Probiotics and Health Claims

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Probiotics and Health Claims

Probiotics and Health Claims Edited by Wolfgang Kneifel and Seppo Salminen © 2011 Blackwell Publishing Ltd. ISBN: 978-1-405-19491-4

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Probiotics and Health Claims Edited by

Wolfgang Kneifel and Seppo Salminen

A John Wiley & Sons, Ltd., Publication

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This edition first published 2011 © 2011 Blackwell Publishing Ltd. Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing programme has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell. The right of the authors to be identified as the authors of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Probiotics and health claims / edited by Wolfgang Kneifel, Seppo Salminen. p. cm. Includes bibliographical references and index. ISBN 978-1-4051-9491-4 (hardback : alk. paper) 1. Probiotics–Health aspects. 2. Probiotics–Law and legislation. I. Kneifel, Wolfgang. II. Salminen, Seppo. [DNLM: 1. Probiotics–therapeutic use. 2. Food Industry–legislation & jurisprudence. QU 145.5 P9214 2011] RM666.P835P78 2011 615′.329—dc22 2010020489 A catalogue record for this book is available from the British Library. This book is published in the following electronic formats: ePDF (9781444329391); Wiley Online Library (9781444329384); ePub (9781444329407) Set in 10/12pt Times by SPi Publisher Services, Pondicherry, India

1

2011

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Contents

Preface Contributors 1 Probiotics and Health: From History to Future Barry R. Goldin 1.1 Early history of the use of microorganisms for human benefit 1.2 Overview of probiotic studies and results for the past 35 years 1.3 Current evidence for probiotic health benefits 1.3.1 Lactose intolerance 1.3.2 Inflammatory bowel disease 1.3.3 Treatment of gastroenteritis 1.3.4 Cholesterol lowering 1.3.5 Treatment for urogenital infections 1.3.6 Treatment of allergic reactions 1.3.7 Prevention of dental caries 1.3.8 Treatment and prevention of cancer by probiotics 1.3.9 Additional health benefits attributed to probiotics 1.3.10 Conclusions based on past and present use of probiotics for health applications 1.4 Nutritional effects of probiotics 1.5 Future development and uses of probiotics for health application 1.5.1 Probiotics as a platform for delivery of drugs, enzymes, hormones, nutrients and micronutrients 1.5.2 Toxin sequestration 1.5.3 Carcinogen detoxification 1.5.4 Antibody production 1.5.5 Treatment for enzyme deficiencies 1.5.6 Other potential future directions for probiotics for medical use 1.6 Conclusions

xiv xv 1 1 2 2 2 3 4 6 6 6 7 7 8 8 9 9 10 10 11 11 11 13 13

2 The World’s Oldest Probiotic: Perspectives for Health Claims Tomoyuki Sako

17

2.1 From theory to practice: the challenge of Dr Minoru Shirota 2.1.1 The discovery of Lactobacillus casei strain Shirota

17 17

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2.2

2.3 2.4 2.5

2.1.2 Early studies in Japan: the first clinical study era for Yakult and L. casei Shirota 2.1.3 Probiotic definition and the L. casei Shirota strain Health benefits of Yakult and L. casei Shirota 2.2.1 Identification and characterisation of L. casei Shirota 2.2.2 Beneficial modulation of the intestinal microbiota 2.2.3 Improvement of stool consistency 2.2.4 Protection from infection 2.2.5 Immune modulation activity 2.2.6 Prophylactic effect of L. casei Shirota on cancer development Safety Health claims for L. casei Shirota and the product Yakult Current perspectives

3 Probiotics: from Strain to Product Arthur C. Ouwehand, Lisbeth Søndberg Svendsen and Gregory Leyer 3.1 3.2 3.3 3.4

Introduction Isolating a potential probiotic strain Producing probiotic strains on a large scale Producing products containing probiotics 3.4.1 Fermented milk products 3.4.2 Cheese 3.4.3 Non-fermented milk drinks 3.4.4 Fruit and vegetable juices 3.4.5 Dried products 3.5 Probiotic products and feeding trials 3.6 Conclusion 4 Probiotics and Health Claims: Challenges for Tailoring their Efficacy Christophe Chassard, Franck Grattepanche and Christophe Lacroix 4.1 Introduction 4.2 Current selection of probiotics: setting the scene for tailoring probiotics 4.2.1 Safety considerations 4.2.2 Technological considerations 4.2.3 Functionality and health benefits 4.3 Improving the assessment of probiosis 4.3.1 In vitro models for the assessment of probiosis 4.3.2 In vivo models for the assessment of probiosis 4.3.3 Clinical trials for the assessment of probiosis 4.4 Improving the discovery of probiotic strains 4.4.1 Exploring and isolating bacterial diversity 4.4.2 New generations of probiotics from new bacterial genera and with new targeted functions 4.5 Improving probiotic specificity 4.5.1 Future therapeutic strategies: combination of strains? 4.5.2 Nutritional manipulation

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4.5.3 Genetic engineering 4.6 Conclusions 5 Probiotics: from Origin to Labeling from a European and Brazilian Perspective Célia Lucia Ferreira, Marcos Magalhães, Miguel Gueimonde and Seppo Salminen 5.1 5.2 5.3 5.4 5.5 5.6

Introduction Terminology and probiotics Health claim regulation in the European Union Health claims in Europe Health claim regulation in Brazil Defining health claims 5.6.1 Characterization of probiotic bacteria 5.6.2 Safety assessment 5.6.3 Human intervention studies for health claims 5.6.4 Totality of supporting evidence 5.7 Specific challenges for probiotics 5.7.1 Viability 5.7.2 Clinical studies demonstrating efficacy of probiotics in healthy subjects 5.7.3 Challenges in regulatory areas 6 Substantiating Health Benefit Claims for Probiotics in the United States Mary Ellen Sanders 6.1 Introduction 6.1.1 Probiotics and health benefits 6.1.2 Probiotics: a term often misused 6.2 Health benefit claims allowable in the United States 6.2.1 FDA and FTC standards 6.2.2 Structure/function claims 6.2.3 Health claims 6.2.4 Medical food claims 6.3 Substantiation of health benefit claims for probiotics 6.3.1 Overriding considerations 6.3.2 Specific issues related to human efficacy studies 6.3.3 Key considerations for probiotic efficacy substantiation 6.4 Bridging the gap between the US consumer, probiotic science and commercial products 6.5 Conclusions 7 Health Claims and Dietary Guidance in the United States: Case “Reduced Cardiovascular Disease Risk” Alice H. Lichtenstein 7.1 Introduction 7.2 Types of health claims

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66 67

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75 76 76 76 77 78 79 81 82 83 84 84 84 85 88 88 88 89 90 90 90 91 92 92 92 96 97 97 98

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7.3

7.4

7.5 8

10

103 103 103 104 104 106 106 106 107 108 109 110 111 112 114 114 114 116

Probiotics and Health Claims: a Japanese Perspective Fang He and Yoshimi Benno

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8.1 8.2

118 119 120 121 122 124 124

8.3 9

7.2.1 Definition 7.2.2 Authorized health claims 7.2.3 Qualified health claims 7.2.4 Structure/function claims 7.2.5 Nutrient content claims Legislation governing US health claims 7.3.1 Nutrition Labeling and Education Act (NLEA 1990) 7.3.2 Food and Drug Administration Modernization Act (FDAMA 1997) 7.3.3 Consumer Health Information for Better Nutrition Initiative (2003) Dietary guidance to reduce cardiovascular disease risk 7.4.1 Dietary Guidelines for Americans 7.4.2 National Cholesterol Education Program 7.4.3 Dietary Reference Intakes 7.4.4 American Heart Association 7.4.5 American Diabetes Association 7.4.6 American Cancer Society 7.4.7 Case study: evolution of Dietary Guidelines for Americans Current challenges

Introduction FOSHU health claims 8.2.1 History of FOSHU 8.2.2 Specifics of FOSHU health claims 8.2.3 Procedure for obtainining permission for FOSHU 8.2.4 FOSHU health claim for probiotics: gastrointestinal conditions Non-FOSHU health claims for probiotics in Japan

Regulation of Probiotics in China Anu Lahteenmäki-Uutela

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9.1 9.2 9.3 9.4

126 126 127 131

Introduction Health food or medicine? Health food regulations Novel food regulation

Probiotics and Health Claims: an Indian Perspective Jashbhai B. Prajapati and Nagendra P. Shah

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10.1 The background 10.2 The status 10.3 Animal studies 10.3.1 Chicken 10.3.2 Albino rats 10.3.3 Pigs

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10.4

10.5 11

10.3.4 Sheep 10.3.5 Calves 10.3.6 Fish 10.3.7 Post-larvae Human studies 10.4.1 Probiotics in gut microbiology 10.4.2 Probiotics in diarrheal diseases 10.4.3 Effects on lipid profile 10.4.4 Morbidity and nutritional status An Indian perspective on regulation of probiotics

The Role of Meta-analysis in the Evaluation of Clinical Trials on Probiotics Hania Szajewska 11.1 11.2 11.3

Introduction What is a systematic review? What is a meta-analysis? How to conduct a systematic review 11.3.1 Formulation of the review question (the problem) 11.3.2 Searching 11.3.3 Selecting studies and collecting data 11.3.4 Assessment of methodological quality (i.e. the risk of bias in included trials) 11.3.5 Analysing the data and presenting the results 11.4 Why perform a meta-analysis? 11.5 Heterogeneity 11.6 How to interpret a forest plot 11.7 Critical appraisal of a systematic review 11.8 Published meta-analyses on the effects of probiotics 11.8.1 Acute gastroenteritis 11.8.2 Antibiotic-associated diarrhea 11.8.3 Clostridium difficile-associated diarrhea 11.8.4 Traveler’s diarrhea 11.8.5 Necrotizing enterocolitis 11.8.6 Helicobacter pylori infection 11.8.7 Functional gastrointestinal disorders 11.8.8 Irritable bowel syndrome 11.8.9 Inflammatory bowel disease 11.8.10 Functional constipation 11.8.11 Allergy prevention 11.8.12 Respiratory tract infections 11.9 Is a meta-analytical approach appropriate for assessing the efficacy of probiotics? 11.9.1 Arguments for pooling data 11.9.2 Arguments against pooling data 11.10 What could be the solution? 11.11 Unpublished data 11.12 Quality of included trials

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139 139 140 140 141 141 142 143 144 145

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11.13 Inconclusive systematic reviews and meta-analyses 11.14 Opposite conclusions 11.15 Summary and key messages

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Applied Studies with Probiotics: Fundamentals for Meeting the Health Claims Hannu Mykkänen, Silvia W. Gratz and Hani El-Nezami

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12.1 12.2 12.3 12.4 12.5

13

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Probiotics Research: the Pediatric Perspective Karl Zwiauer

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13.1 13.2 13.3

178 178 181 181 182 183 184 184 185 187 188 190 192 192 193 193 194

13.4

13.5 14

Introduction Mycotoxin problem Lactobacillus rhamnosus strain effectively binds aflatoxin: in vitro findings Animal models for studying the aflatoxin–probiotic interaction Field studies with Lactobacillus rhamnosus strain in aflatoxin-exposed populations

Introduction Development of the gastrointestinal flora postnatally Probiotics in infant nutrition 13.3.1 Growth of healthy infants 13.3.2 Probiotics in preterm infants 13.3.3 Safety concerns Clinical effect of probiotics in children 13.4.1 Prevention of allergic disease: food hypersensitivity 13.4.2 Atopic dermatitis 13.4.3 Prevention of antibiotic-associated diarrhea 13.4.4 Acute gastroenteritis and community-acquired diarrhea 13.4.5 Irritable bowel syndrome and constipation 13.4.6 Infantile colic 13.4.7 Inflammatory bowel disease 13.4.8 Oral health effects: caries 13.4.9 Other clinical conditions Summary and key messages

Probiotics and Health Claims Related to OTC Products and Pharmaceutical Preparations Frank M. Unger and Helmut Viernstein 14.1 14.2 14.3

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Introduction Production, processing and formulation of probiotic cultures for pharmaceutical purposes Clinical studies 14.3.1 Gastroenterology 14.3.2 Gynecology 14.3.3 Dentistry/stomatology

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14.4 Evaluation and outlook 14.4.1 Antibiotic-associated diarrhea and Clostridium difficile disease 14.4.2 Traveler’s diarrhea 14.4.3 Helicobacter pylori infection 14.4.4 Lactose intolerance 14.4.5 Irritable bowel syndrome 14.4.6 Ulcerative colitis 14.4.7 Pouchitis 14.4.8 Crohn’s disease 14.4.9 Bacterial vaginosis 14.4.10 Gingivitis, reduction of plaque and alleviation of gum bleeding 14.4.11 Selected experimental approaches to probiotic products with new properties and in new indications

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Probiotics and Health Claims: the Perspective of the Feed Industry Anja Meieregger, Elisabeth Mayrhuber and Hans Peter Lettner

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15.1 Introduction and history 15.2 Feed probiotics versus food probiotics 15.2.1 Gram-positive non-sporulating bacteria 15.2.2 Bacillus species 15.2.3 Yeasts 15.2.4 Filamentous fungi 15.3 Efficacy 15.4 Feed probiotics 15.4.1 Fundamentals 15.4.2 Industrial production 15.5 Authorisation processes 15.6 Probiotics as performance enhancers: conclusions

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Developing LGG®Extra, a Probiotic Multispecies Combination Maija Saxelin, Eveliina Myllyluoma and Riitta Korpela

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16.1 Introduction 16.2 Strain selection 16.3 Probiotic characteristics of the strains 16.3.1 Gastrointestinal persistence and colonisation 16.3.2 Influence on human intestinal microbiota 16.3.3 Immunological effects in vitro 16.3.4 Potential for reducing dietary toxins 16.3.5 Safety aspects 16.4 Clinical studies on the probiotic multispecies LGG®Extra combination 16.4.1 Relieving symptoms of irritable bowel syndrome 16.4.2 Eradication of Helicobacter pylori and Candida 16.4.3 Other research areas 16.5 Conclusions

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Probiotics and Health Claims: How to Be Met by SMEs? Miguel Gueimonde and Sampo J. Lahtinen

263

17.1 17.2 17.3 17.4

263 265 267

18

19

20

Introduction Developing proprietary probiotic strains Probiotic research by SMEs using strains from larger companies Example of successful probiotic research program by an SME company: the development of probiotic strains Bifidobacterium longum 46 and B. longum 2C

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Probiotic Products: How Can They Meet the Requirements? Wolfgang Kneifel

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18.1 Introduction 18.2 Quality criteria of probiotics 18.2.1 Basic composition and nutrient profile 18.2.2 Nature, identity and safety of probiotic strains 18.2.3 Viability and probiotic viable count 18.3 Future perspectives

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Probiotics and Health Claims: Hurdles for New Applications? Lorenzo Morelli

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19.1 Introduction 19.2 Identifying the hurdles 19.2.1 Characterisation 19.2.2 Relationship to health 19.2.3 Scientific substantiation 19.3 Approaching the hurdles 19.3.1 Hurdle characterisation 19.3.2 Relationship to health 19.3.3 Scientific substantiation 19.4 New perspectives 19.4.1 General considerations 19.4.2 Functional genomics 19.5 Conclusions

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Probiotics and Innovation Jean-Michel Antoine, Jean-Michel Faurie, Raish Oozeer, Johan van Hylckama Vlieg, Jan Knol, Herwig Bachmann and Joël Doré

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20.1 Introduction 20.1.1 Early history 20.1.2 Recent history 20.2 Not all probiotics are the same: genomic perspective 20.3 Not all probiotic foods are the same: functional perspective 20.4 Not all probiotics are cross-talking in the same way: dialogue with the host

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20.4.1 Dialogue with the human intestinal microbiota: a logical trigger for innovation 20.4.2 Novel functional targets for the human intestinal microbiota 20.5 European regulatory perspective: a threat or an opportunity? 20.5.1 European regulatory perspective: a threat? 20.5.2 For innovation in probiotics, the present regulatory requirements are an opportunity 20.6 Conclusion Index

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Preface

Probiotics have a long history in nutrition and medicine, but their health benefits have been demonstrated only more recently, when proven standards of clinical assessment have been applied. These findings have contributed to the scientific basis for the establishment of health claims associated with some products. Concomitantly, the need for objective regulation of these claims has arisen. Today, health claim regulations are subject to intensive discussions in the public as well as by experts, and new legislative developments have been implemented in the European Union, the United States, Australia and New Zealand, China and Japan. Moreover, the Codex Alimentarius organization is still working on guidelines for global health claim definitions and assessment. Probiotic microorganisms as well as probiotic products can be regarded as the most prominent pacemakers in the area of functional foods and have always been important components providing demonstrated health benefits for various parts of the population. This development has enormously stimulated targeted research in the area of food and feed as well as in medical and pharmaceutical science. Historical developments, from classical food fermentation to today’s highly defined areas of functional foods and even clinical foods, have had a major impact on nutritional and adjuvant therapy in many gastrointestinal-associated diseases and their risk reduction. Moreover, in addition to preventive measures, new fields of probiotic applications have gradually emerged worldwide during the last few years. Thus, it is important to illuminate and to evaluate the differences in health claim requirements and assessment procedures in major global market areas by the help of experts in various but cooperating disciplines. The information collected in this book covers different scientific areas and viewpoints and will furnish food developers and scientists involved in the work on food, health and nutrition with current multidisciplinary expertise in this field. It is also intended to be used by researchers, consulting experts and regulators who need to compare the systems and guidance used in different parts of the world. The readership may also include nutrition professionals, physicians and teachers. Additionally, the contents have been designed to be valuable not only for food science but also for students in human and animal nutrition and microbiology as well as those studying pharmaceutical sciences and drug development. The chapters are written by renowned experts and will comprise a compendium on most up-to-date developments and associated requirements as well as assessment procedures. This enables the reader to develop probiotics and new probiotic research programmes for characterizing new strains, verifying health claims and understanding the food and health relationships with specific focus on probiotics. Wolfgang Kneifel and Seppo Salminen Vienna and Turku

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Contributors

Jean-Michel Antoine Danone Research RD 128 Palaiseau Cedex, France Herwig Bachmann Vrije Universiteit Amsterdam Systems Bioinformatics IBIVU Amsterdam, The Netherlands Yoshimi Benno Benno Laboratory, Riken, Wako Saitama, Japan Christophe Chassard ETH Zurich Laboratory of Food Biotechnology Institute of Food, Nutrition and Health Zürich, Switzerland Joël Doré INRA Domaine de Vilvert Jouy en Josas, France

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Célia Lucia Ferreira Federal University of Viçosa, Viçosa, Minas Gerais, Brazil Barry R. Goldin Department of Public Health and Community Medicine Tufts University School of Medicine Boston, Massachussetts, USA Franck Grattepanche ETH Zurich Laboratory of Food Biotechnology Institute of Food, Nutrition and Health Zürich, Switzerland Silvia W. Gratz Rowett Institute of Nutrition and Health University of Aberdeen Aberdeen, UK

Hani El-Nezami School of Biological Sciences University of Hong Kong Pokfulam, Hong Kong, China

Miguel Gueimonde Instituto de Productos Lácteos de Asturias Consejo Superior de Investigaciones Científicas Villaviciosa Asturias, Spain

Jean-Michel Faurie Danone Research RD 128 Palaiseau Cedex, France

Fang He Technical Research Laboratory Takanashi Milk Products Co., Ltd Yokohama, Kanagawa, Japan

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Contributors

Wolfgang Kneifel Department of Food Science and Technology Boku – University of Natural Resources and Life Sciences Vienna Vienna, Austria

Elisabeth Mayrhuber Lactosan GmbH & Co. KG Kapfenberg, Austria

Jan Knol Danone Research Wageningen, The Netherlands

Lorenzo Morelli Istituto di Microbiologia Università Cattolica del Sacro Cuore Piacenza, Italy

Riitta Korpela University of Helsinki Institute of Biomedicine, Pharmacology, University of Helsinki, Finland Christophe Lacroix ETH Zurich Laboratory of Food Biotechnology Institute of Food, Nutrition and Health Zürich, Switzerland Anu Lahteenmäki-Uutela Turku School of Economics Turku, Finland Sampo J. Lahtinen Danisco Oyj Kantvik, Finland Hans Peter Lettner Lactosan GmbH & Co. KG Kapfenberg, Austria Gregory Leyer Danisco USA Madison, Wisconsin, USA

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Anja Meieregger Lactosan GmbH & Co. KG Kapfenberg, Austria

Hannu Mykkänen School of Public Health and Clinical Nutrition Department of Clinical Nutrition/ETTK University of Kuopio Kuopio, Finland Eveliina Myllyluoma Valio Ltd, Research and Development Helsinki, Finland Also: University of Helsinki, Institute of Biomedicine, Pharmacology University of Helsinki, Finland Raish Oozeer Danone Research RD 128 Palaiseau Cedex, France Arthur C. Ouwehand Health & Nutrition Danisco Sweeteners Kantvik, Finland

Alice H. Lichtenstein Friedman School, Tufts University Boston, Massachussetts, USA

Jashbhai B. Prajapati Department of Dairy Microbiology SMC College of Dairy Science Anand Agricultural University Anand, Gujarat, India

Marcos Magalhães Federal University of Viçosa, Viçosa, Minas Gerais, Brazil

Tomoyuki Sako Yakult Europe BV Almere, The Netherlands

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Contributors

Seppo Salminen Functional Foods Forum University of Turku Turku, Finland Mary Ellen Sanders Dairy & Food Culture Technologies Centennial, Colorado, USA Maija Saxelin Valio Ltd, Research and Development Helsinki, Finland Present address: Kiesikuja, Vantaa, Finland Nagendra P. Shah Faculty of Health Engineering and Science Victoria University, Werribee Campus Melbourne, Victoria, Australia Lisbeth Søndberg Svendsen Danisco A/S Brabrand, Denmark

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Hania Szajewska Department of Paediatrics The Medical University of Warsaw Warsaw, Poland Frank M. Unger Department of Pharmaceutical Technology & Biopharmaceutics University of Vienna Vienna, Austria Johan van Hylckama Vlieg Danone Research RD 128 Palaiseau Cedex, France Helmut Viernstein Department of Pharmaceutical Technology & Biopharmaceutics University of Vienna Vienna, Austria Karl Zwiauer Department of Paediatrics Landesklinikum St Pölten St Pölten, Austria

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Probiotics and Health: From History to Future

Barry R. Goldin

1.1

EARLY HISTORY OF THE USE OF MICROORGANISMS FOR HUMAN BENEFIT

There is evidence from wall carvings that cultured milk products were made at least 4500 years ago. Written evidence for fermented milks appears in Genesis 18: 8, “He then brought some curds and milk that had been prepared and set these before them”. The production of wine is referred to in Genesis 9: 20, “Noah a man of the soil, proceeded to plant a vineyard, where he drank some of its wine, he became drunk and lay uncovered inside his tent”. In Exodus 12: 39 the use of microorganisms to prepare bread is cited: “They baked the dough which they had brought out of Egypt into cakes of unleavened bread. For it had not become leavened, since they were driven out of Egypt and could not delay.” The exodus from Egypt is believed to have occurred approximately in 1440 bc. Homer in the Iliad, written between 900 and 800 bc makes numerous references to wine and cheese. In book 11 of the Iliad there is the following passage: “Pours a large portion of Pramnian wine; with goats milk cheese a flavourous taste bestows, and last with flour the smiling surface stows”. The ancient production of wine, cheese and bread served a number of useful purposes. It altered the flavor and texture of the natural foods and in the case of milk products extended the time of edible use by preventing rapid spoilage by random bacterial or fungal growth. In the case of wine, in addition to its pleasurable mind-altering properties, wine was used as an anesthetic. In a 10th-century Persian work, the Shahnameh, the use of wine is described for performing Caesarean sections. In India wine was used as an anesthetic by the surgeon Sushruta around 600 bc. Therefore a long history exists for the use of microorganisms to benefit the human condition. In more recent times an early reference to the use of microorganisms for a specific medical condition was proposed by Doderlein (1892), in which year he proposed to treat vaginal infections with lactobacilli. In 1900 Henry Tissier at the Pasteur Institute isolated a Bifidobacterium from a breast-fed infant (Tissier, 1905). This bacterium is now designated Bifidobacterium bifidus. Tissier also showed that bifidobacteria are the predominant organism found in breast-fed infant feces and recommended administering this organism to infants with diarrhea. In 1907 the use of a specific class of microorganisms to benefit human health was introduced to the general public by the Nobel Prize winner Elie Metchnikoff. In his book The Prolongation of Life (1907), Metchnikoff stated his belief


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that bacteria in the colon were responsible for adverse health in adults and that consuming sour milk or yogurt would counteract these harmful bacteria. He proposed that the strain “Bulgaricus Bacillus”, later named Lactobacillus bulgaricus, was the strain responsible for conferring better health and longer life in humans. In 1911 Douglas published The Bacillus of Long Life, which supported the concept of human longevity and the consumption of fermented milk. In 1917 Alfred Nissle isolated an Escherichia coli that he used to treat acute intestinal diseases such as salmonellosis and shigellosis, with a significant success rate. This organism is now designated E. coli Nissle and is still used as a probiotic and is an example of a non-lactic acid bacteria probiotic. In 1935, Retteger at Yale University proposed that Lactobacillus acidophilus would be an appropriate species to use for human clinical trials (Retteger et al., 1935). This approach was followed by a study demonstrating positive results for patients with chronic constipation. The use of specific bacteria for human disorders dates to the 1920s but the term “probiotic” was not used in this context until 1974. Parker (1974) described probiotics as “organisms and substances, which contribute to intestinal microbial balance”. In 2002 a European Expert Committee (FAO, 2006) defined probiotics as “living microorganisms, which upon ingestion in adequate amounts exert health benefits beyond inherent general nutrition”.

1.2

OVERVIEW OF PROBIOTIC STUDIES AND RESULTS FOR THE PAST 35 YEARS

Based on the definitions for a probiotic expressed in 1974 and modified in 2002, a significant number of microorganisms have been isolated and identified as probiotics. Some of these probiotics have been fed to humans and animals to test, treat or prevent various diseases, disorders and syndromes. The approximate number of different bacterial strains in each genera that have been attributed as probiotics are as follows: Lactobacillus, 23; Bifidobacterium, 5; E. coli, 2; and one strain each of Bacillus, Streptococcus, Enterococcus and Lactococcus. In addition there is one yeast, namely Saccharomyces boulardii, that has probiotic attributes (Sanders, 2007). The list is increasing yearly and as will be discussed later in this chapter propionibacteria will most certainly be added to the list of genera. With the corresponding isolation and identification of probiotic microorganisms there has been an increasing number of basic research, clinical research, clinical trial and intervention studies published. Year to year, since the mid-1980s, the number of papers has increased exponentially. It will not be possible in a chapter or a book to cover all the studies in print and therefore the following sections describe the highlights of the findings on health benefits.

1.3

CURRENT EVIDENCE FOR PROBIOTIC HEALTH BENEFITS

1.3.1

Lactose intolerance

Worldwide many millions of people experience lactose malabsorption. The frequency of the disorder increases with age. The cause for this disorder is a decline in the activity of the enzyme lactase in the intestinal brush border mucosa. This decline in activity results in lactose malabsorption. This incomplete absorption causes flatus, bloating, abdominal

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cramps, and moderate to severe diarrhea. A major consequence of this sequence of events is a severe limitation in consumption of dairy products, which is particularly pronounced in the elderly. Several studies have demonstrated that during the fermentation of milk to make yogurt lactase is produced and on consumption of yogurt this lactase is active in the intestinal tract (Kim & Gilliland, 1983; Kolars et al., 1984; Savaiano et al., 1984; de Vrese et al., 2001). The organisms used for the production of yogurt are Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Kim and Gilliland (1983) found that feeding yogurt to participants who were lactose-intolerant caused a significant reduction in the levels of hydrogen found in the breath compared with feeding milk to subjects with the same condition. The level of hydrogen in the breath reflects the intestinal microflora metabolism of lactose not absorbed in the small intestine and thus present in the colon, where the microflora are present in high concentrations. Kolars et al., (1984) found that subjects who ingested 18 g of lactose in yogurt had 67% less hydrogen in their breath compared with the same lactose dose delivered in milk. An analysis of intestinal duodenal aspirates obtained from the subjects consuming yogurt indicated that there were significant levels of lactase in the duodenum. A systematic review of the published literature in 2005 analysing studies of probiotic treatment of adult lactose intolerance concluded that the evidence does not support the effectiveness of probiotics for treatment of this disorder (Levri et al., 2005). However, the authors conclude that this may result from the variation in the nature or type of probiotic used in the specific study. For example, lactobacilli that have low levels of lactase could be potential confounder. The strains selected for yogurt production have high lactase levels, required for the efficient preparation of yogurt.

1.3.2

Inflammatory bowel disease

Inflammatory bowel disease (IBD) is a major medical problem. IBD is a general term used for intestinal inflammation, and the specific diseases and disorders that fall into the IBD category include Crohn’s disease, ulcerative colitis, and irritable bowel syndrome. One of the important potential medical applications for probiotics is the treatment and prevention of IBD relapses. There have been a limited number of reports of the beneficial effects of probiotics in treating or alleviating IBD symptoms. It has been shown that E. coli Nissle is helpful in maintaining the remission phase for patients with Crohn’s disease (Malchow, 1997). Administration of Lactobacillus salivarius in milk to interleukin (IL)-10 knockout mice significantly reduced inflammation in the cecum and colon compared with the same knockouts fed milk alone (O’Mahoney et al., 2001). IL-10 is an anti-inflammatory cytokine that causes progressive colonic inflammation when levels are low or absent, as is the case for these knockout mice. These results suggest that probiotics, alone or by interaction with the existing intestinal flora, can influence the colonic immune system and counteract low IL-10 levels. IL-10 is normally expressed in T cells in the lamina propria of the colon. In another murine model study, IL-10 knockout mice treated with a combination of L. salivarius and Bifidobacterium longum subsp. infantis in a dairy product resulted in a decrease in disease severity. The severity of disease was evaluated by weight loss, colon pathology and general appearance over a 6-week period (McCarthy et al., 2003). Control animals fed only dairy products exhibited a chronic wasting disease during the same time period. VSL#3 is a product containing multiple probiotic strains (Sheil et al., 2007). VAL#3 was tested in patients with ulcerative colitis; 15 of 20 patients treated remained in remission over the 12-month period of the study, suggesting the mixture may be useful in maintaining remission in patients with ulcerative colitis (Venturi et al., 1999). A study involving 32 patients

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with Crohn’s disease in clinical remission and given either mesalamine or mesalamine plus S. boulardii showed that 37% of patients given the drug alone relapsed in 6 months while 6.5% of patients receiving drug plus S. boulardii relapsed (Guslandi et al., 2000). These data suggest that S. boulardii could be a useful adjuvant for preventing symptomatic relapse in patients with Crohn’s disease. The sum total of the existing human and animal probiotic IBD literature is preliminary and equivocal; however, it does suggest that specific probiotics could be useful in preventing symptomatic relapse for patients with ulcerative colitis and/or Crohn’s disease.

1.3.3

Treatment of gastroenteritis

The most extensive probiotic medical literature is in the area of diarrheal diseases (gastroenteritis). The treatment and prevention can be further categorized by etiologic agent or by the type of disease. 1.3.3.1

Antibiotic-associated diarrhea

There have been numerous studies investigating the efficacy of probiotics for preventing or reducing the frequency and severity of diarrhea associated with the clinical use of antibiotics (Siitonen et al., 1990; Arvola et al., 1995; Vanderhoof et al., 1999; Armuzzi et al., 2001a,b; Cremonini et al., 2002). When studying 119 children who received antibiotics for respiratory infections and concomitant Lactobacillus rhamnosus GG (LGG) or placebo during the antibiotic treatment period, investigators found a 70% reduction in diarrheal symptoms for the group administered LGG compared with a placebo arm (Arvola et al., 1995). In a larger study involving 202 children treated with oral antibiotics, 8% of the children who were given LGG concurrently with antibiotic experienced diarrheal symptoms compared with 26% of the placebo group (Vanderhoof et al., 1999). In two studies with 60 and 120 adult patients respectively receiving antibiotic treatment to eliminate a Helicobacter pylori infection, investigators found that a significantly lower number of patients who received concurrent LGG experienced nausea and diarrhea compared with a group given placebo (Armuzzi et al., 2001a,b). Helicobacter pylori has been identified as an etiologic agent for gastric ulcers. Saccharomyces boulardii has also been shown to reduce antibioticassociated diarrhea (Marchand & Vandenplas, 2000). A meta-analysis of the effect of probiotic administration on antibiotic-associated diarrhea comprising 22 placebo-controlled studies found a combined relative risk of 0.39 for diarrhea among the probiotic-treated cohorts (D’Soriza et al., 2002). The investigators concluded that a strong benefit exists for probiotic administration for antibiotic-associated diarrhea, although they cautioned that the evidence is not yet definitive and more studies are required. 1.3.3.2

Acute diarrhea

Numerous studies have reported the use of probiotics to prevent or treat acute diarrhea (Hochtes et al., 1990; Cetina-Savri & Sierra, 1994; Raza et al., 1995; Sepp et al., 1995; Pant et al., 1996; Shornikova et al., 1997a,b; Oberhelman et al., 1999; Guandalini et al., 2000; Mastretta et al., 2002; Szajewska et al., 2001; Allen et al., 2003). The majority of the studies involved infants or children and the etiologic agent was rotavirus or of unknown cause. Probiotics that have been shown to be effective for the treatment of acute gastroenteritis include LGG, Lactobacillus reuteri and S. boulardii (Hochtes et al., 1990;

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Cetina-Savri & Sierra, 1994; Raza et al., 1995; Sepp et al., 1995; Pant et al., 1996; Shornikova et al., 1997a,b; Oberhelman et al., 1999; Guandalini et al., 2000; Mastretta et al., 2002; Szajewska et al., 2001; Allen et al., 2003). A multicenter European based trial with 287 children aged 1–36 months from 10 countries is one of the most extensive trials investigating probiotic treatment for acute diarrhea reported (Guandalini et al., 2000). The children were experiencing moderate to severe diarrhea. The patients were randomized to be given placebo or LGG along with oral rehydration solution. The children receiving LGG had a shorter duration and decreased severity of disease along with a shorter hospital stay. Another important finding was that on follow-up the probiotictreated children had a decreased likelihood of persistent diarrheal illness. There are other examples of findings similar to those described above in children with diarrheal disease (Pant et al., 1996; Shornikova et al., 1997b). A review of the double-blind randomized literature for probiotic biotherapeutic agents found that LGG and S. boulardii had the most favorable effect for treatment of acute diarrhea in children and adults (Marchand & Vandenplas, 2000). 1.3.3.3

Traveler’s diarrhea

Visitors from countries with temperate climates to areas with tropical or subtropical climates experience a high incidence of diarrhea. The incidence rate often approaches 50%. There have been a few published studies that have investigated the efficacy of probiotic treatment for lowering the diarrheal incidence rate. A study that tracked Finnish travelers to Turkey showed that in one of two resorts oral ingestion of LGG conferred a significant protection rate of 30.5% and 27.9% in weeks 1 and 2 of the study respectively (Oksanen et al., 1990). In another study, 245 travelers from New York were followed for 1–3 weeks after arriving in various developing countries. The travelers were provided with LGG or a placebo prior to their trip and LGG afforded a protection rate of 47% (Hilton et al., 1997). McFarland (2007) performed a meta-analysis of studies designed to investigate probiotics for the prevention of traveler’s diarrhea. The analysis included 12 studies that met the inclusion and exclusion criteria. The results of the analysis showed that the pooled relative risk was 0.85 (P < 0.001) and that probiotics significantly prevent traveler’s diarrhea. The meta-analysis investigator also concluded that S. boulardii and a mixture of L. acidophilus and Bifidbacterium bifidum had significant treatment efficacy. 1.3.3.4

Treatment of relapsing gastroenteritis caused by Clostridium difficile toxin

Often as a result of antibiotic treatment, the normal intestinal microflora can be altered. The disturbance to the microflora can result in C. difficile growth from existing spores, with the concomitant production of toxin in the intestinal tract. Several studies have shown that treatment with LGG prevents relapse of gastroenteritis (i.e. recurrent C. difficileassociated disease, RCDAD) after use of antibiotics. Clinical observations have indicated a 60% relapse rate after therapy with metronidazole or vancomycin. Only 16% who had received LGG had a relapse and after a second course of treatment with LGG, there was a 94% overall cure rate (Gorbach et al., 1987; Biller et al., 1995; Bennet et al., 1996). There have been several recent studies that have cast doubt on these earlier findings. No benefit was found for a yogurt/LGG formulation for patients with RCDAD (Pochapin, 2000). In a small study using capsules containing lyophilized LGG there again was no benefit noted

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with the probiotic, although the study had too few subjects to provide statistical power (Lawrence et al., 2005). It is therefore not clear if probiotics are beneficial for patients with RCDAD.

1.3.4

Cholesterol lowering

There is some evidence based on human studies that probiotics may lower total serum cholesterol and/or low-density lipoprotein (LDL) cholesterol. The results are not definitive and often conflicting. The lowering of LDL cholesterol would have important implications for decreasing the risk of coronary artery disease and for fatal myocardial infarction. The human studies that have shown an effect for fermented milk products on plasma cholesterol levels found a lowering of total cholesterol between 5.4 and 23.2% and for LDL between 9 and 9.8% (Anderson & Gilliland, 1999). A recent study of 14 subjects in a randomized crossover trial involving ordinary yogurt or yogurt plus L. acidophilus and B. animalis subsp. lactis for 6-week feeding periods and a 4-week washout period found a significant decline in serum total cholesterol when comparing the yogurt plus probiotics to the yogurt alone (Atoie-Jafari et al., 2009). The cholesterol studies have had small numbers of subjects and were limited in duration, generally 6 weeks. Based on in vitro and animal studies, several mechanisms for the probiotic lowering of serum cholesterol have been proposed. These involve absorption or assimilation of cholesterol by probiotics (Walker & Gilliland, 1993). There has been a study showing optimal removal of cholesterol from growth media in the presence of L. casei plus a prebiotic (Liong & Shah, 2005). A separate mechanism that has been proposed for probiotic-induced cholesterol lowering is the ability of bifidobacteria and lactobacilli to deconjugate bile acids. The deconjugation would lead to more rapid excretion of bile acids in the feces and since cholesterol is a precursor for bile acid synthesis, the lower bile acid concentration would act as a positive feedback for increasing synthesis from cholesterol to bile acids (Walker & Gilliland, 1993).

1.3.5

Treatment for urogenital infections

Vaginal infections are caused by such agents as Candida, Trichomonas, or bacterial organisms such as Gardnerella vaginalis and Mycoplasma hominis. Urinary tract infections are far more common in women and are generally caused by E. coli, Chlamydia and Candida. There are approximately 300 million urogenital infections reported per year. Normal healthy women have approximately 50 different species of microorganisms in the vaginal flora. Reid et al. (1995) reported that weekly intravaginal instillation of lactobacilli in 10 premenopausal women reduced urinary tract infections from 6.3 per patient per year before treatment to 1.3 per patient per year during treatment. Hilton et al. (1992) found that yogurt containing L. acidophilus reduced Candida-caused vaginitis by threefold in a crossover-designed trial. The results of studies using probiotics for treatment or prevention of urogenital infections are very limited, although there are investigators attempting to design specific probiotics to be administered orally to prevent or reduce the incidence of urogenital infections.

1.3.6

Treatment of allergic reactions

The most extensive studies directed at probiotic modulation of the immune response to food allergens has been done with LGG for preventing and treating atopic eczema. In a study of 159 pregnant women with a family history of atopic disease the subjects were

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given either LGG or placebo for 2–4 weeks prior to their expected delivery (Kalliomaki et al., 2001). Women who breast-fed their infants received LGG or placebo for 6 months and women who bottle-fed their newborns fed them LGG or placebo for 6 months. A 50% reduction in the incidence of atopic eczema was noted in the first 2 years of the child’s life for the group receiving LGG compared with the placebo group. In a follow-up to this study, after 4 years the children given LGG had a significantly lower incidence of atopic eczema compared with the placebo group (Kalliomaki et al., 2003). In another study 27 infants with atopic eczema were randomized into three groups and given LGG, Bifidobacterium animalis subsp. lactis or placebo (Isolauri et al., 2000). After 2 months the clinical score for the severity and extent of the eczema indicated a significant improvement in the skin condition of the infants fed the probiotics (P = 0.002). A similar study in which 31 infants with atopic eczema had their exposure to cows’ milk terminated and were treated with LGG showed a significant improvement compared with a group who were not fed cows’ milk and were fed placebo (Majamaa & Isolauri, 1997). Bifidobacterium animalis has also been shown to reduce the severity of atopic eczema in young children (Majamaa & Isolauri, 1997).

1.3.7

Prevention of dental caries

After oral ingestion, probiotics can be isolated from the oral cavity. Therefore it would be logical to study their efficacy in preventing dental caries. In addition, LGG has been shown to have antimicrobial activity against the Streptococcus spp., an organism involved in causing tooth decay (Silva et al., 1987). Children in a multicenter daycare trial were given LGG-containing milk or non-supplemented milk and examined before and after the 7-month intervention study. The children receiving the probiotic had a lower rate of clinical development of dental caries, which was most pronounced in the group aged 3–4 years (Nase et al., 2001). More studies are needed to see if this observation can be repeated and if other probiotics will have the same beneficial effect.

1.3.8

Treatment and prevention of cancer by probiotics

By virtue of their metabolic activity, probiotics can influence the etiology of colon cancer and possibly tumors at other sites. Probiotics have been shown to reduce intestinal bacterial enzymes involved in the activation of procarcinogens (Hosoda et al., 1996). Probiotics also can produce short-chain fatty acids that may also be protective in the colon. Animal studies in rats have shown that probiotics can inhibit the formation of aberrant crypt foci in the colon. A combination of inulin plus B. longum reduced chemically induced aberrant crypt foci by 74% (Rowland et al., 1998). Inulin alone reduced the aberrant crypts by 21%. Rats fed a mixture of oligofructose, inulin, LGG and B. animalis subsp. lactis had significantly lower azoxymethane-induced colon tumors (Marotta et al., 2003). Mice genetically bred to be susceptible to colitis and colon cancer had a 10% incidence rate of adenocarcinoma when fed L. salivarius compared with the 50% rate for control animals (O’Mahoney et al., 2001). Rats injected with DMH and fed LGG had a significantly lower colon cancer incidence than animals receiving DMH alone (Goldin et al., 1996). Human colon cancer trials have not been conducted with probiotics, primarily due to the difficulty of conducting a preventive intervention trial. There is one report in the literature of a human trial of patients with superficial bladder cancer. The patients receiving L. casei had an 80% longer diseasefree period, with a mean of 350 days compared with 195 days for the control group.

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1.3.9

Additional health benefits attributed to probiotics

There are a number of other health benefits that have been observed for probiotic use over the past number of years. Some are included in this section. A study conducted in Italy with children suffering from cystic fibrosis and given LGG for the chronic abdominal pain often associated with the disease indicated that the frequency and severity of abdominal problems were reduced and that intestinal inflammation as judged by the fecal marker calprotectin and rectal nitric oxide was also decreased (Bruzzese et al., 2004). Rheumatoid arthritis is a systemic inflammatory disease. Animal studies using experimental arthritis model in Lewis rats showed that these rats improved when fed LGG compared with placebo (Baharav et al., 2004). The findings of a preliminary study involving 21 patients with rheumatoid arthritis receiving either placebo or LGG showed that the LGG group had a decreased number of swollen joints and lower overall arthritic activity, although the difference did not reach statistical significance (Hatakka et al., 2003). Nanji et al. (2005) studied the ability of probiotics to prevent alcohol-induced liver disease in a rat model. Rats were conditioned to drink ethanol and one group was administered LGG orally. The rats fed LGG had reduced liver disease and lower plasma endotoxin levels. In a related study rats were given carbon tetrachloride to induce chronic liver disease as a model to study the efficacy of probiotics in spontaneous bacterial peritonitis (Bauer et al., 2002). LGG was not effective and did not prevent bacterial overgrowth or bacterial translocation from the colon into mesenteric lymph nodes or portal blood. The effect of probiotics on radiation exposure has been studied in a mouse model (Dong et al., 1987). Mice were either fed LGG or maintained on a normal diet and then exposed to 14 Gy of total body irradiation. The LGG-fed rats had a significantly lower mortality rate at 48 hours after irradiation. Of the 21 control mice 10 had Pseudomonas aeruginosa bacteremia, compared with 1 of 21 mice fed LGG. None of the LGG-fed mice had LGG bacteremia. There is a preliminary study from Japan using a streptozotocin-induced diabetic mouse model which showed that feeding LGG lowered hemoglobin A1c blood levels and improved glucose tolerance compared with controls (Tabuchi et al., 2003). Bone marrow transplantation patients can develop graft-versus-host disease (GVHD). Bacterial lipopolysaccharide (LPS) is believed to be involved in this process. A mouse model of GVHD has been developed where the disease is induced by employing a major histocompatibility mismatch (Gerbitz et al., 2004). The animals show serious damage to the bowel mucosa and high levels of serum LPS and inflammatory cytokines. The animals were divided into three groups, receiving in their drinking water LGG, ciprofloxacin or no additive for 7 days prior to transplantation. Treatment with LGG reduced mortality, which was most prominent in the early post-transplantation period and was reflected in a lower GVHD score compared with the other groups. Mesenteric lymph nodes of LGG treated animals had a lower concentration of translocated intestinal organisms.

1.3.10

Conclusions based on past and present use of probiotics for health applications

This section has outlined the current knowledge regarding the application of probiotics for preventing and treating medical diseases and disorders. Table 1.1 lists the medical applications for probiotics that have been studied in the past and which are currently under investigation.

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Table 1.1 Past and current applications for probiotics. Medical condition

Example of probiotic used or studied

Antibiotic-associated diarrhea

Lactobacillus, Saccharomyces boulardii

Lactose malabsorption

Lactobacillus, Streptococcus thermophilus, Streptococcus salivarius

Acute diarrhea

Lactobacillus, Bifidobacterium, S. boulardii

Traveler’s diarrhea

Lactobacillus

Vaccine adjuvant

Lactobacillus

Vaginitis

Lactobacillus

Dental caries

Lactobacillus

Relapsing C. difficile colitis

Lactobacillus

Inflammatory bowel disease

Lactobacillus, Bifidobacterium, S. boulardii

Rheumatoid arthritis

Lactobacillus

Cirrhosis of the liver

Lactobacillus

Cystic fibrosis abdominal side effects

Lactobacillus

Food allergies

Lactobacillus

Diabetes

Lactobacillus

Graft-versus-host disease

Lactobacillus

Cancer

Bifidobacterium, Lactobacillus

Nasal pathogen colonization

Lactobacillus

Radiation side effects

Lactobacillus

Hypercholesterolemia

Bifidobacterium, Lactobacillus

1.4

NUTRITIONAL EFFECTS OF PROBIOTICS

There are numerous reports showing that probiotics can influence nutritional status. Bifidobacteria have been shown to produce the water-soluble vitamins thiamine, nicotinic acid, folic acid, pyridoxine, biotin and B12 (Lee et al., 1999). Additional nutritional effects have been noted for L. acidophilus, which increases iron bioavailability (Lee et al., 1999), and numerous lactobacilli species deconjugate bile acids (Walker & Gilliland, 1993). As noted earlier probiotics can hydrolyse lactose in milk products.

1.5

FUTURE DEVELOPMENT AND USES OF PROBIOTICS FOR HEALTH APPLICATION

Sections 1.1–1.4 have reviewed the past and current development and applications of probiotics for nutrition and health purposes. In this section a review of possible new probiotic development and uses will be discussed. The future is always harder to evaluate then the

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past; however, with current probiotic projects and goals in mind, this section will attempt to predict the future for probiotics. The major thrusts in future health applications for probiotics will be based on the development of new organisms, through genetic modification (GM) or by natural selection, that specifically exhibit activities that would, from a mechanistic approach, apply to specific diseases, disorders or nutritional or drug requirements. The capability to achieve this objective will define current uses and additional future applications for probiotics. This section will not attempt to cover the area of bacterial genetics or techniques for gene insertion and current knowledge of bacterial genomics. Given current progress, this area would require at a minimum an entire book or possibly a multivolume series of books.

1.5.1

Probiotics as a platform for delivery of drugs, enzymes, hormones, nutrients and micronutrients

One of the intriguing areas of current research and future development of probiotics is their use as delivery systems for health-related compounds, enzymes, toxin inhibitors, carcinogen detoxifiers and immune modulators. Lothar Steidler has developed one of the best examples of using GM probiotics to deliver anti-inflammatory agents to the colon. A strain of Lactococcus lactis has been modified to express murine and human IL-10, a potent anti-inflammatory cytokine (Steidler et al., 2003). Knockout IL-10 mice rapidly develop colonic inflammation and subsequently adenocarcinomas (Scheinin et al., 2003 ). When introduced orally the recombinant L. lactis has been shown to have a positive effect by reducing intestinal inflammation in mice treated with colitis-inducing dextran sulfate (Steidler et al., 2000). These investigators have succeeded in replacing the thymidylate synthetase gene with the human IL-10 gene. This replacement results in a probiotic that can produce human IL-10 but which is not capable of synthesizing thymidine. The inability to make thymidine assists in biocontainment of the mutant, since the L. lactis now requires thymidine for growth and would not thrive in an outside environment. The GM L. lactis have been fed to a small number of patients with Crohn’s disease in a Phase I human clinical trial (Braat et al., 2006). This type of GM probiotic is a model for future development of organisms that can decrease local inflammation at the site where the probiotic resides. Probiotics with IL-4 or IL-12 producing genes can expand the array of organisms to combat colon inflammation and treat IBD and possible lower the subsequent risk for developing adenocarcinomas. Future directions for probiotics could include insertion of higher plant genes responsible for multistep synthesis pathways leading to antiinflammatory products. An example would be curcumin or flavonoids and their analogues, which have been shown to be beneficial in treating inflammatory disease and dermatological disorders. These compounds are believed to act through inhibition of inducible nitric oxide synthetase. Recombinant bacteria with herb or plant genes directed toward the synthesis of a variety of medicinal products can have great potential for future probiotic development. This approach is dependent on methods of biocontainment that would prevent survival of the organism outside the human or animal and this should act to prevent environmental contamination.

1.5.2

Toxin sequestration

Studies have shown that various strains of Lactobacillus and Bifidobacterium have the ability to bind and inactivate aflatoxins (Gratz et al., 2005). Investigators have constructed strains of GM E. coli that can bind Shiga toxin (STX) produced by toxigenic E. coli or by Shigella dysenteriae (Paton et al., 2001). The possibility of selecting

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probiotics that can bind a variety of bacterial toxins, either naturally (as for aflatoxin) or by GM (as for STX) is feasible in the future.

1.5.3

Carcinogen detoxification

A major challenge for understanding the causes of human cancers is identifying dietary or environmental agents involved in the etiology of cancer at different organ sites. Most agents are believed to be procarcinogens that require enzymatic or other types of catalysis to generate the direct-acting carcinogens. Therefore inhibitors of the activating enzymes that convert procarcinogens to carcinogens or the introduction of enzymes that deactivate the direct-acting carcinogens can interfere with chemical carcinogenesis. Probiotics that express enzymes such as NADPH cytochrome P450 reductase, aldehyde reductase, glutathione-S-transferase or N-acetyltransferase, among others, can deactivate procarcinogens such as benzpyrene, heterocyclic amines, nitrosamines and heterocyclic amines. A recombinant strain of Saccharomyces cerevisiae that overexpresses NADPH cytochrome P450 reductase has been produced (Blanquet et al., 2001). This strain has been shown in vivo in the intestine to convert trans-cinnamic acid to p-coumaric acid.

1.5.4

Antibody production

Recombinant probiotics can be designed to produce single-chain antibodies that can be processed downstream to generate neutralizing antibodies against microbial pathogens, toxins and inflammatory cytokines. An example of this GM technology has been used to create a recombinant Lactobacillus zeae that expresses a surface-bound single chain that recognizes the SA I/II adhesion molecule of Streptococcus mutans (Lehner et al., 1985). In vivo studies in which rats were orally inoculated with L. zeae that expressed the singlechain antibody resulted in a marked decrease in Strep. mutans counts in the oral cavity and a concomitant decline in the development of dental caries (Lehner et al., 1985). This type of study verifies in vitro studies showing recombinant L. zeae with SA I/II surface single antibody as capable of causing coagulation with Strep. mutans in suspensions. A similar approach has been used to combat Candida albicans a major cause of acute vaginitis. Two strains of Strep. gordonii have been produced to express and secrete and surface bind a single-chain antibody that exhibits candidacidal activity over a wide concentration range (Beninati et al., 2000). Both Strep. gordonii strains colonize the vagina and cleared a C. albicans infection in rats. The single-chain secretor strain of Strep. gordonii showed a faster reduction of the pathogenic load of C. albicans. This type of technology can be used in the future to deactivate toxins and cytokines such as tumor necrosis factor (TNF)-α.

1.5.5

Treatment for enzyme deficiencies

Probiotics can be used to produce enzymes that are lacking or abnormally low in humans or animals. Lactococcus lactis expressing a lipase gene from Staphylocccus hyicus has been constructed (Drouault et al., 2002). The L. lactis could potentially be used to treat pancreatic insufficiency. The L. lactis strain requires a nisin promoter. Upon induction it was shown that lipase accumulated to account for up to 15% of total protein intracellularly. Pigs that had their pancreatic duct ligated and then treated with the recombinant L. lactis strain had 10% higher fat absorption than untreated controls (Drouault et al., 2002). This type of enzyme replacement technology can be used in the

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future to treat a wide variety of enzyme deficiencies resulting from disease, surgical procedures or genetic conditions. As stated in section 1.5.1, it is desirable to engineer recombinant probiotics with a biocontainment factor. Removing the thymidylate synthetase gene concomitant with adding a gene that expresses a desired product is one example. Another example is to remove the gene that converts l-alanine to d-alanine. Since d-alanine is an essential component of most bacterial cell walls, the ability to inhibit the conversion of l-alanine to d-alanine would limit the growth of the bacteria in the environment. There are numerous other strategies that can be used for biocontainment of probiotics and some of these are discussed in an article on genetically engineered probiotics (Steidler, 2003). Box 1.1 shows some of the future developments for selecting probiotics for medical applications.

Box 1.1 Selected future probiotic medical applications Production of anti-inflammatory agents Genetically modified to produce IL-10, IL-4, IL-12 Genetically modified to produce curcumin and flavonoids and their analogues Detoxification activity Toxin sequestration ● Natural selection of strains that bind mycotoxins ● GM strains constructed to bind E. coli Shiga toxin or Shigella dysenteriae Shiga toxin Carcinogen detoxification: deactivation via enzymatic expression ● NADPH cytochrome P450 reductase ● Aldehyde reductase ● Glutathione-S-transferase ● N-Acetyltransferase Antibody production by GM probiotics Streptococcus mutans Candida albicans Other pathogens Treatment for enzymatic deficiencies Lipase expression Enzyme deficiencies resulting from surgical procedures, diseases, genetic disorders Treatment for hormone deficiencies Nasal introduction of GM probiotics expressing for insulin or proinsulin GM probiotics producing promoter-controlled growth hormone or thyroid hormone Natural selection of new probiotic genera Propionibacteria Other genera that are non-pathogenic

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1.5.6

13

Other potential future directions for probiotics for medical use

The use of new genera of microorganisms for probiotic purposes is an additional future direction that has to be considered. The propionibacteria are one such example and their attributes have been discussed in a review article (Ouwehand, 2004). In summary, some of the propionibacteria produce antimicrobial substances such as propionic acid and bacteriocins. In addition, propionibacteria have been shown to have antivirial activity and the ability to adhere to intestinal surfaces. Propionibacteria are capable of stimulating the growth of Bifidobacterium, which is an established probiotic and therefore makes propionibacteria a candidate component of probiotic mixtures. The number of different genera of bacteria, yeast, molds and fungi is large and many of these different organisms have not been tested for beneficial health effects, an area that will be subject to future research.

1.6

CONCLUSIONS

This chapter has outlined the early history of bacterial use of probiotics for the benefit of humankind and the current medical uses and the evidence supporting these health applications. The chapter has also attempted to predict future developments of probiotics based on the latest technological advances in the field of microbial genetics. These future developments will provide new applications for probiotics and an important place for them in the armamentarium against the multiple diseases and disorders that afflict humankind.

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Blanquet S, Marol-Bonnin S, Beyssac E, Pompon D, Renard M, Alric M (2001) The biodrug concept: an innovative approach to therapy. Trends Biotechnol 19:393–400. Braat H, Rottiers P, Hommes DW et al. (2006) A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin Gastroenterol Hepatol 4:754–759. Bruzzese E, Raia V, Gauidiello L et al. (2004) Intestinal inflammation is a frequent feature of cystic fibrosis and is reduced by probiotic administration. Aliment Pharmacol Ther 20:813–819. Cetina-Savri G, Sierra BG (1994) Evaluation therapeutique de Saccharomyces boulardii chez des enfants souffrant de diarrhea aigue. Ann Pediatr 41:397–400. Cremonini F, Di Caro S, Cavino M et al. (2002) Effect of different probiotic preparations on antiHelicobacter pylori therapy-related side effects: a parallel group, triple blind, placebo-controlled study. Am J Gastroenterol 97:2744–2749. de Vrese M, Stegelmann A, Richter B, Fenseau S, Love C, Schrezenmeir J (2001) Probiotics compensate for lactase insufficiency. Am J Clin Nutr 73:421s–429s. Doderlein A (1892) Das Scheidensekret und seine Bedeutung für das Puerperolfieber. Zenralbl Bakteriol 11:699–700. Dong MY, Chang TW, Gorbach SL (1987) Effect of feeding Lactobacillus GG on lethal irradiation in mice. Diagn Microbiol Infect Dis 7:1–7. Drouault S, Juste C, Marteau P, Renault P, Corthier G (2002) Oral treatment with Lactococccus lactis expressing Staphylococcus hyicus lipase enhances lipid digestion in pigs with induced pancreatic insufficiency. Appl Environ Microbiol 68:3166–3168. D’Soriza AL, Rajkumar C, Cooke J, Bulpitt CJ (2002) Probiotics in prevention of antibiotic associated diarrhoea: meta-analysis. Br Med J 324:1361–1366. FAO (2006) Guidelines for the evaluation of probiotics in food. Report of a Joint FAO/WHO Working Group, London, Ontario, Canada, 30 April to 1 May 2002. In: Probiotics in Food: Health and Nutritional Properties and Guidelines For Evaluation. FAO Food and Nutrition Paper no. 85, Rome, Italy, pp. 1–56. Gerbitz A, Schultz M, Wike A et al. (2004) Probiotic effects on experimental graft-versus-host disease: let them eat yogurt. Blood 103:4365–4367. Goldin BR, Gualtieri LJ, Moore RP (1996) The effect of Lactobacillus GG on the initiation and promotion of DMH-induced intestinal tumors in the rat. Nutr Cancer 25:197–204. Gorbach SL, Chang TW, Goldin BR (1987) Successful treatment of relapsing Clostridium difficile colitis with Lactobacillus GG. Lancet 2:1519. Gratz S, Mykkanen H, El-Nezami H (2005) Aflatoxin B1 binding by a mixture of Lactobacillus and Propionibacterium in vitro versus ex vivo. J Food Protect 68:2470–2474. Guandalini S, Pensabene L, Zikri MA et al. (2000) Lactobacillus GG administration in oral rehydration solution to children with acute diarrhea: a multicenter European trial. J Pediatr Gastroenterol Nutr 30:54–60. Guslandi M, Mezzi G, Sorghi M, Testori PA (2000) Saccharomyces boulardii in the maintenance treatment of Crohn’s disease. Dig Dis Sci 45:1462–1464. Hatakka K, Maitio J, Korpela M et al. (2003) Effects of probiotic therapy on the activity and activation of mild rheumatoid arthritis: a pilot study. Scand J Rheumatol 32:211–215. Hilton E, Isenberg HD, Alperstein P, France K, Borenstein MT (1992) Ingestion of yogurt containing Lactobacillus acidophilus as prophylaxis for candidal vaginitis. Ann Intern Med 116:352–357. Hilton E, Kolakowski P, Singer C, Smith M (1997) Efficacy of Lactobacillus GG as diarrheal preventive in travelers. J Travel Med 4:41–43. Hochtes W, Chase D, Hegenhoff G (1990) Saccharomyces boulardii in treatment of acute adult diarrhea: efficacy and tolerance of treatement. Munch Med Wochenschr 132:188–192. Hosoda M, Hashimoto H, He F, Morita H, Hosono A (1996) Effect of administration of milk with Lactobacillus acidophilus LA-2 on fecal mutagenicity and microflora in the human intestine. J Dairy Sci 79:745–749. Isolauri E, Arvola T, Sutas Y, Moilanaen E, Salminen S (2000) Probiotics in the management of atopic eczema. Clin Exp Allergy 30:1604–1610. Kalliomaki M, Salminen S, Arvilomni H, Kero P, Koskinen P, Isolauri E (2001) Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial. Lancet 357:1076–1079. Kalliomaki M, Salminen S, Poussa T, Arvilonmi H, Isolauri E (2003) Probiotics and prevention of atopic disease: 4-year follow-up of a randomized placebo-controlled trial. Lancet 361:1869–1871. Kim HS, Gilliland SE (1983) Lactobacillus acidophilus as dietary adjunct for milk to aid lactose digestion in humans. J Dairy Sci 66:959–966.

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Kolars JC, Levitt MD, Aouj M, Savaino DA (1984) Yogurt an antidigesting source of lactose. N Engl J Med 310:1–3. Lawrence SJ, Korzenik JR, Mundy LM (2005) Probiotics for recurrence of Clostridium difficile disease. J Med Microbiol 54:905–906. Lee YK, Nomoto K, Salminen S, Gorbach SL (eds) (1999) Handbook of Probiotics. New York: John Wiley & Sons. Lehner T, Caldwell J, Smith R (1985) Local passive immunization by monoclonal antibodies against streptococcal antigen I/II in the prevention of dental caries. Infect Immun 50:796–799. Levri KM, Ketvertis K, Derano M, Merenstein J, D’Amico F (2005) Do probiotics reduce adult lactose intolerance? A systematic review. J Fam Pract 54:613–620. Liong MT, Shah NP (2005) Optimization of cholesterol removal by probiotics in prebiotics by using a response surface method. Appl Environ Microbiol 71:1745–1753. Majamaa H, Isolauri E (1997) Probiotics: a novel approach in the management of food allergy. J Allergy Clin Immunol 99:179–185. McCarthy J, O’Mahoney L, Sheil B et al. (2003) Double-blind, placebo-controlled trial of two probiotic strains in IL-10 knockout mice and mechanistic links with cytokine balance. Gut 52:975–980. McFarland LV (2007) Meta-analysis of probiotics for the prevention of traveler’s diarrhea. Trav Med Infect Dis 5:97–105. Malchow HA (1997) Crohn’s disease and Escherichia coli: a new approach in therapy to maintain remission of colonic Crohn’s disease. J Clin Gastroenterol 25:653–658. Marchand J, Vandenplas Y (2000) Microorganisms administered in the benefit of the host: myths and facts. Eur J Gastroenterol Hepatol 12:1077–1088. Marotta F, Naito Y, Minelli E et al. (2003) Chemopreventive effect of a probiotic preparation on the development of preneoplastic and neoplastic colonic lesions: an experimental study. Hepatogastroenterology 50:1914–1918. Mastretta E, Lonzo P, Laccisasaglia A et al. (2002) Effect of Lactobacillus GG and breast-feeding in the prevention of rotavirus nosocomial infection. J Pediatr Gastroenterol Nutr 35:527–531. Metchnikoff E (1907) The Prolongation of Life. Heinemann, London. Nanji AA, Kheltry V, Sadrzadeh SM (2005) Lactobacillus feeding reduces endotoxemia and severity of experimental alcoholic liver disease. Proc Exp Biol Med 3:243–247. Nase L, Hatakka K, Savilahti E et al. (2001) Effect of long-term consumption of probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental carries and caries risk in children. Caries Res 35:412–420. Nissle A (1918) Die anatagonistische Behandlung chronischer Darmstörungen mit Colibakterien. Med Klin 2:29–30. Oberhelman RA, Gilman RH, Sheen P et al. (1999) A placebo-controlled trial of Lactobacillus GG to prevent diarrhea in undernourished Peruvian children. J Pediatr 134:15–20. Oksanen PJ, Salminen S, Saxelin M et al. (1990) Prevention of traveler’s diarrhea by Lactobacillus GG. Ann Med 22:53–56. O’Mahoney L, Feeney M, O’Hallaran S et al. (2001) Probiotics impact on microbial flora, inflammation and tumor development in IL-10 knockout mice. Aliment Pharmacol Ther 15:1219–1225. Ouwehand AC (2004) The probiotic potential of propionibacteria. In: Salminen S, von Wright A, Ouwehand AC (eds) Lactic Acid Bacteria. New York: Marcel Dekker, pp. 159–174. Pant AR, Graham SM, Allen SJ et al. (1996) Lactobacillus GG and acute diarrhea in young children in the tropics. J Trop Pediatr 42:162–165. Parker RB (1974) Probiotics, the other half of the antibiotic story. Animal Nutr Health 29:4–8. Paton AW, Morona R, Paton JC (2001) Neutralization of Shiga toxin Stx1, Stx2c and Stx2e by recombinant bacteria expressing bacteria mimics globotriose and globotetraose. Infect Immun 69:1967–1974. Pochapin M (2000) The effect of probiotics on Clostridium difficile diarrhea. Am J Gastroenterol 95:11s–13s. Raza S, Graham SM, Allen SJ, Sultana S, Cuevas L, Hart CA (1995) Lactobacillus GG promotes recovery from acute non-bloody diarrhea in Pakistan. Pediatr Infect Dis J 14:107–111. Reid GA, Bruce W, Taylor M (1995) Instillation of Lactobacillus and stimulation of indigenous organisms to prevent recurrence of urinary tract infection. Microecol Ther 23:32–45. Retteger LF, Levy WN, Weinstein L, Weiss JE (1935) Lactobacillus acidophilus and its Therapeutic Application. New Haven: Yale University Press. Rowland IR, Rumney CJ, Coutts JT, Lievense LC (1998) Effect of Bifidobacterium longum and inulin on gut bacterial metabolism and carcinogen induced aberrant crypt foci in rats. Carcinogenesis 19:281–285.

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Sanders ME (2007) Probiotics, strains matter. Functional Foods and Nutraceuticals Magazine June:36–41. Savaiano DA, Abou El Anouar A, Smith DE, Levitt MD (1984) Lactose malabsorption from yogurt, sweet acidophilus milk and cultured milk in lactose deficient individuals. Am J Clin Nutr 40:1219–1223. Scheinin T, Butler DM, Salway F, Scallon B, Feldmann M (2003) Validation of interleukin-10 knockout mouse model of colitis. Clin Exp Immunol 133:38–43. Sepp E, Tamm E, Torm S, Lustar I, Mikelsaar M, Salminen S (1995) Impact of a Lactobacillus probiotic on the fecal microflora in children with Shigellosis. Microecol Ther 23:74–80. Sheil B, Shanahan F, O’Mahoney L (2007) Probiotic effects on inflammatory bowel disease. J Nutr 137:819s–824s. Shornikova AV, Cosos J, Mykkanen H, Salo E, Vesikari T (1997a) Bacteriotherapy with Lactobacillus reuteri in rotavirus gastroenteritis. Pediatr Infect Dis J 16:1103–1107. Shornikova AV, Isolauri E, Burkanova L, Lukovnikova S, Vesikari TA (1997b) A trial in the Karelian Republic of oral rehydration and Lactobacillus GG for treatment of acute diarrhea. Acta Pediatr 86:460–465. Siitonen S, Vapaatalo H, Salminen S et al. (1990) Effect of Lactobacillus GG yogurt in prevention of antibiotic-associated diarrhea. Ann Med 22:57–59. Silva M, Jacobus NV, Deneke C, Gorbach SL (1987) Antimicrobial substance from a human Lactobacillus strain. Antimicrob Agents Chemother 31:1231–1233. Steidler L (2003) Genetically engineered probiotics. Best Pract Res Clin Gastroenterol 17:861–876. Steidler L, Hans W, Schotte L, Neriynck S, Remaut E (2000) Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289:1352–1355. Steidler L, Neriymck S, Huyghebaert N et al. (2003) Biological containment of genetically modified Lactocococcus lactis for intestinal delivery of human interleukin-10. Nat Biotechnol 21:785–789. Szajewska H, Kotowska M, Murkowicz JZ, Armanska M, Mikolajczyk W (2001) Efficacy of Lactobacillus GG in prevention of nosocomial diarrhea in infants. J Pediatr 138:361–365. Tabuchi M, Ozaki M, Tomura A et al. (2003) Antidiabetic effect of Lactobacillus GG in streptozotocininduced diabetic rats. Biosci Biotechnol Biochem 67:1421–1424. Tissier H (1905) Taxonomy and ecology of bifidobacteria. Bifidobacteria Microflora 3:11–28. Vanderhoof JA, Whitney DB, Antoson DL, Hanner TL, Lupo JV, Young RJ (1999) Lactobacillus GG in the prevention of antibiotic associated diarrhea in children. J Pediatr 135:564–568. Venturi A, Gionchetti P, Rizzello F et al. (1999) Impact on the composition of the faecal flora by the new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Aliment Pharmacol Ther 13:1103–1108. Walker DK, Gilliland SE (1993) Relationship among bile tolerance, bile salt deconjugation, and assimilation of cholesterol by Lactobacillus acidophilus. J Dairy Sci 76:956–961.

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The World’s Oldest Probiotic: Perspectives for Health Claims

Tomoyuki Sako

2.1 2.1.1

FROM THEORY TO PRACTICE: THE CHALLENGE OF DR MINORU SHIROTA The discovery of Lactobacillus casei strain Shirota

The discovery of this well-known health-promoting microbe dates back to 1930, when Dr Minoru Shirota isolated the acid- and bile-tolerant Lactobacillus strain from a collection of about 300 strains of lactic acid bacteria. In 1924 Shirota had started research in Professor Kiyono’s lab in the Faculty of Medicine at Kyoto University in Japan, working on establishing a biological means to inhibit the growth of pathogenic bacteria. His work initially focused on the ‘Bulgarian’ yogurt strain (Lactobacillus delbrueckii subsp. bulgaricus) which, based on Metchnikoff’s theory of the longevity of Bulgarian peasants, was believed to prevent harmful bacteria colonising the human gut. According to Shirota’s own short memoir, however, the results were disappointing: in their tests the strain could not survive passage through the human gastrointestinal tract. Their conclusion was that Metchnikoff’s theory was too optimistic. Shirota changed the focus of his research to analysing in more detail the human intestinal microbiota. He observed that babies completely lack lactobacilli at birth, but that within days different species of lactobacilli colonise their intestines, particularly in breast-fed babies. Shirota then screened the research group’s collection of 300 lactobacilli for strains tolerant of acid and bile. From 18 strains selected on this basis, he discovered a particularly robust strain of Lactobacillus casei (then classified as L. acidophilus), which was able to survive in a highly acidic environment. This strain was later named L. casei strain Shirota in his honour. Shirota’s challenge was not just to discover and culture this strain, but to devise a way in which it could benefit people. In 1935, Yakult was first developed and manufactured, a result of the collaboration between Shirota and his colleagues and fulfilment of the philosophy of Shirota, who wanted to develop a product that would support intestinal health and be available to everyone at an affordable price. He was convinced that a healthy gut was absolutely essential for the enjoyment of a long and healthy life. This unique fermented milk product allowed Metchnikoff’s theory to become a reality. It is not the aim of this chapter to give a detailed description of the process of Yakult production, except to emphasise that its long fermentation involving just this one strain is key to the process and reflects Shirota’s philosophy. This production process has remained basically unchanged since the product was first made. Probiotics and Health Claims Edited by Wolfgang Kneifel and Seppo Salminen © 2011 Blackwell Publishing Ltd. ISBN: 978-1-405-19491-4

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2.1.2

Early studies in Japan: the first clinical study era for Yakult and L. casei Shirota

In the 1960s and 1970s several clinicians and scientists conducted a large number of clinical studies with Yakult and L. casei Shirota (LcS). At this time, Yakult drink was a completely unique concept: a fermented milk delivering an active Lactobacillus strain that was a practical realisation of Metchnikoff’s hypothesis. This was why, in Japan, clinicians and scientists wanted to confirm for themselves the health benefits associated with LcS. In 1961, Kotani et al. published an extensive report evaluating the potential health benefits of Yakult, which included numerous studies using a range of different approaches, for example in vitro studies, epidemiological studies, and intervention studies. Kotani et al. studied the effect of Yakult on the incidence of shigellosis in soldiers of the Japanese Self Defence Force who were all housed in the same billet. A total of 2000 soldiers were enrolled and randomised to two groups for a crossover trial involving two 45-day periods. For the first trial period, Yakult was given daily to one group but not the other group; for the subsequent 45-day period, only the second group received Yakult. No shigellosis patients or carriers of Shigella flexneri were detected in the volunteers during their period of Yakult consumption. In contrast, when Yakult was not consumed, two shigellosis patients and 10 subclinically infected persons (0.6% in total) carrying S. flexneri were detected. This finding was statistically significant (P = 0.01). In addition, when a 3-month epidemiological survey was conducted on a total of 2890 soldiers, the incidence of illness was significantly less for soldiers who habitually drank Yakult compared with those who did not. Open-label intervention studies were also carried out with kindergarten children (N = 160) and elementary school pupils (N = 600); these studies also showed that the incidence of illness was less in participants who drank Yakult compared with those who did not. By including other observational studies, the effect of Yakult on the maintenance of health was investigated in a total of 7600 people. Taken as a whole, this conclusively demonstrated that habitual consumption of Yakult could decrease the incidence of infection and other illnesses. Aritaki and Ishikawa (1962) and Kikuchi (1962) reported the effect of Yakult consumption on the intestinal microbiota of humans. In babies, this was associated with an increase in numbers of lactobacilli and a decrease in numbers of Gram-negative rods (Aritaki & Ishikawa, 1962) and also in adults admitted in hospitals for various reasons (Kikuchi, 1962). In both cases, this microbial change was accompanied by a decrease in faecal pH. A reduction in intestinal gas production and in faecal pH after Yakult intake was further reported by Shimizu and Shibamoto (1964). In a later placebo-controlled study, Shirota and colleagues conducted a more detailed analysis of the probiotic effect on the faecal microbiota of children aged from 2 to 6 years old (Shirota et al., 1966). As before, they found that Yakult consumption for 4 weeks was associated with significant increases in lactobacilli and bifidobacteria-like anaerobic rods, and significant decreases in Gram-negative rods and enterococci, as well as a decrease in faecal pH. However, when the Yakult was heattreated, there was no detectable change in the intestinal microbiota or the faecal pH. All these results can be interpreted as follows: the LcS strain in Yakult influences the composition and function of the intestinal microbiota thereby beneficially changing the intestinal environment, by decreasing faecal pH, etc. The pH reduction might be an important factor in helping to reduce the risk of infection by opportunistic and food-borne pathogens. The effect of LcS on constipation and bowel discomfort was investigated in several clinical studies, most of which used a freeze-dried preparation of the strain. Ogawa et al. (1974)

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performed a multicentre, double-blind, placebo-controlled crossover trial with 50 constipated patients admitted to several hospitals for various reasons. The volunteers were given 1 g of the LcS preparation (1 × 1010 cells) or placebo three times daily for 30 days, and then the groups were crossed to the alternate intervention for an additional 30-day period. There was a statistically significant response rate of 67.7% for improvement in constipation for completed 36 subjects. In addition, the response rate of the younger subjects (aged 20–59 years) was significantly higher (P < 0.05) than that of older subjects (aged 60–78 years). The subjects in this study did not have chronic and/or organic constipation, but had developed constipation as a result of their hospital admission and change in living conditions. Therefore these results can be extrapolated to healthy people in the general population, who can sometimes develop a suboptimal bowel habit for various reasons, not necessarily known. Other open trials in constipated patients with different complications yielded similar positive results (Ohta, 1972; Morita, 1973; Numata, 1973; Kawamura et al., 1981). In the 1980s a new trend emerged in the development of biological immune modulators from bacteria and mushrooms. Two new cancer drugs of this type were authorised in Japan: Picibanil (a Streptococcus pyogenes preparation) in 1975, and Krestin (a mushroom extract) in 1976. The active components were thought to be polysaccharides from the microbial and plant cell walls. Following this line of thinking, Yokokura et al. (1981) screened the anticancer activity of various Lactobacillus strains (including LcS), in mouse models, and found promising results with several strains. Of the strains tested, LcS showed strong inhibition of tumour growth when administered to the mice by a non-oral route. Numerous subsequent experiments confirmed that heat-killed LcS, administered non-orally in different animal models, had a strong preventive effect in vivo on the progression of various types of cancers. The underlying mechanism to explain this effect was stimulation of the antitumour immune responses, including activation of neutrophils and macrophages, stimulation of natural-killer cell activity, and increase in various cytokine production. Overall, the studies in this era strongly supported the idea that LcS could influence the immune responses of host animals, even when administered perorally.

2.1.3

Probiotic definition and the L. casei Shirota strain

The probiotic definition encompasses the basic ideas of Shirota and Metchnikoff: delivery of live beneficial bacteria to the intestines to maintain a well-balanced intestinal microbiota and thus promote healthy longevity. In every respect, LcS and Yakult fit the definition of a probiotic, as a microbial strain and product respectively. Thanks to the work of Shirota and the research that followed, LcS holds the unique status of being the world’s oldest probiotic strain.

2.2 2.2.1

HEALTH BENEFITS OF YAKULT AND L. CASEI SHIROTA Identification and characterisation of L. casei Shirota

LcS was initially classified as belonging to the species L. acidophilus. However, modern taxonomy now classifies it in the L. casei/paracasei group. There is still debate on naming the species in this group but, for the purposes of this chapter, the species L. casei is used to

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describe the Shirota strain due to the length of time that most of the L. casei/paracasei group were classified as L. casei. The microbiology, biochemistry, and physicochemistry of LcS have all been thoroughly studied; the structure and composition of the cell wall have also been extensively analysed. Identification of LcS at strain level was based on its reactivity to a monoclonal antibody (A. Mike, unpublished result; Yuki et al., 1999), which is highly specific for this strain. Recently the entire genome sequence of the strain was elucidated, which means that DNA sequence-based identification for this strain is now possible.

2.2.2

Beneficial modulation of the intestinal microbiota

Shirota’s initial idea was to eliminate harmful bacteria that colonise the intestines. His own study (Shirota et al., 1966) and several other preceding clinical studies (Aritaki & Ishikawa, 1962; Kikuchi, 1962; Shimizu & Shibamoto, 1964) showed this effect: Yakult consumption by children and various other population groups was associated with an increase in lactobacilli and/or bifidobacteria and a decrease in Enterobacteriaceae and enterococci. A decrease in faecal pH values was also observed, which correlated with the observed reduction in harmful bacteria. Later double-blind placebo-controlled studies (Tanaka & Ohwaki, 1994; Spanhaak et al., 1998; Matsumoto et al., 2006) confirmed that bifidobacteria and lactobacilli increase after ingestion of Yakult, although not all these studies detected a change in faecal pH. This latter observation might be explained by the apparent differences between initial faecal pH values for subjects investigated in the early studies and more recently. In the early studies, subjects had faecal pH values above 7.0, which now seems rather high as an average value. What are the actual benefits in terms of microbiota composition from drinking Yakult? A decrease in the number of Enterobacteriaceae and enterococci could be considered beneficial, because they are often implicated in opportunistic infections and some strains of these genera produce toxins. In addition, bacterial enzyme activities that lead to carcinogen production have been shown to be suppressed by LcS ingestion (Tanaka & Ohwaki, 1994; Spanhaak et al. 1998). Modification of gut metabolism by LcS was first reported by Shimizu and Shibamoto (1964), who showed a decrease in gas production associated with Yakult drinking. Analysis by Tohyama et al. (1981) of urine samples from healthy adult volunteers demonstrated that concentrations of indol/indican, p-cresol and phenol (bacterial metabolites in the intestines) inversely correlated with numbers of faecal lactobacilli (Fig. 2.1a). Mean levels of indican, p-cresol and phenol were significantly reduced in the urine of healthy volunteers during a 5-week intervention with milk containing LcS (Fig. 2.1b). Since production of these phenol compounds completely depends on the activity of microbial enzymes in the gut, this change could be attributed to a decrease in the number or activity of bacteria that produce these enzymes. Recent in vivo studies using stable isotope-labelled substances clearly showed suppression of ammonia and p-cresol production, and suppression of β-glucuronidase activity in the faeces associated with ingestion of LcS (De Preter et al., 2004, 2007, 2008).

2.2.3

Improvement of stool consistency

Early Japanese studies had already shown an LcS-associated improvement in the defecation frequency of constipated patients as well as patients who developed constipation following hospital admission and/or a change of lifestyle. As described above, Ogawa et al. (1974)

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Indican (log mg/dl)

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Fig. 2.1 (a) Correlation between faecal resident lactobacilli level and urinary indican. The data were obtained from 28 healthy male adult samples. The regression equation is y = −0.0898x + 1.478, and the correlation coefficient r = −0.532 (P < 0.01). (b) Effect of feeding of L. casei Shirota on the excretion of indican and phenols in seven healthy adults. The values for indican, p-cresol, and p-cresol + phenol during feeding periods were significantly less than before feeding (P < 0.05, respectively). (From Tohyama et al. 1981 with permission.)

also demonstrated a statistically significant improvement in defecation frequency and stool consistency for Yakult drinkers in a double-blind, placebo-controlled, crossover clinical trial. In a recent study by Matsumoto et al. (2006), 40 healthy volunteers were selected on the basis of their slightly low stool frequency (mean stool frequency: 4.1 times or less per week). In the double-blind placebo-controlled trial, the effect of Yakult containing 4 × 1010 LcS cells per bottle on their stool frequency and faecal microbiota composition was investigated. A significant improvement in stool frequency was found in the Yakult group compared with the placebo group. When a more constipated subgroup (mean stool frequency: 3.2 and 3.3 times a week) was analysed, the improvement associated with Yakult became even more obvious. In addition, total lactobacilli, total bifidobacteria, and the relative content of bifidobacteria in the faeces were significantly higher in the probiotic group during the intake period; other bacterial groups analysed showed little change during the study period.

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(a)

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Week of intervention

Final examination

Fig. 2.2 (a) Occurrence of severe and moderately severe constipation in treatment (•) and placebo (o) groups. (b) Occurrence of hard lumpy stools in treatment (•) and placebo (o) groups. (From Koebnick et al. 2003 with permission.)

A study in Germany by Koebnick et al. (2003) is another good example that shows the effect of Yakult in improving defecation frequency. This trial examined 70 subjects with chronic constipation [mean defecation frequency: three (two to five) times a week] that was not of organic or neurological origin; most participants suffered moderately severe constipation. Subjects drank one daily bottle of Yakult (6.5 × 109 cells a bottle) or placebo for 4 weeks, and recorded their symptoms throughout the study period. Occurrence of severe and moderately severe constipation as well as the occurrence of hard or lumpy stools significantly decreased for those in the treatment group compared with the control group, an effect that was apparent from the second week of intervention until the end of the study (6 weeks) (Fig. 2.2). As a result, the stool frequency of the treatment group was significantly improved at the end of the study. The mechanism underlying this constipation benefit is not yet fully understood. Animal studies with pigs showed an increase in organic acids after LcS intake that would stimulate bowel movement, but no clear indication of this was found in recent human studies (Tanaka & Ohwaki, 1994; Spanhaak et al., 1998; Matsumoto et al., 2006).

2.2.4

Protection from infection

Evaluation of an effect of LcS on infectious diseases is more difficult, but the study of Kotani et al. (1961) is a good example of an early trial. This investigated the incidence of shigellosis and Shigella carriers among 2000 members of the Japanese Self Defence Force, all of whom had the same lifestyle and diet over a long period. A significant difference in infection rates was found between the treatment group and non-treatment group, indicating that habitual intake of Yakult had a prophylactic effect on Shigella infection. Sugita and Togawa (1994) administered an LcS preparation in addition to standard treatment for acute

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infantile rotavirus infection, and found this reduced the duration of clinical illness. Cats et al. (2003) analysed the effect of Yakult intake on patients with Helicobacter pylori infection and found some effect in reducing levels of the urease breath test, a marker for H. pylori gastric bacterial load. Animal and in vitro studies on the effect of LcS have also been reported; these include infection models for enterotoxigenic Escherichia coli and Vibrio cholerae (Jacalne et al., 1990), Listeria monocytogenes (de Waard et al., 2002, 2003), Salmonella (Paubert-Braquet et al., 1995), E. coli 0157:H7 (Ogawa et al., 2001), H. pylori (Sgouras et al., 2004), and influenza virus (Hori et al., 2002; Yasui et al., 2004). All gave varying degrees of positive results, indicating potential in humans.

2.2.5

Immune modulation activity

Natural killer (NK) cell activity is thought to be a good indicator of the general strength of the human immune response; NK cells target tumours and viral-infected cells (Imai et al., 2000; Hori et al., 2002; Andoniou et al., 2005; Kalinski et al., 2005). When Morimoto et al. (2005) investigated the effect of drinking Yakult containing at least 4 × 1010 live LcS cells per bottle on NK cell activity, they found an inverse correlation between NK cell activity and the number of cigarettes smoked daily by healthy people. They further found that Yakult intake led to significant restoration of NK cell activity in this study population. The effect of Yakult was also demonstrated in two double-blind placebo-controlled crossover studies in healthy elderly people and healthy middle-aged people with relatively low NK cell activity (Takeda et al., 2006; Takeda & Okumura, 2007). Before the start of the trial, the mean NK cell activity of both treatment and placebo groups was quite similar. However, the NK cell activity of the treatment group just after the Yakult drinking period was significantly higher than that of the placebo group (Fig. 2.3). In addition, enhancement of NK cell activity was greater in subjects who had the lowest NK cell activity at baseline (Takeda et al., 2006; Takeda & Okumura, 2007). Following this work, Takeda et al. (2006) and Shida et al. (2006a) showed that interleukin (IL)-12 was involved in activation of NK cells in human peripheral blood mononuclear cells ex vivo (Fig. 2.4), which confirmed

NK cell activity (% of specific lysis)

NK cell activity (% of specific lysis)

50 40 30 20 10 0

10 20 E/T ratio

(c) 50 40

*

30 20

*

10 0

20 10 E/T ratio

Change of NK cell activity by intake (%)

(b)

(a)

30 P = 0.09

20 10 0 –10 P < 0.001

–20 –30

0

80 20 40 60 NK cell activity before intake (%)

Fig. 2.3 Change of NK cell activity in peripheral blood mononuclear cells by intake of fermented milk drink containing L. casei Shirota or a placebo. NK cell activity in peripheral blood mononuclear cells was measured (a) before and (b) after intake of fermented milk containing LcS (•) or a placebo (o). Asterisks indicate P < 0.05. (c) Relationship between change of NK cell activity 3 weeks after the beginning of intake of fermented milk containing LcS (•) or a placebo (o) and the level of NK cell activity before intake. (From Takeda et al. 2006 with permission of Blackwell Publishing Ltd.)

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24

Probiotics and Health Claims (a)

(b) 4 IL-6 (ng/ml)

4 3 2 1 0

None

Cont IgG

NK cell activity (LU/106 cells)

(c)

3 2 1 0

αIL-12

None

αIL-12

Cont IgG

(d) 40 30 20 10 0

None

Cont IgG

αIL-12

NK cell activity (LU/106 cells)

IFN-γ (ng/ml)

5

3 2 1 0

0

0.1

1

10

rIL-12 added (ng/ml)

Fig. 2.4 Involvement of IL-12 in LcS-triggered enhancement of NK cell activity. Peripheral blood mononuclear cells were cultured with LcS for 6 days in the absence (None) or presence of control mouse IgG (Cont IgG) or anti-IL-12 monoclonal antibody (αIL-12). (From Takeda et al. 2006 with permission of Blackwell Publishing Ltd.)

in vitro and in vivo animal studies indicating lactobacilli activation of macrophages and dendritic cells to produce IL-12 (Shida et al., 2006b). Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM) is a disease specific to patients with HTLV-1 infection, who present with neuronal paralytic symptoms. Matsuzaki et al. (2005) demonstrated that Yakult intervention in HAM patients could ameliorate their neuronal as well as urinary symptoms with simultaneous activation of NK cell activity. The aetiology is still not understood for chronic fatigue syndrome (CFS), a disease that presents as continual fatigue and severe anxiety, often with bowel discomfort. Recently Maher et al. (2005) and Mihaylova et al. (2007) demonstrated that the syndrome strongly correlates with a decrease in NK cell and cytotoxic T-cell activity or in blood perforin levels, which are important for NK cell activation. Rao et al. (2009) conducted a double-blind placebo-controlled pilot study in 35 CFS patients investigating the effect of Yakult intake on CFS-related scores as well as the faecal microbiota. The Beck Anxiety Inventory score, but not the Beck Depression Inventory score, was significantly decreased in the Yakult group compared with the placebo group, and fecal bifidobacteria and lactobacilli also increased significantly with Yakult intake. Immune modulatory activities associated with LcS have been documented in a variety of animal studies. The following paragraphs summarise the effect of LcS in different animal models for human autoimmune disorders and allergy. Matsuzaki and his colleagues focused on diabetes mellitus, using type 1 diabetes model NOD mice (Matsuzaki et al., 1997a) and type 2 diabetes model KK-Ay mice (Matsuzaki et al., 1997b) to study the effects

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(a) Insulin

600 400

**

200 0

Cont

Plasma insulin (µU/ml)

Plasma glucose (mg/dl)

Glucose 800

150 100

LC

**

50 0

Cont

LC

(b) CD3 % of CD3

20

*

10 0

Cont

% of CD45R

CD45R

30

40 30 20 10 0

LC

Cont

CD8

CD4 10

10

*

5 Cont

LC

% of CD8

% of CD4

15

0

LC

5 0

Cont

LC

Fig. 2.5 Effect of L. casei Shirota on (a) plasma glucose and insulin and (b) cell differentiation in spleen cells. Heat-killed L. casei Shirota (2 mg/mouse) was orally administered to 4-week-old KK-Ay mice (N = 5) five times a week for 8 weeks, and plasma glucose and insulin as well as spleen cell surface markers were analysed. ** and * indicate significant difference from control (P < 0.01). (From Matsuzaki et al. 1997b with permission.)

of oral administration of heat-killed LcS. In both studies, the researchers found a dramatic improvement in diabetic symptoms after oral administration of LcS, as seen by a decrease in plasma glucose and insulin compared with placebo administration (Fig. 2.5a). Destruction of Langerhans β cells was clearly reduced in NOD mice that had consumed LcS. In addition, there were signs of modulation of immune responses that might result in suppression of the rise in number of CD4+ T cells, which are thought to be involved in autoimmunity (Fig. 2.5b). Inhibition of Langerhans β-cell reduction by LcS was also detected in an alloxan-induced diabetes model (Matsuzaki et al., 1997c), suggesting that LcS may be more generally effective in suppression of autoimmune β-cell destruction. Kato et al. (1998) analysed the effect of LcS on type II collagen-induced arthritis (CIA) using DBA/1 mice, a model for human rheumatoid arthritis, and found that orally administered LcS at doses between 0.5 × 108 and 2 × 109 cells per mouse significantly reduced the cumulative CIA development rate and mean arthritis scores after immunisation with type II collagen. CIA is caused by a humoral immune response to collagen, so anti-collagen antibody titres were analysed during the course of the study, which revealed that the expected rise in anti-collagen antibody titre was significantly suppressed at all the test doses. All these data indicate the suppressive effect of LcS on detrimental antibody production against collagen in the DBA/1 mouse.

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Human systemic lupus erythematosus is another example of autoimmune disease, and MRL/lpr mice are a model for disease progression and symptoms. It is known that antiThy-1.2 monoclonal antibody and immune suppressors such as FK506 prevent disease development and prolong the lifespan of MRL/lpr mice. Mike et al. (1999) gave LcS orally to MRL/lpr mice for 1 year and found a significant increase in their survival rate compared with a saline control group. This improvement was accompanied by an increase in I-Ak− macrophages and a decrease in the B220+ T-cell population that produces interferon (IFN)-γ, a cytokine known to have a key role in the development of the disease (Haas et al., 1997). Matsumoto et al. (2001, 2005) reported the suppressive effect of LcS on the development and exacerbation of inflammatory bowel disease (IBD)-like symptoms in different mouse models (Fig. 2.6). Recent biochemical (Matsumoto et al., 2008) and genetic (Yasuda et al., 2008) analyses have clearly shown that this immune modulation is achieved by means of a specific polysaccharide structure on the LcS cell wall. It should be noted that the inflammatory process suppressed by LcS has been linked to the development of colitisassociated cancer (Matsumoto et al., 2008). In addition, the genes responsible for the synthesis of this polysaccharide moiety on the cell wall appear to be unique to LcS (Yasuda et al., 2008). Therefore, it is possible that the immune modulation activities demonstrated for LcS are specific for this strain, and perhaps for its close relatives. Matsuzaki et al. (1998) and Shida et al. (2002) evaluated the effect of LcS on allergic reactions in different ovalbumin-induced allergy models, and both research groups demonstrated that oral administration of the probiotic was associated with a Th1-skewed immune reaction and reduction of ovalbumin-specific IgE levels in the blood. A few trials have been conducted on human allergic conditions, particularly seasonal pollen allergy, although all of these were relatively small. Ivory et al. (2008) conducted a double-blind placebo-controlled study with 20 seasonal grass pollen allergic rhinitis sufferers consuming Yakult or placebo daily for 5 months. The researchers found that LcS intervention resulted in suppression of the allergen-specific response, specifically by reduction of IL-5, IL-6, IFN-γ and antigen-specific IgE levels.

2.2.6

Prophylactic effect of L. casei Shirota on cancer development

Microorganisms such as OK-432 and BCG have been used as anticancer biological response modifiers. The first report on the effect of LcS against cancer development was published in 1981 (Yokokura et al., 1981). This led researchers to investigate the idea that oral administration of lactic acid bacteria could help prevent cancer development and/or its progression. After Asano et al. (1987) had reported the inhibitory effect of orally administrated LcS on bladder cancer cell proliferation in a mouse model, Aso et al. (1992) conducted the first human trial to investigate the effect of LcS on superficial bladder cancer development. They enrolled patients with superficial bladder cancer who had undergone transurethral resection of the cancer, gave them oral LcS or no intervention for 1 year in addition to their normal medication, and monitored any recurrence of the bladder cancer for over 1 year. The study showed that the cumulative recurrence-free rate of tumours after 430 days in the LcS group was significantly lower compared with the control group. Aso et al. (1995) conducted a second similar double-blind placebo-controlled study, and again demonstrated a statistically significant reduction in the cumulative recurrence-free rate for an LcS-treated subgroup that initially had primary multiple lesions or recurrent single lesions (Fig. 2.7). There was no

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27

(a) Control

LcS

(b)

(c) *

20

160 SAA conc. (μg/ml)

15 Scores (point)

*

200

10

120

80

5

LcS

0

Control

LcS

0

Control

40

Fig. 2.6 Effect of oral administration of L. casei Shirota on inflammatory symptoms in murine model of Crohn’s disease. SAMP1/yit mice were fed MF chow with or without L. casei Shirota. Disease activities were determined from both histological scores (a, b) and serum amyloid A protein (c). Asterisks indicate significant difference between groups (P < 0.05). (From Matsumoto et al. 2005 with permission of Blackwell Publishing Ltd.)

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Recurrence-free rate (%)

100 BLP

80 60

Placebo

40 20 0 0

100

200

300

400

500

600

700

Days Fig. 2.7 Corrected cumulative recurrence-free rates for the placebo-treated and BLP-treated patients in sum of subgroups A and B, as determined by Cox multivariate analysis. Subgroup A consists of patients with primary multiple lesions and subgroup B consists of patients with recurrent single lesions. (From Aso et al. 1995 © 1995 with permission of Elsevier.)

effect of LcS on the group which had recurrent multiple lesions. Naito et al. (2008) also conducted a prospective, randomised, double-blind, placebo-controlled trial to investigate whether LcS could enhance the prevention of recurrence during intravesical instillation of epirubicin after resection of superficial bladder cancer, and found a significant higher 3-year recurrence-free rate in the LcS group. Ohashi et al. (2002) conducted a case–control epidemiological study to assess the relationship between bladder cancer development and regular consumption of Yakult in 180 cases and 445 controls, and showed a significant correlation between regular Yakult intake and reduced risk of bladder cancer development. The data also showed that smoking positively correlated with bladder cancer development. All these results indicate the potential for LcS in reducing incidence of bladder cancer. The underlying mechanism behind this effect is not yet fully understood, but several hypotheses have been proposed with some evidence to support them. Superficial bladder cancer often recurs in patients, which indicates a possible inherited susceptibility; on the other hand, the observed efficacy of LcS administration in reducing this cancer’s recurrence strongly suggests that other factors are involved. Firstly, as Imai et al. (2000) reported, NK cell activity inversely correlates with cancer incidence and LcS can stimulate NK cell activity in various conditions, as described above. There are further reports of LcS stimulation of NK cell activity in compromised persons such as colon cancer patients (Sawamura et al., 1994) and HAM patients (Matsuzaki et al., 2005). Secondly, the mutagenic/carcinogenic activity in caecal and urinary contents can be reduced by oral intake of LcS. Hayatsu and Hayatsu (1993) demonstrated that LcS administration reduced urinary mutagenicity that was a result of eating fried ground beef. A mechanism to explain this reduction of mutagenic substances was revealed in an in vitro study by Morotomi and Mutai (1986), in which LcS was shown to be able to strongly adsorb and inactivate food-borne mutagens and carcinogens such as Trp-P-1 and Trp-P-2 (Table 2.1 and Fig. 2.8). HernandezMendoza et al. (2009) recently reported that LcS also has a strong ability to bind aflatoxin B1, a well-known carcinogen produced by fungi. Thirdly, consumption of Yakult has been linked to reduction of harmful microbial enzymes found in faeces that can deconjugate inactivated carcinogenic compounds such as conjugated bile acids (Tanaka & Ohwaki, 1994; Spanhaak et al., 1998; De Preter et al., 2008).

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Table 2.1 In vitro binding of mutagenic pyrolysates to faeces, various bacteria, and dietary fibres. Unbound mutagenic pyrolysates (%) Agent

Trp-P-1

Trp-P-2

Glu-P-1

Glu-P-2

IQ

MeIQ

MeIQx

Freeze-dried faeces #1

15

17

81

88

93

73

83

Freeze-dried faeces #2

11

12

53

64

46

30

41

6

9

86

93

64

39

59

10

9

80

90

61

31

44

6

7

73

81

49

31

50

11

19

72

80

45

24

45

7

10

58

68

26

14

27

L. casei Shirota

10

10

84

90

52

30

45

L. fermentum

14

22

79

85

54

32

47

Enterococcus faecalis

4

6

95

95

89

78

73

Bacteroides thetaiotaomicron

9

8

57

76

23

11

21

E. coli

38

34

90

98

69

55

62

Cellulose

59

68

94

96

99

93

93

1

1

5

17

4

2

3

14

16

90

91

98

93

94

Bifidobacterium breve Bifidobacterium adolescentis Eubacterium eligens Lactobacillus acidophilus L. delbrueckii subsp. bulgaricus

Corn bran Soy bean fibre

Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3-amino-1-methyl-5H-pyrido[4,3-b]indole; Glu-P-1, 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole; Glu-P-2, 2-aminodipyrido[1,2-a:3′,2′-d]imidazole; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Source: Morotomi & Mutai (1986) with permission from Oxford University Press.

100

60

60

40

40

20

20

0

0.5 1 2 4 LC9018 or corn bran (mg)

8

Corn bran LC9018

80

Trp-P-2 (% of Control)

80

bUnbound

LC9018 Corn bran aRemaining mutagenicity (% of Control)

100

0

Fig. 2.8 Inhibitory effect of L. casei Shirota and corn bran on the mutagenicity of Trp-P-2 for S. typhimurium TA98 and the binding of Trp-P-2 to L. casei Shirota and corn bran under the Ames assay conditions. (From Morotomi & Mutai 1986 with permission from Oxford University Press.)

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LcS inhibition of colon tumour development has also been reported in human trials. Ishikawa et al. (2005) examined the recurrence rates of colon tumours in patients who had previously had resection of at least two such tumours. Throughout a monitoring period of 4 years, the patients were given LcS freeze-dried preparation, wheat bran, both preparations, or nothing. While no improvement was observed with the wheat bran group compared with the controls, after 2 years the cumulative recurrence rate for the LcS group was significantly reduced to 76% of the control group’s rate. After 4 years the recurrence rate was still 85% of the control group, although this value was no longer statistically significant. The aetiology of colon cancer is also not yet fully understood. There is strong evidence to suggest a genetic link for development of certain types of colon cancer but it is also widely accepted that environmental factors may have a greater influence. As mentioned above, cancer development may be influenced by mutagenic/carcinogenic compounds, ingested as foods or produced in the gut by the metabolic activity of the intestinal bacteria. A strong immune response involving, for instance, NK cell activity and dendritic cells could help delay or prevent cancer progression, and LcS may stimulate the innate immune system. It has also been established that patients with IBD have an increased risk of developing colorectal cancer. LcS has been shown to reduce inflammatory responses and IBD symptoms in animal models and in human peripheral mononuclear cells taken from IBD patients (Matsumoto et al., 2005). More recently, it has become widely accepted that chronic inflammation can lead to the development of various different cancers. Colitisassociated cancer is a typical example; LcS can reduce the development of colitis-associated cancer by suppressing the activated immune system (Matsumoto et al., 2008). The anticancer effect of LcS has been demonstrated in several animal models (Matsuzaki & Yokokura, 1989; Tomita et al., 1994; Takagi et al., 1999, 2001). More recently, it was reported that enterotoxigenic Bacteroides fragilis, a human commensal, promotes colitis and subsequent colon tumorigenesis by activating the Th17 response (Wu et al., 2009). This finding indicates the importance of both a balanced immune system and a balanced intestinal microbiota in reducing the risk of cancer development.

2.3

SAFETY

Fermented foods containing live bacteria and yeast have been consumed for thousands of years, showing clearly that fermented foods have a long history of safe use. The United States Food and Drug Administration has declared that foods that have been consumed for over 30 years can be considered as safe. Yakult is a fermented food containing live cells of L. casei, a lactic acid bacterial species that occurs widely in traditional fermented foods. The particular strain in Yakult, LcS, has been consumed in Japan since 1935. Currently, more than 28 million bottles of this probiotic drink containing LcS are consumed daily by people around the world, without any problem. Therefore there are no safety concerns with regard to this probiotic strain. However, to ensure consumer protection, the Yakult company has continuously accumulated as much safety data for LcS as possible. The strain has passed a range of safety tests, including studies for general acute and chronic toxicity, genotoxicity, mutagenicity, antibiotic resistance, and immunology. In a pathogenicity study involving a rabbit experimental endocarditis model, LcS was shown to be one of the safest strains tested (Asahara et al., 2003). In addition, no adverse effects have been reported in the high number of human trials in which LcS has been used, which include an increasing number of studies

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that have used probiotic and synbiotic LcS preparations in critically ill patients (Candy et al., 2001; Kanamori et al., 2002, 2004; Sugawara et al., 2006; Shimizu et al., 2009).

2.4

HEALTH CLAIMS FOR L. CASEI SHIROTA AND THE PRODUCT YAKULT

Based on the scientific evidence that supports LcS and its associated product Yakult, health claims for Yakult and related products have been approved in five different countries. The exact content and wording of these approved health claims differ according to each country’s regulations and policies for functional foods (Table 2.2). Japan was the first country where Yakult received such an approval, with the granting of Food for Specified Health Use (FOSHU) status in 1998. After this health claim legislation

Table 2.2 Health claims for Yakult in different countries. Country

Product

Conditions

Date of approval

Health claim

Japan

Yakult and its related products

1 bottle per day

20 May 1998

L. casei strain Shirota in this product reaches the intestine alive, makes our intestinal microbiota improved by increasing beneficial bacteria and decreasing harmful bacteria, and makes our intestinal environment better.

Brazil

Yakult

15 February 2001

L. casei Shirota in Yakult improves the gut microbiota balance and improves bowel habit.

Taiwan

Yakult

2 bottles per day

22 January 2003

L. casei Shirota in Yakult reaches the intestines alive, increases beneficial bacteria in the intestines, and stimulates the bowel movement.

Yakult300LT

1 bottle per day

22 May 2008

L. casei Shirota in Yakult reaches the intestines alive, increases beneficial bacteria in the intestines, and stimulates the bowel movement. L. casei Shirota stimulates immune function by activating NK cell and macrophage activities and by stimulating antibody production.

China

Yakult

2 bottles per day

30 December 2004

Yakult supports the improvement of gut microbiota, and stimulates immune functions.

The Netherlands

Yakult

1 bottle per day

10 November 2006

Yakult may improve bowel habit in subjects who are susceptible to constipation. Yakult may support a well-balanced gut microbiota through an increased number of lactobacilli.

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was established in Japan in 1991, Yakult was one of the first probiotic products to be given approval for a health claim. The detailed claim wording is as follows: ‘L. casei strain Shirota in this product reaches the intestine alive, makes our intestinal microbiota improved by increasing beneficial bacteria and decreasing harmful bacteria, and make our intestinal environment better’. Other countries where Yakult has been given health claim approval are Brazil (2001), Taiwan (2003), China (2004), and the Netherlands (2006). Most of the claims relate to improvement of the intestinal microbiota and digestive function. In addition, a claim for immune stimulation has been approved in China and Taiwan, based on the total evidence available and studies published in scientific journals as well as studies conducted in China.

2.5

CURRENT PERSPECTIVES

About 100 years after Metchnikoff presented to the world his vision on the role of lactic acid bacteria for a healthy gut microbiota balance and longevity, the ‘probiotic’ concept has become well established, with LcS holding a position of honour as the world’s oldest probiotic strain. Over its long history of research, various health benefits of LcS and its product Yakult have been investigated and demonstrated in a broad range of clinical and animal studies. These benefits include normalisation of bowel habit, improvement of the gut microbiota and its metabolism, risk reduction of certain infectious diseases, risk reduction of cancer development, immune modulation, and amelioration of autoimmunity. Recent research has clearly shown how the gut and its surroundings play a central role in the body, which is absolutely in line with the ideas first proposed by Metchnikoff and Shirota more than 100 years and 75 years ago respectively. As well as this, there is a growing body of evidence that the gut microbiota, particularly certain bacterial species, genera and/or phyla, influence various cellular and body processes that directly influence our health and ability to respond to disease (Turnbaugh et al., 2006; Goehler et al., 2007; Strober et al., 2007; Gasbarrini et al., 2008; Ou et al., 2009; Wu et al., 2009). Therefore it is vital that our knowledge of probiotic effects is further increased, not only in how these ‘friendly’ microorganisms directly influence the body’s various mechanisms but also to better understand how they affect the gut microbiota. In this respect, a more detailed analysis of our gut microbiota and detection of individual specific microorganisms in health and disease states could be critically important in understanding the role and mechanisms of probiotics in health maintenance. The health benefits of LcS are multiple; some may involve direct interaction/cross-talk between LcS and the host cells, while others may involve bacteria–bacteria interaction/cross-talk or modulation of the gut microbiota. Our goal is to use existing and emerging analytical techniques to further elucidate the mechanisms of activity for LcS by which this strain exerts its various health benefits. Through such sound scientific evidence, LcS will continue to be acknowledged as the world’s oldest probiotic with several established health claims in different countries.

REFERENCES Andoniou CE, van Dommelen SL, Voigt V et al. (2005) Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity. Nat Immunol 6:1011–1019. Aritaki S, Ishikawa S (1962) Application of a fermented milk drink containing Lactobacillus acidophilus in the field of paediatrics [in Japanese]. Jpn J Paediatrics 66:125–130.

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Asahara T, Takahashi M, Nomoto K et al. (2003) Assessment of safety of Lactobacillus strains based on resistance to host innate defence mechanisms. Clin Diagn Lab Immunol 10:169–173. Asano M, Karasawa E, Takayama T (1987) Antitumor activity of Lactobacillus casei (LC 9018) against experimental mouse bladder tumor (MBT-2). J Urol 136:719–721. Aso Y, Akazan H (1992) Prophylactic effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer. BLP Study Group. Urol Int 49:125–129. Aso Y, Akaza H, Kotake T et al. (1995) Preventive effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer in a double-blind trial. Eur Urol 27:104–109. Candy DC, Densham L, Lamont LS et al. (2001) Effect of administration of Lactobacillus casei Shirota on sodium balance in an infant with short bowel syndrome. J Pediatr Gastroenterol Nutr 32:506–508. Cats A, Kuipers EJ, Bosschaer MAR, Pot RGJ, Vandenbroucke-Grasus CMJE, Kusters JG (2003) Effect of frequent consumption of a Lactobacillus casei-containing milk drink in Helicobacter pylori-colonized subjects. Aliment Pharmacol Ther 17:429–435. De Preter V, Geboes K, Verbrugghe K et al. (2004) The in vivo use of the stable isotyope-labelled biomarkers lactose-[15N]ureide and [2H4]tyrosine to assess the effects of pro- and prebiotics on the intestinal flora of healthy human volunteers. Br J Nutr 92:439–446. De Preter V, Vanhoutte T, Huys G et al. (2007) Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans. Am J Physiol 292:G358–G368. De Preter V, Raemen M, Cloetens L, Houben E, Rutgeerts P, Verbeke K (2008) Effect of dietary intervention with different pre- and probiotics on intestinal bacterial enzyme activities. Eur J Clin Nutr 62:225–231. de Waard R, Garssen J, Bokken GCAM, Vos JG (2002) Antagonistic activity of Lactobacillus casei strain Shirota against gastrointestinal Listeria monocytogenes infection in rats. Int J Food Microbiol 73:93–100. de Waard R, Claassen E, Bokken GCAM, Buiting B, Garssen J, Vos JG (2003) Enhanced immunological memory responses to Listeria monocytogenes in rodents, as measured by delayed-type hypersensitivity (DTH), adoptive transfer of DTH, and protective immunity, following Lactobacillus casei Shirota ingestion. Clin Diagn Lab Immunol 10:59–65. Gasbarrini A, Lauritano EC, Garcovich M, Sparano L, Gasbarrini G (2008) New insights into the pathophysiology of IBS: intestinal microflora, gas production and gut motility. Eur Rev Med Pharmacol Sci 12(Suppl 1):111–117. Goehler LE, Lyte M, Gaykema RP (2007) Infection-induced viscerosensory signals from the gut enhance anxiety: implications for psychoneuroimmunology. Brain Behav Immun 21:721–726. Haas C, Ryffel B, Le Hir M (1997) IFN-gamma is essential for the development of autoimmune glomerulonephritis in MRL/lpr mice. J Immunol 158:5484–5491. Hayatsu H, Hayatsu T (1993) Suppressing effect of Lactobacillus casei administration on the urinary mutagenicity arising from ingestion of fried ground beef in the human. Cancer Lett 73:173–179. Hernandez-Mendoza A, Guzman-de-Peña D, Garcia HS (2009) Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria. J Appl Microbiol 107:395–403. Hori T, Kiyoshima J, Shida K, Yasui H (2002) Augmentation of cellular immunity and reduction of influenza virus titer in aged mice fed Lactobacillus casei strain Shirota. Clin Diagn Lab Immunol 9:105–108. Imai K, Matsuyama S, Miyake S, Suga K, Nakachi K (2000) Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence. Lancet 356:1795–1799. Ishikawa H, Akedo I, Otani T et al. (2005) Randomized trial of dietary fiber and Lactobacillus casei administration for prevention of colorectal tumors. Int J Cancer 116:762–767. Ivory K, Chambers SJ, Prieto C, Pin E, Arquez JL, Nicoletti C (2008) Oral delivery of Lactobacillus casei Shirota modifies allergen-induced immune responses in allergic rhinitis. Clin Exp Allergy 38:1282–1289. Jacalne AV, Jacalne RR, Hirano H, Suetomi T, Villahermosa CG, Castaneda I (1990) In vivo studies on the use of Lactobacillus casei (Yakult strain) as biological agent for the prevention and control of diarrhea. Acta Medica Philippina 26:116–122. Kalinski P, Mailliard RB, Giermasz A et al. (2005) Natural killer–dendritic cell cross-talk in cancer immunotherapy. Expert Opin Biol Ther 5:1303–1315. Kanamori Y, Hashizume K, Sugiyama M, Moritomi M, Yuki N, Tanaka R (2002) A novel synbiotic therapy dramatically improved the intestinal function of a pediatric patient with laryngotracheo-esophageal cleft (LTEC) in the intensive care unit. Clin Nutr 21:527–530.

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Kanamori Y, Sugiyama M, Hashizume K, Yuki N, Morotomi M, Tanaka R (2004) Experience of long-term synbiotic therapy in seven short bowel patients with refractory enterocolitis. J Pediatr Surg 39:1686–1692. Kato I, Endo-Tanaka K, Yokokura T (1998) Suppressive effects of the oral administration of Lactobacillus casei on type-II collagen-induced arthritis in DBA/1 mice. Life Sci 63:635–644. Kawamura T, Ohnuki K, Ichida F (1981) Clinical study of BLG-01 including Lactobacillus casei on irregular movement and abdominal discomfort [in Japanese]. Pharmacol Ther 9:4361–4370. Kikuchi K (1962) Fluctuation of number of coli bacilli and lactobacilli in human stool by peroral administration of Yakult [in Japanese]. Teishin Medical J 14:65–66. Koebnick C, Wagner I, Leitzmann P, Stern U, Zunft HF (2003) Probiotic beverage containing Lactobacillus casei Shirota improves gastrointestinal symptoms in patients with chronic constipation. Can J Gastroenterol 17:39–48. Kotani S, Chiba H, Takeuchi K, Uchida K, Shimizu T, Sonoguchi T (1961) A study of the influence of cow’s milk fermented by some kind of ‘Family Lactobacillaceae’ upon human body [in Japanese]. Jpn J Public Hygiene 8:29–53. Maher KJ, Klimas NG, Fletcher, MA (2005) Chronic fatigue syndrome is associated with diminished intracellular perforin. Clin Exp Immunol 142:505–511. Matsumoto K, Takada T, Shimizu K et al. (2006) The effects of a probiotic milk product containing Lactobacillus casei strain Shirota on the defecation frequency and the intestinal microflora of sub-optimal health state volunteers: a randomized placebo-controlled cross-over study. Biosci Microflora 25:39–48. Matsumoto S, Watanabe N, Imaoka A, Okabe Y (2001) Preventive effects of Bifidobacterium- and Lactobacillus-fermented milk on the development of inflammatory bowel disease in senescenceaccelerated mouse P1/Yit strain mice. Digestion 64:92–99. Matsumoto S, Hara T, Hori T et al. (2005) Probiotic Lactobacillus-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines in lamina propria mononuclear cells. Clin Exp Immunol 140:417–426. Matsumoto S, Hara T, Nagaoka M et al. (2008) A component of polysaccharide peptidoglycan complex on Lactobacillus induced an improvement of murine model of inflammatory bowel disease and colitisassociated cancer. Immunology 128:e170–e180. Matsuzaki T, Yokokura T (1989) Antitumor effect of oral administration of Lactobacillus casei in newborn mice [in Japanese]. Igaku no Ayumi 150:745–746. Matsuzaki T, Nagata Y, Kado S et al. (1997a) Prevention of onset in an insulin-dependent diabetes mellitus model, NOD mice, by oral feeding of Lactobacillus casei. APMIS 105:643–649. Matsuzaki T, Yamazaki R, Hashimoto S, Yokokura T (1997b) Antidiabetic effects of an oral administration of Lactobacillus casei in a non-insulin-dependent diabetes mellitus (NIDDM) model using KK-Ay mice. Endocrine J 44:357–365. Matsuzaki T, Nagata Y, Kado S, Uchida K, Hashimoto S, Yokokura T (1997c) Effect of oral administration of Lactobacillus casei on alloxan-induced diabetes in mice. APMIS 105:637–642. Matsuzaki T, Yamazaki R, Hashimoto S, Yokokura T (1998) The effect of oral feeding of Lactobacillus casei strain Shirota on immunoglobulin E production in mice. J Dairy Sci 81:48–53. Matsuzaki T, Saito M, Usuku K, Nose H, Izumi S, Arimura K (2005) A prospective uncontrolled trial of fermented milk drink containing Lactobacillus casei strain Shirota in the treatment of HTLV-1 associated myelopathy/tropical spastic paraparesis. J Neurol Sci 237:75–81. Mihaylova I, DeRuyter M, Rummens JL, Bosmans E, Maes M (2007) Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells. Neuroendocrinol Lett 28:477–483. Mike A, Nagaoka N, Tagami Y et al. (1999) Prevention of B220+ T cell expansion and prolongation of lifespan induced by Lactobacillus casei MRL/lpr mice. Clin Exp Immunol 117:368–375. Morimoto K, Takeshita T, Nanno M, Tokudome S, Nakayama K (2005) Modulation of natural killer cell activity by supplementation of fermented milk containing Lactobacillus casei in habitual smokers. Prev Med 40:589–594. Morita M (1973) Clinical use of a high-concentration Lactobacillus casei preparation [in Japanese]. J New Remed Clin 22:1351–1353. Morotomi M, Mutai M (1986) In vitro binding of potent mutagenic pyrolyzates to intestinal bacteria. J Natl Cancer Inst 77:195–201. Naito S, Koga H, Yamaguchi A et al. (2008) Prevention of recurrence with epirubicin and Lactobacillus casei after transurethral resection of bladder cancer. J Urol 179:485–490.

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Numata K (1973) Clinical effect of a high concentrate lactobacilli preparation on chronic constipation [in Japanese]. Clinical Report 7:1856–1857. Ogawa M, Shimizu K, Nomoto K et al. (2001) Protective effect of Lactobacillus casei strain Shirota on Shiga toxin-producing Escherichia coli 0157:H7 infection in infant rabbits. Infect Immun 69:1101–1108. Ogawa T, Hirai R, Nakakuni H et al. (1974) Clinical experience with the use of the high-concentration lactic acid bacteria preparation LP-201 to treat habitual constipation: double-blinded placebo-controlled crossover study [in Japanese]. Clinical Report 8:1085–1092. Ohashi Y, Nakai S, Tsukamoto T et al. (2002) Habitual intake of lactic acid bacteria and risk reduction of bladder cancer. Urol Int 68:273–280. Ohta Z (1972) Clinical treatment to bowel discomfort of SMON patients with high-concentration Lactobacillus preparation [in Japanese]. Nihon Iji Shinpo No. 2514:25–31. Ou G, Hedberg M, Hörstedt P et al. (2009) Proximal small intestinal microbiota and identification of rodshaped bacteria associated with childhood celiac disease. Am J Gastroenterol 104:3058–3067. Paubert-Braquet M, Gan X-H, Gaudey C et al. (1995) Enhancement of host resistance against Salmonella typhimurium in mice fed a diet supplemented with yogurt or milks fermented with various Lactobacillus casei Shirota. Int J Immunotherapy 11:153–161. Rao AV, Bested AC, Beaulne TM et al. (2009) A randomized double-blind placebo-controlled pilot study of a probiotic in emotional symptoms of chronic fatigue syndrome. Gut Pathogens 1:6. Sawamura A, Yamaguchi Y, Tohge T et al. (1994) Enhancement of immuno-activities by oral administration of Lactobacillus casei in colorectal cancer patients [in Japanese]. Biotherapy 8:1567–1572. Sgouras D, Maragkoudakis P, Petraki K et al. (2004) In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota. Appl Environ Microbiol 70:518–526. Shida K, Takahashi R, Iwadate E et al. (2002) Lactobacillus casei strain Shirota suppressed serum immunoglobulin E and immunoglobulin G1 responses and systemic anaphylaxis in a food allergy model. Clin Exp Allergy 32:563–570. Shida K, Suzuki T, Kiyoshima-Shibata J, Shimada S, Nanno M (2006a) Essential roles of monocytes in stimulating human peripheral blood mononuclear cells with Lactobacillus casei to produce cytokines and augment natural killer cell activity. Clin Vaccine Immunol 13:997–1003. Shida K, Kiyoshima-Shibata J, Nagaoka M, Watanabe K, Nanno M (2006b) Induction of interleukin-12 by Lactobacillus strains having a rigid cell wall resistant to intracellular digestion. J Dairy Sci 89:3306–3317. Shimizu K, Ogura H, Goto M et al. (2009) Synbiotics decrease the incidence of septic complications in patients with severe SIRS: a preliminary report. Dig Dis Sci 54:1071–1078. Shimizu S, Shibamoto G (1964) Clinical observation of the effect of a strain of acidophilic lactic acid bacteria (Yakult strain) on the intestinal gas production [in Japanese]. Proc Tokyo Medical College 21:1–5. Shirota M, Aso K, Iwabuchi A (1966) Study on the intestinal microflora: effect of the administration of Lactobacillus acidophilus strain Shirota on the composition of the intestinal microflora of healthy children [in Japanese]. Proc Jpn Soc Microbiol 21:274–283. Spanhaak S, Havenaar R, Schaafsma G (1998) The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal microflora and immune parameters in human. Eur J Clin Nutr 52:899–907. Strober W, Fuss I, Mannon P (2007) The fundamental basis of inflammatory bowel disease. J Clin Invest 117:514–521. Sugawara G, Nagino M, Nishio H et al. (2006) Perioperative synbiotic treatment to prevent postoperative infectious complications in biliary cancer surgery: a randomized controlled trial. Ann Surg 244:706–14. Sugita T, Togawa M (1994) Efficacy of Lactobacillus preparation Biolactis powder in children with rotavirus enteritis [in Japanese]. Jpn J Pediatr 47:2755–2762. Takagi A, Matsuzaki T, Sato M, Nomoto K, Morotomi M, Yokokura T (1999) Inhibitory effect of oral administration of Lactobacillus casei on 3-methylcholanthrene-induced carcinogenesis in mice. Med Microbiol Immunol 188:111–116. Takagi A, Matsuzaki T, Sato M, Nomoto K, Morotomi M, Yokokura T (2001) Enhancement of natural killer cytotoxicity delayed murine carcinogenesis by a probiotic microorganism. Carcinogenesis 22:599–605. Takeda K, Okumura K (2007) Effects of a fermented milk drink containing Lactobacillus casei strain Shirota on the human NK-cell activity. J Nutr 137:791S–793S. Takeda K, Suzuki T, Shimada S-I, Shida K, Nanno M, Okumura K (2006) Interleukin-12 is involved in the enhancement of human natural killer cell activity by Lactobacillus casei Shirota. Clin Exp Immunol 146:109–115.

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Tanaka R, Ohwaki M (1994) Effect of consumption of fermented milk drink containing Lactobacillus casei strain Shirota on the intestinal microflora and its metabolic activity [in Japanese]. Proc Int Flora Symposium 12:85–104. Tohyama K, Kobayashi Y, Kan T, Yazawa K, Terashima T, Mutai M (1981) Effect of Lactobacillus on urinary indicant excretion in gnotobiotic rats and in man. Microbiol Immunol 25:101–112. Tomita K, Akaza H, Nomoto K et al. (1994) Influence of Lactobacillus casei on rat bladder carcinogenesis [in Japanese]. Nippon Hinyokika Gakkai Zasshi 85:655–663. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027–1031. Wu S, Rhee K-J, Albesiano E et al. (2009) A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses. Nat Med 15:1016–1022. Yasuda E, Serata M, Sako T (2008) Suppressive effect on activation of macrophages by Lactobacillus casei strain Shirota genes determining the synthesis of cell wall-associated polysaccharides. Appl Environ Microbiol 74:4746–4755. Yasui H, Kiyoshima J, Hori T (2004) Reduction of influenza virus titer and protection against influenza virus infection in infant mice fed Lactobacillus casei Shirota. Clin Diagn Lab Immunol 11:675–679. Yokokura T, Kato I, Mutai M (1981) Antitumor effect of Lactobacillus casei (LC 9018) [in Japanese]. In: Mitsuoka T (ed.) Intestinal Flora and Tumor Development. Tokyo: Gakkai Shuppan Center, pp. 125–137. Yuki N, Watanabe K, Mike A et al. (1999) Survival of a probiotic, Lactobacillus casei strain Shirota, in the gastrointestinal tract: selective isolation from faeces and identification using monoclonal antibodies. Int J Food Microbiol 48:51–57.

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3

Probiotics: from Strain to Product

Arthur C. Ouwehand, Lisbeth Søndberg Svendsen and Gregory Leyer

3.1

INTRODUCTION

The most widely accepted definition of a probiotic is the one suggested by a joint FAO/ WHO working group (2002): ‘Live microorganisms which when administered in adequate amounts confer a health benefit on the host.’ Although non-viable ‘probiotics’ have been shown to mediate certain health benefits (Ouwehand & Salminen 1998), the definition indicates that probiotics will have to be live at the moment of consumption. The definition does not stipulate that the microbes have to be viable in the gastrointestinal tract. However, the general interpretation is that this is desirable as metabolic activity by the probiotic microbes can be expected and may be (partially) responsible for the health benefits. Viability is more difficult to define in microbes than one would think. In general, culturability is taken as a sign of viability in the case of probiotics (Zitz et al., 2007). However, microbes may become damaged and enter a so-called ‘viable but non-culturable’ state; probiotics are no exception to this (see also Chapter 18). Other techniques are available to investigate the viability of microbes, including staining techniques to determine cell membrane integrity or enzyme activity (Lahtinen et al., 2006). Membrane integrity can also be determined by combining polymerase chain reaction (PCR) with propidium monoazide (Kramer et al., 2009). The discussion on methods to determine the viability of probiotics and what the different states of microbial viability mean falls outside the scope of this chapter, but it is important to bear this in mind. More details regarding viability properties are dealt with in Chapter 18. Formulating the food product in such a way that it facilitates the survival of an included probiotic is one strategy. Another strategy is to select robust strains or protect sensitive strains by technological means such as microencapsulation of the strain or the use packaging that protects the strain from the food until the moment of consumption. This strategy is not only of relevance for food products but also for probiotic feeds (for details see Chapter 15).

3.2

ISOLATING A POTENTIAL PROBIOTIC STRAIN

All development of a probiotic starts with the isolation of the strain. For this, it is good to take into account the selection criteria that are commonly mentioned in probiotic reviews, and their relevance (Box 3.1).


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Box 3.1 ● ● ● ● ● ● ●

Common selection criteria for probiotics

Human origin, if intended for human use Safe for food and clinical use Acid and bile resistance Adhesion behaviour to mucosal surfaces Accurate taxonomic identification and characterisation Clinically validated and documented health effects Good technological properties

Source: Modified after Ouwehand et al. (1999).

Human origin is often mentioned as a selection criterion. It is difficult to determine whether an isolate is really part of the core microbiota of the human it was isolated from. At best it can be said it was isolated from a particular source. It is recommended that the date and site of the first isolation of a particular strain is recorded, but it should be well understood that this may not reflect the usual or natural habitat of the isolate. Neither does isolation from a particular host guarantee probiotic properties. For safety, it would nevertheless be advised not to use isolates from diseased tissues or patients. Safety should be a prime selection criterion, but is often taken for granted in the case of lactobacilli and bifidobacteria. Indeed, lactobacilli and bifidobacteria are rarely involved in disease (Salminen et al., 2002). Specific species of Lactobacillus and Bifidobacterium have therefore received qualified presumption of safety (QPS) status in the European Union (Barlow et al., 2007) (Table 3.1). Nevertheless, in rare cases, probiotics may also be involved in infections (Boyle et al., 2006). However, it does seem that not so much the probiotic but rather the patient is the risk. Severely reduced immune function is often associated with these rare cases of Lactobacillus infection (Sharp et al., 2009). Most probiotics will be consumed orally. In this case, acid and bile resistance are preferable traits since the general perception is that it is desirable for a probiotic to survive gastrointestinal transit. Comparison of in vitro and in vivo survival does indicate that acid and bile resistance indeed correlates with a higher faecal recovery (Dunne et al., 2001). Of course, for probiotics that are administered into the urogenital tract or which are functioning in the oral cavity, these criteria or not relevant. Originally, adhesion to mucosal surfaces was considered important as it would facilitate colonisation. However, it has repeatedly been shown that probiotics do not colonise. It still remains to be shown whether a non-adherent strain would have a shorter persistence then an isogenic adherent strain. Nevertheless, adhesion may be important for immune modulation and competitive exclusion, but this remains to be documented (Ouwehand & Salminen, 2003). Furthermore, a non-adherent strain such as Lactobacillus casei Shirota has many documented health benefits (Matsuzaki, 2003). The importance of correct taxonomic identification is exemplified by the European QPS strategy. It is also important for the manufacturer as it enables better protection of the strain in which substantial investments have been made. Current molecular techniques

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Table 3.1 Species of commonly used food microorganisms with qualified presumption of safety (QPS) status in the European Union. Genus

Species

Bifidobacterium

adolescentis animalis bifidum breve longum

Corynebacterium Lactobacillus

glutamicum acidophilus amylolyticus amylovorus alimentarius aviaries brevis buchneri casei coryneformis crispatus curvatus delbrueckii farciminis fermentum gallinarum gasseri helveticus hilgardii johnsonii kefiranofaciens kefiri mucosae panis paracasei paraplantarum pentosus plantarum pontis reuteri rhamnosus sakei salivarius sanfranciscensis

Lactococcus

lactis

Leuconostoc

citreum lactis mesenteroides

Pediococcus

acidilactici dextrinicus pentosaceus

Propionibacterium Streptococcus

freudenreichii thermophilus

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Remark

Only for production purposes

Includes L. zeae

Remains under surveillance

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Table 3.1 (cont’d ). Genus

Species

Bacillus

amyloliquefaciens atrophaeus clausii coagulans fusiformis lentus licheniformis megaterium mojavensis pumilus subtilis vallismortis

冎

Geobacillus

stearothermophillus

Hanseniaspora

uvarum

Kluyveromyces

lactis marxianus

Pichia

angusta anomala

Saccharomyces

bayanus cerevisiae

Remark

Absence of emetic food poisoning toxins with surfactant activity and absence of enterotoxic activity

S. cerevisiae subtype S. boulardii is contraindicated for patients of fragile health and for patients with a central venous catheter in place

pastorianus Schizosaccharomyces

pombe

Xanthophyllomyces

dendrorhous

Source: Andreoletti et al. (2008) and Barlow et al. (2007).

may not allow strain identification, with the exception of whole-genome sequencing (Barrangou et al., 2009). It is therefore of major importance that the strain is deposited in a public strain collection, for safe-keeping and future reference, though not for general public access. By definition, microbes are only probiotics when they have documented health effects. Appropriate testing is therefore of main importance. Also, regulation is becoming increasingly strict in this respect, as health claims can only be made on the basis of scientific evidence (European Parliament and the Council of the European Union, 2006). The challenges in conducting human feeding trials will be discussed in more detail in section 3.5. Last but not least are the technological properties of the strain. It is not uncommon that academic researchers isolate a promising strain that in the end appears to be difficult to grow on a large scale. Probiotics are often intestinal (faecal) isolates and may have very specific growth requirements (fastidious, absolute anaerobicity, etc.). The challenges in the large-scale production of probiotics are discussed in section 3.3.

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Probiotics: from Strain to Product Fermentation

Concentration

Stabilisation

Freezing

Drying

Blending

Packaging

41

Storage

Fig. 3.1 Schematic representation of the manufacture of probiotics.

3.3

PRODUCING PROBIOTIC STRAINS ON A LARGE SCALE

As consumers become increasingly interested in their personal health and expect the products they eat to be healthy or even capable of preventing illness, those consumer needs and preferences are translated into a product format concept. In order to provide consumers with products that provide meaningful levels of probiotics, several strain development considerations are warranted that are complementary to a functional probiotic dairy product. Prior to manufacturing a probiotic strain, there are several critical product development considerations that impact the scale-up process development: the shelf-life requirements of the intended finished product, delivery product matrix formulation (especially pH), and daily probiotic dosage required. Most refrigerated food products have shelf-lives of less than 60 days, whereas a probiotic delivered into a dietary supplement can have a non-refrigerated shelf-life of more than 2 years. The activity of the targeted strain or strains needs to be carefully monitored during the developmental process to ensure that the probiotic and the product matrix are compatible. A probiotic strain manufacturing process can vary from supplier to supplier, and a simplified version (post strain selection) of the process is provided in Figure 3.1. Because probiotic strains can vary widely in their tolerance to processing conditions, a consideration of the inherent probiotic traits along with an understanding of finished product matrix formulation can be critical in developing a suitable process. One of the critical factors to consider is whether activity of the probiotic is expected in the finished product. For example, in most dietary supplement or powder applications, a long shelf-life, tolerance to desiccation, and elevated storage temperatures and cell dormancy are required. In a different product example, like a dairy matrix, cell activity and resistance to low pH and oxygen are required. After selecting the strain and performing some initial processability experiments, such as determining cell yield and concentration capability in small laboratory trials, one would embark on a fermentation optimisation exercise. The formulation of the fermentation medium will be a critical factor in determining downstream processability (i.e. stability or activity, depending on the intended application). Careful examination and optimisation of the fermentation ingredients, evaluating multiple lots of the same ingredients, should be employed in any fermentation optimisation exercise. When selecting fermentation ingredients, an understanding of the allergen status requirements of the final product is important to properly select for alternative ingredients. Many ingredients can be selected to be void of milk, soy, gluten, or other allergens. Other requirements, like the kosher and halal status, may impact fermentation ingredient selection. Additional parameters, such as the pH set-point the organism is grown at, the temperature of growth, the oxygen tension, agitation speed in the fermenter and even light (Kiviharju et al., 2004), all need to be tailored to the particular organism. After fermentation, a large amount of biomass has been produced that, for most downstream processes, needs to be concentrated. The traditional methods of concentration would involve centrifugation and/or membrane filtration. These methods should be carefully selected in order to achieve the desired biomass concentration factor, while not

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imparting a negative impact on the organism. The concentration step should be done under cold conditions in order to better preserve the physiological state of the cells. Once concentrated, an important decision needs to be made regarding preservation of the cells. Preservation of biological materials is a complex undertaking which is relevant to many industries, including pharmaceutical, biotechnology and food. Basic studies with bacteria have shown microscopically that disruption in cellular membranes (leakage, fusion and rupture) is correlated with cell death. Much of the bacterial preservation technology employed industrially is centred on preserving the state of biological membranes and associated proteins through the drying process (freeze-drying, spray-drying, vacuum-drying) in order to maximise cell recovery following rehydration. To do so, compounds called cryoprotectants are used to minimise the degrading effects caused by freezing and drying. The literature has described many compounds that can provide this benefit. Some include dimethyl sulfoxide (DMSO), larger polymeric compounds and disaccharides. Cryoprotectants used in preserving probiotic bacteria have more restrictions placed on them in order to be compliant with regulatory and marketing demands of the dietary supplement and food industry. However, from a kinetic perspective, when dried the compounds used form amorphous or ‘glassy’ solids that mimic the hydrogen bonding behaviour of water at a lipid or protein surface. Both of these properties help stabilise membranes and hence the probiotic bacterium during dry storage. The stabilised material is then ready to be frozen for the freeze-drying process, and this can take place either inside the freeze-drier (tray freezing) or before entering the freezedrier. In this step, care should be taken to minimise ice crystal formation and size. After freezing, the material is ready for drying, and a traditional method would be freeze-drying. In freeze-drying, solid water is removed via sublimation, i.e. the change of state from a solid to a gas, while bypassing the liquid phase. This is performed under vacuum and at very low initial temperatures. Optimisation of the freeze-drying step is a critical process involving careful consideration of ramp-up temperature, chamber vacuum, finishing temperature, and desired residual moisture. The residual moisture content along with the stabiliser components chosen can have a very positive impact on increasing the glass transition temperature of the amorphous material and corresponding shelf-life. After the powder has been carefully milled to produce a product with appropriate particle size distribution, it is ready for blending (if the cell materials need to be diluted to achieve a particular cell count) and packaging. A variety of commercial blending options are available for use in designing a mixing process that maximises homogeneity and minimises mixing time. Careful blending tests help determine the minimum time needed to ensure adequate product homogeneity. Some blending parameters to consider include (i) avoiding prolonged exposure to the blending operation and processing environment before the product is packaged and (ii) incorporation of a pre-blend may be necessary to ensure homogeneity. Selection of the final packaging material should be made with the following considerations in mind. ● ● ● ●

To minimise oxygen and moisture transfer into the product, the use of material with low moisture vapour transmission rate is preferred. Inclusion of a desiccant can be beneficial, especially for packaging with tablets and veggie-caps. Process in a cool room with humidity control (90%) and good stability up to 6 months is very often observed. The benefit of cheese is that is provides a protective matrix that facilitates the survival of the probiotics both in the product and during digestion (Mäkeläinen et al., 2009).

3.4.3

Non-fermented milk drinks

Addition of probiotics in non-fermented milk drinks is often challenging. First, the inoculation process must be absolutely aseptic to avoid the risk of contaminants. Furthermore, it is absolutely vital to choose a strain without growth capacity as even a slight growth may result in an off taste. The milk must be stored at refrigerated temperatures as otherwise growth will take place.

3.4.4

Fruit and vegetable juices

Fruit and vegetable juices provide an alternative vehicle for the administration of probiotics (Luckow & Delahunty, 2004). They are particularly attractive for those who do not like dairy products or are milk intolerant. However, including probiotics in fruit juices and maintaining their viability is challenging as the pH is usually low (256 μg/mL) were detected in L. rhamnosus GG and LC705 strains (Klein et al., 2000). The vancomycin resistance is an intrinsic property of this species and no transferable van genes were found in the GG and LC705 strains (Tynkkynen et al., 1998; Klein et al., 2000; Kankainen et al., 2009). For dairy propionibacteria no MIC distribution data are available, but PJS was shown to be susceptible to all relevant antibiotics, except to a lower extent to aminoglycosides, to which anaerobic bacteria, as well as lactobacilli and bifidobacteria, are more resistant (Bryan & Kwan, 1981). The MICs for Bb12 (S. Tynkkynen, Valio Ltd, Finland, personal communication, and Chr. Hansen, Denmark) are within the range for other Bifidobacterium species (Mättö et al., 2007). Like many other bifidobacteria strains, Bb12 contains a tetracycline resistance gene, tet(W), but its transfer has been evaluated as highly improbable (Masco et al., 2006; Saarela et al., 2007).

16.4

CLINICAL STUDIES ON THE PROBIOTIC MULTISPECIES LGG®EXTRA COMBINATION

16.4.1

Relieving symptoms of irritable bowel syndrome

When used as a single strain probiotic, Lactobacillus GG has great potential to enhance immune responses, thus reducing the risk of infections and helping in their treatment. It has also been used with varying success to relieve the symptoms and reduce the risk of atopic dermatitis (Doron et al., 2005; Prescott & Bjorksten, 2007). However, Lactobacillus GG had only minor benefits for treatment of common intestinal discomforts in adults, such as IBS (O'Sullivan & O'Morain, 2000; Bausserman & Michail, 2005), for which current medical treatments are regarded as unsatisfactory. Thus, effective dietary therapy would be a most welcome means of relieving the symptoms of IBS. The present probiotic multispecies combination was primarily developed in order to relieve non-infectious functional gastrointestinal symptoms, especially in the adult population, although some studies were conducted in infants and children as well. The probiotic combination was tested in two long-term clinical trials for IBS patients (Kajander et al., 2005, 2008). In the first study, 103 patients fulfilling the Rome I or II criteria took part in a 6-month, randomised, double-blind placebo-controlled trial using the multispecies probiotic combination or placebo (Kajander et al., 2005). The study products were in capsule form: the active product contained the GG, LC705, PJS and Bb99 strains, with a total daily dose of 8–9 × 109 CFU (equal amount each strain), and the placebo capsules only the inert filling material. The symptoms of IBS were significantly alleviated with the probiotic supplementation. At the end of the study, the treatment difference in the baseline-adjusted total IBS symptom score was –7.7 points when the probiotic group was compared with the placebo group (P = 0.015). In the second study, 86 patients with similar criteria participated in a

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5-month intervention (Kajander et al., 2008). The study volunteers were randomised to take the study product, a milk drink containing the probiotic combination (GG, LC705, PJS and Bb12, total dose about 109 CFU, each strain, respectively), or a similar placebo drink without live bacteria. At the end of the intervention the total IBS score had decreased 14 points in the probiotic group compared with 3 points in the placebo group (P = 0.0083). Altogether, the results corresponded to an average score reduction of 40% for the probiotic supplementation, and less than 10% for the placebo supplementation. Rumbling and distension were the most markedly alleviated individual symptoms (P = 0.008 and P = 0.023, respectively). Quality of life of the study patients was also recorded, using the Health Related Quality of Life (HRQL) questionnaire in the first study (Kajander et al., 2005) and a more defined IBSQL questionnaire in the second study (Kajander et al., 2008). In general, IBS patients have lower HRQOL scores than healthy persons for physical role functioning, pain, general health, energy and social functioning. This questionnaire demonstrated no effect of the probiotic intervention as analysed by the mean change of the total HRQOL score, or the eight individual scales. Conversely, the specified IBSQL, dividing the quality of life into four domains (bowel symptoms, fatigue, activity limitations and emotional function), showed that the bowel symptoms domain was significantly improved by the probiotic supplementation (P = 0.045). An especially important finding in these studies was that the strain combination gave similar results when used in two different formulations, capsules and dairy drink form. There has been some concern about the validity of extrapolating the results of studies made with probiotic bacteria in one formulation to another. These two studies clearly showed that a highly similar efficacy is obtained when the four probiotic strains are used in combination, in two totally different product forms but in the same amounts. Furthermore, two different bifidobacteria strains were used in the two different studies, and the results were similar. This confirmed that the effect is not dependent on the Bifidobacterium strain only, and various bifidobacteria strains may be used in the combination. However, all the combinations must be studied separately. Modern culture-independent methods have also given us new insights to study human intestinal microbiota. Although there is no clear and identified aberrancy in the microbiota of IBS patients compared with healthy persons, several studies have indicated alterations and some degree of microbiota instability in IBS patients (Malinen et al., 2005; Mättö et al., 2005; Maukonen et al., 2006; Kassinen et al., 2007). The consumption of the present probiotic combination stabilised the composition of the microbiota (Kajander et al., 2008), which may be of particular importance in conditions like IBS, where increased instability of the microbiota appears to be typical (Mättö et al., 2005; Maukonen et al., 2006).

16.4.2

Eradication of Helicobacter pylori and Candida

Gastritis-inducing Helicobacter pylori is commonly carried by major populations in many parts of the world. Standard eradication therapy of H. pylori comprises antibiotics and proton pump inhibitors. The eradication therapy commonly causes severe gastrointestinal symptoms, resulting in treatment failures. Quite frequently the treatment is not efficient enough, and a person can remain a carrier of the pathogen. The LGG®Extra combination was used as an adjunct to a 7-day triple therapy (lansoprazole, clarithromycin, amoxicillin) for adult carriers in order to diminish the rate of symptoms of the therapy and to improve the eradication frequency (Myllyluoma et al., 2005). The participants (N = 47) were randomised

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to receive a dairy drink enriched with the probiotic multispecies combination (GG, LC705, PJS and Bb99, total daily dose 1 × 1011 CFU during the eradication period, and 6 × 1010 CFU during the following 3 weeks) or an identical drink without bacteria as a placebo. The participants recorded their daily symptoms in a standardised questionnaire, for 1 week before, during the therapy and the following 3-week intervention periods. Treatment-related total intestinal symptom score decreased during the eradication treatment (P = 0.038); in particular, epigastric pain and bloating were less common in the probiotic group compared with placebo. The probiotic supplementation also had a non-significant tendency to enhance the eradication of H. pylori compared with placebo (91% vs. 79%, respectively). In another trial, 12 patients waiting for a diagnostic gastroduodenoscopy were recruited into a study where gastroduodenal biopsy samples were taken before and after the 8-week probiotic intervention, during which period they consumed a dairy drink supplemented with GG, LC705, PJS and Bb12 strains (2.5 × 109 CFU/day, each strain, respectively) (Myllyluoma et al., 2007b). A paired study design in which each subject served as his or her own control was used. The aim was to evaluate whether the probiotic strains adhere to the upper gastrointestinal mucosa and modify H. pylori colonisation and influence the inflammation it produces. The colonisation and inflammation were evaluated by urease activity (13C-urea breath test), histology and serum pepsinogen I, II and gastrin-17 measures. Lactobacillus GG was occasionally isolated in gastric biopsy samples at the end of the intervention; no other strains of the combination were observed. Treatment with the probiotics decreased serum gastrin-17 levels (P = 0.046) and had a non-significant tendency to reduce the results of urease breath tests (P = 0.063), indicating some level of decrease in acid output and some reduction in the risk for gastric atrophy. Thus, although antibiotic treatments are needed to eradicate H. pylori, the probiotic multispecies combination is effective in reducing treatment-related symptoms and in improving the results of eradication. Elderly people are especially vulnerable to Candida infections due to chronic diseases, medication, poor oral hygiene and reduced salivary flow. Since earlier studies showed that the combination of LC705 and PJS strains had yeast-suppressive effects in dairy products (Suomalainen & Mäyrä-Mäkinen, 1999), a study to try to eradicate high oral Candida counts in elderly people was conducted, using semi-hard cheese enriched with the three strains of the combination (GG, LC705 and PJS) (Hatakka et al., 2007a). During this 16-week, randomised, double-blind, placebo-controlled study, 276 elderly people consumed daily 50 g of either probiotic or control cheese. In the probiotic cheese, Lactococcus lactis and Lactobacillus helveticus were used as starter cultures in the probiotic cheese, and the final count of each probiotic strain was 107 CFU/g. Control cheese contained only Lactococcus lactis as a starter culture, and no probiotic strains were added. The primary outcome measure of the participants was the prevalence of a high salivary yeast count (>104 CFU/mL). The results showed that the prevalence of high yeast counts decreased in the probiotic group from 30% to 21% (32% reduction) but increased in the control group from 28% to 34%. Thus, the probiotic intervention reduced the risk of high yeast counts by 75% (P = 0.004). Also, the risk of hyposalivation was reduced by 56% (P = 0.05). These results indicate that the probiotic cheese can be used to improve the oral health and hygiene of the elderly.

16.4.3

Other research areas

The probiotic multispecies combination of GG, LC705, PJS and Bb99 has also been tested in children for treatment of recurrent acute otitis media. Otitis media is a continuous problem especially in children under 3 years of age who attend daycare outside their home and

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are heavily exposed to the microbiota of their playmates. During this double-blind, placebo-controlled, randomised, 24-week intervention, 309 otitis-prone children (aged 10 months to 6 years) consumed either one probiotic (8–9 × 109 CFU, equal level of each strain) or placebo capsule daily. The parents were instructed preferably to open the capsule and empty the powder into milk product, or alternatively to give the capsule intact, if the child was able to swallow it. Parents recorded the symptoms of infection in a diary, and clinical examinations were carried out by a specialist doctor and nasopharyngeal samples taken three times during the study. Results showed that the probiotic combination did not reduce the occurrence of otitis or upper respiratory tract infections (Hatakka et al., 2007b). However, another study showed that infants who were administered the multispecies combination (GG, LC705, PJS and Bb99) together with galacto-oligosaccharides (GOS) during their first 6 months of life had less frequent respiratory tract infections during the 2-year follow-up period, and needed fewer prescriptions of antibiotics during the intervention period (Kukkonen et al., 2008). Also, the immune response against Haemophilus influenzae was enhanced, as noticed in a vaccination study (Kukkonen et al., 2006). Risk reduction and treatment of allergic diseases in infancy is under intensive research in many countries (Prescott & Bjorksten, 2007). Lactobacillus GG has so far been the most successful strain in these studies. In a randomised, double-blind, placebo-controlled study, 230 infants with atopic eczema/dermatitis syndrome and suspected cows' milk allergy were administered the multispecies probiotic combination (GG, LC705, PJS and Bb99, total daily dose 1.2 × 1010 CFU), Lactobacillus GG (total daily dose 5 × 109 CFU) or placebo, during a milk elimination diet and topical skin treatment, for 4 weeks (Viljanen et al., 2005a). The main clinical outcome was that Lactobacillus GG alleviated the skin symptoms but the probiotic combination did not. One potential mechanism for the effect of Lactobacillus GG in alleviating atopic dermatitis/eczema is stimulation of a Th1-type directed immune response in IgE-mediated atopic children, as measured by only slightly, but statistically significantly, higher plasma C-reactive protein (CRP) and IL-6 levels in sera compared with the groups treated with the placebo or the multispecies combination. Both GG and the strain combination increased faecal IgA level compared with placebo (Viljanen et al., 2005b), but the inducing effect of the multispecies combination on the Th1-directed immune response seemed to be too weak to evoke it, and the combination was ineffective in treating the symptoms of all types of atopic dermatitis/eczema syndrome (Pohjavuori et al., 2004; Viljanen et al., 2005a,c). However, the administration of the probiotic combination together with prebiotic GOS to pregnant mothers of high-risk infants for 4 weeks before the expected delivery and for the infants 6 months after birth reduced the risk of IgE-associated eczema in a 2-year prospective study (Kukkonen et al., 2007). At the age of 6 months faecal IgA tended to be higher (P = 0.085) and faecal α1-antitrypsin was significantly increased (P = 0.001) in the probiotic group compared with the placebo group. High intestinal IgA in early life is associated with minimal intestinal inflammation and indicates reduced risk for IgE-associated allergic diseases (Kukkonen et al., 2010). The intervention also induced an increased level of IL-10, total IgE, and CRP in plasma, without induction of an allergen-specific IgE response (Marschan et al., 2008a). Changes in immune response were similar to those seen in helminth infections, which stimulate Th2type reactions, but reduce the risk of atopic diseases (Marschan, 2007; Marschan et al., 2008b). However, in the 5-year follow-up the beneficial effects vanished to be limited only to infants having IgE-associated allergic diseases and who were delivered by Caesarean section (Kuitunen et al., 2009). Altogether, more studies are needed to understand the overall mechanism of atopic diseases and their possible risk reduction by probiotics.

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16.5

CONCLUSIONS

The present multispecies probiotic combination, LGG®Extra, has been found to effectively reduce the symptoms of intestinal discomfort, including IBS. Similar unparalleled efficacy of the probiotic combination in the treatment of IBS was not found with Lactobacillus GG alone (O'Sullivan & O'Morain, 2000; Bausserman & Michail, 2005). Although the clear mechanism of action is not known, the multispecies combination seemed to stabilise the composition of intestinal microbiota (Kajander et al., 2008). It is interesting that the two separate bifidobacteria strains used in the combination, B. breve 99 and B. animalis subsp. lactis Bb12, gave similar results in the IBS studies. The multispecies combination also seemed to be more effective than Lactobacillus GG as an adjunct in standard H. pylori eradication therapy; both Lactobacillus GG and the combination significantly reduced treatment-induced gastrointestinal symptoms, but the combination only had a tendency to enhance pathogen eradication (Cremonini et al., 2002; Myllyluoma et al., 2005). We also augmented the therapeutic application of the probiotic combination with a dairy-compatible prebiotic substrate. While the combination was less immunogenic than Lactobacillus GG alone, combined with the prebiotic GOS it resulted in enhancement of the formation of vaccine antibodies (Kukkonen et al., 2006) and reduced the risk of respiratory infections and IgE-associated eczema in infants (Kukkonen et al., 2007, 2008). To the best of our knowledge, this is the first case where the symbiotic concept has successfully been applied to a multispecies probiotic combination. These results encourage and open up possibilities for further expanding the activity of probiotics by strain combinations and by using them with other active components. In conclusion, this chapter illustrates the potential of probiotic foods that directly influence our intestinal well-being. The multispecies combination has potential to become a probiotic with focused use, especially for the relief of complex disorders such as IBS, which leads to intestinal discomfort with large societal and economic impacts. Furthermore, consumption of the probiotic combination may provide a safe, easy and an effective dietary means superior to many pharmaceutical formulations that are presently used and that may cause unnecessary adverse effects. Questions that need to be addressed in future studies include the ways the different probiotic bacteria interact with the host, stabilise the intestinal microbiota and affect the gut–brain axis, visceral nerves and other potential mechanisms underlying IBS as well as other functional disorders.

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Marschan E (2007) Immunological effects of probiotic bacteria in prevention and treatment of allergic diseases in children. PhD thesis, Department of Viral Diseases and Immunology, National Public Health Institute Helsinki, Finland and Hospital for Children and Adolescents, University of Helsinki, Finland. Marschan E, Kuitunen M, Kukkonen K et al. (2008a) Probiotics in infancy induce protective immune profiles that are characteristic for chronic low-grade inflammation. Clin Exp Allergy 38:611–618. Marschan E, Honkanen J, Kukkonen K, Kuitunen M, Savilahti E, Vaarala O (2008b) Increased activation of GATA-3, IL-2 and IL-5 of cord blood mononuclear cells in infants with IgE sensitization. Pediatr Allergy Immunol 19:132–139. Masco L, Van Hoorde K, De Brandt E, Swings J, Huys G (2006) Antimicrobial susceptibility of Bifidobacterium strains from humans, animals and probiotic products. J Antimicrob Chemother 58:85–94. Mättö J, Maunuksela L, Kajander K et al. (2005) Composition and temporal stability of gastrointestinal microbiota in irritable bowel syndrome: a longitudinal study in IBS and control subjects. FEMS Immunol Med Microbiol 43:213–222. Mättö J, Suihko ML, Saarela M (2006) Comparison of three test media for antimicrobial susceptibility testing of bifidobacteria using the Etest method. Int J Antimicrob Agents 28:42–48. Mättö J, Van Hoek AHAM, Doming KJ et al. (2007) Susceptibility of human and probiotic Bifidobacterium spp. to selected antibiotics as determined by the Etest method. Int Dairy J 17:1123–1131. Maukonen J, Satokari R, Matto J, Soderlund H, Mattila-Sandholm T, Saarela M (2006) Prevalence and temporal stability of selected clostridial groups in irritable bowel syndrome in relation to predominant faecal bacteria. J Med Microbiol 55:625–633. Mikkola H, Myllyluoma E, Ahlroos T, Tynkkynen S (2006) Quantification of probiotic strains in faecal samples by real-time PCR. In: Proceedings of IUFOST, 13th World Congress of Food Science and Technology: Food is Life, p. 286. INRA (National Institute for Agricultural Research), Nantes, France, 17–21 September 2006. Mykkanen H, Zhu H, Salminen E et al. (2005) Fecal and urinary excretion of aflatoxin B1 metabolites (AFQ1:AFM1 and AFB-N7-guanine) in young Chinese males. Int J Cancer 115:879–884. Myllyluoma E, Veijola L, Ahlroos T et al. (2005) Probiotic supplementation improves tolerance to Helicobacter pylori eradication therapy: a placebo-controlled, double-blind randomized pilot study. Aliment Pharmacol Ther 21:1263–1272. Myllyluoma E, Ahlroos T, Veijola L, Rautelin H, Tynkkynen S, Korpela R (2007a) Effects of antiHelicobacter pylori treatment and probiotic supplementation on intestinal microbiota. Int J Antimicrob Agents 29:66–72. Myllyluoma E, Kajander K, Mikkola H et al. (2007b) Probiotic intervention decreases serum gastrin-17 in Helicobacter pylori infection. Dig Liver Dis 39:516–523. O'Sullivan MA, O'Morain CA (2000) Bacterial supplementation in the irritable bowel syndrome. A randomised double-blind placebo-controlled crossover study. Dig Liver Dis 32:294–301. Ouwehand AC, Isolauri E, Kirjavainen PV, Tolkko S, Salminen SJ (2000) The mucus binding of Bifidobacterium lactis Bb12 is enhanced in the presence of Lactobacillus GG and Lact. delbrueckii subsp. bulgaricus. Lett Appl Microbiol 30:10–13. Ouwehand AC, Suomalainen T, Tölkkö S, Salminen S (2002a) In vitro adhesion of propionic acid bacteria to human intestinal mucus. Lait 82:123–130. Ouwehand AC, Lagstrom H, Suomalainen T, Salminen S (2002b) Effect of probiotics on constipation, fecal azoreductase activity and fecal mucin content in the elderly. Ann Nutr Metab 46:159–162. Peltonen K, El-Nezami H, Haskard C, Ahokas J, Salminen S (2001) Aflatoxin B1 binding by dairy strains of lactic acid bacteria and bifidobacteria. J Dairy Sci 84:2152–2156. Pohjavuori E, Viljanen M, Korpela R et al. (2004) Lactobacillus GG effect in increasing IFN-gamma production in infants with cow's milk allergy. J Allergy Clin Immunol 114:131–136. Prescott SL, Bjorksten B (2007) Probiotics for the prevention or treatment of allergic diseases. J Allergy Clin Immunol 120:255–262. Riedel CU, Foata F, Philippe D, Adolfsson O, Eikmanns BJ, Blum S (2006) Anti-inflammatory effects of bifidobacteria by inhibition of LPS-induced NF-kappaB activation. World J Gastroenterol 12:3729–3735. Saarela M, Maukonen J, Von Wright A et al. (2007) Tetracycline susceptibility of the ingested Lactobacillus acidophilus LaCH-5 and Bifidobacterium animalis subsp. lactis Bb-12 strains during antibiotic/probiotic intervention. Int J Antimicrob Agents 29:271–280.

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Salminen MK, Tynkkynen S, Rautelin H et al. (2002) Lactobacillus bacteremia during a rapid increase in probiotic use of Lactobacillus rhamnosus GG in Finland. Clin Infect Dis 35:1155–1160. Saxelin M, Tynkkynen S, Mattila-Sandholm T, De Vos WM (2005) Probiotic and other functional microbes: from markets to mechanisms. Curr Opin Biotechnol 16:204–211. Suomalainen T, Mäyrä-Mäkinen A (1999) Propionic acid bacteria as protective cultures in fermented milks and breads. Lait 79:165–174. Suomalainen T, Sigart-Mattila P, Mättö J, Tynkkynen S (2008) In vitro and in vivo gastrointestinal survival, antibiotic susceptibility and genetic identification of Propionibacterium freudenreichii ssp. shermanii JS. Int Dairy J 18:271–278. Szajewska H, Skorka A, Ruszczynski M, Gieruszczak-Bialek D (2007) Meta-analysis: Lactobacillus GG for treating acute diarrhoea in children. Aliment Pharmacol Ther 25:871–881. Tiihonen K, Suomalainen T, Tynkkynen S, Rautonen N (2008) Effect of prebiotic supplementation on a probiotic bacteria mixture: comparison between a rat model and clinical trials. Br J Nutr 99:826–831. Timmerman HM, Koning CJ, Mulder L, Rombouts FM, Beynen AC (2004) Monostrain, multistrain and multispecies probiotics: a comparison of functionality and efficacy. Int J Food Microbiol 96:219–233. Tuomola EM, Ouwehand AC, Salminen SJ (2000) Chemical, physical and enzymatic pre-treatments of probiotic lactobacilli alter their adhesion to human intestinal mucus glycoproteins. Int J Food Microbiol 60:75–81. Tynkkynen S, Singh KV, Varmanen P (1998) Vancomycin resistance factor of Lactobacillus rhamnosus GG in relation to enterococcal vancomycin resistance (van) genes. Int J Food Microbiol 41:195–204. Vaarala O (2003) Immunological effects of probiotics with special reference to lactobacilli. Clin Exp Allergy 33:1634–1640. Veckman V, Miettinen M, Pirhonen J, Siren J, Matikainen S, Julkunen I (2004) Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells. J Leukoc Biol 75:764–771. Viljanen M, Pohjavuori E, Haahtela T et al. (2005a) Induction of inflammation as a possible mechanism of probiotic effect in atopic eczema-dermatitis syndrome. J Allergy Clin Immunol 115:1254–1259. Viljanen M, Kuitunen M, Haahtela T, Juntunen-Backman K, Korpela R, Savilahti E (2005b) Probiotic effects on faecal inflammatory markers and on faecal IgA in food allergic atopic eczema/dermatitis syndrome infants. Pediatr Allergy Immunol 16:65–71. Viljanen M, Savilahti E, Haahtela T et al. (2005c) Probiotics in the treatment of atopic eczema/dermatitis syndrome in infants: a double-blind placebo-controlled trial. Allergy 60:494–500. Zoetendal EG, Rajilic-Stojanovic M, De Vos WM (2008) High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota. Gut 57:1605–1615.

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Probiotics and Health Claims: How to Be Met by SMEs?

Miguel Gueimonde and Sampo J. Lahtinen

17.1

INTRODUCTION

The European Regulation (EC) No. 1924/2006 on Nutritional and Health Claims made on food has been in force in European Union member states for 2 years. This regulation will have a great impact on the European probiotic sector. The need for approval of any health claim made on foods, on the basis of scientific evidence, promises to modify the marketing strategies used to communicate the beneficial effects attributed to probiotic products within Europe. While the regulations on probiotic health claims in other parts of the world may vary greatly, there is a clear trend for more scientific substantiation of health claims, especially with regard to human studies. On the basis of this European regulation, high-quality clinical intervention studies are needed to substantiate a specific health claim for a certain product. This requirement for a number of human studies applies to both large multinational companies providing probiotic strains as well as small and medium-size enterprises (SMEs) either producing specific strains or using them in their products. This regulation will give new directions to the research efforts aiming at developing and manufacturing probiotic products with health claims. Given the increase in research and development (R&D) resources required for carrying out expensive clinical studies necessary to substantiate a claim, the new regulations may be considered a great challenge and, at least a priori, a very difficult task for SMEs due to their often limited R&D budgets. However, despite the need of reallocation of resources by SMEs, the new regulatory framework may also constitute a source for new R&D opportunities for SME companies with high ability to rapidly identify new market niches and adapt to new situations. The often higher flexibility of SMEs compared with larger companies (Menrad, 2003), together with a closer knowledge of their local market and needs of the local customers, may constitute an advantage for SMEs within the rapidly moving field of functional foods. In addition, the market for functional foods in Europe is estimated to reach about 5% of total food expenditure (Stanton et al., 2001). Therefore, SMEs need to find ways to enter this large and growing market. However, it is important to emphasize that having a strong health claim is not enough to ensure consumer acceptance and purchase of the product – it is also necessary to understand consumer attitudes and local sensory/organoleptic preferences as well as local dietary habits in order to develop new successful functional products with excellent quality and taste. Compared with larger multinational companies, it


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may be an easier task for SMEs to develop products with superior sensory qualities and added health benefits which fit local consumer habits. This may even be a specific benefit for SMEs in the long run. Additionally, the European community offers possibilities for SMEs to join forces in the development of functional foods and this should enable them to have some advantages in the case of probiotics and specific probiotic products. Having a functional health benefit adds value to a product but this may not outweigh the sensory characteristics of the product (Bech-Larsen & Scholderer, 2007). The distribution channel is also of critical interest as consumers demand convenience and easy access to the products. Last but not least, in some instances the development of probiotic products not for use as foods but as drugs may constitute an option to be considered as a source of future business opportunities (Hoffman, 2008) for SMEs specialized in pharmaceutical R&D. The specific knowledge of SMEs on their local markets, together with the acceptance by the local consumers who may regard local small businesses as more reliable than large multinational companies, and the desire of some consumers to favor locally produced foods, may constitute a key success factor for SMEs. It is known that there are differences in consumer interest and attitude towards health claims in different European countries (Cathro & Hilliam, 1993) and around the world, and these differences may be used by the SMEs to focus on those health issues more widely accepted by the consumers in their specific markets. In general, consumers show the greatest interest in the areas of digestive health, immune enhancement and heart disease. Probiotic products should be targeted towards those health effects more relevant for the potential consumers in the specific target market. The ability to detect changes in their local consumer demand, which is easier locally than internationally, constitutes another advantage for SMEs, which usually have more direct communication with the local consumers and better understanding of their preferences and needs. This will allow identification of new market niches with target consumer groups that are too small for larger companies to have interest in, or which are regionally too limited to attract global companies (Fig. 17.1). Examples of such products could include products targeted at preventing or treating a very specific health condition which affects only a limited proportion of customers, probiotic versions of local and traditional food varities, or for example probiotic strains isolated from locally produced traditional fermented products. In addition, in order to develop products that are convenient and which satisfy the needs of the target consumers, fitting in with their lifestyle and shopping habits, good communication with consumers is required to allow SMEs to “educate” consumers on the specific properties of the product. Such communication is critical for increasing consumer understanding of the health claim and product acceptance. In this regard, the need for standards for information on probiotics to both healthcare workers and consumers has been noted (Hoffman et al., 2008). It is important to emphasize that within the new and stricter regulatory framework, only those products backed up by good research data are likely to succeed. Well-designed preclinical and clinical trials are therefore needed for each specific strain (Hoffman et al., 2008). Therefore, extensive R&D effort will be required from those SMEs interested in the fast-growing market of probiotic products, at least if the SMEs aim at developing their own proprietary strains. In recent years several R&D-focused SMEs have emerged, very often from the academic environment (e.g. spin-off companies from universities). For these companies the new regulations will not cause a need for a big change in their operating procedures, since the target of such SMEs has been to focus on R&D to start with. However, other SMEs, such as many traditional food manufacturers, may need to change their R&D operations to focus more on the scientific substantiation of functional properties and health claims of the product, if they wish to compete within this field. Regardless of the R&D

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Probiotics and Health Claims: How to Be Met by SMEs? All food industries

Pioneer food industries

Extraintestinal

Current Scientific Knowledge

Immune Gut

Current Market Niche

Current Consumer Acceptance

Current applications

Traditional (cultural) knowledge

Need for Consumer Information

Future Market Niche

New (scientific) knowledge

Market Competence

Traditional applications

265

Future applications

Fig. 17.1 Current and future market niches and needs for the development of probiotic foods.

background of SME companies, the new regulatory situation brings new opportunities for companies given that they understand the need not only for increasing their own knowledge but also for collaboration and networking with other companies and academic partners. The complexity of the area of functional foods (Fig. 17.2) and the need for clinical trials to substantiate health claims for the development of probiotic food products require substantial economic and personnel resources, which may be challenging for SMEs. However, at the same time many opportunities arise. For an SME alone, it may be difficult to develop products with clinically proven health benefits. Therefore, the new regulatory requirements may stimulate collaboration with other partners, such as larger companies, other SMEs and academic collaborators. In this regard, once the consumer needs and the market potential for a new probiotic product have been identified by an SME, the company may have to make a difficult decision at the very beginning of the process: whether to develop proprietary strains or whether to use commercially available probiotic strains from larger strain manufacturers.

17.2

DEVELOPING PROPRIETARY PROBIOTIC STRAINS

Networking and collaboration with other companies and collaboration with research organizations may constitute the sole possibility for development of proprietary strains by many SMEs whose resources are limited. In this regard different networking projects have been

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Probiotics and Health Claims Market studies to determine suitability and profitability of the product

Identify consumer needs and target population

Obtain/indentify suitable strains for the desired application. In vitro, in vivo and human intervention studies needed

Successful Functional Probiotic Product Claim

Increase nutritional knowledge of consumers for understanding health benefit of the product Develop appropriate marketing strategies for communication of the health claim

Develop a tasty and nutritionally adequate product retaining functionality at competitive price

Scientific Dossier preparation-Health Claim application

Fig. 17.2 Probiotic product development process.

launched and funded by the EU in the different framework programs, such as the Functional Food Network Project (www.functionalfoodnet.eu) finished in 2008, which involved over 185 companies. In the future, it is expected that similar programs will allow and even increase networking and collaboration. A number of Functional Foods research centers, offering services to the food and food supplement sector, have also been established within different European universities. In-depth knowledge of both national and international networking and funding programs as well as identification of potential research partners is essential and should be a major target for SMEs interested in developing probiotic products. Dedicated universities and research centers may be of help in gaining this knowledge, as such partners may have the experience, skills and networks required for identifying the existing possibilities for external funding. Collaboration between a food SME and a pharmaceutical SME may bring additional advantages due to their complementary competences and may also constitute an interesting possibility (Bech-Larsen & Scholderer, 2007). When collaborating with other SMEs in the development of new proprietary probiotic strains, the proper management of intellectual property rights becomes a critical issue. An open-minded approach is then needed, because for a fruitful collaboration the results should be beneficial to everyone and not only to a specific partner. Thus it is important to consider these issues from the very beginning of the collaborative work to reach agreement on exploitation of the developed strains. Different options, such as licensing by geographical areas or by food application, may be identified as mutually beneficial by the partners. It is important to understand that benefits for the participating SMEs will very likely be seen in mid or long term rather than in the short term. Developing proprietary probiotic strains, although very challenging, may constitute an opportunity for SMEs to join the functional foods market and, as indicated below, there are some examples showing that SMEs may succeed in this task. It is notable that successful development of proprietary strains may also lead to business opportunities outside the company’s local market area or company’s own product range; proprietary

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strains can be licensed out to other companies, for example to be used in other geographical regions or within other application areas.

17.3

PROBIOTIC RESEARCH BY SMEs USING STRAINS FROM LARGER COMPANIES

Developing proprietary strains from scratch may seem like an unbearable burden for many SME companies due to the high cost and duration of the research required for transforming new bacterial isolates into technologically applicable probiotic strains with clinical documentation on health benefits. However, the development of the company’s own strain is not the only way for SMEs to carry out original probiotic research and to accumulate scientific evidence for probiotic products in order to substantiate health claims. Instead of using proprietary strains, most SMEs producing probiotic products rely on well-documented probiotic strains from larger culture providers. While SMEs may rely on the existing health benefit documentation of the commercially available probiotic strains, it may also be in the interest of the SMEs to develop new science with the company’s own products containing commercial probiotic strains. Collaborative studies between probiotic strain providers and their customers, including SMEs, benefit both the provider of the probiotic strain and the SME. The former gets additional documentation for the portfolio of the probiotic strain, while the latter gets productand region-specific scientific back-up data to support health claims related to its own product(s). Carrying out such studies is in the interest of both parties, and thereby SMEs may get support from large strain providers for the trials, especially for new and innovative products. In addition to a possible direct contribution to study costs or providing research material for the study, large companies have many other ways of contributing to the costs involved with clinical trials. Large companies often have expertise, either in-house or through academic or other contacts, required for planning, organizing and carrying out clinical and other trials, and may offer these services to the collaborating SMEs. Large companies with their own research facilities may also participate in the laboratory analysis of biological samples from the study, for example microbiological or immune parameters. Moreover, companies with expertise in R&D focusing on health and nutrition can contribute to the publication of the results of the trials by means of journal articles and conference presentations, a crucial and time-consuming part of scientific research aiming at documentation of health effects. All the above-mentioned contributions by larger strain providers may help to significantly ease the burden of the SMEs in carrying out scientific research with their own products. Although commercially available “mainstream” probiotic strains already have scientific documentation for health effects, thus providing opportunities for all companies using the strains including SMEs to seek opportunities for health claims, it may still be beneficial for SMEs to carry out clinical trials with their own product(s) in order to augment the documentation supporting health claims. For example, the scientific documentation generated in this way is directly related to the product itself. This may be seen as a benefit for the SME, for example because such documentation clarifies the question of dosage: the strain itself may have sufficient evidence of a health benefit when administered at a certain dose, and it is up to the company producing the probiotic product to ensure that the product contains a sufficient level of the probiotic to ensure the beneficial effect. When the health benefit has been demonstrated using the final product itself, it testifies that the product

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contains a sufficient level of probiotics to carry out the health effect. On the other hand, in some countries the regulators may prefer scientific substantiation of health claims based on clinical trials carried out within the local population. This is despite the fact that current scientific evidence suggests that strain selection is the most important factor contributing to the health benefits of probiotic products, more so than genetic background, intestinal microbiota composition or dietary habits of the targeted consumers (all of which are likely to be quite variable among individuals even within the same geographical region). For example, very similar beneficial effects on innate immune system function (e.g. phagocytic activity) have been demonstrated for the probiotic strain B. lactis HN019 in different parts of the world, such as New Zealand (Gill et al., 2001), Taiwan (Chiang et al., 2000) and Canada (Arunachalam et al., 2000). Taken together, for SMEs, collaboration with larger strain producers to obtain scientific documentation for their own product may in many cases be the most feasible option. Unlike with proprietary strains, using commercially available probiotic strains, although not exclusive to the SME, allows immediate focusing of research efforts on to product-specific health efficacy studies – the laborious tasks of isolating new strains, purifying and identifying the strains properly, ensuring and optimizing technological feasibility of the strains, demonstrating the safety of the strains, and carrying out the preclinical in vitro and animal studies have all been carried out already. In addition, SMEs may obtain significant help from the larger strain providers to find the resources needed for clinical trials. Last but not least, even in the case of successful development of proprietary strains, SMEs still require collaboration with strain producers to make the proprietary strains commercially viable: very few SME food companies have the facilities and resources needed for commercialscale production of probiotic strains, and therefore a toll producer for the proprietary strains is almost always needed.

17.4

EXAMPLE OF SUCCESSFUL PROBIOTIC RESEARCH PROGRAM BY AN SME COMPANY: THE DEVELOPMENT OF PROBIOTIC STRAINS BIFIDOBACTERIUM LONGUM 46 AND B. LONGUM 2C

A case example of an SME company successfully developing and applying a probiotic research program is that of the small family-owned food manufacturer Bioferme Ltd (Kaarina, Finland). The company’s more than decade-long probiotic research program enabled the commercial launch of proprietary strains, developed from human isolates into patented probiotics with more than 20 scientific publications, including human intervention studies. The development work for the new probiotic strains targeted for adult and elderly consumers began with collaboration between Bioferme Ltd, the University of Turku (Finland) and Raisio Ltd, a larger Finnish food company. However, the key driver in the development process was the SME. The research project was funded partly by the Finnish Funding Agency for Technology and Innovation (TEKES) and the companies. In addition, close collaboration with the University of Turku and other universities and international research institutes as well as hospitals in Finland greatly contributed to the research efforts and the project funding, for example in the form of student thesis research projects.

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The two proprietary probiotic strains developed during the collaboration were named Bifidobacterium longum 46 (BL46) and B. longum 2C (BL2C). The two strains were originally selected among a large number of candidate strains, based on in vitro selection criteria including adhesion to human mucus, tolerance to acid, toxin binding and safety (Salminen et al., 2002; Laine et al., 2003). The candidate strains were originally isolated from healthy elderly subjects within a Finnish–Japanese collaboration project. The health effects of BL2C and BL46 have been assessed in a number of scientific in vitro studies as well as clinical interventions, all of which have been carried out as collaborative studies between the SME, academic researchers and hospitals/elderly nursing homes. In vitro studies have demonstrated the antimicrobial activity of BL46 against Staphylococcus aureus (Lahtinen et al., 2007) as well Helicobacter pylori and Escherichia coli (Hütt et al., 2006). Laboratory studies have shown that both BL2C and BL46 are capable of binding dietary toxins such as aflatoxin B1, a potent carcinogenic contaminant present in certain foods around the world (Salminen et al., 2002). BL2C and BL46 are also able to bind heavy metals. Halttunen et al. (2007) have shown that both BL2C and BL46 are capable of effectively binding lead and cadmium. Although strains of B. longum are regarded as extremely safe and therefore have qualified presumption of safety (QPS) status in Europe, the safety of these strains has also been demonstrated in a human intervention study (Mäkeläinen et al., 2003). In the trial, the product containing BL2C and BL46 was well tolerated and did not cause adverse effects or symptoms in healthy adult volunteers. The ability of BL2C and BL46 to stabilize gut function has recently been demonstrated scientifically. In a clinical study by Pitkälä et al. (2007), the consumption of BL2C and BL46 resulted in more frequent bowel movements in elderly subjects consuming the probiotic strains compared with the placebo group. The study showed that it is possible to normalize bowel movements in frail nursing home residents by including BL46 and BL2C in the diet. BL46 and BL2C may induce positive changes in the gut microbiota of elderly subjects. Two recent clinical intervention trials have shown that consumption of BL2C and BL46 leads to synergistic effects on the Bifidobacterium species naturally present the gut microbiota (Ouwehand et al., 2008; Lahtinen et al., 2009). These effects correlate with changes in immune function (Ouwehand et al., 2008). The results of the study indicated that the consumption of BL2C and BL46 influences serum cytokine levels. The net effect of the modulation of the immune system by BL2C and BL46 was shown to be anti-inflammatory rather than proinflammatory. Today, the new probiotic strains developed by Bioferme Ltd have matured into a new probiotic product, an oat-based smoothie-type fermented drink, launched in Finland in 2008. The commercial launch of the product was facilitated by the scientific documentation of the health effects of the two probiotics, but also by the excellent sensory qualities of the product fermented with the strains. In 2009, the probiotic smoothie was awarded the “Star Product of the Year” title by an independent Finnish Expert Assessment Board. The quantity of scientific research and the number of peer-reviewed publications with these two proprietary probiotic strains demonstrate that it is indeed possible for SMEs to successfully participate in probiotic research and the development new probiotic strains with clinical documentation on health benefits. Such efforts require countless hours of work and long-term intellectual, R&D and financial commitment from SMEs. Collaboration with other companies and/or academic collaborators is essential for success. Moreover, external funding, originating for example from national or international funding programs including the European Research agencies targeted for research and development specifically focusing on SMEs, is critically important. It should be noted that research programs

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aiming at the development of new proprietary strains take several years to mature, and therefore commitment and patience is required from all parties involved, including the funding agencies.

REFERENCES Arunachalam K, Gill HS, Chandra RK (2000) Enhancement of natural immune function by dietary consumption of Bifidobacterium lactis (HN019). Eur J Clin Nutr 54:263–267. Bech-Larsen T, Scholderer J (2007) Functional Foods in Europe: consumer research, market experiences and regulatory aspects. Trends Food Sci Technol 18:231–234. Cathro JS, Hilliam MA (1993) Future opportunities for functional and healthy foods in Europe. An in depth consumer and market analysis. Leatherhead Food RA special report. Leatherhead, Surry, UK. Chiang BL, Sheih YH, Wang LH, Liao CK, Gill HS (2000) Enhancing immunity by dietary consumption of a probiotic lactic acid bacterium (Bifidobacterium lactis HN019): optimization and definition of cellular immune responses. Eur J Clin Nutr 54:849–855. Gill HS, Rutherfurd KJ, Cross ML, Gopal PK (2001) Enhancement of immunity in the elderly by dietary supplementation with the probiotic Bifidobacterium lactis HN019. Am J Clin Nutr 74:833–839. Halttunen T, Salminen S, Tahvonen R (2007) Rapid removal of lead and cadmium from water by specific lactic acid bacteria. Int J Food Microbiol 114:30–35. Hoffman FA (2008) Business considerations in the development of probiotics. Clin Infect Dis 46:S141–S143. Hoffman FA, Heimbach JT, Sanders ME, Hibberd PL (2008) Executive summary: Scientific and regulatory challenges of development of probiotics as foods and drugs. Clin Infect Dis 46:S53–S57. Hütt P, Shchepetova J, Loivukene K, Kullisaar T, Mikelsaar M (2006) Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol 100:1324–1332. Lahtinen SJ, Jalonen L, Ouwehand AC, Salminen SJ (2007) Specific Bifidobacterium strains isolated from elderly subjects inhibit growth of Staphylococcus aureus. Int J Food Microbiol 117:125–128. Lahtinen SJ, Tammela L, Korpela J et al. (2009) Probiotics modulate the Bifidobacterium microbiota of elderly nursing home residents. Age (Dordr. ) 31:59–66. Laine R, Salminen S, Benno Y, Ouwehand AC (2003) Performance of bifidobacteria in oat-based media. Int J Food Microbiol 83:105–109. Mäkeläinen H, Tahvonen R, Salminen S, Ouwehand AC (2003) In vivo safety assessment of two Bifidobacterium longum strains. Microbiol Immunol 47:911–914. Menrad K (2003) Market and marketing of functional food in Europe. J Food Eng 56:181–188. Ouwehand AC, Bergsma N, Parhiala R et al. (2008) Bifidobacterium microbiota and parameters of immune function in elderly subjects. FEMS Immunol Med Microbiol 53:18–25. Pitkälä KH, Strandberg TE, Finne-Soveri UH, Ouwehand AC, Poussa T, Salminen S (2007) Fermented cereal with specific bifidobacteria normalizes bowel movements in elderly nursing home residents. A randomized, controlled trial. J Nutr Health Aging 11:305–311. Salminen S, Ouwehand A, Salminen E, Isolauri E (2002) Method for screening probiotic strains of the genus Bifidobacterium. World Patent WO 02/38798. Stanton C, Gardiner G, Meehan H et al. (2001) Market potential for probiotics. Am J Clin Nutr 73(Suppl):476S–483S.

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18

Probiotic Products: How Can They Meet the Requirements?

Wolfgang Kneifel

18.1

INTRODUCTION

In general, probiotics differ markedly from other food products as they either are or contain live cultures of individual organisms with different properties. For this reason probiotic products, to some extent, are positioned on the cusp where microbiology, technology, human and animal physiology, food and nutrition meet. According to current requirements, not only the cultures themselves but also their corresponding product matrices need to be adequately characterised and assessed based on several quality and safety criteria. Probiotic bacteria exhibit strain-dependent properties with regard to their health benefits, and their characterisation has to be in agreement with current requirements on identity/taxonomy, safety, physiological nature, microbial stability and viability. Hence probiotic products can only be successfully developed and marketed if these quality parameters are well proven, characterised and all prerequisites met.

18.2

QUALITY CRITERIA OF PROBIOTICS

Usually, probiotic products are the result of extensive screening and selection procedures involving isolation, strain typing, applied experiments and assessment in model systems, subsequently leading to clinical trials performed under conditions that have to meet current scientific standards (for details see Chapters 3 and 4). The outcomes of these studies comprise the scientific basis for probiotic strategies. In practice, three main issues can be distinguished regarding the desired properties of probiotics (Fig. 18.1). As probiotics should be administered on a regular basis (i.e. daily), only those foods that are consumed regularly and frequently are useful (De Vuyst et al., 2008; Rodgers, 2008). In addition, it is important to have the different disciplines (technology, microbiology, nutrition, medicine) involved in order to facilitate the development of a successful product. According to economic reviews, milk-based products play a major role in the probiotic market as they are part of the usually preferred diet of all human age groups. While probiotic dairy products, like any other kind of food, should meet the sensory requirements of consumers (also taking into account hedonic preferences in different countries and regions), the realisation of particular probiotic features attributed to specific


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Sensory properties

Meeting consumer needs

Technical properties

Functional properties

Tailored strain-matrices combination

Pronounced tolerance during gastrointestinal passage

Viable probiotic bacteria, optimum dose

Beneficial effects

Probiotic strain stability during storage

Scientific approval, dossiers, meta-analyses

Physical product stability during storage Controlled hygiene properties

Fig. 18.1 Quality criteria of probiotic products.

bacterial strains as identified and assessed by health professionals and nutritionists will be a specific challenge for the food technologist. In principle, the individual beneficial effects of a bacterial strain, as well as its stability under different circumstances, can be regarded as the most relevant factors determining the quality of a probiotic.

18.2.1

Basic composition and nutrient profile

Products like probiotics beneficial to human health have to fulfil fundamental requirements in terms of their nutrient composition. The European Food Safety Authority (EFSA) has stated that the nutrient profile of such a product has to be in accordance with current nutritional recommendations. As outlined in Regulation 1924/2006, the importance of meeting the requirements regarding nutrient profile is shown by the insistence that claimed benefits should not mask the overall nutritional status of a food product, which could mislead consumers. Scientific knowledge about overall composition, diet and nutrition and their relation to health should be taken into consideration because it is unfair to the consumer to market, for example, a drink with defined health benefits but overloaded with sugar, fat and calories. Neither can nutritionally adverse products be transformed into positive ones just by adding beneficial ingredients. In parallel, there are ongoing attempts to investigate the role of probiotic food matrices in conferring the beneficial effects of the bacteria (Mattila-Sandholm et al., 2002; Anal & Singh, 2007; Champagne et al., 2009). With the exception of lyophilised and/or coated or microencapsulated bacterial preparations (as found in capsules, tablets or powdered products), it is well known that the inclusion of probiotic microorganisms in diverse food matrices poses different challenges to the organism. Traditionally, fermented milk products are the most frequently used vectors for probiotics. However, these products require refrigerated storage and cooled distribution conditions. Their survival and stress resistance mainly depend on factors like acidity, pH and redox potential tolerance, and cooling temperature, but also on the nature of the protein matrix itself (Laine et al., 2003; Reid, 2008). This means, again, that individual strain characteristics play a dominant role in

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interaction with the matrices and the environmental conditions (Boylston et al., 2004; Madureira et al., 2005; Phillips et al., 2006).

18.2.2

Nature, identity and safety of probiotic strains

Because the beneficial effects of a probiotic product depend on the individual properties of the specific bacterial strain, information on the nature and identity of the microorganism is of utmost relevance, as it clearly defines the key subject of a product. However, it should not be forgotten that many probiotic cultures do not necessarily act as pure fermentation bacteria. For this purpose, in many cases regularly used starters such as thermophilic yogurt-type cultures consisting of Lactobacillus delbrueckii subsp. bulgaricus and/or Streptococcus thermophilus are applied in order to biologically acidify the milk without negatively influencing the performance and stability of the probiotic microflora. This means that today the majority of probiotic dairy products possess a dual-type microflora with separate targets. These properties need to be taken into account in the qualitative as well as quantitative assessment of a product (see also section 18.2.3). The EFSA expects application dossiers for health claims of probiotics to contain a clear definition of the bacterial strain and an extensive description of the inherent culture, based on current taxonomy, which necessarily has to meet modern scientific standards. Moreover, probiotic strains are unique organisms and need to be deposited in an internationally recognised culture collection in order to allow control as well as comparison with other strains. Correspondingly, taxonomic identity should take account of the qualified presumption of safety (QPS) concept (EFSA, 2004). This concept is the counterpart to the GRAS (generally recognised as safe) status used in the United States. Besides its exact nomenclature, there should also be detailed information on familiarity with the strain, which in addition forms some basis for evaluating its safety (Felis & Dellaglio, 2007). Reliable identification by means of adequate methods is an important prerequisite for probiotic strains. Moreover, pathogenicity criteria, such as diverse virulence factors, toxigenic potential or transferability of antibiotic resistance, play an important role in the safety evaluation of a strain. Finally, the target product where the strain is to be used also determines some relevant criteria of the QPS status (EFSA, 2007). Figure 18.2 depicts a simplified decision tree for assessment of a probiotic as well as any other food-grade fermentation microorganism. Not unexpectedly, well-established probiotic strains with a proven safe history over the long term possess considerable advantages over new probiotic candidate strains, which need to undergo comprehensive safety assessments before they can be used. During the last two decades, the strategies for bacterial identification and differentiation have undergone pronounced methodological modifications, and this has led to differing assignments and nomenclature of some microorganisms. This development was mainly due to the technical advances in molecular biology, which have increasingly enabled improved analytical characterisation of these bacteria (Ben Amor et al., 2007; Pineiro & Stanton, 2007). A current and continuously updated weblink provides some useful information about recent advances in bacterial taxonomy (Euzeby, 1997). It should be mentioned here that a considerable proportion of commercial probiotic cultures have not yet been correctly identified and are therefore mislabelled (Huys et al., 2006). According to review articles of the most relevant genera of lactic acid bacteria, Lactobacillus and Bifidobacterium comprise the major probiotic microorganisms (Thomsen, 2006;

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Taxonomic basis (exactly defined nomenclature)

Sufficient (further) information about the strain? No

Yes Are there any objections regarding the safety of the microorganism? Yes Can they be excluded? Yes No

QPS status

No

Literature studies, experience products Literature studies, cases experience Literature studies, special tests

Not QPS qualified

Fig. 18.2 Evaluation tree for the assessment of qualified presumption of safety (QPS) status of a microorganism. (Modified from EFSA 2004.)

Table 18.1 Historical development of the nomenclature of bacteria of potential probiotic relevance: example Bifidobacterium spp. 1899

Bacillus bifidus: isolate from a breast-fed infant

Tissier (1899)

1920

Renamed Lactobacillus bifidus

Holland (1920)

1963

New genus Bifidobacterium established

Reuter (1963), Mitsuoka (1969), Scardovi & Trovatelli (1969)

1997

Regrouping of Bifidobacterium into Family Bifidobacteriacea based on 16SrRNA sequence data

Stackebrandt et al. (1997)

Today

38 species and 9 subspecies using polyphasic taxonomy

Biavati & Mattiarelli (2006), Felis & Dellaglio (2007), Euzeby (1997)

Kneifel & Domig, 2008). For species identification, DNA hybridisation or 16S rRNA sequence analysis can be regarded as the gold standard, while strain typing needs to be based on macrorestriction methodologies like pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD)-PCR analysis, amplified ribosomal restriction analysis (ARDRA) and repetitive genomic element PCR (repPCR). In general, bacteria sharing 16S rRNA gene homology of more than 97% are considered as members of the same species. According to the results elaborated in an EU research project published by Vankerckhoven et al. (2008), this technique has been recommended as the most useful and the most comparable method for species identification. Table 18.1 shows the developments in the taxonomic assignment of Bifidobacterium spp. over the last century. Although these modern techniques allow detection of the identity and individuality of a microorganism to a high degree of accuracy, the individual behaviour of probiotic bacteria under applied conditions, for example during the shelf-life of a product or passage through the stomach and intestinal tract, needs special consideration based on traditional physiological and biochemical methods, which prevailingly involve traditional analytical techniques.

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18.2.3

275

Viability and probiotic viable count

Another major criterion is the number of living microorganisms contained in the product or the probiotic preparation, and this has to be guaranteed for a certain shelf-life by the producer or marketing institution. The number of living microorganisms in a food product is routinely examined using culture count techniques with selective media and under defined cultivation conditions. However, culturability of a microbial strain depends on many factors. Today, speculation about the capability of culture methods to sufficiently describe the status of the product and the bacterial strains has led to a situation where it may be almost considered philosophical to rely on these analytical methods. Advances in microbiology have taught us that we may distinguish among the different stages of the transition between multiplication, growth, dormancy and death inherent in the life cycle of microorganisms (Volkert et al., 2008; Garcia-Cayuela et al., 2009). Moreover, the postulate that probiotics contain living bacteria must increasingly be viewed with caution, as we are presently unable to satisfactorily monitor the various phases of viability and sublethal damage, especially during passage through the different segments of the gastrointestinal tract (Gueimonde et al., 2004a,b). As a result, we cannot exclude the possibility that even bacteria which are not (fully) detected by culture techniques may definitely interact with the human and animal organism to some extent, and it is not known whether these forms exhibit beneficial effects on the organism or not. For example, it has been shown in comparison experiments with live and heat-killed Lactobacillus rhamnosus GG (LGG) cells that high doses of intact LGG cells significantly increase the production of inflammatory markers in intestinal epithelial cells, while heat-killed LGG only cause a slight increase, when an inflammatory response had not been stimulated (Zhang et al., 2005). Another study demonstrated that 7 days after oral administration to mice, the cellfree fraction of milk fermented by a Lactobacillus helveticus strain induced a significant increase in the number of IgA-producing cells in the small intestinal lamina propria (Vinderola et al., 2007). Further health-promoting effects observed in fermented products with non-viable microflora is the enhanced concentration of B vitamins produced during fermentation, the bioactive peptides released from milk proteins, or even the cell wall fragments of lactic acid bacteria which may activate the mucosal immune system (Vinderola, 2008). In their review, Kataria et al. (2009) have compiled the various concerns related to the potential risks that live probiotic bacteria may exert under some circumstances. The authors were investigating if cell fragments were sufficient to initiate some effects on or in the human organism. In particular, it should not be forgotten that every living bacterium may pose particular problems for severely ill patients as well as for very young individuals, as unwanted side effects and translocation to the locally draining tissues and the blood cannot be excluded in these fringe groups where special precautions should be taken (Jack, 2009). The viability of probiotic bacterial strains in powdered milk products like infant formulas or animal feed requires special attention. There are different technological means to accomplish the appropriate survival of the bacteria during spray-drying as well as during storage of the dried product, which physically should represent a glassy stage with low water activity values (Teixeira et al., 1995; Ananta et al., 2005). According to a recent review, a number of conditions have to be met in order to ensure protection of probiotics (Chavez & Ledeboer, 2007). Based on the intrinsic stress tolerance of the individual strain, the carrier matrices should provide optimum protection during processing and shelf-life. As probiotic bacteria are heat-sensitive, mortality during heat treatment should be reduced

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Product Phase Fresh product Product storage loss

Stored product

Body Passage Phase Consumption Digestion loss

Probiotic effect Fig. 18.3 Survival hurdles for probiotics.

by applying tailored drying temperatures and correspondingly short exposure times. Furthermore, osmotic, oxidative and mechanical stress should be minimised by, for example, supplementation with osmoprotectants and antioxidants. Even pretreatment can be applied to fortify bacterial stress tolerance (Ananta et al., 2004). With regard to optimum stability during gastrointestinal passage, the most useful protection is achieved by means of encapsulation and coating. Viable count enumeration methods, which are rather easy to perform, allow us to observe two phases in the lifespan of a probiotic product (Fig. 18.3). The fresh product necessarily should contain a defined number of probiotic microorganisms, at least as many as have been demonstrated in clinical studies to exert a defined effect when administered to the human or animal organism. Furthermore, it should be guaranteed that this viable count does not undergo severe reduction in the product during the given shelf-life under refrigerated conditions or, in the case of pharmaceutical preparations like capsules or tablets, even at ambient storage temperatures. This entire period comprises the so-called ‘product phase’. At some stage of storage, the product will be consumed or administered (if pharmaceutical preparations are considered); then it is relevant that the targeted number of microorganisms possesses the ability to survive gastrointestinal passage in high numbers until that segment of the gut where local interaction with the organism takes place, possibly followed by other systemic effects. This comprises the so-called ‘body passage phase’ of the probiotic. Hence, in summary, the viability and survival of a microorganism strongly depends on the specific nature of the strain and on extrinsic factors exerted by the environment. Moreover, interactions with other microorganisms and corresponding metabolites (e.g.

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antimicrobial substances, bacteriocins) may complete the array of influencing criteria. The individual survival behaviour of the probiotic microorganism is of crucial importance, as decreed in accordance with the definition of probiotics. However, taking into account the above-described critical comments on viability, it is not totally certain if different probiotic approaches definitely require cultivatable microorganisms, as even autolysed bacterial products are known to exert certain effects (Lopez et al., 2008; RigonZimmer et al., 2008). Because of the huge demand for suitable methods to assess the quality of probiotic (fermented) milk products, the International Dairy Federation (IDF) has taken over responsibility for developing standardised routine techniques for enumerating the specific cultures used in probiotic dairy products. These methods should also allow clear differentiation of the probiotic from the commonly used fermenting bacteria such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Lactococcus lactis. Methods are based on viable count culture techniques and are usually applied in assessment of product quality. Suitable routine methods have been sought, screened and optimised by IDF expert panels, and finally evaluated in collaborative trials with international laboratories. IDF emphasised that the suggested methods should be able to be performed in conventionally equipped dairy and food analytical laboratories. Moreover, corresponding media and chemicals need to be globally available and of proven quality. An important challenge for the working groups was also to find and/or to develop methods that can be applied to the various bacterial strains of the same species used worldwide and not only to suit the requirements of selected single cultures of certain culture producers or suppliers. Table 18.2 gives an overview of selected relevant culture methods that have been demonstrated as useful for these purposes. Unfortunately, there are still also several in-house methods that have not fully taken into account drawbacks like (1) competitive effects and interference with other microbial components in the product, (2) retarded growth of the taget microorganisms due to the inclusion of antibiotics or antibiotic mixtures in the media and (3) lack of selectivity and electivity because of insufficient basic composition of the substrate. As a result of the IDF work, two standardised method protocols (one for the enumeration of Lactobacillus acidophilus and one for the enumeration of bifidobacteria) have been published (International Standards Organization, 2002, 2008). Today, they form the basis for viable count assessments of most commercially available probiotic dairy products. In parallel, alternative instrumental methodologies able to assess viable microorganisms in probiotic products have been promoted (Table 18.3). Among these, flow cytometric methods have been shown to possess high practical relevance due to their rapidity. However, although some of the results of the methods seem to be in agreement, there is some evidence that, as for culture techniques, even the choice of alternative enumeration method influences the results of the analysis (Lahtinen et al., 2006). With regard to the application of real-time PCR methods, it is generally accepted that DNA levels are not associated with viability, as dead cells may also retain significant amounts of genetic material, which can be detected in molecular tests. On the other hand, fluorescent in situ hybridisation (FISH) may offer the advantage of detecting the 16S rRNA of bacterial cells. This 16S rRNA possesses a shorter half-life than that of DNA, so it obviously better reflects the viability status of the bacteria than these other techniques. Nevertheless, the suitability of the FISH method for viability testing depends on the decay of rRNA after cell death. Interestingly, in a storage stability study performed with probiotic dairy products, both FISH and real-time PCR produced comparably constant results, while culture counts showed a reduction in certain bifidobacterial strains during the observation period (Lahtinen et al., 2006).

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Table 18.2 Methods and media (selection) used for the selective enumeration of probiotics based on the viable count technique. Method

Reference

Bifidobacterium spp. BIF Agar MRS medium containing selective and elective additives: cystein-HCl, lactulose, vitamin mixture, sheep blood, human milk whey, antibiotics (aztreonam, nalidixic acid, netilmycin, paromomycin sulphate) Bifidobacterium Selective Medium MRS-based medium containing cystein-HCl and antibiotic (mupirocin) MRS-LP MRS agar supplemented with lithium chloride and sodium propionate

Pacher & Kneifel (1996)

Leuschner et al. (2003), Simpson et al. (2004)

Vinderola & Reinheimer (1999), Van de Casteele et al. (2006)

MRS-NPNL Agar MRS supplemented with antibiotics (neomycin, paromomycin, nalidixic acid)

Dave & Shah (1996), Roy (2001)

RB Medium Raffinose–Bifidobacterium medium with lithium chloride, propionate and raffinose

Hartemink et al. (1996)

RCM-BIM 25 RCM medium supplemented with TTC, iodoacetic acid and antibiotics (nalidixic acid, polymyxin B sulphate, kanamycin sulphate)

Munoa & Pares (1988)

TPY-MUP Agar Tryptone phytone yeast agar supplemented with antibiotic (mupirocin)

Rada & Petr (2000)

TOS-MUP Agar Propionate agar containing TOS (galactooligosaccharide mixture) and antibiotic (mupirocin)

International Standards Organization (2008), Zitz et al. (2007)

TOS-propionate Agar Propionate agar supplemented with TOS oligosaccharide mixture

Sonoike et al. (1986), Japanese Association of Fermented Milks and Fermented Milk Drinks (2000)

Lactobacillus acidophilus group (L. acidophilus, L. johnsonii, L. gasseri, L. crispatus) MRS-Clindamycin Agar MRS agar supplemented with antibiotic (clindamycin)

International Standards Organization (2002)

XGlu Agar Rogosa agar supplemented with X-Glu (5-bromo-4-chloro-3-indolyl-b-D-glucopyranoside)

Kneifel & Pacher (1993)

Lactobacillus casei group (L. casei, L. paracasei, L. rhamnosus) LC Medium MRS Vancomycin Agar

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Su et al. (2006), Van de Casteele et al. (2006)

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Method

Reference

MRS agar supplemented with antibiotic (vancomycin)

Tharmaraj & Shah (2003)

279

Lactobacillus plantarum Lactobacillus plantarum Selective Medium Medium containing sorbitol and bromocresole purple with antibiotic (ciprofloxacin)

Bujalance et al. (2006)

Table 18.3 Non-cultural methods (selection) used for the quantification of probiotic bacteria. Method

Reference

Fluorescence in situ hybridisation (FISH)

Lahtinen et al. (2006)

Fluorescence staining and microscopic counting (viability assay)

Lahtinen et al. (2006), Maukonen et al. (2006)

Flow cytometry

Maukonen et al. (2006), Sunny-Roberts et al. (2007) Volkert et al. (2008)

Quantitative real-time PCR

Furet et al. (2004), Lahtinen et al. (2005, 2006), Gueimonde et al. (2004b), Garcia-Cayuela et al. (2009), Bogovic-Matijasic et al. (2010)

18.3

FUTURE PERSPECTIVES

At present, approximately 30 bacterial strains can be classified as probiotics according to observed and more-or-less proven beneficial effects. There are several promising probiotic candidates on hold that may offer advantageous application but which have not been implemented in real products. In many countries, meanwhile, numerous test phases and hurdles have to be passed by a new strain or new probiotic product before it can be marketed as a safe and effective food or preparation. Besides the technological boundaries encountered by innovative probiotic food product developments, novel applications ranging from new strain combinations to products tailored for certain age groups and certain preventive or therapeutic applications may pose another challenge for product developers. In the area of food science, cultures possessing multifaceted benefits such as protective plus probiotic effects may be of increasing interest (Hatakka et al., 2008; Rodgers, 2008) as they not only contribute to improved safety and shelf-life of the product itself by acting as biopreservatives, but are also beneficial to body functions of the consumer on ingestion. This synergistic combination of strains is a rather new scenario which, though having been demonstrated during the last decade, could even better bridge the divide between food science and medical science than classical probiotics ever did. As most fermented foods usually contain large numbers of fermentation microorganisms, the question of whether viable probiotic bacteria may, under given circumstances, be detrimental to the consumer is of minor relevance to normal and even ill persons suffering from different diseases. Like most other food-grade microoorganisms, probiotics usually have a long history of safe use, most of them even complemented with proven clinical efficacy. As far as pharmaceutical applications of probiotic preparations are considered, a more differentiated assessment of the product seems to be useful if targeted application to severely ill patients is considered.

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Probiotic products for this particular category of consumer should probably be of non-viable bacterial content in order to avoid undesired complications and adverse effects. Interestingly, there are several pharmaceutical and food products on the market that contain identical bacterial strains with defined probiotic properties, some of them even with comparable microbial doses per portion. So, in some cases it may be up to the consumer to decide which of the products should be taken. This can be seen as a nice example for strong development of the so-called functional food area and Hippocrates dictum ‘Let food be your medicine’.

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Reuter G (1963) Vergleichende Untersuchungen über die Bifidus-Flora im Säuglings- und Erwachsenenstuhl. Zentralbl Bakteriol (A) 191:486–507. Rigon-Zimmer K, Mullie C, Tir-Toul-Meddah A, Buisson P, Leke L, Canarelli, JP (2008) Impact of colostomy on intestinal microflora and bacterial translocation in young rats fed with heat-killed Lactobacillus acidophilus stain LB. Folia Microbiol 53:89–93. Rodgers S (2008) Novel applications of live bacteria in food services: probiotics and protective cultures. Trends Food Sci Technol 19:188–197. Roy D (2001) Media for the isolation and enumeration of bifidobacteria in dairy products. Int J Food Microbiol 69:167–182. Scardovi V, Trovatelli LD (1969) New species of bifidobacteria from Apis mellifica and Apis indica F: a contribution to the taxonomy and biochemistry of the genus Bifidobacterium. Zentralbl Bakteriol (A) 123:64–88. Simpson PJ, Fitzgerald GF, Stanton C, Ross RP (2004) The evaluation of a mupirocin-based selective medium for the enumeration of bifidobacteria from probiotic animal feed. J Microbiol Method 57:9–16. Sonoike K, Mada M, Mutai M (1986) Selective agar medium for counting viable cells of bifidobacteria in fermented milk. J Food Hyg Soc Japan 27:238–244. Stackebrandt E, Rainey FA, Ward-Rainey NL (1997) Proposal for a new hierarchic classification system. Actinobacteria classis nov. Int J Syst Bacteriol 47:479–491. Su P, Henriksson A, Mitchell H (2006) Survival and retention of the probiotic Lactobacillus casei LAFTI® L26 in gthe gastrointestinal tract of the mouse. Lett Appl Microbiol 44:120–125. Sunny-Roberts EO, Ananta E, Knorr D (2007) Flow cytometry assessment of Lactobacillus rhamnosus GG (ATCC 53103) response to non-electrolytes stress. Nutrition Food Sci 37:184–200. Teixeira PC, Castro MH, Malcata FX, Kirby RM (1995) Survival of Lactobacillus delbrueckii spp. bulgaricus following spray-drying. J Dairy Sci 78:1025–1031. Tharmaraj N, Shah NP (2003) Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and propionibacteria. J Dairy Sci 86:2288–2296. Thomsen M (2006) Probiotics: enhancing health with beneficial bacteria. Alternative Complemenary Therapies 12:14–21. Tissier MH (1899) La reaction chromophile d’Escherich et Bacterium coli. C R Soc Biol 51:269–271. Van de Casteele S, Vanheuverzwijn T, Ruyssen T, van Assche P, Swings J, Huys G (2006) Evaluation of culture media for selective enumeration of probiotic strains of lactobacilli and bifidobacteria in combination with yoghurt or cheese starters. Int Dairy J 16:1470–1476. Vankerckhoven V, Huys G, Vancanneyt M et al. (2008) Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE project. Trends Food Sci Technol 19:102–114. Vinderola G (2008) Dried cell-free fraction of fermented milks: new functional additives for the food industry. Trends Food Sci Technol 19:40–46. Vinderola CG, Reinheimer JA (1999) Culture media for the enumeration of Bifidobacterium bifidum and Lactobacillus acidophilus in the presence of yoghurt bacteria. Int Dairy J 9:497–505. Vinderola CG, Matar C, Palacios J, Perdigon G (2007) Mucosal immunomodulation by the non-bacterial fraction in milk fermented by Lactobacillus helveticus R389. Int J Food Microbiol 115:180–186. Volkert M, Ananata E, Luscher C, Knorr D (2008) Effect of air freezing, spray freezing, and pressure shift freezing on membrane integrity and viability of Lactobacillus rhamnosus GG. J Food Eng 87:532–540. Zhang L, Li N, Caicedo R, Neu J (2005) Alive and dead Lactobacillus rhamnosus GG decrease tumor necrosis factor-a induced interleukin-8 production on Caco-2 cells. J Nutr 135:1752–1756. Zitz U, Kneifel W, Weiss, H, Wilrich P-T (2007) Selective enumeration of bifidobacteria in dairy products: development of a standard method. Bull Int Dairy Fed 411:2–30.

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19

Probiotics and Health Claims: Hurdles for New Applications?

Lorenzo Morelli

19.1

INTRODUCTION

The initial outcomes of Regulation (EC) No. 1924/2006 are in some cases encouraging but in others of some concern to microbiologists, and there are now both new and old hurdles for the research community to consider in the future. The positive aspects of this evolution include the introduction of a regulatory framework within which research and development activities can operate more safely in order to generate new knowledge and products. The negative aspects are that the scenario is not yet fully developed and there is concern that excessive regulation could hinder innovative research. This chapter reviews some of the hurdles posed by the first European Food Safety Authority (EFSA) opinions on probiotic claims and some perspectives for future basic and applied research able to overcome these hurdles are presented.

19.2

IDENTIFYING THE HURDLES

A detailed analysis of the opinions issued by the NDA panel clearly outlines the three areas that will be evaluated in a scientific dossier submitted to EFSA for approval: (1) characterisation of the product/ingredient, (2) relationship to health, and (3) scientific substantiation. All these considerations have been shown to be severe hurdles for the use of probiotics, at least for applications submitted in the first months since Regulation 1924/2006 came into effect.

19.2.1

Characterisation

In assessing probiotic foods, the EFSA panel requests a well-detailed characterisation of the bacterial strains, their shelf-life and period of use, and their interaction with the food matrix. An insufficient characterisation of at least one of the above traits will result in a statement from the EFSA that the ‘food for which the health claim is made, has not been sufficiently characterised’. An insufficient characterisation has been reported for most of the applications dealing with probiotics, but this is not the case for other types of foods or food ingredients. It seems therefore that there is a specific problem with probiotics, even though several regulatory bodies have set up recommendations for identifying them since 1999 (Box 19.1). Probiotics and Health Claims Edited by Wolfgang Kneifel and Seppo Salminen © 2011 Blackwell Publishing Ltd. ISBN: 978-1-405-19491-4

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Box 19.1 Guidelines for probiotics as issued by international and national organisations European Commission, Health and Consumer Protection Directorate-General, Directorate C: Scientific Opinions Guidelines for the assessment of additives in feedingstuffs: Part II enzymes and microorganisms, October 1999 ● The name and taxonomic status of each microorganism according to the latest published information in the international Codes of Nomenclature should be provided ● All microorganisms, whether used as a product or as a producer strain, should be deposited in an internationally recognised culture collection (preferably in the European Union) and maintained by the culture collection for the authorised life of the additive ● In addition, all relevant morphological, physiological, and molecular characteristics necessary to identify the strain and confirm its genetic stability should be described ● Microorganisms intended as active agents should not be capable of the production of antimicrobial substances relevant to the use of antibiotics in humans or animals ● Strains of bacteria intended for use as an additive should not contribute further to the reservoir of antibiotic resistance genes already present in the gut flora of animals and the environment. Consequently, all strains of bacteria should be tested for resistance to at least one representative of each of the antibiotic families in use in human and veterinary medicine. Where resistance is detected, the genetic basis of the resistance and the likelihood of transfer of resistance to other gut-inhabiting organisms should be established Report of a joint FAO/WHO expert consultation, 2001 Probiotics in food: health and nutritional properties and guidelines for evaluation ● The Consultation recommended that probiotics be named according to the International Code of Nomenclature to ensure understanding on an international basis ● The Consultation strongly urged that for the sake of full disclosure, probiotic strains be deposited in an internationally recognised culture collection ● Since probiotic properties are strain related, it is suggested that strain identification (genetic typing) be performed, with methodology such as pulsed field gel electrophoresis (PFGE). It is recommended that phenotypic tests be done first, followed by genetic identification, using such methods as DNA/DNA hybridisation, 16S RNA sequencing or other internationally recognised methods

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Box 19.1

285

(cont’d)

Italian Ministry of Health, 2005 Guidelines for probiotics and prebiotics Determination of taxonomic order should be of primary importance. In fact, before putting on the market new probiotic products, we need to acquire accurate and complete knowledge of the specific properties of the selected bacterial strains. Phenotypic taxonomy has represented for a long time the foundation of the species classification. The integration of phenotypic and genotypic characterisation allows today to work with good consistency; this procedure should be considered as a fundamental requirement for the probiotic food introduction on the market

It is noteworthy also that following on from European Union (EU) Regulation No. 1831/2003 on additives used in animal nutrition, the EFSA has established procedures to both identify and characterise bacteria that need to be approved as feed additives. Under these guidelines, clear indications are provided about the methodology to be used for bacterial identification and the depositing of these strains into an international culture collection is described as ‘mandatory’. However, even before the EFSA had been established, guidelines for the use of viable bacteria as feed additives released by the Standing Committee in Animal Nutrition in accordance with Council Directive 93/113/EC (concerning the use and marketing of enzymes, microorganisms and their preparations in animal nutrition) stated that: All microorganisms, whether used as a product or as a producer strain, should be deposited in an internationally recognised culture collection (preferably in the European Union) and maintained by the culture collection for the authorised life of the additive. Evidence of deposition in the form of a certificate from the culture collection specifying the accession number and name under which the strain is held, must be provided. In addition, all relevant morphological, physiological, and molecular characteristics necessary to identify the strain and confirm its genetic stability should be described.

It is thus surprising the lack of characterisation found by the EFSA panel in most of the applications for probiotic health claims. It is also quite surprising that the viability of the probiotic strains during the shelf-life of the end product has often not been reported by applicants. The definition of probiotics clearly states that they are ‘viable bacteria’ and it is therefore mandatory to have a sound assessment of their viability during the storage and eventual consumption of the food product. In this regard, since 2001 the FAO/WHO document recommends that ‘viability and probiotic activity must be maintained throughout processing, handling and storage of the food product containing the probiotic, and verified at the end of shelf-life’.

19.2.2

Relationship to health

Identification of a measurable health effect seems to be a quite challenging task in the case of probiotics and several opinions state that ‘the claimed effect is not sufficiently defined’. A large number of probiotic products are sold as a tool to support the perpetuation of ‘healthy’

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Box 19.2 Negative statements on health relationships (‘claimed effect’) for probiotic products/ingredients released by EFSA ‘The claimed effect is to support, stimulate and modulate the immune system of children during growth. However no definition is provided of what constitutes a healthy immune system or how the support, stimulation or modulation of the immune system can be assessed’ (EFSA Journal 2008;782:1–9) ‘The claimed effect is improving the general immunity and the proposed biological mechanism of maintaining the microbiological balance have not been sufficiently defined by the applicant to allow an adequate evaluation of the effect and its impact on health’ (EFSA Journal 2008;860:2–8) ‘The claimed effect is to maintain the natural intestinal microflora during travel, changing the climatic zone or a diet, especially in poor hygiene conditions. The applicant has not sufficiently defined “natural intestinal microflora” to allow an adequate evaluation of the effect and its impact on health’ (EFSA Journal 2008;863:2–8) ‘ “Helps to bring back the normal functioning of the alimentary tract during its microflora disturbances (for example in case of loose stools, after taking antibiotics, in case of intestinal disorders caused by enteric pathogens).” The target population for the food supplement is children. The applicant has not defined “microflora disturbances”. Furthermore, the applicant has not sufficiently defined the abnormal function of the alimentary tract to be corrected’ (EFSA Journal 2008;861:7–9) ‘The claimed effect is that product X contains living probiotic bacteria which have a strong ability for intestinal tract colonisation and have been isolated from healthy, naturally fed infants. Intestinal tract colonisation can be a property of any (resident) gut bacterium (also pathogens). The Panel therefore concludes that the applicant has not shown the relevance of the claimed effect to human health’ (EFSA Journal 2008;862:2–8) ‘The claimed effect is to reduce arterial stiffness in mildly hypertensive subjects, and consequently the risk of cardiovascular disease. The Panel considers that it has not been established that reducing arterial stiffness is beneficial to the health of mildly hypertensive subjects by reducing their risk of cardiovascular disease’ (EFSA Journal 2008;824:2–12)

microflora. Even before Metchnikoff (1907), this was the original suggestion of Tissier, a French paediatrician who observed that children with diarrhoea had a low number of bacteria in their stools characterised by a ‘bifid’ morphology. In contrast, stools of healthy children had an abundance of such bacteria (Tissier, 1906). Tissier thus suggested that bifid bacteria could be administered to patients with diarrhoea to help restore a healthy gut flora. Unfortunately, as reported by Agence Française de Sécurité Sanitaire des Aliments (AFSSA, 2005), it is not possible to define a ‘good flora profile’ and the flora in healthy subjects and certain types of patients, particularly those with bowel diseases, are significantly different, without it yet having been possible to establish whether these modifications occurred before or after development of the disease. This second statement has probably been in the minds of the NDA panellists when providing some of their opinions

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Box 19.3 Positive statements on health relationships (‘claimed effect’) for probiotics released by EFSA ‘Protecting the “alimentary system” against enteric pathogens is potentially beneficial for human health’ (EFSA Journal 2008;859:2–9) ‘The claimed effect is to improve iron absorption and the target population is adults at risk for iron deficiency. Iron deficiency is one of the most common micronutrient deficiencies. The Panel considers that improving iron absorption might be beneficial to human health’ (EFSA Journal 2009;999:2–9)

(Boxes 19.2 and 19.3). Further problems have been raised by one of the most studied probiotic activities, the modulation of the immune system. The lack of useful in vivo biomarkers is definitely hampering this link between probiotics and health. On the other hand, a reduction in gastrointestinal discomfort and the promotion of antioxidant activities are accepted benefits of their use.

19.2.3

Scientific substantiation

The regulatory requirement for a cause-and-effect relationship between the consumption of a particular food product and its claimed effects in humans, including strength, consistency, specificity, dose–response and biological plausibility of the relationship is, at the moment, the most challenging hurdle for all probiotic products submitted for approval to the EFSA. A lack of human studies, the use of doses that differ from those contained in the final products for which approval is sought, and data obtained using bacterial strains not present in the product are among the most common criticisms of the NDA panel. Also mentioned by the panel is the lack of a mechanism of action for the suggested health benefit. Such a mechanism, according to the panel, should at least be proposed on the basis of strainspecific scientific evidence. Further issues in this specific section of the evaluation scheme used by EFSA include the paucity of dose–response studies in the area of probiotic efficacy and lack of statistical power to back up claims.

19.3 19.3.1

APPROACHING THE HURDLES Hurdle characterisation

The identification and characterisation of a single probiotic strain in a food product does not seem to be a significant hurdle, as molecular biology provides a full range of tools for these tasks. However, this is not the case for multistrain products. These kinds of preparations represent the large majority of the food supplement markets throughout Europe and they are also commercially available worldwide. Unfortunately, as reported in several studies (HamiltonMiller et al., 1999; Hamilton-Miller & Shah, 2002; Bertazzoni-Minelli et al., 2002; Weese, 2002, 2003; Temmerman et al., 2003; Drago et al., 2004; Elliot & Teversham, 2004; Szajewska et al., 2004; Sheu et al., 2009), a rather high percentage of these probiotic products are substantially different from what is listed on the label and what is yielded in bacterial counts.

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An important first hurdle to overcome in properly characterising a probiotic product containing more than one live bacterial strain is the development of robust strain-specific identification protocols. This will not be easy if the strains belong to the same or closely related species. Insensitivity (so-called ‘intrinsic resistance’) to antibiotics or other selective agents could assist with this if the mixture is formed by strains with a different profile in this regard. As an example, a food supplement containing Lactobacillus rhamnosus and Lactobacillus acidophilus could be characterised at the species level by plating on two different agar media, the first containing vancomycin to count L. rhamnosus and the latter, specific for L. acidophilus, supplemented with ciprofloxacin (International Standards Organization, 2006) (see also Chapter 18). However, classical microbiology cannot provide solutions to the discrimination of the strains found in most of the complex food supplements present on the market. There is for example no way of selectively plating L. rhamnosus and L. paracasei. The culture-independent approach, generally based on the extraction of bacterial DNA followed by PCR amplification, could provide an answer to this characterisation issue. Qualitative approaches (presence/absence of a species listed on a label) have been realised by using variable regions of the 16S ribosomal DNA to obtain species-specific amplicons (Ben et al., 2007; Sul et al., 2007; Tsai et al., 2008; Youn et al., 2008; Sheu et al., 2009). This is followed by visualisation using an electrophoretic gel. This kind of approach was developed mainly for scientific purposes, but has now been added to the quality control of food supplements by national bodies such as the Italian Istituto Superiore di Sanità (Aureli et al., 2008). Methods are also currently available that can identify individual members of microbial consortia by means of gene amplification followed by separation on a denaturing gradient gel. Digital capture and the processing of denaturing gradient gel electrophoresis (DGGE) band patterns facilitate the direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 hours. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for the detection of microbial strains in probiotic products in a fast, reliable and reproducible manner (Fasoli et al., 2003; Temmerman et al., 2003; Theunissen et al., 2005). An additional bottleneck in this activity is the need to achieve a strain-specific characterisation. This problem originates directly from the accepted definition of a strain: ‘a population of organisms that descends from a single organism or pure culture isolate. Strains within a species may differ slightly from one another in many ways’ (Willey et al., 2007). Other researchers, taking into consideration the accumulation of spontaneous mutants during the reproduction of the first isolated bacterial cell, prefer to use the expression ‘axenic culture’ instead of ‘pure culture’ but this remains the scientific definition. Both of these terms appear to be acceptable definitions for scientific purposes but what about regulatory needs involving live bacteria as food supplements? Some of the potential hurdles posed by these definitions and the identification of what the term ‘strain’ really could mean for regulatory purposes are discussed below. For epidemiologists, bacterial isolates are defined as being genetically ‘indistinguishable’ if their pulsed-field gel electrophoresis (PFGE) profile is 100% similar, and as being ‘closely related’ if they show PFGE profiles with at least 85% similarity, typically a two to three band difference, which is consistent with a single genetic event (Tenover et al., 1995). However, this does not seem to be applicable to probiotic strains. In 2001 in our laboratory (Cesena et al., 2001) we showed that a spontaneous mutant, obtained simply by culturing, plating and re-isolating an L. crispatus strain, was indistinguishable using PFGE (three enzymes) used,

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ribotyping and randomly amplified polymorphic DNA (RAPD) from the wild parent strain. However, the co-aggregation phenotype, present in the wild type, was missing in the mutant. This difference in phenotype was shown to be responsible for the two variants, in adhesion to mucus (Kirjavainen et al., 1998) and, moreover, survival and persistence in mice (Castagliuolo et al., 2005) and in the human gut (Cesena et al., 2001). Additionally, the wildtype strain has been shown to protect mice from DSS-induced colitis, whereas this mutant was totally ineffective in conferring such resistance (Castagliuolo et al., 2005). More recently, the group of T. Klaehammer (Goh et al., 2009) has shown that a single gene variation has a dramatic impact on the probiotic aptitudes of an L. acidophilus strain. A two-step plasmid integration and excision strategy was used to achieve an in-frame deletion of the gene encoding a 51-kDa surface (S)-layer protein L. acidophilus NCFM. The resulting mutant, isogenic to wild type with the exception of a single gene encoding the surface protein, exhibited lower growth rates, increased sensitivity to sodium dodecyl sulphate and greater resistance to bile, with the latter mechanisms clearly involved in determining the probiotic functionality of the strain. The same strain and gene manipulation strategy has also been used to study the mechanism of immune modulation as L. acidophilus NCFM attaches to dendritic cells and induces changes in interleukin production (Konstantinov et al., 2008). A knockout mutant of L. acidophilus NCFM lacking the S-layer A protein (SlpA) was significantly impaired in its binding to dendritic cells. This mutant incurred a chromosomal inversion leading to the dominant expression of a second S-layer protein, SlpB. In the SlpB-dominant strain, the results of the immune modulation activities changed dramatically. Higher concentrations of proinflammatory cytokines were produced by the interaction of the SlpB-dominant strain with dendritic cells when compared with the parent NCFM strain. The role of SlpA in immune modulation was further confirmed by using purified SlpA protein ligated directly to dendritic cells. The results clearly and strongly suggested that a single gene mutation can induce major changes in the probiotic activity of a given strain. In addition, the genomes of bacteria also comprise extra-chromosomal elements, such as plasmids. Recently, a cured derivative of a well-known probiotic L. reuteri (Rosander et al., 2008) was obtained in order to obtain a drug-resistant-free derivative of the wild-type strain. All assays confirmed the substantial identity of the cured derivative as wild type but, as shown above, the tools available at present are not sufficiently sensitive to completely establish an identity. These methods can provide robust evidence of a difference but not vice versa. However, tools are available to establish an ‘equivalence’ between two biological compounds such as antibiotics and other drugs. A significant issue to be overcome in the future therefore is how microbiologists can translate the concept of ‘bioequivalence’ used for generic drugs to the issue of bacterial strains for which health claims are made and approval is sought. Bioequivalence is a term used to define the biological equivalence of two pharmaceutical products: if two drugs are said to be bioequivalent, they would be expected to be, for all intents and purposes, the same. In the EU two medicinal products are defined as bioequivalent if they are pharmaceutically equivalent and if their bioavailability is such that their effects, with respect to both efficacy and safety, are essentially the same (EMEACPMP, 20001). The United States Food and Drug Administration (FDA) definition of bioequivalence is: the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available

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at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study (Food and Drug Administration, 2003).

The assessment of bioequivalence is regulated by tight procedure and protocols but no such framework is in place to assess the bioequivalence of probiotic food or food supplements containing probiotic bacteria. To develop a concept of bioequivalence for probiotics seems therefore to be a fundamental challenge in the coming years not only for the reasons listed above but also to overcome issues underpinning many of the claims related to probiotics and collected in the member states under Article 13.1 as most of these properties have been described as ‘strain specific’. This will be one of the major problems to be addressed by EFSA, as it seems that for the specific issue of probiotics it is difficult to link the best available science with the current regulatory issues. It is generally accepted that probiotic properties are strain-specific as, in addition to a scientific consensus, a number of national and international agencies have supported the concept of strain specificity for the characteristics of these bacteria. For example, FAO/WHO (2001) states that ‘probiotic properties are strain related’, while AFSSA (2005) states that: It is therefore generally accepted that the effects of one strain cannot be extrapolated to another. In other words, clinical studies on the strain itself are required before any claim can be made. Producers can use this characteristic to protect the specificities of their products. Advertising or claims referring to similar strains must not be used in scientific or promotional dossiers or brochures, neither in their evaluations.

In addition, the NDA panel of the EFSA has expressed the same opinion regarding the strain specificity of probiotic action ‘probiotic effects are strain-specific and dosedependent’ (EFSA Journal 2008;859:2–9). However, all these statements conflict with the provisions laid down in Article 13.1: Health claims…which are indicated in the list provided for in paragraph 3 may be made without undergoing the procedures laid down in Articles 15 to 19, if they are (i) based on generally accepted scientific evidence. To avoid the procedures cited in these Articles means that specific applications supported by scientific and clinical evidence are not necessary and it also means that the claims allowed under art. 13.1 will be shared by all foods or ingredients of the same category. As an example, a claim approved for Vitamin C under the art. 13, will apply to all products containing the same amount of this vitamin and of the same purity etc. However, if probiotic properties are strain-related and these strains are proprietary, how could these type of ‘generic’ claims be extended to other strains?

Furthermore, several probiotic bacteria are patented on the basis of their individual traits, making it even more difficult to share potential health claims under Article 13.1 with other strains. Again, the need to establish effective procedures for assessing bioequivalence is a barrier that must be surmounted in the future. This creates an additional need for greater clarity regarding the mechanisms of probiotic action. Bioequivalence analysis will also need to be based on affordable in vitro or ex vivo assays as well as animal model experiments. A small but still statistically significant in vivo trial to establish whether strains under evaluation have the same ability as a control strain to survive and reproduce in the human gut will probably also be necessary, but this effort will certainly be less costly and more affordable than testing the probiotic properties of a given strain from the beginning.

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In conclusion, therefore, it is surprising that at present EFSA has not asked for probiotic bacterial strains that are proposed for claims to be deposited with an international deposit authority; this is mandatory when applying for probiotics to be used as feed additives and it is also recommended in Italy for human dietary supplements (Ministero della Salute, 2005). Depositing these strains will be of help in addressing the problem of characterising strains in applications for which health claims are being made.

19.3.2

Relationship to health

This is an area of investigation that represents a clear barrier in verifying the health claims associated with probiotics. The message from the NDA panel is quite explicit and clear: ‘A cause and effect relationship between consumption of the food and the claimed effect in humans, including strength, consistency, specificity, dose–response and biological plausibility of the relationship’ is required. This hurdle is both scientific and regulatory: scientific because it will be necessary to develop probiotic-specific biomarkers based on both microbial and non-microbial assessments of the impact of probiotics on human health; regulatory because it seems that assessments of data supporting the claims in this scientifically fast-moving world have not been established as a permanent framework. European-funded research has provided some guidelines for establishing biomarkers for food and the PASSCLAIM project (Aggett et al., 2005) defines a set of conditions for biomarkers, which should: ● ● ●

have a known relationship to the final outcome; have a known range of variability within the target population; have a methodological validity with respect to their analytical characteristics.

However, this set of features seems not to be applicable to ‘natural intestinal flora’ used as markers to measure the impact of a probiotic on well-being. In this regard, note the following. ●

●

While it is known that a high content of some bacteria is harmful, it is still unclear and a matter of some debate whether the lack of other bacteria could also be dangerous. It is known the some people are naturally lacking in lactobacilli but nothing is known about the link between this ecological situation and a reduced health outcome. On the other hand, the lack of specific bacteria (Sokol et al., 2008) has recently been linked to the presence of a pathological inflammatory condition of the gut, but this observation needs to be confirmed and validated using intervention studies. The range of variability of the composition of human microbiota is still unknown. The techniques available for determining the composition of this microbiota, including non-cultivatable species are now available, from a high-throughput sequencing approach to DGGE or fluorescence in situ hybridisation (FISH). However, a lack of knowledge still exists due to the limited number of subjects investigated, the paucity of information on diet and lifestyle impacts, etc. Long-term studies still need to provide the link between overall microbial composition of the gut ecosystem and health status.

An exception to these situations could be the bacterial composition of the vaginal ecosystem, in which the relevance of lactobacilli in ensuring the well-being of women is

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well described in a large number of studies (for a recent review see Reid, 2008). Moreover for this microbiota, a recognised measurement assay is in place, namely the Nugent score (Sha et al., 2005, 2007), which can be used to validate the impact of a Lactobacillus supplementation (a methodological validity with respect to analytical characteristics ensures a technical validation in terms of accuracy, precision, repeatability, reproducibility, etc.). However, the fact that a result has been accepted at the scientific level (i.e. published in a peer-reviewed journal) does not mean that it will be accepted by national regulatory bodies or an evaluation panel as valid. A specific hurdle to overcome in this regard is to move from the ecological concept of feeding beneficial bacteria to ‘restore’ an ideal equilibrium (scientifically exciting but not measurable) to measurable markers such as frequency of defecation, pain reduction and gastrointestinal discomfort. In addition, the efficient colonisation of the gut by probiotic bacteria is not widely accepted as a health-related trait: one comment by the NDA panel stated that ‘intestinal tract colonisation can be a property of any (resident) gut bacterium (also pathogens). The Panel therefore concludes that the applicant has not shown the relevance of the claimed effect to human health’. It can be concluded therefore that health relationships based on ecological considerations are at the moment not sufficiently measurable to be used in support of probiotic products. Probiotic-specific, robust and validated biomarkers are therefore required. The cross-talk between beneficial bacteria and the mucosal immune system has been proposed as one of the most attractive properties of probiotics. However, the need for ‘measurable’ characteristics represents a serious impediment to claiming this beneficial potential of these bacteria. The panel has expressed this opinion: The claimed effect is to ‘support, stimulate and modulate the immune system of children during growth’. However no definition is provided of what constitutes a healthy immune system or how the support, stimulation or modulation of the immune system can be assessed.

This hurdle is therefore very similar to the previous one as it implies the need to develop and validate specific biomarkers.

19.3.3

Scientific substantiation

Several documents have already been published which can assist in the development of scientific dossiers for the substantiation of probiotic health claims. The first guidelines for assessing the efficacy of probiotics were provided by FAO/WHO (2002). These are useful tools and provide good guidance even though they are not specific for European legislation. The EU has funded a project, PASSCLAIM, which is devoted to the establishment of guidelines for efficacy assessments of functional foods (Contor & Asp, 2004; Aggett et al., 2005; Asp & Bryngelsson, 2008). The International Dairy Federation (IDF) has recently published a document on probiotics (Mercenier et al., 2008) in which a chapter is devoted to the evaluation of the probiotic properties of different bacterial strains. Among the eight points listed in this chapter, it is noteworthy that deposit of probiotic strains in an international culture collection (which is mandatory in the EU for their approval as a feed supplement) is highly recommended. In the same document, good practice guidelines for undertaking in vitro and in vivo studies of probiotics are provided. These guidelines in turn provide a good reference system for researchers who wish to develop new probiotics and

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aim to apply for EU approval within the framework of the health claims legislation. To provide some more details about the hurdles posed by EFSA policies regarding the scientific substantiation of probiotic-related health claims, it seems worthwhile to examine some of the issues that result from their stated positions and opinions. Firstly, in relation to EFSA statements that: the published studies provided to substantiate the health claim relate to a number of different probiotic strains but not to the bacterial strains in product X. As probiotic effects are strain specific and dose-dependent these publications cannot be used to substantiate the health claim.

This is not an insurmountable hurdle but clearly points to the need for strain-specific studies. Secondly, in relation to EFSA statements that: statistical analysis did not exploit the data available for different time-points, particularly for fluctuating symptoms with a high variability within individuals which were assessed through scales that have not been validated, and that, post-randomisation, the study was not sufficiently controlled for confounders that could potentially have affected the outcome (e.g. background diet and use of medications other than antimicrobials).

The first part of this statement indicates the need for a solid statistical framework as an integral and preliminary part of any intervention studies. In the second part, the need for detailed analyses of the ‘environment’ to evaluate possible confounding aspects is required. Thirdly, the sample size and study design are also clearly critical. The number of subjects enrolled needs to be sufficient to provide statistical significance to any final outcomes. Furthermore, the studies need to be designed according to the best clinical guidelines and practices, most preferably in a double-blind, randomised, placebo (or reference food)-controlled manner, and if possible in the intention-to treat way.

19.4 19.4.1

NEW PERSPECTIVES General considerations

There are three major lines of research and development that will be required to address the major hurdles posed by the new EU Regulations and convert them into opportunities. The first of these is multistrain characterisation from the functional point of view. If it is tempting, from the scientific point of view, to speculate on the major beneficial impacts of complex products, it is also very challenging to provide a clear picture of what is happening in vivo. The second line of research needed to overcome regulatory barriers requires a refocusing of scientific attention from the microbiota to the microbiome. The final line will be the development of protocols for efficacy trials that are more suitable for food in general and for probiotics in particular. Among the first nine (all negative) opinions released by EFSA for probiotics claims under Article 13.5 and 14, seven related to multistrain products. The first hurdle/opportunity in this regard could potentially be divided into several subcategories.

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Development of tools that can assess the behaviour in vivo of a mixture of bacterial strains. This could be the aim of a new line of research and even if tools to discriminate bacteria at the strain level are already available, they are generally time-consuming and often require plating steps to obtain colonies to be analysed. Availability at reasonable cost of the nucleotide sequence of the entire chromosome will allow researchers to develop strain-specific primers or probes, thus enabling scientists to monitor the fate of ingested bacteria even if fed as part of a complex mixture. RNA-based detection methods could then be used, yielding information on both the presence and viability of strains. Examples of these methodologies are already available, but they are generally provided by single-strain products. To identify a research strategy that will characterise the mechanisms of action of a product containing more than one strain is an even more challenging barrier in the regulatory context. Some of these products have been supported by rationales based on so-called additive effects, i.e. it is presumed that in a combination of strains belonging to different species and with different probiotic profiles, each of the beneficial properties of individual strains will be maintained and expressed after ingestion in an additive way. However, this effect is not really proven and in fact there is evidence, at least in vitro and ex vivo, that additive effects do not result from combining bacteria with different abilities to modulate the immune system (Castellazzi et al., 2007). In addition, the ecological assumption that as lactobacilli preferably colonise the upper part of the intestinal tract while bifidobacteria mainly inhabit the colon, both of these benefits will be obtained by mixing strains belonging to the two genera in the same product does not seem to be confirmed by the albeit scarce data available from in vivo studies showing that the same strain of lactobacilli has been found throughout the intestinal tract (Morelli et al., 2006).

These are only a few examples of the lack, at least at present, of a solid scientific basis for using mixtures of probiotic bacteria, but they strongly suggest that the rationale for using multistrain products is still to be robustly verified and this will need to be done using a case-by-case approach. Sound science to overcome these issues could be provided by (1) the development of validated animal models to further understand the interactions among strains and between the strains and the host and (2) the exploitation, in terms of functionality, of the wealth of genomic data already available or in progress. Animal models At the moment a consensus does not exist regarding the most suitable model system to be used (such as mouse and pig) and it is also unclear what the optimal doses would be in different animal models that would faithfully reflect the situation in humans. Also unknown are the effects of the indigenous microbiota in these animal models on the ingested allochthonous bacteria. It has been suggested that probiotic strains show a species-specific pattern of gut colonisation. If this is true, it will always be difficult for human-derived strains to fully exert their effects in an animal gut and to dominate an ecosystem for which they are not fully suited. Even if the species-specific pattern of persistence is questionable (Morelli, 2000), it is clear that the overall composition of naturally occurring microbiota is not equivalent in animals and humans and we can therefore reasonably speculate that colonisation resistance (the difficulty encountered by a newcomer bacterium in establishing itself in an already existing bacterial ecosystem) will be different. The animal model may therefore have limited value as a tool for assessing the persistence of probiotic strains to be used in humans.

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A model system provided by germ-free animals is also of little value in assessing the colonisation potential of human strains of bacteria, as the experiments performed in these animals to assess persistence do not produce clear-cut results or outcomes that can be applied to human use. However, germ-free animals could be helpful in assessing the difference between the characteristics (enzymes, histology, immunological profiles) of germfree animals that are due only to the gut (gut-associated characteristics) and those related to the presence of microbiota (microbiota-associated characteristics, MAC) (Midtvedt, 1986). These analyses would be useful in identifying the mechanisms of action of these bacteria, i.e. at the immunological, metabolic or enzymatic level. MAC features are typically measured in ex-germ-free mice, after conventionalisation or inoculation with known groups of bacteria (gnotobiotic mice).

19.4.2

Functional genomics

A new perspective in the comprehension of bacterial mechanisms of action is now provided by what is termed ‘functional genomics’, i.e. the exploitation of nucleotide sequencing to identify and understand the phenotypes responsible for survival and reproduction in the gut, interactions with the intestinal tissues and the whole range of probiotic traits. The genomes of some probiotic bacteria have been sequenced and some others are currently in progress. These data could provide information on the genetic profiles of bacteria that can survive into the intestinal tract and, more importantly, predict some probiotic activities such as immune stimulation. Some examples of functional characterisations of probiotic strains include the following. ●

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Sugar utilisation has been analysed in depth in all probiotic bacteria for whom genomic sequences and information are available, and the results are quite promising. In the Bifidobacterium longum NCC2705 genome (Schell et al., 2002), a large number of genes appear to encode products that catabolise a range of oligosaccharides, some of which are possibly novel hydrolases that are also active against prebiotic-like plant polymers or host-derived glycoproteins and glycoconjugates. For this strain, functional genomics has shown that the potential of a bacterium to utilise a large variety of nutrients is likely to contribute to its persistence in the colon. Moreover, genomic analysis has provided information regarding the potential for this particular strain to adhere to epithelial tissues, i.e. it was possible to identify genes coding for polypeptides with homology to major proteins required for the production of glycoprotein-binding fimbriae, structures that could possibly be important for adhesion and persistence in the gastrointestinal tract. Carbohydrate utilisation by Lactobacillus acidophilus NCFM was characterised using whole-genome cDNA microarrays. Transcriptional profiles were determined for growth on glucose, fructose, sucrose, lactose, galactose, trehalose, raffinose and fructo-oligosaccharides. The overall results obtained by microarray data revealed that the transcription of genes involved in sugar uptake and metabolism is coordinated and regulated by the specific carbohydrate provided. Moreover, the adaptability of L. acidophilus to particular intestinal conditions is likely to contribute to its ability to compete for carbohydrate sources available in the human gut (Barrangou et al., 2003; Altermann et al., 2005). Genes involved in ensuring the persistent intestinal growth of bacteria that have been the most extensively investigated have been analysed by means of genomics and transcriptomics in a single strain of L. johnsonii (Pridmore et al., 2004). Three gene loci,

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expressed in vivo and specific to prolonged gut persistence, have been detected within which genes were identified encoding glycosyltransferase, a sugar phosphotransferase system (PTS) transporter annotated as mannose PTS, and also a gene whose product shares 30% amino acid identity with IgA proteases from pathogenic bacteria. Knockout mutants tested in vivo showed that deletion of the sugar transport system decreases the gut residence time of the bacteria, whereas a mutant with a deleted exopolysaccharide biosynthesis cluster had a slightly increased residence time. Again, the fermentation potential of bacteria seems to play a relevant role in facilitating their persistence in the intestinal tract. In addition to these persistence phenotypes assessed by functional genomics, genome bioinformatics analysis of the Bifidobacterium longum NCC2705 strain has revealed the presence of genes encoding peptides that are homologous to proteins needed for production of glycoprotein-binding fimbriae (Klijn et al., 2005). This suggests that bifidobacteria could use these structures to adhere to intestinal tissues in a similar manner and competitively with Enterobacteriaceae. Using sequence data from B. longum NCC2705, we have also shown in our laboratory the presence of the gene encoding fimbriae in another strain of B. longum, but this gene is absent in all Bifidobacterium breve strains that we have so far assayed, thus revealing a difference between the two species in terms of adhesion and persistence in the gastrointestinal tract (L. Morelli, unpublished results). In L. johnsonii NCC533, genomic analysis has revealed the presence of more than 12 large and unusual cell-surface proteins, including fimbrial subunits, possibly involved in adhesion to intestinal mucins (Pridmore et al., 2004).

From this short list of probiotic features revealed by genomic analysis, it is noteworthy to add that a bioinformatics assessment of Lactobacillus plantarum WCFS1 has provided a wealth of data regarding the behaviour of this strain under bile stress (Bron et al., 2004). Moreover, in B. longum NCC2705 (Schell et al., 2002) a eukaryotic-type serine protease inhibitor (serpin) has been identified which is possibly involved in the reported immunomodulatory activity of bifidobacteria. Genomic analysis is therefore a promising and fast-growing tool for probiotic strain selection which has the capacity to provide not only robust science in this context that is equivalent to the widely accepted ecological selection criteria but also valuable in vitro insights into the functional activities of probiotic strains. A second area in which the EU Regulations regarding probiotic health claims are creating new perspectives and hurdles to surmount is related to the need to investigate complex microbiota and to extrapolate the links between the presence/absence of specific groups of bacteria and health outcomes. This issue is also relevant to the assessment of health benefits provided by prebiotic substances. An FAO report describes a prebiotic as ‘a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota’. To observe a variation (modulation) and obtain robust evidence that this variation confers a health benefit clearly necessitates the need to acquire in-depth knowledge of the composition of the microbiota and the bacteria which are to be favoured. In the same FAO document cited above, it is stated that ‘Bifidogenic effects are not sufficient without demonstrated physiological health benefits’. Hence the simple observation of some changes in the microbiotic profiles does not enable firm conclusions to be drawn regarding beneficial probiotic effects if not coupled to changes in selected biomarker(s). It is noteworthy that the information obtained using molecular tools has significantly modified our view of the intestinal microbial ecosystem in humans. Thus, the composition of human microbiota is now believe to be dominated by Eubacterium rectale, Clostridium

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coccoides and Clostridium leptum groups, along with Bacteroides and related groups, although in proportions which are highly variable between individuals. It is not yet possible to define a set of microbial species as a marker of normal flora. These data are also challenging the concept of ‘good’ and ‘bad’ bacteria, at least for healthy subjects. However, distortions in any one of the functions of the microbiota could potentially contribute to a wide range of diseases, from inflammatory diseases to neoplasias. Studies suggesting associations between microbiota and obesity have also been recently reported and received significant publicity (Ley et al., 2005, 2006) but other studies have refuted the existence of such an association (Duncan et al., 2008) suggesting the need for more in-depth investigations (Turnbaugh et al., 2009). Gut microbiota can be then regarded as an extra-genomic functional unit providing control mechanisms outside the genome of the host that affect nutritional status and health (Mai & Draganov, 2009). To increase the depth of understanding of the issues and regulatory hurdles caused by the relationship between the whole human microbiota composition and probiotic health claims, a fundamental consideration is the fact that the most commonly employed probiotics, Bifidobacterium and Lactobacillus, are not present in the human intestine at high levels. Thus, as there is a wealth of data suggesting that probiotic bacteria impart health benefits to individuals who have normal levels of indigenous bacteria, it is intuitive to conclude that these bacterial strains exert their beneficial actions without promoting any detectable changes in microbiota composition. It is clear therefore that there is a need to develop new tools and conduct further research on the whole bacterial population of the human gut. One very feasible possibility in this regard would be to study the impact of probiotics (and prebiotics) on the whole microbiota and on well-being by using animal models harbouring humanised microbiota (HBM). It has been reported that human neonatal microbiota can be transplanted and maintained in mice (Martin et al., 2008). It was then possible using this mouse model to reveal alterations in carbohydrate and protein fermentation and the subsequent effects of this on host lipid and energy metabolism as a consequence of diet supplementation with Lactobacillus paracasei and, to a lesser extent, L. rhamnosus. This mouse model is also promising, even if it refers to a simple human microbiota, as it consists of seven bacterial strains that were isolated from the stool of a 20-day-old female baby who was born by normal delivery and breast-fed. In a second study, the specific impacts of two probiotics (L. paracasei and L. rhamnosus) on the microbial populations of HBM mice were evaluated. An increase in the presence of bifidobacteria (which were not fed to the mice) and a reduction in Clostridium perfringens was observed in a more pronounced way when combining these prebiotics with L. rhamnosus. In addition, and of particular relevance in terms of health claims, it was possible to associate the ecological effects of probiotic and prebiotic supplementation with the modulation of a range of host metabolic pathways such as lipid profiles, gluconeogenesis, and amino acid and methylamine metabolism associated with the fermentation of carbohydrates and lipid homeostasis. While these results seem promising, it is worth bearing in mind that obtaining and maintaining mice harbouring an adult microbiota is challenging, but this is one of the hurdles to be overcome. A second opportunity to address the problem of characterising the whole microbiota is provided by two recently launched research projects on the human microbiome. The National Institutes of Health and the EU have launched two coordinated projects, namely the Human Microbiome Project (HMP) in the United States and the MetaHIT in Europe. The aims of the HMP are to generate resources for a comprehensive characterisation

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of the human microbiota and analysis of its role in human health and disease. In a similar way MetaHIT aims to establish associations between the genes of the human intestinal microbiota and health and disease. Two disorders of increasing importance in Europe, inflammatory bowel disease and obesity, will be specifically addressed by this research project. These studies are both timely and needed to further elucidate the roles of the whole microbiota and/or some of its components in human health and they will almost certainly provide a good framework of support to enable researchers to overcome the hurdle posed by EFSA statements on the ‘healthy microflora’ concept. However, we have to remember that, to date, most metagenomic studies of human gastrointestinal microbiota have analysed DNA sequences rather than whole genomes. From these studies, microbial diversity in the human gut has been found to be represented by members of only two bacterial phyla: the Firmicutes (65% of clones) and the Bacteroidetes (23% of clones). Additional phyla, such as the Proteobacteria, Actinobacteria, Fusobacteria and others, are detected in the intestines but at much lower frequencies. All large-scale studies of human gastrointestinal microbiota have also demonstrated substantial interindividual variation in sequence libraries, although variability is largely manifested at lower taxonomic ranks. Crucially, it is this variability at the species (and even strain) level which will be a major determinant of the support for probiotic health claims, a significant regulatory barrier. The third area of research needed to surmount regulatory barriers to probiotic use in the EU is the design of efficacy trials that can assess the probiotic potential of bacterial strains. Unlike drugs, the effects of foods are generally weak (and this is not a negative connotation but a specific feature that distinguishes them from pharmaceuticals), but this means that to assess the efficacy of probiotics and indeed all foods with a healthy potential using established medical protocols could be extremely difficult. Moreover, as the intended consumer is the general population, which is for the most part not affected by specific acute pathologies, some biomarkers as well as some clinical protocols will not be useful. This regulatory hurdle has been identified by the FAO/WHO document, in which the section ‘Use of probiotics in otherwise healthy people’ states that: many probiotic products are used by consumers who regard themselves as being otherwise healthy. They do so on the assumption that probiotics can retain their health and well being, and potentially reduce their long-term risk of diseases of the bowel, kidney, respiratory tract and heart

and concludes that ‘the Consultation would like studies to be done to give credibility to the perception that probiotics should be taken on a regular basis by healthy men, women and children’. Specific features of efficacy testing for foods (probiotics) may be amenable to some clinical protocols, such as the crossover protocol, in an attempt to minimise the impact of confounding factors. In addition, the intention-to-treat approach could be used to strengthen the statistical power of the whole study. In this context also, the placebo could be the same food containing probiotic strains but lacking active bacteria but developing a real placebo could prove technically challenging. Special attention will probably need to be paid to randomisation, ensuring that the appropriate microbiological parameters are used to ensure this. A final hurdle to consider that does not require research but which is probably the most relevant to human health concerns is the need to discriminate probiotics in food from those

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used in a clinical setting or in patients affected by pathological conditions. There is a need to specify whether the activities being advocated are designed to operate in otherwise healthy people or subjects with known diseases. Reviewing recently published nutritional and clinical trials, it becomes evident that scientists and clinicians are currently unable to draw a borderline between food probiotics and the therapeutic use of beneficial bacteria, but this differentiation has to be made, for both legal and safety reasons. In terms of EU regulations, claims are not applicable to products intended to be used in patients but only to food and food supplements commercialised for the general population. In terms of safety, it must be remembered that there is a long history of safe use of lactobacilli and bifidobacteria in food but the question of safety has been raised with the more recent use of intestinal isolates of bacteria delivered in high numbers to severely ill patients (Besselink et al., 2008). The use of probiotics to treat disease is only acceptable after approval by an independent ethics committee and after a specific and adequate safety assessment of the pathological conditions in which they are intended to be used. An FAO/WHO document issued in 2001 made a clear distinction between ‘biotherapeutics’ and ‘probiotics’ and even if the bacteria in question are the same strains, their intended use affects consideration of the product including safety assessments, dosage, etc. This is an issue that needs to be addressed quickly.

19.5

CONCLUSIONS

The EU is experiencing a revolution in its regulation of claims for food and food supplements. Probiotics represent about 8% of the food ingredients for which applications for health claims have been made under this new legislation. Guidelines for the substantiation of probiotics date back to 2002 (FAO) and, as described above, have been updated regularly via contributions from several bodies, but all on a voluntary basis. As of now, however, the new EU Regulations can undoubtedly restrict ill-defined and poorly characterised products, including probiotics. It would be incorrect to conclude that the regulatory hurdles imposed by the initial negative comments on probiotic products for which health claim applications have been made are the only obstacles for researchers and industry. In fact this legislation could induce a boost in basic research as well as in clinical studies, supporting the launch of new products. A recent publication (Mater, 2007) has pointed out that the supply of probiotic dairy products and related scientific publications showed a parallel increase from 2000 to 2006. It would be of interest to monitor the future development of the science and marketing of probiotic dairy products in Europe after the new legislation has been introduced. To reiterate the close link between regulatory and scientific issues, it is important to remember that Article 15.5 of Regulation 1924/2006 states that ‘The Commission, in close cooperation with the Authority, shall make available appropriate technical guidance and tools to assist food business operators, in particular SMEs, in the preparation and presentation of the application for scientific assessment’. It is thus tempting to speculate that the ‘appropriate technical guidance and tools’ will also in the future comprise guidelines for the number of efficacy studies necessary to support data obtained in vivo regarding probiotic health claims. This kind of guideline exists to substantiate the efficacy of viable bacteria used as feed supplements and applying for approval to another panel (FEEDAP) of the same European authority (EFSA).

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Szajewska H, Fordymacka A, Bardowski J, Gorecki RK, Mrukowicz JZ, Banaszkiewicz A (2004) Microbiological and genetic analysis of probiotic products licensed for medicinal purposes. Med Sci Monit 10:BR346–BR350. Temmerman R, Scheirlinck I, Huys G, Swings J (2003) Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis. Appl Environ Microbiol 69:220–226. Tenover FC, Arbeit RD, Goering RV et al. (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–2239. Theunissen J, Britza TJ, Torriani S, Witthuhn RC (2005) Identification of probiotic microorganisms in South African products using PCR-based DGGE analysis. Int J Food Microbiol 98:11–21. Tissier H (1906) Traitement des infections intestinales par la méthode de la flore bactérienne de l’intestin. CR Soc Biol 60:359–361. Tsai CC, Lai CH, Yu B, Tsen HY (2008) Use of specific primers based on the 16S–23S internal transcribed spacer (ITS) region for the screening Bifidobacterium adolescentis in yogurt products and human stool samples. Anaerobe 14:219–223. Turnbaugh PJ, Hamady M, Yatsunenko T et al. (2009) A core gut microbiome in obese and lean twins. Nature 457:480–484. Weese JS (2002) Microbiologic evaluation of commercial probiotics. J Am Vet Med Assoc 220:794–797. Weese JS (2003) Evaluation of deficiencies in labeling of commercial probiotics. Can Vet J 44:982–983. Willey JM, Woolverton CJ, Sherwood LM (eds) (2007) Klein’s Microbiology. New York: McGraw-Hill Higher Education. Youn SY, Seo JM, Ji GE (2008) Evaluation of the PCR method for identification of Bifidobacterium species. Lett Appl Microbiol 46:7–13.

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20

Probiotics and Innovation

Jean-Michel Antoine, Jean-Michel Faurie, Raish Oozeer, Johan van Hylckama Vlieg, Jan Knol, Herwig Bachmann and Joël Doré

20.1 20.1.1

INTRODUCTION Early history

The modern history of probiotics started with a fascinating innovation. When Metchnikoff arrived in Paris to work in the Pasteur Institute, he was impressed by the hectic life of Paris and the bad health of the Parisian population. They were dying much younger than people in Ukraine where he came from. It would have been logical for Metchnikoff, like all researchers at the Pasteur Institute at that time, to look for the potential pathogen killing Parisians. Instead he had a wise innovative thought: he would look for the potential protective factor helping Ukrainians to live longer and potentially missing in the Parisian lifestyle and, more specifically, in Parisians’ diet. He hypothesised that the gut microbiota could be a critical target and that the diet could be a way to improve the gut microbiota by either restoring the good bacteria or cleansing the bad ones. He invented the concept of probiotics: a live microorganism that can bring a specific benefit for the host (Metchnikoff, 1907). At the same time two others innovations occurred. One was the observation, again in Paris, by Tissier (1900), a paediatrician, that a specific microorganism was present in the stools of healthy breast-fed babies, whereas it was absent from the stools of formula-fed babies suffering from diarrhoea. He called that specific Y-shaped bacterium Bifidus. This specific microorganism was a marker for the healthy status of babies, reinforcing the view that some microorganisms could be active contributors to health. The other innovation was the first human test performed by Nissle in 1916 with a live bacterium. He demonstrated that ingestion of a specific Escherichia coli, able to survive in the human gut, was able to cleanse healthy typhoid carriers of their Salmonella. He explored one of the first functions of the gut microbiota, namely its capacity to resist colonisation of the gut biotope by newcomers, even pathogenic microorganisms. Ingestion of a live microorganism was able to provide benefit to the host, thus illustrating the definition of probiotics.

20.1.2

Recent history

Improvement in technologies and tools are the usual triggers for innovation, and the case of probiotics is no exception. Kolars et al. (1984) used a modern test, the breath test, to


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assess the capacity of yogurt to improve lactose digestion in lactose malabsorbers (Savaiano et al., 1984). The breath test is able to monitor the amount of sugar not digested in the small intestine of humans; these sugars are fermented in the large intestine by the gut microbiota. One of the end products of the fermentation is hydrogen that is exhaled partially in the breath. Therefore monitoring the hydrogen concentration in the breath reflects the amount of sugar fermented, and a decrease in that concentration indicates that less sugar has been fermented and absorbed earlier in the small intestine. Savaiano et al. (1984) demonstrated that when lactose malabsorbers were drinking milk, there was a sustained rise in the breath hydrogen concentration during the 8 hours following milk ingestion, indicating that some lactose has not been digested. When the same subjects drank the same amount of lactose as yogurt, there was a dramatic decrease in the kinetics of hydrogen concentration in the breath, indicating a significant increase in digestion of lactose due to the yogurt. Savaino et al. (1984) demonstrated that the living conditions of the yogurt symbiosis was essential and that other species were not able to provide similar benefit (Martini et al., 1991). It was the starting point of a modern age: a probiotic is a living microorganism that, when ingested in adequate amounts, is able to provide a health benefit to the host. Different probiotics will provide different benefits. The radical innovation was that while classic starter cultures have been used for centuries and are still used to improve taste, nutritional values or to preserve foods from spoilage, probiotics open up a new field of food microbiology by aiming to provide health benefits beyond nutrition. Several probiotic strains have been tested in humans for their ability to provide specific health benefits, to improve physiological functions and risk factors, or to affect the risk of diseases. When innovating in probiotics, at least three different aspects need to be considered and these are briefly introduced below. I

II

III

It is easy to understand that not all species have the same potential. Within a species all strains share a number of characteristics specific for this species, e.g. their capacity to hydrolyse certain sugars, and this is the basis of the classic API identification technique. However, within the same species different strains will have some differences and these include a wide variety of functions, ranging from the capacity to cope with acidic stress to interaction with intestinal cell receptors. The discovery of the functional capacities of microorganisms and the selection of strains that combine a set of desired activities provides a huge window for innovation. The analysis and understanding of the genomic machinery underlying desired effects has facilitated screening for similar capacities in other species or strains as well as deciphering the potential for suitable application in food products. The expression of the functional potential of a given strain also depends on the growth conditions. Microbes adapt their physiology to their environment and respond to the growth medium, interacting with other components of the symbiosis used to ferment the milk into an enjoyable food. The anticarcinogenic activities of some strains depends on the growth medium they are cultivated on (Tavan et al., 2002). The interactions of a probiotic with the host can occur via multiple pathways and may include direct interactions with the gut epithelium, the mucosal immune system or on the activity of the host gut microbiota. The physiological consequences can reach far beyond the primary site of interaction and affect a broad range of physiological processes, including the immune and nervous systems that are interconnected throughout the whole body. It is fascinating to observe that prokaryotes are cross-talking to eukaryotes, in a dialogue beyond biological systems.

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20.2

305

NOT ALL PROBIOTICS ARE THE SAME: GENOMIC PERSPECTIVE

A first target for innovation is the microorganism itself. The availability of the full genome sequences of important food microorganisms has radically changed academic and industrial research on food microorganisms and probiotics. What can we expect in the future, when we will have at our disposal functional genomics technologies that can help elucidate the mechanisms of food fermentation and probiotic activity? In this section we briefly review how specific molecular approaches developed for post-genomic analysis in food microbiology, especially in situ analysis in fermented foods, can facilitate research in this field. The full genome sequences of more than 20 bacterial strains of dairy-related lactic acid bacteria and probiotics have been published, comprising several strains of Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesenteroides as well as several bifidobacteria (Siezen & Bachmann, 2008). The rapid increase in the number of available genome sequences has fuelled the emergence of various functional genomics technologies, allowing monitoring of physiological responses at the level of the transcriptome, where the DNA code is translated into instructions for the microorganism to produce some specific proteins, or at the level of the proteome, where newly produced proteins are synthesised under transcriptome instructions, and at the level of the metabolome, the end result at the metabolic level. These technologies have facilitated novel ways of studying the physiological responses of microorganisms to environmental stimuli. However, this very detailed analysis is providing huge amounts of data, and deciphering which are the key elements is still a challenge. A common feature of functional genomics approaches is that they include elaborate procedures for sample preparation and purification that are poorly compatible with the complex matrices encountered during food processing or in the gastrointestinal tract. Most of these technologies are therefore well adapted for studies in laboratory media and under laboratory conditions. However, understanding of bacterial responses under real-life conditions is ultimately required to control, predict and improve their performance in applications as food products or probiotics. Fluctuating and diverse microbial interactions as well as unstable physicochemical conditions result in very different bacterial responses that affect the process outcome. Biological material from environmental samples of food products or from the gastrointestinal tract are difficult to prepare for analysis of bacterial RNA, proteins or metabolites. Depending on the nature of the sample one could first try to isolate bacteria followed by the envisioned functional genomics approach (Gitton et al., 2005). However, depending on the isolation protocol, this process might lead to a distinct cellular response during isolation and provide data not representative of the physiological state of a cell in situ. While there is abundant use of functional genomics technologies in a laboratory environment, few studies are carried out under circumstances that resemble in situ conditions more realistically. Examples include transcriptome analysis of Lactobacillus helveticus and Lactococcus lactis after growth in milk or proteome analysis of Lactococcus lactis during growth in milk (Smeianov et al., 2007). However, there is increasing interest in studying various lactic acid bacteria in the dairy environment as illustrated by the proceedings of the 2008 symposium on lactic acid bacteria which contained numerous abstracts and reports on this topic. The global measurement of bacterial metabolites is a promising and highly relevant approach for investigating the functionality of food-fermenting dairy cultures. Many volatile

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bacterial metabolites act as potent flavour compounds and, because of their volatility, they can be sampled from the headspace of a sealed vial without elaborate sample preparation. Strains of Lactococcus lactis vary significantly in their metabolic capabilities, especially with regard to secondary metabolite production. Notably, some of the variable secondary metabolites are key flavour compounds in dairy products and it has been shown that growth conditions strongly influence metabolite production in a strain-dependent manner, indicating that screening conditions have to be carefully selected in order to obtain meaningful results (Bachmann et al., 2008). Analysis of the metabolome can be carried out in highthroughput systems; however, if the objective is to screen starter cultures in complex application habitats like a cheese or yoghurt, appropriate high-throughput protocols are often lacking. This is also the case for screening probiotic activity. It is due to this lack of costeffective screening in a system with a high predictive value that industrial culture collections remain poorly characterised in terms of their true application potential. For the investigation of the transcriptional response in situ, a number of strategies have been developed that have potential use in food applications. Signature tagged mutagenesis (STM) investigates the survival of random insertion mutants which are individually tagged, allowing the identification of genes involved in the survival of an organism in a particular environment (Hensel et al., 1995). Selective capture of transcribed sequences (SCOTS) allows the isolation of transcribed sequences from complex environmental samples and differential fluorescence induction (DFI) uses fluorescence-activated cell sorting (FACS) to enrich active promoter sequences that are transcriptionally coupled to a fluorescent reporter protein (Valdivia & Falkow, 1996; Graham & Clark-Curtiss, 1999). One technology that has been frequently applied to identify promoters that are specifically induced in situ is designated in vivo expression technology (IVET). It is based on the transcriptional fusion of random DNA fragments to a selectable marker gene (Merrell & Camilli, 2000). A variation of the IVET approach is recombinase-based in vivo expression technology (R-IVET), which uses a recombinase as the primary reporter. On expression of the recombinase, a chromosomally localised selective marker that is flanked by two recombination sites will be excised, leading to an irreversible phenotypic change that can be readily detected (Rediers et al., 2005). Initially, IVET systems have been used primarily to investigate the bacterial response of human pathogens in animal models, with one recent study of a Vibrio cholerae R-IVET system ingested by human volunteers. Over the past few years it has been applied to many organisms besides human pathogens, including soil bacteria. In addition, studies on human commensals and candidate probiotic bacteria have allowed the identification of genes specifically induced in the gastrointestinal tract that are potentially involved in probiotic activity (Bron et al., 2004). R-IVET has also been applied to food-fermenting bacteria, in particular starters involved in sourdough and meat fermentation. These studies have led to the identification of a variable number of genes that might be of importance for performance of the investigated strain in a particular ecological niche, which in several cases could be confirmed by the evaluation of specific knockout mutant strains. Despite all the advantages of IVET assays, there are clear shortcomings: only upregulated genes can be detected and the validation of identified target sequences requires subsequent, often very laborious, strategies. Recently, an improved R-IVET system has been reported that allows more rapid validation of target sequences by using the luciferase gene as a secondary reporter (Bachmann et al., 2008). In addition to the fundamental characterisation of bacteria in their natural/application biotope, the industrial relevance of such approaches was recently demonstrated. Kringelum et al. (2006) performed a transcriptome analysis of samples directly obtained from an

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industrial fermenter and revealed a purine limitation during the later phases of growth in a batch culture. The addition of extra purine sources increased the bacterial yield by more than 150%, thereby increasing the cost-effectiveness of the production process. In summary, there is large functional genomics tool box available that allows analysis of specific physiological responses of food-fermenting and probiotic microorganisms in food matrices and the gastrointestinal tract. These are powerful tools that allow us to increase our understanding of specific metabolic traits relevant for in situ behaviour and potentially serve as biomarkers for process development. One fascinating application of these techniques is the demonstration by Corthier et al. (1998), in a mouse model harbouring a human gut microbiota, that some probiotics ingested as a fermented milk were metabolically active in the gut within a few hours after ingestion as shown by the fluorescence generated by the added luciferase gene.

20.3

NOT ALL PROBIOTIC FOODS ARE THE SAME: FUNCTIONAL PERSPECTIVE

The second target for innovation is the impact of the process as well as the matrix on the potential of microorganisms. During industrial processes, the starters are inoculated in a rich medium suitable for their growth. Bacterial metabolism is focused on growth and acidification in order to provide a competitive advantage in a challenging environment. During exponential growth many genes that do not support rapid growth on the primary substrate are not expressed. The most well-known molecular mechanism controlling gene expression in this respect is catabolite repression. Most species show this type of behaviour when grown on glucose: many genes involved in use of less favourable sugars are repressed. This phenomenon is triggered by regulatory effects mediated by glycolytic intermediary protein catabolites like CcpA (Luesink et al., 1998). Upon entering the stationary phase of growth, glycolysis slows down, and with the decrease in intracellular concentrations of these compounds, several formerly repressed genes are expressed and another class of metabolites, called secondary metabolites, is produced. This illustrates the fact that during the major phase of fermentation a huge part of the microbial metabolic potential is not available due to regulatory events at the genetic level, and depending on the growth conditions different genetic potential will or will not be expressed. Therefore optimisation of the microbial metabolic potential can be achieved by creating conditions that allow the expression of desired activities encoded in the genomes of lactic acid bacteria by applying appropriate process conditions. This will give us access to new metabolic functions that could be translated into innovative functional products. This is exemplified by the vitamin K production by lactic acid bacteria and other food microorganisms. Vitamin K is involved in carboxylation reactions. Its role is well known in the production of blood clotting factors in humans. Recently, several studies have reported its role in bone metabolism (Weber, 2001) and its nutritional importance in improving health in human populations. It was shown that even in developed countries, the daily recommended intake is not achieved by all consumers (Booth & Suttie, 1998) and the vitamin K status is related to bone status and structure during growth of children. A possible innovation could be increasing vitamin K intake through dairy products fermented by specific strains that produce high levels of vitamin K. The association between a dairy matrix rich in protein and calcium, often enriched in vitamin D, and vitamin K in fermented milk should be of great interest for bone health and growing children.

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Menaquinone, the microbial form of vitamin K, is widespread among aerobic Gram positive bacteria. Menaquinone is a component of the respiratory chain. In lactic acid bacteria, only a few species are able to produce menaquinone. Morishita et al. (1999) showed that Lactococcus lactis and Leuconostoc lactis are part of this small group. Leuconostoc lactis is not able to produce haem but when haem is present in the medium, the bacteria is able to respire during aerated growth. This phenomenon starts at the end of the exponential growth phase. Following this work, we showed that menaquinone is mainly produced by Leuconostoc lactis during a very short period of the growth phase, during the transition from exponential to stationary growth. A common approach for increasing the production of secondary metabolites is to decrease the growth rate of the bacteria, and this has been successfully applied for a range of metabolites. Sybesma et al. (2004) used a similar strategy and presented convincing results for production of folates. The growth rate reduction could be achieved by using suboptimal growth conditions (temperature, nutrient limitations, antibiotic) or by using continuous fermentation operated at low growth rate. These approaches allowed a significant increase in folate production levels. Interestingly, there are only few applications of continuous fermentation in dairy production and, when used, increasing the productivity is the application advantage. The extreme point of growth rate reduction is zero growth, not far from resting cells. In this condition the cells are metabolically active but they are unable to grow. It is also possible to condition the metabolic behaviour of the strain in the fermentation at the strain production stage. We applied this strategy for vitamin K production. The strain is produced using respiration and inoculated at 2.5 × 109 CFU/mL in whole milk. This innovative process increases production of vitamin K 10-fold compared with a classical fermentation in skim milk. Another innovative way to overcome decreased production due to regulatory events during growth could be to use immobilised cells. Cells can be embedded in various biopolymers to create a specific microenvironment. Fermentative substrates and product transfers are then buffered into the polymeric gel. Leuconostoc lactis immobilised at high cell density shows fermentative patterns normally observed at the end of fermentation when reaching the stationary phase. Glucose is fermented to lactate, formate, acetate, ethanol and 2,3-butanediol instead of solely lactate during the growth phase with free cell cultures. A similar kind of fermentative modification is observed with E. coli immobilised in alginate beads. Inada et al. (1996) studied the sequential use of a carbon source (called diauxie) in E. coli entrapped in k-carrageenan grown on glucose and lactose. At low cell densities, glucose is consumed prior to lactose. At high cell densities, lactose and glucose are used at the same time. The hypothesis is that, due to transfer limitation in the gel, glucose concentration becomes too low for triggering catabolite repression. Cell immobilisation is a way to relieve catabolite repression during fermentation and gives access to metabolic activities usually repressed during normal fermentation processes. Different kinds of biopolymers (alginate, locus bean gum, carrageenan) could be used similarly for protecting probiotics during passage through the gut via encapsulation. The protective effect is supposed to be linked to the permeability of the coating, which consequently reduces the level of stress induced by the gut environment. Perrot et al. (2001) subjected gel-entrapped E. coli to cold shock. In this study proteome profiles obtained with immobilised cells using two-dimensional gel electrophoresis were compared with those of free cells during exponential growth and stationary phase after cold shock. The two-dimensional gel protein patterns obtained with resting cells are similar but showed a

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few significant differences to those of immobilised cells. Non-trapped resting cells show less resistance to cold shock. This behaviour is probably more related to a different physiological status, making them more resistant, rather than to a protective effect of the coating itself. The same type of observations have been seen with biofilm-grown cells. Cell immobilisation is an innovative way to access physiological status that is not achievable with free cell culture; it also enhances the survival rate of microorganisms in variable environments. Whatever the probiotic strain and its benefit, it typically needs to be preserved in the dairy matrix, and the cold chain that is commonly used in our countries requires a costly process. It starts with sterilisation of the raw milk before fermentation, and requires energy to ensure the cold chain after fermentation. There is a huge need for innovation to provide new starters able to reduce the endogenous microbes in raw milk, and able to survive for days or weeks at room temperature without loss of viability and activity while ensuring good organoleptic properties of the food products. Some technologies have been assessed as ways to overcome this challenge, including encapsulation, freeze-drying and spraydrying of the active probiotic, with the objective of increasing the shelf-life. Alone or in combination these techniques are very promising, enabling increased survival without post-acidification, and preserving the benefits of the probiotic, including its ability to survive in the human gut. However, these technologies are still expensive and technologically challenging, and they require a lot of energy. Another area for innovation is to explore the potential of fermentation to improve the nutritional qualities of non-dairy ingredients, such as some traditional vegetal products. Vegetal raw materials contain some antinutritional compounds like phytates or some toxic compounds like cyanosides found in cassava. Some strains of lactic acid bacteria are able to metabolise these compounds and reduce their adverse effects. Bering et al. (2006) demonstrated very nicely the advantage of oat gruel fermentation by Lactobacillus plantarum, compared with acidified oat gruel, on iron absorption in relation to a phytase activity. Iron deficiency is widespread among the populations in emerging countries. In these countries, cassava and cassava-derived foods form an important part of the daily diet. Cassava contains cyanoglycosides that have to be removed during a long processing. The use of defined starters leads to the production of better organoleptic products with reduced residual cyanoglycoside concentrations, higher microbial quality and a significant decrease in the process time. Modification of processes is a powerful lever for delivering innovative functionalities in food, for proposing existing benefits in new countries, and for improving the nutritional value of traditional products.

20.4

NOT ALL PROBIOTICS ARE CROSS-TALKING IN THE SAME WAY: DIALOGUE WITH THE HOST

The third target for innovation is the dialogue between the probiotic and/or its byproducts and the host. This huge area can be split into three sections: (1) the dialogue between prokaryotes, (2) direct cross-talk between probiotic (prokaryote) and the host (eukaryote), mainly the digestive wall, and (3) remote effects of the probiotic on host functions, mainly the immune system.

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20.4.1

Dialogue with the human intestinal microbiota: a logical trigger for innovation

The human intestinal microbiota constitutes a complex ecosystem that has been ignored for a very long time, mainly due to the low accessibility of this internal ‘organ’. The microbiota plays an important role in human health and disease, and there are 10 times more microorganisms in our gut than we have cells in our body. The easiest way to analyse the microbiota is sampling of the faeces, and in previous decades this was only possible through laborious culture techniques, mainly limited to basic population analysis by CFU enumeration and complex enumeration of the enzymatic capacities of the various cultures. However, only a small fraction of the intestinal microbiota can be cultivated under laboratory conditions and therefore this approach provides only a limited view of the diversity of the intestinal microbes. In the past 10 years, culture-independent molecular approaches have resulted in a complete reunderstanding of the phylogenetic composition of the dominant human intestinal microbiota. Novel technologies such as pyrosequencing-based phylogenetic mapping are today far more appropriate for assessing the dynamics of dominant species profiles and reveal the existence of conserved core species. The information gathered is still mainly limited to the population structure of the ecosystem, answering the question ‘Who is there?’ It has already led to a description of the phylogenetic core of the human intestinal microbiota, leading to the concepts of eubiosis and dysbiosis, the two states of the gut microbiota formerly identified as balanced or unbalanced, or ‘good’ and ‘bad’ microbiota, where lactobacilli and bifidobacteria were considered as classical examples of good microbes. Current developments using metagenomic approaches allow analysis of the complete gene repertoire of the human intestinal microbiota, providing for the first time informative insights at the level of encoded functionalities, even though a significant gap still remains between the presence of genes and expression of the corresponding gene products. The gene repertoire of the human intestinal microbiota will constitute a unique reference allowing the design of a new repertoire of tools for functional mapping at the level of the metagenome, meta-transcriptome and meta-proteome, where ‘meta’ refers to the totality of the microbiota. It will thereby permit the question ‘Who is doing what?’ in the ecosystem. This will allow the identification of diagnostic or prognostic biomarkers, as well as determining the contributing factors at these various levels of ‘omic’ integration. This will also provide models to cluster selected cohorts of patients and healthy subjects with similar microbial potential, and/or functional deficit, as the basis of intervention studies. It is as important to identify factors contributing to a beneficial or a deleterious effect on the host when they are present or when they are absent. It looks like the revolutionary view of Metchnikoff (i.e. protective factors) has been validated in some intestinal disorders where the microbiota signature is the absence of certain functions and not the presence of a never-identified pathogen. For example, is there a specific microbe generating Crohn’s disease or is a specific gut microbial function lacking that allows the imbalance to end in disease? Another expectation of microbiomic studies is a description of the complete metabolic map of the trophic interactions within the intestinal ecosystem. These complex networks allow the microbiota to cope with the huge number of different components of our diet and are of particular importance in the production of short-chain fatty acids and other metabolic end products that fuel the host. Such a metabolic framework will allow novel perspectives

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in terms of mathematical modelling of functional interactions between microbial actors (ecotypes) in different ecological niches. As in many physiological pathways, there are cascades of different microorganisms that depend on metabolites from others higher in the chain, right through to the end product. This complexity allows the microbiota to cope with the huge number of different components of our diet and to produce a limited number of metabolites for the benefit of the host. In turn, it will tentatively permit predictions about the impact of modulating biotic or abiotic components via a system’s ecology approach and to look for potential dietary or microbial modulators of gut microbiota functions and their consequences on human health. The microbiota is highly resilient and its ability to prevent newcomers becoming part of the microbiota is critical in maintaining health. This ability, also known as colonisation resistance, contributes to the barrier separating the luminal environment, part of the outside world, from the milieu interieur described by Claude Bernard. It prevents colonisation by pathogens by keeping them at such low population levels that they are unable to express pathogenicity. In addition, the high resilience of the microbiota ensures that it remains largely intact when challenged, either from dietary sources (i.e. fiber intake) or iatrogenic ones, mainly antibiotic treatment. The second function of the colonic microbiota is a metabolic one, ranging from facilitating energy extraction from the digestive bolus to detoxification of xenobiotics and endogenous toxic molecules. The enzymatic diversity of the gut microbiota is larger than that of the liver and has the capacity to digest many compounds not metabolised in the small intestine, such as endogenous secretions including mucins and digestive enzymes as well as molecules recirculated via the enterohepatic cycle, including bile acids, cholesterol and hormones. Some glycolytic activities as well as azoreductase may unravel the glycation achieved by liver enzymes in order to transfer some compounds from blood to bile. This will free some aglycone part of the then-activated molecules. One of the benefits is also to detoxify some components of the diet. Goldin and Gorbach (1976) have been able to show that in this way the carcinogenicity of a westernised diet in rats can be reduced with probiotics. This bioconversion of food-borne compounds unabsorbed in the upper parts of the digestive tract has recently resulted in novel insights about the impact of the gut microbiota on the regulation of host energy metabolism (Flier & Mekalanos, 2009) and fat storage (Turnbaugh et al., 2009). Although humans are essentially highly homogeneous in term of gut functionalities, it remains possible to divide the human population into ‘producers’ and ‘non- producers’ or ‘high metabolisers’ and ‘low-metabolisers’ for a whole range of activities. The reasons for such a dichotomy remain unclear. These activities include: ● ● ● ● ●

methanogenesis, i.e. production of methane, which is due to the presence of dominant methanogenic Archaea; conversion of bilirubin to urobilinogen; conversion of daidzein to equol; conversion of cholesterol to coprostanol; ability to reduce absorption of oxalate in the gut and reduce the risk of hyperoxaluria and urolithiasis due to the presence of Oxalobacter formigenes.

As in the case of methanogenesis, where the microorganisms involved are well known, it would be appropriate to identify the dominant actors responsible for the other activities in order to modulate them.

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Box 20.1 Dysbiosis of the human intestinal microbiota in the context of immune, metabolic or degenerative disorders ● ● ● ● ● ● ● ● ● ● ● ●

Frailty in seniors (Van Tongeren et al., 2005) Crohn’s disease (Seksik et al., 2003; Sokol et al., 2006, 2008a,b) Irritable bowel syndrome (Kassinen et al., 2007) Ulcerative colitis (Martinez et al., 2008; Sokol et al., 2008a,b) Obesity (Kalliomäki et al., 2008; Ley, 2010) Type 1 diabetes (Wen et al., 2008; Dessein et al., 2009) Type 2 diabetes (Cani & Delzenne, 2009) Coeliac disease (Nadal et al., 2007; Collado et al., 2009) Allergy (Kirjavainen et al., 2002) Autistic spectrum disorders (Finegold et al., 2002; Song et al., 2004) Clostridium difficile (Hickson et al., 2007) HIV infections (Gori et al., 2008)

The third function of the microbiota relates to its impact on host responses beyond metabolism. Examples are its contribution in maturation and modulation of the immune system, angiogenesis and development of gut tissues. The possible role of the human intestinal microbiota in immune, metabolic and/or degenerative diseases currently increasing in prevalence in industrialised societies has been addressed by several studies (Box 20.1). Although in several cases there are correlations between microbiota composition and activity and impaired health or disease, it remains to be established whether this altered composition is cause or effect. A proper understanding of these interactions may allow rational modulations of the microbiota to prevent dysbiosis, disruption of homeostasis and increased risk of disease.

20.4.2

Novel functional targets for the human intestinal microbiota

Mechanisms of interaction between microorganisms and human cells have been extensively studied for pathogens, especially bacteria. Conversely, very little is known about the mechanisms and signal molecules involved in the interactions between commensal bacteria and intestinal epithelial cells and other human cells. This knowledge is key to deciphering the role of the microbiota in development and maintenance of immune tolerance and general homeostasis. In addition, it is essential to understand the actual role of the microbiota in the onset and/or maintenance and/or prevention of diseases in industrialised countries. These questions represent major challenges and it is likely that not all disease contexts will lead to a mechanistic description and identification of key signal molecules. Significant innovations in exploratory tools will be necessary to address these questions. One emerging and promising technology is the cloning of genome fragments of intestinal bacteria in large metagenomic libraries combined with functional screening for bacteria–cell signalling in order to seek functions after heterologous expression in E.coli. By providing access to totally unexplored biological resources, this technology, already applied to the

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Table 20.1 Interfaces involving the microbiota and source of potential knowledge building and innovations in preventive nutrition and health. Domains of interface

Knowledge and/or societal relevance

Domain of application

Microbe–food

Reconstruction of the microbial food chain (models: hydrolysis, fermentations)

Cognitive and modelling

Bioconversion of polymeric and aromatic substances (food and beyond)

White and green chemistry

Bioavailability of active food constituents, beneficial or deleterious

Functional foods

Prevention of development and dispersal of pathogens

Food safety

Epithelial barrier, angiogenesis, mucus, peptides, defensins

Infection or cancer prevention

Immune development, immune maturation, immune senescence

Allergy, inflammation

Metabolism–absorption

Obesity, type II diabetes

Neuroactive signalling to local or central nervous system

IBS, autism

Antimicrobial activity: bacteriocins, colonisation resistance

Food safety

Quorum sensing in vivo, biofilm, gene regulation

Food safety

Synergistic–mutualistic–antagonistic

Cognitive and modelling

Microbe–host

Microbe–microbe

IBS, irritable bowel syndrome.

identification of novel catabolic functions and characterisation of novel enzymes, should prove very efficient for exploring cross-talk mechanisms. The example of diseases given above suggests that cross-talk mechanisms may be involved in regulation of the immune response, cell proliferation/differentiation, modulation of the sensation of pain, of gut motility and of hunger/satiety signalling, metabolic regulations, etc. Overall, as summarised in Table 20.1, the domains of expected innovation relate to the exploration of interfaces, i.e. domains of interaction between the microbiota and food, microbiota and host cells, and microbiota and other microbes. It is clearly impossible to provide an exhaustive list of all the unanswered questions about the microbial ecology of the human intestinal ecosystem, but a selected list can be proposed in order to highlight the gaps in knowledge likely to benefit from current and upcoming methodological developments. ●

As the stable adult microbiota appears to have only an extremely limited ability to allow allochthonous bacteria to establish dominance, it is conceivable that the digestive tract offers ‘a window of permissivity’ for colonisation early in life. This concept has yet to

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Table 20.2 The place of the human intestinal microbiota in innovation: summary of knowledge gaps and expectations. Knowledge or expectation

Corresponding innovation

Phylogenetic view, stability in composition and core, modelling

Ability for fine tracking of modulation Strategies to modulate composition

Eubiosis vs. dysbiosis, composition, metagenome, metaproteome, metabolome

Diagnostic tools and biomarkers for healthy gut Strategies to modulate biomarkers and restore eubiosis

Metabolic functions, ecotype identification, modelling and systems ecology

Strategies to modulate functions of commensal bacteria

Mechanistic understanding of bacteria–cell cross-talk

Preventive and/or therapeutic applications: gut–immunity, gut–brain (pain, satiety)

● ● ●

● ● ●

be validated and, furthermore, the duration of the period during which the ecosystem would remain permissive and thereby fragile but at the same time potentially amenable to manipulation, to the benefit of the host, has yet to be determined. The respective impacts of ecology (encountering microbes) and host genotype as determinants of the development of the adult microbiota have not been clearly assessed. The impact of the location of birth and/or mode of weaning worldwide (major staple food used) on the development of the intestinal microbiota remains unexplored. Since only 20% of the dominant bacterial species of the human intestinal microbiota are represented in culture collections, the addition of as-yet uncultured microorganisms to these collections is bound to remain an essential goal in order to characterise selected strains, especially those representative of new functional groups. Mechanisms responsible for the dynamic behaviour of intestinal microbiota in terms of resistance and resilience are unknown. More specifically, the stability of the intestinal microbiota over time in terms of phylogenetics and functions remains an open question. Under well-characterised stress conditions the microbiota is resilient, i.e. capable of recovering its original dominant species profile. Limits beyond which the human intestinal microbiota will no longer be spontaneously resilient have yet to be determined.

A summary of expected knowledge gaps to be filled and ensuing innovations is given in Table 20.2.

20.5

EUROPEAN REGULATORY PERSPECTIVE: A THREAT OR AN OPPORTUNITY?

A tremendous world of opportunities (microbiome, probiotics and prebiotics) has been recently identified and can be explored with the help of modern tools. Even if it looks like science is entering a level of mechanistic understanding, the gene level, that is too complex or detailed, full of bypass and redundancy that generates huge amounts of data and which requires an integrative capacity that may go beyond the human brain without the help of computerised intelligence, it is a fascinating source of innovations. On the other hand, thanks to progress in

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science and education, more and more convincing arguments are requested before a potential benefit can be claimed, and this is the case also for the relationship between food and health. The time for legends and beliefs is gone, and scientific demonstration is requested before a benefit can be claimed. It may be useful to remember the young history of food and health and to learn from the example of the discovery of the anti-scurvy activity of some foods. In 1750, James Lind explored the benefit provided by acidic beverages on a disease affecting sailors. Using six groups of two sick sailors he tested five different acidic beverages and, as a control, sea water, added to the same diet. Two beverages improved the health status: cider provided some benefit and a mixture of lemon and orange was quite successful. Lind went on looking for acids to improve beverages. It was one of his colleagues, Blane, who, 50 years later, made the real experiment during a trip from the United Kingdom to India. He split the team into two groups, one receiving an addition of lime in their daily diet, the other acting as a control. The result was convincing: those receiving the limes survived well and were able to remain on duty until the end of the trip, whereas the control group suffered from what came to be called scurvy. However, the mechanism(s) of action of lime were unknown. The use of lime was one of the key elements fostered by the British navy and led to the successful voyages of Cook. However, the Royal Academy of Medicine needed more than half a century of discussion before agreeing on the benefits of lime, despite their use in the Navy! The active factor, vitamin C, was discovered in 1928 by Szent-Gyorgyi, providing the ultimate part of the demonstration. At what point was the science strong enough to allow the use of lime in reducing the risk of scurvy? Interestingly, we still do not have the final answer today, as the efficacy of lemon juice goes beyond its vitamin C content. The multiple impacts of vitamin C on different tissues and metabolic pathways make precise understanding of how vitamin C is able to prevent scurvy a complex and difficult issue. It was fortunate for the sailors that the practical observations were taken into account as a rationale for improving their diet far before official medical agreement and the scientific deciphering of the mechanisms. There is endless debate in science. When is emerging science strong enough to be communicated to a non-expert audience? It is also interesting to consider that this food was used to prevent a disease, even if this disease is a nutrient-related one.

20.5.1

European regulatory perspective: a threat?

Today European regulations suggest that a food cannot prevent, cure or mitigate a disease, an area that is restricted to drugs. This European limitation seems to forget that, from the beginning of human existence, medicinal ingredients were extracted from foods, and may also be ignoring the situation in Asia, where some foods are used to prevent diseases, others are used to cure diseases, and these therapeutic foods are integrated in the daily diet. The present European requirements may become a threat when regulation starts to take over science and common sense and suggest that foods cannot be used to prevent or cure diseases. The first argument comes from a historical perspective: Hippocrates used food as a first medicine many centuries ago, using for example liver to provide vitamin A or sea foods to provide iodine and more. The second, and more modern, argument is in two parts. Foods provide energy and nutrients to the body and there are requirements that must be satisfied daily to prevent nutrient deficiency-related diseases, and the only way to provide these nutrients is an adequate diet. Diet must be used to prevent nutritional diseases, that is common sense, and no drug can substitute for these nutrients nor their effects. A stimulating area was opened by Norwegians some three decades ago when they discovered that muscular function may benefit from specific nutritional practices: they invented the

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sequential carbohydrate feeding plan to improve muscular strength and endurance. This was successful and helped, among other benefits, Ron Hill to win the Athens marathon in 1969. The gut is another organ that may benefit from an adapted diet. It is a very demanding organ with a high rate of cell renewal and the requirement for branched-chain amino acids is higher for the gut than for the whole body. Dietary supplements enriched in branchedchain amino acids are used for gut recovery (McCauley et al., 1996). This illustrates that beyond global nutrition to address the needs of every organ, there is room for specific feeding of functions. This has been called ‘functional feeding’: it is an exploding area where probiotics have great potential. The list of essential nutrients is not complete, and scientists regularly re-evaluate the recommended daily allowances and identify some new requirement, like the essential fatty acids in the last decade, as well as discovering some semi-essential ones, and exploring trace elements like selenium or arsenic. Nutritionists are also discovering, within the list of nutrients, some non-nutrients that are also important for health: a now classical one is fibre. The beneficial effect of bran on gut transit has been confirmed many times and expanded beyond wheat bran. Some of these fibres also act via the gut microbiota, and are called prebiotics. A new category of non-nutrients is probiotics. It may look like probiotics are at the stage where vitamins were a century ago. Various effects have been reported, depending on the probiotic, without clear mechanisms of action. We have one advantage: we can use modern tools to rapidly explore physiological and pathophysiological effects in humans. Increasingly, these tools also allow us to decipher the mechanisms of cross-talk between microbes and host that are important for exerting the mechanism of action. Foods are partners in the care of patients. Energy and nutrients are essentials for the body to cope with disease and to ensure adequate functioning of the defence systems. One of the markers of malnutrition is a deficit in the immune system and an increased sensitivity to infectious diseases. Sometimes food is the only practical way to cure a disease and the World Health Organization is promoting oral rehydration as a cure for diarrhoea in children and is saving millions of lives. Similarly, refeeding is the first step in fighting infections in malnourished consumers, and a new role of vitamin A in restoring proper functioning of the defence systems has been discovered in the last decades. For a long time physicians were willing to avoid feeding their patients and the first rule was: when you are sick, fasting is best. Physicians have killed millions of patients like that. Surgeons like Jeejeeboy led the way in the 1960s in using nutrition to strengthen the recovery of their patients, and nowadays it is agreed that one major prognostic criterion is the nutritional status of the patient. It is obvious that proper feeding is an essential part of the management of disease, apart from drugs targeting pathogenic factors and dysfunctioning organs. It is scientific nonsense not to use food to manage patients and their diseases, and this is becoming more and more important with the increase in chronic diseases and in healthcare expenditure. Some probiotics will have beneficial effects on the recovering gut and at least on recovering microbiota. This will be another innovative field.

20.5.2

For innovation in probiotics, the present regulatory requirements are an opportunity

The modern scientific assessment of an effect is a clear opportunity to discriminate real efficacy from misleading fantasy and to select probiotics that bring proven health benefits.

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A well-conducted, randomised, controlled human trial is the cornerstone of a successful innovation. Blane was right in conducting his human experimental trial in controlled conditions (a boat), with an identified product (lime), even if the control group was not drinking a real ‘placebo’. Another logical requirement is clear identification of the strain(s) selected to provide a given benefit, as well as the food matrix used to deliver the probiotic. It is well known that not all microorganisms are similar, and the matrix may modulate the efficacy of a given strain. The beneficial effect provided by a probiotic can be measured either directly by the improvement in a global function (e.g. improvement in slow gut transit time, improvement in gut comfort) or by monitoring significant changes in relevant marker(s) if the improvement in a function cannot be measured directly. The selection of the relevant marker(s) is a little trickier, as the effect of a probiotic is often a multitarget one. Most of the time there is no one single marker for assessing the effect of a probiotic on a function. The improvement in a given function may result from improvement in different factors modulating that function. For example, improvement in gut transit time can be generated by (1) a bulking effect of non-digestible components, like the amount of bacterial cells in the stool, or by improvement in the texture of the stool by a reduction in the amount of non-digested mucin or by an increase in the water content, (2) improvement in the muscular function of the gut, (3) a reduction in the hyperinflammatory status of the mucosa, or (4) a change in the composition of short-chain fatty acids, and so forth. The global benefit may result from small, sometimes undetectable, improvements in multiple mechanisms that result in a common significant improvement of a function. It is a common feature in nutrition that a nutrient is modulating multiple pathways and/or at different steps of a cascade, ending in a change of a function. Therefore, most of the time, investigators use multiple primary criteria, sometimes integrated in a common benefit, sometimes exploring different benefits that converge on a more global health effect. One extreme of this is the improvement in well-being that is often reported spontaneously by consumers. Recently, we were fortunate to be able to compare the effect of Bifidobacterium lactis DN-173 010 on gut comfort (Agrawal et al., 2009) measured by self-reported improvement in gut comfort (on a subjective 10-point scale) and an objective measure of abdominal circumference during the day. There was a significant correlation between the objective change in abdominal circumference and the reported subjective improvement in well-being. It is also important to use multiple markers to assess a beneficial effect on a normal function. First, because foods, including probiotics, often induce small, therefore not easily detectable, changes. This is expected when dealing with physiological conditions. Second, because a similar improvement in a global function, like gut comfort, may be due to a change in multiple different factors either in the same subject on different days or in different subjects on the same day. This is common practice in nutrition and physiology, but is not common in pharmacology, where physicians target unique mechanisms more and more precisely to improve the specificity and efficacy of drugs. Among the complex global functions like natural defences, digestion, elimination, reproduction and so forth, one is very new: the gut and its microbiota functions. This organ has long been ignored despite the fact that it is the most potent enzymatic organ in the body, with roughly 10 times more enzymes than the liver, and is linked to the whole body through the immune and nervous systems. This organ is a logical key partner for probiotics. Probiotics may interact and/or modulate the gut microbiota, or interact with the gut mucosa or the gut immune system.

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●

●

● ●

●


Some probiotics can provide additional enzymatic capabilities to the gut microbiota, e.g. glucosidases, glycosidases or azoreducases. Others can substitute for a missing enzymatic function in the gut mucosa. A classic example is the capacity of yogurt probiotics to compensate for the natural decrease in lactase activity in the human gut, and to digest dietary lactose ingested in a yogurt providing glucose and galactose that can be absorbed by the epithelium. The understanding of the role of the gut microbiota in human digestion will open the door for new probiotics to either add or compensate for some impaired enzymatic function of the human gut microbiota. Some probiotics can help the first basic function of the gut microbiota, namely resistance to colonisation by new external microorganisms. This benefit was described nearly a century ago by Nissle. Others may improve the gut barrier effect, by interacting with the gut mucus, or with specific gut defence cells like Paneth cells and their defensins; or by strengthening the tight junction between enterocytes; or by improving the gut immune system and its different components. Some probiotics will interact with gut transit and gut comfort. Some probiotics will interact with the inflammation system. This is a complex issue where an adaptive level of inflammation is necessary to maintain gut barrier capacity, although an excess of inflammation will end up in digestive diseases. It is interesting to note that some inflammatory diseases may be due to the lack of specific microorganism(s), which is not obvious when we have been taught that every disease is generated by a pathogen. This impact on inflammation may also result in changes in global well-being, and even mood perception. Some probiotics may also modulate the global food efficiency of our digestive system. Recent reports on related changes in gut microbiota composition and weight gain or loss is opening a fascinating door for deciphering part of the modern challenge of obesity.

20.6

CONCLUSION

Innovation requires two conflicting elements: first, a capacity to detect and trust early findings outside the norm, which means also exploring some areas not within bounds of commonly agreed science; second, a willingness to confirm the robustness of the early finding, because they are the roots of sustainable business. However, a sustainable business requires accessible foods, and those who will benefit the most from probiotics may not be able to offer the costs of sophisticated science. The challenge is to weigh the uncertainty between the benefit and the cost of not using it, taking into account that foods are daily requirements. Should we be on Cook’s side, who used lime before it was cleared by Science and sailed around the globe, or on the academic side waiting for more evidence? There is no definite answer and a step-by-step approach can be useful. As long as there is no harm, which is the case for food, the door to innovation should be open. The long history of fermentation in our food systems supports an open-mind approach. The requirements of an increasing worldwide population, with a dramatic increase in the elderly and their healthcare needs, as well as the quest for more comfort, and the double challenge of feeding the undernourished and managing the obesity of the malnourished, need innovative solutions and probiotics will part of them. The challenge is to require a fair amount of science and not to overload innovators with expensive requirements, neither to let fantasy offer unsustainable dreams to naive consumers.

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In between, the ethical committees are wondering whether the risks of every human exploration are worth the additional knowledge. It is also important to consider that academic scientists are looking for challenging hypotheses that satisfy Karl Popper’s golden rule: science exists when it is refutable. On the other hand, consumers are looking for practical information and ways to improve their diets and health. Therefore what is a ‘real’ demonstrated benefit, a clinically proven argument or a biologically plausible hypothesis? How can these different lines of evidence be conveyed to consumers in a way that they understand? The assessment of the potential of probiotics in foods, and beyond, needs expertise in nutrition, microbiology, gastroenterology and integrative physiology. Common sense and ethical considerations will also be needed to prevent an overly expensive quest for more evidence, as we know that this is never-ending search. In the probiotic field, there is enough convincing evidence for some strains to allow academics and industrialists to look for innovation that will benefit our modern world.

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Index

16S rRNA gene homology, 79, 274 2,3-butanediol, 308 2,4,6-trinitrobenzene sulfonic acid, 57, 61, 138 a1-antitrypsin, 257 abdominal, 8–9, 157, 183, 204, 317 cramps, 2–3 distension scores, 191 distension symptoms, 249 pain(s), 8, 65, 157–8, 163 Acceptable Macronutrient Distribution Range, 112 accuracy, 91, 274, 292 acetogenesis, 61 acute intestinal disease(s), 2 adaptive response, 63 additives, 77, 128, 172, 179, 224–6, 229–30, 232–4, 242–7, 278, 284–5, 291 nutritional additives, 242 sensory additives, 243 zootechnical additives, 234, 243 adenocarcinoma, 7, 10 adhesion, 11, 38, 51, 55, 64, 82, 174, 250, 269, 295–6 to human (intestinal) mucus, 46, 55, 250, 269, 289, 296 adjuvant, 4, 9, 216, 218 Aeromonas, 202 Aeromonas hydrophila, 140 aflatoxin(s), 10–11, 28, 171–6, 252–3 exposure, 172, 175–6, 253 aflatoxin-B1, 28, 172–6, 252, 269 Agence Française de Sécurité Sanitaire des Aliments (AFSSA), 286 a-glucosidase, 60 alanine, 12

alanine transaminase, 140, 253 alcohol, 8, 113–15, 225, 235 aldehyde reductase, 11–12 allergen status, 41 allergic disease, 161, 184–6, 257 allergic reactions, 6, 26 allergy cedar pollen, 124 prevention, 160 a-linolenic acid, 112 allochthonous bacteria, 294, 313 Alternaria, 171 American Cancer Society, 108 American Diabetes Association, 108, 113–14 American Heart Association, 108, 112–13 Aminoglycosides, 254 Aminopenicillins, 187 Ammonia, 20, 193, 224, 241 Amoxicillin, 181, 200, 203–4, 255 amplified ribosomal restriction analysis (ARDR), 82, 274 amylase, 139–140 anaerobes, 60, 179, 212 anesthetic, 1 animal studies, 6, 7–8, 22, 24, 32, 95, 122–3, 135, 268 Anthropometric, 144 indicators, 143 antibiotic(s), 4–5, 55, 64, 94, 127, 136, 155–6, 179, 181, 187–9, 194, 200–201, 203–4, 207, 209, 213, 223–5, 245–7, 251, 254–5, 257, 277–8, 284, 286, 288–9, 319 antibiotic-associated diarrhea, 4, 9, 52, 58, 94, 155, 187, 200, 215


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Index

antibiotic(s) (cont’d) resistance(s), 30, 51, 82–83, 95, 227, 236, 254, 273, 284 transferability, transmissible antibiotic resistance, 82, 236, 273 treatment, 4–5, 51, 155, 187, 201, 251, 256, 311 antibody, 11, 20, 24, 26 production, 11–12, 25, 31, 136–7 titre (titer) 25, 135, 232 anticancer biological response modifiers, 26 anticarcinogenic activities, 304 anti-collagen antibody, 25 anti-flatulence properties, 65 anti-inflammatory, 10, 12, 46, 55, 65, 138, 163, 207, 209, 269 cytokines, 55, 138, 252 properties, 60–61 antimicrobial(s), 7, 52, 54, 64, 163, 193, 203, 213, 223, 246, 293, 313 activity, 7, 269, 313 properties, 54 substances, 13, 45, 235, 277, 284 antioxidant(s), 129, 138, 242, 276 activities, 287 antitumour immune responses, 19 antivirial activity, 13 ANVISA, 77–8 API identification, 304 apoptosis, 55, 61, 219 ARDRA, see amplified ribosomal restriction analysis arthritis, 8–9, 25 aspartate, 140 Aspergillus, 171 Aspergillus flavus, 252 Aspergillus oryzae, 137 atopic, 161, 192, 257 dermatitis, 46, 161–2, 164, 185–7, 194, 249, 254, 257 diseases, 49, 52, 58, 257 eczema, 6, 7, 46, 85, 161, 164, 184–7, 257 authorisation process, 225, 234, 242–5, 247 autoimmunity, 25 amelioration, 32 autopsy, 56–57 azoreductase, 252, 311 azoxymethane-induced colon tumors, 7 B vitamins, 275 Bifidobacterium, 1, 2, 9–10, 13, 38–9, 49, 54, 59–61, 78, 81, 98, 119, 144,

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157–9, 180–181, 204, 207, 227, 250, 254–5, 269, 273–4, 278, 317 Bifidobacterium adolescentis, 29, 39, 63, 128, 141, 147, 227 Bifidobacterium animalis, 6–7, 47, 160, 192, 203, 215–17 Bifidobacterium animalis Bb12, 204 Bifidobacterium animalis subsp. lactis (Bb12), 46, 250–251, 253, 258 Bifidobacterium bifidum(s), 1, 137, 128, 156, 166, 186–7, 215 Bifidobacterium bombi, 59–60 Bifidobacterium breve, 29, 62, 64, 119, 128, 210, 218, 250, 251, 258, 296 Bifidobacterium infantis, 64, 128, 158, 163, 207, 210, 217–18 Bifidobacterium longum, 7, 64, 187, 268–9, 296 Bifidobacterium thermophilum, 55 Bacillus Bacillus subtilis, 138, 140, 157, 227, 233–4 Bacillus circulans, 140 Bacillus spores, 234–5 Bacillus toyoi, 225, 242 bacteremia, 8, 56, 183–4 bacterial biomass, 63, 200 bacterial, 1–2, 6, 8, 10, 13, 20–21, 23, 30, 32, 42, 44, 50, 52, 54, 56, 58–64, 67, 79, 81, 89, 99, 128, 136, 139–42, 154–6, 173–5, 178–82, 193, 200–201, 205, 207, 210, 212–13, 214, 217, 218, 223–5, 230–232, 236, 238, 240–241, 247, 249, 251, 272–3, 275–7, 279–80, 283, 285, 287–9, 291–5, 298, 305–7, 314, 317 cell wall, 12, 173 diversity, 57, 59–60 enzyme(s), 7, 20 isolates, 267, 288 toxins, 11 translocation, 8, 51, 56 bacteriocins, 13, 54, 277, 313 Bacteroides, 29, 60, 179, 297 Bacteroides fragilis, 30, 180 Bacteroides–Prevotella, 60 Bacteroidetes, 298 batch fermentation, 54, 239 benzpyrene, 11 b-galactosidase, 53, 60, 138, 206 b-glucosidase, 60, 140, 251 b-glucuronidase, 20, 137–8, 251–2 b-glucuronidase activity, 20, 137–8, 251

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Index

Bifantis®, 217 bile acids, 6, 9, 28, 311 bile resistance, 38, 95 bioactive peptides, 275 biochemical medicines, 127 biodiversity, 145 bioequivalence, 289–90 biomarkers, 47, 93, 96–7, 175, 287, 291–2, 298, 307, 310, 314 biopolymers, 308 biopreservatives, 279 biotherapeutic agents, 5 bloating, 2, 65, 157–8, 163, 190, 206–7, 209, 217, 256 blood mononuclear cells, 23–4, 251 blood products, 127 body passage phase, 276 bone density, 96, 129–30 bone marrow transplantation, 8 bovines, 229, 245 bowel discomfort, 18, 24 bowel dysfunction, 65 bowel movement, 22, 31, 158, 207, 217, 269 branched-chain amino acids, 316 Brazil, 31–2, 76–8, 85 bread, 1, 250 breast milk, 60, 181, 204 breast-fed, 1, 7, 17, 179–81, 192, 274, 297, 303 breath hydrogen excretion, 205–6 broilers, 136–137 Brucella abortus, 67 Bulgarian yogurt, 17 business opportunities, 264, 266 butyrate, 57, 61 Clostridium difficile, 5, 155, 180, 187, 199–200, 215, 312 C. difficile-associated diarrhea, 155 Caco-2 cells, 55 cadmium, 269 Caesarean section, 1, 179–80, 185, 257 calcium, 91, 104–105, 124, 131, 307 calprotectin, 8 calves, 135, 139–40, 230–231, 233, 245 cancer, 7, 9, 11, 19, 26, 28, 30, 32, 49, 52, 91, 107–8, 113–15, 124, 129, 171, 209, 218, 313 cancer cell proliferation, 26 Candida, 6, 11–12, 228, 255–6 Candida celanoides, 228 infections, 256

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325

capsules, 5, 45–6, 64, 78, 89, 113, 120, 123, 175, 191, 200, 203, 213, 250, 254–5, 272, 276 carbohydrate metabolism, 51, 54 carbohydrate sweeteners, 91 non-cariogenic, 91 carbohydrate-induced hypercholesterolemia, 111 carbon tetrachloride, 8 carboxymethyl cellulase, 140 carcinogen, 11, 28 compounds, 28, 30 cardiovascular disease (health), 102, 104, 106–10, 112–13, 115, 122, 286 caries, 7, 9, 11, 66, 83, 85, 91, 193, 214 carrier matrices, 275 case-by-case basis, 126 cassava, 309 catabolite repression, 307–8 catalase, 62 catalytic activities, 63 catheter, 40, 183–4 caustic, 241 CBER, see Center for Biologics Evaluation and Research cecum, 3, 136, 205 cell aggregates, 64 Cell bank, 237–8, 240 cell immobilization, 63–4 cell injury, 241 cell layer integrity, 55, 252 cell membrane integrity, 37 cell proliferation, 26, 313 cell wall fragments, 275 cellular stress response, 62 cellulase activity, 139 Center for Biologics Evaluation and Research, 94 cephalosporins, 187, 200 CFU, see colony-forming units chaperone, 62 cheese, 1, 44–6, 48, 250, 253, 256, 306 Gouda, 44 chemotherapy-induced diarrhea, 65 chewing gum, 85, 218 chicks, 135–7, 172 children, 4, 7, 8, 18, 47, 52, 55–6, 82, 85, 94, 130, 141–4, 152, 154–5, 157–8, 160, 162, 164, 166–7, 178–9, 181, 183–94, 254, 256–7, 286, 292, 298, 307, 316 children’s development, 77

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China, 31–2, 85, 126–7, 129–32, 175–6 Chinese health claim categories, 129 Chinese medicine, 126–7, 130 Chinese Medicine Administration Law, 126 Chinese regulations, 126 Chlamydia, 6 cholesterol, 6, 46, 52, 82–3, 85, 91, 104–5, 107–15, 121–2, 124, 136, 143–4, 311 chronic abdominal pain, 8, 190, 206, 217 chronic fatigue syndrome, 24 chronic inflammation, 30, 207, 209 chronic liver disease, 8 claims, 32, 75, 76–81, 84–5, 88–94, 96–100, 102–4, 106, 108–9, 118–19, 121–4, 126–31, 263, 266, 283, 287, 291, 293, 299 disease claim, 93, 99, 124 health/food claim, 2, 31–2, 40, 46, 49, 75–85, 89–93, 96, 102–4, 106–9, 118–32, 134, 145, 171, 173, 175–6, 199, 215, 223, 226, 263–8, 273, 283, 285, 289–93, 296–9 nutrient content claim, 93, 102, 104–6 off-label claim, 124 structure/function claim, 90–4, 96, 100, 102, 104 clarithromycin, 203–4, 255 clavulanate, 187 clindamycin, 178, 200, 278 clinical, 2, 4, 5, 7, 10, 21, 23, 38, 49, 53, 56, 58, 67, 82, 93–4, 99, 123, 134, 136–7, 143, 145, 149–50, 152, 154, 156, 158–9, 161, 166, 171–2, 182–90, 192–4, 199, 203, 210, 212, 215–18, 249, 252–4, 257, 264–5, 267–9, 271, 279, 290, 293, 299 indications, 199–200 protocols, 298 research, 2, 46, 58 score, 7, 186–7 study(ies), 18, 20, 51, 84, 99, 122, 135, 141, 145, 199–200, 207, 212, 214–15, 226, 254, 263, 276, 290, 299 cloning, 312 genome fragments, 312 Clostridium, 180 C. coccoides, 60 C. difficile, 5, 9, 155, 180, 188, 200–201, 215 disease(s), 199–200, 215 C. leptum, 60, 297

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C. perfringens, 124, 297 clue cells, 212–13 coadministration, 163 coated, 45, 272 Cochrane Collaboration, 149–50 Cochrane Handbook, 151, 165 Cochrane Neonatal Review Group, 182 Cochrane review, 154, 159, 160, 162, 187, 189, 218 Codex, 78, 85 co-fermentation, 43 cohorts, 4, 310 coliform count, 141, 143 colitis, 3, 4, 7, 9, 10, 26, 30, 56–7, 61, 138, 155, 159–60, 187, 192, 194, 199, 201, 206–8, 217, 289, 312 colon, 2, 3, 7, 8, 10, 28, 30, 54, 61, 65, 122, 138, 160, 207–11, 216–17, 250–251, 294–295 cancer (tumour), 7, 28, 30, 209 colonisation, 38, 250, 256, 286, 292, 294–5, 303, 311, 313, 318 colony-forming units (CFU), 44, 99, 136–42, 144, 154, 163, 174–5, 185, 188–9, 201, 207, 210, 212–14, 229, 246, 253–7, 308, 310 enumeration, 44, 276–8, 310 colorectal cancer, 30 commensal gut microflora, 161 commercial-scale production, 268 Community Register of Feed Additives, 232, 243–5 community-acquired diarrhea, 188–91, 194 competitive exclusion, 38, 79, 137, 182 confidence interval, 144, 151–3, 156, 183 constipated patients, 19, 20, 160 constipation, 2, 18–20, 22, 31, 104, 129–30, 160, 182, 190, 192, 206–7, 217 consumer guidelines, 99 Consumer Health Information for Better Nutrition Initiative, 106–7 consumer protection, 30, 78, 284 consumer watchdog organizations, 98 conventional food, 88–90, 93, 118 cooling temperature, 272 coronary artery disease, 6 heart disease, 85, 91–2, 96, 109, 113 Corynebacterium, 39, 227 cosmetic claims, 130 cows’ milk, 7, 186–7, 257 C-reactive protein, 144, 257

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Index

Crohn’s disease, 3–4, 10, 27, 57, 61, 159, 192–3, 199, 206, 209–11, 218, 310, 312 crossover protocol (trial), 6, 19, 190, 298 cross-protection phenomena, 63 cross-talking, 304, 309 CRP, see C-reactive protein crude fiber, 139 crude protein, 139 cryoprotectants, 42 crypt foci, 7 culturability, 37, 75, 84, 275 culture methods (techniques), 59, 250, 275, 277, 310 cultured milk products, 1 culture(s), 1, 17, 43–4, 54, 59, 62–3, 81, 84, 94, 120, 134–5, 137, 139, 141, 145, 183, 199–200, 213, 237–41, 249–50, 252–3, 255–6, 267, 271, 273, 275, 277, 279, 284–5, 288, 292, 304–10, 314 yogurt-type, 273 curcumin, 10, 12 cyanoglycosides, 309 cystic fibrosis, 8, 9 cytochrome P450 reductase, 11, 12 cytokine, 3, 8, 10–11, 19, 26, 51, 55, 57, 66, 138, 163, 192, 207, 209, 251–2, 269, 289 cytotoxic T-cell activity, 24 dahi, 134, 138, 141, 142, 144 daily feed intake, 137, 231 daily weight gain, 139, 230, 231 daily-dose, 43 dairy matrix, 41, 309, 309 dairy products, 3, 45, 49, 59, 111, 113, 119, 130–131, 173, 228, 253, 256, 271, 273, 277, 299, 306, 307 d-alanine, 12 data, 4, 21, 25, 28, 30, 58, 67, 81–4, 92, 94–6, 103, 106, 108–10, 114–15, 122–4, 131, 141, 145, 149–52, 154, 156–8, 160–166, 171, 176, 181–4, 187–91, 214, 225, 229, 232, 234, 249, 254, 267, 274, 287, 291, 293–7, 299, 305, 314 pooling, 163, 164 Debaryomyces hansenii, 228 deconjugation, 61 deconjugate(d) bile acids, 6, 9 defecation frequency, 20–2, 124, 160

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327

defense mechanisms, 62–3 defensins, 207, 209, 313, 318 deficiencies, 11–12, 108, 199, 287 deficiency-related diseases, 315 dehydration, 143, 187, 190, 209, 237 delivery matrix, 97 denaturing gradient gel electrophoresis (DGGE), 288, 291 dendritic cells, 24, 30, 192, 252, 289 dental caries, 7, 9, 11, 66, 85, 91, 193 Department of Health and Human Services, 108 dextran sulfate, 10 DGGE, see denaturing gradient gel electrophoresis diabetes, 9, 24–5, 57, 108, 113–14, 122, 138, 312, 313 diabetic symptoms, 25 diagnostic aids, 127 diarrhea (diarrhoea), 1, 4, 5, 9, 52, 58, 65, 79, 88, 94, 134, 136–7, 139, 142–4, 150–151, 153–7, 163–4, 166, 182, 187–91, 194, 199–207, 209, 213, 215–17 antibiotic-associated, 4, 9, 52, 58, 94, 155, 187, 200, 215 diarrheal diseases, 4, 142, 154 dietary, 11, 89, 91, 92, 100, 102–4, 106, 108–16, 135–6, 138–9, 157, 171, 173, 175–6, 191, 206, 254, 258, 263, 268, 311, 318 fiber (fibre),29, 65, 107 guidance, 102, 103, 108, 116 lipids, 91 supplements, 65, 88–90, 92–3, 99–100, 107, 112, 118, 228, 291, 316 toxins, 252, 269 Dietary Guidelines Advisory Committee, 110 Dietary Guidelines for Americans, 108 Dietary Supplement Health and Education Act, 96, 104 differential fluorescence induction, 306 digestive, 49, 56, 59–60, 92, 97, 98, 205, 229–30, 264, 309, 311, 318 ecosystems, 59, 60 function, 32, 194 tract, 54, 56, 59–60, 210, 235, 311, 313 digestive tract, bumblebee, 60 dimethyl sulfoxide, 42 disease risk, 77, 79–80, 83, 102, 108, 110, 119

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Index

disorders, 2–3, 8, 10, 12–13, 24, 49, 52, 67, 85, 145, 157, 159–61, 190, 236, 258, 286, 298, 310, 312 disruption in cellular membranes, 42 d-lactate, 54 DNA fragments, 238, 306 DNA–DNA hybridization, 79, 82 docosahexaenoic acid, 112 dormancy, 41, 43, 275 dosage, 41, 44, 127–8, 130–131, 154, 163, 183, 199–201, 267, 299 dose–response studies, 287 double-blind randomized, 5, 207, 293 downstream processing, 41, 50–51, 62–3, 236–7, 241 Dr. Minoru Shirota, 17 drugs, 10, 19, 88–90, 93–4, 118, 120, 127, 179, 207, 223–4, 252, 264, 289, 298, 315–17 drug therapies, 58 dual-type microflora, 273 dullness, 136–7 duodenum, 2, 136, 203 dysbiosis, 65, 310, 312, 314 dysentery, 134, 144 dyslipidemia, 138 Escherichia coli (E. coli), 2, 23, 54, 124, 135–6, 156–7, 160, 173, 179–80, 187, 202, 209, 269, 291, 303, 308, 312 E.coli Nissle (1917), 2, 3, 160, 209, 217 enterotoxigenic, 23, 30, 156, 202–3 early infancy, 186 EC Regulation No. 1924/2006, 76 eczema/atopic dermatitis, see atopic effectiveness, 3, 58, 121–2, 140–141, 149–50, 159, 188, 191, 307 efficacy, 5, 7, 28, 49–53, 56, 58, 65–7, 79, 81, 84–5, 88–9, 92–7, 100, 103, 118, 124, 126, 128–30, 137, 141, 143–5, 154, 156, 158–60, 163, 165–6, 173–4, 183, 189, 192, 202, 207, 209–10, 215–17, 224–5, 228, 231, 234, 236, 245–7, 249, 255, 258, 268, 279, 287, 289, 292–3, 298–9, 315–17 of probiotics, 4, 8, 67, 84, 138, 159, 162, 166, 185, 188, 218, 232, 292, 298 EFSA, see European Food Safety Authority egg-laying performance, 135 Egypt, 1, 175, 176, 203 eicosapentaenoic acid, 112

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EMBASE, 150 encapsulation, 64, 276, 308–9 endocarditis model, 30 enhancement, 23–4, 124, 130, 249, 258, 264 of anoxia endurance, 130 of sleep, 130 Enterobacteriaceae, 20, 296 Enterococcus (enterococci), 2, 18, 20, 81, 224, 227, 235, 251 Enterococcus faecalis, 29, 136 Enterococcus faecium, 137, 187, 224, 232–3, 239 enterohepatic cycle, 311 enzyme(s), 2, 7, 10–11, 20, 28, 52–53, 65–6, 79, 82, 140, 204–5, 217, 251, 284–5, 288, 295, 311, 313, 317 activity, 20, 37, 140, 252 deficiency (ies), 11–12 replacement technology, 11 epirubicin, 28 eradication, 157, 193, 203–4, 216, 255–6, 258 therapy, 216, 251, 255, 258 essential nutrients, 111, 112, 178, 316 ETEC, see E.coli, enterotoxigenic ethanol, 8, 234, 308 ethics committee, 299 etiology, 7, 11, 24, 30, 159, 190, 192, 203, 206 European Food Safety Authority (EFSA), 77, 79, 81, 83–5, 227–8, 232, 243–5, 253, 272–4, 283, 285–7, 290–291, 293, 298–9 European Union (EU), 38–40, 75–6, 178, 224, 263, 284–5 Eubacterium rectale, 296 eukaryotes, 304, 309 European Regulation (EC) No. 1924/2006, 76, 263 European Society for Paediatric Gastroenterology, Hepatology and Nutrition, 154, 189 European Society of Paediatric Infectious Diseases, 154, 189 Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 108, 110–111, 113–14 faecal pH, 18, 20 Faecalibacterium prausnitzii, 60 FAO, see Food and Agriculture Organization fatal myocardial infarction, 99, 226 FDA, see Food and Drug Administration

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Index

faecal (fecal) flora, 135, 137–8, 141, 181 faecal (fecal) marker, 8 Federal Trade Commission (FTC), 90, 98, 102 feed, 3, 21, 37, 40, 84, 135, 137, 139–40, 161, 172, 184–5, 223–32, 235–8, 240–243, 245–7, 252, 275, 292 additives, 224–6, 229, 232–3, 242–7, 285, 291 consumption, 134 conversion ratio, 135–6, 140, 231 management, 229 supplement(s), 223, 229, 232, 292, 299 utilization, 139–40 FEEDAP panel, 243 fermentation, 3, 17, 41, 43–4, 50, 52–5, 59, 61–3, 81–2, 139, 199, 224–5, 228, 230, 235–7, 239–41, 273, 275, 279, 296–7, 304–9, 313, 318 fermented milks, 1, 2, 6, 17–18, 23, 43, 45, 59, 134–5, 141–2, 167, 214, 272, 277–8, 307 fermenter, 41, 54, 241, 307 ferritin levels, 144 Firmicutes, 227, 298 fish, 106, 110, 112–14, 130, 140, 229, 245 FISH, see fluorescent in situ hybridisation flatus, 2, 206 fluidised bed-drying, 241–2 fluorescent in situ hybridisation (FISH), 277, 279, 291 fluorescent reporter protein, 306 fluoroquinolones, 200 FNFC, see Foods with Nutrient Function Claims folic acid, 9, 91, 109, 124 food allergens, 6 Food and Agriculture Organization (FAO), 2, 37, 75, 79, 81–2, 88–9, 94, 98, 134, 145, 226, 249, 284–5, 290, 292, 296, 298–9 Food and Drug Administration, 30, 83, 85, 88–94, 96, 98–100, 102–4, 106–9, 126, 227, 253, 289–90 Food and Drug Administration Modernization Act, 106 Food and Nutrition Board, 108, 111 Foods for Specified Health Use (FOSHU), 31, 78, 85, 118–124 Food Guide Pyramid, 115 Foods with Nutrient Function Claims (FNFC), 118, 121 forest plot, 152, 153, 163

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Formula(s), 65, 66, 88, 152–3, 178, 180–184, 190, 275, 303 probiotic (fortified), 189–90 formulation, 5, 41, 44, 46, 130, 150, 163, 199, 214, 237, 242, 255, 258 FOSHU, see Food for Specified Health Use FOSHU logo, 120 freeze-drying, 42, 62, 64, 200, 237, 241, 309 frequency, 2, 4, 8, 20–22, 124, 143, 160, 189–92, 203, 207, 255, 292 fructo-oligosaccharides, 122, 180, 295 fructose-6-phosphate phosphoketolase, 60 fruit and vegetable juices, 45 FTC, see Federal Trade Commission function, 18, 31–2, 38, 43, 49–50, 52, 60, 62, 65, 67, 77–8, 90–92, 94, 96–7, 100, 102, 104, 118–22, 124, 126–31, 138, 171, 181, 184, 194, 200, 207, 217, 219, 224, 230, 232, 255, 268–9, 279, 286, 297, 303–4, 307, 309–18 body-enhancing, 120 taste function, 120 functional, 46, 49, 51–2, 54, 58, 64, 93, 97, 118, 120–123, 129, 134, 157, 160, 191, 206, 226, 234, 236, 242–3, 249, 254, 258, 263–4, 272, 293, 295–7, 304, 307, 310–312, 314, 316 claim,77 food, 31, 45, 47, 49, 58, 66, 75, 77–8, 88, 93, 120, 126, 129, 131, 134, 178, 263–4, 265–6, 280, 292, 313 gastrointestinal disorders, 157, 190, 254 genomics, 295, 296, 305, 307 probiotic dairy product, 41, 266 Fusarium, 171 Galactomyces geotrichum, 228 Gardnerella vaginalis, 6 gas production, 18, 20 gastric, 23, 54, 62, 64, 82, 129–30, 203–4, 236, 256 gastric carcinoma, 203 gastric ulcer, 4, 203 gastroenteritis, 4–5, 154, 189 acute, 4, 154, 188–9, 194 gastrointestinal, 38, 46, 51, 54, 56, 64–5, 121–2, 124, 129–30, 141–2, 152–3, 157, 174, 178, 184, 187, 192, 194, 206–7, 209, 236, 250, 254–6, 258, 272, 276, 298 discomfort, disorders, disorders, alleviation, 52, 141, 157, 191, 236, 287, 292

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gastrointestinal (cont’d) health, 121–2, 144 tract, 17, 37, 49, 51–4, 59–60, 62, 64–6, 137, 141–2, 145, 173–4, 178–9, 181, 184, 206, 223, 230, 234–5, 237, 250–251, 275, 295–6, 305–7 gastrostomy tube, 183 GC content, 67 gene expression, 61, 307 Generally recognized as safe, 50, 90, 194 Genesis, 1, 66 genetic modification (GM), 10, 62, 66 genetically modified organism (GMO), 62, 245 GenMont Biotech, 132 genome, 66, 81, 289, 295–8, 307, 312 sequence, sequencing, 20, 40, 51, 81, 250, 305 genotoxicity, 30, 226 genus, 39–40, 43, 59, 89, 94–5, 97, 99, 173, 235, 274 germ-free animals, 51, 57, 295 gestational age, 179 gingivitis, 214, 218 gluconeogenesis, 297 glutathione-S-transferase, 11, 12 gluten, 41 glycation, 311 glycerol, 54 glycine betaine uptake transporter, 62 glycolytic activities, 311 gnotobiotic animal models, 56 goats, 233, 245 goats milk, 1 Good Manufacturing Practice (GMP), 50, 95, 127 Good Supply Practice (GSP), 127 grain products, 91, 92, 107, 115 Gram-negative rods, 18 GRAS, see Generally recognized as safe, 50, 83, 85, 90, 194, 226–7, 273 growth, 1, 5, 6, 8, 10, 12–13, 17, 19, 40–41, 44–5, 53–4, 59, 63, 65–6, 77–8, 81–2, 97, 120–121, 129, 134–40, 171, 180–182, 202, 223, 228–30, 235–6, 240, 242, 246, 250, 275, 277, 286–9, 292, 295, 304–8 medium, 237, 239, 304 promoters, 44, 223–4, 230, 246–7 GSP, see Good Supply Practice guava-polyphenols, 122 gut, 17, 20, 30–32, 49, 51–6, 58–66, 79, 89, 97, 129–30, 136–7, 140–141, 145,

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159, 161, 179–81, 183–4, 188, 192, 199–200, 208–9, 211–12, 216–17, 223, 225–6, 230–2, 234–6, 243, 250, 265, 269, 276, 284, 286, 289–92, 294–8, 303–4, 307–14, 316–18 gut–brain axis, 258 gut comfort, 317–18 ecosystems, 50, 59 transit time, 205, 317 H. pylori, see Helicobacter pylori habitual intake, 22 haem, 308 Haemophilus influenzae, 257 Hanseniaspora, 40, 228 humanised microbiota (HBM), 297 harmful, 66, 141, 171–4, 152, 291 bacteria, 2, 17, 20, 31–2, 124 microbial enzymes, 28 HDL cholesterol, 144 health advocacy organization, 103 health, 1, 2, 8–10, 13, 17–18, 31–2, 38, 40–41, 47, 49–50, 52, 58, 60–61, 66–7, 75–84, 88–9, 91, 93–4, 96–100, 102–4, 106–8, 110, 112, 114, 116, 118–24, 126–31, 134, 139, 141, 144, 156, 171–72, 178–9, 193, 201, 223, 225–6, 228, 230–232, 235–6, 243, 247, 252, 255, 264, 267–9, 272, 275, 283–7, 291–2, 296–8, 303, 307, 310–313, 315–17 benefit(s), 2, 8, 18–19, 32, 37–8, 46, 49–50, 52–3, 58, 64, 66, 75, 78–80, 85, 88–90, 92, 95–100, 106, 115, 119, 145, 178, 199, 249–50, 264–9, 271–2, 287, 296, 297, 304, 316 claims, 2, 31–2, 40, 46, 49, 75–85, 89–93, 96, 102–4, 106–9, 118–32, 134, 145, 171, 173, 175–6, 199, 215, 223, 226, 263–8, 273, 283, 285, 289–93, 296–9 claims, Article 13, 77 claims, Article 14, 77 drinks, 132 food(s), 118, 120, 126–130, 132, 134 food advertisement, 127, 130 maintenance, 32 professionals, 272 -related quality of life (HRQOL), 255 healthcare expenditure, 316 healthy volunteers, 20–21, 58, 60, 251 heat-shock proteins, 62, 67 heat-treatment, 173

11/1/2010 6:33:30 PM

Index

Helicobacter pylori, 4, 23, 157, 193, 203–4, 216, 251, 255, 269 helminth infections, 257 hemoglobin, 8, 144 hen-day egg production, 135, 137 HEPA filtration, 43 hepatic encephalopathy, 193 hepatitis B virus, 253 hepatotoxicity, 174 heterocyclic amines, 11 heterologous expression high-density cholesterol, 62, 312 high-density lipoprotein, 110 Hippocrates, 280, 315 homeostasis, 297, 312 hormones, 10, 311 horses, 233, 245 hospital, hospitalization, 5, 18–20, 45, 141–5, 179–80, 183, 190, 200–201, 268–9 host functions, 309 HPV types, 67 human, 1, 2, 4, 6–7, 10–11, 13, 17–18, 22–23, 25–26, 30, 38, 40, 46, 49–61, 64–67, 75–80, 82–5, 88–9, 94–7, 99, 104, 106, 108, 114, 118–19, 122–4, 126–8, 130–131, 134–5, 141, 143, 145, 171–6, 180–181, 184, 194, 200, 203, 207, 209, 219, 223–6, 228–9, 247, 250–251, 253, 255, 263, 268–9, 271–2, 275–6, 278, 284, 286–7, 289–92, 294–8, 303–4, 306–7, 309–18 autoimmune disorders,24 feeding trials, 40, intervention studies, 76, 82–3, 85, 121, 252, 254, 266, 268 intestinal tract chip, 251 origin, 38, 51, 200, 236 peripheral blood mononuclear cells, 23, 251 T-cell lymphotrophic virus, 24 Human Microbiome Project, 297 humidity control, 42 humoral immune response,25, 232 hydrogen 3, 42, 61–2, 112–13, 205–6, 304 metabolism, 61 hydrolyse lactose, 9 hygiene hypothesis, 184 hyperglycemia, 138 hyperinflammatory status, 317 hyperinsulinemia, 138 hypertension, 92, 97, 129 hypocholesterolemic supplement, 144

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331

IBD, see inflammatory bowel disease, 3, 4, 9–10, 26, 30, 57–8, 60–61, 64–6, 138, 159, 192, 199, 206–7, 209–10, 219, 298 IBS, see Irritable Bowel Syndrome ice crystal formation, 42 identification, 2, 19–20, 38, 40, 50, 56, 61, 79–82, 94, 131, 152, 164, 173, 264, 266, 273–4, 284–5, 287–8, 304, 306, 310, 312–14, 317 IDF, see International Dairy Federation IFN-g, 24, 26 IgA, 232, 257, 275, 296 mucosal responses augmentation of, 182 IgE, 26, 161, 186, 232 hypersensitivity, 187 response, 257 IL-10, 57, 66, 138, 207, 219, 257 levels, 3 IL-5, 26 IL-6, 24, 26, 138 levels, 257 IL-8, 219 production, 61 Ileum, 136, 205, 209–10 ileo-anal pouch, 209–10 illness, 5, 18, 23, 41, 58, 94, 99, 144 ILSI, see International Life Sciences Institute immobilised (immobilized), 308 cells, 308–9 human feces, 54–5 immune, 10, 19, 23–4, 26, 31–2, 38, 47, 49, 65, 97, 124, 129–30, 138, 171, 207, 232, 252, 264–7, 269, 289, 312–13 response, 6, 19, 23, 25, 30, 57, 135, 137, 182, 186, 188, 230, 232, 249, 254, 257, 313 stimulation, 32, 53, 252, 295 system, 3, 30, 49, 51, 58, 64, 66–7, 92, 99, 160–161, 184, 192, 232, 235, 268–9, 275, 286–7, 292, 294, 304, 309, 312, 316–18 immunocompetence, 136 immunocompromised, 184 immunomodulatory effect, 135, 138, 163 immunosuppression, 184 immunosuppressive agent, 51, 209 in situ conditions, 305 in vitro experiments, 50, 61, 235 inactivation kinetics, 43 inappetence, 136 inconclusive results

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Indian Council of Agricultural Research, 145 Indian Council of Medical Research, 145 industrial fermenter, 307 infant(s), 3, 4, 7, 46, 65, 88, 90, 142, 152–4, 156–7, 161, 176–92, 194, 204, 253–4, 257–8, 274–5, 286 breast-fed, 1, 180, 181, 274 feces (faeces), 1 nutrition, 179, 181 infantile, 200 colic, 192 rotavirus infection, 23, 199, 200 infectious diseases, 22, 32, 100, 154, 189, 316 inflammation, 3, 8, 10, 30, 56, 61, 66, 159–60, 184, 187, 207, 209–10, 218, 256–7, 313, 318 inflammatory, 10, 12, 27, 46, 60–61, 65, 138, 163, 185, 207, 209, 214, 269, 275, 291, 297 bowel disease (syndrome), see IBD cytokines, 8, 11, 55, 57, 138, 209, 252, 289 diseases, 297, 318 process, 8, 26 response, 30, 184, 275 Influenza virus, 23 Institute of Medicine, 108 insulin, 12, 25 intention-to-treat analysis, 151, 155, 158, 165, 188 interferon (IFN)-g, 26, 56, 138, 186 Interleukin, 23, 138, 186, 207 interleukin (IL)-10, 3, 56, 207, 251 production, 289 International Code of Nomenclature, 80, 284 international culture collections, 81, 94, 285, 292 International Dairy Federation, 81, 277, 292 International Food Information Council, 92 International Life Sciences Institute, 75, 88 International Standards Organization (ISO), 277–8, 288 intervention trial, 7, 96, 106, 153, 249, 269 intestinal, 2–3, 5, 7–8, 10, 13, 17–18, 30–32, 40, 51, 54–7, 60–61, 64–6, 75, 78, 94, 97, 121, 124, 137, 141, 179, 182, 184–5, 187, 192, 194, 204, 206, 217, 230–231, 249–51, 254, 256–8, 275, 286, 291, 295–6, 299, 304, 310, 312–13 ailments, disorders, 134, 141, 142, 286, 310

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gas production, 18 lumen, 174, 252 microbial balance, 2, 49, 229 microbiota, 17–20, 30–32, 84, 89, 156–60, 164, 182, 250–251, 255, 258, 268, 298, 310, 312, 314 mucus, 46, 55, 174, 250, 252–3 origin, 51–52, 236 tract, 3, 5, 17, 37, 49, 51–54, 59–60, 62, 64–6, 79, 141, 231, 235, 251, 274, 286, 292, 295–6 intravaginal instillation, 6 intrinsic stress tolerance, 63, 275 inulin, 7, 60, 207, 218 iron, 104–5, 144, 287, 309 absorption, 287, 309 bioavailability, 9 irradiation, 8, 129, 179 irritable bowel syndrome (IBS), 3, 49, 58, 60–61, 64–5, 157–9, 163, 190–192, 199, 206–7, 217, 249, 251, 254–5, 258, 312–13 IBS patients, 61, 65, 207, 251, 254–5 IBS symptom score, 244 ischemic heart disease, 49, 58 ISO, see International Standards Organization isolation, 2, 37, 38, 59, 60–61, 95, 236, 271, 305, 306 Isomalt, 132 Issatchenkia, 228 Japanese Pharmaceutical Affairs Law, 118 Japanese Self Defence Force, 18, 22 jejunum, 136 Joint Research Centre, 243 JRC, see Joint Research Centre, 243 Kluyveromyces, 40, 228, 234 Kluyveromyces marxianus, 228, 234 knockout, 3, 10, 57 mice, 3 mutant, 289, 296, 306 rodents, 57 Lactobacillus, 9–11, 17–19, 38–39, 49, 54, 59–61, 67, 78, 81, 98–9, 121, 135–7, 141–2, 154–5, 157–61, 163–4, 174, 181, 183–93, 201–4, 206–7, 210, 212, 215–17, 227, 249–54, 256–8, 273–5, 292, 297, 305 Lactobacillus acidophilus, 2, 29, 32, 39, 55, 128, 144, 166, 185, 187, 203–4, 206–7,

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Index

210, 215–18, 224, 227, 233–4, 277–8, 288, 295 L. acidophilus C2, 135 L. acidophilus La5, 204 L. acidophilus P, 135 Lactobacillus animalis, 60 Lactobacilus casei, 6, 7, 17, 19–20, 30, 38–9, 62, 64, 66, 121, 137–9, 142, 157, 160, 173, 183, 187, 201, 207, 210, 215, 218, 278 L. casei Shirota, 18–19, 21, 23, 25–7, 29, 31, 160 Lactobacillus casei/paracasei group, 19–20 L. paracasei, 63, 278, 288, 297 L. paracasei F19, 186 Lactobacillus crispatus, 29, 227, 278, 288 Lactobacillus (delbrueckii subsp.) bulgaricus, 2, 3, 17, 29, 39, 44, 46, 64, 121, 128, 137–9, 142, 144, 210, 218, 227, 273–7, 305, 142 Lactobacillus fermentum, 29, 39, 60, 227, Lactobacillus gasseri, 39, 54, 62, 227, 278, Lactobacillus johnsonii, 39, 157, 227, 278, 295, 296 L. johnsonii LA 1, 210 Lactobacillus jugurti, 139 Lactobacillus murinus, 60 Lactobacillus plantarum, 39, 55, 59, 62, 65, 135, 155, 190–191, 207, 210, 218, 227, 279, 296, 309, Lactobacilus reuteri, 4, 39, 54, 60, 128, 181, 187, 190, 192, 212–14, 218–19, 227, 289 Lactobacillus rhamnosus, 25, 39, 46–47, 138, 173–5, 212–13, 218, 227, 233–4, 249–50, 253, 278, 288, 297 L. rhamnosus GG, 4, 46, 143, 155, 173, 179, 185, 187, 193, 207, 215, 252–4, 275 L. rhamnosus GR-1, 212–13, 218 Lactobacillus salivarius, 3, 7, 62, 163, 207 Lactobacillus sporogenes, 137, 140, 188 labeling of products, 79 lactase, 2, 3, 53, 98, 204–6, 217, 318 levels, 3, 142 lactate, 54, 60–61, 139, 234, 308 lactic acid bacteria, 2, 17, 26, 30, 32, 52, 81, 139, 163, 172–3, 224, 226, 231, 234–6, 242, 273, 275, 305, 307–9 Lactinex, 202 lactococci, 44 Lactococcus, 2, 67, 135, 227

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333

Lactococcus lactis, 10, 11, 39, 66–67, 135–6, 142, 157, 186, 256, 227, 277, 305–6, 308 lactose 3, 9, 52–3, 60, 204–6, 216–17, 241, 295, 304, 308, 318 hydrolysis, 205–6 intolerance, 2, 3, 52, 58, 79, 199–200, 205–6, 216–17 malabsorbers, malabsorption, 2, 9, 199, 204–6, 304 maldigestion, maldigestors, 53, 98, 205, 206, 217 lactose-free diets, 192 lamina propria, 3, 275 Langerhans b-cell, 25 large-scale production, 40, 64 lead, 6, 20, 30, 43, 46, 52, 61, 64, 97, 129, 165, 188, 193, 199, 205–6, 209, 227, 229, 231–2, 235, 238, 241, 253, 258, 266, 269, 305, 309, 312 lemon, 315 leucocrit values, 140 Leuconostoc, 227, 308 L. mesenteroides, 39, 142, 227, 305 life cycle, 201, 275 lifestyle, 20, 22, 111–13, 116, 129–30, 185, 264, 291, 303 line of no effect, 152 lipid profile(s), 143, 297 lipopolysaccharide, 8 Listeria, 54 L. monocytogenes, 23 live beneficial bacteria, 19 liver, 8, 56, 136, 171, 253, 311, 315, 317 chemical injury, 129, 130 enzymes, 311 livestock husbandry, 229 living microorganisms, 2, 50, 226, 229, 241, 234, 275, 304 number, enumeration of, 275 l-lactate, 54 longevity, 2, 17, 19, 32 low-birth-weight neonates, 142 low-density blood lipids, 96 lipoprotein (LDL), 6, 110, 136 low-fat diets, 110 luciferase gene, 306, 307 Lung and Blood Institute, 108, 110 Lupus erythematosus, 26 Lyophilisation, lyophilized, 5, 138, 175, 203, 225, 240, 241

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Maastricht III Consensus Report, 216 Macrobrachium rosenbergii, 140 macrophages, 19, 24, 26, 138, 232 macrorestriction methodologies, 274 maltodextrin, 185 mannan, 137 manufacturing process, 41 marker gene, 306 marketing strategies, 76, 263, 266 maternal diet, 76, 263, 266 matrix, 22, 41, 43, 45–6, 94, 97, 272, 283, 307, 309, 317 measles, 144 meat(s), 59, 105, 110, 113–14, 130, 228, 230–232, 306 media and chemicals, 277 medical, 1, 3, 4, 9, 13, 68, 89, 92, 116, 142–145, 159, 183–4, 190, 202, 254, 279, 298, 315 applications, 3, 8, 12 food, 92 Medline, 150, 202 megacolon, 187, 209 menaquinone, 308 mesalamine, 4, 209 meta-analysis, 4, 5, 149–67, 182–3, 185, 187–9, 215–16 metabolic, 7, 30, 37, 51, 54, 56–7, 60, 78, 97, 157, 204, 224, 234–5, 252, 295, 305–8, 310–314 pathways, 61, 297, 315 potential, 307 metabolism, 3, 20, 32, 51, 54–5, 61, 66, 122, 173, 187, 205, 251–3, 295, 297, 307, 311–13 metabolome, 305, 306, 314 metaplasia, 203 meta-proteome, 310 meta-transcriptome, 310 Metchnikoff, 1, 17–19, 32, 286, 303, 310 methane, 61, 311 method(s), 10, 37, 41–2, 44, 50–51, 55–6, 59, 75, 79–81, 84, 94, 123, 128, 149, 152, 155–6, 158, 165, 181, 188, 205, 219, 223, 226, 231, 236, 238, 241–4, 250–251, 254–5, 273–9, 284, 288–9, 294 microarray-based, 251 Metschnikowia, 228 mice, 3, 7, 8, 10, 19, 24–7, 53, 67, 130, 135, 138, 219, 275, 289, 295, 297 microbial ecosystem, 54, 60–61, 296

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microbiota, 17–21, 24, 30–32, 38, 49, 52–6, 58, 60–61, 64–5, 79, 82, 84, 89, 92, 97, 156, 159–60, 164, 172–3, 179–82, 250–252, 255, 257–8, 268–9, 291–8, 303–4, 307, 310–314, 316–18 microencapsulation, 37, 62, 64, 242, 272 microencapsulated bacterial preparations, 272 microorganisms, 1, 2, 6, 13, 26, 32, 37, 39, 49–51, 56, 59–61, 63, 66, 67, 75, 79–81, 85, 89, 92, 94, 99, 131, 134–5, 145, 154, 156, 163–4, 180–182, 223–9, 231–2, 234–6, 238, 240–243, 245–7, 253, 272–3, 275–7, 284–5, 303–5, 307, 309–12, 314, 317–18 cultivatable microorganisms, 227 fermentation microorganisms, 279 food-grade microorganisms, 279 milieu interieur, 311 milk, 1–3, 6–7, 9, 17–18, 20, 23, 41, 43–5, 59–60, 110, 119, 121, 131, 134–5, 137, 139, 141–4, 167, 178, 180–181, 184, 186–7, 190, 193, 200, 204–6, 214, 217, 226, 229, 245, 253, 255, 257, 272–3, 275, 277–8, 304–5, 307–9 milk-based products, 271 milky vaginal discharge, 212 minimum level, 52 misuse, 68, 78, 89 model(s), 8, 10, 19, 23–6, 30, 49–51, 53–8, 76, 135, 138, 151, 155, 160, 172, 174, 230, 271, 290, 294, 297, 306, 310, 313 mouse model, murine model, 3, 8, 19, 26–7, 56, 297, 307 systems, 271 modulation of the immune response, 6, 186 molecular techniques, 38, 79, 94 monoclonal antibody, 20, 24, 26 morphology, 286 mortality, 8, 58, 135–7, 156, 182–3, 275 mortality rate, 8, 139 mucins, 296, 311 mucosa, 2, 8, 39, 55, 61, 129–30, 174, 182, 227, 231, 236, 256, 317–18 multiple probiotic strains, 3 multispecies, 249–58 muscular function, 315, 317 mutagenicity, 28, 29, 30 Mycoplasma hominis, 6 mycotoxin, 12, 171–4 bioavailability, 172

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Index

NADPH, 11–12 National Cholesterol Education Program, 108, 110 National Heart, 108, 110 National Institutes of Health, 93, 110, 297 National People’s Congress, 126, 127 natural, 1, 10, 12, 38, 45, 60, 89, 112, 130, 138, 157, 199, 204, 218, 225, 234–5, 247, 286, 291, 306, 318 defense, 78, 317 killer cells (NK) (activity), 19, 23–4, 28, 30–31, 47 nausea, 4, 94, 157, 202, 204, 209 necrotizing enterocolitis, 156, 182, 194 neoplasias, 297 nervous systems, 304, 317 neural tube defects, 91, 124 neurological, 22 New York, 5 NF-kB, 61 NH3, see Ammonia nisin promoter, 11 nitric oxide, 8 synthetase, 10 nitrosamines, 11 NK, see natural, killer cells Nobel Prize, 1 nocturia, 91 non-adherent strain, 38 non-cultivatable, 68, 291 non-lactic acid bacteria probiotic, 2 non-urease-producing bacteria, 193 Novel Food Regulation, 128, 131–2 Novel foods, 78, 126, 128, 131–2 Nugent score, 212–13, 292 Nulab, 224 nutrition function, 120 Nutrition Labeling and Education Act, 104, 106 nutritionally adverse products, 272 oat gruel, 309 obesity, 49, 114, 297–8, 312–13, 318 observational studies, 18, 83 ochratoxin A, 171 odds ratio, 144, 151, 186 oligofructose, 7, 207 opportunistic infections, 20, 216 oral antibiotics, 4, 18 oral cavity, 7, 11, 38, 59, 214, 218 orange, 315 osmoprotectants, 276

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osteoporosis, 91, 96, 104, 124 outcomes, 58, 94, 98, 108, 112, 115, 150, 152, 154, 158–9, 161, 185, 189, 214, 271, 283, 293, 295–96 continuous, 151, 158 dichotomous, 150–151, 158 oxidative, 62, 64, 138, 276 oxygen, 41, 42, 44, 51, 62, 64, 179, 230, 235–7, 239, 241 Propionibacterium freudenreichii, 39, 157, 174, 227, 250 packaging, 37, 41–3, 99, 118, 122 pancolitis, 207–8 pancreatic insufficiency, 11 pancreatitis, 58 acute pancreatitis, 58 Paneth cells, 318 papillomavirus, 67 parenteral nutrition, 183 particle size distribution, 42 PASSCLAIM project, 291 Pasteur Institute, 303 pathogen invasion, 49 pathogenic activity, 95 pathophysiological effects, pathophysiology, 61, 316 patients, 2–8, 10, 18–20, 23–4, 26, 28, 30–31, 38, 40, 59–61, 65–7, 91, 123, 141–3, 154–5, 157, 159–60, 162, 188, 191, 193, 200–201, 204, 207, 209–10, 212–14, 251, 254–6, 275, 279, 286, 299, 310, 316 patulin, 171 p-coumaric acid, 11 PCR amplification, 288 PCR, see polymerase chain reaction p-cresol production, 20 Pediococcus, 39, 227, 233 peer-reviewed, 99, 122, 269, 292 Penicillium, 171 performance enhancers, 225, 246 peripheral blood mononuclear cell, 23, 24, 55, 251 persistence, 38, 55, 137, 204–5, 217, 250, 289, 294–5, 296 pesticides, 179 pets, 229, 231–2 PFGE, see pulsed-field gel electrophoresis pH, 18, 20, 41, 44–5, 54, 63, 82–3, 136, 140, 142, 160, 182, 202, 212, 235, 241–2, 272

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336

Index

pH (cont’d) reduction, 18 ruminal pH, 139 phagocytic activity, 138, 268 phagocytic ratio, 140 Pharmaceutical Affairs Law, 118, 120, 122 pharmaceutical preparations, 199, 276 pharmacology, 317 phenotype, phenotypic, 50, 79, 82, 284–5, 289, 295–6, 306 physical fatigue alleviation of, 129–130 physiological, 50, 54–5, 64, 78, 92–3, 95, 97, 105, 122, 127, 200, 230, 235, 271, 274, 284–5, 296, 301, 305, 307, 309, 316 capacity, 97 methods, 50 pathways, 311 phytase activity, 309 Pichia, 40, 228 PICO, 150 Piglets, pigs, 11, 22, 66, 138, 224, 229–31, 233–4, 245–6 placebo, 4–5, 7–8, 19, 22–3, 25–6, 28, 46, 58, 66, 96, 142–4, 155, 158–63, 175–6, 186, 188–91, 193, 201–2, 204, 207, 209–10, 212–14, 216–17, 253–7, 293, 298, 317 group, 4, 7, 21–4, 143, 185–6, 188, 190–191, 193, 201–4, 207, 210, 213–15, 251, 254–5, 257, 269 -controlled, 4, 18–21, 23–4, 26, 28, 46, 58, 122, 142–4, 181, 187–8, 190, 192–3, 201–2, 204, 207, 210, 212–14, 254, 256–7 plants, 59, 131, 234 plaque, 83, 85, 214, 218 plasma, 8, 25, 138, 253, 257 cholesterol, 6 glucose, 25 pollen allergy, 26, 124 polymerase chain reaction (PCR), 37, 238, 251, 274, 277, 279 polymeric compounds, 42 polyphasic approach, 50 polysaccharide(s), 19, 26, 60, 173, 252 pooled relative risk, 5, 156 Popper, K., 319 pouchitis, 65, 160, 192, 199, 206, 209–10, 218 poultry, 105, 110, 114, 130, 224, 229–31, 235, 245–6 powdered products, 43, 272

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prebiotic(s), 6, 45, 65–6, 91–2, 99, 142, 144, 161, 257–8, 285, 295–7, 314, 316 supplementation, 297 precision, 151, 152, 292 pre-cultures, 237 predominant gut bacteria, 54 pregnancy, 181, 186 premature neonates, 184 premenopausal women, 6 preservation, preservatives, 42, 179, 242, 251, 279 prevention, 3–8, 28, 49, 52, 58, 63, 66–7, 79, 84, 94, 108, 112, 114, 118, 120–121, 124, 126, 128–9, 137, 142, 145, 149, 152, 155–6, 160–162, 164, 166, 172, 184–8, 194, 201–2, 210, 212, 215–16, 218, 312, 313 Prevention of Food Adulteration Act, 145 primary endpoint, 188, 210 probiosis, 49–50, 53–8 probiotic(s) –aflatoxin interaction, 171–2, 176 cheese, 44, 256 definition, 19 mixtures, 13 efficacy, 53, 84, 97, 236, 287 feed(s), 37, 224, 229, 232, 241, 245–7 smoothie, 269 properties, 38, 61–2, 65, 172, 280, 284, 290, 292 vectors for, 272 procarcinogens, 7, 11, 79, 82, 252 proctitis, 207 proctosigmoiditis, 207–8 product phase, 276 production of folates, 308 prognostic biomarkers, 310 proinflammatory, 46, 55, 57, 138, 163, 207, 252, 269, 289 prokaryote, 304, 309 prophylactic, 155, 183, 199, 216 administration, 202, 216 effect, 22, 26, 190, 191, 202 propionibacteria, 2, 12–13, 173, 254 Propionibacterium freudenreichii, 39, 157, 227 P. freudenreichii subsp. shermanii, 174, 250 proprietary strains, 264–8, 270 prostatic hyperplasia, 91 protective, 7, 45, 55–6, 65, 225, 242, 279, 303, 310 effect, 63, 135, 190, 308–9 environment, 46

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Index

protein, 11, 27, 42, 44, 62, 66, 92, 104–5, 107, 112, 122, 131, 138, 190, 140, 144, 173, 181, 224, 228–9, 241, 250, 257, 275, 289, 295–7, 305–8 biosynthesis, 55 matrix, 272 proton-pump inhibitor, 193, 203 pseudomembranous colitis, 155, 187, 201 Pseudomonas aeruginosa bacteremia, 8 psychosomatic involvement, 206 public health, 91, 102, 116, 120 pulsed-field gel electrophoresis (PFGE), 82, 238–9, 274, 284, 288 purine limitation, 307 QPS, see qualified presumption of safety quadruple therapies, 204 qualified presumption of safety (QPS), 38, 39, 79, 81, 83, 85, 226–8, 242, 245, 253, 269, 273–4 questionnaire, 202, 204, 210, 255–6 rabbits, 233, 245 radioactive drugs, 127 randomisation, 293, 298 randomized controlled trials, 103, 149–50, 171, 183 randomly amplified polymorphic DNA (RAPD)-PCR, 82, 238, 274, 289 RAPD-PCR, see randomly amplified polymorphic DNA (RAPD)-PCR RDA, see Recommended Dietary Allowance RDI, see Reference Dietary Intake reactive oxygen, 62 real-time PCR method, 277 recombinant probiotics, 11–12 Recommended Dietary Allowance (RDA), 106, 111–12 redox potential tolerance, 272 reduction of risk of disease, 93 Reference Dietary Intake (RDI), 106 regulated foods, 118 rehydration, 5, 42, 143, 154, 189–90, 316 relationship to health, 283, 285, 291 relative risk, 4, 5, 152–3, 156, 183, 186, 215 remission, 3, 4, 159–60, 193, 209, 212, 217–18 repeatability, 292 repetitive genomic element PCR, 274 repPCR, see repetetitive genomic element PCR reproducibility, 292

9781405194914_6_index.indd 337

337

reproductive toxicity including teratogenicity, 226 research and development, 263, 269, 283, 293 resistance, 30, 38, 41, 50–51, 54–5, 62–4, 82–3, 95, 137, 223, 227, 234–6, 245, 254, 272, 237, 284, 288–9, 294, 309, 311, 313–14, 318 respiratory infections, 4, 258 respiratory tract infections, 162, 257 rheumatoid arthritis, 8–9, 25 ribotyping, 82, 289 risk, 4–6, 10, 18, 28, 30, 32, 38, 44–7, 49, 58, 67–77, 79–80, 82–5, 91–4, 102–4, 107–10, 112–15, 119, 123, 138, 144, 150–156, 160, 162, 164–5, 172, 179, 182–9, 193–4, 201, 203, 214–16, 226, 249, 245, 253–4, 256–8, 275, 286–7, 298, 301, 311–12, 315, 319 assessment, 13, 243 ratio, 153, 183, 151 robust strains, 37 role of diet, 134, 206 Rome criteria, 190 rotaviral gastroenteritis, 189 rotavirus, 4, 52, 58, 143, 189, 194 infection, 23, 199–200 Royal Academy of Medicine, 315 Saccharomyces, 40, 228, 235 S. bayanus, 40, 228 S. boulardii, 2, 4–5, 9, 40, 154–7, 163, 166, 184, 187–9, 201–2, 204, 215–16, S. carlsbergensis, 136 S. cerevisiae, 11, 40, 66, 135–6, 138–40, 228, 233 S. pastorianus, 228 Shigella, 22, 202 S. dysenteriae, (S. dysenteriae), 10, 12, 138 S. flexneri, 18 Shiga toxin, 10, 12 shigellosis, 2, 18, 22 sachets, 45, 200, 202, 204, 210 safe history, 85, 273 safety, 30, 38–9, 49–51, 56–8, 61, 66–7, 77, 79, 81–3, 85, 88–9, 92, 94–5, 115, 120, 122–4, 126–9, 131–2, 145, 158, 160, 172, 174, 181, 183–4, 209, 212, 217, 225–8, 236, 242–3, 245, 247, 249, 253, 268–9, 271–4, 279, 283, 289, 299, 313 tests, 30 Salmonella, 23, 53–5, 136–7, 202, 235, 303

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338

Index

salmonellosis, 2 sample size, 122, 142, 152, 158, 163, 165–6, 185, 187, 293 sanitary control, 78 scale-up, 41 SCAN, see Scientific Committee for Animal Nutrition Schizosaccharomyces, 40, 228 Scientific Committee for Animal Nutrition (SCAN), 243 scientific substantiation, 76, 263–4, 268, 283, 287, 292–3 screening, 51, 53, 63, 67, 85, 174, 271, 304, 306, 312 sensory/organoleptic preferences, 263 septicemia, 183–4, 209 serum(s), 6, 8, 27, 127, 144, 186, 205, 256, 269 cholesterol, 6, 136, 144 severity, 3–5, 7–8, 151, 161–2, 183, 185–7, 190–191, 194, 206, 210, 212, 217 sheep, 136–7, 139, 233, 245, 278 shelf-life, 41–5, 82, 92, 95, 97, 274–6, 279, 283, 285, 309 SHIME, see Simulator of the Human Intestinal Microbial Ecosystem shopping habits, 264 short-chain fatty acids, 7, 192, 251, 310, 317 short-gut syndrome, 183–4 sigmoidoscopy, 207, 209 signature tagged mutagenesis, 76, 263 Simulator of the Human Intestinal Microbial Ecosystem (SHIME), 54 skim milk, 135, 137, 308 small and medium-size enterprises (SME), 76, 203, 263–9, 299 SME, see small and medium-size enterprises smoking, 28 sodium, 57, 92, 104–5, 107, 114–15, 241, 278, 289 sour milk, 2 soy, 29, 41, 228 protein, 92, 107, 122, 224 Species, 2, 6, 9, 17, 19, 30, 32, 38–40, 43, 49–50, 54, 59–63, 67, 79–83, 89, 94–5, 97, 99, 131, 142, 157, 161, 164, 173, 182, 185, 193, 202–3, 210, 225–9, 234–6, 238, 245–6, 251, 253–4, 269, 274, 277, 285, 288, 291, 294, 296–8, 304, 307–8, 310, 314 spin-off companies, 264 spontaneous fermentation, 81 spray-drying, 42, 200, 237, 240–241, 275

9781405194914_6_index.indd 338

stabilization, 64, 67, 97 of membranes, 62 technologies, 62 standardized mean difference, 151 Standing Committee in Animal Nutrition, 285 stanols/sterols, 92 Staphylococcus Staphylococcus hyicus, 11 Staphylococcus aureus, 269 State Food and Drug Administration, 126 stool(s), 22, 141, 143, 160, 179–82, 189, 201–2, 206–7, 209, 286, 297, 303, 317 consistency, 20–21, 160, 192 frequency, 21–2, 192 strain, 2–3, 10–12, 17–20, 26, 30–32, 37–8, 40–41, 43–7, 49–57, 59–68, 76, 79–84, 88–9, 92, 94–5, 97–100, 119, 121, 124, 128, 131–2, 135–7, 140–142, 145, 154–5, 158, 163–4, 167, 171, 173–9, 181–6, 188–9, 199–201, 206–7, 209, 212–14, 216–19, 224–8, 231–2, 234–42, 245, 249–58, 263–77, 279–80, 283–5, 287–99, 304–9, 314, 317, 319 collection, 40, 238 development, 41 -dependent properties, 271 Streptococcus, 2, 7, 19, 121, 157–8, 227, 242 Streptococcus gordonii, 11 Streptococcus mutans, 11, 12, 66, 83, 85, 193, 214–15, 218 S. (salivarius subsp.) thermophilus, 3, 9, 39, 44, 65, 98, 119, 121, 128, 138, 142, 144, 155, 181, 187–8, 190, 201, 203, 210, 215–18, 227, 273, 277, 305 stress-specific response, 62 structure/function claims, 90–94, 96, 100, 102, 104 studies, 2–8, 10–11, 18–20, 22–5, 30, 32, 42, 47, 50–51, 53–4, 57–8, 62, 64–6, 76–8, 80–85, 88, 93–7, 99, 114, 118, 121–4, 135, 141, 145, 149–53, 155–66, 171–6, 178, 180–185, 187–93, 202, 207, 215–18, 225–6, 229–30, 232, 242, 245, 249, 251–8, 263, 266–9, 271, 274, 287, 291–4, 297–9, 305–7, 310, 312 case–control studies, 83, 93 clinical studies, 18, 20, 51, 84, 99, 122, 135, 141, 145, 199–200, 206–7, 212, 214–15, 226, 254, 263, 276, 290, 299 cohort studies, 83 cross-sectional studies, 83

11/1/2010 6:33:31 PM

Index

human intervention studies, 76, 82–3, 85, 121, 252, 254, 266, 268 human observational studies, 83 study, 2–8, 11, 18–28, 30, 47, 53–9, 67, 83–4, 88, 93–4, 96, 99, 114, 120, 122–3, 135–44, 150, 152–3, 157–8, 161, 163–6, 171, 175–6, 181, 185–7, 190–191, 193, 202–7, 209–10, 213–15, 217, 232, 250–7, 267, 269, 275, 277, 289, 290, 293, 297–8, 306, 308 design, 83, 88, 96, 150, 152, 154, 217, 256, 293 double-blind (placebo-controlled), 20, 142–3, 181, 186, 188, 193, 201–4, 207, 210, 213–14, 256–7 sublethal damage, 275 subtropical climates, 5 sugar utilisation, 295 sulfate reduction, 61 superficial bladder cancer, 7, 26, 28 superoxide dismutase, 62 surface (S)-layer protein, 289 survival, 10, 26, 37–8, 43–6, 51–3, 56–7, 65–6, 82, 99, 140, 224, 250, 272, 275–7, 289, 295, 306, 309 swollen joints, 8 synbiotic(s), 31, 65–6, 68, 207, 209, 217–18 Syndrome(s), 2–3, 24, 49, 64, 115, 157, 183–4, 190–192, 199, 206, 217, 249, 254, 257, 312–13 synergism, 66 synthetic fertilizers, 179 systematic review, 3, 103, 108, 149–51, 153–8, 160, 162, 164–6, 183, 188, 191, 216–17 systemic effects, 276 T lymphocytes, 186 tablets, 42, 120, 123, 200, 272, 276 tailored probiotics, 53, 67 taxonomic identification, 38, 273 taxonomy, 19, 271, 273–4, 285 T-cell subpopulations, 144 technological, 13, 37, 46, 51, 61, 63, 82, 236, 250, 268, 275, 309 additives, 242 boundaries, 279 properties, 38, 40, 49–52, 236 terminology, 76, 79, 80, 127 tetracycline resistance, 254 Th17 response, 30 therapetic purposes, 128 Therapeutic Lifestyle Change Diet, 111

9781405194914_6_index.indd 339

339

therapy, 5, 52, 143, 154–5, 157–60, 189, 192–3, 202–4, 207, 212, 216, 218, 229, 254–6 eradication therapy, 216, 251, 255, 258 probiotic therapy, 155, 158, 160, 218 throat sprays, 88 thymidine, 10, 66 thymidylate synthetase (gene), 10, 12 thymine, 66 tolerance, 8, 41, 54, 62–4, 82, 181, 206, 226, 234, 236, 269, 272, 275–76, 312 tooth decay, 7, 83, 85 Torulaspora delbrueckii, 228 Torulopsis spp., 137 toxic activity, 95 toxicity, 30, 51, 131, 172, 226 acute, 226 chronic, 30 oral, 226 genotoxicity, 30, 226 subchronic oral toxicity, 226 toxigenic potential, 273 toxin sequestration, 10, 12 toxins A and B, 200 trans fatty acids, 108, 110, 112–14 transcription, 201, 295 regulation of, 55 transcriptome, 305–6, 310 transferable antibiotic-resistance genes, 254 translocation, 8, 51, 56, 275 transplantation, 8 transurethral resection, 26 traveler’s diarrhea, 5, 9, 156, 166, 199–202, 215–16 Trichomonas, 6 trichothecenes, 171 triple, 193, 203–4, 255 tropical, 5, 201 trypsin activity, 252 tumor necrosis factor, 11, 56, 138, 209 tumo(u)rs, 7, 23, 26, 30 tumour growth, 19 Turkey, 5, 202, 233, 245 ulcerative colitis, 3–4, 61, 159–60, 192, 199, 206–8, 217, 312 United States Food and Drug Administration, 30, 289 unpublished data, 150, 164–6 unregulated foods, 118 unsaturated fatty acids, 108, 110

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340

Index

urease breath tests, 23, 256 urinary (urogenital) tract infections, 6, 94 urogenital tract, 38 US Department of Agriculture, 108 US regulatory law, 89 vaccination, 66–7, 232, 257 vaccines, 67, 93, 127 vacuum-drying, 42 vaginal, 60, 180, 212, 213, 291 bacteria, 179 delivery, 179–80 flora, 6, 212–14, 218 infections, 1, 6, 100 vaginosis, 212–13, 218 validity, 67, 90, 151, 155, 165, 255, 291–2 vancomycin, 5, 254, 278–9, 288 Vedic culture, 134 vegetables, 59, 91, 92, 107, 113, 115–16, 130, 179 vegetal products, 309 veggie-caps, 42 very low density lipoprotein, 136 veterinary drugs, 179 viability, 37, 43, 45, 50–51, 53, 55, 62–4, 75, 84, 199–200, 236–7, 239, 241–2, 271, 275–7, 279, 285, 294, 309 viable but non-culturable, 37 Vibrio cholerae, 23, 306 virginiamycin, 136 virulence, factors, 51, 83, 273 Vitamin(s), 9, 104–5, 109, 127, 228, 236, 242, 246, 275, 278, 290, 316 C, 290, 315 D, 91, 307 K, 307–8

9781405194914_6_index.indd 340

water-soluble vitamins, 9 vomiting, 143, 182 VSL#3, 3, 64, 65, 143, 157–8, 160, 210, 218 w-3 fatty acids, 106, 109, 112–13 water activity, 43, 45, 200, 242, 275 weaning period, 186 weight, 77, 98, 113–15, 129, 135–40, 142–4, 152–3, 174, 181–4, 228–32, 253, 318 loss, 3, 174, 210, 231 weighted mean difference, 151 weight-for-age (percent), 143 well-being claim, 96 Western lifestyle, 185 WHO, see World Health Organization wine, 1 World Health Organization (WHO), 37, 67, 75, 79–80, 84, 88, 94, 98, 145, 193, 226, 249, 284–85, 290, 292, 298–9, 316 world’s oldest probiotic, 17, 19, 32 Xanthophyllomyces, 40, 228 xenobiotics, 311 xylanase, 140 xylitol, 85 Yakult (drink), 17–24, 26, 28, 30–32, 119, 121, 142 Yarrowia lipolytica, 228 Yeasts, 213, 228, 230, 234–6 yogurt (yoghurt), 2–3, 5–6, 17, 43–4, 52, 88, 94, 96, 98–9, 121, 130–131, 134, 138, 142, 205–6, 214, 216–17, 228, 250, 273, 304, 306, 318 Z-score, 143 Zygosaccharomyces rouxii, 228

11/1/2010 6:33:31 PM

Food Science and Technology GENER AL FOOD SCIENCE & TECHNOLOGY AND FOOD PROCESSING Food Flavour Technology 2E Food Mixing: Principles and Applications Functional Food Product Development Confectionery and Chocolate Engineering Industrial Chocolate Manufacture and Use (4th Edition) Chocolate Science and Technology Essentials of Thermal Processing Calorimetry in Food Processing: Analysis and Design of Food Systems Fruit and Vegetable Phytochemicals Water Properties in Food, Health, Pharma and Biological Systems Nutraceuticals,Glycemic Health and Type 2 Diabetes Nutrigenomics and Proteomics in Health and Disease Food Science and Technology (textbook) IFIS Dictionary of Food Science and Technology 2nd Edition Sensory Evaluation: A Practical Handbook Statistical Methods for Food Science Drying Technologies in Food Processing Biotechnology in Flavor Production Frozen Food Science and Technology Sustainability in the Food Industry Kosher Food Production 2nd Edition Dictionary of Flavors 2nd Edition Whey Processing, Functionality and Health Benefits Nondestructive Testing of Food Quality High Pressure Processing of Foods Concept Research in Food Product Design and Development Water Activity in Foods Food and Agricultural Wastewater Utilization and Treatment Multivariate and Probabilistic Analyses of Sensory Science Problems Applications of Fluidisation in Food Processing Encapsulation and Controlled Release Technologies in Food Systems Accelerating New Food Product Design and Development Chemical Physics of Food Handbook of Organic and Fair Trade Food Marketing Sensory and Consumer Research in Food Product Design and Development Sensory Discrimination Tests and Measurements Food Biochemistry and Food Processing Handbook of Fruits and Fruit Processing Food Processing - Principles and Applications Food Supply Chain Management

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