the storage, manufacturing process, and transit through the stomach and small intestine. Prior to being categorized as probiotics, organisms must follow a process of testing, including strain testing; identification by genotype and phenotype, functionalized characterization, and safety assessment testing; and double-blind, placebo-controlled human trials to verify the subjects’ health benefits. Also, the guidelines for the evaluation of probiotics in food must be followed (FAO/WHO, 2002). Most probiotic foods contain lactobacilli and/or bifidobacteria. Enterococci are infrequently used. Microorganisms used as probiotics are mainly bacterial strains of members of the heterogeneous group of lactic acid bacteria, Lactobacilli (Lactobacillus acidophilus, L. casei, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius); bifidobacteria (Bifidobacterium breve, B. longum, B. lactis), Bacillus (B. subtilis, B. cereus var. toyoi), and Enterococcus (E. faecium), among others. The yeast Saccharomyces boulardii is also used as a human probiotic, although in capsule or powder form rather than in food form. Bacillus and Lactobacillus differ in many characteristics; Bacillus and the yeasts are not usual components of the gut microflora. Most of the species and genera are apparently safe, but certain microorganisms may be problematic. This is especially true for the enterococci (E. faecium and E. faecalis) that have emerged as opportunistic pathogens in hospital environments, causing nosocomial infections such as endocarditis and bacteraemia, as well as intra-abdominal, urinary tract, and central nervous system infections. Some species and genera may also harbor transmissible antibiotic resistance determinants (e.g., vancomycin-resistant Enterococcus strains) and bacilli, particularly those belonging to the B. cereus group that are known to produce enterotoxins and an emetic toxin (Anadón et al., 2006). The selection criteria of new probiotic strains is determined by many factors, such as resistance to pancreatic enzymes, acid and bile, preferably human origin (although the S. boulardii is not of human origin), documented health effects, known

Prebiotics and Probiotics  Chapter | 1  11

safety, and good technological properties, especially the potential probiotics (Ouwehand et al., 2002). It is generally assumed that probiotics are live microorganisms, generally bacteria but also yeasts, that, when ingested alive in sufficient numbers, interact with the gut microflora and host, having a positive effect on the health going beyond the nutritional ones commonly known. In most cases, the safety of novel strains has been deduced mainly from the common occurrence of the species, either in foods or as normal commensals in the human gut. Depending on the country, the same probiotic microorganism(s) may be available as food supplement(s), available as registered medicinal products, and/or incorporated into the food. At present, probiotic human foods are not governed under specific EU regulatory frameworks, but the Regulation (EC) No. 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and food ingredients (OJ No. L 043, 14.02.1997, p.1) may cover other more novel types of probiotics species that need to be discussed and assessed in the light of the novel food guidelines (Jonas et al., 1996). According to this Regulation 258/97, novel foods and food ingredients are those that have not hitherto been used for human consumption to a significant degree within the community. Specifically, foods and food ingredients containing or consisting of, or produced from, genetically modified organisms (GMO), and foods consisting of, or isolated from, micro-organisms, fungi, or algae belong to the category of novel foods. It should be stated, however, that additives and processing aids fall outside the scope of the regulation. The case of a processing aid or additive consisting of live micro-organisms thus remains ambiguous. However, microbial food additives are covered by Regulation (EC) No. 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition (OJ No. L 268, 18.10.2003) and in accordance with the guidelines of the FEEDAP Panel of EFSA, are subjected to the detailed efficacy and safety assessment, the latter with the intention of ensuring that they are innocuous to target animals, users, and consumers (Anadón et al., 2006). Regulation 258/97 is stated in relation to the nutritional information that nutritional consequences should be assessed at normal and maximum levels of consumption, and that the effect of antinutritional factors (e.g., inhibiting mineral absorption or bioavailability) relative to the nutritional value of the whole diet should be also assessed. Also, the numbers involved in study groups should ensure that the study has adequate statistical power, and all studies should comply with relevant elements and ethical principles of guidelines on good clinical practice and good laboratory practice. With respect to the implications of novel food to human nutrition overall, assessment must consider nutritional implications (expected normal intakes and at maximum levels of consumption). The nutritional considerations affecting toxicological testing in animals is of crucial importance. To interpret carefully any adverse effects seen in animal studies and to distinguish toxic effects and those due to nutrition imbalance in the experimental diet and in the design of animal feeding studies, the maximum level of dietary incorporation achievable without causing nutritional imbalance should be the highest dose level. The lowest dose level should be comparable to its anticipated role in human diet. Finally, the toxicological requirements for novel food need to be considered on a case-by-case basis. In the worst case, the following elements are needed: consideration of the possible toxicity of the analytically identified individual chemical components; toxicity studies in vitro and in vivo, including mutagenicity studies, reproduction and teratogenicity studies; and long-term feeding studies and studies on potential allergenicity. The Novel Food Regulation defines novel foods as foods and food ingredients that were not used for human consumption to a significant degree within the community before 15 May 1997. “Human consumption to a significant degree within the community,” in this context, has been interpreted as being demonstrated by a food having been generally available within the community. For example, if a food was available only in pharmacies within the community, this would not constitute evidence of use for human consumption to a significant degree. In contrast, if a food was available in general food stores, this would constitute evidence of use for human consumption to a significant degree (SANCO, 2002).

6 SYNBIOTIC Probiotic-containing foods can be categorized as functional foods and are often associated with prebiotics, which are nondigestible carbohydrates that act as food for probiotics. When probiotics and prebiotics are combined, they form a synbiotic. The potential synergies between probiotics and prebiotics have been considered efficient due the improvement of the survival and implantation of live microbial dietary supplement in the GIT. One of the main benefits of synbiotics is the increased persistence of probiotics in the GIT. There are many examples of synbiotics reported in the scientific literature with data from in vitro observations (e.g., L. casei strain Shirota + oligomate 55™; L. acidophilus ATCC 4962 + mannitol, FOS, and inulin; Lactobacillus sakei JCM + FOS and trehalose; L. plantarum and L. acidophilus + xylo- and FOS) or from in vivo observations (i.e., B. longum + OF; B. breve strain Yakult + GOS) Lafti™ B94 + resistant starch; and Lactobacillus gasseri + inulin and unspecified oligosaccharides (Furrie et al., 2005).

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It would appear unlikely that supplementation with a single probiotic strain would be sufficient to have a major influence on the very diverse intestinal microbiota and the complex interaction between the gut bacteria and the host. This has led to an interest in dietary substrates that could have a more global effect on gut microbiota—namely, prebiotics (nondigestible, fermentable oligosaccharides promoted the growth of allegedly “beneficial” bacteria, particularly of Bifidobacterium and Lactobacillus species) to a similar extent to commercial xylo-oligosaccharides. Altering the intake of foods containing these products could conceivably directly influence the composition and activity of intestinal microbiota. Combinations of oligosaccharides and probiotic bacteria have been tried in the treatment of eczema in infants. Treatment of atopic dermatitis in infants with a symbiotic mixture of B. breve and galacto- and FOS has been tested in clinical trials with no clear conclusions. Overall, a part of the efficacy on the safety of synbiotics should be considered.

7  SAFETY ASPECT OF PROBIOTICS Assessment of the safety of probiotics is not an easy task (Anadón et al., 2014). The selection of new probiotic organisms targets new strains and even genera that are more beneficial or specific. When novel microbes and GMO are introduced, their safety and the risk-to-benefit ratio have to be carefully studied and assessed. Also, new probiotics should be of genera and strains commonly found in the healthy human intestinal microflora. The microbes could be classified as nonpathogenic (Lactobacillus, Lactococcus, Bifidobacterium, and Saccharomyces), pathogens (B. cereus), and opportunistic pathogens (Enterococcus and other general lactic acid bacteria). Lactic acid bacteria and bifidobacteria are the most common bacteria that attach to the human intestinal mucosa and are commonly regarded as having the GRAS status. Moreover, certain strains of probiotic bacteria have been proven to be free of risk factors such as transferable antibiotic resistances, cancer-promoting and/or putrefactive enzymes and metabolites, hemolysis, activation of thrombocyte aggregation, and mucus degradation in the mucus layer of the GIT. Despite the absence of a pathogenic potential, lactic acid bacteria were found in 41 °C in cheese analogs, especially in products with lower milk-fat levels and higher inulin content. All of the tested samples exhibited good melting properties.

Prebiotic Addition in Dairy Products  Chapter | 3  43

The addition of inulin to cheese samples did not influence their water activity, but did alter their color. Confocal microscopy revealed that the highest hardness values could be attributed to more intense distribution of fat droplets in the samples with the highest fat content. Results suggest that milk fat in processed cheese analogs can be partially replaced with inulin to improve the functional properties of the final product. Morais et al. (2014b) evaluated the addition of prebiotics (inulin and FOS) in chocolate dairy desserts. The optimal concentration was 7.5% (w/w) prebiotic and the ideal sweetness analysis revealed that the ideal concentration of sucrose was 8.13%. The relative sweetness analysis showed that Neotame (NutraSweet Corp., Chicago, IL, USA) had the highest sweetening power compared with the prebiotic chocolate dairy dessert containing 8% sucrose, followed by sucralose, aspartame, and stevia. The study of sweetness in this product was important because consumers desire healthier functional products with no added sugar. Dave (2012) studied the effects of inulin as a fat replacer on the rheological and textural properties of low-fat processed cheese spread. The author found that low-fat cheese spreads with 7% and 8% inulin had significantly higher yield-stress values than either the full-fat cheese spread control (20% fat without inulin) or low-fat spreads with relatively low inulin contents. Inulin also showed a positive effect on the spreadability of the low-fat cheese spreads. The results indicated that the low-fat processed cheese spreads with 7% and 8% inulin achieved yield-stress values and spreadability similar to the full-fat processed cheese spread. However, the low-fat cheese spread with a 6% level of inulin resulted in a mushy product. Fadaei et al. (2012) studied the chemical characteristics of low-fat, whey-less cream cheese containing inulin as a fat replacer. No significant difference was found in the pH and salt values of cream cheeses. The authors observed that an inulin proportion of 10% was enough to obtain a low-fat cream cheese with chemical attributes near to those of high-fat cream cheese that did not contain inulin. They also reported that inulin presented an excellent WBC, which inhibits syneresis in spreads and fresh cheeses. The influence of probiotic bacteria, prebiotic compounds (FOS and inulin), and ripening time on the free-fatty acid profile of cheese, with special emphasis on the conjugated linoleic acid (CLA) content, was investigated by Rodrigues et al. (2012). The addition of FOS alone or combined with inulin did not significantly affect probiotic strain growth and viability during the ripening period. However, the advantage of the addition of prebiotic compounds in probiotic cheese manufacture is that it may allow the production of cheeses with improved performance as far as functional CLA compounds are concerned, as well as an improved nutritional quality reflected in a lower atherogenicity index.

16 PERSPECTIVES The importance of consuming functional foods for improvement of the quality of life is clearly described in the scientific literature, and the number of foods to which they can be applied is increasing, as is the diversification of the agents that provide these characteristics in the products. Dairy products are considered an excellent food matrix for incorporation of functional ingredients, including prebiotics. The number of studies evaluating the addition of prebiotics in dairy products is increasing. This shows the importance of updating these food segments, dairy, and functional. Results of the research presented in this chapter, which focused on the addition of prebiotics in dairy products, are very useful for dairy processors and dairy researchers who wish to participate in the competitive market of functional and prebiotic foods.

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Aryana, K.J., McGrew, P., 2007. Quality attributes of yogurt with Lactobacillus casei and various prebiotics. LWT—Food Sci. Technol. 40 (10), 1808–1814. Bayarri, S., Chuliá, I., Costell, E., 2010. Comparing lambda-carrageenan and an inulin blend as fat replacers in carboxymethyl cellulose dairy desserts. Rheological and sensory aspects. Food Hydrocoll. 24, 578–587. Betoret, N., Puente, L., Díaz, M.J., Pagán, M.J., García, M.J., Gras, M.L., 2003. Development of probiotic-enriched dried fruits by vacuum impregnation. J. Food Eng. 56, 273–277. Bhatia, A., Rani, U., 2007. Prebiotics and health: clinical implications. J. Clin. Diagn. Res. 1 (6), 546–554. Biedrzycka, E., Bielecka, M., 2004. Prebiotic effectiveness of fructans of different degrees of polymerization. Trends Food Sci. Technol. 15, 170–175. Bird, A.R., Lopez-Rubio, A., Shrestha, A.K., Gidley, M.J., 2009. Resistant starch in vitro and in vivo: factors determining yield, structure, and physiological relevance. 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Symbiotic potential of fresh cream cheese supplemented with inulin and Lactobacillus paracasei in co-culture with Streptococcus thermophiles. Food Chem. 104, 1605–1610. Cardarelli, H.R., Saad, S.M.I., Gibson, G.R., Vulevic, J., 2007. Functional petit-suisse cheese: measure of the prebiotic effect. Anaerobe 13, 200–207. Cardarelli, H.R., Aragon-Alegro, L.C., Alegro, J.H.A., Castro, I.A., Saad, S.M.I., 2008. Effect of inulin and Lactobacillus paracasei on sensory and instrumental texture properties of functional chocolate mousse. J. Sci. Food Agric. 88, 1318–1324. Castelli, F., Sarpietro, M.G., Micieli, D., Ottimo, S., Pitarresi, G., Tripodo, G., Carlisi, B., Giammona, G., 2008. Differential scanning calorimetry study on drug release from an inulin-based hydrogel and its interaction with a biomembrane model: pH and loading effect. Eur. J. Pharm. Sci. 35, 76–85. Castro, F.P., Cunha, T.M., Ogliari, P.J., Teófilo, R.F., Ferreira, M.M.C., Prudêncio, E.S., 2009. 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Rheological and physico-chemical characterization of prebiotic microfiltered fermented milk. Journal of Food Engineering 99, 128–135. Debon, J., Prudêncio, E.S., Petrus, J.C.C., Fritzen-Freire, C.B., Muller, C.M.O., Amboni, R.D.D.M.C., Vieira, C.R.W., 2012. Storage stability of prebiotic fermented milk obtained from permeate resulting of the microfiltration process. LWT—Food Sci. Technol, 47 (1), 96–102. Delgado, G.T.C., Tamashiro, W., Pastore, G.M., 2010. Immunomodulatory effects of fructans. Food Res. Int. 43, 1231–1236. Di Bartolomeo, F., Startek, J., Van den Ende, W., 2013. Prebiotics to fight diseases: reality or fiction? Phytother. Res. 27, 1453–1473. Fadaei, V., Poursharif, K., Daneshi, M., Honarvar, M., 2012. Chemical characteristics of low-fat wheyless cream cheese containing inulin as fat replacer. Eur. J. Exp. Biol. 2, 690–694. FDA (Food and Drug Administration), 2008. 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Chapter 4

Low-Lactose, Prebiotic-Enriched Milk Francisco J. Plou, Barbara Rodriguez-Colinas, Lucia Fernandez-Arrojo and Antonio O. Ballesteros Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain

1  HUMAN MILK OLIGOSACCHARIDES In addition to its role as a source of nourishment, human milk provides various bioactive substances to the infants that modulate their immune and cognitive systems and participate in the development of their microbiota (Zivkovic et al., 2011). The two most abundant components of breast milk are lactose and lipids, followed by the HMOs (Figure 4.1). Human milk oligosaccharides (HMOs) constitute a family of more than 100 carbohydrates—with varying composition and structure—that exert numerous benefits to breast-fed infants (Sela and Mills, 2010). In this context, it is worth mentioning that the microbiota of babies fed with breast milk is more dominated by bifidobacteria than those fed on cow’s milk (Locascio et al., 2007). The concentration of functional oligosaccharides in human milk varies between 5 and 15 g/L, a value nearly 100 times higher than in cow’s milk. The amount of HMOs is even higher in the colostrum, reaching up to 24% of total colostrum carbohydrates (Bode, 2006). HMOs contain lactose at their reducing end, which is normally elongated with lacto-n-biose or N-acetyl-lactosamine and further fucosylated or sialylated (Bode, 2012). The different combinations of monosaccharides and glycosidic linkages between the sugar units give rise to a structurally complex array of linear and branched glycoderivatives. Due to their complex structures, the synthesis of HMOs is a difficult task. However, since the early 2000s, there has been a notable progress in the preparation of HMOs by chemical and biotechnological methodologies. In particular, recent developments on sialidases (EC 3.2.1.18) and α-l-fucosidases (EC 3.2.1.51) will contribute to expand the synthesis of HMOs (Zeuner et al., 2014). To mimic the multiple benefits of HMOs, infant formulas are often supplemented with structurally related carbohydrates, particularly galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) (Moro and Arslanoglu, 2005; Shadid et al., 2007). Both GOS and FOS have been used as functional ingredients in foods for more than 35 years, especially in Japan and Europe (Tzortzis and Vulevic, 2009). In fact, GOS are minor components of HMOs, as human milk contains several GOS with β(1 → 3), β(1 → 4), and β(1 → 6) linkages between the galactosyl moieties, in amounts ranging from 2.0 to 3.9 mg/L (Boehm et al., 2005).

2  GALACTO-OLIGOSACCHARIDES (GOS) AND FRUCTO-OLIGOSACCHARIDES (FOS) IN DAIRY PRODUCTS GOS are formed by various galactosyl moieties linked to a terminal glucose, or exclusively by galactose units—called galactobioses, galactotrioses, etc.—(Gosling et al., 2010). They are obtained by transgalactosylation reactions in which lactose, as well as the glucose and galactose released by hydrolysis, serve as galactosyl acceptors (Tzortzis and Vulevic, 2009). FOS are fructose oligomers with a terminal glucose unit in which 2-8 fructosyl moieties are linked via β(2 → 1) glycosidic bonds (Antosova and Polakovic, 2001). They are produced from sucrose using fungal-transfructosylating enzymes (EC 2.4.1.9) or by hydrolysis of inulin catalyzed by endoinulinases (EC 3.2.1.7) (Gimeno-Perez et al., 2014; Plou et al., 2014). The main components of commercial FOS are 1-kestose (GF2), nystose (GF3), and 1F-fructofuranosyl-nystose (GF4), although derivatives with higher polymerization degree are present, especially in samples obtained by controlled hydrolysis of inulin. Both GOS and FOS are included in various foods as health-promoting ingredients to emulate the bifidogenic properties of HMOS—despite their structural differences—and also to inhibit the adherence of pathogens to the gut epithelium due to their resemblance to glycostructures on cell surface receptors (Miniello et al., 2003). It has been demonstrated that the incorporation of GOS and FOS into baby foods improves the microbiota composition in the feces and reduce allergenic Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00004-6 © 2016 Elsevier Inc. All rights reserved.

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FIGURE 4.1  Composition of human milk.

manifestations (e.g., atopic dermatitis) and infections during the first years of life (Boehm et al., 2005; Kukkonen et al., 2007). A 9:1 ratio (w/w) between short-chain GOS and long-chain FOS is typically used in infant formulas to mimic the molecular size distribution of the neutral fraction of HMOs (Fanaro et al., 2005; Moro et al., 2003). GOS and FOS belong to the so-called prebiotics, which are nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or the activity of certain types of bacteria in the colon, basically of the genera Bifidobacterium and Lactobacillus (Gibson and Ottaway, 2000). The metabolism of such bacteria releases short-chain fatty acids (acetate, propionate, and butyrate) and l-lactate (Roberfroid, 2007; Rodriguez-Colinas et al., 2013b), which produce positive effects on human health. In particular, they exert protective effects against colorectal cancer and bowel-infectious diseases by inhibiting pathogen bacteria, reduce the level of cholesterol in serum, improve the bioavailability of essential mineral components such as calcium, and modulate the immune system (Rastall et al., 2005; Sabater-Molina et al., 2009; Tuohy et al., 2005). Depending on the source of the enzyme used, commercial GOS may contain predominantly β(1 → 3), β(1 → 4), or β(1 → 6) linkages (Park and Oh, 2010; Torres et al., 2010; Villamiel et al., 2014). The complete identification of the GOS synthesized by a particular enzyme is a difficult job due to the multiple combinations of monomers and linkages. Thus, considering only the formation of β(1 → 2), β(1 → 3), β(1 → 4), and β(1 → 6) bonds, the theoretical number of linear GOS accounts for 7 disaccharides, 32 trisaccharides, and 128 tetrasaccharides (Rodriguez-Colinas et al., 2013a). Figure 4.2 depicts several of the most common compounds encountered in commercial GOS powders and syrups. Considering that commercial GOS are mixtures of various oligosaccharides and that they present different percentages of residual lactose and monosaccharides (glucose, galactose), their physicochemical properties may vary from one product to another. Table 4.1 summarizes the main properties of GOS products. In general, they are very soluble in water-forming colorless viscose solutions, which are very stable toward pH and temperature. For example, they remain stable after treatment at 120 °C for 10 min at pH 3.0; this fact explains their numerous applications in liquid and solid food matrices. Their sweetness, their low-caloric value (50% compared with sucrose), and their noncariogenicity promote the use of GOS as sugar substitutes. At a daily dose of 1.5 g/kg of body weight, GOS are nontoxic. Some of their physicochemical properties (moisture-retaining capacity, low aw, modification of freezing point, etc.) are very convenient for processed foods (Konar et al., 2011; Tzortzis and Vulevic, 2009). Their unique properties along with their health-promoting effects (e.g., bifidogenic) make GOS very attractive ingredients for the food industry, especially in infant formula, growing-up milk, and even in dairy products for elderly people.

3  ENZYMATIC SYNTHESIS OF GOS Apart from lactose hydrolysis, β-galactosidases (EC 3.2.1.23, β-gal) are able to catalyze a transgalactosylation reaction in which lactose or other carbohydrates present in the mixture serve as galactosyl acceptors, yielding GOS with different glycosidic bonds and polymerization degrees (Hsu et al., 2007; Park and Oh, 2010). The enzyme mechanism is based on a double-displacement pathway (Figure 4.3) in which a covalent galactosyl-enzyme intermediate is initially formed. This intermediate can be subsequently attacked by water (resulting in lactose hydrolysis) or by a carbohydrate (forming GOS). The enzymatic transformation of lactose into GOS is thus controlled by kinetics. The enzyme origin and the experimental conditions (lactose concentration, water activity, temperature, pH, and time) notably influence the yield and composition

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FIGURE 4.2  Structure of representative components of commercial GOS.

TABLE 4.1  Main Properties of GOS (Konar et al., 2011; Macfarlane et al., 2006; Playne and Crittenden, 2009; Sako et al., 1999; Torres et al., 2010; Tzortzis and Vulevic, 2009) Water solubility

Approx. 80% (w/w)

Sweetness

0.3-0.6 times referred to sucrose

Appearance

Colorless

Viscosity

Similar to high fructose corn syrup (HFCS)

Stability

High thermal and acidic pH stabilities

Humectant properties

High moisture retaining capacity

Water activity (aw)

Low value (minimizing microbial contamination)

Cariogenicity

Low

Digestibility

Nondigestible

Fermentability

Caloric value between 1 and 2 kcal/g

Toxicity

Negligible for 1.5 g/kg body/day during 6 months

Prebiotic properties

Well established

50  PART | I  Prebiotics in Health Promotion

FIGURE 4.3  Hydrolysis/transgalactosylation processes catalyzed by β-galactosidases.

of the synthesized GOS (Gosling et al., 2011; Iqbal et al., 2010; Urrutia et al., 2013). In general, the GOS yield is higher with increasing lactose concentration (Vera et al., 2012). The intrinsic enzyme properties—that is, its ability to exclude H2O and to bind the sugar acceptor to which a galactosyl moiety is transferred—are crucial for a satisfactory GOS production. The time required to get the maximum GOS concentration depends inversely on the amount of enzyme; however, this maximum GOS yield is not affected by the dosage of biocatalyst (Ballesteros et al., 2006). Under the optimal conditions, GOS yields do not surpass 30-40% (w/w) referred to the total amount of sugars in the mixture (Gosling et al., 2010). The use of milk whey permeant to synthesize GOS has also been investigated (Lopez Leiva and Guzman, 1995; Lorenzen et al., 2013). In this context, Chen et al. (2002) developed a multistep process to increase GOS production by ultrafiltration of milk to separate lactose from proteins, followed by a concentration of the permeant and a further transgalactosylation reaction with β-galactosidases. Several companies, most of them located in Japan and Europe, produce different GOS powders and syrups with various degrees of purity (Gosling et al., 2010; Park and Oh, 2010). In addition, commercial GOS contain derivatives with different linkages between the galactosyl moieties depending on the source of the enzyme. For example, the product Oligomate® (Yakult Honsha, Japan) is manufactured with Aspergillus oryzae β-gal and contains mainly β(1 → 6) bonds, whereas the major linkages in Bimuno® GOS (Clasado, UK) are β(1 → 3) as it is produced with Bifidobacterium bifidum β-gal (Tzortzis and Vulevic, 2009).

4  IN SITU FORMATION OF GOS IN MILK Due to the deficiency of intestinal β-galactosidase, about 70% of the world population is intolerant to lactose at a certain degree; the problem is currently solved by means of the removal of lactose in dairy products with lactases from microorganisms generally recognized as safe (GRAS) (Adam et al., 2004; Mlichova and Rosenberg, 2006). Instead of adding GOS to dairy products, an attractive strategy could be to form such oligosaccharides in situ during the treatment of milk with β-galactosidases. Despite its technological interest, the formation of GOS in situ directly in milk has been scarcely studied (Kim et al., 1997; Mlichova and Rosenberg, 2006; Puri et al., 2010; Ruiz-Matute et al., 2012). This is probably due to the fact that lactose concentration in bovine milk is around 45 g/L, a value much lower than that typically employed in lactosebuffered solutions (200-500 g/L) to promote the transglycosylation reaction (Guerrero et al., 2011; Prenosil et al., 1987). In our work, we have investigated GOS formation during lactose hydrolysis in bovine milk catalyzed by several β-galactosidases with different specificity (Rodriguez-Colinas et al., 2014). In particular, β-gal from Bacillus circulans and Kluyveromyces lactis—both of which possess the GRAS status and are widely employed in the dairy industry to eliminate lactose—were assessed. Our objective was to develop a strategy for obtaining dairy products with a significant content of GOS and, concomitantly, a low content of lactose. Such family of products with double functionality could be of interest in the dairy market, not only for infants but also for adults and elderly people to take advantage of the prebiotic and other health benefits of GOS.

5  GOS FORMATION IN MILK WITH β-GALACTOSIDASE FROM B. CIRCULANS The β-galactosidase from B. circulans is an extracellular enzyme with a high thermostability and a notable transgalactosylation activity, even in the presence of organic solvents (Bridiau et al., 2010; Wei et al., 2009). Different isoforms of the β-gal from B. circulans have been identified (Song et al., 2011). A novel commercial preparation of β-gal from B. circulans (Biolactase®, Biocon) was tested in this study.

Low-Lactose, Prebiotic-Enriched Milk  Chapter | 4  51

FIGURE 4.4  Depletion of lactose and GOS formation in skim milk during treatment with β-galactosidase from B. circulans (Biolactase). Conditions: 0.1% (v/v) enzyme dosage, 40 °C. Adapted from Rodriguez-Colinas et al. (2014).

The optimum pH for this β-gal is 5.5, which is about one unit lower than the pH of bovine milk (6.7). However, an advantage of the B. circulans enzyme for its use in the dairy industry is that it is hardly inhibited by calcium ions present in milk compared with other β-galactosidases (Mozaffar et al., 1984). In our experiments, we incubated skim milk with the β-gal from B. circulans at 40 °C using an enzyme dosage of 0.1% (v/v), a similar amount to that employed in the manufacture of lactose-free milk. The lactose concentration in milk varied between 44 and 46 g/L as measured by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). We analyzed the disappearance of lactose and the formation of GOS during the treatment with this β-gal at 40 °C (Figure 4.4). Interestingly, we observed the typical pattern with a point of maximum GOS concentration followed by a progressive decrease in the amount of total GOS. This profile is the consequence of the competition between hydrolysis and transglycosylation reactions (Mozaffar et al., 1985). The maximum GOS concentration during B. circulans treatment was approx. 7.6 g/L, which corresponded to 16% (w/w) of total carbohydrates in milk. The highest value was achieved when around 50% of the initial lactose had been removed. Mozaffar et al. (1985) reported a maximum production of GOS in milk close to 5.5% (w/w) using a purified β-gal from B. circulans, which was obtained at 39% conversion of lactose. Figure 4.5 shows the HPAEC-PAD chromatogram of the treated milk at the point of maximum GOS concentration. Apart from galactose, glucose, and lactose, three main GOS were identified in the milk treated with B. circulans β-gal: (1) the disaccharide 4-galactobiose [Gal-β(1 → 4)-Gal]; (2) the trisaccharide 4′-O-β-galactosyl-lactose [Gal-β(1 → 4)-Gal-β(1 → 4)-Glc]; and (3) the tetrasaccharide Gal-β(1 → 4)-Gal-β(1 → 4)-Gal-β(1 → 4)-Glc, confirming the specificity of this enzyme for the formation of β(1 → 4) linkages (Rodriguez-Colinas et al., 2012; Yanahira et al., 1995). Using a buffered 400 g/L lactose solution, other minor GOS containing β(1 → 3) bonds have been also detected with this enzyme (Rodriguez-Colinas et al., 2013a).

6  GOS FORMATION IN MILK WITH β-GALACTOSIDASE FROM K. LACTIS One of the major commercial sources of β-galactosidase is the yeast K. lactis. Since it is an intracellular enzyme, the production of soluble β-gal is expensive due to the complex downstream processing and its low stability (Chockchaisawasdee et al., 2005). On the contrary, the optimum pH for the K. lactis β-gal (6.8) almost coincides with the pH of milk (6.7). A novel commercial preparation of β-gal from K. lactis (Lactozym pure®, Novozymes) was tested in this study. For a fixed enzyme dosage (0.1%), K. lactis β-gal was more active than the B. circulans enzyme, as lactose was almost completely depleted in 1.5 h (Figure 4.6), whereas B. circulans required 4.5 h (Figure 4.4). It is worth noting that maximum GOS concentration with K. lactis β-gal (7.0 g/L, 15% of total sugars, Figure 4.6) was obtained at a significantly higher lactose conversion (95%) than with B. circulans. The HPAEC-PAD chromatogram in Figure 4.7 illustrates that the major GOS synthesized by β-gal from K. lactis were the disaccharides 6-galactobiose [Gal-β(1 → 6)-Gal] and allolactose [Gal-β(1 → 6)-Glc], and the trisaccharide

FIGURE 4.5  HPAEC-PAD chromatogram of skim milk treated with β-galactosidase from B. circulans at 40 °C, at the point of maximum GOS concentration (0.75 h). The synthesized GOS correspond to (1) 4-galactobiose; (2) 4′-O-β-galactosyl-lactose; and (3) Gal-β(1 → 4)-Gal-β(1 → 4)-Gal-β(1 → 4)Glc. Gal: Galactose; Glc: Glucose; Lact: Lactose. Adapted from Rodriguez-Colinas et al. (2014).

FIGURE 4.6  Elimination of lactose and GOS formation in skim milk during the treatment with β-galactosidase from K. lactis. Conditions: 0.1% (v/v) enzyme dosage, 40 °C. Adapted from Rodriguez-Colinas et al. (2014).

FIGURE 4.7  HPAEC-PAD chromatogram of skim milk treated with β-galactosidase from K. lactis at 40 °C, at the point of maximum GOS concentration (1 h). The synthesized GOS correspond to (4) 6-Galactobiose; (5) Allolactose; (6) 6′-O-β-galactosyl-lactose. Gal: Galactose; Glc: Glucose; Lact: Lactose. Adapted from Rodriguez-Colinas et al. (2014).

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TABLE 4.2  Carbohydrate Composition of UHT Skim Milk Treated at 40 °C with 0.1% (v/v) of B. circulans and K. lactis β-Galactosidases, at the Point of Maximum GOS Concentration Composition (g/L)

B. circulans

K. lactis

Lactose

28.1

2.1

Galactose

3.4

16.7

Glucose

6.9

20.2

6-Galactobiose

-

2.2

Allolactose

-

2.8

4-Galactobiose

0.3

-

6′-Galactosyl-lactose

-

1.7

4′-Galactosyl-lactose

6.7

-

Tetrasaccharide

0.6

-

Other GOS

-

0.3

Total GOS

7.6

7.0

a

a

Gal-β(1 → 4)-Gal-β(1 → 4)-Gal-β(1 → 4)-Glc.

6′-O-β-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc]. These results confirm that this enzyme exhibits a clear tendency to form β(1 → 6) linkages (Martinez-Villaluenga et al., 2008; Rodriguez-Colinas et al., 2011). Table 4.2 summarizes the carbohydrate composition of the β-galactosidase-treated milk at the point of maximum GOS concentration. As stated before, one of the main differences between K. lactis (Lactozym pure) and B. circulans (Biolactase) refers to the concentration of residual lactose when maximum GOS yield is achieved: 2.1 and 28.1 g/L, respectively. This could be related with the fact that the β(1 → 6) bonds are more resistant to enzymatic hydrolysis than β(1 → 4) linkages, and it is in agreement with the discovery that GOS with β(1 → 6) bonds have been detected as residual components in lactosefree ultra-heat treatment (UHT) milk and dairy drinks (Ruiz-Matute et al., 2012). In this context, Ruiz-Matute et al. (2012) analyzed the formation of GOS in milk at 30 °C treated with β-gal from K. lactis. The researchers reported that a residual lactose content lower than 1000 ppm (1 g/L) can be achieved with a GOS content of nearly 7.8 g/L. In our study, a similar GOS concentration (7.0 g/L) was obtained at 40 °C with K. lactis β-gal, but the remaining lactose was 2100 ppm. Further reduction of the lactose content to 360 ppm would lower the GOS concentration to 4.9 g/L (Figure 4.6). Figure 4.8 depicts the profile of GOS concentration versus lactose conversion determined for each enzyme at 40 °C. These profiles correlate well with those already published with buffered lactose solutions (Rodriguez-Colinas et al., 2011, 2012).

FIGURE 4.8  GOS formation versus lactose conversion using skim milk catalyzed by β-galactosidases from different sources at 40 °C. (a) B. circulans and (b) K. lactis. Adapted from Rodriguez-Colinas et al. (2014).

54  PART | I  Prebiotics in Health Promotion

With B. circulans β-gal, the maximum amount of GOS was produced at approximately 45-50% of lactose conversion. In contrast, when K. lactis β-gal was tested, the maximum GOS yield was achieved when 95% of the lactose had disappeared. However, after this point, GOS concentration suffered a sharp decrease at higher lactose conversions. The preceding results indicated that with K. lactis β-gal, it was possible to obtain treated milk with a GOS concentration similar to that of HMOs in human milk, and at the same time with a low content of lactose (2100 ppm).

7  EFFECT OF TEMPERATURE ON GOS FORMATION IN MILK From the industrial point of view, some dairy processes are preferably performed at 4 °C to prevent thermal degradation. Low temperatures are particularly appropriate for processing ice cream, in which β-galactosidase treatment gives a sweeter and creamier product that avoids lactose crystals when frozen. We analyzed the GOS formation at this temperature with the two enzymes (Figure 4.9), and data was compared with that obtained at 40 °C. Figure 4.9 illustrates that at lower temperatures, the time required to reach the maximum production of GOS was higher. The maximum GOS yield at 4 °C was obtained with B. circulans β-gal (8.1 g/L, 18% of total carbohydrates) and it was reached again at 50% of lactose conversion. Gosling et al. (2009) assayed the B. circulans β-gal preparation Biolacta in milk in the temperature range of 4-60 °C; they observed that GOS yield increased with temperature, as has been described

FIGURE 4.9  Kinetics of GOS formation in skim milk at 4 °C and 0.1% enzyme dosage using β-galactosidase from (a) B. circulans and (b) K. lactis. Adapted from Rodriguez-Colinas et al. (2014).

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FIGURE 4.10  Proposed process to obtain milk with low content of lactose and enriched in GOS.

in other transglycosylation processes (Linde et al., 2012; Ning et al., 2010). However, in our experiments, the GOS yield with B. circulans β-gal was similar at both temperatures. In the case of K. lactis, the maximum GOS concentration was lower at 4 °C compared to that obtained at 40 °C (4.8 vs. 7.0 g/L). In addition, the reaction time required to reach the maximum GOS yield was five fold higher (5 h at 4 °C vs. 1 h at 40 °C). When studying the effect of lactose conversion on GOS synthesis, the behavior was similar at 4 and 40 °C. The amount of residual lactose at the point of maximum GOS concentration was only 2.7 g/L.

8  PROPOSED METHOD TO OBTAIN LOW-LACTOSE, MILK-ENRICHED IN GOS In the dairy industry, lactose hydrolysis can be performed before or after thermal treatment. Heating prior to enzymatic treatment offers some advantages because the monosaccharides formed in the hydrolysis are more susceptible to suffer Maillard reactions during thermal treatment, contributing to the loss of essential amino acids such as lysine. However, Ruiz-Matute et al. (2012), analyzing the presence of several markers (furosine, tagatose, etc.) in various lactose-free UHT milk products, noticed that, in fact, UHT treatment is normally carried out after the enzymatic treatment with β-galactosidases. From the data obtained in our work, we suggest that enzymatic treatment be carried out before thermal treatment (Figure 4.10) because the β-galactosidase can be heat inactivated and thus the reaction stopped when a maximum GOS concentration is reached. On the contrary, if the enzymatic depletion of lactose was performed—under aseptic conditions—after thermal treatment of milk, it would not be possible to stop the reaction at will, which will cause hydrolysis of most of the synthesized GOS. The β-gal from K. lactis possesses another advantage that reinforces its use for the preparation of low-lactose, milkenriched in GOS, specifically its high susceptibility to heat inactivation. We demonstrated that this enzyme loses most of its activity in a short time even at moderate temperatures (Rodriguez-Colinas et al., 2011). Consequently, any of the thermal treatments employed by the dairy industry (UHT, pasteurization, etc.) will assure inactivation of the lactase, thus stopping the reaction at the point of maximum GOS production.

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Dairy J. 9, 69–80. Sela, D.A., Mills, D.A., 2010. Nursing our microbiota: molecular linkages between bifidobacteria and milk oligosaccharides. Trends Microbiol. 18, 298–307. Shadid, R., Haarman, M., Knol, J., Theis, W., Beermann, C., Rjosk-Dendorfer, D., Schendel, D.J., Koletzko, B.V., Krauss-Etschmann, S., 2007. Effects of galactooligosaccharide and long-chain fructooligosaccharide supplementation during pregnancy on maternal and neonatal microbiota and immunity—a randomized, double-blind, placebo-controlled study. Am. J. Clin. Nutr. 86, 1426–1437. Song, J., Abe, K., Imanaka, H., Imamura, K., Minoda, M., Yamaguchi, S., Nakanishi, K., 2011. Causes of the production of multiple forms of βgalactosidase by Bacillus circulans. Biosci. Biotechnol. Biochem. 75, 268–278. Torres, D.P., Goncalves, M., Teixeira, J.A., Rodrigues, L.R., 2010. Galacto-oligosaccharides: production, properties, applications, and significance as prebiotics. Compr. Rev. Food Sci. Food Saf. 9, 438–454. Tuohy, K.M., Rouzaud, G.C.M., Bruck, W.M., Gibson, G.R., 2005. Modulation of the human gut microflora towards improved health using prebiotics— assessment of efficacy. Curr. Pharm. Des. 11, 75–90. Tzortzis, G., Vulevic, J., 2009. Galacto-oligosaccharide prebiotics. In: Charalampopoulos, D., Rastall, R.A. (Eds.), Prebiotics and Probiotics Science and Technology. Springer, New York, pp. 207–244. Urrutia, P., Rodriguez-Colinas, B., Fernandez-Arrojo, L., Ballesteros, A.O., Wilson, L., Illanes, A., Plou, F.J., 2013. Detailed analysis of galactooligosaccharides synthesis with β-galactosidase from Aspergillus oryzae. J. Agric. Food Chem. 61, 1081–1087. Vera, C., Guerrero, C., Conejeros, R., Illanes, A., 2012. Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose. Enzyme Microb. Technol. 50, 188–194. Villamiel, M., Montilla, A., Olano, A., Corzo, N., 2014. Production and bioactivity of oligosaccharides derived from lactose. In: Moreno, J., Sanz, M.L. (Eds.), Food Oligosaccharides: Production, Analysis and Bioactivity. Wiley-Blackwell IFT Press, Chichester, pp. 137–167. Wei, L., Xiaoli, X., Shufen, T., Bing, H., Lin, T., Yi, S., Hong, Y., Xiaoxiong, Z., 2009. Effective enzymatic synthesis of lactosucrose and its analogues by β-galactosidase from Bacillus circulans. J. Agric. Food Chem. 57, 3927–3933. Yanahira, S., Kobayashi, T., Suguri, T., Nakakoshi, M., Miura, S., Ishikawa, H., Nakajima, I., 1995. Formation of oligosaccharides from lactose by Bacillus circulans β-galactosidase. Biosci. Biotechnol. Biochem. 59, 1021–1026. Zeuner, B., Jers, C., Mikkelsen, J.D., Meyer, A.S., 2014. Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics. J. Agric. Food Chem. 62, 9615–9631. Zivkovic, A.M., German, J.B., Lebrilla, C.B., Mills, D.A., 2011. Human milk glycobiome and its impact on the infant gastrointestinal microbiota. In: Proc. Natl. Acad. Sci. U. S. A., 108, pp. 4653–4658.

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Chapter 5

Intestinal Microbiota in Breast-Fed Infants: Insights into Infant-Associated Bifidobacteria and Human Milk Glycans Nina Kirmiz*,† and David A. Mills*,†,‡ *Food Science & Technology, University of California, Davis, California, USA, †Foods for Health Institute, University of California, Davis, California, USA, ‡Viticulture & Enology, University of California, Davis, California, USA

1 INTRODUCTION Formation of the human host-microbe symbiosis during early life is a complex and important biological process. In humans, the intestinal microbiota plays a key role in host physiology, and understanding the establishment of this symbiosis is of significant interest (Scholtens et al., 2012). The intestinal microbiota is dynamic during the first years after birth. The infant gastrointestinal tract (GIT) is rapidly colonized through events related to the process of giving birth to the offspring (Adlerberth and Wold, 2009; Sela and Mills, 2014; Thum et al., 2012). Exposure to vaginal, fecal, epidermal, and milk microbiota are among the various routes by which microbial inoculation may occur (Cabrera-Rubio et al., 2012; DominguezBello et al., 2010; Sela and Mills, 2014). The mode of delivery, type of feeding, cultural influences, and geographical factors also affect the establishment of bacterial communities in the gut. Furthermore, the development of the intestinal microbiota is highly dependent on the diet, which changes from milk to a multifaceted adult diet (Scholtens et al., 2012). Breast-feeding is associated with numerous positive effects on the neonate (Smilowitz et al., 2014). Studies have linked breast-feeding with a reduction in the risk of asthma, obesity, type 1 and type 2 diabetes, and necrotizing enterocolitis, among other improved health outcomes (Ip et al., 2007). Human milk is a complex and unique fluid shaped by evolution to provide nutrients to the developing infant. In addition to providing nourishment to the infant, human milk has numerous bioactive components that provide protection against pathogens, direct enrichment of beneficial microorganisms, and modulation of the immune system (Field, 2005; Hamosh, 2001; Lonnerdal, 2013; Sela and Mills, 2014).

2  INTESTINAL MICROBIOTA IN BREAST-FED INFANTS The infant GIT is quickly colonized through events associated to the process of the birth of the offspring (Adlerberth and Wold, 2009). Over 1000 species of bacteria will colonize the intestine during the first year (Weng and Walker, 2013). Early colonizers of the gut typically include facultative anaerobes, which are followed by strict anaerobes such as Bifidobacterium, Bacteroides, and Clostridium (Matamoros et al., 2013). Within the first 3 years after birth, the microbiota of the infant matures and moves toward an adult-like microbiota (Groer et al., 2014; Palmer et al., 2007; Yatsunenko et al., 2012). In the early stages of life, development of the gut microbiota is dependent on the diet (Scholtens et al., 2012). Various studies have shown that Bifidobacterium is a predominant genus in the microbiota of breast-fed infants (Harmsen et al., 2000; Roger and McCartney, 2010; Yatsunenko et al., 2012). The species of Bifidobacterium that are most frequently found in breast-fed infants are Bifidobacterium breve, Bifidobacterium longum subsp. infantis (B. infantis), Bifidobacterium longum subsp. longum (B. longum) and, to a lesser extent, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum (Avershina et al., 2013; Roger et al., 2010; Ruiz-Moyano et al., 2013; Turroni et al., 2012a). Bifidobacterium adolescentis and Bifidobacterium animalis are more commonly associated with the intestinal microbiota of adults (Mangin et al., 2006; Sela and Mills, 2010; Turroni et al., 2012a). Variations in infant intestinal microbiota due to geographical factors have been found (Grzeskowiak et al., 2012; Huda et al., 2014; Yatsunenko et al., 2012). An examination of the human microbiome from individuals from the Amazons of Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00005-8 © 2016 Elsevier Inc. All rights reserved.

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Venezuela, rural Malawi, and U.S. metropolitan areas revealed significant differences in the phylogenetic composition of the fecal microbiota between individuals from different countries (Yatsunenko et al., 2012). Fallani and colleagues showed that the country of birth is believed to influence infant fecal microbiota composition, with Stockholm (Sweden) and Glasgow (UK) having overall higher proportions of bifidobacteria, Atopobium, and Clostridium perfringens and Clostridium difficile than Düsseldorf (Germany), Reggio Emilia (Italy), and Granada (Spain) (Fallani et al., 2010). Additionally, Grzeskowiak and colleagues reported that in Malawian and Finnish infants, bifidobacteria were dominant at 6 months of age; however, Malawian infants had greater proportions of bifidobacteria than Finnish infants (Grzeskowiak et al., 2012). Furthermore, Young and associates compared the feces of infants born in Ghana, New Zealand, and the United Kingdom and determined that the majority of the fecal samples from Ghana contained B. infantis, whereas the fecal samples from infants from New Zealand and the United Kingdom did not (Young et al., 2004). The mode of delivery is another factor that can affect the establishment of the infant gut microbiota. Dominguez-Bello and colleagues examined nine Mestizo and Amerindians women and ten newborns for the influence of delivery mode and body habitat on the microbiota of the neonate (Dominguez-Bello et al., 2010). They found that vaginally delivered infants had bacterial communities comparable to their mother's vaginal microbiota and were abundant in Lactobacillus, Prevotella, or Sneathia spp. However, C-section-delivered infants acquired fecal bacterial communities in the first few days of life that were similar to communities found on the skin of the mother, including taxa such as Staphylococcus (Dominguez-Bello et al., 2010). How long these microbiota differences persist is unclear. In a different study, Huda and colleagues reported that in a cohort of 48 Bangladeshi infants, 83% of which were born by cesarean delivery, the stool microbiota were still dominated by bifidobacteria (primarily B. infantis) by the 15th week (Huda et al., 2014). Other researchers have noticed a lower abundance of Bacteroides colonization in C-section babies over time and have suggested these differences may be a contributing factor to differences in the Th1-associated chemokines (Jakobsson et al., 2014).

3  HUMAN MILK COMPOSITION AND COMPLEXITY Human breast milk is a unique and complex fluid shaped by years of evolution and is produced at the mother's expense. It is believed that lactation evolved to maximize energy and nutrient utilization by the developing infant while simultaneously minimizing the mother's energy expenditure (Hernell, 2011). Milk is made of components that meet the numerous demands of the developing infant, including lactose, fatty acids, human milk oligosaccharides (HMOs), proteins, vitamins, minerals, and nucleotides (Hernell, 2011; Petherick, 2010; Picciano, 2001).

4  ANTIMICROBIAL ACTIVITIES IN HUMAN MILK Human breast milk is well known to contain a combination of direct-acting antimicrobial factors that can provide protection against infection (Isaacs, 2005). These defense factors have very diverse antimicrobial activities (Lonnerdal, 2013). Proteins in milk such as lysozyme, lactoferrin, immunoglobulins, lactoperoxidase, bile salt-stimulated lipase, and α-lactalbumin have antimicrobial activity (Lonnerdal, 2003, 2013). A well-known antimicrobial protein, lactoferrin, is associated with antimicrobial processes via multiple activities, including iron depletion and cell membrane disruption (Embleton et al., 2013; Lonnerdal, 2003; Sanchez et al., 1992). Another antimicrobial factor, lysozyme, is capable of degrading the outer cell wall of Gram-positive bacteria and, with lactoferrin, can kill Gram-negative bacteria (Ellison and Giehl, 1991; Lonnerdal, 2003). Antimicrobial lipids, antimicrobial peptides, and antibodies are also important in pathogen inactivation and removal (Isaacs, 2005). Recently, Dallas and associates used a peptidomics approach to analyze peptides naturally occurring in freshly expressed human milk (Dallas et al., 2013). These assays showed that growth of Escherichia coli and Staphylococcus aureus was inhibited by endogenous milk peptides (Dallas et al., 2013). Another important component of human breast milk that may serve as a defense against pathogens is glycans found in milk. Many enteric pathogens use cell surface glycans to recognize and bind to their target cells (Newburg et al., 2005). Human epithelial cell surface glycans and glycans found in milk have a structural resemblance, and milk glycans can therefore serve as soluble receptors that block pathogen attachment to host cells (Bode and Jantscher-Krenn, 2012). Pathogenesis of Campylobacter jejuni, which can cause diarrhea, involves adherence to the intestinal mucosa, and fucosylated HMOs appear to block C. jejuni from binding and infection (Newburg et al., 2005; Ruiz-Palacios et al., 2003). In the case of HIV-1, entry across the infant's intestinal mucosal barrier is mediated partially by binding of HIV-1 glycoprotein gp120 to dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) on human dendritic cells, and HMO reduce HIV-1 glycoprotein gp120 binding to DC-SIGN (Hong et al., 2009). HMOs have been linked to inhibiting

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adhesion to intestinal cells of diarrheal pathogens such as E. coli, Vibrio cholerae, and Salmonella fyris (Coppa et al., 2006). According to Manthey and colleages, HMOs were shown to protect against enteropathogenic E. coli attachment to cultured epithelial cells and reduce colonization in suckling mice (Manthey et al., 2014). In another study, bladder epithelial cells pretreated with HMO had a significant reduction in uropathogenic E. coli (UPEC) internalization; however, interestingly, this particular study did not show HMO pretreatment having a significant effect on UPEC binding to bladder epithelial cells (Lin et al., 2014). Free glycans in milk are not alone in their ability to inhibit pathogen binding to host cells, as glycoconjugates in milk also have inhibitory activities against various pathogens (Liu and Newburg, 2013). Human milk mucins can inhibit pathogen binding, and examining the roles of specific glycan constituents such as sialic acid found in milk mucins is of significant interest to elucidate the mechanisms of pathogen interaction (Liu and Newburg, 2013; Liu et al., 2012; Yolken et al., 1992). For example, rotavirus can bind to milk mucin, however, binding is significantly reduced after removal of the sialic acid (Yolken et al., 1992). Another glycoprotein found in milk, lactadherin, has been reported to protect against rotavirus (Newburg et al., 1998). Secretory IgA (sIgA) is an immunoglobulin found in human milk, and it is believed that the glycans in sIgA can serve as decoys to prevent pathogen binding to host surfaces (Arnold et al., 2007; Liu and Newburg, 2013; Murthy et al., 2011). An example of this is demonstrated in the inhibition of V. cholerae biofilm formation by the mannose glycans of sIgA (Murthy et al., 2011).

5  HUMAN MILK GLYCANS A feature of one class of bioactive agents found in human breast milk is that they are glycosylated (Garrido et al., 2013a). Glycans in milk can be found as free HMOs or conjugated to proteins or lipids (Garrido et al., 2013a). It is suggested that there is minimal degradation of milk glycoproteins in the upper GIT (Dallas et al., 2012). One very interesting feature of HMOs is they are indigestible to the infant and can reach the large intestine (Coppa et al., 2001; Smilowitz et al., 2014).

6  HMO STRUCTURES AND PROPERTIES HMOs are the third most abundant component of human breast milk after lactose and lipids (Garrido et al., 2013a; Petherick, 2010). Hundreds of different oligosaccharide structures have been identified in human milk (Wu et al., 2010, 2011). With a few exceptions, lactose is found at the reducing end of these oligosaccharides (Kunz et al., 2000). This lactose core can be modified by fucose or sialic acid to make 2′-fucosyllactose (2′-FL), 3′-fucosyllactose (3′-FL), 3′-sialyllactose (3′-SL), and 6′-sialyllactose (6′-SL) (Bode and Jantscher-Krenn, 2012). The lactose core can also be modified by the addition of repeating units of lacto-N-biose (Galβ1-3GlcNAc; LNB) or N-acetyllactosamine (Galβ1-4GlcNAc) (Bode and Jantscher-Krenn, 2012; Garrido et al., 2013a). These additions can be further decorated with fucose bound in an α1-2, α1-3, or α1-4 linkage or sialic acid bound at an α2-3 or α2-6 linkage (Bode and Jantscher-Krenn, 2012). Pooled HMOs contain both neutral and acidic glycans, the latter depending on the presence of a negatively charged sialic acid (Bode and Jantscher-Krenn, 2012; Wu et al., 2011). Figure 5.1 presents examples of select HMO structures. There is some variation among HMO composition between different women, as well as from the same woman, d­ epending on the stage of lactation (Bode and Jantscher-Krenn, 2012; Davidson et al., 2004; De Leoz et al., 2012). Between different women, there can be large variation in terms of HMO fucosylation (Bode and Jantscher-Krenn, 2012). A main difference is related to the secretor status of the mother. The “secretor” gene, fucosyltransferase 2 (FUT2), catalyzes the transfer of fucose residues to glycans via an α1-2 linkage to form α1-2 fucosylated glycans found in HMOs such as 2′-­FL and lacto-N-fucopentaose I (LNFP I) (Bode and Jantscher-Krenn, 2012; Kumazaki and Yoshida, 1984). Because “non-secretor” women do not possess a functional FUT2 gene, they do not express a significant level of α1-2 ­fucosylated glycans. Even among “secretor” mothers, the concentration of α1-2 fucosylated HMOs such as 2′-FL can vary across mothers and over the course of the lactation period (Castanys-Munoz et al., 2013; Chaturvedi et al., 2001; Erney et al., 2000; Thurl et al., 2010). Another variation is seen in the FUT3 gene, which is associated with α1-3/4 fucosyltransferase activity and encodes an enzyme that generates the Lewis a and Lewis b antigens (Bode and Jantscher-Krenn, 2012; Johnson and Watkins, 1992; Totten et al., 2012). To summarize, FUT2 and FUT3 gene status can generate four possible phenotypes: Se+Le+, Se−Le+, Se+Le−, and Se−Le− (Se = secretor; Le = Lewis). Therefore, there can be significant variation among the fucosylation of HMOs based on the woman's secretor and Lewis blood group status (Bode and Jantscher-Krenn, 2012; Totten et al., 2012). For example, a Se+Le+ woman can have a complex composition of fucosylated HMOs based on the many possible fucosylated linkages (Bode and Jantscher-Krenn, 2012).

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FIGURE 5.1  Examples of several select human milk oligosaccharide structures are shown. Variability in linkages and monosaccharide components creates the high structural diversity that HMOs possess. Human milk oligosaccharides contain lactose at the reducing end and can be elongated with lacto-Nbiose or N-acetyllactosamine. Lactose or oligosaccharide chains can be decorated with fucose or sialic acid. Human milk oligosaccharides are categorized as neutral HMOs if they lack sialic acid or acidic HMOs if they contain sialic acid.

There are differences between milk oligosaccharides found in human milk and milk oligosaccharides found in other animal species, including bovine, porcine, and primate milks (Tao et al., 2008, 2010, 2011). Bovine milk contains oligosaccharides that are complex and structurally related to oligosaccharides found in humans (Aldredge et al., 2013). However, HMOs are highly fucosylated, but bovine milk oligosaccharides (BMOs) do not exhibit fucosylation at appreciable levels (Aldredge et al., 2013; Tao et al., 2008, 2009). Aldredge et al. recently annotated and elucidated the structures of BMOs from pooled bovine milk colostrum samples and showed that while fucosylated oligosaccharides are present, the total amount of fucosylation present is less than 1%, which correlated with previous studies (Aldredge et al., 2013; Tao et al., 2008, 2009). Interestingly, BMOs are significantly more sialylated than HMOs (Ninonuevo et al., 2006; Tao et al., 2008; Wu et al., 2011). Similarly, porcine milk oligosaccharides also are highly sialylated (Tao et al., 2010). Milk oligosaccharides from different primates have also been characterized (Tao et al., 2011). In general, oligosaccharide pools from primate milk, including humans, are more varied and more complex than oligosaccharide pools from nonprimate milks such as bovine and porcine (Tao et al., 2011).

7  STRUCTURE-FUNCTION RELATIONSHIPS OF HMOS There is a major interest in understanding and elucidating the numerous functional implications of the structure and diversity of HMOs (Smilowitz et al., 2014). In addition to their association with the deflection of numerous pathogens, HMOs evade digestion and enrich commensals that can utilize these carbohydrates (Coppa et al., 2001; Sela and Mills, 2010; Smilowitz et al., 2014). Ward and colleagues first showed that B. infantis ATCC 15697 could grow to high cell densities in vitro on HMOs as a sole carbon source, and subsequent studies showed that other strains of B. infantis are similar (Locascio et al., 2009; LoCascio et al., 2007; Ward et al., 2006, 2007). Additionally, strains of B. bifidum have the ability to grow well on HMOs as a sole carbon source (Asakuma et al., 2011; Kitaoka, 2012). Strains of B. breve and B. longum are able to consume HMOs; however, there appears to be more strain-to-strain variability in this phenotype (Asakuma et al., 2011; Locascio et al., 2009; Ruiz-Moyano et al., 2013). Marcobal and colleagues examined 16 bacterial strains of different genera for the ability to consume HMOs and found that Bacteroides fragilis and Bacteroides vulgatus were also able to metabolize HMOs and reach high cell densities

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d­ uring growth on HMOs (Marcobal et al., 2010). This study also showed that Enterococcus, Streptococcus, Veillonella, Eubacterium, Clostridium, and E. coli were either unable to grow on HMOs or grew poorly (Marcobal et al., 2010). Glycoprofiling was employed to examine HMO consumption patterns and revealed that B. infantis and B. vulgatus have a preference for fucosylated HMOs (Marcobal et al., 2010). Further analysis confirmed various Bacteroides species possess the ability to grow on HMOs (Marcobal et al., 2011). In a recent study, Yu and colleagues tested 25 of the major isolates of human intestinal microbiota and showed that strains of Bifidobacterium and Bacteroides grew on select fucosylated and sialylated HMO components, whereas strains of Lactobacillus delbrueckii, Enterococcus faecalis, and Streptococcus thermophilus exhibited only slight growth on 2′-FL or 3′-FL (Yu et al., 2013). In this study, several strains of Bifidobacterium and Bacteroides induced α-L-fucosidase activity and produced short-chain fatty acids when grown on the fucosylated HMO 2′-FL, 3′-FL, and lactodifucotetraose (Yu et al., 2013). Patterns have been identified in the relationship between HMOs and the infant gut community. De Leoz and colleagues examined infant fecal HMOs in relation to fecal bacterial population in two healthy infants over the first few weeks of life. They used bacterial DNA sequencing and mass spectrometry, which showed by week 13 that there was a decrease in fecal HMOs, whereas Bacteroides spp. and Bifidobacterium spp. had increased (De Leoz et al., 2014). This study showed that HMO consumption appears to be structure specific with certain isomers being consumed (De Leoz et al., 2014). For example, data suggested that among four fucosylated lacto-N-fucopentaose isomers, LNFP II, which contains α-1,4-fucosylation, was not consumed over the first 13 weeks of life, whereas the other three lacto-N-fucopentaose isomers, which contain α-1,2-fucosyl and α-1,3-fucosyl linkages, were consumed (De Leoz et al., 2014). In a recent study, Wang and associates assessed the microbiota of the feces of 16 breast-fed and 6 formula-fed infants and examined HMO content of collected human milk (Wang et al., 2015). Partial least squares regression of HMOs and the infant gut microbiota showed several bacterial genera such as Bifidobacterium, Bacteroides, and Enterococcus could be predicted by their mother's HMO profiles (Wang et al., 2015). Another study suggests that mother’s secretor status drives differences in the infant microbiota. Bifidobacteria are established at an earlier time and more often in infants fed by secretor mothers than infants fed by nonsecretor mothers (Lewis et al., 2015). Moreover, a higher percentage of bifidobacterial isolates from secretor mothers were able to grow on 2′-FL as a sole carbon source than isolates from nonsecretor mothers (Lewis et al., 2015). Lewis and colleagues showed that bifidobacteria-dominated feces have lower absolute amounts of fucosylated HMOs than Bacteroidesdominated feces (Lewis et al., 2015).

8  BIFIDOBACTERIAL STRATEGIES OF HMO CONSUMPTION A number of studies have provided insight into specific bifidobacterial strategies of HMO transport and catabolism, ­reviewed in various venues (Garrido et al., 2012a, 2013a; Kitaoka, 2012; Marcobal and Sonnenburg, 2012). Genome analysis, coupled with functional studies, has helped to elucidate the catabolic pathways B. infantis ATCC 15697 employs to grow on HMOs (Garrido et al., 2011, 2012c; Sela et al., 2008, 2011, 2012; Yoshida et al., 2012). B. infantis ATCC 15697 has several HMO-related clusters that are shared among other B. infantis isolates but are absent in other bifidobacteria such as B. longum DJO10A and B. adolescentis ATCC 15703, which grow weakly, on or do not grow, on HMOs (respectively) (Zivkovic et al., 2011). Notably, B. infantis ATCC 15697 possesses a unique 43-kbp cluster, HMO cluster 1, encoding transport systems and glycosyl hydrolases necessary for HMO import and metabolism (Sela and Mills, 2010). A range of family 1 solute-binding proteins (SBPs) with affinity for HMOs and intracellular glycosyl hydrolases with activity on HMO in B. infantis support the model that B. infantis imports HMOs intact prior to intracellular degradation (Garrido et al., 2011, 2012a, 2013a; Sela et al., 2011, 2012; Sela and Mills, 2010; Yoshida et al., 2012). Garrido and colleagues examined the binding specificity of a number of family 1 SBPs purified from B. infantis to an array of mammalian glycans and demonstrated several SBPs have a preference for glycans similar to HMOs (Garrido et al., 2011). For example, Blon_2177 bound to type 1 polymers such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc; LNT) and lactoN-hexaose (LNH), which are abundant structures in HMOs (Garrido et al., 2011). SBPs Blon_2344 and Blon_2347 bound to type 2 glycans found in HMOs such as lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc; LNnT) (Garrido et al., 2011). SBP recognition of fucosylated structures was also determined for both Blon_0343 and Blon_2202 recognizing Fucα1-2Gal, which is a fucosylated structure found in the fucosylated HMO 2′-FL (Fucα1-2Galβ1-4Glc) and also in the ABO blood group (Garrido et al., 2011). This binding specificity also correlated with expression of these family 1 SBPs during growth on HMOs (Garrido et al., 2011). A proteomic approach designed to target B. infantis ATCC 15697 cell wall/ surface proteins also showed that expression of certain family 1 SBPs with affinity for HMOs are expressed during growth on HMOs; however, significant expression of these family 1 SBPs is not observed during growth on glucose or lactose (Kim et al., 2013).

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In addition to family 1 SBPs, B. infantis ATCC 15697 has an array of glycosyl hydrolases such as β-galactosidases, α-fucosidases, α-sialidases, and N-acetyl-β-d-hexosaminidases that are involved in HMO utilization (Garrido et al., 2012c; Sela et al., 2011, 2012; Yoshida et al., 2012). These glycosyl hydrolases hydrolyze HMOs into monosaccharides, which are routed into the fructose-6-phosphate phosphoketolase pathway, also known as the “bifid-shunt” (Kim et al., 2013; Sela et al., 2008). Various studies have examined glycosyl hydrolases in B. infantis ATCC 15697 in relation to HMO hydrolysis (Garrido et al., 2012c; Sela et al., 2011, 2012; Yoshida et al., 2012). Fucosyl moieties in fucosylated HMOs as well as sialyl residues in sialylated HMOs can act as a “shield” on HMO preventing enzyme degradation and HMO digestion (Ashida et al., 2009; Sela et al., 2011, 2012). The release of these fucosyl and sialyl residues is the first step in the catabolism of fucosylated and sialylated HMOs by bacteria that are able to consume HMOs (Ashida et al., 2009; Sela et al., 2011, 2012). Five α-fucosidases are present in B. infantis ATCC 15697. Blon_2335 and Blon_2336 are α-fucosidases located in the HMO cluster 1 and belong to glycosyl hydrolase families 95 and 29, respectively (Sela et al., 2012). In B. infantis ATCC 15697, Blon_2335 is an efficient α1-2 fucosidase that also has activity on α1-3 and α1-4 fucosyl linkages and is able to release fucose from fucosylated HMOs such as 2′-FL, 3′-FL, and lacto-N-fucopentaoses (Sela et al., 2012). Blon_2336 is an α-fucosidase with α-1-3/4 specificity and has activity on fucosylated HMOs with these linkages such as 3′-FL and lacto-N-fucopentaose III (Sela et al., 2012). During growth on purified HMO sugars, the expression of both Blon_2335 and Blon_2336 are induced relative to growth on lactose (Sela et al., 2012). Interestingly, of the five α-fucosidases, the two located in HMO cluster 1 have the most evidence for involvement in removing fucose from fucosylated HMO (Sela et al., 2012). Two α-sialidases are present in B. infantis ATCC 15697 (Sela et al., 2011). Sialidase NanH2, which is located in HMO cluster 1, removes sialic acid from α2-3 and α2-6 sialyl linkages that are found in sialylated HMOs (Sela et al., 2011). This sialidase is induced during growth on HMOs relative to lactose and also is active on sialylated LNT (Sela et al., 2011). Sialidase NanH1 does not appear to be involved in HMO degradation. B. infantis ATCC 15697 has five genes encoding β-galactosidases, and two are used to degrade type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) HMO (Yoshida et al., 2012). Located distant from the HMO cluster 1, Blon_2016 encodes β-galactosidase Bga42a with specificity for type 1-like linkages such as that found in LNT (Yoshida et al., 2012). Located within the HMO cluster 1 is Blon_2334, which encodes β-galactosidase Bga2a, with specificity for lactose and type 2-like linkages (Yoshida et al., 2012). Three N-acetyl-β-d-hexosaminidases from B. infantis are induced during growth on HMOs and can cleave linkages found in HMOs (Garrido et al., 2012c). Blon_0459, Blon_0732, and Blon_2355 encode N-acetylβ-d-hexosaminidases and are expressed primarily during early growth on HMOs (Garrido et al., 2012c, 2013a). All three of these enzymes are active on the GlcNAcβ1-3 linkage that is found in LNT (Garrido et al., 2012c). Additionally, Blon_0459 and Blon_0732 have activity on GlcNAcβ1-6 linkages that are found in LNH (Garrido et al., 2012c). Another species of bifidobacteria, B. bifidum, uses HMOs by employing a strategy where extracellular glycosyl hydrolases are used (Kitaoka, 2012; Turroni et al., 2010). B. bifidum JCM1254 has two different membrane-associated fucosidases that are able to release fucose from fucosylated milk oligosaccharides (Ashida et al., 2009). An extracellular membrane anchored exo-α-sialidase, SiaBb2, from B. bifidum JCM1254 has activity on 3′-sialyllactose, 6′-sialyllactose, and disialyllacto-N-tetraose (DSLNT) (Kiyohara et al., 2011). SiaBb2 can liberate sialic acid from glycoproteins such as porcine gastric mucin containing sialylated O-glycoproteins (Kiyohara et al., 2011). An extracellular membrane bound β-galactosidase, BbgIII, and two extracellular membrane bound N-acetylhexosaminidases, BbhI and BbhII, have also been characterized from B. bifidum JCM1254 (Miwa et al., 2010). BbgIII is able to hydrolyze LNnT into galactose and lacto-N-triose II, and BbhI is able to hydrolyze lacto-N-triose II into N-acetylglucosamine (GlcNAc) and lactose (Miwa et al., 2010). Additionally, in B. bifidum JCM1254, a membrane lacto-N-biosidase, LnbB, has been characterized and found to have the ability to liberate LNB from LNT (Wada et al., 2008). The lacto-N-biosidase from B. bifidum acts on unmodified structures, but a novel lacto-N-biosidase with activity on more modified substrates such as LNFP I and sialyllacto-N-tetraose has recently been characterized from B. longum JCM1217 (Sakurama et al., 2013). Different species of bifidobacteria, such as strains of B. infantis, B. bifidum, B. longum, and B. breve, can grow in vitro on LNB as a carbon source (Kiyohara et al., 2009). A key difference between the B. infantis and B. bifidum modes of HMO consumption is that B. infantis imports HMO intact and utilizes intracellular fucosidases and sialidases to release fucose and sialic acid, whereas B. bifidum appears to extracellularly process HMOs (Garrido et al., 2012a, 2013a; Kitaoka, 2012; Sela and Mills, 2010; Turroni et al., 2010). Ward and colleagues first showed that B. bifidum ATCC 29521 left degraded HMO monomers such as fucose and sialic acid outside of the cell during in vitro growth on HMO (Ward et al., 2007). B. bifidum JCM1254 and B. infantis JCM1222 both grow robustly on HMOs; however, B. bifidum JCM1254 was shown to leave monosaccharide constituents of HMOs such as fucose outside of the cell during in vitro growth on HMOs whereas B. infantis JCM1222 did not (Asakuma et al., 2011). This makes sense from a genomic perspective, as B. bifidum PRL2010 has a limited number of genes encoding

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FIGURE 5.2  Models of consuming human milk oligosaccharides by bifidobacteria are shown. Importing HMOs and intracellular digestion of HMOs is the most common mode of consumption by infant-borne bifidobacteria. The model for B. longum subsp. infantis consumption of HMOs is that HMOs are imported intact prior to degradation by intracellular glycosyl hydrolases. Transporters with affinity for HMOs and glycosyl hydrolases with activity on HMOs support this model. Strains of B. longum subsp. longum and B. breve that can consume HMOs appear to have a mode of consumption similar to that of B. longum subsp. infantis. B. bifidum uses HMOs by employing extracellular glycosyl hydrolases to process the HMOs followed by import and degradation of select components.

carbohydrate transporter systems in contrast to other infant-associated bifidobacteria such as B. infantis, B. breve, and B. longum (Roger et al., 2010; Turroni et al., 2012a,b). Ward and colleagues also examined strains of each of B. infantis, B. bifidum, B. breve, B. longum, and B. adolescentis for the ability to grow on the monosaccharide constituents of HMOs and found that, although all strains tested were able to ferment glucose and galactose (components of HMOs), only B. ­infantis and B. breve were able to ferment glucosamine, fucose, and sialic acid (Ward et al., 2007). Recent work reveals that B. bifidum growth on FL and SL induces a transcriptome profile similar to that induced during growth on lactose, providing further evidence that fucose and sialic acid are not consumed (Garrido et al., in press). Strains of B. breve and B. longum that can consume HMOs appear to have a mode of glycan consumption similar to that of B. infantis involving importing HMOs whole and subsequently employing intracellular glycosyl hydrolases to degrade the imported HMOs (Ruiz-Moyano et al., 2013) (D. A. Mills, unpublished data). Thus, intracellular import and digestion of HMOs appears to be a more common mode of consumption by most infant-borne bifidobacteria, a process that is clearly and dramatically different in B. bifidum. A comparison of the models of utilizing HMO by B. infantis, B. breve, B. longum, and B. bifidum is shown in Figure 5.2.

9  HUMAN MILK GLYCOPROTEINS AND GLYCOLIPIDS Protein glycosylation is a posttranslational modification where a glycan is covalently linked to amino acids in the protein structure (Garrido et al., 2013a; Moremen et al., 2012). The structure of the glycan attached to the protein can be very complex (Moremen et al., 2012). In eukaryotes, there are two major types of protein glycosylation: N-linked and O-linked (Moremen et al., 2012). In N-linked glycans, the glycan is linked to an asparagine and has a standard consensus sequence of Asn-X-Ser/Thr; however, there are known nonstandard sequences such as Asn-X-Cys (Moremen et al., 2012). O-glycosylation occurs at a serine or threonine residue but does not have a common consensus sequence for attachment (Moremen et al., 2012). Glycoproteins present in human milk are an important component in numerous aspects. Significant protein glycosylation in milk suggests structure-specific roles, and it is estimated that 70% of abundant milk proteins are glycosylated (Froehlich et al., 2010). Human milk glycoproteins can serve as a defense against infection (Lonnerdal, 2003; Peterson et al., 1998). Glycoproteins found in human milk include mucins, secretory immunoglobulin A (sIgA), bile salt-stimulated lipase (BSSL), lactoferrin, lactoperoxidase, lactadherin, butyrophilin, and a subunit of casein (Newburg, 2013). Mucins are high-molecular-weight glycoproteins linked to diverse functions, and human milk mucins have been associated with protection against infection from pathogens (Newburg, 2013; Yolken et al., 1992). Immunoglobulin A is found at highest concentrations in the colostrum and is an important means of passive immunity (Froehlich et al., 2010; Hanson, 1998). Glycoprotein, BSSL, is an enzyme with lipolytic activity and has a broad substrate specificity (Hernell and Olivecrona, 1974).

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Lactoferrin is another major milk antimicrobial glycoprotein that is a member of the transferrin family and has a twofold internal homology (Anderson et al., 1987). Lactoferrin was recently shown to inhibit pathogen adhesion to intestinal cells and reduce Salmonella invasion of colonic epithelial cells (Barboza et al., 2012). One aspect in which protein glycosylation affects the protein is by providing a defense against proteolysis (Smilowitz et al., 2014; van Berkel et al., 1995). An example of this is seen in lactoferrin where glycosylated and unglycosylated lactoferrin differ in their resistance to tryptic proteolysis (van Berkel et al., 1995). Additionally, variation in expression and glycosylation of the glycoproteome of human milk have been observed (Froehlich et al., 2010). For example, analysis of lactoferrin during different time periods over lactation reveals changes in glycosylation (Froehlich et al., 2010). Glycolipids are another type of glycoconjugates present in human milk. These glycolipids are located almost exclusively in the outer part of the milk fat globule membrane (Newburg and Chaturvedi, 1992; Smilowitz et al., 2014). Similar to glycoproteins, milk glycolipids are associated with pathogen deflection (Garrido et al., 2013a; Newburg, 2013; Otnaess et al., 1983). Human milk glycolipids occur mainly in the form of glycosphingolipids (Newburg, 2013). Gangliosides are glycosphingolipids that contain sialic acid and are present in human milk (Newburg, 2013). Gangliosides have a role as receptors for bacterial adhesion (Rueda, 2007; Smilowitz et al., 2014). They also function in cell-cell recognition, modulation of immunity, and modulation of membrane protein function (Rueda, 2007; Smilowitz et al., 2014).

10  CONSUMPTION OF HUMAN MILK GLYCOCONJUGATES BY BIFIDOBACTERIA Breast milk glycoproteins can play a role in shaping the intestinal microbiota (Garrido et al., 2013a). Different researchers have examined the ability of bacteria to degrade and modify glycoconjugates (Garrido et al., 2013a; Hoskins et al., 1985; Variyam and Hoskins, 1981). Interestingly, it has been shown that milk glycoproteins enrich bifidobacteria (HernandezHernandez et al., 2011; Kim et al., 2004; Petschow et al., 1999; Smilowitz et al., 2014). For example, lactoferrin has growth-promoting effects on Bifidobacterium spp. (Kim et al., 2004). Additionally, specific milk peptides derived from lactoferrin purportedly stimulate the growth of bifidobacteria (Liepke et al., 2002). Some studies have focused on elucidating mechanisms involved in degradation of glycoproteins by bifidobacteria (Garrido et al., 2012b, 2013a; Kiyohara et al., 2012). Infant-associated bifidobacteria endo-β-N-acetylglucosaminidases release N-glycans from glycoproteins (Garrido et al., 2012b). Specifically, endo-β-N-acetylglucosaminidase’ presence in isolates of B. longum, B. infantis, and B. breve correlates with the ability of these strains to deglycosylate the model glycoprotein bovine ribonuclease B (Rnase B) (Garrido et al., 2012b). From B. infantis ATCC 15695, endoglycosidase EndoBI-1 (glycosyl hydrolase family 18) has activity on all major types of N-linked glycans that are found in glycoproteins (Garrido et al., 2012b). This enzyme has activity on human milk glycoproteins such as human lactoferrin, IgA, and IgG and activity on glycoproteins with different glycosylation types (Garrido et al., 2012b). Furthermore, some strains of bifidobacteria are capable of degrading mucin (Crociani et al., 1994; Hoskins et al., 1985). From B. bifidum JCM1254, a novel α-Nacetylgalactosaminidase, NagBb (glycosyl hydrolase family 129), was identified and shown to cleave specific O-linked glycans that can be found in mucin (Kiyohara et al., 2012).

11  BIFIDOBACTERIA AND HEALTH BENEFITS TO THE INFANT Understanding the specific role glycans play in the enrichment of bifidobacteria in the infant GIT is of significant interest. Findings indicate that HMO and the enrichment of a bifidobacterial-dominant microbiota can support intestinal barrier function and modulate immunity (Chichlowski et al., 2012; Smilowitz et al., 2014). Enrichment of a beneficial microbiota containing bifidobacteria and fermentation of HMO results in production of lactic acid and acetate (Garrido et al., 2013b). It has been shown that acetate production by bifidobacteria protects against enteropathogenic infection. Fukuda and colleagues used mice associated with certain bifidobacterial strains and a model of lethal infection with enterohaemorrhagic E. coli O157:H7 to explore and elucidate molecular mechanisms of bifidobacterial modulation of host responses and protection from infection. One finding of this work was that the increased production of acetate was in part responsible for protection of mice from enteropathogenic infection by improving barrier function (Fukuda et al., 2011). Furthermore, short-chain fatty acids regulate colonic regulatory T cell homeostasis (Smith et al., 2013). Chichlowski and colleagues examined the relationship between HMO-grown bifidobacteria and intestinal epithelial cells and found that certain bifidobacteria, when grown on HMOs, positively modulate intestinal epithelial function (Chichlowski et al., 2012). In this study, B. infantis grown on HMOs had a significantly higher rate of adhesion to HT-29 compared with B. bifidum (Chichlowski et al., 2012). Binding of both B. infantis and B. bifidum grown on HMOs caused a higher level of antiinflammatory cytokine, interleukin-10 in Caco-2 cells compared to the same strains grown on lactose (Chichlowski et al., 2012). Another finding of this study was that both B. infantis and B. bifidum grown on HMOs caused less occludin

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relocalization than bacteria grown on lactose, which indicates a beneficial effect in maintaining epithelial barrier structure (Chichlowski et al., 2012). It has been shown that secreted bioactive factors from bifidobacteria are also effective in improving epithelial cell barrier function (Ewaschuk et al., 2008). Another study showed administration of bifidobacteria increased IgA levels in the feces (Fukushima et al., 1998). Various studies provide insight into the association between bifidobacteria and positive effects on the host (Fukuda et al., 2011; Huda et al., 2014; Lievin et al., 2000; Underwood et al., 2014). Probiotic studies using bifidobacteria can be a proxy for understanding how infant-borne bifidobacterial benefits might be relayed. Bifidobacteria are believed to provide protection to the newborn from pathogens, and this effect has been demonstrated using mice challenged with Salmonella typhimurium or E. coli O157:H7 (Gagnon et al., 2006; Lievin et al., 2000; Russell et al., 2011; Silva et al., 2004). Various studies have investigated the effects of bifidobacterial supplementation on infectious diarrhea (Chouraqui et al., 2004; Plummer et al., 2004; Qiao et al., 2002; Saavedra et al., 1994). B. bifidum and S. thermophilus supplementation can lessen the incidence of infectious diarrhea and rotavirus shedding in infants (Saavedra et al., 1994). Bifidobacteria are also associated with the formation of a healthy microbiota in preterm infants (Kitajima et al., 1997; Russell et al., 2011). For example, it has been reported that B. breve improves weight gain in very low birthweight infants (Kitajima et al., 1997). Furthermore, a recent study examined if the composition of stool microbiota correlated with specific infant vaccine responses (Huda et al., 2014). The stool microbiota was characterized at different points in time, and the response to oral polio virus (OPV), bacille Calmette-Guérin (BCG), tetanus toxoid (TT), and hepatitis B virus vaccines were measured (Huda et al., 2014). A positive association was found between the abundance of Actinobacteria and T cell responses to OPV, BCG, and TT, and B. infantis had positive associations with several vaccine responses (Huda et al., 2014). In another recent study, Underwood and colleagues examined the impact of B. infantis in a rat model of necrotizing enterocolitis (NEC) (Underwood et al., 2014). Administration of B. infantis reduced the impact of NEC, and inflammation associated with NEC was attenuated with B. infantis in a rat model (Underwood et al., 2014).

12  INFANT FORMULA A major goal in the production of infant formula is to closely resemble the numerous critical components found in breast milk (Hernell, 2011). The composition of infant formulas has changed throughout the years (Lonnerdal, 2014). Infant formulas often include bovine milk components. Bioactive components found in human milk are a noteworthy difference between human breast milk and bovine-based formulas (Garrido et al., 2013a; Hernell, 2011; Le Huerou-Luron et al., 2010). Prebiotics are defined as: “a selectively fermented ingredient that allows specific changes, both in the composition and/ or activity in the gastrointestinal microflora that confers benefits upon host well-being and health” (Roberfroid, 2007). Prebiotics such as fructo-oligosaccharides, galacto-oligosaccharides, and inulin are commonly added to infant formula; however, these compounds are structurally very different than HMOs (Garrido et al., 2013a; Gibson et al., 2004; Rycroft et al., 2001). Commercial production of simple HMO sugars is possible; however, the diversity and complexity of the constellation of glycan components delivered in human milk make commercial replication a daunting task. Furthermore, to add bioactive proteins to infant formula, various factors such as protein purity and possible contamination need to be considered (Lonnerdal, 2014). For example, it has been reported that commercial sources of bovine lactoferrin contain lipopolysaccharide, which may impede certain bioactivities of lactoferrin (Lonnerdal, 2014). Numerous studies have provided insight into differences between the bacterial colonization of breast-fed infants and formula-fed infants (Bezirtzoglou et al., 2011; Harmsen et al., 2000; Roger and McCartney, 2010; Sakata et al., 2005). The microorganisms that are found in the gut of breast-fed infants differ from those found in the gut of formula-fed infants. Formula-fed infants harbor a microbiota that is overall more diverse than that of breast-fed infants (Bezirtzoglou et al., 2011; Fallani et al., 2010; Penders et al., 2006). Formula-fed infants have lower numbers of Bifidobacterium and higher numbers of Bacteroides in their feces compared to breast-fed infants (Bezirtzoglou et al., 2011). It has also been reported that Atopobium is found in higher numbers in the feces from formula-fed infants compared to the feces from breast-fed infants (Bezirtzoglou et al., 2011). It is clear that additional research to identify and annotate the multifold functions of human milk components will simultaneously drive an increased search for structural and functional mimics that can improve infant formulas in the future.

13 CONCLUSIONS In conclusion, breast milk is a unique fluid shaped by evolution to provide nutrition and beneficial components to the neonate (Smilowitz et al., 2014). Numerous studies have provided insight into the relationship between components of milk, such as glycans and bioactive proteins, and the developing infant. The composition of breast milk shapes the intestinal

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microbiota. The interaction between human milk glycans and infant-associated bifidobacteria can ultimately be translated to allow for more persistent colonization of bifidobacteria. Investigating prebiotic effects and characterizing bifidobacterial strains can allow for development of synbiotic formulas and understanding probiotic strains. Further advancements in elucidating linkages between breast milk components, the intestinal microbiota, and the developing infant can give rise to treatment of intestinal maladies and modulation of infant health.

ACKNOWLEDGMENTS We acknowledge all of the researchers in the University of California—Davis Foods for Health Institute and the Milk Bioactives Program for their enthusiasm, imagination, and collective contribution to this subject matter. Work by the Milk Bioactives Program has been supported by the University of California—Davis Research Investments in the Sciences and Engineering Program; the Bill & Melinda Gates Foundation; and National Institutes of Health awards R01HD059127, R01HD065122, R01HD061923, R21AT006180, R01AT007079, and R01AT008759. Author Nina Kirmiz is supported in part by a Wine Spectator scholarship and author David A. Mills acknowledges support as the Peter J. Shields Endowed Chair in Dairy Food Science.

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Turroni, F., Bottacini, F., Foroni, E., Mulder, I., Kim, J.H., Zomer, A., Sanchez, B., Bidossi, A., Ferrarini, A., Giubellini, V., Delledonne, M., Henrissat, B., Coutinho, P., Oggioni, M., Fitzgerald, G.F., Mills, D., Margolles, A., Kelly, D., van Sinderen, D., Ventura, M., 2010. Genome analysis of Bifidobacterium bifidum PRL2010 reveals metabolic pathways for host-derived glycan foraging. Proc. Natl. Acad. Sci. U. S. A. 107, 19514–19519. Turroni, F., Peano, C., Pass, D.A., Foroni, E., Severgnini, M., Claesson, M.J., Kerr, C., Hourihane, J., Murray, D., Fuligni, F., Gueimonde, M., Margolles, A., De Bellis, G., O’Toole, P.W., van Sinderen, D., Marchesi, J.R., Ventura, M., 2012a. Diversity of bifidobacteria within the infant gut microbiota. PLoS One 7, e36957. Turroni, F., Strati, F., Foroni, E., Serafini, F., Duranti, S., van Sinderen, D., Ventura, M., 2012b. Analysis of predicted carbohydrate transport systems encoded by Bifidobacterium bifidum PRL2010. Appl. Environ. Microbiol. 78, 5002–5012. Underwood, M.A., Arriola, J., Gerber, C.W., Kaveti, A., Kalanetra, K.M., Kananurak, A., Bevins, C.L., Mills, D.A., Dvorak, B., 2014. Bifidobacterium longum subsp. infantis in experimental necrotizing enterocolitis: alterations in inflammation, innate immune response, and the microbiota. Pediatr. Res. 76, 326–333. van Berkel, P.H., Geerts, M.E., van Veen, H.A., Kooiman, P.M., Pieper, F.R., de Boer, H.A., Nuijens, J.H., 1995. Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis. Biochem. J. 312 (Pt 1), 107–114. Variyam, E.P., Hoskins, L.C., 1981. Mucin degradation in human colon ecosystems. Degradation of hog gastric mucin by fecal extracts and fecal cultures. Gastroenterology 81, 751–758. Wada, J., Ando, T., Kiyohara, M., Ashida, H., Kitaoka, M., Yamaguchi, M., Kumagai, H., Katayama, T., Yamamoto, K., 2008. Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure. Appl. Environ. Microbiol. 74, 3996–4004. Wang, M., Li, M., Wu, S., Lebrilla, C.B., Chapkin, R.S., Ivanov, I., Donovan, S.M., 2015. Fecal microbiota composition of breast-fed infants is correlated with human milk oligosaccharides consumed. J. Pediatr. Gastroenterol. Nutr. 60, 825–833. Ward, R.E., Ninonuevo, M., Mills, D.A., Lebrilla, C.B., German, J.B., 2006. In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri. Appl. Environ. Microbiol. 72, 4497–4499. Ward, R.E., Ninonuevo, M., Mills, D.A., Lebrilla, C.B., German, J.B., 2007. In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteria. Mol. Nutr. Food Res. 51, 1398–1405.

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Weng, M., Walker, W.A., 2013. The role of gut microbiota in programming the immune phenotype. J. Dev. Orig. Health Dis. 4, 203–214. Wu, S., Tao, N., German, J.B., Grimm, R., Lebrilla, C.B., 2010. Development of an annotated library of neutral human milk oligosaccharides. J. Proteome Res. 9, 4138–4151. Wu, S., Grimm, R., German, J.B., Lebrilla, C.B., 2011. Annotation and structural analysis of sialylated human milk oligosaccharides. J. Proteome Res. 10, 856–868. Yatsunenko, T., Rey, F.E., Manary, M.J., Trehan, I., Dominguez-Bello, M.G., Contreras, M., Magris, M., Hidalgo, G., Baldassano, R.N., Anokhin, A.P., Heath, A.C., Warner, B., Reeder, J., Kuczynski, J., Caporaso, J.G., Lozupone, C.A., Lauber, C., Clemente, J.C., Knights, D., Knight, R., Gordon, J.I., 2012. Human gut microbiome viewed across age and geography. Nature 486, 222–227. Yolken, R.H., Peterson, J.A., Vonderfecht, S.L., Fouts, E.T., Midthun, K., Newburg, D.S., 1992. Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. J. Clin. Invest. 90, 1984–1991. Yoshida, E., Sakurama, H., Kiyohara, M., Nakajima, M., Kitaoka, M., Ashida, H., Hirose, J., Katayama, T., Yamamoto, K., Kumagai, H., 2012. Bifidobacterium longum subsp. infantis uses two different beta-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology 22, 361–368. Young, S.L., Simon, M.A., Baird, M.A., Tannock, G.W., Bibiloni, R., Spencely, K., Lane, J.M., Fitzharris, P., Crane, J., Town, I., Addo-Yobo, E., Murray, C.S., Woodcock, A., 2004. Bifidobacterial species differentially affect expression of cell surface markers and cytokines of dendritic cells harvested from cord blood. Clin. Diagn. Lab. Immunol. 11, 686–690. Yu, Z.T., Chen, C., Newburg, D.S., 2013. Utilization of major fucosylated and sialylated human milk oligosaccharides by isolated human gut microbes. Glycobiology 23, 1281–1292. Zivkovic, A.M., German, J.B., Lebrilla, C.B., Mills, D.A., 2011. Human milk glycobiome and its impact on the infant gastrointestinal microbiota. Proc. Natl. Acad. Sci. U. S. A. 108 (Suppl. 1), 4653–4658.

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Chapter 6

Probiotics and Prebiotics for Promoting Health: Through Gut Microbiota Manoj Kumar*,#, Ravinder Nagpal†,#, Rajkumar Hemalatha*, Hariom Yadav‡ and Francesco Marotta§ Microbiology and Immunology Division, National Institute of Nutrition, Hyderabad, India, †Division of Laboratories for Probiotics Research, Juntendo University Graduate School of Medicine, Tokyo, Japan, ‡Diabetes, Endocrinology and Obesity Branch, Clinical Research Center, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA, §ReGenera Research Group for Aging Intervention, Milano, Italy *

1 INTRODUCTION The past decade has witnessed an increasingly great deal of quest and zest being dedicated to explicate the role of the gastrointestinal microbiota in health and diseases as well as explore and exploit novel ways to investigate and manipulate the gut microbial composition for an improved health and well-being. The gut microbiota has been observed to play a significant role in numerous metabolic and immunological functions, and a disturbed gut microbial balance has been underscored as an instigating factor for various metabolic, lifestyle, and diet-related maladies such as obesity, endotoxemia, insulin resistance, type 2 diabetes (T2DM), metabolic syndrome (MetS), inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), nonalcoholic fatty liver disease (NAFLD), colorectal cancer, atopic diseases, and more. The gut microbial ecosystem has been observed to be influenced by numerous factors such as host physiology, age, antibiotics, disease, diet, immune health, environment, prenatal exposure, and so on. Of these, diet has particularly been the subject of much interest, since diet is the major nutrient source for gut bacteria and dietary constituents could directly have a potential influence on the populations of gut microbiota and intestinal environment. Of various dietary options as an effective means to improve and/or restore the gut health and microbial balance, probiotics and prebiotics (or their combination as synbiotics) have attracted the special limelight owing to their research-backed safety aspects and myriad of potential health attributes.

2  HUMAN GUT MICROBIOTA: COMPLEXITIES, DIVERSITIES, FUNCTIONALITIES The human gastrointestinal tract (GIT) is sterile at birth; however, soon after birth, it is colonized quickly by a swarming and diverse bacterial population (Walter et al., 2011). These commensal microbes hail from various sources such as the mother, mother’s milk, nutrition, and the contiguous environment. The gut microbial composition differs significantly between individuals, and is modified and influenced by an individual’s diet, lifestyle habits, and the surroundings (Dominguez-Bello et al., 2011). An adult human body contains approximately 10 times more microbial cells than somatic cells and about 150 times more genes than our own genome (Backhed et al., 2005). There are more than 1000 bacterial species living in and on the human body, most of which are harbored by the gut, with hundreds of trillion organisms, including all three domains of life (i.e., bacteria, archaea, and eukaryotes) (The Human Microbiome Project Consortium, 2012), thereby making the human gut a significant vital “organ” and making us “superorganism.” Although characterizing the healthy gut microbial array and ascertaining the precise functions of thousands of different bacterial species still remains a challenge due to its extreme diversity and complexity; the importance of gut microbiota in maintaining and promoting health is well realized and recognized. The complex ecosystem of GIT involves a dynamic interplay between diet, host cells, and microbiota (Ley et al., 2006a,b; Dethlefsen and Relman, 2011). The gut microflora plays a significant role in numerous metabolic and immunological f­ unctions such as vitamin synthesis, immuno-modulation, maintenance of the epithelial homeostasis, intestinal Disclosure: Ravinder Nagpal is a postdoctoral fellow at Juntendo University; however, this manuscript does not represent any scientific or monetary views of Juntendo. The authors declare no competing financial interests or conflict of interests for writing and publishing this manuscript. # Equal contribution. Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00006-X © 2016 Elsevier Inc. All rights reserved.

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p­ ermeability and mucosal integrity, prevention of colonization of pathogens in the GIT, dietary energy harvest and metabolism, regulation of host fat storage, degradation of the indigestible dietary polysaccharides, fermentation of monosaccharides to short-chain fatty acids (SCFAs), cholesterol assimilation and reduction, maintenance of bowel health, xenobiotic/ drug metabolism, regulation of gut associated immune system, and, to some extent, cognitive health (Backhed et al., 2004, 2005; Rakoff-Nahoum et al., 2004; Flint et al., 2008; Wallace et al., 2011). In short, the human gut microbiota bestows many life-essential capacities to us that we would otherwise not be able to sustain ourselves without our microbiome. Thanks to the revolutionary advances in molecular, system, and computational biology, tools and technologies have made it possible to decode the compositional mysteries of the gut microbiota by sequencing 16S rRNA genes of numerous uncultivable members of the gut microbiota (Eckburg et al., 2005; Backhed et al., 2005). Recent investigations have revealed that the human gut microbiota is dominated by two major bacterial divisions—Firmicutes and Bacteroidetes—and one single Archaea phylotype Methanobrevibacter smithii (Backhed et al., 2005; Eckburg et al., 2005; Ley et al., 2006a). Additional bacterial phyla of the phylogenetic core gut microbiota are Actinobacteria, Proteobacteria, and Verrucomicrobia (Tap et al., 2009). Although huge deviations are found among Bacteroidetes phylotypes, majority of the Firmicutes are observed to be the members of the Clostridia class (Eckburg et al., 2005). Based on 16S rRNA-based sequences, nine phyla are observed with inconsistent dominance in the human large intestine, including Actinobacteria, Bacteriodetes, Cyanobacteria, Firmicutes, Fusobacteria, Proteobacteria, Spirochaeates, Lentisphaerae, and Verrucomicrobia in fecal and mucosal tissue samples from healthy adults (Eckburg et al., 2005; Ley et al., 2006a). In people consuming typical western diets high in carbohydrates and fats, fecal bacteria may comprise about 40-55% of solid stool (Stephen and Cummings, 1980). In humans, the distal gut (including cecum, colon, and rectum) is the most densely colonized part of the GIT with about 1012 to 1014 bacterial and yeast cells per gram of wet feces (Whitman et al., 1998). Although the diversity of gut microbiota varies significantly according to the genetics, nutrition, lifestyle, health, diseases, and other environmental factors, most people share a common core gut microbiota within a population of definite size (Qin et al., 2010). Nevertheless, it may be arguable, since the gut microbiota is highly host specific and may vary within an individual during the life span or in response to medication, relocation, disease-states, antibiotics, dietary changes, physical activities, and so on (Dethlefsen and Relman, 2011). Arumugam et al. (2011) have classified three robust bacterial clusters of typical human gut microbiota that are known as enterotypes. It is also proposed that a core gut microbiome could exist at the level of metabolic functions (Turnbaugh et al., 2009).

3  GUT MICROBIOTA BALANCE IN THE TRIANGLE OF NUTRITION, HEALTH, AND DISEASE Generally, the gut microbiota in a healthy state is mutualistic to the host and contributes significantly in several important functions (Backhed et al., 2005). The particular distribution of microbes along the different regions of GIT also hints at the metabolic adaptableness attained during human evolution. The upper gut is lightly occupied with bacteria so as to prevent competition with the host for digestible monosaccharides, whereas the lower gut is colonized with plentiful bacteria for fermentation of complex carbohydrates and proteins. It is observed that conventionally raised rats can metabolize about 80% of their dietary Kcal intake, but germ-free rats metabolize only about 72% of intake, thereby suggesting that gut microbiota is probably devouring about 8-10% of energy of the host’s dietary intake (Wostmann et al., 1983). Furthermore, the fermentation of glucose by gut inhabitants could provide up to 60% ATP as SCFAs to be utilized by colonocytes (Bergman, 1990). Backhed et al. (2004) revealed that inoculating germ-free mice with gut microbes from conventional mice can increase body fat content, carbohydrate absorption from gut lumen and de novo lipogenesis, and reduce food intake, which indicate that gut microflora can aid in maintaining colon health by harvesting and supplying energy required for the host health but may also enhance the predisposition to obesity by increasing body fat. Thanks to these intriguing studies in the past decade, the field of gut microbiota has emerged as a hot research topic for researchers, nutritionists, and clinicians, especially for its involvement in obesity, T2DM, MetS, and other associated risks. Besides obesity risk, a disturbed gut microbiota has also been found to exhibit harmful effects such as toxin/carcinogen production, intestinal putrefaction, diarrhea/constipation/bowel diseases, nonalcoholic liver damage, and other intestinal infections (Wallace et al., 2011). Gut pathogens such as Salmonella and Listeria may occasionally enter less-competitive niches, escape the lumen, and enter into epithelial cells. Other opportunistic pathogens such as C. difficile and C. perfringens may also flourish and lead to chronic ailments under disturbed homeostasis of gut microbiota. Under healthy conditions, however, gut commensals such as lactobacilli and bifidobacteria are able to check the overgrowth of pathogens and their entry to the host cells by producing antimicrobial metabolites, by competitively prohibiting the pathogens from inhabiting receptor mucosal sites, and by competing for sharing dwelling space and nutrients (Rastall, 2004). But, under “dysbiosis” settings, which may be caused by antibiotic use, dietary alterations, chemotherapy, contaminated foods, and the like, the balance of

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beneficial, commensal, and pathogenic microbes may get disturbed and lead to one or the other ill effects leading to intestinal infections, diarrhea, constipation, obesity, endotoxemia, insulin resistance, T2DM, MetS, IBD, IBS, colorectal cancers, NAFLD, and more (Ley et al., 2005; Cani et al., 2007, 2008; Qin et al., 2008; Schwiertz et al., 2010; Wallace et al., 2011; Boleij and Tjalsma, 2012; Nagalingam and Lynch, 2012).

4  FACTORS INFLUENCING THE GUT MICROBIOTA The gut microbiota composition is unique for each individual and the dominant gut microbial communities exhibit a notable steadiness over time during early adulthood until early old age (Eckburg et al., 2005; Ley et al., 2006a). However, various extrinsic factors—such as host genetics, maternal microbiota and nutrition, mode of delivery, breast/formula feeding, family environment, ageing, dietary and lifestyle habits, geographical impact, stress, disease, antibiotics/drugs, and intake of probiotics and prebiotics—could affect the composition and diversity of the gut microbiota (Salminen et al., 2004; Sartor, 2004; Lay et al., 2005; Mueller et al., 2006; Ley et al., 2006a; Woodmansey, 2007; Booijink et al., 2007; Jernberg et al., 2007; Abrahamsson et al., 2007; Kajander et al., 2008; Canani et al., 2007). Nevertheless, it remains to be seen which confounding factors play the main dynamic role in influencing the gut microbial structure and how and to what degree.

5  MODULATION OF GUT MICROBIOTA COMPOSITION Modulation of the gut microbiota is rapidly emerging as a prospective frontier for improved gut and cardio-metabolic health. Many approaches are being explored for positive modulation of GI microbiota; however, of these approaches, probiotics and prebiotics have drawn most attention owing to their safety and promising health benefits. Many probiotic strains, mostly from genera Lactobacillus and Bifidobacterium, have been characterized, clinically validated, and even commercialized for their health attributes, including improvement of lactose intolerance, reduction in pathobionts, improvement of diarrhea and constipation, immuno-modulation, cancer prevention, maintenance of gut microbial homeostasis and mucosal barrier integrity, and improvement of microbial metabolic activity (Salminen et al., 1999; Czarnecki-Maulden, 2008; Nagpal et al., 2012). In addition, various prebiotic oligosaccharides, such as fructo-oligosaccharides (FOS), galactooligosaccharides (GOS), and inulin, have received abundant interest because of the beneficial functions, such as production of favorable SCFAs (e.g., butyrate, maintenance of lower colonic pH, reduced pathogens, and immune modulation) (Gibson and Roberfroid, 1995; Gibson, 1998; Macfarlane et al., 2008; Czarnecki-Maulden, 2008).

6  PROBIOTICS: FOUNDATION AND DEFINITION The term probiotic was originally derived by the combination of a Latin preposition pro (meaning “for” or “in support”) with a Greek noun bios (meaning “biotic” or “life”). Thus, the word denotes “for life,” “in favor of life,” or “in support of life.” However, ever since the first proposed definition of probiotics as “substances secreted by one microorganism that stimulate another microorganism,” proposed by Lilly and Stillwell in 1965, the term has undergone an extensive modification over the subsequent years. A brief chronicle of evolution in the definition of the term probiotics is presented in Table 6.1. As in the case of antimicrobials and functional foods, the safety of probiotics has also been a concern for the consumers. However, after scrutiny of the extensive literature, it can be presumed that safety is not a major issue for probiotics because these microbes, especially lactic acid bacteria, have been used for many decades for making fermented food products. Furthermore, they are a regular element of the normal intestinal flora of humans (Fox, 1988). Consequently, since the pathogenicity linked with these probiotics has been highly uncommon, the probiotics are generally considered as safe to the host as well as to the environment. However, it must be noted that not all the strains or species of lactic acid bacteria or other probiotics are alike; and for that reason, particular characteristics have been proposed as a criteria for a good probiotic candidate to be used for any health use, particularly in humans (Fuller, 1989). The criteria that make a microbial strain eligible to be considered as a probiotic are as follows: ●

● ● ●

●

It should be a strain that is able to exert a beneficial outcome on the host animal—for instance, increased growth or disease resistance. It should be nonpathogenic, nonallergic, nontoxic, and noncarcinogenic. It should be present in viable form, and, if possible, in large numbers. It should be able to survive and metabolize in the gut environment—for example, it should be resistant to low pH, bile salts, organic acids, and so on. It should be stable and able to remain viable for longer periods under storage and field conditions.

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TABLE 6.1  Chronology of Proposed Definitions of the term Probiotics Definition

References

Substances secreted by one microorganism that stimulate another microorganism

Lilly and Stillwell (1965)

Tissue extracts that stimulate microbial growth

Sperti (1971)

Organisms and substances that have a beneficial effect on the host animal by contributing to its intestinal microbial balance

Parker (1974)

A live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance

Fuller (1989)

A viable mono- or mixed culture of microorganisms that, applied to animals or humans, beneficially affects the host by improving the properties of the indigenous microflora

Havenaar and Huis in’t Veld (1992)

Live cultures of microorganisms that are intentionally added into the rumen in order to improve the animal health or nutrition (Rumen probiotics)

Kmet et al. (1993)

A live microbial feed supplement that improves the intestinal microbial balance of the host animal

Cruywagen et al. (1995)

A live microbial culture of cultured dairy product that beneficially influences the health and nutrition of the host

Salminen (1996)

Viable bacteria, in a single or mixed culture, that have a beneficial effect on the health of the host

Donohue and Salminen (1996)

Living microorganisms that on ingestion in certain numbers exert health benefits beyond inherent basic nutrition

Guarner and Schaafsma (1998)

A microbial dietary adjuvant that beneficially affects the host physiology by modulating mucosal and systemic immunity, as well as improving nutritional and microbial balance in the intestinal tract

Naidu et al. (1999)

A preparation of or a product containing viable, defined microorganisms in sufficient numbers that alter the microflora (by implantation or colonization) in a compartment of the host and exert beneficial health effects in this host

Schrezenmeir and De Vrese (2001)

Natural live organisms either of bacteria or fungal cultures used as feed additives in livestock feeding and in human diets

Todd (2001)

Specific live or inactivated microbial cultures that have documented targets in reducing the risk of human disease or in their nutritional management

Isolauri et al. (2002)

Preparation of viable microorganisms that is consumed by humans or other animals with the aim of inducing beneficial effects by qualitatively or quantitatively influencing their gut microflora and/or modifying their immune status

Fuller (2004)

A preparation or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host, and exert beneficial health effects in this host

Roselli et al. (2005)

Live microorganisms when administered in adequate amounts confer a health benefit on the host

FAO/WHO (2009)

Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host

Hill et al. (2014)

Furthermore, in order for an eligible probiotic strain to be capable of exerting its beneficial effects, it should be able to demonstrate certain desirable characteristics, such as (1) acid and bile tolerance, (2) adhesion to mucosal and epithelial surfaces, (3) antimicrobial activity against pathogenic bacteria, and (4) bile salt hydrolase activity.

7  HEALTH BENEFITS OF PROBIOTICS Even though the health benefits of fermented foods have been recognized for centuries—long before the discovery of microorganisms—the notion of administering microbes for a positive health effect started over a century ago when Nobel laureate Elie Metchnikoff introduced for the first time the concept of probiotics to the scientific population (Metchnikoff, 1908).

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Competitive exclusion of enteric pathogens by blocking adhesion sites and competing for nutrients

Inhibition of enteric pathogens by producing lactic acid, hydrogen peroxides, bacteriocins, and other antimicrobial metabolites Maintenance of gut permeability integrity

Immune modulation and reduced inflammation

Enhancement of bowel motility

Lactose intolerance alleviation

Probiotics Mechanisms

Cholesterol lowering by assimilation and deconjugation

Maintenance of normal intestinal pH Suppression of toxin production, degredation of toxin receptor on the intestinal mucosaand neutralization/ detoxification of dietary carcinogens

Maintenance of normal gut microbiota and microbial homeostasis, and correction of dysbiosis

FIGURE 6.1  Proposed mechanisms of actions underlying the health effects of probiotics.

Ever since this foundational observation, probiotics have been extensively explored, researched, endorsed, and consumed worldwide. Some of the proposed mechanisms of action for probiotics as outlined in Figure 6.1 are maintenance or positive modulation of gut microbial communities, control of opportunistic pathogens, immuno modulation, stimulation of epithelial cell proliferation and differentiation, and strengthening of the intestinal barrier integrity. Although lactobacilli and bifidobacteria are the most common genera studied and exploited as probiotics, certain species/strains of other bacterial genera have also been tagged as probiotics in several reports (Figure 6.2) (Fijan, 2014). Nevertheless, it has always been heavily emphasized that the health effects of probiotics are highly species and strain specific (Azaıs-Braesco et al., 2010). Numerous animal and human trials have demonstrated the health benefits of specific probiotic strains on the risk prevention and reduction and management of various diseases. As outlined in Figure 6.3, some of the therapeutic targets where probiotics are proposed to be helpful are intestinal homeostasis, diarrhea, intestinal microbiota dysbiosis, IBD, IBS, constipation, lactose intolerance, food allergies, necrotizing enterocolitis, hypercholesterolemia, hypertension, Helicobacter pylori infections, colorectal carcinogenesis, breast cancer, NAFLD, T2DM, MetS, and viral-associated pulmonary damage (McFarland, 2007; Yadav et al., 2007, 2013; Doron et al., 2008; Chmielewska and Szajewska, 2010; Choi et al., 2011; Johnston et al., 2011; Wilhelm et al., 2011; Hemsworth et al., 2012; Bernardo et al., 2013; Fitzpatrick, 2013; Lee et al., 2013; Mohania et al., 2013; Orlando and Russo, 2013; Serban, 2013; Zelaya et al., 2014; Kumar et al., 2011, 2012, 2013; Hemalatha et al., 2014). More studies are anticipated to provide in-depth mechanisms and evidences underlying these benefits.

Lactobacillus L. acidophilus L. rhamnosus L. gasseri L. casei L. reuteri L. bulgaricus L. plantarum

Bifidobacterium

Lactococcus

B. bifidum

Lactococcus lactis subsp. lactis

B. animalis

Lactococcus lactis subsp. cremoris

B. breve B. infantis B. longum

Others

B. lactis

Streptococcus thermophilus

B. adolascentis

Propionibacterium freudenreichii Pediococcus acidilactici

L. salivarus L. johnsonii

Enterococcus

L. fermentum

Enterococcus faecalis Enterococcus faecium

L. helveticus

Saccharomyces cerevisiae Saccharomyces boulardii Leuoconostoc mesenteroides E. coli strain nissle

FIGURE 6.2  Some most commonly used probiotic species.

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Intestine Inflammatory bowel disease, Irritable bowel syndrome, diarrhea, acute gastroenteritis, lactose intolerance, food allergy, constipation, colorectal cancer, dysbiosis, gut permeability perturbation

Stomach H. pylori; gastritis, ulcers

Urogenital tract Urinary tract infection, vaginitis, vaginal candidosis

Probiotics Therapeutic targets

Liver Hepatic steatohepatitis, nonalcoholic fatty liverdisease, nonalcoholic steatohepatitis

Oral cavity Dental caries, cavities, periodontitis

Systemic Atopy/Allergy; respiratory tract infection hypercholesterolemia, hypertension, breast cancer, AIDS, hyperinflammation, obesity, T2 diabetes, insulin resistance, Metabolic Syndrome, osteoporosis

FIGURE 6.3  Potential therapeutic targets for probiotic application.

8  PROBIOTICS’ EFFECTS ON INTESTINAL MICROBIOTA AND ENVIRONMENT Of various proposed mechanisms of action of probiotics’ health effects, most are directly or indirectly linked to the positive influence on the composition and function of the gut microbiome, be it the production of antimicrobial compounds to restrain the growth of other microbes or competing with them for receptors and binding sites on the intestinal mucosa as well as for nutrients (O’Shea et al., 2011). Some probiotic strains can also improve the integrity of the intestinal barrier, thereby maintaining the immune tolerance and preventing the leakage of bacteria or their products (e.g., LPS) across the intestinal mucosa, which could prevent gastrointestinal infections, IBD, IBS, endotoxemia, T2DM, and so on (Lee and Bak, 2011; Bron et al., 2011). For instance, ingestion of a probiotic beverage containing L. plantarum has been found to decrease pain and flatulence in IBS patients, along with reduced counts of fecal enterococci (Nobaek et al., 2000). In a similar study, a probiotic blend of L. acidophilus, L. rhamnosus, L. plantarum, B. breve, B. longum, B. lactis, and S. thermophilus conferred symptomatic relief in IBS patients, along with a more stable intestinal microbiota composition (Ki Cha et al., 2011). Cox et al. (2010) observed an improved and more robust fecal microbiota in 6-month-old infants taking L. rhamnosus GG supplements. An altered gut microbiota with an increased community consistency, stability, and diversity was also observed in a neonatal mouse model treated with probiotic L. reuteri (Preidis et al., 2012). A reduced microbial diversity and/or dysbiosis is correlated with various maladies such as eczema, Crohn’s disease, IBD, MetS, colorectal cancer, and other diseases or disorders (Artis, 2008; Forno et al., 2008). Hence, in this context, probiotics may be useful in stimulating positive alterations in the gut microbiota by stabilizing the microbial communities and also by modulating the overall metabolic activities and functions of the gut microbiome. Nevertheless, the reports of altered gut microbiota and its metabolic function by probiotics have been inconsistent; thus, additional studies are anticipated in order to further validate these effects and benefits. In addition to microbial modulations, several probiotic strains can also alter the intestinal immune system by secreting metabolites that influence the growth and activities of intestinal epithelial and immune cells and hence may influence both innate and adaptive immunity (Liu et al., 2010; Hemarajata and Versalovic, 2013). Some of the proposed mechanisms in this intestinal immuno-modulation include regulation of cytokine production by immune cells, induced production of antiinflammatory cytokines, regulation of Treg cells, reduced recruitment of monocytes and macrophages to the intestines, amelioration of rota-virus induced pro-inflammatory immune cell recruitment to intestinal and systemic lymphoid tissues, inhibited production of pro-inflammatory cytokines and signaling immune cells, and inhibition of TNF production by LPS-activated macrophages (Thomas et al., 2012; Hemarajata and Versalovic, 2013). Since the GI tract also comprises an extensively complex neural network of the enteric nervous system, which regulates the communication across the gutbrain axis (Mayer, 2011), the gut microbial dysbiosis is also linked with psychopathological symptoms such as anxiety, hypertension, high blood pressure, and functional intestinal problems such as IBS (Neufeld and Foster, 2009; Hemarajata and Versalovic, 2013). In this context, in addition to contributing to immune homeostasis and development, gut microbes

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are also speculated to cross-talk with the gut-brain axis by producing various neuroactive molecules (Jarchum and Pamer, 2011; Bravo et al., 2011; Hemarajata and Versalovic, 2013). Hence, probiotic-induced alterations in the microbiota may produce positive results in patients suffering from psychiatric or autoimmune disorders.

9 PREBIOTICS The recent awareness and popularization of health benefits associated with low-glycemic index fiber-rich diets has immensely stimulated the research on the potential role of nondigestible oligosaccharides as food supplements on human health, particularly on the gut health. Of all these dietary substrates, prebiotics have undoubtedly been given preferred interest and consideration, especially for their ability to positively modulate the gut microbiota and impart various health benefits (Figure 6.4). In view of the fact that the complex nondigestible oligosaccharides are not hydrolyzed by our small intestinal enzymes, these dietary ingredients reach the colon nearly intact, where these are fermented by and promote the growth of colonic bacteria, hence justifying their designation as prebiotics (i.e., “foods for promoting the growth of probiotics”) (Alles et al., 1999; Gibson et al., 2004). Prebiotics were initially defined as “non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria already residing in the colon, and thus improves host health” (Gibson and Roberfroid, 1995). However, their definition has subsequently been modified to “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host welling-being and health” (Gibson et al., 2004). Generally, the term prebiotics is also defined as “non-digestible (by the host) food ingredients that have a beneficial effect through their selective metabolism in the intestinal tract.” Most of the accepted prebiotic compounds are mainly carbohydrates, especially polysaccharides or oligosaccharides. Various substrates that fulfill the criteria of being considered as “prebiotic” are inulin, FOS, GOS, isomalto-­oligosaccharides, lactulose, lactosucrose, soybean oligosaccharides, and xylo-oligosaccharides (Rastall and Maitin, 2002). These prebiotic criteria generally require that the substrate (1) exhibits resistance to hydrolysis and absorption in the upper part of the GI tract; (2) acts as a selective substrate for fermentation by one or a limited number of beneficial bacteria in the colon, thereby increasing their growth and/or metabolic activity; (3) modulates the composition of the colonic microbiota toward a healthier composition; and (4) imparts beneficial effects on the host’s health (Gibson and Roberfroid, 1995; Gibson et al., 2004). Any ingested food ingredient could theoretically be prebiotic in nature; however, the required and preferred modulation by prebiotics is specific, mainly targeting the population of lactobacilli and bifidobacteria in the colon. Some of the major sources of naturally occurring prebiotics are vegetables and fruits such as chicory, artichokes, garlic, onions, bananas, and leeks (Macfarlane and Cummings, 1999). Although various carbohydrates (including dietary fiber) can have prebiotic activity, the most researched and endorsed prebiotics are the fructans, inulin, FOS, and GOS. Inulin and FOS have been investigated rigorously in numerous studies over the past decade; however, potential studies are also emerging to provide promising data on the use of GOS as potential prebiotics, owing to their similarity with human milk oligosaccharides and their ability to favor the growth of bifidobacteria and lactobacilli. Numerous studies have reported an increase in the abundance of lactobacilli and bifidobacteria in the GI tract (Alles et al., 1999; Gibson et al., 2004; Langlands et al., 2004; Tannock et al., 2004; Ramirez-Farias et al., 2009; Davis et al., 2011; Joossens et al., 2011; Walton et al., 2012).

FIGURE 6.4  Some proposed health benefits of prebiotics.

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Furthermore, supplementation of milk-based infant formulas with GOS and FOS has also been found to enhance the growth of bifidobacterial population in a way similar to that observed in breast-fed infants (Rinne et al., 2005).

10  FUTURE PROSPECTS AND EXPECTATIONS Most of the gastrointestinal ailments such as IBS, IBD, necrotizing enterocolitis, diarrhea, obesity, T2DM, MetS, and so on, are associated with an imbalance of gut microbiota. Probiotics may be helpful in re-establishing the composition and homeostasis of the gut microbiota and intestinal microenvironment, thereby aiding in prevention or improvement of various systemic disease phenotypes ignited by dysbiosis and inflammation of the gut. The rapidly growing data supporting the role of gut microbiota in health/disease and positive influence of probiotics on gastrointestinal health is anticipated to stimulate novel breakthroughs related to the application of probiotics for improved human health. Nevertheless, further studies are anticipated to reveal molecular insights into the pathophysiological and pathobiological aspects of the gut microbiota, and also to further decipher the precise role of probiotics and prebiotics in promoting health and preventing diseases. Indeed, advanced biological tools and emerging studies on metagenomic, metatranscriptomic, and metabonomics are providing novel insights into the interface between probiotics and the gut microbiome.

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Schrezenmeir, J., de Vrese, M., 2001. Probiotics, prebiotics, and synbiotics: approaching a definition. Am. J. Clin. Nutr. 73, 361S–364S. Schwiertz, A., Taras, D., Schäfer, K., Beijer, S., Bos, N.A., Donus, C., Hardt, P.D., 2010. Microbiota and SCFA in lean and overweight healthy subjects. Obesity (Silver Spring) 18, 190–195. Serban, D.E., 2013. Gastrointestinal cancers: influence of gut microbiota, probiotics and prebiotics. Cancer Lett. 345, 258–270. Sperti, G.S., 1971. Probiotics. AVI Publishing Co., West Point, CT. Stephen, A.M., Cummings, J.H., 1980. Mechanism of action of dietary fibre in the human colon. Nature 284, 283–284. Tannock, G.W., Munro, K., Bibiloni, R., Simon, M.A., Hargreaves, P., Gopal, P., Harmsen, H., Welling, G., 2004. Impact of consumption of ­oligosaccharide-containing biscuits on the fecal microbiota of humans. Appl. Environ. Microbiol. 70, 2129–2136. Tap, J., Mondot, S., Levenez, F., Pelletier, E., Caron, C., Furet, J.P., Ugarte, E., Munoz-Tamayo, R., Paslier, D.L., Nalin, R., Dore, J., Leclerc, M., 2009. Towards the human intestinal microbiota phylogenetic core. Environ. Microbiol. 11, 2574–2584. The Human Microbiome Project Consortium, 2012. Structure, function and diversity of the healthy human microbiome. Nature 486, 207–214. Thomas, C., Hong, T., Van Pijkeren, J., Hemarajata, P., Trinh, D., Hu, W., Britton, R.A., Kalkum, M., Versalovic, J., 2012. Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of Pka and Erk signaling. PLoS One 7, e31951. Todd, R.K., 2001. The probiotic concept. In: Michael, P.D., Larry, R.B., Thomas, J.M. (Eds.), Food Microbiology, Fundamental and Frontiers, second ed. ASM Press, Washington, DC, USA, pp. 128–139. Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel, B.L., Duncan, A., Ley, R.E., Sogin, M.L., Jones, W.J., Roe, B.A., Affourtit, J.P., Egholm, M., Henrissat, B., Heath, A.C., Knight, R., Gordon, J.I., 2009. A core gut microbiome in obese and lean twins. Nature 457, 480–484. Wallace, T.C., Guarner, F., Madsen, K., Cabana, M.D., Gibson, G., Hentges, E., Sanders, M.E., 2011. Human gut microbiota and its relationship to health and disease. Nutr. Rev. 69, 392–403. Walter, J., Britton, R.A., Roos, S., 2011. Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. Proc. Natl. Acad. Sci. U. S. A. 108, 4645–4652. Walton, G.E., van den Heuvel, E.G., Kosters, M.H., Rastall, R.A., Tuohy, K.M., Gibson, G.R., 2012. A randomised crossover study investigating the effects of galactooligosaccharides on the faecal microbiota in men and women over 50 years of age. Br. J. Nutr. 107, 1466–1475. Whitman, W.B., Coleman, D.C., Wiebe, W.J., 1998. Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. U. S. A. 95, 6578–6583. Wilhelm, S.M., Johnson, J.L., Kale-Pradhan, P.B., 2011. Treating bugs with bugs: the role of probiotics as adjunctive therapy for Helicobacter pylori. Ann. Pharmacother. 45, 960–966. Woodmansey, E.J., 2007. Intestinal bacteria and ageing. J. Appl. Microbiol. 102, 1178–1186. Wostmann, B.S., Larkin, C., Moriarty, A., Bruckner-Kardoss, E., 1983. Dietary intake, energy metabolism, and excretory losses of adult male germfree Wistar rats. Lab. Anim. Sci. 33, 46–50. Yadav, H., Jain, S., Sinha, P.R., 2007. Antidiabetic effect of probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei in high fructose fed rats. Nutrition 23, 62–68. Yadav, H., Lee, J.H., Lloyd, J., Walter, P., Rane, S.G., 2013. Beneficial metabolic effects of a probiotic via butyrate-induced GLP-1 hormone secretion. J. Biol. Chem. 288, 25088–25097. Zelaya, H., Tsukida, K., Chiba, E., Marranzino, G., Alvarez, S., Kitazawa, H., Agüero, G., Villena, J., 2014. Immunobiotic Lactobacilli reduce viralassociated pulmonary damage through the modulation of inflammation-coagulation interactions. Int. Immunopharmacol. 19, 161–173.

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Chapter 7

Prebiotics in Human Milk and in Infant Formulas Jose M. Moreno Villares Servicio de Pediatría, Madrid, Spain

ABBREVIATIONS ESPGHAN European Society of Pediatric Gastroenterology, Hepatology, and Nutrition FOS fructo-oligosaccharides GOS galacto-oligosaccharides HMO human milk oligosaccharides RCT randomized-controlled trial SCFA short-chain fatty acids WHO World Health Organization

1 INTRODUCTION The human intestinal microbiota is composed of 1013-1014 microorganisms whose collective genome (microbiome) contains at least 100 times as many genes as our own genome. In a way, we can say that humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes. Evidence is accumulating that the interaction of the intestinal microflora with the intestinal mucosal cells plays a significant role in subsequent health, including autoimmune diseases as well as allergies and gastrointestinal diseases. A deeper knowledge of this close relationship between host and microflora will help us to better understand health status, to develop new ways for optimizing our personal nutrition, and to think of new ways to forecast our individual and societal predispositions to some diseases (Gill et al., 2006). Now is the time to consider the role of microbes in the gut through the lens of the evolutionary history of prokaryotic–eukaryotic relation. Depart from the usual paradigm of microbes as presumptive pathogens, and assume that prokaryotic–eukaryotic interactions in the gut are generally mutually beneficial (Neish, 2009). Newborn babies and infants possess a functional but immature immune system that has the function of protecting against infections. The maturation of this immune system is closely related to the acquisition of an appropriate gut microflora. Breast milk contains a number of biological, active compounds that can improve an infant’s immune system directly or through components that help to establish a determined intestinal flora. Although it is impossible to produce infant formulas having identical composition and properties to breast milk, potential health benefits could arise from the supplementation of these products with one and/or combinations of functional food ingredients (Meyer and Shah, 2013). Increasing evidence shows that such dietary modulation could be beneficial for the host by effecting a health-promoting modification in the composition and the activities of gut microflora. This chapter reviews the strength of evidence regarding the immune-stimulating effects of one of these components, prebiotics, and how they are used in infant formulas. The risks associated to its use and the regulatory status of prebiotics in infant formulas will be also reviewed. Distinguishing Prebiotics from Probiotics Prebiotics are food ingredients, typically oligosaccharides that are selectively fermented by beneficial bacteria in the gut (such as Bifidobacterium), stimulating the growth and/or activity of those bacteria and thereby contributing to host health and well-being. Prebiotics are resistant to gastric acidity, enzymes, and absorption. Their purpose in infant formula is to stimulate the growth and colonization of naturally occurring beneficial bacteria.

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Probiotics are bacteria that pass through the gastrointestinal tract and have beneficial effects on the health of the host. Probiotic bacteria typically have a history of safe consumption; examples include Lactobacillus rhamnosus GG and Bifidobacterium animalis ssp. lactis. Their purpose in infant formula is to substitute for naturally occurring beneficial bacteria and thereby influence the mucosal immune system.

2  DEVELOPMENT OF THE IMMUNE SYSTEM IN INFANTS At birth, the gastrointestinal tract is essentially germ free, with intestinal colonization occurring during birth or shortly afterwards. Within the first days of life, mucosal surfaces of the gastrointestinal as well as the respiratory tract become colonized with bacteria. The first colonization of the intestine is one of the most critical immunologic exposures faced by the newborn infant because microbial niches become established, allowing long-term colonization as part of the biofilm located in the glycocalyx of the epithelial layer (Sonnenburg et al., 2004). The lymphoid system is not yet mature, although it is developed. The fetal immune system develops at least partial functional competence before birth, but lacks full capacity to generate sustained immune responses. Lymphocytes T and B are naïve. Activation of T lymphocytes results in a type Th2 response—that is, production of cytokines IL-4 and IL-5 and very low Th1 cytokine γ-interferon (Szépfalusi, 2008). Although after birth there is an immense exposition to a wide spectrum of commensal and pathogenic microorganisms, the immune system does not respond to every stimulus. The corresponding pathogen-associated molecular patterns are recognized by receptors of the immune system, and this shapes the direction of the immune system’s development through childhood to adulthood. During pregnancy, the immune system of the fetus coexists with the mother’s immune system. After birth, the immune system must switch in order to protect the infant against pathogens and to develop tolerance to harmless nonself antigens, such as food antigens. At birth, T-lymphocytes exhibit a Th2-profile, characterized by a limited ability to produce cytokines. Until this immune defense is already set, infants are at risk for serious infection. Throughout the first months after birth, these Th2-skewed responses are modified toward a low-level immunity, predominantly Th1-cytokines, and IgG antibodies, particularly IgG1 (Holt and Jones, 2000). On the other side, the immune system is tightly controlled by its own regulatory network to prevent inappropriate immune reactions from pathologic conditions. If this system fails, the result can be an allergy or autoimmune disease (Calder et al., 2006). The close relationship between colonic microflora and host cells has a central role in health and disease. Dietary modulation is important for improved gut health, especially during the highly sensitive stage of infancy (Bach, 2002; Renz et al., 2006). Marked differences in the composition of gut flora have been recognized in response to the infant-feeding regimen. The process of bacterial colonization of the gut begins just after birth and includes three phases: delivery, breast-feeding, and weaning. Differences in gut microflora composition and incidence of infections exist between breast-fed and formula-fed infants, with the former thought to have improved protection. By the age of 18 months, the colonic bacterial microbiota is considered complete. Although there are different elements in infant feeding that can play a role in modifying gut microflora, this chapter will discuss only the role of prebiotics, especially when added to infant formulas.

3  BREAST MILK AND DEFENSE AGAINST INFECTIONS AND ALLERGIC MANIFESTATIONS Breast-feeding is the ideal mode of feeding for the newborn infant. The World Health Organization (WHO) identifies breastfeeding as providing the optimum nutrition and protection for infants (WHO, 2009). The benefits of human milk in terms of infant development and protection have been well documented (Eidelman et al., 2012). Breast milk confers passive immunity to the newborn. Clearly, the effect of human milk on the postnatal development of the intestinal flora cannot be attributed to a single ingredient. Various factors present in breast milk are known to modulate the developing microbiota in the gastrointestinal tract. Breast milk contains 0.4-1.0 g/L secretory IgA, other immunoglobulins, antimicrobial proteins (lactoferrin, lysozyme), leukocytes, cytokines, and chemokines, hormones, bioactive lipids, fatty acids, oligosaccharides, glycans, as well as minerals, vitamins, and other components that may contribute to the defense against infections (Table 7.1) (Ballard and Morrow, 2013). Bioactive components in breast milk come from different sources; some are produced and secreted by the mammary gland, some are produced by cells contained in human milk, and others are produced anywhere, carried into serum and secreted through the mammalian epithelium into the milk. Many of these factors act synergistically.

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TABLE 7.1  Bioactive Components in the Human Milk Component

Function

Cells Macrophages

Protection against infection, T-cell activation

Stem cells

Regeneration and repair

Immunoglobulins IgA/sIgA

Pathogen-binding inhibition

IgG

Antimicrobial, activation of phagocytosis (IgG1, IgG2, IgG3); antiinflammatory response to allergens (IgG4)

IgM

Agglutination, complement activation

Cytokines IL-6

Stimulation of the acute phase response, B-cell activation, proinflammatory

IL-7

Increase thymic size and output

IL-8

Recruitment of neutrophils, proinflammatory

IL-10

Repressing Th 1-type inflammation, induction of antibody Production and facilitation of tolerance

IFNγ

Proinflammatory, stimulates Th 1 response

TGFβ

Antiinflammatory, stimulation of T-cell phenotype switch

TNFα

Stimulates inflammatory immune activation

Chemokines G-CSF

Trophic factor in gut

MIF

Macrophage Migratory Inhibitory Factor; Prevents macrophage movement, increases antipathogen activity of macrophages

Cytokine inhibitors TNFRI and II

Inhibition of TNFα, antiinflammatory

Growth factors EGF

Stimulation of cell proliferation and maturation

HB-EGF

Protective against damage from hypoxia and ischemia

VEGF

Promotion of angiogenesis and tissue repair

Modified from Ballard and Morrow (2013).

Breast-feeding protects against atopy (Gdalevich et al., 2001) and infections (Pettigrew et al., 2003). In classical long-term epidemiological studies, it has been demonstrated that breast-fed infants are better protected against infections of the gut, respiratory, and urinary tract, when compared with those who are formula fed (Levy, 1998; López Alarcón et al., 1997). The review of all bioactive factors in human milk is far beyond the scope of this chapter; rather, the focus will be on human milk oligosaccharides (HMO). Breast milk contains more than 200 types of oligosaccharides, ranging from 3 to 32 sugars, and differs in composition from those of other mammals. They act through several defense mechanisms (Table 7.2). Many of these oligosaccharides act as analogs of receptors in gut epithelial cells inhibiting the binding of bacterial and viral pathogens as well as toxins. Oligosaccharides also promote the proliferation of commensal Bifidobacterium spp. and lactobacilli in the intestinal tract (Niers et al., 2007).

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TABLE 7.2  Mechanisms of Defense of Human Milk Oligosaccharides Action

Mechanism

Prevention of Pathogen Adhesion

- Serve as ligands, analogs, and block pathogens adhesion - Change the expression of intestinal epithelial cell surface ligands

Development of the immune system

- Interact with selectins and Toll-like receptors - Affect leukocyte-endothelial cell and leukocyte-platelet interactions

Growth factor for bifidobacteria

- Induces intracellular processes, including differentiation and apoptosis of intestinal epithelial cells - Some acidic fractions have direct immunomodulatory effects

4  WHAT ARE PREBIOTICS? Three different approaches toward modifying the development and balance of infant intestinal microflora can be taken: first is the addition of live bacteria, such as bifidobacteria (probiotics); second is the addition of oligosaccharides that survive passage through the small intestine and reach the colon where they are used by colonic bacteria, involving the manipulation of its energy sources (prebiotics); and third is that both pre- and probiotics can be added (symbiotic) (Hord, 2007). A prebiotic is “a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/ or activity of one or a limited number of bacterial species already resident in the colon, and thus improves host health” (Gibson and Roberfroid, 1995). Although any dietary component that reaches the colon intact is a potential prebiotic, most of the interest is focused on the nondigestible oligosaccharides (Delzenne, 2003). Fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) have demonstrated beneficial effects on the intestinal microflora. Oligosaccharides are sugars containing between 2 and 20 units. They can occur naturally in fruits and vegetables or be produced by the hydrolysis of polysaccharides. Because prebiotics are not digestible, they are fully available to the bacteria that reside in the intestinal tract and interact with the intestinal microbiota. Prebiotic consumption shifts the composition of the intestinal microbiota toward those associated with a healthy condition in the host (Gibson et al., 1995). As the composition of the microbiota is modified, the types of bacterial metabolites into which prebiotics are converted are also modified (e.g., producing a greater amount of short-chain fatty acids, SCFAs). The SCFAs have important effects in the intestinal tract. Butyrate has an essential role in maintaining the metabolism, proliferation, and differentiation of the different epithelial cell types. Many of these metabolites are absorbed into the blood and enter the systemic circulation interacting with many physiologic processes. In this way, prebiotics (1) improve intestinal transit time (Cherbut, 2003); (2) increase the absorption of minerals, mainly calcium, and manganese (Tahiri et al., 2003); (3) have anticancer effects, mainly in the prevention or progression of colon cancer (Pool-Zobel et al., 2002; Wollowski et al., 2002); (4) modify lipid metabolism (López et al., 2001); and (5) modulate various systemic immune markers (Van Loo, 2004). The possible therapeutic application of some prebiotics in specific clinical conditions are inflammatory bowel diseases (Guarner, 2007; Leenen and Dieleman, 2007; Duggan et al., 2002), and others (Lenoir-Wijnkoop et al., 2007) that are further analyzed in other chapters in this book.

5  HUMAN MILK OLIGOSACCHARIDES Shortly after birth, the previously sterile infant gut begins to be colonized by bacteria—facultative anaerobes and strict anaerobes from the birth canal and its surroundings (Figure 7.1). Microbial flora of the female genital tract, sanitary conditions, and the type of delivery has an effect on the level and frequency of various species colonizing the infant gut. But the main factor contributing to the establishment of a particular microflora is the type of feeding. In the gastrointestinal system of breast-fed babies, bifidobacteria are soon selected and become predominant (Figure 7.2). Formula-fed babies harbor a varied flora consisting of Bifidobacteria, Escherichia coli, and Bacteriodes (Harmsen et al., 2000). The fecal bacterial population shifts from non-HMO-consuming microbes to HMO-consuming bacteria during the first few weeks of life (De Leoz et al., 2014). When complementary feeding is introduced, a further diversification of the flora occurs. The bifidogenic effect of human milk has been ascribed to oligosaccharides, lactoferrin, and nucleotides (Mountzouris et al., 2002). But the two last components seem to have more of an inhibitory effect of the p­ athogenic

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FIGURE 7.1  Colonization of infant gut at birth and in the first days of life.

FIGURE 7.2  Intestinal colonization according to age.

flora rather than a direct stimulus to the development of bifidobacteria. One could say, then, that breast milk stimulates the growth of Bifidobacteria because of its high oligosaccharide content (approximately 8% of total carbohydrate content) (Sherman et al., 2008). They are also part of glycolipids as well as glycoproteins (milk glycans) (Pacheco et al., 2014). After ingestion, HMOs pass unabsorbed through the gut and reach the colon, where they are fermented and where SCFA are produced. An acidic environment is then created and favors the establishment of a bifidogenic flora. HMO are a combination of five monosaccharides: glucose, galactose, sialic acid, fucose, and N-acetyl-glycosamine. The HMOs are formed by the attachment of a single glucose molecule at the reducing end of a galactose, to form a lactose core. Then a linear chain is formed, and afterwards a branched chain and this structure repeated multiple times (Wu et al., 2010) (Figure 7.3). There are at least 12 different types of glycosidic bonds in HMOs. Although there are small HMOs, they are generally less abundant than the larger, more complicated structures. New tools are being implemented for a precise and rapid analysis of HMOs in order to get a better knowledge of their properties and possible applications to infant feeding (Totten et al., 2014). HMO are synthesized in the mammalian gland by specific enzymes, the glycosyltransferases, in sequences of different numbers of monosaccharides (Coppa et al., 2004). Human milk contains at least 200 various kinds of oligosaccharides

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FIGURE 7.3  Structure of fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS).

composed of many different molecules (Chichlowski et al., 2011). These oligosaccharides are predominantly neutral, lowmolecular-weight molecules, and depending on the Lewis blood group of the mother. The oligosaccharides represent the third largest component (after lactose and lipids) in breast milk, occurring at a concentration of 12-14 g/L in mature milk and 20-23 g/L in colostrum (Coppa et al., 1999). On the contrary, cow’s milk, commonly used to manufacture infant formulas, contains less than 1 g/L of oligosaccharides. HMO are highly resistant to enzymatic hydrolysis. Besides their role into the intestinal lumen, they can be absorbed and cross the brush border membrane of the intestine (Kunz et al., 2000). In this case, they may have a systemic effect and their properties are not restricted to the mucosal environment. Recently, it was found that the presence of ingested HMOs in urine as well as in infants’ circulation and their concentration correlates with levels of the corresponding oligosaccharide in mothers’ milk. This may be a rational explanation of the postulated systemic benefits of the presence of HMOs in human milk (Goehring et al., 2014). Experimental studies have shown that the human milk-derived acidic oligosaccharide fraction is able to enhance the production of certain cytokines as well as γ-interferon (Eiwegger et al., 2004). The same authors also demonstrated that some plant-derived oligosaccharides have a similar effect. Substantial differences exist in the quality and quantity of HMOs among different nursing mothers (Smilowitz et al., 2013), but it has not been determined whether there is a relationship between the quantity and quality of HMOs and the presence of different bacterial species in the composition of intestinal microflora. It is generally accepted that the mother’s diet, physiology, and feeding behavior may have an impact on the daily HMO production. Human milk already has a probiotic effect because it also contains lactic acid bacteria. In this sense, we could more properly talk of the symbiotic effect of breast milk (Martín et al., 2003, 2006). The review on this topic is further the scope of this chapter.

6  PREBIOTICS IN INFANT FORMULAS Due to their complexity, oligosaccharides with identical structure to HMOs are not available as dietary ingredients to be added to infant foods (Figure 7.4). It was shown previously that the bioactivity of oligosaccharides from bovine and human milk is similar (Gopla and Gill, 2000), and therefore they could be used as bioactive components in human nutrition. Searching for alternatives, several mixtures of GOS ± FOS have been tested, although a relatively low number of mixtures have been clinically probed. FOS are linear fructose polymers, whereas the basic structure of GOS incorporates lactose at the reducing end and contains different branching. Inulin and oligofructose are safe inducers of a Bifidus flora, so it appears clear when it is used in infant feedings (Vandenplas, 2002; Fanaro et al., 2005a,b; Vitoria Miñana, 2007). The most exten-

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FIGURE 7.4  Bifidogenic effect of a prebiotic mixture in an infant formula. Standard: no prebiotics. Formula A: GOS + FOS 0.4 g/dL. Formula B: GOS + FOS 0.8 g/dL. Modified from Moro et al. (2002).

sive experience is available for long-chain FOS obtained from chicory extract, and GOS gained from enzymatic synthesis of lactose (Boehm et al., 2005). In Europe, these are the most common prebiotics added to infant formulas (10% inulin with 5-fructose monomers and 90% galacto-oligosaccharides with 2-7 monomers), whereas in Japan, isomalto-oligosaccharides and xylo-oligosaccharides are used. Also acidic oligosaccharides such as pectin hydrolysate are under investigation (Fanaro et al., 2005a,b). Structurally, the acidic oligosaccharides of human milk are characterized by their content in sialic acid. The formulas supplemented with a prebiotic mixture are reported to have multiple effects mediated through changes in the flora, the immune system, and other mechanisms (Veereman, 2007; Boehm et al., 2004). It has been demonstrated that the Bifidobacteria and Lactobacilli content in feces of term infants after 28 days of supplementation with a mixture FOS-GOS, in a dose-related mode (with 0.4 and 0.8 g/dL), increases to the levels seen in breast-fed infants (Moro et al., 2002). This change in flora was correlated with an increase in the metabolic activity (pH, lactate, and SCFAs production) (Knol et al., 2005a,b). Nineteen infants who received a prebiotic mixture (GOS/FOS) 6 g/L presented in the feces a higher fecal acetate ratio and lactate concentration and lower pH after 16 weeks than did the group receiving a standard formula or a formula supplemented with B. animalis (6.0 × 1010 viable cells per liter) (Bakker-Zierikzee et al., 2005). Using molecular biology techniques, it was observed that the species of Bifidobacteria present in infant-fed FOS-GOS supplemented formula corresponded with the patterns seen in breast-feeding. That is, Bifidobacterium infantis, Bifidobacterium breve, and Bifidobacterium longum were dominant in breast-fed and supplemented infants, whereas the infants receiving a standard formula had lower levels of B. breve and higher levels of Bifidobacterium catenulatum and Bifidobacterium adolescentis (Haarman and Knol, 2005). This shift in microflora was accompanied by a reduction in potential pathogens. In several of these studies, a positive effect on stool characteristics such as stool consistency and stool frequency (Scholtens et al., 2014) was found. In preterm infants of about 31 weeks’ gestational age and about 1 week old, a double-blind, randomized controlled study was performed comparing standard formula with a formula containing 1 g/dL of a prebiotic mixture. During the 28-day study period, the number of fecal bifidobacteria and lactobacilli increased in the prebiotic formula group to levels seen in the breast-fed group, used as a control. The difference in composition of the fecal flora between the standard formula and the prebiotic formula group was highly significant. At the same time, Knol et al. (2005a,b) found a significant reduction in the total number of relevant pathogens in the fecal flora. Moreover, stool consistency and stool frequency were similar in the breast-fed and the supplemented groups (Boehm et al., 2002, 2003). Stool characteristics in the group fed the supplemented formula were close to those found in the human milk group. Boehm et al. (2003) postulated that prebiotic mixtures may help in improving intestinal tolerance to enteral feeding in preterm infants. The prebiotic mixture might also have improved calcium absorption, as indicated by a similar urinary Ca/P ratio in prebiotic-fed and breast-fed babies (Marini et al., 2003).

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Early evidence led to the publication of a statement by the Scientific Committee on Food of the European Commission on December 13, 2001, in which the addition of the prebiotic mixture (10% FOS, 90% GOS) at a concentration of 0.8 g/dL to infant formula was considered safe (EC Scientific Committee on Food, 2001). A few other studies have been done using only FOS. The Committee on Nutrition of the European Society of Pediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) pointed in 2004 that at that moment no conclusive recommendation could be done on the benefits of the addition of a prebiotic mixture to an infant formula. They suggested to performing prospective clinical trials designed to show the clinical benefits of such an approach (Agostoni et al., 2004). Since then, several new trials have been published (Table 7.3). The putative effect of prebiotic formula on the immune system has been demonstrated by recent studies on the incidence of infections and on atopic dermatitis during the first year of life. In a prospective, randomized, placebo-controlled open trial, infants receiving the prebiotics mixture during 12 months had significantly fewer episodes of GI and respiratory tract infections (Bruzzesse et al., 2009; Arslanoglu et al., 2007). In other study in infants at risk for atopy, the use of the prebiotic formula demonstrated a protective effect at 6 months (Moro et al., 2006). In a follow-up 2 years later, there was still a lower incidence of allergic manifestations in the group of infants who received a prebiotic supplemented formula when compared with a standard one (Arslanoglu et al., 2008). Potential mechanisms of the prebiotic effect may be by improving gut barrier and also an enhanced fecal secretory IgA levels. The same effect on secretory IgA, butyric acid concentration, and increase in bifidobacteria counts were found in a randomized controlled clinical trial performed on 365 healthy infants randomly assigned to a formula with or without GOS (0.44 g/dL), until 12 months of age (Sierra et al., 2014). In another study with a quite similar formula (GOS 0.4 g/dL), a similar bacterial pattern was found in the stools as well as clinical improvement in colic and constipation (Giovannini et al., 2014). Gastrointestinal tolerance as well as stool characteristics are well demonstrated in all studies using both an oligosaccharides mixture or only GOS, if the amount is under 1 g/dL (Williams et al., 2014). When used in infant formula at a concentration of 1.5 g/L or 3.0 g/L for 5 weeks, no significant differences in fecal Lactobacillus or Bifidobacterium counts were indicated (Euler et al., 2005). Furthermore, at that higher level there were an increased number of adverse events (flatulence, spit-ups, and loose stools). A 2008 paper published the results on the supplementation with GOS (5 g/L) on follow-up formula for 18 weeks. The data indicated that this supplementation positively influenced the bifidobacteria flora and the stool consistency during the supplementation period (Fanaro et al., 2008). Despite the fact of these new data, the ESPGHAN Committee on Nutrition, in their last statement (2011) on supplementation of infant formula with prebiotics, pointed that “at present, there is insufficient data to recommend the routine use of probiotic- and/or prebiotic-supplemented formula” and recalls for further clinical trials (ESPGHAN Committee on Nutrition et al., 2011). The Committee on Nutrition of the American Academy of Pediatrics stated in 2010 that “there may be some long-term benefit of prebiotics for the prevention of atopic eczema and common infections in healthy children” (Thomas et al., 2010). Mihatsch et al. (2006) demonstrated in 20 preterm infants that the addition of 1 g/100 ml of GOS-FOS reduced significantly stool viscosity and accelerated gastrointestinal transport when compared with placebo (maltodextrin). This may mean an advantage if one could prove if GOS-FOS facilitates enteral feeding advancement in these preterm infants. Further trials are required. Inulin and oligofructose have been also studied in special infant formulae as well as in weaning foods in toddlers. Tolerance to increased fibre intake in the form of FOS as part of a weaning food has been well documented. Its consumption led to more regular and softer stools as well as decreased frequency of symptoms associated with constipation (Moore et al, 2003). A double-blind study comparing a formula containing partially hydrolyzed protein, a high β-palmitic acid level, and nondigestible oligosaccharides demonstrated that, when compared with standard infant formula, led to higher counts of bifidobacteria in the feces and was well tolerated and supported satisfactory growth (Schmelzle et al., 2003). Combinations of prebiotic oligosaccharides with pectin-derived acidic oligosaccharides also appeared to be clinically safe and effective on modifying infant microbiota (Magne et al., 2008), even in the recovery after antibiotic treatment (Brunser et al., 2006). Although these initial results are promising, additional studies are needed in order to confirm the evidence of clinical benefits (Osborn and Sinn, 2008). Besides this, there is a need to research the use of sialyllactose and other sialylated milk oligosaccharides added to infant nutrition (ten Bruggencate et al., 2014) as well as other new GOSs structurally more closely related to HMOs (Intanon et al., 2014). As more recent studies support the hypothesis that human milk has a greater symbiotic effect than exclusively a prebiotic one, there is an increased interest in demonstrating safety and efficacy of the combination or prebiotics plus probiotics,

TABLE 7.3  Randomized controlled trials on the effects of supplemented infant formulas with prebiotics Subgrups

Prebiotic mixture

Length of the study

Results

Brunser et al. (2006)

110 (12-24 m)

SF: 66 PF: 64

Oligofructose + inulin 0.45/dL

3 weeks

↑ Bifidobacteria in feces after amoxicilin treatment

Moro et al. (2006)

259

SF: 104 PF: 102

GOS + FOS 0.8 g/dL

6 months

↑ Bifidobacteria in feces ↓ atopic dermatitis (9.8 vs 23.1%)

Bruzzese et al. (2006)

281

SF: 145 PF: 136

GOS + FOS 0.8 g/dL

12 months

↓ Acute diarrhea episodes (0.15 vs 0.28 episodes/infant) ↓ Infants with diarrhea (17 vs 34%) ↓ Respiratory tract infections (19% vs 35% infant with ≥3 infections) ↓ Infants needing antibiotics (30% vs 49%)

Arslanoglu et al. (2007))

206

SF: 130 PF: 129

GOS + FOS 0.8 g/dL

6 months

↓ Number of infectious episodes ↓ Respiratory tract infections ↓ Infants needing antibiotics

Arslanoglu et al. (2008)

134

SF: 68 PF: 66

GOS + FOS 0.8 g/dL

24 months

↓ Number of allergic manifestations ↓ Respiratory tract infections ↓ Infants needing antibiotics

Mihatsch et al. (2006)

20 prematures (GE: 24-31 weeks)

SF: 10 PF: 10

GOS + FOS 1 g/dL

14 days

Laxative effect. Decreased transit time

Bruzzese et al. (2006)

342

SF: PF:

GOS + FOS 0.8 g/dL

12 months

↓ Gastroenteritis ↓ Use of antibiotics

Sierra et al. (2014)

365 (0-12 m)

SF: 188 PF: 177

GOS 0.44 g/dL

12 months

Lower fecal pH ↑ sIgA ↑ Bifidobacteria in feces Softer stools

Giovannini et al. (2014))

163 (0-4-6 m)

SF: PF:

GOS 0.4 g/dL

SF: study formula; PF: control formula; sIgA: secretory IgA.

↓ Colic episodes ↓ Clostridum in feces ↑ Bifidobacteria

Prebiotics in Infant Formulas  Chapter | 7  95

Number of infants

Authors (year)

96  PART | I  Prebiotics in Health Promotion

both in the prevention of gastrointestinal infections and diarrhea, in the prevention of the onset of allergies, and the usefulness in the treatment of atopic disease. Initial studies are on the way (Chouraqui et al., 2008).

7  SIDE EFFECTS Because the neonatal period is a critical period for development and it is also the period when microbes become established in the gastrointestinal tract, the long-term effects of manipulation of gut microbiota may have more deleterious effects than when these modifications occur later in life (Neu, 2007). Oligosaccharides are, in general, considered as very safe. Infants fed a prebiotic inulin/GOS mixture in an infant formula grew well, had a stable water balance, and did not show undesirable effects. Prebiotics are mostly not absorbed in the small bowel, exerting an osmotic effect in the intestinal lumen, and are fermented in the colon in SCFAs and gas. Prebiotics are usually well tolerated, but if supplied in excessive amounts they may have undesirable effects such as excessive flatus, borborygmi, abdominal pain, and diarrhea (Marteau and Flourié, 2001). It has been reported in adult patients with gastroesophageal reflux a worsening of the symptoms after the administration of up to 20 g per day of FOS (Piche et al., 2003). It seems clear that the tolerance to prebiotics is related to their nature, dose, individual sensitivity factors, and adaptation to chronic consumption. Total doses of less than 20 g per day are well tolerated (Marteau and Seksik, 2004).

8  REGULATION OF THE ADDITION OF PREBIOTICS TO INFANT FORMULAS Although the majority of published papers are funded by the food industry, a recent systematic review on the topic could not find any significant association between the source of funding and sequence generation, allocation concealment, blinding, and selective reporting, majority of reported clinical outcomes, or authors’ conclusions. In randomized-controlled trials (RCTs) on infants fed infant formula containing probiotics, prebiotics, or symbiotics, the source of funding did not influence the majority of outcomes in favor of the sponsors’ products. Nevertheless, more nonindustry-funded research is needed to further assess the impact of funding on methodological quality, reported clinical outcomes, and authors’ conclusions (Mugambi et al., 2013). The Scientific Committee on Food of the European Union considered the addition of an oligosaccharide mixture (GOS 90% + FOS 10%) at 0.8 g/dL safe when added to an infant formula (Commission Directive, 2006). This was confirmed in the last European Union Directives of December 2006 (Commission Directive, 2006/141/EC on infant formula and followup formula). The Scientific Panel on Dietetic Products, Nutrition, and Allergies of the European Commission considered in 2004 that there is no evidence of benefits to infants from the addition of FOS (1.5-3.0 g/L) to infant formula, and there are reasons for safety concerns (prevalence of adverse events, including loose stools) (Opinion of the Scientific Panel on Dietetic Products, 2004). The Panel on Dietetic Products, Nutrition and Allergies of the European Food Safety Authority (EFSA) concluded in 2010 that a cause-and-effect relationship had not been established between the consumption of prebiotics or probiotics or mixtures and a beneficial physiological effect related to increasing numbers of gastrointestinal microorganisms (VeeremanWauters, 2009). The EFSA does not allow one to include this health claim in the advertisement of these products, and it requests new evidence.

9 CONCLUSIONS One of the most challenging current research areas is the potential beneficial effect of prebiotics on the immune system of young infants (Parracho et al., 2007; Niers et al., 2007). Prebiotics in early nutrition may have profound effects on the intestinal barrier, internal milieu, and defense mechanism. It has been well established that the addition of prebiotics to infant formula has a bifidogenic effect. Are there long-term health benefits related to an early intervention? A few recent clinical studies report encouraging data on immune-mediated effects of prebiotic supplementation: fewer gastrointestinal and respiratory infections and less atopic dermatitis in the first years of life. It is probable that both effects are related. Clearly, additional research is still needed on the optimal composition, dosage, and combinations of different oligosaccharides (Aggett et al., 2003). Selective manipulation of the intestinal microbiota might be an approach to novel prophylactic and therapeutic interventions in atopy, by redirecting allergic Th-2 responses in favor of Th-1 responses (Miniello et al., 2003). Moreover, should prebiotics be used in case of illness? What are the effects of adding prebiotics to infants’ formulas? The functional effects of prebiotics on infant health and the long-term effects of different dietary prebiotics on adult health and gastrointestinal diseases need to be further studied in controlled intervention trials.

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Tahiri, M., Tressol, J.C., Arnaud, J., Bornet, F.R., Bauteloup-Demange, C., Feillet-Coudray, C., Brandolini, M., Ducros, V., Pépin, D., Brouns, F., Roussel, A.M., Rayssiguier, Y., Coudray, C., 2003. Effect of short-chain fructooligosaccharides on intestinal calcium absorption and calcium status in postmenopausal women: a stable-isotope study. Am. J. Clin. Nutr. 77, 449–457. ten Bruggencate, S.J., Bovee-Oudenhoven, I.M., Feitsma, A.L., van Hoffen, E., Schoterman, M.H., 2014. Functional role and mechanisms of sialyllactose and other sialylated milk oligosaccharides. Nutr. Rev. 72, 377–389. Thomas, D.W., Greer, F.R., Thomas, D.W., Greer, F.R., Committee on Nutrition, Section on Gastroenterology, Nutrition Hepatology, 2010. Clinical reports. Probiotics and prebiotics in pediatrics. Pediatrics 126, 1217–1231. Totten, S.M., Wu, L.D., Parker, E.A., Davis, J.C., Hua, S., Stroble, C., Ruhaak, L.R., Smilowitz, J.T., German, J.B., Lebrilla, C.B., 2014. Rapid-throughput glycomics applied to human milk oligosaccharide profiling for large human studies. Anal. Bioanal. Chem. 406, 7925–7935. Van Loo, J.A.E., 2004. Prebiotics promote good health. The basis, the potential, and the emerging evidence. J. Clin. Gastroenterol. 38, S70–S75.

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Vandenplas, Y., 2002. Oligosaccharides in infant formula. Br. J. Nutr. 87, S293–S296. Veereman, G., 2007. Pediatric applications of inuline and oligofructose. J. Nutr. 137, 2585S–2589S. Veereman-Wauters, G., 2009. Application of prebiotics in infant foods. Br. J. Nutr. 93 (Suppl. 1), S57–S60. Vitoria Miñana, I., 2007. Oligosacáridos en nutrición infantil: fórmula infantil, alimentación complementaria y del adolescente. Acta Pediatr. Esp. 65, 175–179. WHO, 2009. Infant and Young Child Feeding: Model Chapter for Textbooks for Medical Students and Allied Health Professionals. WHO, Geneva pp. 1–111. Williams, T., Williams, T., Choe, Y., Price, P., Choe, Y., Price, P., Katz, G., Suarez, F., Paule, C., Mckey, A., Katz, G., Suarez, F., Paule, C., Mckey, A., 2014. Tolerance of formulas containing prebiotics in healthy, term infants. J. Pediatr. Gastroenterol. Nutr. 59, 653–658. Wollowski, I., Rechkemmer, G., Pool-Zobel, B., 2002. Protective role of probiotics and prebiotics in colon cancer. Am. J. Clin. Nutr. 73, 451S–455S. Wu, S., Tao, N., German, J.B., Grimm, R., Lebrilla, C.B., 2010. Development of an annotated library of neutral human milk oligosaccharides. J. Proteome Res. 9, 38–51.

Chapter 8

Prebiotics and Probiotics in Infant Nutrition Antonio Alberto Zuppa*, Giovanni Alighieri*, Antonio Scorrano† and Piero Catenazzi* *Division of Neonatology, Catholic University of the Sacred Heart, Rome, Italy, †Division of Pediatrics, Neonatal Intensive Care Unit, Cardinale G. Panico Hospital, Tricase (Lecce), Italy

1 INTRODUCTION In the past few years, prebiotics and probiotics have gained a more central role in the nutritional scientific panorama for their important therapeutic “alternative” role in the treatment of some pathologies that affect both adults and children, as early as the first few days of life. According to the criteria proposed in a report of the Conseil de l'Europe of 2004 titled “The Quality of Life and Management of Living Resources Program,” prebiotics and probiotics can be called “functional foods” (Conseil de l'Europe, 2001). According to the most recent definitions, the term functional refers to a food—not a dietary supplement—that, in addition to its intrinsic nutritional value, can also positively affect specific functions of the organism, improve a person's health and well-being, and reduce risks of diseases (Giorgi, 2002).

2  DEVELOPMENT AND PHYSIOLOGY OF THE GASTROINTESTINAL ECOSYSTEM The gastrointestinal bacterial flora, the intestinal epithelium, and the mucosal immune system constitute a highly integrated unit called the gastrointestinal ecosystem. Intrinsic disorders (genetic) and acquired alterations to any component can bring about pathological changes to the digestive system (Premysl, 2007). During fetal life, the intestine is sterile; at birth, the gastrointestinal tract is being progressively colonized by commensal bacteria, creating the so-called microflora that is essential to the development of intestinal structures and functions (Underwood et al., 2005). Such bacteria can be classified into three groups, depending on their impact on the person's health: beneficial bacteria, potentially harmful bacteria, and bacteria that can have both pathogenic and beneficial effects (Gibson, 1995) (see Table 8.1). Intestinal colonization is strongly influenced by genetic factors, by the type of delivery, by the maternal bacterial flora, by the type of nutrition, and by exposure to the external world (Jose and Saavedra, 2007; Martin and Walker, 2008). In babies born by spontaneous delivery, microbial colonization begins with the passage of the fetus through the birth canal. The microbial colonization pattern is the same as that of the mother's vaginal and perineal microflora (microbial heredity) (Palmer et al., 2007; Mandar and Mikelsaar, 1996). Enterococcus, Streptococcus, Staphylococcus and Lactobacillus bacteria are the first to colonize (Parracho et al., 2007). This microbial flora is transitory and its role is simply to create a favorable environment for the true intestinal flora (Conway, 1997). On the contrary, the microflora of a child born through a cesarean section depends on the surrounding environment and is characterized by low levels of Bifidobacterium and Bacteroides type bacteria, and higher levels of Clostridium (sp. difficile) (Penders et al., 2006; Neut et al., 1987; Fanaro et al., 2003). During the first 2 days of a baby's life, a high oxidoreductive intestinal potential facilitates the development of facultative aerobic bacterial strains such as the more prevailing Escherichia (sp. coli), Streptococcus, and Enterococcus (VeeremanWauters et al., 2011; Walker, 2008; Saavedra, 2007). Only later, the progressive decrease in oxidoreductive potential, induced by the aforementioned strains, creates conditions that favor the development of obligate anaerobes belonging to the genera Bifidobacterium, Bacteroides and Clostridium, which after the first week of life represent about 80% of all the bacteria that make up the intestinal flora (Orrhage and Nord, 1999). The intestinal tract of a healthy full-term infant continues to host a simple and unstable pattern of microorganisms through the first few days of life. After the first week, colonization becomes more complex but also more stable and persistent, with about 109 to 1010 organisms per gram of feces (Palmer et al., 2007; Favier et al., 2002). Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00008-3 © 2016 Elsevier Inc. All rights reserved.

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TABLE 8.1  Classification of Indigenous Intestinal Bacteria Beneficial bacteria

Bifidobacterium spp. Lactobacillus spp. Eubatterium spp.

Potentially harmful bacteria

Staphyilococcus spp. Clostridium spp. Proteus spp. Pseudomonas (sp. aeruginosa) Veillonella spp.

Opportunistic bacteria

Escherichia (sp. coli) Streptococcus spp. Bacteroides spp. Enterococcus spp.

From the second week on, and regardless of the type of delivery, the development of the gut microflora is heavily influenced by nutrition (Tissier et al., 1900). Formula-fed infants seem to develop a more complex microflora, represented mostly by anaerobes such as Enterococcus spp., Klebsiella spp., Enterobacter spp., Clostridium spp., and lesser amounts of Bifidobacterium spp., Bacteroides spp., and Lactobacillus spp. On the other hand, the intestinal bacterial flora of breast-fed infants is characterized by the predominant presence of Bifidobacterium spp. and by lesser quantities of Staphylococcus spp., Streptococcus spp., and Lactobacillus spp. In fact, 85% of breast-fed infants harbor bifidobacteria as predominant microorganisms (Balmer and Wharton, 1989; Benno et al., 1984; Fanaro et al., 2003; Harmsen et al., 2000a,b; Hopkins et al., 2005; Penders et al., 2005, 2006; Stark and Lee, 1982a,b; Veereman-Wauters et al., 2011; Yoshioka et al., 1983). It appears that this difference in intestinal colonization between formula-fed and breast-fed infants could be related to the influence that some breast milk components have on the microbial flora, especially oligosaccharides and some humoral mediators of the immune response, such as secretory IgAs, cytokines, and growth factors such as IL-1, IL-6, IL-8, G-CSF, M-CSF, TNF-α, IFN-γ, in which breast milk is particularly rich (Agostoni et al., 2004a,b). In particular, human milk stimulates the growth of bifidobacteria because of its high oligosaccharides (10-12 g/L) content. These oligosaccharides are predominantly neutral, low-molecular-weight molecules, whose composition depends on the Lewis blood group of the mother (Stahl et al., 1994). Some data reported in the literature suggest that differential exposures to the outside world may also play an important role in the development of the endogenous flora; this is particularly important in the case of premature babies. In premature babies, delayed tube feeding, frequent wide-range antibiotic treatments, and the exposure to hospital microbial flora contribute to a delayed colonization by nonpathogenic commensal bacteria and to an increased risk colonization by pathogens (Agarwal et al., 2003; Butel et al., 2007; Claud and Walker, 2001; Lundequist et al., 1985; Magne et al., 2005; Walker, 2002). The most prevalent genera found in the feces of preterm babies are Enterococcus, Enterobacter, Escherichia (sp. coli), Staphylococcus, Streptococcus, Clostridium, and Bacteroides (Millar et al., 2003; Schwiertz et al., 2003; Stark and Lee, 1982a,b). This colonization pattern, although very similar to that of formula-fed term babies, seems to persist longer in preterm infants, and Bifidobacterium spp. bacteria establish themselves much later and at a much slower rate in preterm infants (Sakata et al., 1985; Stark and Lee, 1982a,b; Walker, 2013). The peculiarity of a preterm baby's intestinal ecosystem is due to the fact that those bacteria, which appear early in the intestinal flora, tend to stay longer than those introduced at a later time (Holman et al., 1989; Hooper and Gordon, 2001; Neu, 2007), as well as to the fact that once this colonization pattern has established itself, it is very difficult to change it. In fact, it is believed that inappropriate colonization by pathogens plays an important role in the pathogenicity of necrotizing enterocolitis (NEC) (Claud and Walker, 2001). When the intestinal flora becomes stabilized, it does not undergo any further qualitative changes. As the neonatal age ends, the composition of the bacterial flora changes further at the beginning of weaning, and especially among breast-fed infants (Stark and Lee, 1982a,b). When solid foods are introduced, the composition of the microflora gradually reaches its final pattern, which is characterized by a relatively stable prevalence of anaerobes; and after the second year of life, it shows all the characteristics typical of an adult microflora (Collins and Gibson, 1999) (see Table 8.2).

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TABLE 8.2  Factors That Influence the Gut Microflora Composition Age Gut microflora First few days of life

After first week of life

After weaning

Mode of delivery

Type of diet

High levels of anaerobic bacteria

Vaginal

Breast-fed infants

Bacteroides spp.

 Streptococcus spp.

High levels

Bifidobacterium spp.

 Staphylococcus spp.

Bifidobacterium spp.

Eubacterium spp.

 Enterococcus spp.

Low levels

Clostridium spp.

 Lactobacillus spp.

Staphylococcus spp.

Peptostreptococcus spp.

Cesarean section

Streptococcus spp.

Streptococcus spp.

 High levels

Lactobacillus spp.

Fusobacterium spp.

Clostridium spp.

Formulafed infants

Veillonella spp.

 Low levels

High levels

Low levels of aerobic bacteria

Bifidobacterium spp.

Enterococcus spp.

Escherichia spp.

Bacteroides spp.

Enterobacter spp.

Enterobacter spp.

Klebsiella spp.

Enterococcus spp.

Clostridium spp.

Klebsiella spp.

Low levels

Lactobacillus spp.

Bifidobacterium spp.

Proteus spp.

Bacteroides spp.

Streptococcus spp.

Lactobacillus spp.

Staphylococcus spp.

Preterms Enterococcus spp. Escherichia spp. Enterobacter spp. Klebsiella spp. Staphylococcus spp. Bacteroides spp. Streptococcus spp. Clostridium spp. Hospitalization Klebsiella spp. Enterobacter spp. Bacteroides spp. Clostridium spp.

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The microflora of different parts of the gastrointestinal tract differs from one another quantitatively and qualitatively (proximal-distal gradient). Anaerobes such as Bacteroides spp., Eubacterium spp., Streptococcus spp., and Fusobacterium spp. are found mostly in the large intestine and their rate reaches up to 99% in the rectum. The microflora of the colon is also horizontally stratified and shows a difference between luminal and mucosal microflora, which is further subdivided into flora of the mucosal layer, flora of the crypts, and flora that adheres to the colonocytes (Lee, 1984; Rozee et al., 1982; Swidsinski et al., 2002). Numerous studies have shown how the “bifidogenous flora,” which is comprised of bacteria of the genera Bifidobacterium and Lactobacillus, can benefit an individual's health by stimulating the immune system, inhibiting the development of the pathogenic flora, improving nutrients and mineral absorption, and allowing for vitamin synthesis and gas production (Grizard, 1999; Roberfroid, 2000; Salminen et al., 1998). The primary role of the microflora found in the colon is to obtain energy from food not digested in the upper gastrointestinal tract through fermentation. Approximately 8-10% of the total daily energy requirement derives from bacterial fermentation in the colon (Gibson et al., 2000). Short-chain fatty acids (SCFAs), such as acetic acid, butyric acid, and proprionic acid, are the main products of fermentation in the colon. Butyrate is metabolized by the epithelium of the large intestine's mucosa and plays an essential role in its trophism (Barcenilla et al., 2000; Cummings, 1981). The gut microflora also plays an important immunoregulatory role by promoting the proper development of the lymphoid tissue of the intestinal mucosa (referred to as gut-associated lymphoid tissue, or GALT), as documented by studies on germ-free animals (Moreau, 2001; Sudo et al., 1997). The so-called microbial-epithelial cross talk between the intestinal epithelium and the commensal bacteria allows for a suitable regulation of the intestinal immune and inflammatory response (Vanderhoof and Young, 2002). The proper interaction between the microflora and the intestinal epithelium is guaranteed by the presence of an intact mucosal barrier, a suitable bacterial colonization, an adequate activation of intestinal immune defenses, and modulation of intestinal inflammation (Caplan and Jilling, 2000; Millar et al., 2003). On the whole, the interaction between the microflora and the intestinal immune system allows the latter to develop a “suppressive” immune response, like oral tolerance, as well as an “inductive” response, such as the synthesis of IgA class antibodies. The role of oral tolerance is to inhibit immune responses against food antigens and antigens of commensal bacteria, enabling one to avoid inflammatory intestinal diseases and hypersensitivity reactions to food. Meanwhile, the role of secretory IgAs is to protect the intestinal mucosa from enteropathogenic organisms and to block resident bacteria and food antigens from entering into systemic circulation. There is quite a bit of evidence proving that, by sending signals via specific receptors, especially the toll-like receptors, intestinal bacteria can affect the function of epithelial cells, determine T-cell differentiation and antibody responses to T-cell dependent antigens, and regulate the intestinal immune response. The production of secretory IgAs is the primary component of the antibody response to pathogenic antigens. In addition, the colonization of the bacterial flora causes modulation of the Th2 response (proallergic) to a Th1 response (suppressive), which could reduce immune hyperreactivity, as occurs with allergic pathologies (Isolauri, 2004; MacDonald and Gordon, 2005). The ability of bifidogenic flora to inhibit the growth of pathogenic microorganisms, and consequently to reduce the incidence of intestinal infections, has been documented (Koletzko et al., 1998; Sandine, 1990). It is believed that the fundamental mechanism of this inhibitory process is directly related to a reduction of intestinal pH, caused by a substantial presence of lactic acid and acetic acid that are produced during carbohydrates' fermentation. Furthermore, gut microflora cells can produce active bactericides, defined as bacteriocidines, which can attack Clostridium spp., Escherichia (sp. coli), and other potentially pathogenic microorganisms (Gibson and Wang, 1994). Given this situation, it is easy to recognize how a change in the delicate balance between intestinal resident flora, the epithelium, and GALT is essential to understanding the physiopathology of numerous gastrointestinal and systemic diseases in both pediatric and adult age (Falk et al., 1998; O'Hara and Shanahan, 2007; Shanahan, 2002).

3 PREBIOTICS 3.1 Definition Generally, prebiotics are described as “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health” (Roberfroid, 2007a,b). The term refers to organic substances capable of facilitating the growth of intestinal microbial flora by acting as a nutritional substrate for endogenous microorganisms. According to the definition proposed by ENDO (European Project

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on Non-Digestible Oligosaccharides), prebiotics are “non-digestible oligosaccharides that can stimulate and promote the growth and/or metabolism of bifidobacteria and lactobacilli in the human intestine” (Jeurink et al., 2013; Van Loo et al., 1999). Accordingly, prebiotics must possess the following characteristics: l l l

They cannot be hydrolyzed or absorbed in the upper gastrointestinal tract. They must be a selective substrate for one or a few bacteria found in the colon, such as lactobacilli and bifidobacteria. They must be able to change the gut microflora into a healthier and more beneficial composition to the host organism (Gibson, 1995).

Prebiotics should make up about 10% of the total energy requirement and about 20% of the total volume of food ingested by humans.

3.2 Characteristics Every dietary component that reaches the colon in its intact form can potentially be a prebiotic. Prebiotics include various oligosaccharides (fructo-, galacto-, isomalto-, xylo-, and soyo-oligosaccharides), as well as lactulose and lactosucrose. When talking about prebiotic substances, the literature has focused specifically on nondigestible oligosaccharides (NDOs) (Parracho, 2007). Although they are part of a complex heterogeneous group of substances with different chemical compositions and prebiotic qualities, all NDOs have strong bonds that are resistant to the hydrolytic action of enzymes found at the beginning of the digestive tract, such as lactase, saccharase-isomaltase, maltase-glucoamylase, trealase, and amylase; because of this, they all arrive at the large intestine virtually untouched. The most studied NDOs are those found in breast milk, called human milk oligosaccharides (HMOS) and nonmilk-derived NDOs, such as the galacto-oligosaccharides (GOS) and the fructo-oligosaccharides (FOS), which are sold commercially. They are carbohydrates comprised of 3-10 monosaccharide units such as galactose, fructose, N-acetyl-glucosamine, and sialic acid, and they are linked to one another by their characteristic glucosidic bonds. Oligosaccharides are considered to be the most important prebiotic substrate because they meet all of the prebiotics' current classification criteria (Ouwehand et al., 2005; Rycroft et al., 1999).

4  HUMAN MILK OLIGOSACCHARIDES Human milk is considered the gold standard nutrient in infant nutrition, especially during the first 6 months of life (Cuthbertson, 1999). Human milk offers all the necessary nutrients needed for a baby's healthy growth and development. The HMOS found in breast milk play their prebiotic role by facilitating an intestinal microenvironment rich with Bifidobacterium spp. and Lactobacillus spp. (Garofalo and Goldman, 1999; Goldman et al., 1997; Hamosh, 1996; Oddy, 2002). After lactose (about 6g%) and lipids (about 4g%), these oligosaccharides represent the third most important component of human milk. Their highest concentration is found in the colostrum (>2%). In mature milk (about 10 g/L), they stabilize at 1.2-1.4%. HMOS are synthesized in the mammary gland by specific enzymes called the glycosiltransferases. These enzymes catalyze the sequential addition to the basic lactose molecule (glucose-galactose) of monosaccharide units that form linear and branched molecules thanks to the β-glycosidic bond in d-glucose, d-galactose, and N-acetyl-glucosamin molecules, and thanks to the α-glycosidic bond in l-fucose and sialic acid. More specifically, the l-fucose bond to the basic molecule is correlated to the secretory component of the Lewis antigen of the maternal blood group (Thurl et al., 1997). Lacto-N-tetraose is the most prominent oligosaccharide found in breast milk (Kunz et al., 1999). Evidence indicates that the HMOS fraction is characterized by substantial structural diversity, including over 1000 different identified molecules (Bode, 2006; Boehm and Stahl, 2003). Their concentration and composition differs among people and during the breastfeeding period. These HMOS are also present in their free form or linked to macromolecules such as glycol-proteins, glycol-lipids, and others (Chaturvedi et al., 2001). Since the human gut does not release luminal enzymes that can cleave α-glycosidic or β-glycosidic bonds, HMOS become resistant to intestinal enzymatic digestion (Engfer et al., 2000; Gnoth et al., 2000; Newburg and Neubauer, 1995; Rivero-Urgell and Santamaria-Orleans, 2001). Because of their low digestibility, HMOS can be easily traced in the feces of breast-fed infants (Coppa et al., 2001), despite the fact that some intestinal bacteria release glycosidases capable of metabolizing them (Hill, 1995). Since HMOS have been identified as functional components of human milk, many efforts have been made to mimic these functions with other alternative compounds.

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5  NONHUMAN MILK OLIGOSACCHARIDES 5.1  Oligosaccharides from Animal Milks The concentration of oligosaccharides found in the milk of other animals is very low and by far inferior to that of human milk. In addition, oligosaccharides found in animal milks have a very simple, much less complex molecular structure than that of HMOS (Bode, 2006; Chaturvedi et al., 2001). However, the preparation of these compounds is quite difficult and mass production is not commercially available. This is why clinical trials using nonhuman oligosaccharides as prebiotics are not yet available.

5.2  Nonmilk Oligosaccharides Nonmilk NDOs can be obtained from bacteria, yeasts, and plants. They can be extracted from natural sources, synthesized from monomers and/or small oligosaccharides, or produced by natural polymer hydrolysis. In fact, some NDOs, such as inuline, xylo-oligosaccharides, and maltose-oligosaccharides, are extracted from plant products (soy, chicory) and subsequently undergo partial enzymatic hydrolysis; others, like the FOS and GOS, are obtained from enzymatic synthesis, through glycosil-transferase, and from simple sugars, such as sucrose and lactose. The most commonly used nonmilk NDOs in pediatric trials are: l l l l l l l l

GOS, particularly the short chain ones (scGOS) Both short-chain and long-chain FOS (scFOS and lcFOS) Inulin Lactulose Blends of lactulose and scGOS Blends of scFOS and lcFOS Blend of galacturonic acid oligosaccharides combined with scGOS and lcFOS Blends of scGOS and lcFOS (Alliet et al., 2007; Arslanoglu et al., 2007a,b; Bongers et al., 2007; Brunser et al., 2006; Bruzzese et al., 2006; Costalos et al., 2008; Euler et al., 2005; Indrio et al., 2007; Kapiki et al., 2007; Moore et al., 2003; Rinne et al., 2003, 2005a,b; Savino et al., 2003, 2005; van Hoffen et al., 2009; Waligora-Dupriet et al., 2007; Ziegler et al., 2007)

The amount of fecal bifidobacteria, their percentage compared to the total number of bacteria, and the production of SCFA are generally used in evaluating their prebiotic effect. On the basis of these markers, there is sufficient evidence to classify only GOS, FOS, and inulin as prebiotics (Gibson et al., 2004; Roberfroid, 2007a,b). Inulin and FOS, a polymer and olygomer of fructose, respectively, are food components found as carbohydrates in nature in some plant species such as chicory, garlic, onion, leeks, radicchio, artichoke, banana, and cereal. They are classified as β(2 → 1) fructans, a term that refers to carbohydrates that have mostly fructosyl-fructose type glucosidic bonds. Inulin is a blend of polydisperse β-fructans, whose chains vary in length from 2 to 60 units, and has an average polymerization level equal to 10 monosaccharide units. The inulin available on the market is extracted through a hot water process from chicory root (Cichorium intybus), which contains 15-20% inulin and 5-10% FOS. The final product is a powder comprised of inulin with an average degree of polymerization of 10 to 12 monosaccharide units and a small quotient (about 6-10%) of monosaccharides and disaccharides, such as glucose, fructose, and sucrose. A more refined type of inulin, a “high performance inulin,” has recently become commercially available. It has an average degree of polymerization of 25 monosaccharide units and the advantage of causing less gastrointestinal side effects, such as flatulence and abdominal tension (Frank, 2002). FOS can be produced in two ways: through enzymatic hydrolysis of inulin extracted from chicory, using the inulase enzyme of Aspergillus niger, or through enzymatic synthesis from sucrose. The resulting FOS show an average degree of polymerization of 4 monosaccharide units and can be made up of only fructose chains or of a combination of fructose and terminal glucose. Meanwhile, GOS are a blend of olygominerals made up of one glucose molecule and a few galactose molecules. They are naturally found in foods such as legumes, dairy, and some fermented milk products. They are obtained from lactose biosynthesis induced by β-galactosidase of Aspergillus oryzae (6′-galactosyl-lactose), which catalyzes trans-galactosylation reactions, differently from human β-galactosidase, which hydrolyzes lactose into glucose and galactose. GOS are characterized by a degree of polymerization that ranges between 2 and 8 monosaccharide units and by β1-6 linkages. Among them

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are galactose β(1-6) glucose, galactose β(1-6) galactose, galactose β(1-3) glucose, and galactose β(1-2) glucose. The first two are found in yogurt and in some fermented milk products and, unlike lactose, they can resist the digestive action of human lactase because of the β(1-6) bond. Toxicology investigations have excluded any mutagenic, carcinogenic, or teratogenic action by the previously described NDOs (Carabin and Flamm, 1999). Furthermore, inulin and FOS have been classified as food ingredients and not as additives and have obtained the GRAS acronym (Generally Recognized as Safe). The GOS have also been approved as natural ingredients and are exempted from the limitations provisioned by the European Community on new foods (novel food) (De Bruyn et al., 1992). Companies in both Europe and the United States tend to use more inulin, GOS, and FOS; whereas those in Japan utilize mostly isomalt-oligosaccharides and xylo-oligosaccharides extracted from plants and synthesized from lactose or sucrose. A blend of scGOS/lcFOS (9:1 ratio) was recently suggested for neonatal formulas, with the intent of offering a prebiotic effect comparable to that of human milk (Haarman and Knol, 2005; Knol et al., 2005a,b; Moro et al., 2002; Schmelzle et al., 2003; Scholtens et al., 2006a,b). There are many other reasons for wanting to evaluate the effectiveness of NDOs rather than single components (Boehm and Stahl, 2003). One is that the composition of the bacterial flora is extremely complex and therefore various substrates could be needed for its development (Harmsen et al., 2000a,b). Another reason is the great structural variability of HMOS, which seems to be necessary in order to adequately stimulate the unique intestinal flora of breast-fed babies (Bode, 2006).

5.3  Mechanisms of Action The mechanisms of action of the most studied and best-known prebiotics are those of the oligosaccharidic fraction of breast milk, and can be summarized as the following four main effects. (1) Biomass effect. A number of HMOS found in the large intestine (equal to 40-60%) have a “biomass effect” that promotes the selective development of the bifidogenous flora by reducing the percentage composition of bacterioids, clostrides, and fusobacteria. The consequent fermentative metabolism determines the production of SCFAs (of which butyric acid is the most important); some amino acids (such as arginine, cysteine, and glutathione); as well as polyamines, growth factors, vitamins, and antioxidants. These substances play a crucial role in the nutritional needs of those species of bacteria that colonize the intestinal mucosa and participate in numerous metabolic processes. Even nonmilk oligosaccharides, like FOS, GOS, and inulin, stimulate bifidobacteria and lactobacilli's growth and activity to the detriment of bacteria of the genera Clostridium, Klebsiella, Enterobacter, and Bacteroides (Langlands et al., 2004; Rastall, 2004). Also, in addition to being used as a source of energy, SCFAs may have a trophic effect on the mucosa, can help reabsorb water, reduce intestinal pH, and make it less favorable for pathogenic germs to grow (Cummings et al., 1989; Rechkemmer et al., 1988). (2) Fiber effect. Many HMOS in the large intestine (equal to about 30-50% of the total) have a “fiber effect”; they are expelled through the feces, increasing fecal mass and the number of defecations (Coppa et al., 2001). (3) Immunomodulant effect. HMOS also play an important “immunomodulant effect.” In fact, their fermentation by anaerobes produces the previously described SCFAs, such as butyrate, that can reduce epithelial cells' glutamine requirements, in favor of immunocompetent cells (Salvini, 2003). (4) Anti-infective effect. The anti-infective effect is expressed through a direct and an indirect mechanism. The direct mechanism is linked to the chemical structure of HMOS, which is similar to that of the bonding sites recognized by the bacteria on the epithelium of the enteric mucosa. As a result, they act as “soluble receptors,” able to competitively bind to the pathogenic agents and their toxins and blocking their actions (Kunz et al., 1999). For example, mannose-rich glycoprotein can compete for the bond with type 1 fimbriae of Escherichia coli, whereas sialo-galactoside can bind to the S. fimbriae of the same germ. Concerning this, protective effects of HMOS against enteropathogenic E. coli, Campylobacter jejuni, Shigella spp., and Vibrio colerae gastroenteritis have been reported (Beachey, 1981; Mirelman, 1986). This protective action of the oligosaccharide fraction of breast milk is also present in the upper respiratory tract, blocking the adhesion of some strains of S. pneumoniae and H. influenzae. Table 8.3 shows a number of breast milk oligosaccharides that are able to act as specific ligands (receptors) that bind to pathogenic microorganisms, both bacteria and viruses. On the other hand, the indirect anti-infective effect is determined by a previously described decrease in intestinal pH (see Figure 8.1).

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TABLE 8.3  Pathogenic Bacteria and Oligosaccharide Receptors of Breastmilk Bacteria

Receptors

• E. coli (type 1 fimbria)

Glycoproteins with mannose

• E. coli (thermostable enterotoxin)

Fucosylated oligosaccharides

• E. coli

Fucosylated tetra and penta-saccharides

• E. coli (S. fimbria)

Sialyl (α2-3) lactose and glycoproteins Mucins' sialyl (α2-3) galactosides

• S. pneumoniae

Neutral oligosaccharides

• Pseudomonas aeruginosa

Gal(β1-4) GlcNac o Gal (β1-3) GlcNac

• C. pilory

Sialyl-lactose

• Streptococcus sanguis

Sialyl-lactose

• C. pillory

Sialyl-lactose and sialyl glycoproteins

• M. pneumonia

Sialyl (α2-3) glycoproteins

• M. pneumoniae

Sialyl p-N-acetyl-lactosamine

• Influenza virus A

Sialyl (α2-6) lactose

• Influenza virus B

Sialyl (α2-6) lactose

5.4  Side Effects A daily dose of prebiotics  4 are usually obtained at different time points during the synthesis reaction (Albayrak and Yang, 2002; Neri et al., 2009). The different sources of β-galactosidases produce oligosaccharides with different glycosidic linkages. For instance, the β-galactosidase from L. reuteri preferred to form β-(1→3) and β-(1→6) linkages (Maischberger et al., 2008), while mainly β-(1→4) linkages were formed using the β-galactosidase from Cryptococcus laurentii (Ohtsuka et al., 1990) and B. circulans (Mozaffar et al., 1986). Moreover, A. oryzae β-galactosidase preferred to form β-(1→6) linkages mainly, while the GOS produced by B. bifidum NCIMB 41171 consisted primarily of β-(1→3) linkages, with less amounts of β-(1→4) and β-(1→6). The different glycosidic linkages in the obtained GOS mixtures are formed and hydrolyzed at different rates, so the reaction conditions and time influence the structures present in GOS mixtures. An example of this was observed using the β-galactosidase from B. circulans, as the percent of β-d-Galp-(1→4)-β-dGalp-(1→4)-d-Glc decreased from ~95% to ~30-35% of the total trisaccharides between 1 and 23 h, while the percent of β-d-Galp-(1→4)-β-d-Galp-(1→3)-d-Glc increased to ~18% of the total trisaccharides after 23 h of the reaction (Yanahhira et al., 1992). The different DP and glycosidic linkages in the GOS mixture might affect its prebiotic efficacy. In vitro studies already showed that some Bifidobacterium species preferentially metabolized GOS with DP 3 and 4 over transgalactosylated disaccharides (Gopal et al., 2001). The different glycosidic linkages were also shown to affect the growth of probiotic bacteria differently (Depeint et al., 2008; Sanz et al., 2005, 2006a,b). Human trials, for instance, confirmed that GOS produced using B. bifidum NCIMB 41171, which consisted mainly of β-(1→3) linkages and to a lesser extent β-(1→4) and β-(1→6) linkages, had better prebiotic efficacy compared to GOS mixture containing mainly β-(1→4) and β-(1→6) linkages (Depeint et al., 2008).

5  TYPES OF BIOCATALYSTS USED IN GOS SYNTHESIS 5.1  Whole Cell Biocatalysts Whole cells can be used in their viable or nonviable (resting) forms to conduct GOS synthesis. The use of whole cells is the preferred option when the isolation of the β-galactosidase from its natural producing source is tedious and costly (Fukuda et al., 2008). Whole cells are also chosen as biocatalysts for membrane-bound and cofactor-dependent enzymes (Burton et al., 2002) because it is generally less expensive to regenerate cofactors in metabolically active cells compared to their in vitro regeneration (Schmid et al., 2001). This case, however, is not common in GOS synthesis, as β-galactosidases usually use metal ions as cofactors. Using viable whole cells has the advantage that additional metabolic functions can be included in the GOS synthesis process to remove glucose and galactose and thus improve the GOS yield and purity. These monosaccharides are by-­products, have no prebiotic effects, and contribute to raising both the caloric and glycemic index of foods containing nonpurified GOS. An example of this approach was reported using the whole viable cells of S. elviae, Sirobasidium magnum, and R. minuta, which grew on lactose to produce GOS, and consumed glucose, in particular, as a carbon source for cell growth (Onishi et al., 1995, 1996; Onishi and Tanaka, 1996, 1997). These fermentation systems produced higher GOS yields compared to the use of resting cells or purified β-galactosidases from the same microorganisms. Another interesting example was the synthesis of GOS by expressing a β-galactosidase from Penicillium expansum F3 on the cell surface of Saccharomyces cerevisiae EBY by galactose induction. The anchored β-galactosidase on the yeast cell surface

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produced GOS from lactose; glucose was consumed by the yeast cells as a carbon source, while galactose was used for β-galactosidase expression (Li et al., 2009). Nevertheless, the use of viable whole cells in GOS synthesis is associated with the production of metabolic end products (e.g., ethanol, lactic acid, acetic acid, etc,), the presence of side reactions, and the presence of other remaining components in the culture medium. This inevitably affects the taste and the other characteristics of the final GOS product. Additionally, further complicated purification steps are required to remove these impurities, which add a lot to the cost of GOS production. Another disadvantage of using viable cell biocatalysts is that elevated temperatures, which are desirable during GOS synthesis, should be avoided especially when using nonthermophilic cells, due to the loss of the cells’ viability and ability to conduct GOS synthesis and perform the other desirable metabolic functions. Nonviable cells with high β-galactosidase activity can be also used for GOS synthesis. In many instances, their use is advantageous over viable cells. The use of resting cells protects, to some extent, the β-galactosidases from the external environment and thus whole resting cells can be considered generally as stable biocatalysts. Resting cells can be also used to conduct GOS synthesis at temperatures above the optimum temperature of their growth as long as their β-galactosidases retain their activity. The use of the whole resting cells of B. bifidum NCIMB 41171 at temperatures as high as 60-65 °C is one of the best examples in this regard (Osman et al., 2010, 2012). These cells gave high GOS yields at 55-65 °C compared to 40 °C (Osman et al., 2012). Nonviable cells have been also used in combination with viable cells to conduct GOS synthesis. An example of this was reported by Goulas et al. (2007) who used the resting cells of B. bifidum NCIMB 41171 to perform GOS synthesis and the living cells of Saccharomyces cerevisiae to consume glucose, therefore purifying the obtained GOS mixture. In this case, however, the whole system was operated at 40 °C, as higher temperatures would have stopped the desired metabolic functions of the Saccharomyces cells. Therefore, the advantage of using high temperature to obtain high GOS yields was not achieved in this system.

5.2 Free β-Galactosidases In general, the use of free β-galactosidases can, to a large extent, circumvent all the disadvantages observed when using whole cell biocatalysts. The use of free β-galactosidases usually increases the reaction rates, and thus shorter reaction times are required to obtain the maximum GOS yield, which increases the productivity of the synthesis process. This was clearly observed when the β-galactosidases BbgI, BbgIII, and BbgIV were used free in solution instead of using the whole cells of B. bifidum NCIMB 41171 (Osman et al., 2012). The use of free β-galactosidases also ensures that transgalactosylation can be better controlled compared to the use of whole cells because (1) when free β-galactosidases are used, the possibility of unpredictable side-reactions is diminished and (2) when whole cells containing multiple β-galactosidases with different biochemical characteristics are used, it becomes difficult to control GOS synthesis. The case of the four β-galactosidases BbgI, BbgII, BbgIII, and BbgIV found in B. bifidum NCIMB 41171 is the best example here. Using either free BbgIII or BbgIV, which have high transgalactosylation activity, excluded the hydrolytic activity of BbgII, observed when using the whole Bifidobacterium cells (Osman et al., 2012). Few other examples of whole cells with multiple β-galactosidases include B. circulans with two β-galactosidase (Mozaffar et al., 1984), Aspergillus niger with three β-galactosidases (Widmer and Leuba, 1979), and B. bifidum DSM 20215 with three β-galactosidases (Møller et al., 2001). The combined activity of these β-galactosidases when using the whole cells usually limits the control of the biocatalytic process, highlighting the advantage of using a free β-galactosidase over whole cell biocatalysts. Despite the above advantages, the use of free β-galactosidases in GOS synthesis requires the isolation of the enzyme either from the producing microorganism or from the culture medium. In both cases, the isolation process is tedious and costly as β-galactosidases are found naturally in low quantities in general and are mostly located intracellulary, especially in the case of bacterial β-galactosidases.

5.3 Recombinant β-Galactosidases Recombinant DNA technology offers the possibility to express and optimize the production of β-galactosidases with interesting biochemical properties for GOS synthesis using microbial hosts that are known to produce heterologous proteins efficiently. Compared to native β-galactosidases, the use of recombinant β-galactosidases offers various advantages such as large-scale production, ease of purification due to high expression yields, and the possible improvement in enzyme activity and stability through molecular approaches (Ji et al., 2005). Bacterial expression systems have been widely used for producing recombinant β-galactosidases because (1) they are able to grow rapidly and reach high densities using inexpensive substrates, (2) they have well-characterized genetics, and

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(3) a large number of cloning vectors and mutant host strains are available. The most widely used bacteria for producing β-galactosidases are E. coli and Bacillus subtilis. E. coli fermentation processes are very economical compared to other expression hosts. The progress in understanding the transcription, translation, and protein folding in E. coli along with the availability of modern genetic tools have made E. coli a very valuable tool for protein expression (Baneyx, 1999; Sørensen and Mortensen, 2005). The main disadvantages of E. coli, however, include (1) the production of lipopolysaccharides, generally known as endotoxins; (2) the difficulty in expressing proteins with disulphide bonds; (3) the lack of protein glycosylation, which might affect the activity of some expressed proteins; (4) the formation of acetate, which leads to cell toxicity; and (5) the production of inactive inclusion bodies, which require refolding or the co-expression of chaperones (Terpe, 2006; Demain and Vaishnav, 2009). B. subtilis is another well-used bacterium to produce recombinant β-galactosidases. The genes of B. subtilis have been sequenced and there is no production of harmful exo- or endotoxins. Moreover, the expressed proteins can be secreted into the fermentation medium, which results in easy downstream processing. B. subtilis is generally recognized as safe, efficient, cost-effective, and metabolically robust (Demain and Vaishnav, 2009). In contrast to E. coli, little is known about disulphide bond formation and isomerization. The general disadvantages of Bacillus strains include (1) the secretion of high levels of proteases into the culture medium, which has the potential to degrade other secreted recombinant proteins (this has been recently overcome by developing protease-deficient strains) and (2) the instability of plasmids, and occasionally reduced or absent expression of the proteins of interest (Demain and Vaishnav, 2009; Yin et al., 2007). Lactic acid bacteria are attractive microbial hosts for the production of recombinant enzymes because (1) they are widely used in industrial fermentations and abundant information is available about their nutrient requirements and cultivation conditions; (2) novel genetic engineering tools and well-characterized molecular pathways have been developed in lactic acid bacteria; (3) several inducible and controlled expression systems have been developed, of which the nisin-controlled gene expression system in Lactobacillus lactis is probably the best known and well-studied one (20-22); (4) they are completely food-grade expression systems that do not produce any harmful compounds; and (5) in many cases, the production of recombinant proteins does not require any unwanted selection markers (e.g., antibiotic resistance genes) or any use of antibiotics in the fermentation medium (Maischberger et al., 2010; Schwab et al., 2010; Iqbal et al., 2010; Wegmann et al., 1999; Hickey et al., 2004; Bron et al., 2002; Platteeuw et al., 1996). These food-grade expression systems, however, require stable processes in large industrial-scale applications to be implemented for the commercial production of recombinant β-galactosidases. Yeast expression systems, particularly S. cerevisiae and Pichia pastoris, also have been used to produce β-galactosidases. The advantages of yeast expression systems include (1) high cell densities and high protein yield and productivity; (2) cost-effectiveness, durability, and stable production strains; (3) production of disulphide-rich proteins; (4) proper protein folding; and (5) genetically well-characterized strains known to perform many posttranslational modifications (Buckholz and Gleeson, 1991; Porro et al., 2005; Demain and Vaishnav, 2009). S. cerevisiae has a long history of use in industrial fermentations and can secrete heterologous proteins into the culture medium when proper signal sequences are attached to the structural genes (Demain and Vaishnav, 2009; Yin et al., 2007; Porro et al., 2005). P. pastoris has become very attractive as a host for the industrial production of recombinant proteins, as the promoters controlling gene expression are among the strongest and the most strictly regulated yeast promoters (Demain and Vaishnav, 2009; Yin et al., 2007; Porro et al., 2005). The major advantage of P. pastoris over E. coli is that the former is capable of (1) producing disulphide bonds and glycosylation for proteins, (2) secreting proteins into the medium, and (3) growing in media containing one carbon and one nitrogen source. The major advantages of P. pastoris over S. cerevisiae include (1) the high protein productivity, (2) the avoidance of hyperglycosylation, (3) the growth in reasonably strong methanol solutions that would kill most other microorganisms, and (4) the integration of multicopies of foreign DNA into chromosomal DNA yielding stable transformants (Demain and Vaishnav, 2009; Yin et al., 2007; Porro et al., 2005). One of the disadvantages of P. pastoris, however, is that it is unable to produce chaperones for the proper folding of some expressed proteins. Other expression hosts such as fungi, insect cells, and mammalian cells have not been widely used for expressing β-galactosidases (Demain and Vaishnav, 2009; Yin et al., 2007; Porro et al., 2005). Examples of several β-galactosidases, expressed in different microbial hosts, are shown in Table 9.2. It should be mentioned that the efficient production of biologically active and soluble recombinant β-galactosidases is affected by a variety of factors related to the used vector, the host strain, the host-vector interaction, and the fermentation conditions. Detailed information of these factors and their influence on producing recombinant β-galactosidases can be read elsewhere in the literature.

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TABLE 9.2  Examples of Several Microbial β-Galactosidases Expressed in Different Expression Hosts Expression host

Origin of the enzyme

References

Escherichia coli ER 2566

Sulfolobus solfataricus

Kim et al. (2006) and Park et al. (2008)

E. coli K-12

Thermus sp. Z-1

Akiyama et al. (2001)

E. coli JM109

Geobacillus stearothermophilus

Placier et al. (2009)

E. coli BL21 (DE3)

Thermotoga maritime

Ji et al. (2005)

E. coli JM105

Lactobacillus bulgaricus B131

Schmidt et al. (1989)

E. coli BL21 star (DE3)

Lactobacillus reuteri L103

Nguyen et al. (2006, 2007)

E. coli TG1

Lactobacillus plantarum FUA3112 and Lactobacillus acidophilus FUA3191

Schwab et al. (2010)

E. coli ER2566

Bifidobacterium breve B24

Yi et al. (2011)

E. coli DH5α

Bifidobacterium bifidum NCIMB 41171

Osman et al. (2013)

E. coli ER 2566

Pseudoalteromonas sp. 22b

Cieslinski et al. (2005)

E. coli LMG 194

Paracoccus sp.32d

Wierzbicka-Woś et al. (2011)

Saccharomyces cerevisiae

Aspergillus niger

Domingues et al. (2002) and Oliveira et al. (2007)

S. cerevisiae

Kluyveromyces lactis

Becerra et al. (2001a,b, 2002, 2004)

Bacillus subtilis

Bacillus stearothermophilus

Chen et al. (2008)

B. subtilis

G. stearothermophilus

Xia et al. (2010)

Lactobacillus lactis subsp. lactis MG1363

L. bulgaricus WCH9901

Wang et al. (2008)

L. lactis MG1363

L. plantarum FUA 3112 L. acidophilus FUA 3191

Schwab et al. (2010)

L. plantarum

L. plantarum WCFS1

Iqbal et al. (2010)

L. lactis NZ23900

L. reuteri, L. acidophilus, Lactobacillus sakei, and L. plantarum

Maischberger et al. (2010)

Pichia pastoris

Alicyclobacillus acidocaldarius ATCC 27009

Yuan et al. (2008)

P. pastoris

Arthrobacter sp. 32c

Hildebrandt et al. (2009)

6  IMPROVING THE GOS SYNTHESIS PROCESS Various approaches have been used to improve GOS synthesis. The most used ones are discussed below.

6.1  Immobilization of β-Galactosidases Immobilization is a process that converts the enzyme into a form that is physically confined or localized in a certain defined region of space, thus hindering the mobility of the enzyme while at the same time retaining its catalytic activity (Chibata, 1978; Lalonde and Margolin, 2002). The biochemical characteristics of the enzyme (e.g., pH and temperature profile, stability, and activity), the properties of the carrier (e.g., particle size and shape, surface area, molar ratio of hydrophilic to hydrophobic groups, mechanical properties, and stability under different conditions) and the chosen immobilization technique and conditions determine the final properties of the immobilized enzyme and the extent to which a robust GOS synthesis process can be developed. Immobilized β-galactosidases can be repeatedly and continuously used in a variety of bioreactors. Besides, the stability of β-galactosidases usually increases after immobilization. This increases the time that the same mass of enzyme can be used, thus

146  PART | I  Prebiotics in Health Promotion

ultimately reducing the cost of the GOS synthesis process. Furthermore, the easy separation of the immobilized β-galactosidase from the reaction medium can, to a great extent, ensure that the final product is enzyme free. Additionally, immobilization might result in improving the enzyme properties and the process productivity (Albayrak and Yang, 2002; van Beilen and Li, 2002; Cao et al., 2003; Hanefeld et al., 2009; Huerta et al., 2011; Mateo et al., 2007; Tsakiris et al., 2004; Osman et al., 2014). On the other hand, there are many drawbacks related to the use of immobilized β-galactosidases. The activity of the β-galactosidases can be sometimes reduced after immobilization, due to the losses caused by the binding procedure and the mass transfer effects; the latter usually reduces the accessibility of substrate molecules to the β-galactosidase active site and leads to lower efficiency in the GOS synthesis process (van Beilen and Li, 2002; Bickerstaff, 1997; Cao et al., 2003; Hanefeld et al., 2009). Nevertheless, this disadvantage can be compensated by the increased stability of the immobilized β-galactosidase. Another disadvantage is the desorption of the immobilized β-galactosidase from the support matrix in the case of immobilization by physical adsorption, and the leakage of the immobilized β-galactosidase from the gel matrix in the case of immobilization by entrapment (van Beilen and Li, 2002; Bickerstaff, 1997; Cao et al., 2003; Hanefeld et al., 2009; Mosbach, 1987). This drawback is usually overcome by using cross-linking reagents in combination with physical adsorption and entrapment. Furthermore, β-galactosidases vary a lot in their biochemical characteristics and their ability to produce GOS. As a result, no universal immobilization technique or support carrier exists for immobilizing different β-galactosidases, and therefore a variety of immobilization techniques and carrier supports should be tested for a given β-galactosidase. This obviously increases the time required for developing GOS synthesis using immobilized β-galactosidases. Despite the above, the immobilization of β-galactosidases is highly desirable, due to the moderate thermal and operational stability of free β-galactosidases, the enhanced performance of GOS synthesis using immobilized β-galactosidases, and the expected savings in the enzyme cost.

6.1.1  Methods of β-Galactosidase Immobilization Immobilization techniques can be divided into two distinctive groups: binding (includes cross-linking and carrier binding) and inclusion. 6.1.1.1 Cross-linking Immobilization by cross-linking is based on the use of bi- or multifunctional reagents to form intermolecular cross-linkages either between the enzyme molecules to produce cross-linked enzyme aggregates (CLEAs), or between the enzyme molecules and the insoluble support materials (this case can be also considered as a covalent binding type of immobilization) (Lalonde and Margolin, 2002; Tanaka and Kawamoto, 1999). The cross-linking method was suggested as an alternative to those methods, resulting in a reduced yield and productivity of biocatalytic reactions due to the presence of the noncatalytic mass of the carrier. Yet, this is not always the case, as forming CLEAs might be impractical due to the fact that low-enzymatic activities can be obtained in CLEAs in case the intermolecular cross-linkages take place at or near the active site. Moreover, the cross-linking reaction might require the use of severe conditions, resulting in undesirable changes to the enzyme and a significant loss of activity. Usually, cross-linking is best used with another immobilization technique, mainly with physical adsorption and entrapment. The use of cross-linking along with these two techniques helps overcome many of the problems faced when using adsorption and entrapment individually; that is, the desorption of the β-galactosidase from the support material in the physical adsorption method and the leakage of the entrapped enzyme due to the small molecular weight of the enzyme compared to the pore size of the gel and/or the matrix used in the entrapment method. There are many examples of using cross-linking as a sole technique for the immobilization of β-galactosidases or in combination with another immobilization technique. For instance, the A. oryzae β-galactosidase was immobilized by forming CLEAs using glutaraldehyde. The CLEAs improved the temperature stability of the enzyme compared to the free enzyme, up to 65 °C. However, CLEAs had mainly hydrolytic activity; CLEAs gave only ~4% GOS compared to ~23% GOS obtained by the free enzyme from 20% (w/v) lactose (Gaur et al., 2006). Also, Neri et al. (2009) immobilized the A. oryzae β-galactosidase on magnetic polysiloxane-polyvinyl alcohol using glutaraldehyde as a cross-linking agent, and conducted GOS synthesis using the immobilized enzyme. The GOS synthesis was not affected by the immobilization, indicating the absence of diffusion limitations in the carrier. Additionally, the immobilized enzyme retained 84% of its initial activity after 10 repeated batches at 25 °C (Neri et al., 2009). 6.1.1.2  Carrier Binding Carrier binding is the oldest technique of immobilization. It is based on binding the enzyme molecule to a water-insoluble carrier. The carrier binding method can be divided into covalent binding and noncovalent binding (physical adsorption and ionic binding), depending on the binding mode.

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6.1.1.2.1  Covalent Binding Immobilization by covalent binding is based on the formation of covalent bonds between the β-galactosidase molecules and the carrier support via certain functional groups such as amino, carboxyl, hydroxyl, and sulfydryl groups. One of the major disadvantages of covalent binding is that the immobilization conditions are more complicated and less mild than physical adsorption and ionic binding. Therefore, covalent binding may alter the conformational structure and the active site of the enzyme, resulting in a major loss of activity due to exposing the enzyme to toxic reagents and/or severe reaction conditions (Tanaka and Kawamoto, 1999). Also, the functional groups of amino acids of the active site or those near the active site might be involved in covalent bond formation, resulting in a major loss of activity. Despite the above, the main advantage of covalent binding is that the binding forces between the enzyme and the carrier are so strong that no leakage of the enzymes occurs (Lalonde and Margolin, 2002). These strong binding forces increase the β-galactosidase stability. The covalently bound A. oryzae β-galactosidase to cotton cloth activated by tosyl chloride, for instance, showed activity retention of 55% and a 25-fold increase in thermal stability compared to the free enzyme. It was successfully used for GOS synthesis in batch and continuous reactors with no diffusion limitation issues (Albayrak and Yang, 2002). The same enzyme was covalently immobilized on magnetic hydrazide-Dacron particles via glutaraldehyde and used for GOS synthesis. The immobilized enzyme retained 90% of its initial activity after 10 repeated uses at 25 °C using 20% (w/v) lactose (Neri et al., 2011). Furthermore, the A. oryzae β-galactosidase immobilized by covalent binding to chitosan was stabilized by 1.6-fold at 60 °C (Gaur et al., 2006), and was able to synthesize GOS with a yield of 17.3% (w/w) compared to only 10% (w/w) using the free enzyme from 20% (w/v) lactose solution at 40 °C (Gaur et al., 2006). Additionally, β-galactosidase from the same source was immobilized by covalent binding to glyoxyl-agarose and used for GOS synthesis. The immobilized enzyme was used for 10 repeated batches and showed improved efficiency by ~200% compared to the free enzyme (Huerta et al., 2011). 6.1.1.2.2  Noncovalent Binding This method can be subdivided into simple physical adsorption and ionic binding. The former is based on the adsorption of β-galactosidase molecules on the surface of water-insoluble carriers by van der Waals forces, hydrogen bonding, and hydrophobic interactions, while the latter relies on binding the β-galactosidase molecules to water-insoluble carriers containing ion-exchange residues via ionic interactions (Hartmeier, 1986; Spahn and Minteer, 2008). The main difference between both techniques is that the linkages between the enzyme molecules and the carrier are stronger in the case of ionic binding than in the case of physical adsorption (Costa et al., 2005). Immobilization via noncovalent binding is easily carried out, and the conditions are much milder than those used in covalent binding. Hence, noncovalent binding methods are less disruptive to the enzyme and cause little change in the conformation and the active site of the enzyme, as the use of chemical reagents is not required. Therefore, this method yields immobilized enzymes with high activity in most cases. The main disadvantage of this method is the desorption of enzyme molecules from the carrier at high ionic strength or upon variation in the pH and/or temperature (Costa et al., 2005; Tanaka and Kawamoto, 1999). For instance, the partially purified Bullera singularis β-galactosidase was noncovalently immobilized on chitopearl BCW 3510 beads. The immobilized enzyme was used for 15 days in a continuous process for GOS synthesis with a GOS yield of about 55% (Shin et al., 1998). Also, immobilization of the β-galactosidase from Thermus sp. T2 was performed using ionic adsorption onto two different supports: Sepabeads® internal surfaces coated with polyethylenimine (PEI) and DEAE-agarose. The PEI Sepabeads® remained fully active at pH 5 and 7 after several weeks of incubation at 50 °C (Pessela et al., 2003). Besides, Osman et al. (2014) immobilized the β-galactosidase from B. bifidum NCIMB 41171 on Q-Sepharose by ionic binding. The yield of immobilization exceeded 90% and the GOS yield was similar to that obtained using the free enzyme (i.e., 49-53%). The immobilized enzyme was used for six repeated GOS synthesis batches (Osman et al., 2014). Another example is the immobilization of A. oryzae β-galactosidase using the ion-exchange resin Duolite A568 as a carrier, followed by cross-linking with glutaraldehyde. The residual activity of the immobilized enzyme without cross-linking was 51% of the original activity after 30 uses, while the residual activity with cross-linking was 90%. As mentioned above, cross-linking, in this case, improved the retained activity of the enzyme and prevented enzyme desorption (Guidini et al., 2010). 6.1.1.3 Inclusion The inclusion (entrapment) of β-galactosidases is based on the physical localization of the enzyme molecules within lattices of a semipermeable gel or in a semipermeable polymer membrane (Spahn and Minteer, 2008). The enzyme is retained within the gel and/or the membrane while the substrates and the products are allowed to diffuse through (Lalonde and Margolin, 2002). This method differs from covalent binding and cross-linking in that the enzyme does not bind to the gel

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matrix or the membrane. The advantages of inclusion are its simplicity and the extremely large surface area between the substrate and the enzyme, within a relatively small volume (Costa et al., 2005). However, the major drawback is the possible leakage of the β-galactosidase molecules during repeated use, due to the small molecular size of the entrapped enzyme compared to the pore size of the gels and/or the membranes. Improvements can be made by using suitable cross-linking reagents. Another disadvantage is the diffusion limitations posed on the mobility of the substrate across the gel and/or the membrane toward the enzyme. For instance, the β-galactosidase from P. expansum F3 was immobilized in calcium alginate beads. The immobilized enzyme was able to produce 28.7% GOS from 380 g/L lactose at 50 °C for seven repeated batches with no observed decrease in the GOS yield and with a good operational stability (Li et al., 2008). Another example is the entrapment of the A. oryzae β-galactosidase in polyvinyl alcohol (Grosova et al., 2008). The immobilized enzyme had improved properties in that it was less inhibited by the products of lactose hydrolysis compared to the free enzyme, in addition to the fact that it was stable after 35 repeated batches at 45 °C. Moreover, the β-galactosidase from A. oryzae was entrapped in fibers composed of alginate and gelatine cross-linked with glutaraldehyde (Tanriseven and Dogan, 2002). The immobilized enzyme retained 56% of its activity after immobilization and was active for 35 days without a decrease in its activity. Glutaraldehyde in this case also stabilized the alginate-gelatine fibers and prevented the leakage of the enzyme.

6.2  Protein Engineering Protein engineering is another approach to improve GOS synthesis. The concept of using protein engineering is mainly based on increasing the transgalactosylation activity of the used β-galactosidase at the expense of its hydrolytic activity through changing specific amino acids at the active site of the enzyme. This approach has been recently attempted using few β-galactosidases. For instance, changing the phenylalanine residue at position 426 to tyrosine in the β-glucosidase (CelB) of the hyperthermophilic Pyrococcus furiosus increased the GOS yield from 40% to 45% (Hansson et al., 2001). Also, changing both the phenylalanine residue at position 426 to tyrosine and the methionine residue at position 424 to lysine of the same enzyme showed better transgalactosylation properties at low lactose concentrations compared to the wild-type. For instance, the GOS yield was 40% using the engineered β-galactosidase compared to only 18% using the wild-type enzyme from 10% initial lactose concentration (Hansson et al., 2001). Another example, in this regard, is the β-galactosidase (BgaB) from Geobacillus stearothermophilus KVE39 (Placier et al., 2009). The change of the arginine residue at position 109 to tryptophan increased the GOS yield and productivity by 12- and 17-fold, respectively (Placier et al., 2009). Furthermore, the deletion of approximately 580 amino acid residues from the C-terminal end of the β-galactosidase (BIF3) from B. bifidum DSM 20215 increased the transgalactosylation activity of the enzyme. The truncated β-galactosidase was able to convert ~90% of the reacted lactose into GOS, while only 10% of the reacted lactose was hydrolysed (Jørgensen et al., 2001). Another example is the β-galactosidase (LacS) from Sulfolobus solfataricus P2, which was subjected to sitedirected mutagenesis and two mutants were obtained. In the first mutant, the phenylalanine residue at position 441 was changed to tyrosine, while in the second mutant the phenylalanine residue at position 359 was changed to glutamine (Wu et al., 2013). The GOS yield was 50.9% using the wild type, 61.7% using the first mutant, and 58.3% using the second mutant (Wu et al., 2013). Protein engineering might also be used to reduce the inhibition effects exerted by galactose and glucose, increase the thermal stability of mesophilic β-galactosidases, and alter the substrate and acceptor specificity to produce novel GOS structures. However, changing and/or removing many amino acids to obtain the desired improvement in GOS synthesis might alter the folding and/or the structure of the used β-galactosidase on the secondary, tertiary, and quaternary levels. These probable structural changes should be taken into consideration, as they might affect the eventual performance of the enzyme used in GOS synthesis. Another point to consider is the possible changes in the catalytic efficiency of the engineered enzyme compared to its wild-type counterpart. One of the remaining challenges in using engineered β-galactosidases is if they will be, at all, allowed for use in the food and biotechnology industry for the production of GOS.

6.3  Reaction Medium Engineering The concept of decreasing the availability of water molecules to act as acceptors of the galactosyl moieties has been studied in organic solvent systems because transgalactosylation can be greatly favored over hydrolysis compared to aqueous systems at the same initial lactose concentration (Chen et al., 2001; Cruz-Guerrero et al., 2006). Additionally, the presence of organic solvent limits the potential for microbial contamination within the process. Wang et al. (2012) performed GOS synthesis in organic-aqueous biphasic media using a novel metagenome-derived β-galactosidase BgaP412. They obtained a maximum GOS yield of 46.6% (w/w) at 75.4% lactose conversion in a cyclohexane/buffer system (95:5) under optimum reaction conditions; the GOS yield was higher than that obtained in aqueous medium. Bankova et al. (2006) also reported

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improved transgalactosylation in organic-aqueous systems compared to aqueous systems only, using a β-galactosidase from A. oryzae. Moreover, the synthesis of GOS using a β-galactosidase from A. oryzae in aqueous-organic co-solvent using enzyme-­ cyclodextrin co-lyophilizate was 1.8 times more than it was in the aqueous system (Srisimarat and Pongsawasdi, 2008). Ionic liquids have been also tried for the synthesis of GOS and GOS derivatives. Kaftzik et al. (2002) produced N-acetyllactosamine using the transgalactosylation activity of the β-galactosidase from B. circulans using 25% (v/v) of 1,3-di-methyl-imidazolmethyl sulfate as a water-miscible ionic liquid. The use of ionic liquids in this example suppressed the hydrolysis of the formed product and doubled the yield to ≈60%. Reverse micelles, which are self-assembling structures of water pools in organic solutions produced by the aid of surfactants, have been also used for GOS synthesis. The catalytic behavior of enzymes entrapped in the water pool of reverse micelles is quite different from that in aqueous media. The water in the pools shows also different properties from the water in bulk aqueous solutions. Another advantage is that the polar core and the surrounding hydrophobic boundary of reverse micelles can include many kinds of substrate molecules; that is, hydrophilic, hydrophobic, or amphiphilic (Chen et al., 2003). Reverse micelles can be used without maintaining high lactose concentrations or high temperatures with minimum loss of β-galactosidase activity. For instance, Chen et al. (2001) stated that GOS synthesis was enhanced in reverse micelles using dioctyl sodium sulfosuccinate/isooctane reverse micelles; that is, 51.2% (w/w) GOS compared to 31% GOS (%, w/w) in aqueous systems using a β-galactosidase from A. oryzae. The same effect was found in GOS synthesis by a β-galactosidase from E. coli under controllable water concentration in reverse micelles (Chen et al., 2003). Nevertheless, there are several disadvantages of using nonaqueous systems for GOS synthesis: (1) some organic solvents have limited use in food applications where concerns can be raised if they are used for GOS production; (2) low quantitative final GOS yields and low volumetric productivities are usually obtained in organic solvent-, ionic liquid-, and reverse micelles systems as lactose solubility is low in such systems compared to aqueous systems; (3) additional complicated purification steps are required, which can add a lot to the cost of production; (4) nonaqueous systems might not be the choice for complex waste streams such as whey permeate; (5) the effect of organic systems on improving transgalactosylation at the expense of hydrolysis is not linear; and (6) not all enzymes have tolerance to nonaqueous systems in terms of maintaining their activity and selectivity (Cruz-Guerrero et al., 2006; Kaftzik et al., 2002).

7  FUTURE DEVELOPMENTS One of the developments that will start taking place and will continue in the near future is the further understanding of the structural features of β-galactosidases known to perform transgalactosylation reactions efficiently. This will help in (1) the in vitro and in silico screening for the identification of novel native β-galactosidases with high transgalactosylation activity, (2) locating interesting mutagenesis sites, (3) comparing the structure and function of different β-galactosidases, (4) comparing mutants and wild types of the same enzyme, (5) comparing different conformations of the same enzyme, (6) studying the relationship between similar folds and expected similar functions, and (7) finally developing bioinformatic tools through the elucidation of the structural features of β-galactosidase, particularly those used for GOS synthesis. This is expected to open the door widely for more understanding of the structure-function relationship of β-galactosidases and for knowing the extent to which transgalactosylation reactions can be controlled to produce tailor-made oligosaccharides of specific structures with a profound health impact. Another area of progress is expected to be the discovery and use of cold-active β-galactosidases with high transgalactosylation activity at  10, short-chain DP  10, short-chain DP  10, short-chain DP  10, agave FOS with DP  10 (LcF), agave FOS with DP  10 (LcF), agave FOS with DP   oatmeal-milk gruel > apple juice > spring water. In another study, ice cream was better than yogurt to maintain the stability of probiotics in simulated GIT conditions (Ranadheera et al., 2012). Three elements of foods seem to affect stability in the stomach: (1) buffering ability, (2) carbohydrates, and (3) fat. The buffering ability of the food matrix is arguably a critical factor. This is why cheese and dairy products in general are considered to be good delivery vehicles in the GIT. In milk, these ingredients contribute to a good buffering ability: caseins, phosphates, and citrate. The presence of a fermentable carbohydrate also improves a culture’s ability to survive a simulated gastric environment (Corcoran et al., 2005). In this instance, the carbohydrate provides the cell with the ability to produce ATP, which is required for pumping out acid from the cytoplasm. Not surprisingly, the fiber/carbohydrate content of the food matrix strongly affects the stability of probiotic bacteria during storage in a fruit juice (Saarela et al., 2006). The type of carbohydrate in the medium when cells are exposed to bile salts also influences their survival (Ziar et al., 2014). The effect is very strain-variable (Ziar et al., 2014), and it can be hypothesized that the protective effect of the carbohydrate would be linked to the strain’s ability to metabolize it. Indeed, active sugar metabolism would presumably enhance the generation of ATP, and there is evidence of an ATP-mediated bile acid efflux (Bustos et al., 2011). The third element appears to be fat. Recent data suggest that the presence of fat is also beneficial (Tompkins et al., 2011).

4.4  Using Encapsulation The most important recent advance in improving the delivery of probiotics has been encapsulation. There are various techniques available (Champagne and Fustier, 2007), but two have attracted the most attention (Table 20.1; Figures 20.1 and 20.2). The microentrapment (ME) technology has been applied mostly to alginate (Figure 20.1), but many other polymers can be used, such as carrageenan, pectin, whey proteins. A further advantage of the alginate ME technology is that it enables a novel biomass production method (Champagne, 2006) which can prevent much of the damage to cells which occurs during the traditional process (Table 20.1). Although ME with alginate has obtained wide interest in the academic

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Bacterial culture

Sodium alginate solution (w/o starch)

Cell suspension in alginate

Calcium carbonate

Cells + carbonate in alginate

Alginate droplets

• CaCl2 solution

Alginate gel bead Microentrapped bacteria

CaCl2 solution

• ••• ••••• Oil

Alginate droplets

• ••• ••••• Oil

Acid solution

Chitosan coating Chitosan solution

FIGURE 20.1  Three methodologies based on extrusion or emulsion to obtain alginate beads. Champagne (with permission).

FIGURE 20.2  Various methodologies of spray-coating. Champagne (with permission).

community, its industrial acceptance has been limited. Rather, industry has preferred SC (Figure 20.2). Cultures encapsulated with the SC technology have much slower rehydration properties than the ME cultures or the standard free-cell cultures. This is very helpful when a short exposure to a very stressful environment, such as stomach acid, is required. Accordingly, the cultures prepared for the supplement market are encapsulated by SC rather than ME. It should be pointed out that important improvements have recently been obtained in alginate-based systems. Thus, beads can be coated with chitosan or poly-l-lysine (Martoni et al., 2011; Zarate et al., 2011) or oil (Ding and Shah, 2009), which significantly improves their protective properties in simulated GIT conditions (Riaz and Masud, 2013). Alginate-based systems can also be improved by using palmitoylated ingredients (Amine et al., 2014). There are reviews on the benefits of encapsulation in the delivery of probiotics in dairy products (Champagne and Kailasapathy, 2008b) and other foods (Goulet and Wozniak, 2002). In summary, ME increases the resistance of probiotic bacteria to rehydration in the presence of spices, heating, freezing, pumping/blending, and storage in yogurt. ME in alginate (Figure 20.1) has often been found to improve survival in the gastric environment (Le-Tien et al., 2004;

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Mandal et al., 2006). However, there are also reports with negative data (Sultana et al., 2000; Truelstrup-Hansen et al., 2002). The reasons for these discrepancies could be method of bead production or coating method (Figure 20.1), particle size, or cell load (Champagne and Kailasapathy, 2008; Lee and Heo, 2000). Although ME is effective in protecting cells in simulated stomach conditions, SC is probably even more effective at that level.

4.5  Simulated GIT Conditions There are many examples where probiotic bacteria need to arrive alive at the site of action in the GIT. It is commonly believed that the lower GIT, via the colon, is the target. Even when product formulation procedures have been used that ensure viability during production and storage as described above, the live bacteria must survive transit of the upper GIT. Ethics, cost, and complexity of tests prevent the testing of foods containing probiotics using human feeding trials. In vitro tests, using models of the GIT, can be used to provide data about the ability of bacteria to survive the harsh conditions of the upper GIT. Many studies have been reported that have used test-tube experiments to simulate the acidic conditions in the stomach, and exposure to bile salts and digestive enzymes that occur in the small intestine (Olejnik et al., 2005; Prasad et al., 1998). However, such tests cannot replicate the dynamic conditions that occur in the human GIT, and are limited to testing the actual bacteria as opposed to testing the (as eaten) food product. The effects of absorption of nutrients, interaction with undigested and partially digested food, and peristalsis cannot be studied in such simple systems. Nevertheless, most laboratories cannot afford the sophisticated dynamic models that will be mentioned below, and a static methodology is required. As a result, a consensus methodology has been proposed (Minekus et al., 2014). This approach incorporates an oral phase where saliva and the foods are blended in a 1:1 ratio. With respect to the gastric phase, the consensus method has the following features: (1) A pH of 3.0 is proposed which is a logical “average” of food and gastric secretions; this is because food influences pH of the gastric content. (2) Foods are gradually transferred to the small intestine, and gastric residence time will typically vary between 30 min and 4 h. The average setting proposed is 2 h. (3) The ratio between food and intestinal secretions is 1:1. If one has included the oral phase in the methodology, then the food represents 25% of the volume. It has been difficult to compare data on the stability of probiotics in simulated GIT conditions in the past because of so many different methodologies. Data from our laboratory, where four in-vitro methods were compared (Champagne et al., 2015b), show that in-vitro methodologies strongly influence viability results of probiotic bacteria, which strongly limit comparisons between data in the literature. Therefore this consensus approach should prove very useful in the future. Ideally, dynamic systems should be used because they better mimic the actual in vivo conditions. Several dynamic in vitro models of the human GIT have been published which simulated both the events that occur in the stomach and the small intestine (Hoebler et al., 2002; Mainville et al., 2005; Minekus et al., 1995). Using gravity, pump or mechanical peristalsis, samples move from one chamber to the next and are exposed sequentially to HCl (stomach), bile salts (intestine), and digestive enzymes (intestine). The TNO system (Minekus et al., 1995) also contains porous filters that allow small molecules to pass out of the model thus simulating absorption. Samples can be taken along the artificial GIT to study how bacteria survive and how the food matrix can protect them (e.g., buffering effects). Such information allows food manufacturers to change conditions in their products that ensure adequate numbers of probiotic bacteria arrive at their site of action. As mentioned previously, such dynamic systems are more expensive, and many research teams cannot afford them. We have developed and tested the IViDIS system (Mainville et al., 2005) and use a simplified method (S’IViDIS) which can be more easily carried out in a laboratory setting, but which nevertheless incorporates the principles of dynamic systems (Champagne et al., 2015a). In spite of their sophistication, even these dynamic in vitro systems are still limited to liquid or puréed samples. However, such in vitro GIT simulators have been used to test the protective characteristics of potential encapsulation techniques (Reid et al., 2005).

5  CONCEPT OF PROBIOACTIVE The beneficial effects resulting from the consumption of probiotic bacteria are believed to be dependent upon the bacteria being administered/consumed being alive (Bansal and Garg, 2008; Kailasapathy and Chin, 2000). Interactions between the probiotic bacteria and the intestinal wall cells and resulting changes to the host’s immune system are possible (Gill, 1998).

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However, in cases where bacteria have been added to a food matrix, and a fermentation has occurred, it is not always evident that the bacteria in the product are solely the responsible agents (Farnworth, 2000, 2008). During the fermentation, bioactives could have been generated due to action of the probiotic bacteria on the food matrix. It is also possible that the probiotic bacteria produce metabolites that are bioactive, as they grow in the food matrix. In both cases, once these “probioactives” have been formed, there would be no further need to have live bacteria in the product. Figure 20.1 shows how these two types of probioactives could be found in probiotic foods. This concept of probioactives is more inclusive, and more clearly defines the different origins of beneficial ingredients in fermented foods than that of biogenics (Mitsuoka, 2000).

5.1  Probioactives from the Food Matrix During bacterial fermentation of many foods, the action of the bacteria on the food matrix can produce a wide variety of compounds from the initial constituents of the food. In some cases, it is the generation of these bioactive compounds or probioactives that give the fermented food its health benefits (Figure 20.3). The most common matrix for probiotic bacteria is cows’ milk, although a wide variety of fermented foods believed to be beneficial to health can be found around the world (Farnworth, 2004). It has been shown, that through bacterial hydrolytic enzyme activity on cows’ milk, a variety of peptides can be produced that have biologic effects including antihypertension effects (from angiotensin conversion enzyme inhibition peptides), opioid agonism, antidiarrheal effects (from casomorhin production), induction of protective immunity against infections and some tumors (from immunomodulatory peptides) (de Moreno de LeBlanc et al., 2005; Vinderola et al., 2008). The release of free amino acids is also possible depending on the bacteria involved and their protease/peptidase activity. Milk glutamic acid is the source of γ-aminobutyric acid (GABA) in cheese due to the action of lactic acid bacteria; GABA has been shown to be useful to improve brain metabolic function and hypertension (Tanasupawat and Visessanguan, 2008). Bioconversion of the isoflavone glucosides (daidzin, genistin) into their corresponding bioactive aglycones (daidzein, genistein) has been reported during soymilk fermentation (Chun et al., 2007; Rekha and Vijayalakshmi, 2008). Included in the list of probioactives would be the short-chain fatty acid butyric acid that is found in many cheeses (Woo et al., 1984). During the production of cheese, bacterial action on the milk fat can result in high levels of butyric acid; butyric acid has been recognized as an anticancer agent and may be implicated in regulation of cholesterol metabolism (Bugaut and Bentéjac, 1993). As the development of fermented functional foods expands to include an ever increasing number of food matrices, and the number of bacteria used to carry out the fermentation of these foods grows, new probioactives will be generated.

5.2  Probioactives from Bacterial Metabolism Microorganisms use the milieu/media that surrounds them to produce a wide variety of metabolites as they grow and reproduce. These metabolites serve many purposes including contributing to the structure of the cells wall, carrying out digestion of nutrients required by the bacteria, providing protection for the bacteria against other bacteria, allowing the bacteria to survive in its environment/niche. Some of these metabolites are found on the outside of the cell wall, some are excreted into the surrounding milieu, while others are only liberated after the bacterial cells wall is ruptured. Some bacterial metabolites could be bioactive and have beneficial effects on the host through which the probiotic bacteria are passing. Food matrix

Fermentation

+

Fermented product

+

Micro-organism(s) Bio-active originating from food matrix

Pro-bioactive

+ Bio-active originating from micro-organisms

FIGURE 20.3  The production of probioactives in foods.

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Enzymes are particularly important probioactives which are linked to functionality. β-Galactosidase is the enzyme responsible for the hydrolysis of lactose into its two constituent sugars, glucose, and galactose; insufficient β-galactosidase activity in the brush border membrane on the mucosa in the small intestine leads to lactose maldigestion. Some bacteria also produce this enzyme, and it has been found that lactose maldigestion can be overcome by eating yogurt that contains bacteria that synthesize β-galactosidase (EFSA, 2010). However, it has been reported that lactose hydrolysis is the same if the bacteria (producing β-galactosidase) consumed are alive or not (de Vrese et al., 2001). In case it is the bacterial enzyme that is the probioactive, similarly, bacterial BSH enables probiotics to survive exposure to bile salts (Jungersen et al., 2014), and has been linked to cholesterol metabolism (Jones et al., 2012). Bacteria have a wide variety of enzymes, and therefore the careful selection of bacteria to be added to a food could target specific metabolic or digestive problems in the host. Bacteria are capable of producing a wide variety of exopolysaccharides that serve many purposes (Farnworth et al., 2007). Several of these complex carbohydrates have also been shown to have potential beneficial effects including antitumour properties, immunostimulatory properties, and possible effects on cholesterol metabolism (Furukawa et al., 2000; Vinderola et al., 2006). These beneficial effects are due to the probioactive exopolysaccharides and not the bacteria that produced them.

5.3  Protection of Probioactives It is apparent that the health benefits of fermented foods can be attributed to probioactives that are derived from the initial food matrix or that can be the result of bacterial metabolism during fermentation. In either case, the bioactive action of the food would not require that the responsible bacteria be alive when consumed. However, to retain their bioactive effect, food producers will have to find ways to protect probioactives during production and storage up to the time of consumption.

6 CONCLUSION Consumers who are eager to include probiotics in their diets need to be aware that the ingredients responsible for the health benefits, whether live bacteria or probioactives, are easily killed or destroyed during production, packaging, and storage. Only products that are produced by companies that have the knowledge and capability to produce such sensitive foods should be eaten. Products need to be formulated so that the live bacteria or probioactives arrive at the site of action in sufficient numbers to be effective. Consumers should read labels carefully to ensure that the product they have purchased has the bacteria (identified to the species or subspecies level) that will produce the effect they want. In the future, more foods will contain live bacteria, as technologies such as encapsulation become widely used in the food industry.

REFERENCES Amine, K.M., Champagne, C.P., Salmieri, S., Britten, M., St-Gelais, D., Fustier, P., Lacroix, M., 2014. Effect of palmitoylated alginate microencapsulation on viability of Bifidobacterium longum during freeze-drying. LWT—Food Sci. Technol. 56, 111–117. Ananta, E., Volkert, M., Knorr, D., 2005. Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG. Int. Dairy J. 15, 399–409. Araya, M., Morelli, L., Reid, G., Sanders, M.E., Stanton, C., Pineiro, M., Ben Embarek, P., 2002. Guidelines for the evaluation of probiotics in food. Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food, London (ON, Canada) April 30 and May 1. ftp:// ftp.fao.org/docrep/fao/009/a0512e/a0512e00.pdf. Bansal, T., Garg, S., 2008. Probiotics: from functional foods to pharmaceutical products. Curr. Pharm. Biotechnol. 9, 267–287. Biasco, G., Paganelli, G.M., Brandi, G., Brillanti, S., Lami, F., Callegari, C., Gizzi, G., 1991. Effect of Lactobacillus acidophilus and Bifidobacterium bifidum on rectal cell kinetics and fecal pH. Ital. J. Gastroenterol. 23, 142. Bonorden, M.J.L., Greany, K.A., Wangen, K.E., Phipps, W.R., Feirtag, J., Adlercreutz, H., Kurzer, M.S., 2004. Consumption of Lactobacillus acidophilus and Bifidobacterium longum do not alter urinary equol excretion and plasma reproductive hormones in premenopausal women. Eur. J. Clin. Nutr. 58, 1635–1642. Bugaut, M., Bentéjac, M., 1993. Biological effects of short-chain fatty acids in nonruminant mammals. Annu. Rev. Nutr. 13, 217–241. Bustos, A., Raya, R., Valdez, G., Taranto, M., 2011. Efflux of bile acids in Lactobacillus reuteri is mediated by ATP. Biotechnol. Lett. 33, 2265–2269. Castro, H.P., Teixeira, P.M., Kirby, R., 1997. Evidence of membrane damage in Lactobacillus bulgaricus following freeze drying. J. Appl. Microbiol. 82, 87–94. Celik, O.F., O’Sullivan, D.J., 2013. Factors influencing the stability of freeze-dried stress-resilient and stress-sensitive strains of bifidobacteria. J. Dairy Sci. 96, 3506–3516. Champagne, C.P., 2006. Starter cultures biotechnology: the production of concentrated lactic cultures in alginate beads and their applications in the nutraceutical and food industries. Chem. Ind. Chem. Eng. Q. 12, 11–17.

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Champagne, C.P., 2009. Some technological challenges in the addition of probiotic bacteria to foods. Chapter 19, In: Charalampopoulos, D., Rastall, R. (Eds.), Prebiotics and Probiotics Science and Technology. Springer-Verlag, New York, pp. 761–894. http://dx.doi.org/10.1007/978-0-387-79058-9_19. Champagne, C.P., 2014. Development of fermented milk products containing probiotics. Chapter 9, In: Ozer, B., Akdemir-Evrendilek, G. (Eds.), Dairy Microbiology and Biochemistry: Recent Developments. Science Publications/CRC Press, Boca Raton, FL, pp. 214–244. Champagne, C.P., Fustier, P., 2007. Microencapsulation for delivery of probiotics and other ingredients in functional dairy products. Chapter 23, In: Saarela, M. (Ed.), Functional Dairy Products. second ed. Woodhead Publishing, London, pp. 404–426. Champagne, C.P., Gardner, N.J., 2008. Effect of storage in a fruit drink on subsequent survival of probiotic lactobacilli to gastro-intestinal stresses. Food Res. Int. 41, 539–543. 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Chapter 21

Probiotics and Other Microbial Manipulations in Fish Feeds: Prospective Update of Health Benefits F.J. Gatesoupe INRA, UR 1067, Nutrition Aquaculture et Génomique, Plouzané, France

1 INTRODUCTION Aquaculture was considered as a marginal activity until recently, but the situation changed at the turn of the century, with the always-increasing demand for seafood, while fisheries’ captures have stagnated. Besides the international and governmental efforts to regulate fisheries’ resources, the share of seafood produced by aquaculture will continue to increase inescapably (De Silva, 2012). The contribution of fish farming in 2012 represented almost half of all fish for human food, with projections over 60% by 2030 (FAO, 2014). This fast increase exerts a striking impact on environment and public health. It implies rearing intensification, which may cause fish disease outbreaks, including bacterial infections. The threat of foodborne diseases caused by fish consumption and handling is concurrently increasing (Haenen et al., 2013), and there is a risk of emergence of new human pathogens. For example, freshwater fish have been identified as a source of Laribacter hongkongensis, a bacterium associated with gastroenteritis (Woo et al., 2004). More recently, Streptococcus hongkongensis has been described as a new infective agent after puncture wounds from marine flatfish (Lau et al., 2013). However, the main concern remains the risk of spreading antimicrobial resistance (Shah et al., 2014). While some countries with important aquaculture production still lack sufficient enforcement rules for antibiotic use, there is a pressing demand for sustainable alternatives (Bondad-Reantaso et al., 2012). Probiotics are considered as one of the most promising alternatives, despite the limited knowledge about the intestinal microbiome in fish, which is briefly reviewed in this chapter. Also presented here are the numerous probiotic candidates that have been tested empirically in fish, with some insight into their modes of action, which are becoming better understood. The emerging prospects for prebiotics and other dietary manipulations that can regulate gastrointestinal microbiota are also discussed. Finally, besides these practical aspects relevant to public health, a less expected benefit from the research on fish microbiome is introduced because fish appear as an interesting model for investigating the basic features of the host-microbe interactions.

2  INTESTINAL MICROBIOME IN FISH The scientific approach to the microbial communities has been transfigured during the last decades due to technological innovation, likely in the early stages of a new era for microbial ecology. If most of the first applications of intestinal microbiota have concerned human medicine, the tools and know-how are expanding into other models, including fish (Llewellyn et al., 2014). Ley et al. (2008) distinguished the microbial communities associated with vertebrates from those associated with invertebrates, which looked more dependent on the environment. In particular, water salinity has a determining influence on the microbiota associated with lower animals. This does not exclude the fact that some primitive metazoans seem able to exert a high selective pressure on the hosted microbes (Fraune and Bosch, 2007). The demarcation drawn by Ley et al. (2008) between vertebrates and invertebrates should also be moderated because the data concerning fish were based on one previous study of the gut microbiota in zebrafish (Rawls et al., 2004). In a meta-analysis, fish gut communities appeared quite dissimilar between species and to some extent, related to the environment and feeding habits (Sullam et al., 2012). Fish have particular features, as compared with land animals. The aquatic environment facilitates microbial influx and renewal. Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00021-6 © 2016 Elsevier Inc. All rights reserved.

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Fish are poikilothermic, and seasonal changes have been observed in their intestinal microbiota (e.g. Hovda et al., 2011). Their immune response is somewhat primitive, mainly innate with limited adaptive capability (Gomez et al., 2013). These characteristics combine to stress the differences that may be expected in the microbial ecology of fish gut, as compared with that of higher vertebrates. A wide diversity of anatomical peculiarities can be observed among the digestive tracts of fish, and that reflects the variety of ecological niches offered to different microbial communities. Most aquacultured species are carnivores, whose short intestine may be extended with pyloric caeca in variable numbers (Guillaume and Choubert, 2001). The intestinal transit time is relatively brief in carnivorous fish (mostly less than 15 h; Clements et al., 2014), thus limiting the potential of direct contribution of bacteria to the host’s digestive activity. However, there are also some herbivorous species that are important for aquaculture, like mullets (fitted with a relatively long intestine) and carp (which are devoid of a stomach, but whose pharyngeal teeth facilitate the digestion of vegetable feeds; Stevens and Hume, 1998). Relatively high bacterial concentrations were observed in the feces of some tropical fish (109-1011 cells g−1, counted with epifluoresence microscopy; Smriga et al., 2010), but moderate counts of bacteria are generally retrieved from the intestine of farm fish (e.g. 106-107 g−1 in Atlantic salmon; Abid et al., 2013). Besides bacteria, yeasts are frequently isolated from fish gut, more especially in freshwater (Gatesoupe, 2007; Raggi et al., 2014). Archaea have been also reported in very few studies (van der Maarel et al., 1998, 1999; Ni et al., 2014; Kormas et al., 2014), but methanogens would need further attention, as methane production was reported in fish intestine (Oremland, 1979). Very little is known about bacteriophages in fish intestine (e.g. Tyutikov et al., 1983) though their role is likely crucial. Waller et al. (2014) compared the human gut virome to the data collected during the Sorcerer II Global Ocean Sampling Expedition, and they stressed the high dissimilarity between both datasets. Marine fish gut virome would be worth being characterized, and compared in this context. However, most studies have dealt with bacteria so far: Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes appeared as the main phyla that are commonly detected in the microbiome in fish intestinal mucus and content, as well as in skin mucus (Sullam et al., 2012; Llewellyn et al., 2014). Xing et al. (2013) compared 10 gut metagenomes, 1 from whole gut (mucus and content) of European turbot, 1 from hybrid striped bass, and 8 others from terrestrial animals. The fish metagenomes clustered at the phylum level with that from a human infant, but were relatively more distant from the other datasets, including that from a human adult. Roeselers et al. (2011) showed the relatively high similarity of the clone libraries collected from the whole intestinal contents of zebrafish reared in distant American laboratories, or caught in an Indian river. The same cluster included the library from wild yellow catfish, while other wild fishes harbored more dissimilar bacterial communities. The authors concluded that zebrafish have a specific core intestinal microbiota, as proposed in human gut (Turnbaugh et al., 2009). In the whole intestinal community of rainbow trout, Wong et al. (2013) defined a core microbiome that was resistant to the influence of two diets, which contained either vegetal or animal protein sources. However, the intestinal contents of 11 specimens of Atlantic cod had highly variable pyrosequencing profiles, though the fish were caught in one location, and then kept in a common tank for at least 1 week of fasting (Star et al., 2013). Starvation can modify the bacterial profile. For example in the whole intestine of Asian seabass, the proportion of Bacteriodetes increased, while that of Betaproteobacteria was depleted after 8 days of fasting (Xia et al., 2014). In zebrafish larvae, the gut microbiota appeared quite variable among replicates, but significantly more diverse in the groups that were fed, compared to the unfed (Semova et al., 2012). Beyond the taxonomic analysis, the hypothesis of a core microbiome seems more pertinent in terms of functional metagenomics (Turnbaugh et al., 2009). By comparing 10 metagenomes classified according to the metabolic subsystems, Xing et al. (2013) observed that the cluster of two gut microbiomes from turbot and striped bass was still relatively close to that of the termite, but more distant from those of terrestrial vertebrates. The genes involved in quorum sensing, biofilm formation, and oxidative stress seemed particularly over-represented in fish metagenomes, possibly in relation with the peculiarities of the immune system. In view of the scarcity of data yet available, the scope of influence of the metagenome cannot be yet clearly delineated in fish. In fasting Asian seabass, the intestinal microbiota was oriented to self-protection, with downregulation of the genes involved in transcription and cell division, while those involved in cell envelope biogenesis and other defense mechanisms were upregulated, like those coding for antibiotic production (Xia et al., 2014). Besides the obvious relationship with the immune system, it seems that other microbial genes may significantly contribute to the digestive function of the host, despite the relatively low concentration of intestinal microbes in most fish. In normal trophic conditions, the functional metagenome seems dietary flexible. In the whole intestine and intestinal content of grass carp that were fed ryegrass, some genes were over-represented, compared to those of the control group fed commercial diet, in particular with respect to the pathways of carbohydrate, fatty acid and amino acid metabolism (Ni et al., 2014). Further insight may be expected by studying the effects of prebiotics on fish gut metagenome. Gnotobiotics may bring valuable information about the roles of the intestinal microbiome. The method has been applied to a variety of alevins and fish larvae (see reviews by Marques et al., 2006; Llewellyn et al., 2014). Most studies

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have dealt with the assays of pathogens and candidate probiotics, but Rawls et al. (2006) also compared the effects of the cross-implantation of whole gut microbiota in axenic zebrafish larvae and in mice, further evidencing the intricate hostmicrobiome relationship. There is a mutual shaping of the intestinal microbiome and the host’s transcriptome, including possible epigenetic regulation. The early nutrition and microbial gut colonization are critical for epigenetic programming in man (Takahashi, 2014). Two bacterial metabolites were proposed by Mischke and Plösch (2013) as potential mediators for epigenetic regulation: folate (which may be produced by intestinal microbiota in carp; Kashiwada et al., 1971) and butyrate (one of the main products of carbohydrate fermentation in fish intestine; Leenhouwers et al., 2008). Galindo-Villegas et al. (2012) have recently shown the bacterial epigenetic regulation of immunity in zebrafish by comparing axenic and conventionally reared animals. The short-chain fatty acids produced by intestinal microbes from dietary carbohydrates and proteins may contribute to the host’s nutrition (Carmody and Turnbaugh, 2012), while lipid absorption is stimulated in the enterocytes of conventionally reared zebrafish, compared to what happens in axenic animals (Semova et al., 2012). Many bioactive metabolites are produced by the intestinal microbiota, like putrescine and spermidine in mice (Matsumoto et al., 2012). Intestinal polyamines are essential to human health, with main effects on gut maturation and regeneration, and on the antioxidative status (Kalac, 2014). In fish, such effects were demonstrated with dietary live yeast as candidate probiotics (Debaryomyces hansenii; Tovar-Ramirez et al., 2010), but bacteria may also release polyamines like putrescine, produced by staphylococcal isolates from rainbow trout intestine (Pleva et al., 2012).

3  PROBIOTICS IN FISH In spite of many unanswered questions about gut microbiota in fish, the investigation of probiotics for fish has fairly progressed, mainly over the last decade, as testified by the growing number of scientific articles (Figure 21.1). Probiotics are now integrated in the life cycle assessment of aquaculture practice (Iribarren et al., 2012). However, an official registration is required before application to fish farming. The number of probiotics available on the market depends on the governmental regulations, and on the local production. Recent reports have listed about 20 or more products registered in the Philippines, 119 in Vietnam, and more than 100 companies that have sold many probiotics in China (Qi et al., 2009; Bondad-Reantaso et al., 2012). For extended information, one can refer to the numerous reviews on the subject (including some of the most recent, like De et al., 2014; Newaj-Fyzul et al., 2014; Perez-Sanchez et al., 2014). To the author’s knowledge, the first trial explicitly referring to probiotics for fish was an internal report, which dealt with the limitation of mortality in Japanese eel elvers medicated with spores of Bacillus toyoi, a bacterium of soil origin, commercialized as a probiotic for land animals (Shimizu et al., 1981). During the 1980s, there were few trials on marine fish, including the first attempts on larvae, via live feed organisms (Gatesoupe et al., 1989). Antagonisms among fish gut bacteria have been known for a long time (e.g. Schrøder et al., 1979), and host-derived bacteria with antagonistic behavior

FIGURE 21.1  Annual production of peer-reviewed articles indexed with explicit reference to probiotics and/or prebiotics, and application to finfish or live feed organisms (rotifers and Artemia; search based on titles, keywords and abstracts). The total numbers of items published each year were broken down into specific reviews and experimental reports about either prebiotics, or probiotics, or both (which did not necessarily correspond to a synbiotic approach). The counts were stopped by the end of June 2014, and simply doubled for the last year (thus underestimated). The search was not exhaustive, but sufficient to illustrate the trend of fast increase in the recent years.

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to pathogens were tested as fish probiotics since the early 1990s (Westerdahl et al., 1991). Yeast have been concurrently evaluated as candidate probiotics in fish (Andlid et al., 1995). In the meantime, bacteriophages were tested to inhibit some specific bacterial infections of fish, but there has not been practical application of phage therapy in aquaculture yet (see review by Oliveira et al., 2012). Reciprocally, the screening of bacteria capable to inhibit fish viruses was investigated (Kamei et al., 1987). For example, some strains of Aeromonas sp. are active against the infectious hematopoietic necrosis virus, and salmonids fed with these bacteria showed an improved resistance to the viral infection in experimental challenges (see reviews by Yoshimizu and Ezura, 1999; Maeda, 2004). Mucosal adhesion is an important feature for the intestinal colonization of candidate probiotics (e.g. Grzeskowiak et al., 2011). The strains isolated from fish may have the capacity to persist long after inoculation in the intestine, even when administered to a different fish species. For example, Bacillus amyloliquefaciens was isolated from the gut of marine yellow-fin bream. When this candidate probiotic was introduced in the feed of freshwater Nile tilapia, an effective intestinal colonization was observed, and the strain persisted at least 61 days after stopping its dietary supply (Ridha and Azad, 2012). A first consequence of such colonization is the modulation of intestinal microbiota, which has been frequently observed after probiotic introduction (see review by Mohapatra et al., 2013). Probiotics can benefit fish health either indirectly by regulating gut microbiota, or by direct signals that target mainly the digestive and immune functions of the host (Figure 21.2). An original feature of probiotic treatments in fish is that the route of administration may not be necessarily oral, due to the mucosal organization of skin and gills (Gomez et al., 2013). Besides gut-targeted probiotics, probiotics have been also used in bath treatments. When probiotics are not incorporated to the feed, they are ingested as well, especially by marine fish, which need to drink constantly to avoid dehydration (Whittamore, 2012). This may also result in competitive exclusion of pathogens by direct contact with external mucosa (Llewellyn et al., 2014), and in immunostimulation outside of the oral route (Lazado and Caipang, 2014). By extension, probiotics have also been applied to enhance water quality, mainly by decreasing ammonia and nitrite concentrations, and improving thus indirectly fish welfare and health. Some Bacillus strains look particularly interesting in this regard (e.g. Zink et al., 2011). Many studies have dealt with this genus, which is the main source of fish probiotics after lactic acid bacteria, but the attention to the effects on water quality remains limited, compared to those on host’s response and direct microbial antagonism (Figure 21.3). It may seem rather artificial to amalgamate into the same term “probiotics,” microbes that can act on microbiota or on the host, either directly or indirectly by improving water quality, but the main advantage of probiotics over traditional treatments lies in their potential to inhibit the infection by a variety of modes of action, which does not leave any chance for the pathogen to develop resistance. Some complex consortia have been proposed to provide the widest range of expected effects, like the preparation of live Bacillus subtilis, Lactobacillus acidophilus, Clostridium butyricum, and Saccaromyces cerevisiae, which was used to act on water quality in recirculated systems, while

FIGURE 21.2  Complex interrelationship between probiotic treatments—either dietary, or “external” by bath immersion, or intended for bioremediation of water quality, and their effects on fish health and microbiota, either associated to mucosa, or transiting in the digestive tract, or surrounding in the culture system. The water environment may thus justify an extended concept of probiotics, in comparison to that commonly accepted for man and land animals.

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FIGURE 21.3  Venn diagrams constructed from a nonexhaustive selection of 475 references, corresponding to the publication of experiments that dealt explicitly with probiotics for finfish or live feed organisms. The items were collected until the end of June, 2014, and selected by searching on titles, keywords, and abstracts. The numbers correspond to the total of articles that referred to one or several types of probiotics (a and b), or effects (c and d). (a) Half of the references dealt with lactic acid bacteria, and only 12% with yeast; (b) the other sources of probiotic bacteria were mainly the genus Bacillus and the phylum Proteobacteria; (c) in total, 444 articles dealt with at least one class of effects, which concerned mainly the host, while 19% considered only the direct antagonism to pathogens, and 4.5% attended to the application of probiotics to improve water quality; and (d) among the 350 papers dealing with the effects on host’s health, 42% considered immunological parameters, and 10% focused on the activity of digestive enzymes, while the other half reported only the general rearing performances, or sometimes particular applications.

s­ imultaneously improving the immune response and disease resistance in fish (Taoka et al., 2006). The multiplicity of agents makes the interpretation of the effects difficult, and this should require further investigation to discriminate the role of each strain, and the possible synergies. The stimulation of the immune defenses of the host is one of the most promising modes of actions (see review by Lazado and Caipang, 2014). The viability of probiotics was proved essential for some instances of immunostimulation, and these specific effects should not be confused with those of dead cells or cell components that are used as immunostimulants (Panigrahi et al., 2011). There is growing evidence that probiotics can improve welfare in farm fish, especially by using stress indicators like the levels of cortisol and heat shock proteins (e.g. Avella et al., 2011). These effects of probiotics may be related to the complex relationships between fish welfare and rearing conditions, including stocking density and water quality (Ellis et al., 2002). The interaction between the neuro-endocrine and immune systems may also be involved, in particular in the hormonal regulation of the inflammatory response in fish tissues (Verburg-van Kemenade et al., 2011). Some probiotics seem able to stimulate neuro-endocrine signals: a strain of Lactobacillus rhamnosus modulated the gene expression of neuropeptide hormones, and increased fecundity in female zebrafish (Gioacchini et al., 2010). Positive effects were also observed on the reproduction and larval growth of killifish fed the same probiotic strain (Lombardo et al., 2011). This probiotic had profound repercussions on the metabolism of zebrafish larvae, including the acceleration of vertebral calcification (Avella et al., 2012). Two other strains of lactic acid bacteria were tested on sea bass larvae, but the impact on osteogenesis differed between the two strains, and the accelerated ossification observed with Lactobacillus casei corresponded to a final increased incidence of vertebral deformities, unlike what was observed with Pediococcus acidilactici (Lamari et al., 2013; strain previously documented to improve the vertebral conformation of rainbow trout alevins by Aubin et al., 2005). The new “omics” technologies could improve the view of the impact of probiotic treatments, with respect to the possible side effects (Sanchez et al., 2013). Much remains to be done to reap the full benefits from the carefully thought-out application of probiotics in aquaculture.

4  PREBIOTICS AND OTHER DIETARY MANIPULATIONS The first attempt to use prebiotics in fish appeared contemporaneously to the introduction of the concept expounded by Gibson and Roberfroid (1995). Although Kihara et al. (1995) did not refer explicitly to prebiotics, their experiment was matched with the concept. Intestinal microbiota from red sea bream fermented lactosucrose in vitro, and the introduction

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of dietary lactosucrose increased the thickness of the muscular intestinal layer in fish. In spite of this early trial, it was during the last decade that prebiotics started to be intensively tested, either alone or in a synbiotic approach (Figure 21.1; see reviews by Ringø et al., 2010; Cerezuela et al., 2011; Ganguly et al., 2013; Song et al., 2014). The term “prebiotics” may be ambiguous, as some reports referred to the concept, but tested feed ingredients as immunostimulants, without considering the possible effects on gut microbiota. A specificity of the application to aquaculture is that bifidobacteria, one of the main target for prebiotics in higher vertebrates, has been seldom reported in fish (e.g. Vlkova et al., 2012; Wu et al., 2014). More attention has been paid to the effect of prebiotics on some members of the class Bacilli, and on other potentially beneficial bacteria that are commonly harbored in fish gut. Many carnivorous fish eat crustaceans in the wild, but digest hardly the crystalline chitin. As by-product of the emerging production of insect protein, chitin may be a prebiotic source of particular interest for fish farming (van Huis et al., 2013). In fish gut, many commensal bacteria can hydrolyze it, possibly producing immunostimulant derivatives (Ringø et al., 2012). Some other new ingredients could be added to fish feed like poly-β-hydroxbutyrate, which is selectively degraded by gut microbes in sea bass, producing short-chain fatty acids, and stimulating fish growth (De Schryver et al., 2010). The combination of immunomodulatory and antimicrobial effects can be obtained by using herb medicines, and there is a growing interest for the application to fish (Reverter et al., 2014). The main difficulty is to find the right combination of herbs, and the right dose to administer, depending on the sensitivity of each species to compounds that become harmful at high concentration. Many tracks remain to be explored to improve fish health and microbial management in aquaculture. For example, a new antivirulence therapy could arise from the research about quorum sensing disruption (Defoirdt, 2014).

5  RELEVANCE OF FISH AS MODEL SPECIES Zebrafish has recently become the most studied animal model for translational research in various fields like genetics, embryology or neurosciences, applied to human health (see many recent reviews, e.g. Babin et al., 2014; Ota and Kawahara, 2014; Pickart and Klee, 2014; Stewart et al., 2014). There have been yet some applications to prospective new probiotic treatments, for example, against alcoholic liver disease (Schneider et al., 2014). Further ones could arise from investigations dealing with gut inflammation (Fleming et al., 2010), or with the influence of gut microbiome on obesity (Carmody and Turnbaugh, 2012). Other fish species have been proposed as models for health issues like carcinogenesis, toxicology, or bacterial infections (e.g. Hinton et al., 2009). Among these alternative fish models, medaka has received sustained attention, including medical applications related to endocrinology, reproduction, and aging (Gopalakrishnan et al., 2013). A recent experiment dealt with osteoporosis (Shanthanagouda et al., 2014). This model could be useful to progress the mutual benefit of medicine and aquaculture, especially to study the effects of probiotics that may be mediated by the neuro-endocrine axis, like those concerning reproduction and bone mineralization (Carnevali et al., 2013).

6 CONCLUSION Though most of these treatments are still experimental, fish health management may benefit as well from medical advances in probiotics as from traditional herbs and other soft medicines. In return, it seems now possible to obtain some basic information about host-microbe interactions by experimenting on fish. The development of new tools in molecular biology, especially about functional metagenomics and single cell genomics, should help to fill the gap still remaining in the knowledge about fish gut microbiota (Walker et al., 2014). This is also crucial for understanding the modes of action of probiotics. The direct observation by confocal imaging and electron microscopy are essential to visualize what happens in situ (Salinas et al., 2008; Del'Duca et al., 2013). Combined with these tools, the recent regain of interest for applying gnobiotic studies to the larval stages of farm fish may lead to significant advances in understanding the roles of probiotics and microbiota in species of interest for aquaculture.

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Chapter 22

Current and Future Applications of Bacterial Extracellular Polysaccharides Adrian Pérez-Ramos, Montserrat Nácher-Vázquez, Sara Notararigo, Paloma López and Mª Luz Mohedano Centro de Investigaciones Biológicas, Madrid, Spain

1 INTRODUCTION The exopolysaccharides (EPS) are heterogeneous long-chain polymers, which are synthesized and released mainly by bacteria and microalgae into their surroundings during growth (Sutherland, 1972). These polymers can form an adherent cohesive layer around the cell surface, when they are called capsular polysaccharides, or can be excreted outside the cell wall when they are called exocellular polysaccharides (Ruas-Madiedo and de los Reyes-Gavilan, 2005). Although the biological function of EPS in the microorganisms is not clear, they nevertheless have been exploited as bio-thickeners in the food industry of dietary products. EPS produced mainly by lactic acid bacteria (LAB) have food applications, as viscosifying agents, stabilizers, emulsifiers, gelling agents, or water-binding agents as well as health applications such as reduction of cholesterol levels, reduction of formation of pathogenic biofilms, modulation of adhesion to epithelial cells, and a prebiotic effect by increasing levels of bifidobacteria in the intestinal tract (Patel and Prajapati, 2013). When it was demonstrated that they also play an important role as modulators of the gut microbiota, and the immune system, as anti-carcinogenic agents and antioxidants, their use was extended to the functional food formula to promote the host’s benefits. Currently, research interest in bacterial EPS is growing owing to the chemical properties that these polymers exhibit. Indeed, their exploitation in pharmaceutical products, medical devices, and cosmetics is becoming a new trend to replace traditional hydrocolloid production (Freitas et al., 2011). These aspects constitute an important field of research that could lead to the production of fermented functional foods which benefit human and animal health. Therefore, in this chapter, in addition to describing the nature, origin, and structure of bacterial EPS, we review the current knowledge concerning the functional properties of EPS. We also describe their actual usages and future potential applications to improve rheology of fermented food and for developing of functional food for humans and animals.

2  CLASSIFICATION OF EPS EPS consist of linear or branched, repeating units of sugar or sugar derivatives. These sugar units are mainly glucose, galactose, mannose, N-acetylglucosamine, N-acetyl galactosamine, rhamnose and l-fucose, in variable ratios, sometimes with other inorganic and organic residues such as phosphate, sulfate, succinate, acetate, pyruvate, and glycerol (Finore et al., 2014; Ruas-Madiedo and de los Reyes-Gavilan, 2005). EPS from microbial sources can be classified, based on their monosaccharide composition and biosynthetic pathways into two groups: homopolysaccharides (HoPS) and heteropolysaccharides (HePS). The HoPS contain a single type of monosaccharide, d-glucose or d-fructose joined by either a single linkage type (e.g., 1,2 or 1,4) or by a combination of a limited number of linkage types (e.g., 1,2 and 1,4). The HePS comprise repeating units of different monosaccharides and they vary in number from tri- to octa-saccharides. They possess a variety of two or more different types of monosaccharides and frequently have a range of different linkage patterns. The HePS may also contain nonsugar molecules. The HoPS usually display high molecular masses (up to 107 Da); many of them are synthesized by LAB and can be classified into four groups: α-d-glucans, β-d-glucans, β-d-fructans and others, like polygalactan (De Vuyst and Degeest, 1999). According to the linkages in the main chain, the α-d-glucans are subdivided into dextrans (α-1,6), mutans (α-1,3), glucans (α-1,2), reuterans (α-1,4), alternans, (α-1,3), and (α-1,6). Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00022-8 © 2016 Elsevier Inc. All rights reserved.

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The dextrans are the HoPS most widely used in industry (Leemhuis et al., 2013), including the production of fine c­ hemicals such as plasma substitutes and Sephadex®. Dextransucrase converts sucrose to produce dextrans and this enzyme is synthesized and secreted by Leuconostoc mesenteroides, Lactobacilli, and Weisella sp. strains (Bejar et al., 2013; Kothari and Goyal, 2013; Ruhmkorf et al., 2013; Shukla et al., 2014). The bacteria produce a high level of 1-10 g L−1 (Notararigo et al., 2013) and this production confers a mucous phenotype to the producer strains, when they are grown in the presence of sucrose (Figure 22.1a), which is not detected in the presence of glucose (Figure 22.1b). Dextrans are a class of EPS composed of α-(1,6) glycosidic linkage and which are branched with α-(1,2), α-(1,3), or α-(1,4) linkages. The degree of these branchings varies according to the origin of the dextransucrase. The dextran most widely used industrially is that synthesized by L. mesenteroides NRRL B-512F, which contains 95% α-1,6 and 5% α-1,3 linkages (Korakli and Vogel, 2006), like the dextran produced by Weissella cibaria (Bounaix et al., 2010). Mutans are the glucans synthesized by various serotypes of Streptococcus mutans and S. sobrinus, which differ from dextrans in that they contain a high percentage of α-(1,3) linkages (Asem et al., 1986; Ferretti et al., 1987). Reuterans are water-soluble glucans with mainly α-(1,4) glucosidic linkages synthesized by reuteransucrase. The strains producing reuteransucrase are Lactobacillus reuteri species (Kralj et al., 2004). The EPS produced by alternansucrases contains alternating α-(1,6) and α-(1,3) glucosidic linkages. These HoPS are produced by L. mesenteroides strains and have the characteristics of high solubility, low viscosity, and resistance to enzymatic hydrolysis (Cote and Robyt, 1982). The β-glucans are a class of EPS composed of linked (1,3)-β-d-glucopyranosyl residues that may also contain side chains of β-d-glucopyranosyl units attached by (1,2) linkages. The prototype of bacterial β-glucan is the curdlan produced by Agrobacterium species, a linear (1,3)-β-d-glucan, which may have a few inter- or intra-chain (1,6) linkages. Curdlan has unique rheological and thermal-gelling properties, with applications in the food industry and other sectors (McIntosh et al., 2005). In addition, O2 substituted (1,3)-β-d-glucans are synthesized by the Tts β-glucosyltransferase in Streptococcus pneumoniae serotype 37 (Llull et al., 2001) and the GTF glycosyltransferase (Dols-Lafargue et al., 2008; Werning et al., 2006, 2008, 2012) in LAB strains belonging to the Pediococcus, Lactobacillus, and Oenococcus genera isolated from cider and wine (Dols-Lafargue et al., 2008; Dueñas-Chasco et al., 1997, 1998; Garai-Ibabe et al., 2010; Ibarburu et al., 2007; Llaubères et al., 1990) as well as in Propionibacterium freundenreichii subs shermanii TL34 (Deutsch et al., 2012). The production of this EPS is not very high, around 100–500 mg L−1 (Notararigo et al., 2013). However, it confers to the producing bacteria a ropy phenotype, which allows its distinction from the isogenic nonproducing strain (Figure 22.1c vs. d). Moreover, this EPS generates a thread, when a colony (Figure 22.1e) is lifted (Figure 22.1f). In addition, another LAB belonging to Lactobacilli and Lactococci genera produce polygalactans. Among them, Lactococcus lactis subsp lactis H414 is able to produce a polygalactan containing only galactose residues, but with different linkages that form a pentasaccharide repeating unit (Deveau et al., 2002). According to the linkages in the main chain, the β-d-fructans are subdivided into levans and inulins. Levan is a fructan mainly linked by β-(2-6)-glycosidic bonds with some β-(2,1) linked branch chains. Levansucrase catalyzes the synthesis of levan by transferring the fructosyl group of nonactivated sucrose into the fructan chain. Levan is a natural HoPS with various industrial applications. It is used in the food industry as a source of fructose, as an emulsifier, as a texturizing agent, and as an encapsulating agent. In medicine, it has application as an immunomodulator and as a substitute of blood plasma, among other uses (Han, 1990; Kim et al., 2005; Yamamoto et al., 1999). The bacteria mainly producing levan include Zymomonas mobilis (Silbir et al., 2014), Acetobacter xylinum (Tajima et al., 1997), Bacillus subtilis (Dogsa et al., 2013), Microbacterium laevaniformans (Bae et al., 2008), Bacillus amyloliquefaciens (Tian et al., 2011), Bacillus methylotrophicus (Zhang et al., 2014), Lactobacillus sanfranciscensis (Tieking et al., 2005), L. mesenteroides (Morales-Arrieta et al., 2006), Leuconostoc kimchii (Torres-Rodriguez et al., 2014), and Pseudomonas syringae (Khandekar et al., 2014). In addition, other microorganisms belonging to the genera Streptococcus, Pseudomonas, Xhanthomonas, and Aerobacter are also known to produce levan but their productivities are low (Laue et al., 2006; Simms et al., 1990; Takeshita, 1973). Inulin-type EPS are fructans or fructo-oligosacharides containing β-(2,1) linkages. The enzymes that polymerize the fructose moiety into inulin fructans are the fructansucrases. Inulin-type is a natural storage polysaccharide with a large variety of food and pharmaceutical applications (Apolinario et al., 2014). It is widely distributed in plants and its bacterial production has only been found in a few species of LAB, namely, S. mutans, Leuconostoc citreum, L. reuteri, and Lactobacillus johnsonii (Anwar et al., 2008; Olivares-Illana et al., 2003; Rosell and Birkhed, 1974; van Hijum et al., 2002). An inulin-producing enzyme from Bacillus sp. has also been characterized (Wada et al., 2003). The HePS are produced from sugar nucleotides by the activity of intracelular glycosyltransferases (Welman and Maddox, 2003; Werning et al., 2012) of a variety of mesophilic and thermophilic bacteria (Finore et al., 2014; Mozzi et al., 2006; Nicolaus et al., 2010; Notararigo et al., 2013), and they are composed of repeated subunits that are linear or branched, with variable molecular masses (up to 106 Da). Each one of these subunits can contain between three and eight different

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FIGURE 22.1  Detection of HoPS production. Colonies of the dextran-producing L. mesenteroides RTF10 strain isolated from a processed meat product (Notararigo et al., 2013) in media containing sucrose (a) or glucose (b). Colonies of the 2-substituted β-d-glucan-producing P. parvulus 2.6R (c) and the isogenic nonropy strain P. parvulus 2.6NR (d) (Fernández de Palencia et al., 2009; Fernández et al., 1995). Colony of P. parvulus 2.6R, prior (e) or after lifting (f).

monosaccharides and frequently has a combination of different linkage patterns. The linkages between monosaccharides that are most commonly found are β-(1,4) or β-(1,3) linkages in the backbones characterized by strong rigidity and α-(1,2) or α-(1,6) linkages in the more flexible ones. The components most commonly found in HePS are monosaccharides such as pentoses (d-arabinose, d-ribose, d-xylose), hexoses (d-glucose, d-galactose, d-mannose, d-allose, l-rhamnose, l-fucose), amino sugars (d-glucosamine and d-galactosamine), or uronic acids (d-glucuronic acids, d-galacturonic acids). Organic or inorganic substituents such as sulfate, phosphate, acetate, succinate, and pyruvate may also be present. Growth conditions (pH, temperature, and incubation time) and medium composition (carbon, nitrogen sources, and other nutrients) can affect the polymer yield and the sugar composition (Davey and Amos, 2002; Rinker and Kelly, 2000). The most important HePS are gellan gum, xanthan gum, and kefiran. Gellan is an anionic EPS produced by the bacteria Spingomonas paucimobilis (originally designated Pseudomonas elodea; also referred to as Spingomonas elodea) and Azotobacter chroococcum (Sutherland and Kennedy, 1996). This HePS is composed of repeating tetrasaccharide units of two residues of β-d-glucose, one of β-d-glucuronate and one of α-l-rhamnose (Zhu et al., 2013). Gellan gum has a wide range of applications in the pharmaceutical and other industries, such as the human and animal food industries, as a stabilizing, thickening, emulsifying, and gelling agent because of its appropriate rheological characteristics (Douglas et al., 2014; Prajapati et al., 2013). Xanthan gum is an anionic polysaccharide produced by Xanthomonas campestris (Vorholter et al., 2008). It is made up of pentasaccharide subunits, forming a cellulose backbone with trisaccharide side chains composed of mannose β-(1,4)-glucuronic-acid, β-(1,2)-mannose attached to alternate glucose residues in the backbone by α-(1,3) linkages (Jansson et al., 1975). Due to its rheological properties such as high viscosity and pseudoelasticity, xanthan is used in oil drilling, in building products to optimize material properties and in textile, pharmaceutical, cosmetics, food, and other industries as thickener, emulsifier, and stabilizer (Becker et al., 1998; Crockett et al., 2011). Kefiran is a HePS produced by Lactobacillus kefiranofaciens and several other unidentified species of Lactobacilli (L. kefirgranum, L. parakefir, L. kefir, and Lactobacillus delbrueckii ssp. bulgaricus) (Frengova et al., 2002). It has been reported that kefiran is composed of a branched hexa- or hepta-saccharide repeating unit containing approximately equal

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amounts of d-glucose and d-galactose residues (Wang et al., 2008). Kefiran is reported to have antimicrobial properties, antitumor activities, to modulate gut immune systems, and to have important rheological properties of interest for health and industrial applications (Nielsen et al., 2014).

3  CURRENT APPLICATIONS OF EPS IN THE FOOD INDUSTRY Polysaccharides are widely used in the food industry as additives. They belong to a group of hydrophilic compounds known as hydrocolloids, which are used in many food formulations to improve organoleptic qualities and shelf life, due to their properties as thickening and gelling agents (Saha and Bhattacharya, 2010), forming a viscous suspension or gels when dispersed in water.

3.1  Usage of EPS as Food Additives Although the most important of the hydrocolloid polysaccharides are extracted from plants like celluloses, hemicelluloses, pectins, exuded gums; and from seaweeds like alginate, agar, carrageenan; there are two very important commercialized bacteria-derived polysaccharides, xanthan gum, and gellan gum. These polymers are from Gram-negative bacteria, the xanthan gum synthesized by X. campestris (Becker et al., 1998), and the gellan gum synthesized by S. paucimobilis (Prajapati et al., 2013). The United States Food and Drugs Administration (FDA) has approved xanthan gum for use in human food, where it has an important role as stabilizer and thickening agent, improving viscosity, texture, mouthfeel, flavor release, appearance, and water-binding properties. Xanthan gum is, therefore, applied in bakery products, dairy products, beverages, dressings, sauces, gravies, syrups, and toppings (Palaniraj and Jayaraman, 2011). Thus currently, xanthan gum is the only significant bacterial EPS in the global market of hydrocolloids used as food additives, and its sales account for 6% of the total market value (Freitas et al., 2011). Nevertheless, gellan gum is also finding increased use in the food industry, partly due to its functional properties that provide excellent thermal and acid stability, adjustable gel elasticity and rigidity, high transparency, and good flavor release. Gellan gum is mainly used as a stabilizer, suspending agent, structuring and versatile gelling agent in a wide variety of applications in food products that include bakery fillings, confections, dairy products, dessert gels, frostings, icings and glazes, jams and jellies, low-fat spreads, microwavable foods, puddings, sauces, structured foods, and toppings (Prajapati et al., 2013). In recent years, certain plant polymers have started to be substituted by similar bacterial polymers, such as bacterial alginate (Freitas et al., 2011) and bacterial cellulose (Shi et al., 2014). Several advantages have been described for the latter when compared with plant-derived cellulose. Bacterial cellulose does not contain any significant contaminants, as it is a highly pure form of cellulose, hence does not require harsh chemical treatments for its isolation and purification. This polymer is nontoxic, approved by the FDA, it is crystalline by nature, and produces a range of forms and textures suitable for many food applications (Shi et al., 2014).

3.2  In Situ Production of EPS In the food industry, LAB have an important role improving the preservation and functional characteristics of a large variety of products based on milk, meat, and vegetables. As detailed in the previous section, many of these bacteria, belonging to the genera Streptococcus, Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus, produce a broad range of EPS with variable composition and functionality that give them a high value for industrial applications (De Vuyst et al., 2001; Duboc and Mollet, 2001; Patel et al., 2012; Ruas-Madiedo et al., 2002; Ruas-Madiedo and Sánchez, 2012; Werning et al., 2012). However, the relatively low production of EPS by LAB, compared with the industrial polysaccharides mentioned above, means that these EPS are not widely used as food additives and strategies are being sought to increase EPS yield (Patel et al., 2012). Many EPS producing LAB have generally recognized as safe status and are used as starter cultures in many fermented products, especially dairy products. During the fermentation, bacteria can synthesize the biopolymer in situ producing a natural product with improved rheological properties (Leroy and De Vuyst, 2004; Ruas-Madiedo et al., 2002). In addition, consumers now demand natural dairy products with a smooth and creamy texture, low in fat and sugars, and they pay a lot of attention to the perceived relationship between food and health (Patel and Prajapati, 2013). Thus, the use of synthetic food additives is regarded as unnatural and unsafe, even though additives are needed to preserve food products from spoilage and to improve the organoleptic properties (Leroy and De Vuyst, 2004). From this point of view, the LAB producing EPS represent a natural alternative to address these demands. LAB contribute to the preservation of milk products due to the rapid acidification that arises from the fermentation of lactose during the bacterial growth, which protects the milk against the proliferation of spoilage microorganisms and of pathogens (Ruas-Madiedo et al., 2002). Furthermore, LAB produce several natural antimicrobials that also help to combat microbial contaminations. These substances can replace

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synthetic food additives and include organic acids (lactic acid, acetic acid, formic acid, phenyllactic acid, and caproic acid), carbon dioxide, hydrogen peroxide, diacetyl, ethanol, bacteriocins, reuterin, and reutericyclin (Leroy and De Vuyst, 2004). EPS can also improve the organoleptic properties of the product, contributing to the texture, mouthfeel, taste perception, and storage stability. The success of the application of an EPS is determined by its ability to bind water, to interact with proteins, and to increase the viscosity of the milk serum phase. EPS may act both as a texturizer, improving the rheology (viscosity and elasticity) of a final product, and as physical stabilizers by binding hydration water and interacting with other milk constituents (ions and proteins), thus limiting syneresis. Additionally, the desirable structuring properties of EPS have been attributed to factors other than concentration, such as type of linkage, presence of side groups, branched structure, chain stiffness, molecular size, charge and interactions with milk proteins (De Vuyst et al., 2001; Duboc and Mollet, 2001; RuasMadiedo et al., 2002). Moreover, the effect of EPS on the rheological properties of final fermented milk is more pronounced and has a better outcome when EPS are produced in situ rather than when they are added as an additive (Doleyres et al., 2005). LAB also contribute to the flavor and aroma of the fermented products by generating volatile compounds such as acetate, ethanol, diacetyl, and acetaldehyde as a result of bacterial fermentation. Also, the marked production of organic acids during fermentation gives a pleasant fresh and mild acid taste to the fermented products (Leroy and De Vuyst, 2004; Ruas-Madiedo et al., 2002). Although EPS have, themselves, no taste, they increase the viscosity of fermented products, contributing to the mouthfeel, because this causes flavor compounds to remain longer in contact with the palate and taste receptors. Therefore, the overall aim is to obtain an appealing visual appearance of a product, to prevent syneresis, to have a creamy and firm texture, and to give a pleasant mouthfeel (Duboc and Mollet, 2001). The main fermented products where EPS producing LAB have been applied to improve their organoleptic properties are described below. Yogurt is a widely consumed dairy product throughout the world. Traditionally, yogurt is elaborated by starter cultures with the EPS producing bacteria L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These bacteria have a synergistic growth where both bacteria release metabolites stimulating the bacterial growth (Badel et al., 2011). The texture of yogurt develops during the fermentation due to the milk protein casein which forms micelles held together by colloidal calcium phosphate. When the pH decreases below 5.5 due to the production of lactic acid by the lactose fermentation, colloidal calcium phosphate begins to be solubilized into the aqueous phase causing destabilization of casein micelles. Under pH 5.0, the integrity of casein micelles is completely lost, increasing the rearrangement and interconnections of casein particles that results in a three-dimensional protein network (Laws and Marshall, 2001; Purwandari et al., 2007; Rawson and Marshall, 1997). EPS from LAB have the major role as thickening and stabilizer agents, which results in increased viscosity, limited syneresis, improved sensory characteristics such as mouthfeel, taste perception, creaminess, and storage stability of the final product (De Vuyst et al., 1998; Duboc and Mollet, 2001; Folkenberg et al., 2005; Rawson and Marshall, 1997). It has been shown that ropy EPS has a greater ability to retain serum, resulting in lower levels of syneresis (Folkenberg et al., 2006). The EPS interact with the protein matrix with ions in the aqueous phase and, as a consequence, generate a reduction of syneresis by binding water, as was already commented above. In the case of stirred yogurt, the coagulum is homogenized by shearing, the protein network is broken, favors synergesis and at the rheological level becomes a viscoelastic fluid; the greater the shearing the lower the viscosity of the yogurt. The presence of the EPS helps to recover the viscosity and limits syneresis; they give the stirred yogurt a smooth and creamy texture as demanded by the consumers (Duboc and Mollet, 2001; Rawson and Marshall, 1997). Consumers now also demand low-calorie, nonfat and no-additive-added yogurts, and the use of EPS producing BAL takes on greater importance in the manufacture of these products. Nevertheless, depending on the properties of the starter cultures for EPS production, the desired texture or appearance may not always be achieved. Then, the use of other EPS producing bacteria is required. A recent work shows that EPS producing Lactobacillus mucosae DPC 6426, used as an adjunct culture in the manufacturing of a low-fat yogurt fermented with L. delbrueckii bulgaricus and S. thermophilus strains as starter cultures, resulted in the improvement of textural and rheological properties, decreasing syneresis. A correlation between the increase in EPS concentration with the decrease in syneresis was observed (London et al., 2015). Other properties in yogurt are firmness and cohesiveness, and the production of EPS and subsequent interaction with protein network resulted in a loss of firmness in the coagulum. However, an EPS-producing culture of S. ­thermophilus produced creamy, yet firm, yogurt (Folkenberg et al., 2006) that could be commercially attractive. Kefir is a traditional fermented milk from Eastern Europe with natural carbonation and slightly acidic taste. This beverage is prepared by inoculating milk with kefir grains. Kefir grains are aggregates of microorganisms (homofermentative and heterofermentative LAB, yeasts, and acetic acid bacteria) embedded in a kefiran matrix (Ahmed et al., 2013; Duboc and Mollet, 2001). Kefiran is a water-soluble HePS, which is mainly produced by L. kefiranofaciens strains (Patel et al., 2012). The presence of kefiran in the grains protects the microorganisms against desiccation (Badel et al., 2011). The main difference of kefir with other fermented milks is the variable microbiota that could be within the kefir grains, including

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several bacteria and yeasts. Moreover, kefir has received considerable attention from food scientists because of its unique and complex probiotic properties (Ahmed et al., 2013). Other traditional fermented milks also use EPS producing LAB, which improve their rheological properties. Nordic ropy milk and Viili are milks fermented with strains of Lactococcus lactis and Leuconostoc (Duboc and Mollet, 2001). Dahi is an Indian fermented milk to which S. thermophilus strains are added to improve texture and rheology (Patel and Prajapati, 2013). Sweet acidophilus milk is of interest due to its claimed probiotic properties. It is manufactured by inoculating L. acidophilus bacteria in milk (Anjum et al., 2014). Cheeses are a class of dairy products which are strongly judged by their distinctive appearances and textures. Here, we will focus on the opportunities of using EPS-producing strains in the manufacture of reduced-fat cheddar cheese to improve the rheological properties. Consumption of low-fat products has grown steadily due to consumer concerns about health risks associated with obesity, atherosclerosis, coronary heart disease, and elevated blood pressure (Dabour et al., 2005). However, in the dairy industry, low-fat products are often characterized by inadequate flavor, poor textural quality, and lower keeping quality (O’Donnell, 1993; Olson and Johnson, 1990). In traditional full-fat cheddar cheese, fat globules and residual whey are dispersed into a continuous protein matrix of casein that contributes to the cheese’s moisture. Removal of fat results in a denser and compact structure of the protein network eliciting textural defects where hardness, gumminess, and chewiness are undesirably increased (Mistry, 2001; Ustunol et al., 1995). EPS producing cheese starter cultures could therefore be used for increasing moisture retention in reduced-fat cheddar cheese, due to their capacity to bind water and to interact with protein networks, in an attempt to improve texture and flavor. Indeed, the use of a ropy strain L. lactis subsp. cremoris JFR1 in reduced-fat cheddar cheese production improved moisture and produced textural characteristics similar to a full-fat cheddar cheese (Awad et al., 2005b; Dabour et al., 2005). Also, during ripening both full- and reducedfat cheeses followed the same pattern in texture and rheology. However, increased moisture levels in reduced-fat cheddar cheese may cause bitterness due to increased residual chymosin activity and lack of an adequate peptidolytic system to further hydrolyze the bitter peptides to amino acids (Awad et al., 2005a). Therefore, to improve the development of cheese’s sensory quality during manufacturing, adjunct cultures are often used, generally nonstarter LAB. The proteolytic activity of this bacteria combined with starter-culture proteinases produce the breakdown of large bitter peptides to form free amino acids, which are the precursors of many flavor and aroma compounds (Settanni and Moschetti, 2010). Recently, the dextran-producing strain, W. cibaria MG1 has been found to work effectively as an adjunct culture in the manufacture of cheddar cheese, increasing cheese moisture retention without significantly affecting the proteolysis of the cheese and thus the alteration of the characteristic flavor and aroma (Lynch et al., 2014). The elimination of gluten in bakery products presents a great challenge as low gluten flors yield poor rheological properties, and hence products of very low quality and poor mouthfeel and flavor (Moroni et al., 2009). To improve their properties, hydrocolloids such as starch have been used. However, the use of sourdough has played an important role in the bakery industry and this can replace hydrocolloids because EPS produced by Lactobacilli during sourdough fermentation can improve dough rheological parameters and bread qualities such as texture, aroma, flavor, shelf life, and mineral bioavailability (Arendt et al., 2007; Schwab et al., 2008; Tieking and Gänzle, 2005). The latter authors reported that EPS have a beneficial effect and can influence one or more of the following technological properties of dough and bread: (i) water absorption of the dough, (ii) dough rheology and machinability, (iii) dough stability during frozen storage, (iv) loaf volume, and (v) bread staling. Recent studies have shown a significant impact of sourdough rich in dextran on wheat, rye, and gluten-free bread quality. Dextran from L. mesenteroides provoked better viscoelastic properties of bread than reuteran or levan (Tieking and Gänzle, 2005). Furthermore, in situ production of EPS in sourdough was reported to be more effective than addition of EPS. Sorghum is a natural gluten-free cereal. The improvement in rheology by dextran producing W. cibaria and reuteran producing L. reuteri in sorghum bread was compared, with dextran being superior (Galle et al., 2012; Schwab et al., 2008). Also, HePS produced by L. buchneri were shown to have a significant impact on the dough rheology of sorghum sourdough, being the first report of HePS influence in application in cereal fermentation, because in wheat sourdough they were found to have no influence (Galle et al., 2011).

4  BACTERIAL EPS AND HUMAN HEALTH A functional food is any food which, in addition to its nutritive value, has a positive impact on human health. Probiotics (microorganisms, mainly bacteria), prebiotics (indigestible dietary fibre/carbohydrate), and synbiotics (a mixture of proand prebiotics) are today the most frequent components used for the elaboration of functional food. Functional foods are commonly used to modulate the composition of the gut microbiota contributing to the maintenance of the host health or for prevention of disease, and the use of probiotics and prebiotics is useful to re-establish the normal microbial composition in the host (Przyrembel, 2001). The gut microbiota is able to shorten long polysaccharides such as EPS that derives from:

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(i) the host’s diet or (ii) other microbiota EPS-producing strains. This is due to the variety of glycosyl hydrolases that the microbiota strains can produce. The resultant short polysaccharide chains can be fermented to small-chain fatty acids like butyrate and propionate, vitamins, and other compounds that otherwise would not have been extracted by the host due to the lack of key enzymes.

4.1  EPS as Prebiotics and Immunomodulators The definition of prebiotics has evolved over the years to finally reach the following criteria: (i) nondigestible fiber, (ii) fermentable by gut microbiota, (iii) selective stimulation of growth of intestinal bacteria, and (iv) associated to microbiota modulation (de Vrese and Schrezenmeir, 2008). Human gut microorganisms have been tested for their ability to produce and/or ferment EPS in which strains showed a bifidogenic effect (Ruas-Madiedo et al., 2007). It has been also demonstrated in humans that EPS from Lactobacilli (mainly HePS) and Bifidobacteria (HePS) are able to feed commensal bacteria in the host (Charalampopoulos and Rastall, 2012; Salazar et al., 2008). Another study conducted with a β-glucan purified from Pediococcus parvulus 2.6R, isolated from ropy cider, previously named Pediococcus damnosus 2.6 (Dueñas-Chasco et al., 1998; Fernández de Palencia et al., 2009; Fernández et al., 1995; Werning et al., 2006) (Figure 22.1), improved the growth of three probiotic strains Lactobacillus plantarum WCFS1, Lactobacillus acidophilus NCFM, and L. plantarum WCFS1β-gal that overexpress a β-glycosidase enzyme (Russo et al., 2012). In addition, the availability of d-glucose and EPS as sugar sources retarded the entry into stationary phase of the strains suggesting a synergistic effect in promoting metabolic processes (Russo et al., 2012). The relevance of EPS as prebiotics is correlated to the beneficial properties that they exert on the gut microbiota. Normally, healthy microbiota plays an important role in the homeostasis of the intestine, regulation of immune system, avoiding invasion of pathogenic strains, and clearing of infections (Amores et al., 2004; Kamada et al., 2012; Ruas-Madiedo and Sánchez, 2012). EPS molecules can assemble together and adhere to intestinal epithelial cells, therefore impeding pathogen adhesion and/or stimulation of underlying immune system cells (Anadón et al., 2010). It has been proposed that EPS are involved in the immunomodulation of the innate immune response through the interaction with dendritic cells and macrophages and in the modulation of the adaptative immune response enhancing the T cell and Natural Killer proliferation cells. In both cases, the response takes place with a consistent cytokine production by the immune system cells (Bodera, 2008; Klaenhammer et al., 2012; Rizzello et al., 2011). In vitro experiments, in which EPS-producing and nonproducing strains were compared, indicated that the high-­ molecular-weight HePS of the probiotic Lactobacillus casei strain Shirota (Yasuda et al., 2008), L. rhamnosus RW-9595M (Bleau et al., 2010) and Lactobacillus paraplantarum BGCG11 (Nikolic et al., 2012) have a suppressive effect on activation of macrophages. Also, in vitro analysis of the immunomodulatory activity of purifed HePS with different molecular masses from Bifidobacteria strains on human macrophages support that only high-molecular-weight polymers induced the production of a cytokine pattern that leads to a reduction of the immune response (López et al., 2012). In addition, (1,3)-βd-glucans can promote antitumor and antimicrobial activity by activating macrophages, dendritic cells, or other leukocytes (Brown and Gordon, 2001). The immune responses of the bacterial linear curdlan, used for making functional foods (tofu) and sold as an immunostimulant, as well as eukaryote-derived glucans (either linear or O6 subtituted) have been characterized, and the activities of these molecules have been correlated with their chemical structure, molecular weight, and conformation (de Vrese and Schrezenmeir, 2008; McIntosh et al., 2005). Moreover, in vitro experiments with purified O2 substituted (1,3)-β-d-glucan (Notararigo et al., 2014) from P. parvulus 2.6R and L. lactic NZ9000 [pGTF], which expresses the pediococcal GTF glycosytranferase (Werning et al., 2008) as well as with the EPS producing and isogenic nonproducing strains (Fernández de Palencia et al., 2009; Garai-Ibabe et al., 2010) showed that this EPS modulated human macrophages causing an antiinflammatory response.

4.2  Role of EPS Improving Bacterial Probiotic Properties The definition of probiotic can be summarized as a viable microorganism (normally yeast or bacterium) that when ingested in a sufficient quantity, shows a beneficial effect on the host's health, through the interaction with the gut microbiota (Mäyrä-Mäkinen and Bigret, 1993). Probiotics currently belong mostly to Lactobacilli and Bifidobacteria genera, that normally colonize the human intestine, and which produce EPS (Ruas-Madiedo, 2014), but there are some exceptions like the yeast Saccharomyces boulardi, used as a probiotic, as well as Bacilli like B. subtilis among others, that are not usual component of the gut microbiota, although they are able to restore a normal balance between the other species (MäyräMäkinen and Bigret, 1993; Von Wright and Axelsson, 2012).

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The EPS of LAB have been implicated in cellular recognition and the formation of biofilms. For example, the glucans and fructans of S. mutans play an important role in the adhesion of this bacterium to the tooth surface and in the formation of dental plaque (Klein et al., 2009). However, the role of surface polysaccharides in probiotic-host interactions has not yet been studied in great detail. The production of the O2 substituted (1,3)-β-d-glucan confers to the intestinal Lactobacillus paracasei NFBC 338 higher resistance to gastrointestinal and technological stresses (Stack et al., 2010). Other studies have revealed the ability of the O2 substituted (1,3)-β-d-glucan to improve the adhesion properties of naturally producing and recombinant LAB strains, to intestinal epithelial cell, using the Caco-2 cell line as human mucosal in vitro model (Fernández de Palencia et al., 2009; Garai-Ibabe et al., 2010). This and the results showing the contribution of O2 substituted (1,3)-β-d-glucan on biofilm formation by LAB (Dols-Lafargue et al., 2008) support that this biopolymer could play an important role in cellular recognition and intestinal colonization by LAB strains. Nevertheless, the properties of this polysaccharide are not a universal characteristic of EPS species because the presence of the polygalactan-rich EPS produced by the probiotic L. rhamnosus GG reduced its adhesion capability (Lebeer et al., 2009).

4.3  Potential Effect of EPS as Coadjuvant for Treatment of Diseases The health benefit of prebiotics is not only related to the ability to modulate the microbiota and the immune system; EPS have other biological properties that have been investigated in vitro and in vivo and that suggest potential future usages. Some EPS from LAB seem able to regulate serum cholesterol levels, through the inhibition of absorption of this molecule by the host (Ooi and Liong, 2010). This hypocholesterolemic effect was detected when rats were fed with kefiran produced by L. kefiranofaciens WT-2B(T). After treatment, the levels of cholesterol, triglycerides, and free fatty acids decreased markedly (Maeda et al., 2004). Similarly, human consumption of an oat-based food prepared with P. parvulus 2.6R resulted in a decrease of serum cholesterol levels, boosting the effect previously demonstrated for (1,3)(1,4)-β-d-glucans of oat-based products (Martensson et al., 2005). Also, Lactobacillus species producing EPS (kefiran) prevented the onset and development of atherosclerosis in hypercholesterolemic rabbits fed a diet containing 1% kefiran (Uchida et al., 2010). Some probiotic bacteria that synthesize EPS can also eliminate reactive oxygen species that are formed in the gut through diverse metabolic reactions, and hence these organisms exhibit antioxidant activity. Oxidative stress plays a role in inflammatory bowel disease, both in the initial and progressive phases. The effect of bacterial EPS on the oxidative damage to the intestine has been investigated in a rat colitis model. In a comparative study using two strains of L. delbrueckii subsp. bulgaricus, one (B3) a high producer and the other, (A13) a low producer of EPS, it was observed that there was less intestinal oxidative damage in the rats dosed with the B3 strain (Sengül et al., 2011).

5  BACTERIAL EPS AND ANIMAL HEALTH The investigation of bacterial EPS in animal health has been less than in humans, but its potential application in farm animals, as an alternative to the use of antibiotics, as well as the lack of efficient vaccines, has recently caused increased interest in this sector.

5.1  Animal Models to Study the Role of EPS in Vivo Most of the effects of EPS have been investigated in vitro. However, prior to utilization, in vivo testing in animal models is needed to validate the in vitro results, especially for health applications. Currently, rodents are among the most popular animal models for studying the role of EPS in the probiotic properties of the producing bacteria. Thus, the EPS produced by L. brevis KB290, a probiotic strain derived from a Japanese traditional pickle, seems to play an important part in enhancing cell-mediated cytotoxic activity in mouse spleen (Sasaki et al., 2014). In another study, 20 μg/day of EPS produced by L. delbrueckii ssp. bulgaricus OLL1073R-1 were orally administered to BALB/c mice prior to intranasal infection with influenza virus A/PR/8/34 (H1N1). It was shown that the EPS was one of the active ingredients responsible for the anti-influenza virus activity in mice (Nagai et al., 2011). The long galactoserich EPS of the prototypical probiotic strain L. rhamnosus GG (LGG) were also investigated in murine gastrointestinal tract in comparison with the isogenic non-EPS-producer strain. It was found that the mutant had lower persistence than the wild-type strain, and seems to be more sensitive to host innate defense molecules, such as the LL-37 antimicrobial peptide and complement factors. These results suggested that the EPS of LGG form a protective shield against innate immune factors in the intestine (Lebeer et al., 2011). A Wistar rat model has been also used to show that two EPS, produced by Bifidobacterium strains, could modify the systemic inflammatory profile and insulin-dependent glucose homeostasis (Salazar et al., 2014). The digestive tract of pigs is very similar to that of humans and these animals have been used to test

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antibacterial activity of EPS against pathogens. Enterotoxigenic Escherichia coli (ETEC) is a pathogen that infects calves and piglets and cause diarrhea. Chen et al. (2014) established an in vivo small intestinal segment perfusion model and used to study the antiadhesive properties of bacterial EPS (reuteran and levan) and related glycans (dextran and inulin). All glycans reduced fluid loss, but only reuteran also decreased the adhesion of ETEC K88 to the intestinal mucosa. The innate immune response of the zebrafish (Danio rerio) is comparable to that of higher vertebrates. Thus, this model has been used to study the host immune response to microbial infections (Rojo et al., 2007; van der Sar et al., 2004), to analyze the interactions between the host and its natural gut microbiota (Milligan-Myhre et al., 2011), and the effect of probiotics (Carnevali et al., 2013; Gioacchini et al., 2010, 2011; Rendueles et al., 2012; Rieu et al., 2014). In addition, this animal model has been used to test potential immunostimulant substances, such as β-glucans from cereal origin (Oyarbide et al., 2012) and can be used to test bacterial EPS.

5.2  Unhealthy Effects of EPS in Animals Many bacteria pathogens produce EPS, often forming a capsule, which may play an important role in the organisms’ pathogenicity, because the EPS may enable evasion of phagocytosis in the host. The chemical structures of the EPS may mimic the host cell surface components and not activate the immune system. Streptococcus iniae is an important pathogen in both marine animals (salmonids and other fish species) and humans, which causes systemic infections in these hosts. The ability of S. iniae to provoke an invasive disease depends on the immune status of the fish and on a variety of mechanisms, such as the capability of the bacteria to express a different amount of capsular polysaccharide during the various stages of the disease (Lowe et al., 2007). Furthermore, new strains of S. iniae producing large amounts of extracellular polysaccharide that is different from its capsular polysaccharide, have emerged in rainbow trout farms where the entire fish population was routinely vaccinated (Eyngor et al., 2008). The biofilms also play an important role in pathogenic bacteria and their ability to cause disease. Salmonella enterica serovar Typhimurium colonizes the chicken intestinal tract and invade the intestinal epithelium and oviducts. Its establishment over time is due to its ability to adhere and form biofilms. It has been shown that species of EPS contribute to biofilm formation on HEp-2 cells and chicken intestinal epithelium (Ledeboer and Jones, 2005). Vibrio anguillarum is a pathogen of organisms reared in marine aquaculture (fish and shellfish) which causes a fatal haemorrhagic septicaemia. A DNA locus encoding an EPS transport and biosynthetic system is required for its attachment to the fish skin (Croxatto et al., 2007). EPS deficient mutants are unable to colonize the fish skin, they penetrate skin mucus less efficiently than the EPS-producing wild type strain, and they are significantly more sensitive to lysozyme and antimicrobial peptides (Weber et al., 2010).

5.3  Beneficial Effects of EPS in Animals Oligosaccharides produced by extracellular glycansucrases (glucan- or fructan-sucrases) may have potential applications as anti-adhesive agents in preventing pathogen colonization. Milk oligosaccharides have been proposed to play an important role in newborn defense, blocking bacterial adhesion to the intestinal mucosa and preventing infections. Binding of bovine milk oligosaccharides inhibited the hemaglutination activity of enterotoxigenic E. coli (ETEC) isolated from calves (Martín et al., 2002). Wang et al. (2010) tested for the antiadhesive properties of LAB synthesized EPS (reuteran, levan, dextran, and an uncharacterized glucan), and commercially available prebiotics against porcine ETEC strains. Their results indicated that the LAB reuteran, levan, and the glucan can interfere with ETEC adhesion to erythrocytes and therefore have the potential to benefit the swine industry. The dextran and commercially available oligo- and poly-saccharides did not show anti-hemagglutination activities at the maximum concentration tested (10 mg mL−1). These results suggested a dependence of the activity on structure and molecular weight of the polymers, but further experiments are needed to demonstrate if this is the case. Dextran is a glucose polymer (well known as a plasma expander) that has been investigated as a prebiotic in animals. This EPS is not digestible by gastric juices nor in the calf rumen. Thus, it can reach the intestine and promote the growth of beneficial microbiota in this organ. It has been reported that when dextran is mixed with food, it improves milk production in Holstein dairy cows during hot and humid seasons in Japan (Yasuda and Fukata, 2004) and also when it is used together with L. casei subsp. casei as a symbiotic (Yasuda et al., 2007). In addition, recent in vitro (Nácher-Vázquez et al., 2013) and in vivo (Nácher-Vázquez et al., 2015) experiments have shown that high molecular weight dextrans produced by LAB exhibit antiviral activity against salmonid viruses. Shrimp and salmon aquaculture has been accompanied by a significant use of antibiotics, which has led to a transfer of antibiotic resistance determinants among aquatic bacteria, including pathogens. This fact makes it necessary to drastically reduce the use of antibiotics in aquaculture (Cabello, 2006). Thus, for several years, the immunostimulants have been used as feed additives as this is a better approach to control disease

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losses in this sector. Numerous polysaccharides from a variety of sources have the ability to stimulate the immune system (Ringø et al., 2012). The most widely used is the β-glucan with 1,3-linkages from Saccharomyces cerevisiae yeast cell wall (Immunogen® is a commercialized product) containing β-glucan and mannanoligosaccharides (Yar Ahmadi et al., 2014). Currently, there are no bacterial EPS used as immunostimulants or prebiotics directly in animal health, and this is a new field to explore and to exploit. For example, EPS-producing LAB have been isolated from the gastrointestinal tracts of fish, shellfish and shrimp and include strains belonging to W. cibaria, Weissella confusa, L. plantarum, and Pediococcus pentosaceus (Hongpattarakere et al., 2012). According to the authors, these EPS are potential prebiotic ingredients for the animal feed industry, with a potential application in fish farming. Finally, probiotic bacteria producing EPS have also the potential to be used as coadjuvants for animal health. Thus, in a study of 18 strains of lactobacilli isolated from the gastrointestinal tract of chicken, two strains L. delbrueckii ssp. delbrueckii BAZ32 and L. acidophilus BAZ29 were selected as potential probiotics for chickens due to their high tolerance to acid, bile, antibiotic resistance, high antimicrobial activity, aggregation ability, and EPS production (Yuksekdag et al., 2014). Examination of 96 LAB isolates from feces and oral cavity of calves showed that only a few strains produced capsular polysaccharide, and only one showed a ropy phenotype (Maldonado et al., 2012).

6  CONCLUSIONS AND PERSPECTIVES Research carried out to date has shown that bacterial EPS are of interest to the food industry as thickeners, gelling agents, and texture improvers. As the natural production of these biopolymers is not quantitatively high, their use in food processing normally requires their in situ production by incorporating the producing bacteria (normally LAB) into the food or beverage. This is often economically advantageous, as starter cultures may be cheaper than additives. It also meets customers’ requirements for natural, low fat products that nevertheless possess the expected texture and consistency. Apart from the interest in the food industry, there are also potential pharmaceutical applications of EPS. In the past, it was believed that activation of the immune system and the adaptive response was exclusively triggered by proteins (antigens), and that bacteria had evolved polysaccharide capsules to elude antigenic recognition. However in more recent times, it is known that bacterial polysaccharides can also interact with the immune system, which makes them candidates as lead compounds for developing useful immunomodulators for both human and veterinary use. There is still much work to do to better understand how EPS produced by LAB interact with the intestinal flora, and to gain further insight into how different EPS structures affect the various facets of the immune response. Nevertheless, it appears evident that there is growing industrial awareness of the potential benefits of incorporating EPS into food and medicinal products.

ACKNOWLEDGMENTS We thank Dr. Stephen Elson for critical reading of the manuscript. This work was supported by the Spanish Ministry of Economics and Competitiveness grant AGL2012-40084-C03-01.

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Chapter 23

Probiotic and Prebiotic Dairy Desserts Flávia C.A. Buriti*, Raquel Bedani† and Susana M.I. Saad† *Department of Pharmacy, Center of Biological and Health Sciences, State University of Paraíba, Campina Grande, Brazil, †Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil

1 INTRODUCTION Consumers are increasingly searching for innovations of the food industry whenever they are able to make their own food choices. Besides, individual choices based on knowledge about the relation between certain food and healthiness are improving widely. In this sense, transforming traditional food products into functional food products with potential to modulate the intestinal microbiota might lead to a promising alternative. Therefore, the supplementation of alternative dairy products, like dairy desserts, with probiotic microorganisms and/or with prebiotic fibers seems to be successful. Probiotics are referred to as live microorganisms, that, when administered in adequate amounts, confer a health benefit on the host (FAO/WHO, 2002). Prebiotics are selectively fermentable ingredients that allow specific changes in the composition and/or activity of gastrointestinal microbiota that allow benefits to the host (Gibson et al., 2004, 2010). A product denoted as a synbiotic is one in which a probiotic microorganism and a prebiotic ingredient are combined (Swennen et al., 2006). On the other hand, according to Kolida and Gibson (2011), an additional required condition to be fulfilled for synbiotic foods is that the chosen prebiotic must selectively support the growth of the probiotic microorganism employed. The United States, Europe, and Japan markets account for over 90% of the total functional foods worldwide, most of which comprising functional dairy foods (Marsh et al., 2014). There was a reported 1.5-fold increase in the functional food and beverage global market between 2003 and 2010, which is estimated to grow around 22.8% between 2010 and 2014, achieving total values of around €21.7 billion (approximately US$ 24.4 billion). Estimates also indicate that this market will be worth around €65 billion (approximately US$ 73 billion) in 2016 (Marsh et al., 2014). According to statistical data available, in 2013, the market for functional foods accounted for an estimated amount of US$ 43.27 billion (around € 38.5 billion) worldwide, which represents an approximate 27% increase relative to 2009 (Leatherhead Food Research, 2014). Among functional foods, dairy-based functional foods accounted for nearly 43% of the market, which is predominantly based on fermented dairy products (Özer and Kirmaci, 2010). Specifically regarding probiotic functional food, the global market totalized US$ 27.9 billion (around €24.8 billion), in 2011, and is predicted to reach US$ 44.9 billion (around €40 billion) in 2018. By 2015, the probiotic worldwide market is expected to reach US$ 31.1 billion (around €27.7 billion), and around 90% results from the functional foods and beverages market (Olivo, 2014). As for prebiotic products, the demand was worth US$ 2.3 billion (around €2 billion) in 2012, and is predicted to achieve US$ 4.5 billion (around €4 billion) in 2018. Europe is the global revenue leader in prebiotics and dominates the demand for these products (Transparency Market Research, 2013). Several studies have shown that probiotic microorganisms and prebiotic fibers might be successfully employed in different milk-based food matrices, such as yogurt, cheese, ice cream, beverages, desserts, and others. Dairy desserts, for example, may be considered interesting options for the incorporation of these functional ingredients due to several reasons (Cardarelli et al., 2008), which will be discussed below. Dairy desserts are widely consumed by all age groups and this consumption is mainly influenced by their nutritional and sensory characteristics (Tárrega and Costell, 2007; Ares et al., 2009b). Moreover, the dairy dessert market has increased in the last years and a broad range of ready-to-eat milk-based desserts has been available to the consumer. In this sense, competition regarding products across the health and wellness categories is increasing and, therefore, companies have the challenge to try, as much as possible, to differentiate their products based on superior functionality, through creative segmentation and positioning strategies (Ares et al., 2008a; Raud, 2008; Bogue et al., 2009). Because Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00023-X © 2016 Elsevier Inc. All rights reserved.

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consumers are increasingly being attracted to healthier and functional products, some kinds of dairy desserts have shown a great market potential (Dyminski et al., 2000; Pinto et al., 2003; Aragon-Alegro et al., 2007). Moreover, a great market potential is being represented by the consumption trend of indulgence products, even though reducing fat and calorie content of consumers’ favorite desserts without compromising taste and mouthfeel has been required (Maltete, 2008; Meyer et al., 2011). Therefore, reduced-fat dairy desserts could be explored, mainly regarding conveniently planned changes in the fat profile and increased protein and dietary fiber contents (Komatsu et al., 2013), because desserts with low fat and functional claims are attractive to consumers (Ares et al., 2009b). In general, these factors may explain the different types of dairy desserts included in well-established brands of probiotic products among consumers (Faron, 2010). Moreover, the dairy probiotics success might be partially explained by their general positive image among consumers. In fact, consumers are increasingly aware of the fact that food has a direct contribution to health (Sangeetha et al., 2005; Siró et al., 2008). According to a study reported by Ares et al. (2008b), consumers interested in functional food considered milk-based desserts as “credible carriers of functional messages.” Therefore, the application of probiotic and prebiotic ingredients for the preparation of milk-based desserts is especially attractive to the consumers interested in healthy food, besides enhancing the products’ image and value. Owing to the chances for this group of food products to carry functional components, this chapter focuses on important studies published in the last years regarding the development of refrigerated probiotic and/or prebiotic dairy desserts and presents the main challenges involved in this field.

2  POINTS TO BE CONSIDERED WHEN DEVELOPING PROBIOTIC AND/OR PREBIOTIC DAIRY DESSERTS 2.1  Regulatory Requirements Global regulatory requirements greatly affect the probiotic market and they have become stricter in the past years (Burgain et al., 2011). Indeed, health claims should be based on cell viability and probiotic function (Jankovic et al., 2010; Burgain et al., 2011). Particularly in Europe, several applications for probiotic health claims have been rejected by the European Food Safety Authority, which has ruled that the word “probiotics” itself carries an implied health benefit (Marsh et al., 2014). In this context, even though several studies support probiotic efficacy, regulatory conditions, particularly in the EU, have limited the claims manufacturers are able to make about their products (Olivo, 2014). On the other hand, in the United States, the Food and Drug Administration (FDA) permits the use of structure/function claims in promoting general health due to nutritive value. However, current law forbids the use of claims related to the treatment of diseases (Olivo, 2014). According to the FDA, “health claims describe a relationship between a food, food component, or dietary supplement ingredient, and reducing risk of a disease or health-related condition.” In contrast, a structure/function claim describes “the process by which the dietary supplement, conventional food, or drug maintains normal functioning of the body and does not need FDA approval before marketing” (Venugopalan et al., 2010). One of the most important aspects of probiotics regarding concern to consumers and regulators is safety (Farnworth, 2008). Strains belonging to the Lactobacillus and Bifidobacterium genera are the most well-known probiotic microorganisms (Douglas and Sanders, 2008; Ross et al., 2010; Whelan and Myers, 2010). It is important to bear in mind that probiotic cultures should be recognized as safe (GRAS status—generally recognized as safe) for human consumption through scientific evidence or experiences based on the history of consumption by a significant number of subjects (Kolida and Gibson, 2011). It is also important to emphasize that the added ingredients and the processing steps employed in the development of probiotic dairy desserts should not result in loss of the probiotic microorganism viability during their production and shelf life or reduction in the product’s sensorial quality. Standards requiring a minimum probiotic dose of 6-7 log colonyforming units (cfu) per gram in dairy products have been introduced by several food organizations worldwide (Talwalkar and Kailasapathy, 2004a; Akalın and Erışır, 2008). In Japan, the Fermented Milks and Lactic Acid Bacteria Beverages Association has required a minimum of 7 log cfu viable bifidobacteria per gram or mL of a product for it be considered a probiotic food for human use (Stanton et al., 2001; Vasiljevic and Shah, 2008). The Canadian Food Inspection Agency (CFIA) establishes a minimum dose of 9 log cfu per daily serving portion of the microorganism to allow using the word “probiotic” or other similar designations and these descriptions should come along with specific, validated statements about the probiotic benefits or effects (Health Canada, 2009; CFIA, 2015a). In Canada, the reference amount considering a ready-to-serve form of fresh dairy desserts is 100 g (CFIA, 2015b). According to the Brazilian legislation, probiotic microorganisms should be ranging from 108 to 109 cfu per daily serving portion of the product ready for consumption during the entire shelf life (ANVISA, 2008). Brazilian standards recommend the daily serving portion of 120 g for milk-based desserts (ANVISA, 2003). The use of the word probiotic for

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food and food supplements was also confirmed by the Italian Ministry of Health provided that the product meets certain criteria, such as a minimum number of viable cells (1 × 109 cfu) administered per day, a full genetic characterization of the probiotic strain, and an established history of safe use in the Italian market (Hill et al., 2014). The most studied prebiotics are nondigestible carbohydrates, such as fructo-oligosaccharides (FOS), inulin, galactooligosaccharides, and lactulose (Ruas-Madiedo, 2014). In accordance with FAO (2007), these and other chemical groups commonly used as prebiotics (soya-oligosaccharides, xylooligosaccharides, pyrodextrins, and isomaltooligosaccharides) have a long history of safe use. Nevertheless, regulations may differ from one country to another, even for the same component. Besides, legislation is very limited regarding the use of the word “prebiotic” on food products, as some of these fibers still need studies to prove their real benefits (Dwyer, 2007; FAO, 2007). Most of the data available in the scientific literature about prebiotics’ effects is related to inulin and FOS, for which several health benefits were repeatedly demonstrated through experimental and human trials (Roberfroid et al., 2010). In some countries in Europe, such as France and the Netherlands, inulin-type fructans are recognized as prebiotic ingredients (Brownawell et al., 2012). Nonetheless, the list of permitted health claims published by the European Commission, in 2012, did not include any claim related to the consumption of an ingredient conferring benefits to the intestinal microbiota. Only a claim related to the contribution of lactulose for increasing the intestinal transit rate was approved (EU, 2012). Because the evaluation process of claims for prebiotics and probiotics is difficult, the Panel on Dietetic Products, Nutrition and Allergies in Europe (NDA) published a guidance document on the scientific substantiation of health claims to assist applicants in preparing and submitting their applications for the authorization of health claims related to the gastrointestinal tract (GIT) and the immune system (EFSA-NDA, 2011). In Brazil, only foods containing FOS and/or inulin are allowed to claim a prebiotic effect (ANVISA, 2008). Nonetheless, the Brazilian legislation restricts the claim for prebiotic and/or probiotic foods only to the contribution for the intestinal microbiota balance. In case this effect is supposed to be stated in milk-based desserts, the prebiotic fiber should be present at concentrations of at least 3 g in the daily recommended serving portion of a product ready for consumption (ANVISA, 2008). In fact, considering only the ability of inulin-type fructans to increase fecal bifidobacteria in humans, daily doses of 4-5 g for at least 2 weeks showed to be effective (Roberfroid et al., 2010). For nutritional labeling, Roberfroid (1999) suggested that inulin-type fructans, as well as all of the nondigestible oligosaccharides that are largely or completely fermented in colon, ought to be given a caloric value of 1.5 (6.3) kcal/g (kJ/g).

2.2  Gel Formation and Prebiotic Gelling Properties Some prebiotic fibers can confer gelling properties to dairy desserts. Opposite to what is observed with the application of several probiotic microorganisms, the preparation of dairy desserts with inulin is advantageous because these prebiotics show good stability during the usual food processing steps, especially during heat treatment. At room temperature, solubility of inulin-type fructans reduces with an increased degree of polymerization (DP); therefore, high temperature might be needed to solubilize long-chain inulin during food product processing. However, the β-bonds between the fructose units in FOS may be partially hydrolyzed in very acidic conditions (Kim et al., 2001; Franck, 2002, 2008; Macfarlane et al., 2008). Others factors associated with the technological properties of these prebiotic fibers will be discussed in another section of this chapter. In general, a gel consists of a three-dimensional lattice of large molecules or aggregates, capable of immobilizing solvent, solutes, and filling material. Soluble polymers become insoluble to form a semisolid structure (gel), due to the association of the polymer molecules with the solution in which it is present. Food gels may be formed by proteins and polysaccharides, which may participate in gel formation in the form of solutions, dispersions, micelles, or even in disrupted tissue structures. Many factors may affect gel formation from polysaccharides, including the food processing steps, other ingredients, heating, temperature, and pH. These factors also contribute for the gel strength, besides other rheological properties (Kim et al., 2001). Most of the milk-based desserts are composed of ingredients which interact with milk proteins and influence their stability and consistency, including starch and/or several hydrocolloid types (Dello Staffolo et al., 2007). Native and modified starches from different sources, especially from maize, rice, and tapioca, are widely employed for the production of probiotic and/or prebiotic milk-based desserts, due to their thickening and gelling properties (Helland et al., 2004; Tárrega and Costell, 2006b; Corrêa et al., 2008; Magariños et al., 2008; González-Tomás et al., 2009b). Starch forms consist of two fractions: amylose and amylopectin (Keskar and Igou, 2011). Amylose is a linear polymer of α-d-glucose units linked by α-1,4 glycosidic bonds, whereas amylopectin is a branched polymer of α-d-glucose units linked by α-1,4 and α-1,6 glycosidic bonds (Singh et al., 2010). The amylose/amylopectin ratio varies according to the source and maturity of the

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starch employed, but it is about 1:3 for most starches (Knill and Kennedy, 2005). When starch is heated to about 50 °C, in the presence of water, the amylose in the granule swells; the crystalline structure of the amylopectin disintegrates and the granule ruptures. Hydrogen bridge bonds are weakened in this process. The polysaccharide chains take up a random configuration, which causes swelling of the starch and thickening of the surrounding matrix, in a process that leads to gel formation. Amylopectin binds large quantities of water, and amylose forms helical structures under water binding (Sajilata et al., 2006; Keskar and Igou, 2011). The starch gelatinization is, therefore, related to the destruction of the crystalline structure in starch granules. This process is irreversible and its main stages consist of granular swelling, native crystalline melting, and, in the end, a molecular solubilization occurs (Liu et al., 2009). When gelatinization takes place, the amylose part is not totally dissolved, leading to the formation of crystallin aggregates, which are linked by hydrogen bonds. The gel thus formed may sometimes loose water, and this process is called syneresis. Because of its branched structure, amylopectin gels are more stable than amylose, and less susceptible to retrogradation (Rapaille and Vanhemelrijck, 1998). Native starches might show undesirable properties when they are submitted to certain process conditions, including temperature, pH, or pressure. Moreover, low resistance to high shear rates, high susceptibility to retrogradation, and syneresis may limit the applicability of native starches (Bertolini, 2010; Huber and BeMiller, 2010). On the other hand, when compared to native starches, modified starches present higher thermomechanical resistance and are more stable, enabling the production of more consistent dairy desserts and less susceptible to syneresis (Tárrega and Costell, 2006a). Starch properties might be positively affected by the insertion of small quantities of ionic or hydrophobic groups in its structure. In this way, properties like the solution viscosity, the association behavior, and the shelf life stability of the final products are improved (Xie et al., 2005). Usual methods applied for obtaining modified starches include acetylation, hydroxypropylation, and cross linking. Cross-linked starches increase the gelatinization temperature, reduce viscosity, and increase stability to acid, heat, and shear. Acetylation and hydroxypropylation contribute significantly for the stabilization of food systems during cold storage (Luallen, 2002; Aziz et al., 2004). Because resistant starch is successful in modifying the composition of fecal bacteria (Martínez et al., 2010), it has been pointed as a prebiotic food constituent (Trujillo-de Santiago et al., 2012). This property was extended to some chemically modified starches (also called type 4 resistant starch), such as cross-linked and acetylated starches (Thanh-Blicharz et al., 2014). However, more human trials are required to assess the potential of these carbohydrates as prebiotics (FAO, 2007; Roberfroid et al., 2010), especially regarding selective properties on microorganisms considered as beneficial. Hydrocolloids are employed as food additives. Due to their technological properties, they are advantageously used for thickening, stabilizing, enlarging, adding viscosity and elasticity, and providing the food product with the desirable texture (Maruyama et al., 2006; Brownlee, 2011). These compounds are predominantly polysaccharides, but some proteins are also employed (Burey et al., 2008). Frequently, the polysaccharides employed as hydrocolloids are long-chain gums constituted of water-soluble high-molecular-weight polymers with gel-forming capacity. In order to select the most appropriate hydrocolloid, the composition of the dessert should be considered, especially its protein content, beside its pH, and the conditions employed during the dessert’s production steps. Hydrocolloids are susceptible to shear and thermal treatment and to acidity and these factors may lead to disadvantageous changes in their technological properties (Rapaille and Vanhemelrijck, 1998). For this reason, according to the product to be obtained and the unit operations and conditions to be employed, combinations of different gums are frequently used by the food industry. Carrageenans (mainly κ- and ι-carrageenans), galactomannans (guar, locust bean, and tara gums), pectin, and xanthan are among the gums most used in food production (Maruyama et al., 2006; Bayarri et al., 2010; Buriti et al., 2014). Collagen proteins are also employed as stabilizing agents in the production of milk-based desserts. Gelatine is composed of animal protein derived from the collagen obtained by acid or alkaline extraction from pigskins, cowhides, or bones. This additive is used as foaming and stabilizing agent in the foam structure of mousses and other aerated creams, and also as gelling agent for puddings (Rapaille and Vanhemelrijck, 1998).

2.3  Preparation of Probiotic Strains for Incorporation into Refrigerated Dairy Desserts The preparation of probiotic cultures may have a significant impact on a successful introduction of these microorganisms in a food product (Champagne et al., 2005). Even though a considerable proportion of the commercial probiotic cultures is available in the freeze-dried form as a direct vat set type culture for direct addition to the products, most studies on dairy desserts report strain activation prior to adding it to the product (Helland et al., 2004; Buriti et al., 2007; Corrêa et al., 2008; Magariños et al., 2008). Despite the fact that the food matrix itself has growth factors for probiotic bacteria, including, for example, sucrose, available proteins, and peptides, at refrigeration temperatures the probiotic metabolism is reduced. Moreover, the growth factor availability is limited during shelf life, when compared to their availability during

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fermentation, for example. Therefore, the microorganism activation is an important step during the production of a dessert. Moreover, it is important for probiotic bacteria to be inoculated in levels which are enough for providing health benefits to the consumers. This requires inoculation level of 7-8 log cfu/g because possible losses during shelf life should be forecasted. Helland et al. (2004) employed Lactobacillus acidophilus La-5, Bifidobacterium animalis Bb12, L. acidophilus NCIMB 701748 (1748), and Lactobacillus rhamnosus GG strains for the production of pudding. For this purpose, each microorganism was cultivated at 1% for 2 days in MRS medium, with incubation at 37 °C. Cell concentrates were obtained by centrifugation of this fermented medium, and cell pellets were washed with a potassium phosphate 0.05 M (pH 7.0) solution. After centrifugation, the pellets were resuspended in 100 mL of Ringers solution with 10% sucrose and stored at –80 °C. After cooling (37 °C) the cooked (>90 °C/20 min) and sterilized (121 °C/15 min) pudding mixtures, isolated or combined cultures were inoculated to obtain initial concentrations of 7 log cfu/g. The pudding mixtures were then incubated at 37 °C for 12 h, cooled and stored at temperatures around 5 °C. After this 12-h fermentation, increased probiotic microorganisms populations were observed, ranging from 8 to 9.1 log cfu/g in the milk-based puddings. For the production of milk-based mousses with fruit juice or pulp, Buriti et al. (2007) employed 20 mL of heat-treated milk for fermentation of a L. acidophilus La-5 culture (0.1-0.2 g culture/kg of product) at 37 °C for 150 min. The culture was inoculated in the mixture employed for mousse preparation, following pasteurizing and cooling at 40 °C. The probiotic populations in the final product (day 1) ranged from 6.5 to 7 log cfu/g. Corrêa et al. (2008) employed Bifidobacterium lactis BL-04 or Lactobacillus paracasei subsp. paracasei LBC 82 (0.05 g each), inoculated into 20 mL of milk, kept at 37 °C for 120 min for individual or co-culture addition, while preparing coconut flan, reaching populations up to 6-7 log cfu/g in the final product stored at 5 °C. Magariños et al. (2008) employed Lactobacillus casei Shirota and B. animalis Bb12 in the production of milk-based desserts. Cultures were individually inoculated (2 g of each) to 60 mL of milk containing 0.05% l-Cys-HCl, 2% glucose, and 1% yeast extract. B. animalis Bb12 and L. casei Shirota were incubated at 38 °C and at 32 °C, respectively, until reaching pH 5.0. The incubation time needed to achieve this pH was around 1.25 and 3.12 h, respectively, for L. casei Shirota and B. animalis Bb12. The inoculates were of 9.17 and 9.54 log cfu/g, respectively, for L. casei Shirota and B. animalis Bb12. Populations of both microorganisms were reduced to 8 log cfu/g in the final product, and maintained so during 14 storage days at 5 °C.

3  PROBIOTIC DESSERTS 3.1  General Effects of the Food Matrix on Physicochemical Characteristics and Probiotic Viability Several factors in the food matrix have been reported to affect the probiotic viability, including acidity, hydrogen peroxide, oxygen content, storage temperature, sugar concentration (osmotic stress), water activity (aw), and metabolites, among others (Champagne et al., 2011; Martín et al., 2014). The physical-chemical features of probiotic desserts usually depend on the employed strain and if this strain will be used as an isolated culture or combined with other microorganisms. Ingredients employed in the formulation may also interfere with the metabolism of the probiotic bacteria, mainly affecting the pH, as a consequence of organic acid production. In yogurt and juice, for example, the pH at the end of fermentation is considered as being the most important factor influencing the growth and viability, especially for bifidobacteria species (Champagne et al., 2011). In general, the acid exposure leads to an intracellular accumulation of protons and structural damages to the cell membrane, DNA, and proteins (Corcoran et al., 2008; Champagne et al., 2011). In this context, Helland et al. (2004) evaluated the generation of organic and volatile compounds in milk-based probiotic puddings stored at around 5 °C for 21 days. At the end of the storage period, the L. rhamnosus GG strain was the strain to produce the highest concentration of lactic acid (close to 10,000 mg/kg), citric acid (1819 mg/kg), acetoin (109.4 mg/kg), and ethanol (9.1 mg/kg). The lowest production of lactic acid at the 21st day was obtained in puddings with L. acidophilus 1748 (around 5000 mg/kg), equivalent to 50% of the content produced by L. rhamnosus GG. While inoculated separately, L. acidophilus La-5 showed the lowest citric acid production (1447 mg/kg) in milk-based puddings. The authors also observed that the use of L. acidophilus La-5 in a co-culture with B. animalis Bb12 resulted in the lowest acetoin (33.6 mg/kg) and ethanol (3.5 mg/kg) contents. Regarding volatile compounds, for all cultures used in pudding production, a reduced acetaldehyde and increased diacetyl contents were reported. Puddings produced with L. acidophilus 1748 and L. rhamnosus GG showed, respectively, the highest (above 2 mg/kg) and the lowest (below 0.5 mg/kg) acetaldehyde contents by the end of storage. On the other hand, a pudding prepared with L. rhamnosus GG showed a very high diacetyl content on the 21st day (18 mg/kg), when compared to the other formulations (between 2 and 5 mg/kg). The authors reported that the addition of

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polydextrose (6.0%) did not influence the production of the evaluated compounds and the pH reduction during the storage of puddings prepared with most of the probiotic strains tested. Aragon-Alegro et al. (2007) reported that the simultaneous addition of the prebiotic fiber inulin (5.01%) and of L. paracasei LBC 82 to chocolate mousses caused a more significant pH reduction during a 28-day period at 4 °C (from 6.21 to 5.37), when compared to control mousses (from 6.22 to 6.01) and mousses containing only L. paracasei (from 6.26 to 5.67). Coconut flan, one of the most traditional Brazilian desserts, was studied by Corrêa et al. (2008) as a vehicle for L. paracasei LBC 82 and B. lactis BL 04 strains, isolated or in co-culture. The authors reported that the use of these strains in co-culture lead to a higher pH during 28 days of storage at 5 °C (reduction from 6.8 to 6.4), when compared to the individual use of L. paracasei (reduction from 6.6 to 6.0). The study results regarding the use of probiotics in co-culture were quite similar to those observed by Magariños et al. (2008) for milk-based desserts containing B. animalis Bb12 and L. casei Shirota, with initial pH of 6.8, later reduced to 6.52 at the 21st storage day at 5 °C, as well. Food ingredients and additives that contribute for specific flavor features, appearance, and consistency are essential in milk-based desserts preparation. These ingredients and additives include sweeteners, fruit, natural and artificial colorings and flavoring agents, thickeners, stabilizers, acidifying agents, among others. These additives should not interfere with the probiotic viability during the products’ storage. Therefore, so as to achieve desirable sensorial properties during the development of a new product, it is important for the food technologist to consider the tolerance of probiotic microorganisms to the ingredient or additive which will contribute to the advantageous features of the products in which they are employed (Vinderola et al., 2002a; Buriti et al., 2007; Komatsu et al., 2008; Tripathi and Giri, 2014). The influence of several food ingredients and additives, widely used in the production of milk-based products, on the viability of probiotic strains of bifidobacteria, L. acidophilus, and of the L. casei group (L. casei, L. paracasei, and L. rhamnosus) were tested by Vinderola et al. (2002a). The authors observed that sucrose, commercial flavorings of strawberry, vanilla, and banana, besides a flavoring-coloring commercial mixture of peach, inhibited the tested cultures when used at high concentrations. Bifidobacteria strains were inhibited with 15-20% sucrose concentrations. Natural colorings, including carmine, curcuma/bixin, and bixin did not affect the growth of probiotic bacteria evaluated. Other flavoring-colorant commercial mixtures, like strawberry and vanilla, showed an important inhibition potential in concentrations normally used by the food industry, mainly for bifidobacteria and L. acidophilus. The L. acidophilus CNRZ 1881 strain was inhibited by strawberry, pineapple, and kiwi juices. Strawberry juice also inhibited Bifidobacterium longum A1 strain. However, when fruit juices were neutralized, they did not affect these strains viability. Inhibition of probiotic bacteria by high sugar concentrations is due to the adverse osmotic effect and low aw (Shah and Ravula, 2000). In fruit juices, the pH and the composition of organic acids, besides other factors, may influence the viability of probiotic bacteria (Kailasapathy et al., 2008; Nualkaekul and Charalampopoulos, 2011). According to Nualkaekul and Charalampopoulos (2011), the pH homeostasis between the intracellular pH of the lactic acid bacteria and the extracellular environment is maintained by the activity of a proton-translocating ATPase. This enzyme requires energy for the extrusion of protons from the cytoplasm. In this way, other essential cellular functions are deprived of ATP at low pH, and cell viability cannot be maintained. Additionally, the authors reported that fruit organic acids are frequently employed as preservatives for their antimicrobial properties; therefore, the probability of a negative impact on probiotic survival is high. Regarding the inhibitory effect of flavoring agents, antimicrobial activity is possibly due to the presence of essential oil, reported as capable of inducing cell lysis, and of phenolic compounds, such as eugenol, cinnamic acid, carvacrol, and thymol (Inouye et al., 2001; Gutierrez et al., 2009). However, Sagdic et al. (2012) reported that supplementation of ice-creams with pomegranate peel extract, peppermint essential oil, ellagic acid, gallic acid, or grape seed extract did not affect the survival of L. casei Shirota. Moreover, the use of ingredients rich in phenolic compounds with antioxidant capacity to improve the viability of probiotic microorganisms in food products has been reported (Marsh et al., 2014; Tripathi and Giri, 2014). In a study conducted with milk-based mousses (Buriti et al., 2007), the addition of passion fruit as concentrated juice or pasteurized frozen pulp reduced L. acidophilus La-5 viability in 4.7 log cycles in 21 days of refrigerated storage at 4 °C. On the other hand, the reduction of the viability of the same microorganism was only of 1 log cycle with the addition of pasteurized frozen guava to the refrigerated mousses studied in the same period. The fruits’ acid effect on the L. acidophilus viability in that study was discarded, differently from what was observed by Vinderola et al. (2002a) because acceptable values of the probiotic population were maintained (above 6 log cfu/g), even with the addition of lactic acid to mousses produced with guava pulp. Hence, the behavior variation among the L. acidophilus strain employed observed for each mousse formulation was attributed to the different compounds present in the two fruits tested. Oxygen sensitivity is also considered as an important problem in the production and storage of probiotic foods, particularly for highly aerated products containing bifidobacteria (Bolduc et al., 2006; Kawasaki et al., 2006). This is in part due to the anaerobic or microaerophilic nature of these microorganisms lacking effective oxygen scavenging cellular mechanisms

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such as catalase production. Toxic oxygen metabolites may accumulate in the cell leading to cell death from oxidative damage (Talwalkar and Kailasapathy, 2004b). As a result, a loss in probiotic viability during production and storage, and a detrimental survival throughout the GIT might take place (Grosso and Fávaro-Trindade, 2004; Kawasaki et al., 2006). To protect probiotic bacteria from the deleterious effects of oxygen toxicity, many strategies have been evaluated and shown to be effective in dairy products. Some methods reported recommend the use of special high oxygen consuming strains, of ascorbic acid as an oxygen scavenger in specific products, of cysteine as a redox-potential reducing agent, of microencapsulation, besides the use of packaging material less permeable to oxygen, and oxidative stress adaptation (Hsiao et al., 2004; Talwalkar and Kailasapathy, 2004b; Bolduc et al., 2006; Güler-Akın and Akın, 2007; Martín et al., 2014). Microencapsulation, for example, has been proven to be one the most effective methods for maintaining probiotic viability because it protects probiotic microorganisms during food processing and storage, as well as toward gastric conditions. Besides the polysaccharides traditionally used as matrix in microencapsulation, new materials are being tested (Martín et al., 2014). Castro-Cislaghi et al. (2012) verified that whey is a promising encapsulating agent for B. animalis Bb-12. When the microcapsules were added to a commercial dairy dessert, the authors verified that the probiotic population remained above 7 log cfu/g for 6 weeks.

3.2  Interactions Among Probiotic Microorganisms During Storage Different combinations of strains allow the production of dairy products with target technological features and potential nutritional and health benefits. However, microbial interactions, either beneficial (protocooperation) or unfavorable (antagonism) among these cultures, may generate undesirable changes in the composition of the bacterial microbiota during the manufacture and cold storage of these products (Vinderola et al., 2002b, 2008). Thus, adequate combinations of probiotic strains should be tested specifically for the product to be used as a vehicle for this combination of microorganisms, as well as the proportion among the different strains should be evaluated during all steps, since its preparation until the end of the storage period (Tamime et al., 2005; Komatsu et al., 2008). In a study with coconut flan, Corrêa et al. (2008) observed that the average B. lactis populations, when used separately or in co-culture with L. paracasei, were always maintained above 7.1 log cfu/g during a 28-day storage period. The authors observed a significant variation in B. lactis populations, whether or not in the presence of L. paracasei (p 85% of total bacteria)] and “potential pathogenic bacteria” (Purchiaroni et al., 2013). The GI tract can separate intraluminal bacteria and their products from the internal milieu of the human body, which is referred to as “gut barrier function.” Treatment of GI cancer often induces changes in intestinal microflora and results in attenuation of the gut barrier function through various factors and mechanisms including decreased motility of the GI tract, atrophy of the GI mucosa by malnutrition, perioperative use of antibiotics, suppression of gut immunity by surgical stress, and injury of the GI mucosa by adjuvant therapy (Bengmark, 2012; Deitch and Bridges, 1987). Changes in the composition of normal intestinal microflora and disruption of gut barrier function can promote “bacterial translocation,” a process by which intraluminal bacteria or their components, such as endotoxin and peptidoglycans, traverse the intestinal epithelium to distant sites. This may often predispose patients to systemic inflammation and septic complications during the postoperative period (Marshall et al, 1993; MacFie et al., 1999; MacFie, 1997). The recent trend of perioperative administration of probiotics, prebiotics, and synbiotics is expected to reduce postoperative infectious morbidity in patients who undergo GI surgery, as these agents are expected to help maintain the normal intestinal microflora as well as the normal gut barrier function (Lundell, 2011; Kinross et al., 2013). In addition to the above-mentioned prophylactic benefits against postoperative infections, several groups have expected other potentially beneficial outcomes from probiotics/synbiotics, such as providing anticarcinogenic action and/or gutprotective activity during adjuvant therapy in patients who undergo GI cancer surgery (Peitsidou et al., 2012).

1  PREVENTION OF INFECTIOUS COMPLICATIONS AFTER GI CANCER SURGERY 1.1  Upper GI Surgery Esophagectomy with extended lymphadenectomy and gastric tube reconstruction is the standard surgical procedure for thoracic esophageal cancer and is considered to be one of the most invasive procedures among many types of GI cancer surgeries (Nagawa et al., 1994; Tsujinaka et al., 1990). Patients who undergo esophageal cancer surgery are more likely to develop systemic inflammatory response syndrome (SIRS), which is characterized by excessive production of proinflammatory cytokines [e.g., tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and IL-6], compared to those who undergo other types of surgery for GI cancer. It is well known that SIRS is associated with the development of serious postoperative morbidities, such as multiple organ failure (Sakamoto et al., 1994). In addition, several conditions that may occur before, during, or after esophageal resection may change the balance of intestinal microflora and result in intestinal barrier dysfunction. These include cancer-bearing status, malnutrition due to malignant obstruction, preoperative chemotherapy and radiotherapy (Wada et al., 2000), use of antibiotics (Nieuwenhuijs et al., 1998), long-term parenteral nutrition (Deitch et al., 1995), operative stress (Bengmark, 1992), and reduction of gastric juice and intestinal motility disorders associated with total vagotomy (Forssell et al. 1988; Gao et al., 2010). To date, there have been only two reports that have evaluated the clinical value of synbiotics in esophageal cancer surgery (Table 38.1). Tanaka et al. investigated the effects of pre- and postoperative administration of synbiotics on intestinal Probiotics, Prebiotics, and Synbiotics. http://dx.doi.org/10.1016/B978-0-12-802189-7.00038-1 © 2016 Elsevier Inc. All rights reserved.

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540  PART | III  Synbiotics: Production, Application, and Health Promotion

TABLE 38.1  Clinical Studies Using Probiotics/Synbiotics in Upper GI Surgery Bacteria, type, and dose

Duration of administration

Microflora/bacterial translocation/ inflammatory response

Clinical benefits

Author (year)

Patients (number)

Groups (number)

Tanaka et al. (2012)

Esophageal cancer (64)

Synbiotics (30) Control (34)

B. breve Yakult 2 × 108 L. casei Shirota 2 × 108

Preop 7 days Postop 21 days

Significantly more beneficial bacteria, less harmful bacteria, and higher total organic acid and acetic acid in feces Significantly shorter duration of SIRS

No differences in postop complications Better abdominal symptoms associated with enteral nutrition in the synbiotics group

Yokoyama et al. (2014)

Esophageal cancer (42)

Synbiotics (21) Control (21)

B. breve Yakult (preop 1 × 1010, postop 3 × 108) L. casei Shirota (preop 4 × 1010, postop 3 × 108)

Preop 7 days Postop 14 days

Significantly lower incidence of bacteria in the MLNs and blood

No differences in infections and related complications

Woodard et al. (2009)

Gastric bypass (41)

Probiotics (64) Placebo (65)

Lb. species 108

Postop 6 months

Reduced bacterial overgrowth and higher vitamin B12 levels in the probiotic group

Greater weight loss in the probiotic group

Preop, preoperative and postop, postoperative.

microflora and surgical outcome in patients with esophageal cancer (Tanaka et al., 2012). In their study, 70 patients were randomly allocated to two groups: one group received Bifidobacterium breve strain Yakult and Lactobacillus casei strain Shirota as well as galacto-oligosaccharides for 7 days before surgery and for 3 weeks after surgery through a feeding catheter placed during surgery, while the second group did not. Of the 70 patients, 64 completed the trial (synbiotics, 30; control, 34). The counts of beneficial bacteria and pathogenic bacteria on postoperative day (POD) 7 were significantly larger and smaller, respectively, in the synbiotics group compared with the control group. Furthermore, the concentrations of total organic acid and acetic acid were higher in the synbiotics group than in the control group (P