Probiotics and Health Claims

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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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

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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

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17.1 17.2 17.3 17.4

263 265 267

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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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1

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

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

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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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)

(a) 1.4 1.0 .6 .2

4.0

5.0

6.0

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Indican

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log μg/dl

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Before Feed After

1.0 Before Feed After Before Feed After Before Feed After

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)

(b)

100 90

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80 70 60 50 40 30 Treatment group Control group

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Week of intervention

0

1

2

3

4

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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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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Probiotics and Health Claims

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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(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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Probiotics and Health Claims

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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Probiotics and Health Claims

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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Probiotics and Health Claims

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).

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 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 (<40% is a must, <35% preferred). For products where modified headspace is possible, a nitrogen flush can give marginal stability improvements. However, often the stability gain is outweighed by the cost and time of adding an additional process step.

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Avoid prolonged exposure to the packaging operation. Avoid creating a powder aerosol. Air handling considerations: the ideal environment incorporates HEPA filtration for incoming air and either releases the spent air outside or leads it through HEPA filtration. Be aware of manufacturing activities in the area with regard to scheduling, e.g. avoid proximity to wet cleaning operations.

Probiotic stability in products where the organism is activity metabolising is largely a function of the inherent capabilities of the organism as well as the physical properties of the food matrix. This is addressed elsewhere in the chapter. Regarding probiotic stability in the dry state, it is important to emphasise the two fundamental factors that impact probiotic viability during probiotic powder manufacturing and storage, i.e. temperature and water activity (Aw), where Aw is a function of temperature. The challenge in maintaining dried microbial viability during shelf-life is preserving the integrity of the microbial cell membrane. Cooler temperatures and low water activities maintain the viability of the probiotic component and retard unfavourable chemical and enzymatic reactions that may lead to cell death. Although keeping a powdered product refrigerated may not be feasible in every market segment, prolonged exposure to elevated temperatures (>30°C) should be avoided. In order to maintain a state of cellular dormancy and integrity, it is necessary to reduce the possibility of chemical reactions. A simple means of achieving this is by ensuring that the water activity of the final system is low, preferably below 0.20. As the product Aw increases above 0.15–0.20, probiotic stability declines, a tendency exacerbated by higher temperatures. The inactivation kinetics vary depending on the strain, but the principle of higher temperature and higher Aw leading to accelerated death is consistent for any probiotic culture.

3.4

PRODUCING PRODUCTS CONTAINING PROBIOTICS

Besides the usual quality criteria, the stability of the specific probiotic strain is the most important parameter when producing a probiotic product. Stability, defined as the ability to survive under given circumstances, is not firmly linked to the characteristics of a particular genus or species, although typically there are overall similarities. However, the exact survival is linked to the specific strain.

3.4.1

Fermented milk products

A wide range of different probiotic-containing fermented milk products is available today and are based on regionally highly variable recipes and processes that may impact probiotic survival. Such products include stirred, set and drinking yogurts, typically with shelf-lives varying from 4 weeks to 8 weeks. In particular, daily-dose dairy drinks or ‘shots’ supporting various aspects of healthy living have enjoyed considerable popularity across the world in recent years and the sector is likely to continue to develop over the coming years. In most products, the fermentation process is based on a co-fermentation of the probiotic strain together with the base thermophilic or mesophilic culture. If this type of production process is chosen, the generally known methodology for the fermented milk product can be modified and applied. A co-fermentation results in a much faster and safer fermentation

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process as the base cultures are more adapted to the milk medium and therefore will multiply faster and reduce possible risks for growth of contaminants. The combination of base culture and probiotic strain requires an adapted inoculation rate of the probiotic part, as the probiotic strains in general need a longer fermentation time. Depending on the probiotic strain, a fermentation process based on the pure strain can take from 15 to 72 hours. In this case, substantial attention must be given to hygienic conditions and possibly the use of growth promoters (e.g. yeast extract) to achieve final pH and the correct cell count are needed. The latter is of great relevance as a product must provide a sufficient dose of probiotics. The following parameters have all been suggested to potentially have an effect on the survival of the specific strain in a dairy product: ● ● ● ● ● ● ● ● ● ●

inoculation dosage; growth during fermentation; other starter cultures used in parallel; fermentation temperature and time; protein type and amount; sugar level; fruit and flavour types; final acidity and evolution of same during storage; storage temperature and time; oxygen content in milk.

All parameters mentioned have some effect on survival and should be considered when developing a probiotic dairy product. Nevertheless, the most important factors seem to be the inoculation rate, the growth rate during fermentation, and sensitivity to acidity. Following general consensus of the scientific community, it is recommended that a minimum count of 1–5 × 109 probiotic colony-forming units (CFU) should be maintained in a daily serving of a dairy product. However, due to differences in local legislation and continuous updating, it is recommended that local legislation is consulted before starting any developments. Probiotic strains are supplied by starter culture suppliers either in defined units or in CFU per gram. The recommended inoculation dosage is a combination of the needed addition of bacterial cells and the ability of the specific strain to survive the above-mentioned formulation and processing conditions. In order to guarantee the target probiotic cell count at end of shelf-life, it is important to use optimal microbiological methods. As the probiotic strains are often associated with other strains, such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, lactococci or other probiotic strains, one must use the most appropriate selective medium and protocol for the enumeration. Such optimised methods, providing good selectivity and best recovery of the probiotic strain, can be obtained from the starter culture supplier.

3.4.2

Cheese

Besides the commonly known yogurt (drink)-like probiotic products, dairy innovations such as probiotic cheese and non-fermented dairy drinks are also appearing in the marketplace. The addition of probiotics in cheese (e.g. cheddar and Gouda varieties) takes place together with the standard cheese cultures. However, there are a few specific processing steps that need to be modified, such as scalding above 50°C. Recovery rate

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from the cheese curd is very good (>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 (<4.0) and fruits, and especially berries, may contain natural antimicrobial substances. In contrast to milk, the shelf-life of fruit juice may be up to 4 weeks or more, adding to the challenge. In addition to selecting a robust probiotic strain, the juice can be formulated such that it has a higher pH and is less rich in antimicrobial substances.

3.4.5

Dried products

In addition to the high water activity products described above, probiotics can be incorporated in products with a low Aw (Ouwehand et al., 2004). In theory, this should provide stable products, but there are nevertheless challenges. First, dry products are often not that dry and may have an Aw over 0.25, where stability of probiotics is reduced. There may also be locally a higher Aw, for example raisins in muesli or energy bars may have a higher Aw. Second, dry products often have long shelf-lives of 1 year or more. Dry products are also often stored at ambient temperatures and humidity. These may vary over time or be high in certain climates. An alternative to inoculation of probiotic strains to a high Aw product is the use of add-on straws or another vehicle containing the probiotic strains in dry forms. In this case, the probiotic can be stored dry in the straw and is released during consumption of a liquid product. Some markets prefer supplements over functional foods (such as the USA) or supplements are the most practical form of administration, for example due to lack of cold storage/ transportation or in hospital settings. Probiotic supplements are usually in the form of capsules, but may also be combined with other ingredients (e.g. prebiotics) and packed as sachets. For stability, it is important that capsules are packed in appropriate bottles, and glass is preferred as it is a better barrier to moisture. Likewise, sachets should preferably be of sealed aluminium-coated foil. In both cases, filling agents should be sufficiently dry. This may be particularly demanding when mixing probiotics with prebiotics, which often have a relatively high Aw.

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3.5

Probiotics and Health Claims

PROBIOTIC PRODUCTS AND FEEDING TRIALS

The gold standard for clinical research is the placebo-controlled, randomised, double-blind intervention. This set-up has been very successful in pharmacological research, but poses a number of challenges in food science; after all, there is no such thing as a placebo carrot. Preparing an appropriate placebo is thus challenging and may require substantial efforts. Furthermore, the inclusion of a test product into a diet may lead to an overall change in diet. If energy intake is to remain the same, something else will have to be excluded from the diet, with unknown consequences, something already noticed in the early days of cholesterol-lowering diets (Mann & Spoerry, 1974). Because of the challenges associated with investigating foods, probiotics have often been tested as supplements; it is easy to prepare identical capsules containing either the probiotic strain or a placebo. As the amounts that can be contained in capsules are relatively small, there is little risk that they affect the overall diet and behaviour. However, the small volume may also pose challenges as it limits the amount that can be contained in it. Excessively high levels of probiotics have been shown to cause formulation problems (Whorwell et al., 2006). However, the extrapolation of results obtained with probiotics in supplements to probiotics in a food matrix is challenging and little investigated. Some researchers have looked at the survival of selected probiotic strains in different matrices. It appears that in general a food matrix, and in particular dairy, provides a protective environment for the probiotic (Conway et al., 1987). Simulated gastrointestinal survival of Lactobacillus rhamnosus HN001 was similar for the bacterium in a cheese matrix or as a supplement (Mäkeläinen et al., 2009). Also, L. rhamnosus GG has been observed to exhibit similar gastrointestinal survival in different food matrices (Goldin et al., 1992; Saxelin et al., 1993). Nevertheless, gastrointestinal survival is not a health benefit and although it is likely that with similar survival similar health benefits can be expected, this still needs to be proven. This is a more than trivial issue as they are food products that will potentially carry a health claim, although the ingredient and not the product may have been tested. In this respect, the combination of a probiotic strain with other strains or other functional ingredients particularly deserves attention. It is often assumed, and without any justification, that when combining probiotic strains their properties will simply ‘add up.’ It is unrealistic to assume that by combining, for example, an anti-inflammatory strain with a proinflammatory strain, both properties would remain present without influencing each other. Similar interaction can of course be expected for combinations with other active ingredients as well. Viljanen et al. (2005) showed that L. rhamnosus GG was able to reduce atopic dermatitis while the combination of L. rhamnosus GG and three other strains did not lead to any improvement compared with the placebo. The combination of Bifidobacterium animalis subsp. lactis Bb-12 and L. rhamnosus GG or L. delbrueckii subsp. bulgaricus was found to increase the adhesion of the former to human intestinal mucus (Ouwehand et al., 2000). This suggests that even the presence of a ‘technological strain’ L. delbrueckii subsp. bulgaricus might influence the functionality of the probiotic. Combining strains and predicting the functionality based on the properties of the components is challenging. Likewise, one cannot extract the properties for one strain from a tested combination of strains. Specific probiotic strains may have documented health benefits. However, in most cases these benefits have been investigated and documented only once. The question arises of course whether this is sufficient. An important investigation showed that L. rhamnosus GG was able to reduce the risk for the development of atopic eczema in at-risk infants (Kalliomäki

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et al., 2001), whereas in a subsequent and almost identical study set-up the same strain failed to exert such effects (Kopp et al., 2008). On the other hand, L. rhamnosus HN001 and B. animalis subsp. lactis HN019 have been show to increase phagocytic and natural killer cell activity in populations in New Zealand (Gill & Rutherfurd, 2001; Gill et al., 2001), Taiwan (Chiang et al., 2000; Sheih et al., 2001) and Canada (Arunachalam et al., 2000). The pharmaceutical industry aims at curing disease or improving biomarkers for disease. Functional foods and probiotics are no exception to that, but aim to maintain health rather than cure disease. This is, of course, a much more challenging goal. How do you show you have prevented disease or shortened its duration in a basically healthy population? This requires prospective studies, with large numbers of participants who are being followed over a relatively long period of time. Even then, disease incidence may be too low to draw a conclusion. It is therefore common to investigate in populations that are more at-risk or have an immune status that allows modulation, such as children (Hatakka et al., 2001) or the elderly (Gill & Rutherfurd, 2001). To what extent results from such populations can be extrapolated to a general (adult) healthy population remains to be determined.

3.6

CONCLUSION

From the above, it is obvious that substantial challenges exist in the study and development of functional foods in general and probiotics in particular. It is a long and costly process for which the outcome is by no means certain.

REFERENCES Andreoletti O, Bukda H, Buncic S et al. (2008) Scientific opinion of the panel on biological hazards on a request from EFSA on the maintenance of the list of QPS microorganisms intentionally added to food or feed. EFSA J 923:1–48. 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. Barlow S, Chesson A, Collins JD et al. (2007) Introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. EFSA J 587:1–16. Barrangou R, Briczinski EP, Traeger LL et al. (2009) Comparison of the complete genome sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04. J Bacteriol 191:4144–4151. Boyle RJ, Robins-Browne RM, Tang ML (2006) Probiotic use in clinical practice: what are the risks? Am J Clin Nutr 83:1256–1264. 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. Conway PL, Gorbach SL, Goldin BR (1987) Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J Dairy Sci 70:1–12. Dunne C, O’Mahony L, Murphy L et al. (2001) In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings. Am J Clin Nutr 73:386S–392S. European Parliament and the Council of the European Union (2006) Regulation (EC) No 1924/2006 of the European Parliament and the Council of 20 December 2006 on nutrition and health claims made on foods. Off J Eur Union L404:9–25. FAO/WHO Working Group (2002) Guidelines for the Evaluation of Probiotics in Food. Available at www.who.int/foodsafety/publications/fs_management/probiotics2/en/ Gill HS, Rutherfurd KJ (2001) Probiotic supplementation to enhance natural immunity in the elderly: effects of a newly characterized immunostimulatory strain Lactobacillus rhamnosus HN001 (DR20TM) on leucocyte phagocytosis. Nutr Res 21:183–189.

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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. Goldin BR, Gorbach SL, Saxelin M, Barakat S, Gualtieri L, Salminen S (1992) Survival of Lactobacillus species (strain GG) in human gastrointestinal tract. Dig Dis Sci 37:121–128. Hatakka K, Savilahti E, Pönkö A et al. (2001) Effect of long term consumption of probiotic milk on infections in children attending day care centres: double blind, randomised trial. Br Med J 322:1327–1329. Kalliomäki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E (2001) Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 357:1076–1079. Kiviharju K, Leisola M, von Weymarn N (2004) Light sensitivity of Bifidobacterium longum in bioreactor cultivations. Biotechnol Lett 26:539–542. Kopp MV, Hennemuth I, Heinzmann A, Urbanek R (2008) Randomized, double-blind, placebo-controlled trial of probiotics for primary prevention: no clinical effects of Lactobacillus GG supplementation. Pediatrics 121:850–856. Kramer M, Obermajer N, Bogovic MB, Rogelj I, Kmetec V (2009) Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry. Appl Microbiol Biotechnol 84:1137–1147. Lahtinen SJ, Ouwehand AC, Reinikainen JP, Korpela JM, Sandholm J, Salminen SJ (2006) Intrinsic properties of so-called dormant probiotic bacteria, determined by flow cytometric viability assays. Appl Environ Microbiol 72:5132–5134. Luckow T, Delahunty C (2004) Which juice is ‘healthier’? A consumer study of probiotic non-dairy juice drinks. Food Qual Pref 15:751–759. Mäkeläinen H, Forssten S, Olli K, Granlund L, Rautonen N, Ouwehand AC (2009) Probiotic lactobacilli in a semi-soft cheese survive in the simulated human gastrointestinal tract. Int Dairy J 19:675–683. Mann GV, Spoerry A (1974) Studies of a surfactant and cholesterolemia in the Maasai. Am J Clin Nutr 27:464–469. Matsuzaki T (2003) Health properties of milk fermented with Lactobacillus casei strain Shirota (LcS). In: Farnworth ER (ed.) Handbook of Fermented Functional Foods. Boca Raton, FL: CRC Press, pp. 145–175. Ouwehand AC, Salminen SJ (1998) The health effects of viable and non-viable cultured milks. Int Dairy J 8:749–758. Ouwehand AC, Salminen S (2003) In vitro adhesion assays for probiotics and their in vivo relevance: a review. Microb Ecol Health Dis 15:175–184. Ouwehand AC, Kirjavainen PV, Shortt C, Salminen C (1999) Probiotics: mechanisms and established effects. Int Dairy J 9:43–52. Ouwehand AC, Isolauri E, Kirjavainen PV, Tölkkö 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, Kurvinen T, Rissanen P (2004) Use of a probiotic Bifidobacterium in a dry food matrix, an in vivo study. Int J Food Microbiol 95:103–106. 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, Ahokas M, Salminen S (1993) Dose response on the fecal colonisation of Lactobacillus strain GG administered in two different formulations. Microb Ecol Health Dis 6:119–122. Sharp RR, Achkar JP, Brinich MA, Farrell RM (2009) Helping patients make informed choices about probiotics: a need for research. Am J Gastroenterol 104:809–813. Sheih Y-H, Chiang B-L, Wang L-H, Liao C-K, Gill HS (2001) Systemic immunity-enhancing effects in healthy subjects following dietary consumption of the lactic acid bacterium Lactobacillus rhamnosus HN001. J Am Coll Nutr 20:149–156. Viljanen M, Savilahti E, Hahtela T et al. (2005) Probiotics in the treatment of atopic eczema/dermatitis syndrome in infants: a double blind placebo-controlled trial. Allergy 60:494–500. Whorwell PJ, Altringer L, Morel J et al. (2006) Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable bowel syndrome. Am J Gastroenterol 101:1581–1590. Zitz U, Kneifel W, Weiss H, Wilrich PTh (2007) Selective enumeration of bifidobacteria in dairy products: development of a standard method. Bull Int Dairy Fed 411:1–20.

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4

Probiotics and Health Claims: Challenges for Tailoring their Efficacy

Christophe Chassard, Franck Grattepanche and Christophe Lacroix

4.1

INTRODUCTION

With regard to probiotics, the assessment of safety is the first requirement in addition to good functional and technological properties and beneficial effects on human health. The main target is often the gastrointestinal tract in relation to maintenance of the intestinal microbial balance. There is now substantial evidence that probiotic microorganisms can also confer other major benefits on the host, for example by modulating immune functions or preventing pathogen invasion. The beneficial effects of probiotics and their biological mechanisms must be well demonstrated, and the underlying mechanisms conferring health benefits are the scope of an increasing number of in vitro and in vivo experiments using conventional and molecular biology approaches as well as advanced models and human clinical trials. Various probiotic microorganisms, mainly Lactobacillus spp. and Bifidobacterium spp., are commonly used in functional foods especially fermented dairy products, although for many strains found in products beneficial health effects are poorly demonstrated or even absent. It is recognized that the properties, functionality, and benefits of each selected probiotic are unique. The benefits of a particular strain therefore cannot be extrapolated to other strains, even within the same species (Pineiro & Stanton, 2007). The understanding and targeted design of probiosis has evolved especially during the past two decades. The main target of probiotics is healthy people and consumption is obviously related to the preventive potential of probiotics incorporated in regular food. Probiotics can positively impact the immune system and stabilize the human gut microbiota equilibrium to prevent the onset of negative health states or pathologies. The prevention of digestive problems and infectious and atopic diseases is already well demonstrated for specific probiotic strains while reduction of certain long-term risks such as cancer or ischemic heart disease could be of major interest in the future. Probiotics are also good candidates for pharmaceutical applications and clinical use. Interest in probiotics has been particularly stimulated by the increasing prevalence of disorders such as cancer, atherosclerosis, cardiac disease, obesity, and irritable bowel syndrome (IBS). Probiotics could provide efficient alternative therapies for some of the most prevalent human diseases.

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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It should again be emphasized that health properties are strain specific and one strain may not harbor all functions. The continuous challenge of probiotic research is to select new probiotic strains for specific use. Although the task is ambitious, the development of advanced technologies and strategies should support the emergence of a new generation of probiotics with well-proven mechanisms and tailored functions. Tailoring of the next generation of probiotics is already in progress and this chapter discusses the latest developments to maximize their efficacy and enhance their beneficial effects on human health. The validation of advanced models for the assessement of probiosis is essential for selecting the best new strains. Numerous probiotic strains remain to be discovered from complex bacterial ecosystems but advanced production strategies to propagate sensitive strains and improve stress resistance and efficacy of current strains are urgently needed. For optimal functionality and broad-spectrum activity, the specificity of probiotics could be improved through combination of strains, nutritional approaches, or genetic engineering in order to enhance their initial potential.

4.2

CURRENT SELECTION OF PROBIOTICS: SETTING THE SCENE FOR TAILORING PROBIOTICS

Probiotic bacteria are becoming increasingly important in the context of human nutrition, as scientific evidence continues to accumulate on the properties, functionality, and benefits of probiotics for promotion of good health. Probiotic preparations contain only one or several different species of microorganism (Fooks & Gibson, 2002). The safety of bacterial strains used as probiotics must be demonstrated first. Microorganisms used in food fermentation have a long history of safe use and are therefore considered to be non-pathogenic with “generally recognized as safe” (GRAS) status. However, the emergence of new strains increases the panel of potential probiotics. The safety of these new candidates needs to be carefully evaluated, especially those microorganisms not commonly used in food and most recently isolated from gut ecosystems. Good manufacturing practices must be applied to assure high viability and functionality of probiotics. Technological properties related to production and downstream processing are required in order to maintain and/or enhance the health-related function of these products (Fig. 4.1). The health properties of probiotics are strain specific and no generalization can be made even between closely related strains belonging to the same species. Strain potential should be established through appropriate in vitro experiments, confirmed in complex in vitro systems and/or animal models, and finally validated in clinical trials.

4.2.1

Safety considerations

Good evidence exists that specific strains of probiotics are safe for human use and able to confer some health benefits on the host. Identification is a critical step in assessment of the safety of potential novel probiotic strains (Holzapfel et al., 2001). It should best be done with a polyphasic approach, combining phenotypic and physiological methods with molecular studies because physiological characterization alone, for example by fermentation profile, is insufficient to achieve reliable identification (von Ah et al., 2007). Even if the safety of the current products is excellent, probiotics could, as living microorganisms, theoretically be responsible for side effects in susceptible individuals and must be investigated (Marteau & Shanahan, 2003). Factors to be addressed in probiotic safety

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Probiotics and Health Claims: Challenges for Tailoring their Efficacy Safety aspects • Human origin • Strain typing • Virulence factors • Antibiotic resistances • Genome sequencing

Current criteria of selection for probiotics

Functional criteria

Technological properties

• Survival in the gastrointestinal tract

• Easy propagation

• Adhesion to epithelial cells

• High viability

• Immunostimulatory with appropriate cytokines stimulation • Inhibit gastrointestinal pathogens

• Stability in products • Good physiology

• Desirable metabolic activities, e.g. carbohydrate metabolism • Antimutagenic and anticarcinogenic properties

• Improve strain discovery • Recombinant technology New perspectives for tailoring new probiotics

51

• Screening of strain efficiency in advanced models • Improve specificity of probiotic products

• Technological improvements and cell physiology programming • Novel cultivation methods for sensitive-fastidious organisms • Protection and retention of viability/functionality • Targeted delivery of probiotics

Fig. 4.1 Current criteria and perspectives for selection of probiotics with targeted efficacy.

evaluation include pathogenicity, infectivity, virulence factors, toxicity, metabolic activity, and intrinsic properties of the microbes (Ishibashi & Yamazaki, 2001). Methods for safety assessment range from in vitro studies to animal and human clinical studies (Donohue & Salminen, 1996; Abe et al., 2010). The absence of pathogenicity and infectivity is required for probiotic safety. In particular, no bacterial translocation should be reported. Since translocation from the intestine is difficult to induce in healthy animals (Berg, 1980), alternative strategies based on antibiotic treatment and/or administration of an immunosuppressive agent are generally used. Germ-free animals provide interesting models, with the additional benefit of an immature immune system (Berg & Garlington, 1979). Finally, possible antibiotic resistances must be carefully checked in probiotics and potential for transmission of genetic elements to other intestinal/food microorganisms needs to be considered in the future (Pineiro & Stanton, 2007).

4.2.2

Technological considerations

There are significant technological challenges for probiotics which, being of intestinal origin, are sensitive to many environmental stresses, such as acidity, oxygen, and heat (MattilaSandholm et al., 2002; Champagne et al., 2005; Lacroix & Yildirim, 2007). They should have good technological properties that allow them to be produced on a large scale and incorporated into food products without losing viability and functionality or creating unpleasant flavors or textures. Additionally, a probiotic must exhibit high survival rates in downstream processes (such as centrifugation and drying) and in food products during storage. High survival through the upper gastrointestinal tract and high viability at the site of action are also required, together with high activity in the gut environment. The viability of probiotics is a key parameter for developing probiotic foods (see also Chapters 5 and 18).

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Probiotic cells must be viable for most of the reported beneficial health effects, even though non-viable forms or cell-wall components of probiotics can also exhibit relevant functional health properties (Salminen et al., 1999). Although the number of cells required to produce therapeutic benefit is not known and might vary as a function of the strain and the health effect desired, in general a minimum level of more than 106 viable probiotic bacteria per milliliter or gram of food product is accepted (Ouwehand & Salminen, 1998). Because they must be developed economically at large scale, commercial strains are currently selected for their technological properties, ruling out some strains with promising health properties. Understanding the mechanisms that enhance cell survival could lead to more efficient applications of probiotics in products with high functionality. Consequently, industry demand for new technologies that enable high cell yield at large scale and ensure probiotic stability in food remains strong, because many strains of intestinal origin are difficult to propagate and high survival is important for both economic reasons and health effects. In addition, more efficient technologies could lead to greater product efficacy and strain diversification, with the development of technologically unsuitable strains into products. The most recent developments in fermentation technologies for producing probiotic bacteria with improved technological properties are discussed later in the chapter.

4.2.3

Functionality and health benefits

A large number of health effects are associated with the consumption of probiotics (Pineiro & Stanton, 2007), although some health properties are less well validated than others (Fig. 4.2). Probiotics are an original way of delivering active constituents to targets in the gastrointestinal tract (Marteau & Shanahan, 2003). These active constituents include mainly enzymes and immunomodulatory and antimicrobial components. Health benefits are especially associated with the alleviation of gastrointestinal tract disorders, such as reduction of acute diarrhea caused by bacterial pathogens (e.g. Clostridum difficile and viruses), diarrhea caused by antibiotic therapy, and reduction of infections (Pineiro & Stanton, 2007). Improvement in lactose digestion by probiotics is also well established. For example, common strains of lactic acid bacteria (LAB) used to make yogurt or fermented

• Prevention and treatment of rotavirus induced diarrhea Very well established

• Prevention and alleviation of antibiotic-associated diarrhea • Alleviation of complaints due to lactose intolerance

Well established

• Modulation of the human gut microbiota • Immunomodulation • Prevention or alleviation of allergies and atopic diseases in children

Lack of proof

• Decrease blood cholesterol • Prevention of cancer

Fig. 4.2 Health-related properties of probiotics.

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milks produce the enzyme b-galactosidase, which can improve lactose utilization in lactose maldigestors with insufficiency of the enzyme lactase in the gut (Kolars et al., 1984). Other health benefits related to immunomodulation have also been demonstrated, including allergy reduction and immune stimulation (de Vrese & Schrezenmeir, 2008). However, most mechanisms supporting health benefits are generally poorly understood. Mechanisms may vary from one probiotic to another and may be a combination of factors, thus making this a complex area of investigation. The new generation of probiotics will be tailored to enhance their current efficacy or exhibit new properties. The basis for reliably assessing probiotic efficacy in humans requires a fundamental understanding of probiotic strains, each of which is unique. Probiotics must be able to exert their benefits on the host through growth and/or activity in the human body. This assessment has to be based on scientific evidence established first in vitro, confirmed in vivo through animal models, and finally validated in clinical trials, illustrated for the development of antipathogenic probiotics in Figure 4.3. Most of the current probiotics were not selected for specific purposes; a broad range of activity was expected and tested empirically. The new generation of probiotics, called tailored probiotics, will be based on target-specific selection, and utilization of advanced in vitro and in vivo models combined with basic tests is then required.

Simple in vitro tests, e.g. In vitro gut fermentation antipathogen activity models, e.g. continous colonic against Salmonella strains in reactor model with immobilized antagonism tests human fecal microbiota for (Zihler et al., 2009) Salmonella infection (Le Blay et al., 2009)

Screening of probiotic strains with broad spectrum of Salmonella inhibition

Antipathogen mechanisms in the complex human gut microbiota

Animal models, e.g. mice model for Salmonella infection (Stecher et al., 2007)

Gut microbiota– host response and mechanisms

Human studies, e.g. intervention study on healthy Salmonella carriers or infected population (combined with antibiotherapy)

Final validation of probiotic efficacy

Fig. 4.3 Probiosis assessment: from simple in vitro tests to clinical trials, illustrated for the development of probiotics with antipathogen activity.

4.3

IMPROVING THE ASSESSMENT OF PROBIOSIS

4.3.1

In vitro models for the assessment of probiosis

4.3.1.1

Probiosis and gut fermentation

Survival in the gastrointestinal tract High viability of probiotic strains is generally required in order to maintain functionality in the gut. Probiotics must overcome the two main biological barriers of stomach acidity and

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bile secretion in the small intestine to reach the lower part of the gastrointestinal tract in a viable state. Probiotic bacteria vary considerably in their tolerance to low pH and bile salts; this can be evaluated in vitro by controlled incubation in gastric juice followed by bile treatment to reflect the physiological conditions found in the upper part of the gastrointestinal tract (Dunne et al., 2001). Tolerance to gastrointestinal stress is generally low and it is important to find new strategies to improve probiotic strain resistance (Noriega et al., 2004). Underlying bacterial mechanisms should be systematically investigated to design future generations of probiotics (Whitehead et al., 2008; Gueimonde et al., 2009). Once probiotics have reached the colon, they should compete efficiently with gut microbiota to survive and temporarily colonize it. It is important to study physiological parameters like substrate utilization and to use such markers to help predict colonization success. The ability of probiotics to use a broad range of substrates provides more opportunities to colonize a specific ecological niche and limit competition with predominant gut bacteria. Furthermore, antimicrobial properties primarily targeted against specific pathogens could be very helpful for colonization of the gut microbiota (Cleusix et al., 2007; Le Blay et al., 2007), whereas production of antimicrobial compounds may also depend on the carbon source available for probiotics (Tzortzis et al., 2004). Organic acids produced by probiotics, especially acetate and lactate, can inhibit bacterial growth; however, specific products such as bacteriocins are also produced to counter bacterial growth and provide a competitive advantage to producer strains. Investigation of these key properties is therefore important to better assess functionality of strains. Functional activity The characterization of antimicrobial properties is an important feature in the assessment of probiosis and is generally performed in vitro using plate inhibition tests (Zihler et al., 2009a). Numerous probiotics, and their corresponding antimicrobial products, are able to inhibit closely related bacteria competing for similar intestinal niches or bacterial invaders such as Listeria or Salmonella (Cleusix et al., 2008). The impact of probiotics on the composition and activity of gut microbiota has largely been studied in vitro. Some advantages are inherent to in vitro models: they are inexpensive, with no ethical requirements, and they are also a good design for mechanistic studies since use of radioactive or toxic components is facilitated (Fig. 4.4) (Macfarlane & Macfarlane, 2007). In vitro modeling of the digestive tract is particularly useful for investigating microbial processes such as carbohydrate metabolism, transformation or production of exogenic substances. Batch fermentation has been used to investigate bacterial metabolism (Stewart et al., 2009). However, these fermentations are not pH-controlled and need to be short to avoid selection of non-representative microbial populations (Macfarlane & Macfarlane, 2007). Continuous fermentation models avoid these problems and provide efficient control of bacterial metabolism and environmental conditions. The specific impact of probiotics, including metabolic and antimicrobial properties, has been successfully investigated using complex continuous fermentation models. For example, changes in the composition of the microbiota and the d-lactate/l-lactate ratio was observed after addition of Lactobacillus and Bifidobacterium species in a complex multistage system of fermentation called Simulator of the Human Intestinal Microbial Ecosystem (SHIME) (Alander et al., 1999). More recently, a continuous culture simulator of the human colonic microbiota was used to evaluate the potential of a Lactobacillus gasseri strain for oxalate degradation (Lewanika et al., 2007). An in vitro model of colonic fermentation with immobilized human feces was successfully used to demonstrate the production and activity of reuterin by Lactobacillus reuteri after glycerol addition to the fermenter (Cleusix et al., 2008). The presence of reuterin changed the composition of gut microbiota, decreasing the Escherichia coli population in the system.

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Advantages Limitations

• No ethical consideration • Easy to set up • Easy to modify–control environmental parameters • Easy to use radioactive, toxic and genotoxic agent

• No host immune and neuroendocrine response • Difficult to reproduce other biotic factors • Require fresh and high quality fecal inoculum

Fig. 4.4 Advantages and limitations of in vitro human gut models.

Another fermentation model with immobilized human feces was recently developed to simulate intestinal Salmonella infection in children (Le Blay et al., 2009). The use of this new model revealed the potential of Bifidobacterium thermophilum RBL67 to inhibit Salmonella without affecting the gut microbiota, in contrast to antibiotics (Zihler et al., 2009b). The viability and functionality of probiotics can both be investigated in such complex gut fermentation models, and set-up and validation of suitable models could provide interesting tools to elucidate mechanisms involved in probiosis. 4.3.1.2

Host–probiotic interaction

Adhesion to the intestinal mucosa is yet another important criterion in the assessment of probiosis. Improving adhesion may enhance the persistence of probiotics in the human gut and ensure the closest contact between probiotics and host. Furthermore, the cell adhesion of probiotics could limit the epithelial damage induced by enteroinvasive pathogens that must first adhere in order to infect host cells (Resta-Lenert & Barrett, 2003). Adhesion capacity is usually evaluated in vitro using human intestinal cell lines such as Caco-2 or HT-29 cells. Other methods including direct adhesion to intestinal mucus have also been used (Collado et al., 2005), although intestinal cell models permit investigation of other physiological parameters potentially modulated by probiotic strains using advanced tools. Microarray technology was recently used to examine the expression of genes of Caco-2 cells incubated with a Lactobacillus plantarum strain (Panigrahi et al., 2007). Addition of the probiotic strain changed the expression of several genes involved in important cellular processes, such as regulation of transcription, protein biosynthesis, metabolism, cell adhesion, and apoptosis. Other methods including transepithelial electrical resistance measurements assessing cell layer integrity have been used to characterize the probiotic potential of a Lactobacillus acidophilus strain and demonstrate its protective role against pathogens (Resta-Lenert & Barrett, 2003). Immunomodulation properties can also be investigated using intestinal cells studying release of proinflammatory or anti-inflammatory cytokines. Although mainly studied in vivo in animals or humans, immunomodulation properties can be tested in vitro using intestinal cell models (Ewaschuk et al., 2008). Peripheral blood mononuclear cell (PBMC) assays are also used to investigate probiotic immunomodulatory properties in vitro (Pochard et al., 2002; O’Mahony et al., 2006; Ghadimi et al., 2008). Probiotics,

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mainly LAB strains, enhanced the secretion of interferon (IFN)-g, interleukin (IL)-10, and tumor necrosis factor (TNF)-a by PBMCs in vitro, but these results were not confirmed in vivo in children (Flinterman et al., 2007). In contrast, Foligne et al. (2007) found a correlation between the in vitro immunomodulatory properties of LAB and an in vivo murine colitis model. These results suggest the key value of in vitro methods in the assessment of probiosis and also show the necessity of confirming in vivo, first in animal models and later in clinical trials, each promising finding observed in vitro.

4.3.2

In vivo models for the assessment of probiosis

The availability of animal models for investigating metabolic processes mediated by intestinal microorganisms is of great value in that animals can be given controlled diets while researchers have direct access to intestinal contents as well as tissues and organs at autopsy (Macfarlane & Macfarlane, 2007). Animal models can be used to investigate safety parameters or disease states in ways that would be unacceptable or unethical in a human. 4.3.2.1

Conventional animals

Animal models with characteristics very similar to those of healthy individuals were first used to understand how probiotics react to the complex digestive environment and impact host as well as gut microbiota. Conventional rodents were especially used to investigate the survival and safety of probiotics (Huang et al., 2003; Lara-Villoslada et al., 2007). The identification of possible bacteremia in different organs such as liver and spleen is critical, so the identification of bacterial translocation to tissues after administration is very important. Gastrointestinal survival of orally administered probiotic strains is evaluated by bacterial counting in the digestive tract and in fecal samples of animals (Huang et al., 2003). Other animal models were then developed to reproduce a disease or injury similar to a human condition. These are useful models for investigating the functionality of probiotics and for demonstrating their efficiency. Many rat and murine models have been developed to evaluate specific properties of probiotics. Conventional laboratory rats can be fed with special diets or treated with chemicals to generate pain or inflammation and mimic human diseases (Table 4.1). Infections of conventional laboratory animals with pathogens also generate interesting models for studying the protective role of probiotics against pathogenic agents. However, infection of healthy animals is difficult because of the barrier effect of the gut microbiota, and antibiotic pretreatment to affect the equilibrium of the gut microbiota is often required for colonization of bacterial invaders (Stecher et al., 2007). It should be remembered, however, that the gut microbiota in humans differs considerably from that found in animal models. Advanced gnotobiotic animal models (initially germ-free and then colonized with human microbiota) provide interesting models for studying the functionality of probiotics in a more controlled environment. 4.3.2.2

Gnotobiotic animals

Germ-free and gnotobiotic animals developed over the last 50 years provide interesting models for studying the mode of action of probiotics and for demonstrating their efficacy. They can be colonized by a complex human gut microbiota to generate “conventionalized”

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Table 4.1 Examples of animal models used to investigate the potential of probiotic treatments. Animal

Specificity of the model

Pathology mimics

Reference

Rat

Streptozotocin injection

Diabetes

Yadav et al. (2008)

Rat

Consumption of highfructose diet

Diabetes

Yadav et al. (2007)

Rat

Consumption of lowriboflavin diet

Riboflavin deficiency

LeBlanc et al. (2006)

Mouse

Intrarectal administration of TNBS

Inflammatory bowel diseases

Zoumpopoulou et al. (2008)

Mouse

Administration of DSS

Inflammatory bowel diseases

Mennigen et al. (2009)

Rat

Maternal deprivation

Visceral pain

Eutamene et al. (2007)

Rat

Intrarectal administration of high amount of butyrate

Visceral pain

Rousseaux et al. (2007)

DSS, dextran sodium sulfate; TNBS, trinitrobenzene sulfonic acid.

animals for investigating survival and metabolic activities of probiotics (Lan et al., 2007). They offer controlled microbial environments for the assessement of probiosis and the opportunity to reduce bacterial diversity or populations to study the mechanisms involved. It is possible to colonize germ-free animals with only one or two bacterial strains, providing monoxenic and dixenic animal models respectively (Sonnenburg et al., 2006; Ménard et al., 2008). However, they require special facilities and are quite expensive, which usually limits study numbers. 4.3.2.3

Knockout animals

Knockout rodents (i.e. animals with a single gene disruption) have been developed to mimic human diseases and evaluate the potential of probiotic treatments. The IL-10 knockout mouse is a unique model currently used to investigate probiotic effects. This provides a useful model for studying the effects of probiotics on the immune response in colitis associated with the secretion of proinflammatory cytokines similar to that seen in Crohn’s disease (McCarthy et al., 2003). However, other knockout rodents such as the ob/ ob mouse, which eats excessively and becomes profoundly obese, should be used in the future to evaluate the potential of probiotic treatments in metabolic diseases (Tennyson & Friedman, 2008). In vivo experiments are essential to establish the safety and functionality of probiotics. Animal models offer direct access to intestinal contents as well as tissues and organs at autopsy, which is of major value for safety considerations. Functionality of probiotic strains should be confirmed in vivo in animals using similar samples prior to human studies. A broad range of animal models is currently used and numerous other models will be available in the future. New animal models are continuously developed for drug testing and could also be used for the assessment of probiosis depending on the disease targeted. The interest in in vivo studies is evident and the number of animal experiments is currently increasing. However, the explosive rise of animal rights laws in most

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occidental countries could limit the development of animal research. Although ethically justified, restrictive laws will complicate the use of animal models and the maintenance of animal facilities.

4.3.3

Clinical trials for the assessment of probiosis

Clinical trials are conducted to provide safety and efficacy data for new pharmaceutical products or functional foods that should exhibit health-related properties. Depending on the type of product and the stage of its development, investigators enroll healthy volunteers and/or patients into small pilot studies initially, followed by larger-scale studies in patients that often compare the new product with a placebo and/or the currently prescribed treatment. As positive safety and efficacy data are gathered, the number of patients is typically increased. Clinical trials for the assessment of probiosis can be divided into prevention and treatment trials. Prevention trials aim to find better ways to prevent disease in people who have never had the disease or to prevent a disease from returning and are mainly used to test the effects of probiotics in foodstuffs. Treatment trials are designed to test experimental treatments including probiotics or a combination of probiotics against diseases in patients. Randomized, double-blind, placebo-controlled human trials should be designed to establish the efficacy of the probiotic product (Pineiro & Stanton, 2007). However, clinical trials for the assessment of probiosis can vary in size from a single center in one country to multicenter trials in multiple countries. Randomized double-blind studies have provided evidence for the effectiveness of probiotics in preventing various diarrheal illnesses, including rotavirus-induced and antibiotic-associated diarrhea, and confirmed the role of certain probiotics in the alleviation of complaints due to lactose intolerance, as recently reviewed by de Vrese and Schrezenmeir (2008). Modulation of the immune system and the gut microbiota by probiotics has been established, while promising effects have also been observed in alleviation of allergies and atopic diseases. The effect of probiotics in many other diseases, including inflammatory bowel disease (IBD), IBS, ischemic heart disease and autoimmune diseases, have also been investigated (de Vrese & Schrezenmeir, 2008; Hoveyda et al., 2009; Prisciandaro et al., 2009). Positive effects have been observed in numerous studies but are insufficient as yet to clearly establish the potential of probiotics as alternative therapeutics. The evidence for efficacy of most drug therapies in the treatment of these complex diseases is also weak and clinical research evaluating probiotics as treatments should be accelerated. Relevant outcomes need to be carefully selected to evaluate the health benefits of probiotics in such diseases and multicenter trials are probably required to demonstrate health benefits. Future clinical trials should focus on determining whether the bacterial and genetic profiles or subgroups of patients influence the effectiveness of treatment. The potential role of probiotics in the treatment of acute diseases such as pancreatitis has also been investigated. Positive effects were generally observed (Timmerman et al., 2007; van Minnen et al., 2007) but Besselink et al. (2008) recently reported that treatment with a probiotic preparation in patients with acute pancreatitis was associated with an increased risk of mortality and gut ischemia. Although the results and the interpretation of this study are highly controversial, it highlights the care that should be taken in the design of all studies using probiotics and especially in critically ill patients. Investigation of the mechanisms involved and characterization of the mode of action should be a prerequisite before use of probiotic treatments in acute diseases.

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59

IMPROVING THE DISCOVERY OF PROBIOTIC STRAINS Exploring and isolating bacterial diversity

The isolation and characterization of new microorganisms provide a continuous source of potential probiotics. LAB, including the largest genus Lactobacillus, and bifidobacteria were isolated mainly from fermented food and gut ecosystems respectively. Lactobacillus strains are associated with food production and many new species have been isolated from fermented food (Claesson et al., 2007). For example, Lactobacillus plantarum is found in a wide variety of food products such as dairy products, meat, vegetables and plants (Cai et al., 1999) but also in the human oral cavity and gastrointestinal tract (Molin et al., 1993). Recently, several species related to the Lactobacillus genus have been described from foodstuffs and gut ecosystems (Table 4.2). The characterization of lactobacilli diversity in various fermented products, using different media and culture methods, will enrich culture collections with new strains and species of lactobacilli. Bifidobacterium species are isolated mainly from digestive ecosystems, especially from the human gut. Bacterial diversity in humans has been extensively investigated recently using culture and culture-independent methods. In contrast, a culture-independent study analysing the diversity of several 16S rRNA gene-based libraries revealed the presence of novel bifidobacterial phylotypes that had not been found earlier and which represent novel taxa within the genus Bifidobacterium (Turroni et al., 2009). This study demonstrates that bifidobacterial diversity is higher than expected using culture methods and suggests that new species could be isolated from the human intestine. Improvement in growth conditions including media and abiotic parameters could provide new tools and strategies for cultivating new Bifidobacterium strains from the human gut.

Table 4.2 New species of Lactobacillus and Bifidobacterium recently identified from foodstuffs or the digestive environment. Bacterial species

Origin

Reference

Lactobacillus senmaizukei

Japanese pickle

Hiraga et al. (2008)

Lactobacillus hordei

Malted barley

Rouse et al. (2008)

Lactobacillus cappilatus

Tofu

Chao et al. (2008)

Lactobacillus equigenerosi

Horse feces

Endo et al. (2008)

Lactobacillus kisonensis

Japanese pickle

Watanabe et al. (2009b)

Lactobacillus cacaonum

Cocoa fermentation

De Bruyne et al. (2009)

Lactobacillus fabifermentas

Cocoa fermentation

De Bruyne et al. (2009)

Lactobacillus nodensis

Rice bran

Kashiwagi et al. (2009)

Lactobacillus equicursoris

Horse feces

Morita et al. (2010)

Lactobacillus similis

Fermented cane molasses

Kitahara et al. (2010)

Bifidobacterium mongoliense

Fermented milk

Watanabe et al. (2009a)

Bifidobacterium bombi

Honey digestive tract

Killer et al. (2009)

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Human milk has been described as an important source of new LAB strains. Bifidobacteria and lactobacilli populations have been recently identified in breast milk samples (Martín et al., 2006; Gueimonde et al., 2007; Perez et al., 2007). Some Lactobacillus isolated from breast milk exhibited key properties for probiotics (Martín et al., 2006; Díaz-Ropero et al., 2007). The origin of these microorganisms is not clearly understood but they could reach breast milk through internal blood carriage from gut and/or vaginal microbiota (Perez et al., 2007). Isolation of potential probiotic strains from canine milk has also been reported (Martín et al., 2010). These strains belonged to four Lactobacillus species (L. reuteri, L. fermentum, L. murinus, L. animalis), demonstrating that milk samples from mammals could provide an interesting source of new probiotics. However, it is the specificity of action not the source of the microorganism that is most important. Many intestinal ecosystems harbor bifidobacteria and could be potentially interesting sources for new strains and even new species (see Table 4.2). Bifidobacterium bombi, a new Bifidobacterium species, was recently isolated from bumblebee digestive tract (Killer et al., 2009). All strains related to the new species utilized various sources of carbohydrate and exhibited b-galactosidase, a- and b-glucosidase and fructose-6phosphate phosphoketolase activites as for most of the Bifidobacterium strains, but were unable to use galactose, lactose, and inulin. Insect digestive ecosystems could be an atypical but interesting new source for the isolation of bifidobacteria. Bifidobacterial and LAB diversity should be evaluated in some other animals including all mammals in order to find new species with potential probiotic traits.

4.4.2

New generations of probiotics from new bacterial genera and with new targeted functions

The large intestine is a complex microbial ecosystem harboring more than 500 different bacterial species, mainly strict anaerobes (Eckburg et al., 2005). Hundreds of amazing properties are exhibited by these microbial consortia but only a few specific strains exhibit potential probiotic activities. Furthermore, only a very small part of the bacterial diversity of the human gut microbiota has been characterized and numerous strains remain to be isolated. Among this highly complex community, populations of bifidobacteria and especially lactobacilli are rather low. The human gut microbiota is composed mainly of bacteria belonging to three other major bacterial groups (Clostridium leptum group, Clostridium coccoides group and Bacteroides–Prevotella group), which represent potentially hundreds of commensal microorganisms never identified in infective lesions or linked to pathologies. They exhibit interesting properties or functions never reported in LAB or bifidobacteria and which are important for gut health, as illustrated in sections 4.4.2.1–4.4.2.3. The ability to use intermediate metabolites such as lactate or to degrade complex polysaccharides could have particular value in restoring metabolic dysbioses reported in digestive pathologies such as IBS or IBD. Certain natural inhabitants of the gastrointestinal tract are also able to metabolize various polyphenols and maximize their health-promoting role. 4.4.2.1

Faecalibacterium prausnitzii with anti-inflammatory properties

Recently, Sokol et al. (2008) showed that Faecalibacterium prausnitzii, a highly prevalent intestinal bacterium, exhibits anti-inflammatory properties. Interestingly, the fecal microbiota of patients with IBD differs from that of healthy volunteers and F. prausnitzii is

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especially underrepresented in IBD patients as in patients with Crohn’s disease (Sokol et al., 2009). In vitro experiments using intestinal cells demonstrated that F. prausnitzii displays high anti-inflammatory properties, which were confirmed in vivo using a trinitrobenzene sulfonic acid (TNBS)-induced colitis. Metabolites produced were able to block NF-kB activation and IL-8 production independently of butyrate. Probiotic strategies using F. prausnitzii could be very useful in the treatment of IBD and especially Crohn’s disease. 4.4.2.2

Lactate-dissimilating probiotics

Lactate and hydrogen are intermediate metabolites whose accumulation is mainly prevented by their metabolism in gut bacteria. Lactic acid can accumulate when the microbial balance of the gut is disturbed. Lactate accumulation is associated with inflammation and diseases, especially ulcerative colitis (Vernia et al., 1988), and can lead to neurotoxicity and cardiac arrhythmia (Chan et al., 1994; Vella & Farrugia, 1998). Identification of bacteria able to convert lactate into butyrate, such as Eubacterium hallii, could provide an opportunity to develop a new probiotic strategy (Duncan et al., 2004; Sato et al., 2008). This metabolic pathway may play a key role in gut health because it is a major energy source for the colonic mucosa and an important regulator of gene expression, inflammation, differentiation, and apoptosis in host cells (Scheppach & Weiler, 2004; Pajak et al., 2007; Hamer et al., 2008). 4.4.2.3

Probiotics for controlling hydrogen accumulation

Hydrogen gas can also accumulate when the microbial balance of the gut is disturbed. Hydrogen produced during intestinal fermentation is essentially used by hydrogenotrophic microorganisms in the gut, whereas hydrogen accumulation can affect gut fermentation and lead to microbial imbalance. Although IBS is very common, its pathophysiology is not fully understood and many factors including gut microbiota imbalance are implicated (Nobaek et al., 2000; Mättö et al., 2005; Kassinen et al., 2007). King et al. (1998) reported abnormal fermentation in IBS patients associated with significant hydrogen excretion, which is indicative of an imbalance in hydrogen metabolism. H2–CO2 acetogenesis is of great interest for human nutrition and health by decreasing the total gas volume in the colon and by producing a non-gaseous metabolite that is an energy source for eukaryotic cells (Bernalier et al., 1996). Isolation and use of hydrogen-utilizing acetogenic bacteria from human feces could provide an opportunity to counter hydrogen accumulation without the production of gas, in contrast with the formation of methane (CH4) or deleterious components such as hydrogen sulfide with dissimilatory sulfate reduction (Bernalier et al., 1996). Although fastidious, isolation from gut microbial ecosystems is the original way to identify new promising probiotics. It represents the past and the future of probiotic identification but microbiologists should continue with the development of alternative media and strategies to cultivate bacteria which are currently uncultivable. New probiotic strains will belong to classical probiotic bacterial genera such as Bifidobacterium or Lactobacillus, since the diversity of these bacterial populations has not been completely explored. However, the potential of new strains related to atypical probiotic species but exibiting new probiotic properties should be especially considered. Safety and technological considerations will establish the real potential of new species for probiotic use in the future.

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4.4.2.4

Improving stress resistance of probiotics

The viability of probiotics is a key parameter for developing probiotic foods. Several stress conditions encountered by probiotics, from production to release into the gastrointestinal tract following ingestion, may severely compromise their viability and thereby reduce their efficiency (Lacroix & Yildirim, 2007). In the past decade, a great deal of research effort has been focused on the optimization of process parameters to improve cell viability during production and downstream processes. More recently, studies have been aimed at developing technologies to improve the intrinsic resistance of probiotic cells against stress conditions through exploitation of the cellular stress response and/or production process using immobilized cell technology combined with continuous culture. Different stabilization technologies based on microencapsulation have also been proposed in order to physically protect cells from external environments, with the potential to control cell release at target sites. Grattepanche and Lacroix (2009) recently discussed the potential of novel fermentation and stabilization technologies for the production of high-quality probiotics. Genetic engineering To a certain extent, bacterial cells, including probiotics, are able to overcome adverse conditions through general or stress-specific response mechanisms. Several stress conditions encountered by probiotics affect essential cellular components such as protein, DNA and RNA (De Angelis & Gobbetti, 2004; Corcoran et al., 2008; Sanchez et al., 2008). One of the major bacterial stress response systems is overexpression of chaperone and small heatshock proteins, which are involved in several cellular functions including de novo folding, refolding of stress-denaturated proteins, oligomeric assembly, intracellular protein transport, assistance in proteolytic degradation, stabilization of membranes and protection of nucleic acids (Georgellis et al., 1995; De Bruyn et al., 2000; Narberhaus, 2002; Hartl & Hayer-Hartl, 2009). Heterologous or homologous overexpression of these chaperones or small heat-shock proteins in LAB enhanced stress resistance of recombinant cells compared with parent cells (Desmond et al., 2004; Corcoran et al., 2006; Fiocco et al., 2007). Most LAB and bifidobacteria are very sensitive to oxidative stress due to the accumulation of reactive oxygen species such as the superoxide radical anion, hydroxyl radical and hydrogen peroxide, which react with proteins, lipids and nucleic acids leading to lethal damage (van de Guchte et al., 2002; Talwalkar & Kailasapathy, 2004). Heterologous production of a non-heme manganese-dependent catalase, a heme catalase or a manganese superoxide dismutase in oxidative stress-sensitive strains of L. casei, L. plantarum, L. jonhsonii, L. reteuri, L. acidophilus or L. gasseri provided protection against hydrogen peroxide and long-term aerated cultures (Bruno-Barcena et al., 2004; Noonpakdee et al., 2004; Rochat et al., 2006). Accumulation of compatible solutes is a well-known defense mechanism of bacterial cells for withstanding osmotic stress, as encountered for example during drying processes. Heterologous expression of betL, a gene encoding a glycine betaine uptake transporter in L. salivarius exhibiting relevant probiotic properties but poor robustness during spraydrying, increased resistance to osmotic, cryogenic and barometric stress and chill tolerance as well as spray- and freeze-drying (Sheehan et al., 2006). Recombinant cells of B. breve expressing betL were more tolerant to gastric juice and conditions of elevated osmolarity mimicking the gut environment compared with the parent cells (Sheehan et al., 2007). In addition to the fact that genetic modification could affect probiotic functionalities and also the general negative perception of genetically modified organisms by consumers, it is

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technical limitations (e.g. the ability to transform bifidobacteria cells) that may hinder the development of the genetic engineering approach to improve stress resistance of probiotics. Application of sublethal stresses The stress adaptive response and cross-protection phenomena of bacterial cells, in particular pathogens, have been extensively studied in the last decade (van de Guchte et al., 2002; Ambur et al., 2009; Wesche et al., 2009). These bacterial defense mechanisms can also be exploited to improve the resistance of probiotics to lethal stress conditions. Probiotic cultures are typically harvested in late-log or stationary growth phase in order to achieve maximum cell yield and viability during downstream processing (Saarela et al., 2004). Cells develop general stress resistance mechanisms that can further improve their tolerance to lethal conditions in response to nutrient starvation and/or accumulation of inhibitory fermentation end products encountered in stationary phase of growth (Teixeira et al., 1994; Gouesbet et al., 2002; Maus & Ingham, 2003; Prasad et al., 2003; De Angelis & Gobbetti, 2004; Saarela et al., 2004). Specific sublethal stresses can also induce an adaptive response that protects cells against further homologous or heterologous (cross-protective effect) stress. For example, heat-adapted cells of L. paracasei NFBC 338 and Bifidobacterium adolescentis survived better than unadapted control cultures to a lethal heat stress (Schmidt & Zink, 2000; Desmond et al., 2002). Acid adaptation protects LAB and bifidobacteria against subsequent exposure to lethal pH levels (Park et al., 1995; Maus & Ingham, 2003; De Angelis & Gobbetti, 2004; Saarela et al., 2004). However, the acid tolerance response is strain dependent (van de Guchte et al., 2002; Maus & Ingham, 2003; Saarela et al., 2004). Conditions during application of the sublethal treatment can also differentially affect tolerance of the cells to lethal challenge (Saarela et al., 2004). A general mechanism cannot be defined for sublethal stress applications since their effects are strain-dependent and may vary with sublethal stress conditions. Application of sublethal stresses can also result in reduced cell yields, cell activity and/or process volumetric productivity depending on the strain/species, growth phase and the mechanism of stress-induced protection (Doleyres & Lacroix, 2005). Therefore, for each probiotic strain, a labor-intensive and costly screening procedure has to be performed to optimize sublethal stress conditions. Additional research is required to elucidate stress response mechanisms. New technological approaches using continuous cultures, eventually combined with cell immobilization, have been proposed in order to more efficiently optimize sublethal stress conditions and produce probiotic cells with controlled cell physiology and enhanced intrinsic stress tolerance (Lacroix & Yildirim, 2007; Mozetti et al., in preparation). Immobilized cell technology and continuous culture Cell immobilization is the retention of microorganisms in a discrete region to limit their free migration and to maintain a high level of viability and/or desired catalytic activities. Physical entrapment in polymeric networks with thermal or ionotropic gelation properties, under mild conditions, is the most widely used technique for immobilization of LAB and bifidobacteria (Lacroix et al., 2005). Application of immobilized cell technology for production of bacterial biomass, especially when associated with continuous culture, offers many advantages over free cell cultures, such as high cell density, high productivity, reuse of biocatalyst, resistance to bacteriophage attack, higher plasmid stability, prevention of washing-out, and physical and chemical protection of cells (Doleyres & Lacroix, 2005; Lacroix et al., 2005; Lacroix & Yildirim, 2007). This technology can be used to cultivate

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fastidious and sensitive microbes such as probiotics. In addition, cell immobilization can lead to physiological modifications with particular relevance for the technological and functional properties of probiotic bacteria. Indeed, increased tolerance to adverse conditions such as simulated gastric and intestinal juices, oxidative stress, freeze-drying and resistance to antimicrobial compounds (bacteriocin and antibiotics) has been reported for probiotic cells continuously cultivated using immobilized cell technology (Lacroix & Yildirim, 2007). Cell immobilization can also induce formation of macroscopic cell aggregates (Bergmaier et al., 2005; Reimann et al., in preparation). The ability of probiotics to aggregate has been associated with functional traits such as adhesion to epithelial cells (Del Re et al., 2000; Kos et al., 2003), physical protection in gastrointestinal tract and coaggregation with pathogens (Schachtsiek et al., 2004; Collado et al., 2007). Stabilization of probiotics using microencapsulation technologies Probiotic cells can be physically protected from their external environment using encapsulation techniques, also known as microencapsulation, referring to the size of the capsules produced. Probiotic microencapsulation is a very active field of research and several techniques have been proposed to encapsulate probiotic cells (Kailasapathy, 2002; Champagne & Fustier, 2007). However, difficulties in the large-scale production of microcapsules containing probiotics and in controlling capsules that must not negatively affect the textural and sensorial properties of food products may limit the implementation of microencapsulation in industry even if it provides efficient protection against oxygen, acidic environments, simulated gastrointestinal juices, and during refrigerated storage in food products (Doleyres & Lacroix, 2005). In this regard, spray-coating, widely used for the preparation of commercial products containing probiotics, is a promising technique for encapsulation (Champagne & Fustier, 2007). To date, research on microencapsulation has mainly focused on preserving cell viability under adverse conditions. Further studies are now required to obtain a better understanding of the delivery of microencapsulated cells in the gastrointestinal tract as well as to develop coating or encapsulating materials to control cell release at target sites.

4.5

IMPROVING PROBIOTIC SPECIFICITY

4.5.1

Future therapeutic strategies: combination of strains?

The mechanisms underlying the beneficial effects of probiotics are not completely understood, but numerous bacterial strains exhibit health benefit properties and they may differ markedly in their mode of action. Specific strains of probiotics have been shown to modulate the human gut microbiota, inhibit colonization of pathogens or modulate the immune system but no single strain possesses all properties. The selective combination of strains could be a valuable approach by providing several microbial characteristics not exhibited by a single strain. This approach could be especially efficient in the treatment of gastrointestinal diseases, which are generally very complex and which comprise a high number of different syndromes and symptoms such as IBD and IBS. The VSL#3 preparation is probably the most famous combination of probiotics. It contains eight different bacterial strains: three strains of bifidobacteria (B. longum, B. infantis and B. breve), four strains of lactobacilli (L. acidophilus, L. casei, L. delbrueckii subsp.

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bulgaricus and L. plantarum) and one strain of Streptococcus salivarius subsp. thermophilus (Venturi et al., 1999). Studies on different pathologies have demonstrated that the administration of multiple probiotic organisms might expand their capacity for immunological modulation (Timmerman et al., 2007). The VSL#3 probiotic preparation has been shown to prevent or ameliorate several gastrointestinal pathologies, such as IBD (Di Giacinto et al., 2005), IBS (Camilleri, 2006), pouchitis (Pronio et al., 2008) and chemotherapy-induced diarrhea (Bowen et al., 2007). This broad therapeutic range could be due to the high number of bacteria included in the preparation. Large combinations of probiotics could then generate generic products to treat a broad range of pathology. However, the mechanisms involved, as well as the role of each strain in the mixture, are very difficult to elucidate. An alternative strategy could be to create new probiotic preparations including a lesser number of well-characterized strains exhibiting complementary properties. This approach could be especially relevant in the treatment of IBS. IBS is a common chronic gastrointestinal disorder characterized by abdominal pain, bowel dysfunction and bloating in the absence of structural abnormality (Spiller & Garsed, 2009). Gut microbiota dysbiosis is also frequently reported and therapies including probiotics are popular and frequently used (Hoveyda et al., 2009). Probiotics may play a role in alleviating the symptoms of IBS but evidence of global efficacy is weak. Symptoms exhibited by IBS patients are rather different and it may be difficult to create a generic formula suitable for all patients. In this context, selective combinations based on specific probiotic properties, such anti-nociceptive, anti-inflammatory or anti-flatulence properties, exhibited by a limited number of strains could be combined and used as an efficient and tailored preparation. The specific association of strain(s) depending on the symptomatology could be the future of probiotic therapies in IBS patients and similar strategies could be investigated for other gastrointestinal diseases, providing specific and advanced treatments. Specific age groups could be targeted as well. Food products have been developed for infants or elderly people and these populations are of particular interest because of the early immune development in infants and the marked decline in immune function in the elderly (Saulnier et al., 2009). Combination of strains exhibiting additive properties could help to improve intestinal microbial composition and immune function in specific age groups.

4.5.2

Nutritional manipulation

Prebiotics are dietary fibers not digested by host enzymes but fermented in the colon by the gut microbiota. They exhibit a major impact on the human microbiota through targeted inhibition of potentially pathogenic microorganims and/or the promotion of growth of potentially protective bacteria such as bifidobacteria and, in part, lactobacilli as well (de Vrese & Schrezenmeir, 2008). The growth of probiotics could be stimulated by selected prebiotics providing a specific substrate. Indeed, once probiotics reach the colon, they must compete for nutrients and an ecological niche with the already established colonic microbiota. These factors can compromise probiotic establishment in the colon and therefore diminish efficacy (Saulnier et al., 2008). Synbiotics are defined as mixtures of probiotics and prebiotics that beneficially affect the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract of the host (Andersson et al., 2001). The synbiotic approach should be able to potentiate the effect of probiotics or prebiotics. Although the synbiotic

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approach has attracted interest, the development and evaluation of synbiotics is still at an early stage (Saulnier et al., 2008). However, promising results were demonstrated in vitro (Saulnier et al., 2008) and in vivo in animals (Frece et al., 2009). Similar results were obtained in vivo in humans, where most of the studies demonstrated that combinations were more effective than placebo and probiotics or prebiotics provided alone (de Vrese & Schrezenmeir, 2008). More work is needed to understand the effects of selected synbiotics on microbial communities in the gut and their interactions with the host’s immune system. However, to elucidate the health benefits of synbiotics is a challenge since these food products associate two components, bacteria and substrate, that are biologically active on their own. The demonstration of synergism, and the mechanisms involved, should be much more evident and the emergence of new prebiotic substrates could improve the specificity of their association, localize their action in the gut and maximize their interest (Grootaert et al., 2009). Scientists could then design new synbiotic formulas to enhance the heath-related properties of functional foods tailored to prevent disease or improve host health.

4.5.3

Genetic engineering

Probiotic microorganisms have numerous beneficial effects and demonstrated efficacy in the prevention and treatment of a number of human diseases. Like all forms of life, bacteria undergo evolution. However, evolution is a slow and gradual process which generates new microorganisms exhibiting new properties often in response to their environment. Genetic engineering makes it possible to accelerate evolution toward defined target properties, leading to the continuous genesis of new microorganisms with better characteristics since chosen properties can be specifically acquired. Although extreme care should be taken in their construction and use, genetically modified microorganisms are promising probiotics. They can be engineered to produce and secrete compounds of interest or perform bioconversion in the gastrointestinal tract and provide completely new strategies of host vaccination and drug delivery. The first goal of genetic modification has been to generate harmless commensal strains that, when fed to a patient, displace harmful pathogens (Saier & Mansour, 2005). This strategy has been found useful for the elimination of Streptococcus mutans from dental caries (Hillman et al., 1990; Anderson, 1992). Recent studies have shown that modified microorganisms can produce enzymes and cytokines locally in the gut to counter diseases. Lactococcus lactis strains have been genetically engineered to produce a lipase that increases intestinal fat absorption in metabolically defective animals (Drouault et al., 2000, 2002) and to deliver human IL-10 to modulate inflammation in IBD patients (Steidler et al., 2003; Braat et al., 2006). A strain growth-control strategy based on deprivation of thymidine or thymine was used to assure safety. The L. lactis thyA gene was replaced with expression constructs driving a synthetic human IL-10 gene, resulting in strains that produce human IL-10 and which are strictly dependent on thymidine or thymine for growth and survival. These strains are then self-limiting because they die rapidly in the absence of their essential growth component (Steidler et al., 2003). The biological containment system and the strain’s capacity to secrete human IL-10 were validated in vivo in pigs and confirmed in humans (Braat et al., 2006). Other microorganisms such as Lactobacillus casei (Hazebrouck et al., 2006) and Saccharomyces cerevisiae (Blanquet et al., 2003; Garrait et al., 2007) have also been successfully modified to deliver protein and demonstrate bioconversions. Rapidly growing microbes with small genomes, simple metabolism

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and industrial relevance are selected for engineering. These criteria are met by LAB, a group of phylogenetically related Gram-positive bacteria with low GC content, including Lactococcus and Lactobacillus species (de Vos & Hugenholtz, 2004). Genetic engineering could be a potent approach to control and target administration of vaccine antigens to the mucosal immune system (Bermúdez-Humarán et al., 2004, 2005; Cortes-Peres et al., 2007). Lactococcus lactis strains were genetically modified to express antigens such as GroEL heat-shock protein from Brucella abortus and could provide in vivo immunization of the host against this bacterial pathogen (Miyoshi et al., 2006). Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. A promising probiotic strategy for papillomavirus-induced tumor protection was validated in vivo in mice (Bermúdez-Humarán et al., 2005). The feasibility of developing live recombinant L. lactis strains expressing cell wall-anchored E7 antigen and a secreted form of IL-12 has been demonstrated and successfully tested to help control human papillomavirus (HPV) type 16 by prophylaxis and treatment, respectively. Indeed, 50% of mice vaccinated with both strains were tumor-free over the test period (~100 days) following tumor induction. Although high, the level of immunization generated in mice was lower than the almost 100% immunization in volunteers receiving three injections of new conventional vaccines protecting against HPV types 16 and 18 (Bornstein, 2009; WHO, 2009). However, an extra study confirmed that intranasal administration provides a better way of host immunization (Cortes-Perez et al., 2007). Vaccination by mucosal, rather than systemic, delivery offers several advantages, such as ease of administration without the need for trained personnel or the hygienic risk associated with the use of syringes. These advantages can considerably reduce costs and increase safety of vaccination campaigns, mainly in developing countries. To conclude, promising strategies involving engineered microorganisms have been tested and validated mainly in vivo in animals. If carefully designed, such microorganisms could revolutionize the development of probiotics for medicine. The recent demonstration of their validity in humans using the most promising modified strain shows the potential for drug delivery and a high level of safety (Braat et al., 2006). However, the future of engineered probiotics will depend on safety considerations and demonstration in humans. The opinion of consumers and patients could also affect the development of such strategies involving modified and live microorganisms.

4.6

CONCLUSIONS

The rapid development of the science of probiotics and related products is offering potential benefits for a wide range of health conditions. The efficacy of probiotics in the prevention and treatment of certain human diseases is now well established. There is great potential to discover new probiotics by screening actual bacterial ecosystems with enhanced efficiency or targeting new functions. Current probiotics can be engineered to enhance their actual benefits or induce new health-related properties. As more probiotic strains are discovered or engineered for tailored efficacy, it is likely that probiotics will be used to treat and prevent new disorders. In any case, the development of “tailored” probiotics will be based on in vitro data, elucidation of mechanisms involved and in vivo demonstration of efficacy in clinical trials. Preferential targets for the development of new strategies invoving probiotics include the treatment of serious disorders for which no conventional, efficient and safe treatments exist. Novel cultivation and stabilization strategies

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are urgently needed to investigate non-cultivatable or difficult strains and widen the range of probiotic candidates. The specificity of probiotics should be enhanced by combining the potential of several strains or by adding prebiotics to generate synbiotics. The future of probiotics will depend on the capacity of the medical, scientific and industrial communities to scientifically demonstrate the potential of new strains for alternative therapies, avoiding misuse of the term for products poorly manufactured and/or without relevant documentation.

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Hiraga K, Ueno Y, Sukontasing S, Tanasupawat S, Oda K (2008) Lactobacillus senmaizukei sp. nov., isolated from Japanese pickle. Int J Syst Evol Microbiol 58:1625–1629. Holzapfel WH, Haberer P, Geisen R, Bjorkroth J, Schillinger U (2001) Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr 73:S365–S373. Hoveyda N, Heneghan C, Mahtani KR, Perera R, Roberts N, Glasziou P (2009) A systematic review and meta-analysis: probiotics in the treatment of irritable bowel syndrome. BMC Gastroenterol 16:9–15. Huang Y, Kotula L, Adams MC (2003) The in vivo assessment of safety and gastrointestinal survival of an orally administered novel probiotic, Propionibacterium jensenii 702, in a male Wistar rat model. Food Chem Toxicol 41:1781–1787. Ishibashi N, Yamazaki S (2001) Probiotics and safety. Am J Clin Nutr 73:465S–470S. Kailasapathy K (2002) Microencapsulation of probiotic bacteria: technology and potential applications. Curr Issues Intest Microbiol 3:39–48. Kashiwagi T, Suzuki T, Kamakura T (2009) Lactobacillus nodensis sp. nov., isolated from rice bran. Int J Syst Evol Microbiol 59:83–86. Kassinen A, Krogius-Kurikka L, Mäkivuokko H et al. (2007) The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects. Gastroenterology 133:24–33. Killer J, Kopecny J, Mrázek J et al. (2009) Bifidobacterium bombi sp. nov., from the bumblebee digestive tract. Int J Syst Evol Microbiol 59:2020–2024. King TS, Elia M, Hunter JO (1998) Abnormal colonic fermentation in irritable bowel syndrome. Lancet 352:1187–1189. Kitahara M, Sakamoto M, Benno Y (2010) Lactobacillus similis sp. nov., isolated from fermented cane molasses. Int J Syst Evol Microbiol 60:187–190. Kolars JC, Levitt MD, Aouji M, Savaiano DA (1984) Yogurt: an autodigesting source of lactose. N Engl J Med 310:1–3. Kos B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic S (2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94:981–987. Lacroix C, Yildirim S (2007) Fermentation technologies for the production of probiotics with high viability and functionality. Curr Opin Biotechnol 18:176–183. Lacroix C, Grattepanche F, Doleyres Y, Bergmaier D (2005) Immobilised cell technologies for the dairy industry. In: Nedovic V, Willaert R (eds) Applications of Cell Immobilisation Biotechnology. Dordrecht: Springer, pp. 295–319. Lan A, Bruneau A, Philippe C et al. (2007) Survival and metabolic activity of selected strains of Propionibacterium freudenreichii in the gastrointestinal tract of human microbiota-associated rats. Br J Nutr 97:714–724. Lara-Villoslada F, Sierra S, Martín R et al. (2007) Safety assessment of two probiotic strains, Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714. J Appl Microbiol 103:175–184. LeBlanc JG, Rutten G, Bruinenberg P, Sesma F, de Giori GS, Smid EJ (2006) A novel dairy product fermented with Propionibacterium freudenreichii improves the riboflavin status of deficient rats. Nutrition 22:645–651. Le Blay G, Lacroix C, Zihler A, Fliss I (2007) In vitro inhibition activity of nisin A, nisin Z, pediocin PA-1 and antibiotics against common intestinal bacteria. Lett Appl Microbiol 45:252–257. Le Blay G, Rytka J, Zihler A, Lacroix C (2009) New in vitro colonic fermentation model for Salmonella infection in the child gut. FEMS Microbiol Ecol 67:198–207. Lewanika TR, Reid SJ, Abratt VR, Macfarlane GT, Macfarlane S (2007) Lactobacillus gasseri Gasser AM63(T) degrades oxalate in a multistage continuous culture simulator of the human colonic microbiota. FEMS Microbiol Ecol 61:110–120. McCarthy J, O’Mahony L, O’Callaghan L et al. (2003) Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut 52:975–980. Macfarlane GT, Macfarlane S (2007) Models for intestinal fermentation: association between food components, delivery systems, bioavailability and functional interactions in the gut. Curr Opin Biotechnol 18:156–162. Marteau P, Shanahan F (2003) Basic aspects and pharmacology of probiotics: an overview of pharmacokinetics, mechanisms of action and side-effects. Best Pract Res Clin Gastroenterol 17:725–740. Martín R, Jiménez E, Olivares M et al. (2006) Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother–child pair. Int J Food Microbiol 112:35–43. Martín R, Olivares M, Pérez M et al. (2010) Identification and evaluation of the probiotic potential of lactobacilli isolated from canine milk. Vet J 185:193–198.

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Probiotics: from Origin to Labeling from a European and Brazilian Perspective

Célia Lucia Ferreira, Marcos Magalhães, Miguel Gueimonde and Seppo Salminen

5.1

INTRODUCTION

The definition of probiotics has evolved over the years and its development has been difficult at times. The history is quite long and is discussed in Chapter 2. As a result of this development, the current definition, used also in the European Union (EU), is mainly based on the work of International Life Sciences Institute (ILSI) Europe and the World Health Organization (WHO) (Salminen et al., 1998; WHO, 2001, 2002). The WHO working group definition of probiotics concludes that probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2006). This differs from the definition often used in Japan, where the term “probiotic” covers live microorganisms but also cells of viable microorganisms, when a health benefit has been demonstrated (Salminen et al., 1998). The definition probably requires further examination now that it is understood that viability of microorganisms is not always directly associated with culturability using traditional methods. It is also understood that the majority of intestinal microorganisms are viable but not culturable and thus probiotics can also be viable without having to be culturable. Because of this development, the term “viability” needs to be redefined in the future (see also Chapters 4 and 18). Because the area is very difficult to define comprehensively, we have taken a terminological approach to clarifying the issues with regard to probiotics and health claims. We have also decided to define the procedures starting with probiotics themselves, proceeding from characterization to health claims. This unified approach allows us to use different terminological viewpoints to clarify the basis and meaning of terms related to probiotics and health claims. This approach also facilitates clarification of the whole process, from characterization of probiotics and their origin to the human health-related claims. For this purpose, we have compared the European and Brazilian regulations in order to develop a basis for understanding and developing probiotics for future functional foods and therapeutic uses with 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

TERMINOLOGY AND PROBIOTICS

The general theory of terminology (GTT) was founded by the Austrian engineer Eugen Wüster (1898–1977) (Felber, 1984). It emerged mainly from the need to describe, order and transfer knowledge, thus creating standards in special fields in order to achieve effective communication. The main purpose of a terminological work is “to improve specialist communication, knowledge transfer and provision of content with a view to facilitate the participation of all in the global multilingual knowledge society” (Infoterm, 2004). Therefore, the terminological principles used in this work aim to identify the main concepts related to probiotics and health claims, model concept systems based on these, as well as establish representations of the concept systems with their terms and definitions through concept diagrams. The concept diagrams are graphically represented by generic relations (tree diagrams ), partitive relations (rake diagrams ) and associative relations (arrow diagrams ). The terminology approach is adjusted in this chapter to make the concept more easily understandable and also for describing two different regulations for probiotic health claims, namely those in Brazil and the EU.

5.3

HEALTH CLAIM REGULATION IN THE EUROPEAN UNION

The regulation on health claims has been in force in EU member states for 2 years, but the practical measures are still developing and the EU Commission is still working on the finalized format of health claims and a common European list of approved health claims. European Regulation (EC) No. 1924/2006 on claims made on foods will have a great impact also 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 regulation 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 human studies. On the basis of the European regulation, high-quality human intervention studies are needed to substantiate a specific health claim for a certain product. This requirement for a number of human studies focuses the research efforts of large multinational companies providing probiotic strains and products as well as small and medium-size enterprises (SMEs) aiming to produce probiotic products with health claims.

5.4

HEALTH CLAIMS IN EUROPE

On 1 July 2007, EC Regulation No. 1924/2006 of the European Parliament and the Council on nutrition and health claims made on foods came into force (European Commission, 2006). This was the first time that a regulation dealt with overall health claims and the reason for implementing the regulation was that more and more products carry health-related

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claims; therefore it should, on the one hand, protect the consumer against misleading information, economic losses or health problems and, on the other, harmonize legislation in member states. Meaningful health claims can help consumers to make healthy food choices. In the Regulation, the terms used in health claims are defined in a number of different categories: first, the so-called Article 13 health claim, also referred to as the “functional claim”, which are claims referring to (1) the role of a nutrient or other substance in growth, development and functions of the body, (2) psychological and/or behavioral functions, and (3) slimming or weight control or a reduction in the sense of hunger or an increase in the sense of satiety or to reduction in the available energy from the diet. The other categories include health claims in the following areas: (1) claims about reduction in disease risk, (2) claims referring to children’s development and health (Article 14 claim), and (3) claims based on new and hitherto not established areas of health claims (including new innovative processes and products with diagnostic measures for the health claim). Additionally, all existing claims in different EU member states were collected by national authorities and submitted to the European Commission. The submitted claims were separately assessed to verify that a scientific basis for such claims exists and, if positive, these were added to the Commission decision and list of allowed European health claims. For the existing Article 13 claims, member states had the opportunity to submit national lists with claim proposals and scientific references to the EC until January 2008. The Commission asked the European Food Safety Authority (EFSA), and specifically the Panel on dietetic products, nutrition and allergies, for their scientific opinions on health claims. The claims and their conditions, as well as rejected claims with the reason for rejection, should be listed in a Community Register. This Register shall be made available to the public (Article 20, EC 2006). Originally, a community list with all approved health claims should have been adopted by the Commission by 31 January 2010 but at the time of writing only a small part of the community list claims had been evaluated. The first Commission decision on approved and rejected claims appeared in October 2009 and these initiate the establishment of the community list of approved claims and rejected claims. The first list of authorized claims was published by the EU Commission in October 2009 (Commission Regulation (EC) No. 983/2009).

5.5

HEALTH CLAIM REGULATION IN BRAZIL

The current Brazilian legislation for functional foods has its roots in 1997, with Act 1549 from October 17 (Brazil, 1997, 1999) related to the categories of different food additives and supplements. It described and defined, among others, foods for special uses. However, the lack of guidelines for labeling such products and misinterpretation of the Act resulted in a large amount of additives and supplements being commercialized “as food” without approved labeling and claims. Moreover, the increasing number of inquiries to the Health Ministry by industries willing to commercialize functional foods induced the development of more specific regulations. Therefore, by Law 9.782 of 26 January 1999, the Brazilian government established the National Health Surveillance Agency (ANVISA). The main goal of this institution is to

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protect and promote the health of the population by means of sanitary control over the production and marketing of products subjected to sanitary surveillance. A few months after the establishment of ANVISA, the Legislation on Functional Foods came into force in Brazil, the first in Latin America (ANVISA, 1999a). The majority of its content was shaped by the draft propositions of the Codex Alimentarius guidelines, still under construction at that time. The legislation approved the technical regulations establishing the basic guidelines for analysis and proof of function and/or health claims on food labels as well as the guidelines for registration of foods carrying these labels (ANVISA, 1999b). According to them, a function claim “is related to the metabolic or physiological role that the nutrient or non-nutrient plays in the growth, development, maintenance and other normal functions of the human organism” while a health claim “states, suggests or implies the existence of a connection between the food or ingredient with disease or condition related to health”. However, the actual probiotic legislation was issued in another regulation dealing with bioactive substances and probiotics with functional and health claims (ANVISA, 2002). This regulation addressed the distinction between a functional food and a bioactive compound (nutraceutical), which are marketed as pills, capsules and under other non-food formats. These products should be registered and approved by the health authority, ANVISA. Most of the claims allowed in Brazil are function claims, such as in the following example: Probiotics (Lactobacillus and Bifidobacterium) “help in maintaining the intestinal flora balance” and/or “help in increasing the beneficial flora”. Claims such as “increased antibodies” and “strengthen natural defenses against daily aggression and stress” were not allowed. It is worth noting that ANVISA is constantly reviewing several misused claims. The agency has instituted the Technical Advisory Scientific Committee on Functional Foods and Novel Foods (CTCAF) to manage unforeseen situations (ANVISA, 1999c). An aspect of concern is that although there is a guideline for health claims, registration of such claims depends on documented information internationally approved, since human studies to substantiate such claims hardly exist in Brazil. As a dynamic process, in 2005, the CTCAF and the Food Advisory Committee revised all the products already on the market with approved functional and/or health claims. In this process, difficulties encountered by consumers in understanding the true meaning of claims were taken into consideration. As a result, many claims were prohibited and others modified.

5.6

DEFINING HEALTH CLAIMS

There has been a long development of health claims with varying results in different countries around the world. Health claims in functional foods were first defined in Japan and came into force with the law on Foods for Specified Health Use (FOSHU). Thereafter, several countries have decided to regulate health messages or other messages about the health benefits of foods or food components. In Europe the development has finally resulted in a common European-wide regulation on health claims in foods and this regulation is binding on all member states. The main objectives of Regulation (EC) No. 1924/2006 of the European Parliament and of the Council of December 2006 on nutrition and health claims made on foods have been to ensure a high level of consumer protection, effective functioning of the internal market within the EU, fair competition within the food industry, and both stimulation and protection of innovations.

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A health benefit is a condition which has been demonstrated to improve the health of the host. In the case of probiotics, the main health benefits of probiotics are based on balancing the gut barrier, balancing of the intestinal tract microbiota, mucosal integrity, and competitive exclusion of pathogens and viruses. Specific probiotics may also be associated with other health benefits, including desirable modulation of lactose intolerance, diarrhea prevention and symptom alleviation, immune and allergy response, mineral absorption, and inhibition of procarcinogen-activating enzymes. A probiotic claim is any claim which states, suggests or implies that a probiotic food has particular characteristics relating to its origin, nutritional properties and health (WHO 2002). A reduction of disease risk claim is formed by “any health claim that states, suggests or implies that the consumption of a food category, a food or one of its constituents significantly reduces a risk factor in the development of a human disease” (EC 2006, Art. 2.2.6, Art. 14). Figure 5.1 illustrates the basic concepts of the terminology of probiotics and health claims. These have also been described in detail by Magalhaes et al. (2010). With regard to the health and probiotic claims for each probiotic strain, the three important areas that require assessment are characterization, safety, and health effects in humans. These three steps establish the basis for evaluating probiotics and the potential human health claims and are discussed in detail in sections 5.6.1–5.6.3.

5.6.1

Characterization of probiotic bacteria

Appropriate identification and nomenclature of microorganisms constitute the starting point for the assessment of microbial properties. Reliable identification by adequate methods confirms the identity of the strain in commercial use and is also necessary for proper labeling of products containing them (Felis & Dellaglio, 2007). In addition, correct identification allows the microorganism to be linked to what is already known about the corresponding microbial group, permitting prediction of some of its properties. During the last few years molecular techniques have replaced or complemented traditional phenotypic methods. DNA–DNA hybridization is the current gold standard for determination of bacterial identification, with two strains being considered to belong to the same species if their DNA–DNA relatedness is 70% or more. However, due to the difficulties associated with this technique, and the need of expertise not normally present in the food industry, phylogenetically based approaches such as sequence analysis of the 16S rRNA gene are currently the most commonly used methods for bacterial species identification. In general, microorganisms sharing a 16S rRNA gene homology higher than 97% are considered members of the same species. Establishing the identity of a microorganism is thus the first step in the assessment of its safety and efficacy. In this respect, the qualified presumption of safety (QPS) approach established by the EFSA considers the identification of microorganisms as the first pillar for safety assessment (EFSA, 2007). However, when evaluating probiotics, in addition to proper species identification, it is very important to take into account that probiotic effects are strain specific, so it is therefore necessary to identify the microorganisms at strain level. According to the FAO/WHO working group (FAO/WHO, 2006), strain typing has to be performed with a reproducible genetic method or using a unique phenotypic trait. In the framework of the EU Regulation on Nutritional and Health Claims made on Foods (Regulation (EC) No. 1924/2006), when assessing applications EFSA has considered appropriate identification at species and strain level a restriction criteria for the further assessment of health claims related to probiotics.

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Probiotics and Health Claims Probiotic “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (WHO, 2002)

Health benefit Condition which has been demonstrated to benefit the health of the host

Probiotic claim Any representation which states, suggests or implies that a probiotic food has particular characteristics relating to its origin, nutritional properties and health

Health claim “Any claim that states, suggests or implies that a relationship exists between a food category, a food or one of its constituents and health” (EC, 2006, Art. 2.2.5)

Reduction of disease risk claim “Any health claim that states, suggests or implies that the consumption of a food category, a food or one of its constituents significantly reduces a risk factor in the development of a human disease” (EC, 2006, Art. 2.2.6, Art. 14)

Fig. 5.1 Terminology of probiotics and health claims: basic concepts.

This emphasizes the need for proper species identification and characterization at strain level (genetic typing) using internationally accepted molecular methods. In addition, strains should be named according to the International Code of Nomenclature. In the context of this Regulation, the purposes of characterization are to confirm the identity of the food/constituent that is the subject of the health claim and to establish that the studies

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provided for substantiation of the health claim were performed with the food/constituent for which the claim is made. Characterization should also be sufficient to allow control authorities to verify that the food/constituent which bears a health claim in the market is the same that was the subject of community authorization (EFSA, 2009a). According to the recommendations of the FAO (2006), and although not mandatory also recommended by EFSA, strains should also be deposited in an internationally recognized culture collection. These are important criteria that will assure the tracking and access of scientist and authorities to the strain and related information in case it is needed. Therefore, proper identification of any investigated strain may constitute the critical starting point for probiotic studies (EFSA, 2009b). In the future, the increasing availability of genome sequences will allow genome-wide and/or multilocus phylogenetic analysis. During the last few years the development of high-throughput sequencing technologies has enormously increased sequencing capability, significantly reducing sequencing costs. Although final genome assembly is still a very time-consuming task, the number of completed bacterial genomes, and particularly of draft genomes, is increasing rapidly (Chain et al., 2009). In fact, some probiotic strain genomes have already been sequenced and in some cases the sequences have been deposited in public databases. In the future the inclusion of genomes of commercial probiotic strains in public or restricted access databases may overcome all the current limitations regarding identification at species and strain level. The genome sequence constitutes the best possible genetic fingerprint of a given strain. In addition, the availability of genomes of commercial strains would allow researchers and authorities very rapid access to the information on potential traits of the strain in case new markers, related to efficacy or safety of probiotics, are identified in the future. It is clear that strains used by the food industry and scientists should be identified using molecular methods and up-to-date taxonomic nomenclature. In this respect, it is also important to make each strain available through international culture collections. Even nowadays, many scientific articles are published without exact characterization of the tested strains, hampering the progress of scientific development in this area and assessment of the efficacy and safety of probiotics.

5.6.2

Safety assessment

Fermented foods containing live microorganisms have been consumed for thousands of years all over the world. Spontaneous fermentation was initially used to preserve foods by inhibiting the growth of spoilage/pathogenic microorganims. A number of species of lactic acid bacteria (LAB) have been used in food fermentation to improve food safety and more recently to improve consumer health, forming the basis of the probiotic concept. Probiotics, mainly from the genera Lactobacillus and Bifidobacterium, have not shown any pathogenicity traits (Vankerckhoven et al., 2008). With the exception of enterococci, LAB and bifidobacteria are rarely involved in infection (Lahtinen et al., 2009). The International Dairy Federation document on microbes with a long history of safe use in foods (Mogensen et al., 2002) and the QPS approach established by the EFSA (2007) support the safety of probiotic lactobacilli and bifidobacteria. Currently used strains are thus considered safe and have a long history of safe use, even with postmarket monitoring data available. Nevertheless, given the importance of this issue, the safety of probiotics, in particular new probiotics that do not have a history of safe use

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Probiotics and Health Claims Probiotic characterization Process in which a strain intended to be used as a probiotic product is identified and aspects of its functionality, safety and technology are evaluated

Probiotic identification Determination of phenotypic aspects and genotypic data of a microorganism that will enable to state that the new isolate belongs to one of the established taxonomic groups based on similarities and relationships (FAO, 2001)

Probiotic functionality Trait inherent to the probiotic strain related to different aspects of human health

Phenotypic characterization by determination of carbohydrate fermentation profile, enzymatic activity profile, lactic acid isomers nature (Ministero della Salute, 2005)

Adhesion properties

Gelatinase negative

Anti-cholesterol

Hemolysin negative

AFLP ARDRA DNA ERIC PFGE RAPD REA

Probiotic technological aspect Trait to be evaluated in the probiotic strain related to different aspects of processing of foods and non-food products

Balance of intestinal microbiota

Procarcinogen enzyme inhibitor

Blood pressure regulation

Lactic acid isomers nature

Pathogen inhibitor

Procarcinogen enzyme inhibitor Genotypic identification by the application of techniques for species (DNA–DNA hybridization, 16s rRNA gene sequence, REA, ARDRA, manual or automated ribotyping) and strain typing (PFGE, RAPD, ERIC, AFLP) (FAO, 2001; Ministero della Salute, 2005)

Probiotic safety Trait to be evaluated in the probiotic strain in order to guarantee a harmless use to the host

Origin of probiotic strain

Acid tolerance Growth in food matrix Process stability Shelf-life Survival in food matrix

Antibiotic resistance/ transferability

Tolerance to gastric pH

Amplified fragment length polymorphism Amplified ribosomal DNA restriction analysis Deoxyribonucleic acid Enterobacterial repetitive intergenic consensus Pulse field gel electrophoresis Random amplified polymorphic DNA Restriction endonuclease analysis

Fig. 5.2 The concepts involved in probiotic characterization.

and those belonging to species for which general assumption of safety cannot be made, requires rigorous assessment (Figs 5.2 and 5.3).

5.6.3

Human intervention studies for health claims

Health claim documentation is mainly based on human intervention studies conducted in the target population using the food and ingredient in the intended dose level. This is especially true in the case of claims intended for children’s health or claims associated with reduction in risk of disease. Prior to human intervention studies it is important to establish the rationale for the probiotic strain or strain combination and to gain information on preclinical properties of the strain or strain combination.

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Probiotic safety

Species / Strain – identity – characteristics

QPS (Europe)

History of safe use Strain properties

– Europe – elsewhere

– lack of virulence factors – lack of antibiotic resistance

GRAS (USA)

– QPS assessment and classification (EFSA)

– GRAS petition (FDA)

Fig. 5.3 Major steps in safety evaluation of specific probiotics or specific probiotic foods.

For human intervention studies it is most important to select the target population that corresponds to the intended claim. It is as important to use the same dose of the food product or ingredient in the studies which is intended to be in the claim. Human intervention studies can be classified in several ways but the following hierarchy of study design forms a common basis. ● ● ●

Human intervention studies, randomized controlled studies, other randomized studies (non-controlled), controlled (non-randomized) studies, other intervention studies. Human observational studies, cohort studies, case–control studies, cross-sectional studies, other observational studies, such as case reports. Other human studies dealing with the mechanisms of action.

Human studies should be conducted according to international guidelines and they should provide information on markers or factors that are important as intermediate markers associated with clear end points in the disease or health area in the claimed effect. Examples could include cholesterol levels in the case of heart disease risk, or numbers of Streptococcus mutans bacteria on dental surfaces, oral pH or dental plaque as risk factors for caries and tooth decay. Guidelines for human studies and other information required for health claim applications are available from EFSA (2009c) (Fig. 5.4).

5.6.4

Totality of supporting evidence

As specified in the European health claim regulation, all claims should be substantiated by taking into account the totality of the available scientific data and by weighing the evidence (European Commission, 2006). This should be conducted considering the specific conditions of use. In particular, the total evidence should demonstrate the following. ● ●

The extent or importance to which the claimed effect of the food/constituent is relevant for human health. Whether a scientific cause-and-effect relationship can be established between the consumption of the food or the food constituent and the claimed effect in humans

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Probiotic efficacy

Strain or combination specific – rationale – concept

Human interventions Preclinical data – mechanism

Human interventions

– Prevention/ treatment pharmaceutia

– Risk reduction nutritional modulation – food

Fig. 5.4 Major areas of preclinical and efficacy studies supporting the establishment of health claims for specific probiotics or specific probiotic foods and/or pharmaceutical products.

●

●

(define, for example, the strength, consistency, specificity, dose–response and biological plausibility of the relationship). The quantity of the food/ingredient and pattern of consumption required to obtain the claimed effect. The quantity and the daily portions should be such that they can be reasonably achieved as part of a recommended balanced diet in European countries. Target population for which the claim is intended should be defined and the specific study populations in which the evidence was obtained should be representative of the target population.

5.7

SPECIFIC CHALLENGES FOR PROBIOTICS

5.7.1

Viability

The future of probiotics has some major challenges to overcome. The first very specific challenge for probiotics in the future is assessing viability. The current WHO definition of probiotics defines them as viable food supplements, but viability is defined by most regulatory authorities as culturability. Culturability itself depends on specific media and culture conditions. As demonstrated in human intestinal microbiota assessment studies, only a small part of the intestinal microbiota is culturable. However, this part is viable and will have an effect on human health. This results in a need on the regulatory side to develop new methods of viability assessment (Gueimonde et al., 2004).Such developments have been seen in the animal feed area in Europe (EFSA, 2008).

5.7.2

Clinical studies demonstrating efficacy of probiotics in healthy subjects

Most probiotic studies in humans have been conducted in subjects who have been either ill or critically ill. Therefore, a further challenge is implicit in the form of the EU regulation, where the health claims are designed for normal healthy populations or populations

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at risk of specific disorders. An example is given by the scientific opinion on xylitol and tooth decay. Dental caries and tooth decay are states that face the normal healthy population of Europe. Dental caries and tooth decay are characterized by factors such as the extent of dental plaque and number of caries-causing organisms such as Streptococcus mutans on dental surfaces. An additional factor is acid production by such bacteria and the impact of acid on dental enamels. All these factors can be defined as risk factors in normal healthy populations. Using xylitol, a 5-carbon polyol, in chewing gum has been demonstrated to impact the risk factors and also the end point (dental caries), resulting in fewer missing or damaged dental surfaces after long-term chewing gum use (EFSA, 2008). Another example is plant sterols and stanols, which have been demonstrated in normal populations to reduce elevated blood cholesterol levels. As there is general consensus that high cholesterol is associated with coronary heart disease, the scientific opinion of EFSA has allowed the claim that plant sterols and plant stanols help to lower cholesterol, which is associated with a reduced risk of coronary heart disease. Similar areas should also be identified for probiotic use and may become associated with health claims in the future. Recent examples on risk reduction include atopic eczema (Kalliomäki et al., 2001; Wickens et al., 2008), flu (Leyer et al., 2009) and infections in daycare children (Rautava et al., 2009).

5.7.3

Challenges in regulatory areas

The current health claim legislation varies significantly around the world. Different safety and efficacy requirements exist in the EU, Japan, China, Canada, Brazil, the USA, and other countries. In Europe, the legislation is in the process of being harmonized throughout all member states of the EU. However, differences may still exist, and elsewhere, in spite of Codex work, other forms of regulations are being developed. For safety assessment of probiotics, general guidelines have been issued by several countries. In the EU systematic assessment has resulted in a process called QPS. This assessment has been conducted by the EFSA, which has also published a list of microorganisms with a safe history of use. This list places microorganisms in food into specific categories according to safety and also allows the first screening for food-grade bacteria for probiotic use. In the USA, the GRAS notification exemplifies another system, where the producer submits an expert dossier and opinion on the safety of a particular product (see also Chapter 6). The US Food and Drug Administration then responds by letter indicating either that it sees no concerns or that there may be challenges. If there are no concerns, the probiotic will be placed on a public list with the letter from the FDA describing the action taken (FDA, 2009). For health claims the systems differ significantly from country to country. In the EU the legislation is now clear, whereas in the USA a more complex system exists. However, both systems require proof of health benefits in human studies. In Japan, the FOSHU system also takes into account smaller human studies complemented with studies in laboratory animals. In China, the system is even more complicated, as the provinces may have regulations that differ from those established by the central government (see Chapter 9). Taken together, health claim regulations require a lot of attention and changes are still underway in several countries around the world. However, the basic principles appear to be similar and proof from well-designed human intervention studies will be more important in the future.

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REFERENCES ANVISA (1999a) Resolution No. 18 of April 30, 1999. Basic guidelines for analysis and proof of functional and or health claims on food labels. National Health Surveillance Agency, Ministry of Health, Brasilia, 1999. Available at www.anvisa.gov.br [Accessed 13 October 2009]. ANVISA (1999b) Resolution No. 19 of April 30, 1999. Regulation on procedures for registration of foods with functional and or health claims on their labels. National Health Surveillance Agency, Ministry of Health, Brasilia, 1999. Available at www.anvisa.gov.br [Accessed 13 October 2009]. ANVISA (1999c) Ministério da Saúde. Secretaria de Vigilância Sanitária. Decreto RDC No. 15. Scientific Advisory Commission on Functional Food and New Foods (CTCAF). Available at www.anvisa.gov.br [Accessed 13 October 2009]. ANVISA (2002) Resolução RDC No. 2, de 7 de janeiro de 2002. Regulamento Técnico de Substâncias Bioativas e Probióticos Isolados com Alegação de Propriedades Funcional e ou de Saúde. Agência Nacional de Vigilância Sanitária, Ministério da Saúde, Brasília. Available at www.anvisa.gov.br Brazil (1997) Ministério da Saúde. Portaria No. 1.549 de 17 de outubro de 1997. D.O.U. n. 203, de 21 de outubro de 1997, págs. 23.756 e 23.757. Brazil (1999) Ministério da Saúde. LEI No. 9.782, de 26 de janeiro de 1999. Define o Sistema Nacional de Vigilância Sanitária, cria a Agência Nacional de Vigilância Sanitária, e dá outras providências. Publicado no D.O.U. de 27.01.1999, Seção 1, pág. 1. Chain PSG, Grafham DV, Fullonn RS et al. (2009) Genome projects standards in a new era of sequencing. Science 326:236–237. EFSA (2007) European Food Safety Authority Scientific Committee (EFSA) public consultation on the Qualified Presumption of Safety (QPS) approach for the safety assessment of microorganisms deliberately added to food and feed. Annex 3: Assessment of Gram positive non-sporulating bacteria with respect to a qualified presumption of safety. Available at: www.efsa.europa.eu/en/science/sc_commitee/ sc_consultations/sc_consultation_qps.html. 2007. EFSA (2008) Safety and efficacy of the product Sorbiflore, a preparation of Lactobacillus rhamnosus and Lactobacillus farciminis, as feed additive for piglets Available at http://www.efsa.europa.eu/en/scdocs/ doc/771.pdf EFSA (2009a) Scientific opinion on the substantiation of health claims related to non-characterized microorganisms pursuant to Article 13(1) of Regulation (EC) No. 1924/20061. Available at www.efsa.europa. eu:80/cs/BlobServer/Scientific_Opinion/nda_op_ej1247_art13(1)_non_characterised_microorganisms_ related_claims_en,0.pdf?ssbinary=true [Accessed 5 December 2009]. EFSA (2009b) Opinion of the Panel on dietetic products, nutrition and allergies (NDA) on a request from the Commission related to scientific and technical guidance for the preparation and presentation of the application for authorization of a health claim. Available at www.efsa.europa.eu/EFSA/efsa_locale1178620753812_1178623592448.htm [Accessed 15 November 2009]. EFSA (2009c). Xylitol chewing gum/pastilles and reduction of the risk of tooth decay. - Scientific substantiation of a health claim related to xylitol chewing gum/pastilles and reduction the risk of tooth decay pursuant to Article 14 of Regulation (EC) No. 1924/2006[1] - Scientific Opinion of the Panel on Dietetic Products, Nutrition and Allergies. Available at www.efsa.euoa.eu/EFSA/efsa_locale-11786207 53812_1211902179398.htm [Accessed 5 December 2009]. European Commission (2006) Corrigendum to Regulation (EC) No. 1924/206 of the European Pment ad of the Council of 20 December 2006 on nutritional and health claims made on foods. Official Journal of the European Union. 18.1.2007, EN, p.1–16, L 12/3. Available at http://eur-lex.europa.eu/LexUriServ/ LexUriServ.do?uri=OJ:L:2007:012:0003:0018:EN:PDF [Accessed 26 August 2009]. FAO/WHO (2006) Probiotics in Food. Health and Nutritional Properties and Guidelines for Evaluation. FAO Food and Nutrition paper 85. FDA (2009) Available at www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=grasListing Felber H (1984) Terminology Manual. Paris: UNESCO/Infoterm. Felis GE, Dellaglio F (2007) Taxonomy of Lactobacilli and Bifidobacteria. Curr Issues Intest Microbiol 8:44–61. Gueimonde M, Delgado S, Baltasar M, Ruas-Madiedo P, Margolles A, los Reyes-Gavilan CG (2004) Viability and diversity of probiotic Lactobacillus and Bifidobacterium populations included in commercial fermented milks. Food Res Int 37:839–850.

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Infoterm (2004) 30 years of INFOTERM, the International Center for Terminology. Available at www. infoterm.info/pdf/about_us/30_years_infoterm.pdf [Acessed 28 October 2009]. Kalliomäki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E (2001) Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial. Lancet 357:1076–1079. Lahtinen SJ, Boyle RJ, Margolles A, Frías R, Gueimonde M (2009) Safety assessment of probiotics. In: Charalampopoulos D, Rastall RA (eds) Prebiotics and Probiotics Science and Technology. New York: Springer, pp. 1193–1235. Leyer GJ, Li S, Mubasher ME, Reifer C, Ouwehand AC (2009) Probiotic effects on cold and influenza-like symptom incidence and duration in children. Pediatrics 124:e172–e179. Magalhaes M, Salminen S, Ferreira C, Tommola J (2010) Terminology: Functional foods, Probiotics, Prebiotics, Synbiotics, Health Claims, Sensory Evaluation, Molecular Gastronomy. University of Turku, Funcrtional Foods Forum, Turku, Finland. Available at http://fff2.utu.fi/media/terminology.html Ministero della Salute, Italy (2005) Guidelines for probiotics and prebiotics. December 2005. Mogensen G, Salminen S, O’Brien J et al. (2002) Food microorganisms: health benefits, safety evaluation and strains with documented history of use in foods. Bull IDF 377:4–9. Rautava S, Salminen S, Isolauri E (2009) Specific probiotics in reducing the risk of acute infections in infancy: a randomised, double-blind, placebo-controlled study. Br J Nutr 101:1722–1726. Salminen S, Bouley C, Boutron-Ruault MC et al. (1998) Functional food science and gastrointestinal physiology and function. Br J Nutr 80(Suppl 1):S147–S171. 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. WHO (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Available at www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf [Accessed 3 December 2009]. WHO (2002) Guidelines for the evaluation of probiotics in food. Available at www.who.int/foodsafety/ fs_management/en/probiotic_guidelines.pdf [Accessed 3 December 2009]. Wickens K, Black PN, Stanley TV et al. (2008) A differential effect of 2 probiotics in the prevention of eczema and atopy: a double-blind, randomized, placebo-controlled trial. Probiotic Study Group. J Allergy Clin Immunol 122:788–794.

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Substantiating Health Benefit Claims for Probiotics in the United States

Mary Ellen Sanders

6.1

INTRODUCTION

The United States is a recent frontier experiencing rapid expansion of commercial probiotic products. This has occurred in the absence of a definition of the term “probiotic” in federal or state law. The scientific community has embraced the FAO/WHO definition of probiotic, which is sufficiently broad that probiotics conceivably can be included in multiple regulatory categories depending on the intention of use of the probiotic, including conventional food (including functional foods), infant formula, dietary supplements, and drugs. Other categories include probiotics for animal foods (direct fed microbials) or for use in non-consumed formats such as cosmetics or throat sprays. Each of these categories has different requirements for substantiation of efficacy and safety. Approaches to claim substantiation as dictated by US regulatory authorities for foods and dietary supplements are discussed. Although the criteria for marketing each category of product are clear, the impact of product categories is often misunderstood or not adequately considered when designing human research on probiotics. Regulatory oversight of manufacturer compliance with efficacy and safety regulations for dietary supplement products is uneven, leading to a less than rigorous approach to claim substantiation on products claiming to be “probiotic”.

6.1.1

Probiotics and health benefits

The commercial success of probiotic products is predicated on research substantiating their impact on human health or disease. This research spans many different probiotic strains, research endpoints, study populations, geographical regions, and study designs. Documentation of health benefits through such research provides the basis for marketing claims (ILSI, 2004; Sanders et al., 2005). The type of claim that is allowable depends on the category of product (conventional food, dietary supplement or drug). Since regulatory categories for products marketed in the United States are determined by the intended use of the product, it is possible for a probiotic-containing conventional food, such as a yogurt, to be considered a drug by the Food and Drug Administration (FDA) if it is being marketed with a drug claim or is the subject of research with a drug endpoint. Therefore, probiotic substances (even if commercially available as foods) that are the subject of studies with endpoints such as reduction of incidence of diarrhea or alleviation of antibiotic-associated

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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symptoms may be considered “investigational new drugs” by the FDA. Although much research worldwide on probiotics is in fact targeted at such endpoints (Sanders et al., 2007), there are no probiotic drug products for human use available in the United States. In addition to this complication of distinguishing between probiotics used as foods or drugs, the US regulatory framework must be interpreted with regard to what degree and type of evidence is required to substantiate health benefit claims for probiotics. After laying the groundwork for understanding what probiotics are, this chapter addresses the nature of substantiation of efficacy for probiotic products in the United States. Keep in mind that the term “health claim” has a specific meaning in US regulatory law, and is not just a general term for all types of health benefit claims.

6.1.2

Probiotics: a term often misused

Probiotics are live microorganisms, which when administered in adequate amounts confer a health benefit on the host (FAO, 2001). An expanded discussion of this definition that provides insight into the complexities of probiotics was recently published (Sanders, 2009). There remains no legal definition of the term “probiotic” in the United States. This definition is quite broad – an umbrella definition, in a manner of speaking – and spans many regulatory categories. Within the regulatory framework of the United States, probiotics could conceivably be components of conventional foods, which provide nourishment to the generally healthy population; drugs, which are designed to cure, treat, mitigate or prevent disease; dietary supplements such as capsules or pills, which supplement the diet of the generally healthy population; foods for use for dietary management of medical conditions; genetically modified products; or direct fed microbials, comprising probiotics for animal use. Each of these categories is regulated differently with regard to efficacy, safety and allowable claims; therefore, a probiotic should meet the minimum criteria stipulated by the definition above, in addition to the criteria stipulated by the specific product category. At a minimum, a probiotic must: ● ● ● ●

be alive when administered; have undergone controlled evaluation to document health benefits in the target host; be a taxonomically defined microbe or combination of microbes (genus, species and strain level); be safe for its intended use.

Frustrating to many in the probiotics field is that the term “probiotic” is frequently misused. It is not uncommon for commercial products to use the term in the absence of substantiation of human health benefits or when adequate levels of live microbes are not present. A legitimate probiotic product has health effects documented for the strain or combination of strains present at a specified dose. Therefore, even claims such as “provides live microorganisms that form part of a natural healthy gut flora” or “promotes balance of intestinal microbiota” must be substantiated for the specific strain(s) and dose. In addition to misuse of the term “probiotic” commercially, there are examples of misuse among scientists who use the term to describe bacterial components, dead bacteria or bacteria with uncharacterized health effects in humans. It is incumbent upon all in the probiotic field – industry, scientists, journal editors, and regulatory agencies – to use the term “probiotic” only when minimum criteria have been met. In this way, the term will retain some significance and consumer confidence can be enhanced.

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6.2

HEALTH BENEFIT CLAIMS ALLOWABLE IN THE UNITED STATES

Since there are no probiotic products in the United States that are marketed as drugs, the focus of this discussion will be on conventional foods and dietary supplements. Claims allowable on food and dietary supplement labels include health claims, nutrient content claims, and structure/function claims. This discussion will not consider nutrient content claims, as they are not relevant to probiotic-specific claims. Depending on the type of claim, the responsibility for ensuring the validity of these claims rests with either the manufacturer or the FDA.

6.2.1

FDA and FTC standards

In the United States, the FDA and Federal Trade Commission (FTC) have jurisdiction over product benefit communications. The FDA oversees claims made on product labeling whereas the FTC oversees claims made in advertising. The standards enforced by these agencies are worded differently but are complementary. The FDA standard is that claims must be truthful and not misleading, as would be interpreted by a reasonable consumer. This is deemed to be consistent with the FTC standard of “competent and reliable scientific evidence”. Furthermore, the FDA and the FTC expect that manufacturers can substantiate all reasonable interpretations of the claims.

6.2.2

Structure/function claims

Structure/function claims may not explicitly or implicitly link the claimed benefit to a disease, but must focus on “maintaining” or “supporting” particular body structures or functions, or on general well-being. To make a structure function claim on a food, the FDA stipulates that the active ingredient must have the following characteristics. ●

● ● ●

“Generally recognized as safe” (GRAS), having been judged by experts in the field as presenting a reasonable certainly of no harm under conditions of use, or it must be an approved food additive. The claims must be truthful and not misleading, based on appropriate scientific criteria. The active ingredient must be contained within a “food”. A “food” is defined as something that is consumed “primarily for taste, aroma, or nutritive value”. The benefit should derive from the nutritive value of the active ingredient, although the FDA has exercised some degree of flexibility with this requirement. This seems to be due, at least in part, to the recognition that certain beneficial food constituents, such as fiber, do not fit this description. The qualifier “primarily” in the definition of food also supports this position.

To make a structure function claim on a dietary supplement the criteria are much the same. However, the benefit need not be derived from the nutritive value of the active ingredient. No FDA approval is required for structure/function claims on foods or dietary supplements, but if making such a claim on a dietary supplement, the FDA must be notified within 30 days of marketing. Furthermore, the dietary supplement label must indicate that “this statement has not been evaluated by the FDA. This product is not intended to

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diagnose, treat, cure, or prevent any disease”. If marketing a food with a structure/function statement, no FDA notification is required and the disclaimer need not be displayed on the label.

6.2.3

Health claims

Health claims describe a relationship between a food, food component, or dietary supplement ingredient to a disease or health-related condition (for further information see www.fda.gov/Food/LabelingNutrition/LabelClaims/ucm111447.htm). In practice, the FDA considers that these claims be limited to reduction in the risk of incurring the disease or condition by the currently healthy population. Although not specifically codified, in practice the FDA also considers that only chronic diet-related diseases are appropriate targets for health claims for foods (see list of approved health claims below). In a letter by Joseph Levitt, Director CFSAN, May 26, 2000, it was made clear that the FDA would not consider the relationship between a food and an existing disease as an appropriate health claim. This letter was in response to a request that FDA consider a health claim for saw palmetto extract to improve urine flow, reduce nocturia and reduce voiding urgency associated with mild benign prostatic hyperplasia. The Director indicated: After thoroughly reviewing the relevant statutory provisions and legislative history, FDA’s past statements on this issue, and the comments we have received on it, we have concluded that claims about effects on existing diseases do not fall within the scope of the health claim provisions in 21 U.S.C, § 343(r) and therefore may not be the subject of an authorized health claim. We have come to this conclusion after carefully considering the language and structure of the NLEA and the FFDCA as well as the public health importance of ensuring that claims to treat disease be substantiated by the appropriate level of evidentiary support to provide protection for patients who are already sick and, therefore, especially vulnerable.

Unlike structure/function claims, health claims must be approved by the FDA or a scientific body of the US government or the National Academy of Sciences. Until 2003, the standard of evidence for an authorized health claim was that there was significant scientific agreement that the relationship described by the claim was accurate. Subsequently, “qualified health claims” were instituted, which are claims where the quality and strength of the scientific evidence falls below significant scientific agreement standard. Such health claims must be qualified to assure accuracy and non-misleading presentation to consumers. Health claims meeting the significant scientific agreement standard are shown below. No such claims on probiotics (or prebiotics) have been established (for further information see www.fda.gov/Food/LabelingNutrition/LabelClaims/HealthClaimsMeeting SignificantScientificAgreementSSA/default.htm): ● ● ● ● ● ● ●

calcium, vitamin D, and osteoporosis; dietary lipids (fat) and cancer; dietary saturated fat and cholesterol and risk of coronary heart disease; dietary non-cariogenic carbohydrate sweeteners and dental caries; fiber-containing grain products, fruits and vegetables and cancer; folic acid and neural tube defects; fruits and vegetables and cancer;

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fruits, vegetables and grain products that contain fiber, particularly soluble fiber, and risk of coronary heart disease; sodium and hypertension; soluble fiber from certain foods and risk of coronary heart disease; soy protein and risk of coronary heart disease; stanols/sterols and risk of coronary heart disease.

Qualified health claims are assigned a rank by the FDA based on the strength of the data supporting the claim. Ranks from A through D are assigned, with A representing significant scientific agreement and D indicating that there is little scientific evidence supporting the claim. A summary of qualified health claims, which also does not contain any claims on probiotics or prebiotics, can be found at www.fda.gov/Food/LabelingNutrition/ LabelClaims/QualifiedHealthClaims/ucm073992.htm. The time and cost of petitioning for health claims equate to a substantial investment by the petitioning company, especially since health claims are not specific to the petitioning company’s product. Furthermore, consumer research conducted by the International Food Information Council (www.ific.org/research/qualhealthclaimsres.cfm) has suggested that consumers perceive the scientific credibility of structure/function claims as equally compelling as an unqualified health claim. This calls into question the value to a company of pursuing an authorized health claim.

6.2.4

Medical food claims

According to the FDA, a medical food is defined as “a food which is formulated to be consumed or administered enterally (i.e., via the digestive system) under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation” [21 U.S.C. 360ee(b)(3)]. The explicit requirement that medical foods function by a nutritional mechanism indicates that this category is not appropriate for probiotic claims.

6.3

SUBSTANTIATION OF HEALTH BENEFIT CLAIMS FOR PROBIOTICS

6.3.1

Overriding considerations

In the United States, the path to substantiation of claims is specific to the regulatory category of the product (food, dietary supplement or drug). There are no probiotic-specific regulations or guidelines. However, being live microorganisms, probiotics present some unique challenges both in substantiation and product marketing. The process of assessing safety prior to conducting research on specific strains requires that characteristics such as impact on colonizing microbiota and immune system development, especially in naive or disrupted subjects, be considered, in addition to any toxicological or opportunistic pathogenic properties of the strains. Keeping probiotics alive through the end of shelf-life of the product can also be difficult, depending on the physiological nature of the probiotic. What might be less evident is that product categories can also impact research. Table 6.1 outlines some specific issues for efficacy substantiation data related to food, dietary supplements

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Table 6.1 Considerations for efficacy substantiation for probiotic products in the United States. Conventional foods (including functional foods)

Dietary supplements

Drugs

Product population

Intended to nourish the generally healthy population

Intended to supplement the diet of the generally healthy population

Intended to cure, treat, mitigate, prevent or diagnose disease

Study population

Should be healthy or representative of the general population; can be an at-risk population

Should be healthy or representative of the target population; can be an at-risk population

Healthy, at-risk or diseased population

Active ingredient

Substance that has its effect through nutritive propertiesa

Functional substance, not limited to nutritive value

Parameters are not limited

Endpoints

Valid biomarkers

Valid biomarkers

Impact on disease incidence, duration, symptoms, progression

Clinical endpoints that provide insight into health promotion or supporting normal physiological and structural parameters Reducing the risk of disease

Clinical endpoints that provide insight into health promotion or supporting normal physiological and structural parameters Reducing the risk of disease

Reduction of risk of disease “health claim”, with FDA or authoritative body approval Structure/function claimc

Reduction of risk of disease “health claim”, with FDA or authoritative body approval Structure/function claim, with FDA notification no later than 30 days after marketing

Type of efficacy claim that is possibleb

Disease claim: substance can cure, treat, mitigate or prevent disease

Currently, there appears to be a broad interpretation by the FDA of what constitutes nutritive properties. Not considered here are nutrient content claims (see www.cfsan.fda.gov/~dms/hclaims.html for description of all claim categories). c Structure/function claims for conventional foods relate to effects derived from nutritive value, while such claims for dietary supplements may relate to nutritive as well as non-nutritive effects. However, the distinction between these effects is not clear. a b

and drugs. A research path to new drug assessment is clear. However, studies designed with endpoints that are considered appropriate targets for drugs by the FDA are very frequently conducted for probiotics that are currently sold as dietary supplements or foods, or which are intended to be used as such. When public funds are sought for such studies from the National Institutes of Health for example, the FDA might request that an investigational new drug application be filed (for further information see www.fda.gov/Drugs/ DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ApprovalApplications/ InvestigationalNewDrugINDApplication/default.htm). It is also not clear what constitutes the difference between reducing the risk of disease (an acceptable endpoint for foods) and preventing (acceptable only for drugs) disease. It is clear that vaccines are considered

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disease prevention. It also seems clear that if the study population already has active disease, substances targeted toward symptom management or curing the disease are considered by the FDA to be drugs. But can probiotics be evaluated in healthy children in a daycare setting to “reduce the risk of developing diarrheal or respiratory illnesses”? Currently, such use of probiotics appears to be seen by the FDA as a drug use. It is evident that foods are traditionally used by people managing health conditions, for example hot chicken soup for symptoms of a cold, saltine crackers for controlling nausea, yogurt for controlling side effects of antibiotics, or cranberry juice for managing urinary tract infections. There are abundant examples of published research on probiotics being used as part of the diet of people with health concerns, with positive outcomes. Yet there does not appear to be a non-drug path within the current FDA approach to probiotics for scientific validation of a product for such use. This situation is likely encouraged by the FDA view that probiotics, as live microorganisms, fall under the jurisdiction of FDA’s Center for Biologics Evaluation and Research (CBER), which also regulates vaccine development. Similar to all other substances in its jurisdiction, CBER seems to view probiotics as drugs, almost regardless of endpoint of research or intent of use. Routing probiotic research proposals that are focused on studies of healthy populations through FDA’s Center for Food Safety and Applied Nutrition (CFSAN) might result in a different perspective on the intended use of these substances. In the event drug-endpoint research has been conducted and is available on a probiotic, it is unclear to what extent it can be used to substantiate claims made for a food or dietary supplement product. Furthermore, translating research with specific clinical endpoints into acceptably worded structure/function claims can be a frustrating exercise. The results of a well-conducted study on managing antibiotic-associated diarrhea might result in a dietary supplement product labeled as “promotes intestinal health”. Such a claim tells the consumer little about how the product was evaluated or what specific benefits might be achieved by taking it. Furthermore, such research may not be viewed by the FDA as being relevant for substantiation of a structure/function claim for a food or dietary supplement. The body of human efficacy data suggests that probiotics can function in a variety of ways. The path to developing a research dossier which provides substantiation for a probiotic food was outlined in an FAO/WHO (2002) document. These guidelines describe a general approach for evaluating the efficacy and safety of probiotics. This document, along with information that has become available since its publication, indicates that the following steps are key to establishing a validated probiotic strain.

Thorough identification of the test product ● ● ●

Genus and species determination of the probiotic strain based on most current genetic methods. Generation of strain-specific identification, with patterns from appropriate molecular techniques (reproducible genetic method). Description of the food or carrier matrix that serves to deliver the probiotic strain.

Deposit in an international culture collection The probiotic strain should be deposited in an international culture collection so that a historical reference substance can be available and to enable research to be repeated by independent investigators.

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Thorough safety assessment (Vankerckhoven et al., 2008; Sanders et al., 2010) ● ● ● ● ● ● ● ●

Description of use (dose, format, stability). Validation that the product is manufactured under good manufacturing practices, specific for the product category. Survey of literature to determine to what extent the strain, species or genus was involved in adverse events. Physiological and genetic capacity for toxic activity. Physiological and genetic capacity for pathogenic/opportunistic pathogenic activity. Genetic stability. Presence of transferable antibiotic resistance markers. Review of data by regulatory authority or a panel of experts qualified in the field to evaluate the safety of the substance, depending on the product category and requirements.

Manufacture under current good manufacturing practice standards The production of probiotics must comply with the standards of good manufacturing practice. Efficacy assessment This will be specific for the particular effects being targeted. The traditional insistence that probiotics be characterized with regard to source of isolation (human, animal, environmental, food, etc.), acid resistance, bile resistance and other such traits may not be relevant for specific applications, and therefore cannot be considered essential. Only in vitro characteristics that are confirmed to be related to in vivo functionality are useful. ● ● ●

Valid in vitro assessments, as dictated by specific probiotic strain and intent of use. Valid animal studies, which might be useful for both safety assessment, mechanistic studies and suggestions of endpoints to confirm in human studies. Human efficacy studies (see below for further discussion).

Product labeling (on product label or in accompanying information on product website) ● ● ● ● ● ● ● ●

Genus, species and strain of each strain included in the product. Level of each strain of probiotic microbes present in the product, through the end of shelf-life. Expiration date. Dose or servings to consume for labeled effects. Health benefits. Proper storage conditions. Company contact number for answering questions and reporting any problems with the product. Product website URL.

Post-market surveillance The product should undergo full post-marketing surveillance.

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6.3.2

Specific issues related to human efficacy studies

The Dietary Supplement Health and Education Act of 1994 requires that a manufacturer of a dietary supplement making a structure/function or general well-being claim have substantiation that the claim is truthful and not misleading. The FDA acknowledges that adequate substantiation is important if consumer confidence is to be maintained in the dietary supplement product category. Guidance documents released by the FDA gives insight into FDA interpretation of what constitutes “substantiation” for dietary supplement claims (FDA 2008) and health claims (FDA 2009), although this guidance is not binding and alternative, scientifically sound approaches will be considered by the FDA. Although perhaps not rigorously enforced, the FDA advises that claims must be substantiated with “competent and reliable scientific evidence”, which include a reasonable assessment of: ● ● ● ●

the meaning of the claim being made; the relationship of the evidence to the claim; the quality of the evidence; the totality of the evidence.

No similar guidance document is available for substantiation of structure/function claims for foods, although it is reasonable to conclude that the four principles above would also relate to substantiation of claims for foods. An article titled “Is my yogurt lying?” provides a legal opinion on what constitutes legitimate substantiation for such claims on foods in the United States (Satine, 2008). The body of evidence assembled to substantiate health benefit claims must be scientifically sound, but no general rule has been issued by the FDA indicating how many studies or what combination of types of evidence are sufficient to support a claim. However, certainly, substantiation of the benefit in humans using validated biomarkers or clinical endpoints is essential. The replication of research results in independently conducted studies makes it more likely that the totality of the evidence will support a claim. If conflicting studies exist, there should be a plausible explanation for the disparate results. The product tested should mimic the marketed product with regard to route of administration (which would be oral for all foods or supplements) and dose or number of servings needed to elicit the effect. A hierarchy of types of evidence has been offered by the FDA, with randomized, controlled, parallel intervention trials topping the list. A crossover design should include randomization of order of product administration and provide effective washout periods. The FDA recognizes that sometimes with studies on foods a blinded placebo is not easily obtained, but proper controls are essential and double-blinding is preferred. Less convincing, but supportive, evidence can be presented from case–control studies and case reports. Attention to conducting a high-quality study with proper study design, execution, data analysis and reporting is important. Launching a study to substantiate the efficacy of a probiotic product is a complicated undertaking. In addition to addressing the issues above, it is often difficult to determine both the appropriate endpoints for human studies and the study population. There are few efficacy biomarkers that are widely considered to be valid indicators of health. Markers such as low-density blood lipids are acceptable indicators of coronary heart disease, bone density is considered a valid predictor of osteoporosis, and blood pressure is a gold standard biomarker

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for hypertension. However, currently no consensus exists on valid biomarkers for optimal immune function or gut microbiota composition, common endpoints for probiotic studies.

6.3.3

Key considerations for probiotic efficacy substantiation

Substantiation for probiotic efficacy provides some unique challenges, not the least of which is that one of the key postulated mechanisms of probiotic action – having a positive impact on colonizing microbiota – is largely unproven. The true nature of native microbe populations and activities is not yet understood, let alone how those populations and activities are altered by adding exogenous microbes to the system. However, resources being devoted globally to characterize the human microbiome may soon make that information available (Mullard, 2008). Probiotic researchers will surely be quick to build on this foundation of scientific discovery. Yet, understanding mechanisms of action of probiotics is key to understanding what benefits they can impart to whom and to what degree. Other important research questions that should be considered as part of an overall process of understanding health benefits include the following. ●

●

●

●

What is the role of the delivery matrix in probiotic function (Sanders & Marco, 2010)? Does it matter if probiotics are delivered in a food, in a dried form or with other functional ingredients? Are the physiological, metabolic or genetic characteristics that impart the benefit of the probiotic strain-specific or can they be generalized to other strains of the same species or genus? What characteristics are important in the consuming host that lead to improved heath upon probiotic use? How do we define responders and non-responders in human trials? Is there a “responsive” microbiological make-up of the consumer? Is there a “responsive” host genotype? What role does physiology of the microbe play in its ability to mediate any specific functional endpoint? ° Does it need to be alive at site of action? ° Do microbes at the end of shelf-life have the same physiological capacity as microbes at the beginning of shelf-life? ° How do conditions of growth, concentration, stabilization and storage impact function?

6.4

BRIDGING THE GAP BETWEEN THE US CONSUMER, PROBIOTIC SCIENCE AND COMMERCIAL PRODUCTS

The commercial reality of probiotics in the United States is that products marketed in the United States as “probiotic” may not be probiotic at all. Many products appear to be marketed with no evident research supporting claims. Claims such as “strengthens immune function”, “improves the balance of intestinal microbes” or “supports digestive health” are common on products or product websites, but often no published research studies on the specific products are offered as evidence in support of the claims. It seems that product

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Table 6.2 Audiences for health benefit claims made for probiotic products. Audience

Question the claim must address

Possible outcomes if claims are “faulty”

Regulatory agencies, including the FDA and FTC

Is the claim allowable under applicable law?

Warning letters from the FDA/ FTC; product seizure

Consumers

Is the claim meaningful to them? Are the benefits substantial enough to justify the price?

Eroding of consumer confidence

Consumer watchdog organizations

Is the claim misleading to consumers resulting in unfair company profit?

Widespread publication of failure to validate claims, eroding of consumer confidence

Competitors/National Advertising Division of the Better Business Bureau

Do your competitors consider your claims fair in the eyes of consumers?

Healthcare professionals

What evidence-based recommendations can be made?

Failure to embrace probiotic options in healthcare recommendations, either for specific product or category as a whole

Litigious elements in society

Is the claim convincingly substantiated or can money be made by bringing a lawsuit?

Lawsuit

manufacturers consider that such claims are “self-evident” and are justified simply by providing live Lactobacillus or Bifidobacterium in a supplement or food. The weight of the scientific community would disagree, however, as the current consensus seems to be that unless otherwise demonstrated, probiotic effects must be considered strain-specific, as reflected in the FAO/WHO guidelines document. One possible exception to this is the ability of most tested strains of yogurt starters (Lactobacillus bulgaricus and Streptococcus thermophilus) to deliver lactase, improving digestion of lactose in lactose maldigesters. Several professional organizations have sought to provide guidance to consumers and healthcare professionals in choosing probiotic products, and the results of their efforts are noteworthy (Fig. 6.1) (American Gastroenterological Association, 2008; World Gastroenterology Organization, 2008). In the end, crafting a health benefit statement must be science-driven and then can be marketing-refined. However, it is useful to be aware that the “audiences” for such statements are quite diverse. Any claims made on a product, or in advertising of this product, can be judged by many components of society (Table 6.2).

6.5

CONCLUSIONS

In the United States, the market potential for probiotics has been growing. The American public has demonstrated its interest in products targeted toward digestive health and the concept that bacteria can be good for you seems to be taking hold. The scientific foundation for probiotic research has expanded tremendously over recent years, providing impetus for this growing category. But it is remarkable how much this literature base contracts

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Probiotics: A Consumer Guide for Making Smart Choices Developed by the International Scientific Association for Probiotics and Prebiotics (www.isapp.net)

A P

The concept of probiotics* has been around for over 100 years, but scientists are just starting to understand their role in maintaining health, regulating the immune system and managing disease. There are hundreds of probiotic products available and an overwhelming amount of information for consumers to sort through. The International Scientific Association for Probiotics and Prebiotics (ISAPP) has developed the following key criteria to help consumers find a credible probiotic product. P

Not all probiotics are created equal ● Different strains of even the same species can be different. A probiotic is defined by its genus (e.g. Lactobacillus), species (e.g. rhamnosus) and strain designation (often a combination of letters or numbers). The concept of a bacterial “strain” is similar to the breed of a dog – all dogs are the same genus and species, but different breeds of dogs have different attributes and different breeds are good for different tasks. So different strains of even the same probiotic species may be different from each other. You cannot assume that different strains of the same species will have the same effects. The names of probiotics sound complicated, but they are important to connecting the specific probiotic strain to the strain’s published scientific literature. ● What about trademarked names? Often, product manufacturers will create a consumer-friendly, trademarked (™) or registered trademark (®) name for the strain in their product. It is just an “alias” for the probiotic strain. These names are not scientific names and they do not reflect product quality. ● Food or supplement – which is better? Probiotics can be found in various foods, yogurts, and supplements. Probiotic content is generally more important than the way in which you consume them. Probiotics must be tested In humans and shown to have health benefits ● Claims - what do they mean? Most probiotics are sold as dietary supplements or ingredients in foods. Their labels cannot legally declare that the probiotic can cure, treat or prevent disease. Claims which relate the product to health are allowable. Any claim made on a product, no matter how general, is supposed to be truthful and substantiated – even though FDA approval is not required to make these claims. ● “Clinically proven.” You might have to do some homework. Product claims of health benefits must be based on sound research done on the particular probiotic. The product should contain the specific strain(s) of bacteria at the same levels as used in published research. The studies should be performed in humans and published in peer-reviewed, reputable journals. Check product websites to see study results. Your pharmacist or healthcare provider should be able to help you sort through the scientific language. ● Just because it says “probiotic,” doesn’t mean it is a probiotic. Some products labeled “probiotic” do not have clinically validated strains or levels in the product. Get your doctor’s OK. Probiotic foods and dietary supplements should be safe for the generally healthy population to consume. But, consult a physician before administering probiotics to infants or to people with compromised immune systems or other major underlying illnesses. Read “Warning” and “Other Information” on the product package and be aware of any expected symptoms or side effects. Choose a product at the right quantity ● What is the minimum CFU I should look for? Probiotics are measured in “CFU.” CFU stands for colony forming units, which is the measure of live microbes in a probiotic. CFU amount should be the same as that shown to be effective in clinical studies. More CFUs does not necessarily mean better. ● Different probiotics have been shown to be effective at different levels. It is not possible to provide one count for all “probiotics.” Scientific literature has documented health benefits for products ranging from 50 million to more than 1 trillion CFU/day. Pick a product from a trusted manufacturer. A responsible manufacturer will make sure its probiotic product has the same strain(s) and is as potent through the end of shelf life as what was used in clinical studies. Here’s what the label should tell you: ● Strain. What probiotic is inside? ● CFU. (Colony Forming Units). How many live microorganisms are in each serving? When does it expire? Packaging should ensure an effective level of live bacteria through the “best by” or expiration date. ● Suggested serving size. How much do I take? ● Health benefits. What can this product do for me? ● Proper storage conditions. Where do I keep it to ensure maximum survival of the probiotic? ● Corporate contact information. Who makes this product? Where to do I go for more information? *Probiotics are defined by the Food and Agriculture Organization of the United Nations as “live microorganisms, which when administered in adequate, amounts confer a health benefit on the host.” http://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf .

Fig. 6.1 Consumer guidelines for choosing probiotics products as prepared by the International Scientific Association for Probiotics and Prebiotics (ISAPP, 2009). Reproduced with permission.

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when searching for specific evidence for a specific claim for a specific strain or strain combination in specific products at specific doses. Often only one or a couple of papers might remain that can be considered relevant to support a health benefit claim. Added to this situation is a regulatory framework which speaks to rigorous approaches to efficacy substantiation for structure/function claims but does not effectively enforce them. Furthermore, the FDA’s rigid interpretations stymie efforts to scientifically investigate the value of dietary supplements or foods with probiotics in the dietary management of acute health conditions (such as antibiotic-associated side effects or recurring vaginal infections) or to improve the ability of healthy people to stay healthy (as indicated by reduced incidence of common infectious diseases) without need for physician oversight or nutritional mechanism. It is incumbent upon all involved in the probiotic field to recognize that science-based claim substantiation is the cornerstone for a sustained industry. Working together to support research which addresses critical unknowns in the field will do much to expand opportunities and understanding.

REFERENCES American Gastroenterological Association (2008) Probiotics. What are they and what they can do for you, a patient’s guide from your doctor and the AGA. Available at http://www.gastro.org/patient-center/ diet-medications/probiotics [Accessed June 8, 2009]. Food and Agriculture Organization of the United Nations (FAO) (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria, Available at www.who.int/ foodsafety/publications/fs_management/en/probiotics.pdf Food and Agriculture Organization of the United Nations (FAO) (2002) Guidelines for the evaluation of probiotics in food. Available at ftp://ftp.fao.org/es/esn/food/wgreport2.pdf Food and Drug Administration (FDA) (2008) Substantiation for dietary supplement claims made under section 403(r) (6) of the Federal Food, Drug, and Cosmetic Act. Available at http://www.fda.gov/Food/ GuidanceComplianceRegulatoryInformation/GuidanceDocuments/DietarySupplements/ucm073200.htm Food and Drug Administration (FDA) (2009) Evidence-based review system for the scientific evaluation of health claims – final. Available at www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/ GuidanceDocuments/FoodLabelingNutrition/ucm073332.htm International Life Sciences Institute (ILSI) Europe (2004) Process for the assessment of scientific support for claims on food (PASSCLAIM). Available at http://www.ilsi.org/Europe/Publications/E2005Pro Asse.pdf International Scientific Association for Probiotics and Prebiotics (ISAPP) (2009) Probiotics: a consumer guide for making smart choices. Available at www.isapp.net/docs/Consumer_Guidelines-probiotic.pdf [Accessed June 8, 2009]. Mullard A (2008) Microbiology: the inside story. Nature 453:578–80. Sanders ME (2009) How do we know when something called “probiotic” is really a probiotic? A guideline for consumers and healthcare professionals. Functional Food Rev 1:3–12. Available at http://journals.bcdecker. com/pubs/FFR/volume%2001,%202009/issue%2001,%20Spring/FFR 2009 00002/FFR 2009 00002.pdf Sanders ME, Marco M (2010) Food formats for effective delivery of probiotics. Annu Rev Food Sci Technol 1:65–85. Available at http://arjournals.annualreviews.org/eprint/k7FW6TJKtWn27zBIBrRr/full/10.1146/ annurev.food.080708.100743 Sanders ME, Tompkins T, Heimbach JT, Kolida S (2005) Weight of evidence needed to substantiate a health effect for probiotics and prebiotics: regulatory considerations in Canada, E.U., and U.S. Eur J Nutr 44:303–310. Sanders ME, Gibson GR, Gill H, Guarner F (2007) Probiotics in food: their potential to impact human health. Council for Agricultural Science and Technology (CAST), Issue Paper 36, CAST, Ames, Iowa. Available at www.cast-science.org/displayProductDetails.asp?idProduct=144. Sanders ME, Akkermans LMA, Haller D et al. (2010) Assessment of probiotic safety for human use. Gut Microbes 1:1–22. Available at http://www.landesbioscience.com/journals/gutmicrobes/article/12127/

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Satine LA (2008) Is my yogurt lying? Developing and applying a framework for determining whether wellness claims on probiotic yogurts mislead. Food and Drug Law Journal 63:537–577. 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. World Gastroenterology Organization (2008) World Gastroenterology Organization Practice Guideline on Probiotics and Prebiotics. Available at www.worldgastroenterology.org/assets/downloads/en/pdf/ guidelines/19_probiotics_prebiotics.pdf [Accessed June 8, 2009].

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7

Health Claims and Dietary Guidance in the United States: Case “Reduced Cardiovascular Disease Risk”

Alice H. Lichtenstein

7.1

INTRODUCTION

The general area of regulating health claims and crafting dietary guidance is challenging. From a public health perspective, it is important for individuals to receive accurate information about a wide range of topics and to have systems in place for communicating those messages in a timely and unambiguous manner. From a technical perspective, it is critical to have adequate evidence for use in crafting accurate recommendations of broad public health significance. Numerous approaches are used to formulate, adjudicate and approve health claims. Each country or economic unit has different regulations for the actual mechanics of this process and distinct approaches to providing dietary guidance. Their features are unique and specifically designed to be consistent with their mandate and intended use of the information. This chapter is limited to the principles of health claims related to a food, food component or dietary ingredient (but not dietary supplement), and dietary guidance currently in use in the United States, with specific emphasis on cardiovascular disease. With certainty, the field of food and nutrition is evolving rapidly, as are the systems used to regulate health claims and disseminate nutrition-related information. Regardless of the form health claims and dietary guidance take, systems must adapt quickly to be able to respond to the emerging science and provide accurate advice.

7.2

TYPES OF HEALTH CLAIMS

In the United States, the Food and Drug Administration (FDA) has jurisdiction for health claims involving food, with the exception of advertisements, for which the Federal Trade Commission (FTC) has jurisdiction. There are three major FDA categories of statements about food, food component or dietary ingredient: health claims, structure/function claims, and nutrient content claims. This chapter deals with the general rules concerning health claims and no probiotic examples are included. However, the legislation also applies to probiotics. 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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7.2.1

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Definition

By definition, a “health claim” has two parts: (1) a food, food component or dietary ingredient and (2) a disease or health-related condition the food, food component or dietary ingredient is intended to mitigate. General statements that go beyond foods, food components or dietary ingredients and refer to dietary patterns or categories of food, for example “balanced diets” or “cereal products”, do not come under the category of heath claims but under the category of dietary guidance (discussed in the second half of this chapter). The implicit assumption of a health claim is that, with regard to the current state of knowledge, they are accurate and non-misleading. It is the responsibility of the FDA to ensure this is the case. The FDA defines a health claim as “any information on the food package that expressly or by implication claims that a relationship exists between the presence or level of a substance in the food and a disease or health-related condition.” It is important to note the distinction between an FDA-sanctioned health claim and other statements. Health claims can only refer to risk reduction for a specific disease. In no way can they imply the intent to diagnose, cure or treat a disease. In general, health claims are intended for a broad base (e.g. general public) or a subgroup of the general public (e.g. women of childbearing age). Prior to use, all health claims on packaged foods undergo an extensive review for approval by the FDA. The two current FDA-sanctioned categories of health claims are authorized health claims and qualified health claims. The FDA conducts systematic reviews of the literature prior to ruling on petitions submitted by manufacturers for a health claim. In the case of either type of health claim, after the data have been reviewed by the FDA, the proposed health claim is published in the Federal Register and there is a 90-day open comment period that is provided for industry, government, health advocacy organizations, and the general public to submit comments. At the end of that period, the comments are taken into consideration by the FDA and they issue a final ruling on the health claim.

7.2.2

Authorized health claims

Authorized health claims are approved on the basis of significant scientific agreement. Significant scientific agreement is determined by the presence of an adequate amount of consistent data, for the most part from randomized controlled trials, to establish the efficacy of the specific health claim. The decision is made on the basis of the amount of data and the degree to which there is general agreement among the available data that the claim is valid.

7.2.3

Qualified health claims

As the name implies, qualified health claims refer to health claims where some but not all of the data support efficacy. Hence, qualified health claims do not meet the standard for significant scientific agreement. By definition, these claims are not as strong as authorized health claims because the data to support the statements are weaker. Qualified health claims contain a qualifying statement or disclaimer associated with the claim. In general, there are three levels of qualified health claims: strongest, indicating that although there is some scientific evidence to support the statement, the evidence is not conclusive; medium, indicating that although there is some scientific evidence to support the statement, the FDA has

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Box 7.1 ● ● ● ●

Examples of structure/function claims

Diets low in saturated fat may help maintain normal cholesterol levels Diets low in sodium may reduce the risk of high blood pressure Diets high in calcium may reduce the risk of osteoporosis Diets high in fiber may provide relief of occasional constipation

determined the evidence is limited and not conclusive; and lastly, weakest, indicating that although there is some scientific evidence to support the statement, the FDA has determined that there is little scientific evidence to support the claim.

7.2.4

Structure/function claims

In 1994, the Dietary Supplement Health and Education Act (DSHEA) established a regulatory process to govern structure/function claims for nutrients or dietary ingredients. Structure/function claims must have two components: (1) a substance to which the claim is attributable, and (2) a disease or health-related condition its intake mitigates. Statements that address the role of a specific substance must be within the context of maintaining health rather than curing or treating a disease. Hence, these claims describe the role of a nutrient or dietary ingredient that affects normal structure or function in humans. Structure/ function claims cannot explicitly or implicitly relate the relationship to a disease or healthrelated condition. These claims may specify a benefit associated with a nutrient deficiency symptom or reduced risk for a disease. In marked contrast to health claims, structure/function claims are not pre-approved by the FDA. Examples of structure/function claims specifically related to cardiovascular disease appear in Box 7.1.

7.2.5

Nutrient content claims

In 1997, the Nutrition Labeling and Education Act (NLEA) provided a process to allow the use of label claims that specifically refer to the level of a nutrient in a food, otherwise termed “nutrient content claims”. The level of a nutrient can be described in terms such as “free” and “low”. Alternatively, levels of a nutrient can be described in terms relative to another food such as “reduced” or “light (lite)”. Box 7.2 contains some examples of standards for nutrient content claims. A food can be characterized as being “healthy” if it meets the following criteria: low in fat, saturated fat, cholesterol and sodium and if it provides at least 10% of one or more of vitamins A or C, iron, calcium, protein, or fiber. A meal-type product (frozen entrees) can be characterized as being “healthy” if it meets the following criteria: low in fat, saturated fat, cholesterol and sodium and provides 10% of two or three of vitamins A or C, iron, calcium, protein or fiber, in addition to meeting the other criteria. NLEA also provided a mechanism for stating the level of a nutrient on a food label. The level cannot be qualified with a term such as “only” or “low” alone if no additional information is provided about the comparison food. Most nutrient content claim regulations apply to those nutrients or dietary substances that have an established Daily Value. A Daily Value can be

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Box 7.2 Standards for some nutrient content claims Free: none, trivial or physiologically inconsequential ● Fat-free: less than 0.5 g per serving ● Saturated fat: less than 0.5 g per serving ● Trans fat: less than 0.5 g per serving ● Cholesterol: less than 5 mg per serving ● Sodium: less than 5 mg per serving ● Sugars: less than 0.5 g per serving ● Calories: fewer than 5 calories per serving Low: low enough so frequent consumption is unlikely to result in exceeding dietary guidelines ● Low-fat: 3 g or less per serving ● Low-saturated fat: 1 g or less per serving ● Low-sodium: 140 mg or less per serving ● Very low sodium: 35 mg or less per serving ● Low-cholesterol: 20 mg or less and 2 g or less of saturated fat per serving ● Low-calorie: 40 calories or less per serving Lean and extra lean: fat content of meat, poultry, seafood ● Lean: less than 10 g fat, 4.5 g or less saturated fat, and less than 95 mg cholesterol per serving ● Extra lean: less than 5 g fat, less than 2 g saturated fat, and less than 95 mg cholesterol per serving Reduced ● Product contains at least 25% less of a nutrient or calories than a comparable regular or reference product Less (fewer) ● Product contains at least 25% less of a nutrient or calories than a reference food (e.g. pretzels compared to potato chips) Light (lite) ● Fat: contains one-third fewer calories or half the fat of the reference food ● Sodium: content has been reduced by at least 50% Healthy ● Low in fat, saturated fat and cholesterol, and contain less than 360 mg per serving of sodium ● Single food: must provide at least 10% of one or more of vitamins A or C, iron, calcium, protein, or fiber ● Meal-type product (frozen entrees): must provide 10% of two or three of these vitamins or minerals or of protein or fiber, in addition to meeting the other criteria. The sodium content cannot exceed that for individual foods and 480 mg per serving for meal-type products

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either a Reference Dietary Intake or Reference Dietary Value. The Reference Dietary Intake is the standard value adopted by the FDA for an individual nutrient based on the highest value of the 1968 Recommended Dietary Allowances for any age or sex group with the exception of values for pregnant or lactating women. The Reference Dietary Value is the standard value adopted by the FDA for nutrients for which Reference Dietary Intake values cannot be established due to lack of Recommended Dietary Allowance values. If no Daily Value can be established, either a Reference Dietary Value or Reference Dietary Intake, a nutrient content claim can still be made for a food, food ingredient or relative amount of a nutrient. In this case, the information can be provided relative to a comparator, for example a serving of a specific food has twice as much of w-3 fatty acids per serving as 100 mg of fish oil. Alternatively, in the absence of a Daily Value, an alternate approach for a nutrient content claim is to carry a disclosure declaration indicating that the statement does not comply with a nutrient claim standard.

7.3

LEGISLATION GOVERNING US HEALTH CLAIMS

There are three major pieces of legislation that govern FDA sanctioned health claims: the Nutrition Labeling and Education Act, the Food and Drug Administration Modernization Act, and the Consumer Health Information for Better Nutrition Initiative. In their own way, each piece of legislation has had a major effect on health claims in the United States.

7.3.1

Nutrition Labeling and Education Act (NLEA 1990)

The intent of NLEA is to provide the general public with guidance about the potential health benefits of certain foods, food components or dietary ingredients, otherwise referred to as a nutrient–disease relationship. This legislation established a mechanism for the FDA to formally review and approve health claims for foods, food components or dietary ingredients and sanction an “authorized health claim”. Although the petitioning manufacturer provides a summary of the data to the FDA, the FDA conducts its own comprehensive review of the available literature, for the most part limited to human intervention trials. This review summarizes and synthesizes the scientific evidence on the topic. Under NLEA the only category of a health claim that is eligible for approval is an authorized health claim. Significant scientific agreement is the FDA's assessment as to whether qualified experts would likely agree that the scientific evidence supports the relationship between foods, food components or dietary ingredients and a disease relationship. An authorized health claim is a claim based on significant scientific agreement. The intent of the legislation is to provide information to consumers, at point of purchase, about good choices based on established health benefits. Examples of FDA-approved authorized health claims specially related to cardiovascular disease are depicted in Box 7.3.

7.3.2

Food and Drug Administration Modernization Act (FDAMA 1997)

The intent of FDAMA was to broaden NLEA by providing the general public with guidance on the potential health benefits of certain foods, food components or dietary ingredients on the basis of an assessment in addition to significant scientific agreement, termed an “authoritative statement”. An authoritative statement is defined as a published statement by

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Box 7.3 Examples of FDA-approved authorized health claims related to cardiovascular disease ● ●

● ● ● ●

Diets low in sodium may reduce the risk of high blood pressure, a disease associated with many factors. Diets low in saturated fat and cholesterol and rich in fruits and vegetables, and grain products that contain some types of dietary fiber, particularly soluble fiber, may reduce the risk of heart disease, a disease associated with factors. While many factors affect heart disease, diets low in saturated fat and cholesterol may reduce the risk of this disease. Soluble fiber (such as whole oats, psyllium seed husk), as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease. Diets low in saturated fat and cholesterol that include 25 g of soy protein a day may reduce the risk of heart disease. Diets low in saturated fat and cholesterol that include two servings of foods that provide a daily total of at least 3.4 grains of plant stanol esters in two meals may reduce the risk of heart disease.

Box 7.4 Examples of FDA-approved health claims based on authoritative statements related to cardiovascular disease ● ●

Diets rich in whole-grain foods and other plant foods and low in total fat, saturated fat, and cholesterol may reduce the risk of heart disease and some cancers. Diets containing foods that are a good source of potassium and which are low in sodium may reduce the risk of high blood pressure and stroke.

a scientific body of the US government or the National Academy of Sciences. This legislation established an additional mechanism by which manufacturers could petition for approval by the FDA. Once an authoritative statement appears, the FDA can be formally requested to authorize the use of a health claim. In contrast to the NLEA, this alternate mechanism for manufacturers to seek approval of a health claim is limited to food and does not include dietary supplements. Examples of FDA-approved health claims related to cardiovascular disease based on authoritative statements are shown in Box 7.4.

7.3.3

Consumer Health Information for Better Nutrition Initiative (2003)

The intent of the Consumer Health Information for Better Nutrition Initiative is to provide a mechanism for the general public to be presented with what are referred to as “qualified health claims”. These are health claims where the quality and strength of the scientific

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evidence does not meet the standards of an authorized health claim on the basis of either significant scientific agreement or an appropriate authoritative statement. Qualified health claims are weaker than authorized health claims because they must be “qualified”. They must provide information on not only the nature of the food, food component or dietary ingredients and reduced disease or health-related condition risk, but also be specific in terms of the strength of the evidence. The intent of such qualifications is to ensure that the general public is not misled by the claims and understands not only the nature of the relationship but also how strong the experimental data are to support the relationship. As with authorized health claims, the FDA rules on petitions for qualified health claims after completing a systematic review. Examples of FDA-approved qualified health claims are depicted in Box 7.5. Depending on the strength of the evidence, some qualified health claims are rarely used on packaged food products. They tend to be longer and more complicated in structure than authorized health claims.

7.4

DIETARY GUIDANCE TO REDUCE CARDIOVASCULAR DISEASE RISK

In developed countries, and more commonly in developing countries, the greatest nutrition challenge in the 21st century is the prevention of chronic disease. Because cardiovascular disease is the leading cause of death, the majority of dietary guidance takes this into consideration (Lichtenstein et al., 2006; Capewell et al., 2010). Fortunately, the general dietary approach to decrease the risk of developing cardiovascular disease encompasses common elements for the prevention of other chronic diseases, including diabetes and cancer. Prior to 2000, one component of most forms of nutrition guidance was to consume a diet with less than 30% of calories from fat, with no lower limit. This was, for the most part, coupled with restrictions in dietary saturated and cholesterol intake. More recent versions of these guidance documents have shifted emphasis from quantity to quality, recommending a moderate fat intake, encouraging the replacement of saturated and trans fatty acids with unsaturated fatty acids, either monounsaturated or polyunsaturated. These include general nutrition recommendations such as the Dietary Guidelines for Americans from the US Department of Agriculture (USDA) and the Department of Health and Human Services (DHHS) (USDA/DHHS, 2005); the Dietary Reference Intakes (DRI) from the Food and Nutrition Board, Institute of Medicine (IOM); and disease-specific recommendations for prevention and treatment such as the National Cholesterol Education Program (NCEP) from the National Heart, Lung and Blood Institute (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001), American Heart Association (Lichtenstein et al., 2006), American Diabetes Association (Bantle et al., 2008) and American Cancer Society (Kushi et al., 2006). While the dietary guidance is generally similar, there are some differences among these recommendations. In most cases, these differences reflect the specific mandate governing each issuing body rather than actual differences in the specific recommendation. It is important to appreciate the differences to avoid confusion or impede their implementation. For example, the Dietary Guidelines for Americans focuses on food patterns associated with optimal health outcomes and the prevention of chronic diseases, one of which is cardiovascular disease, whereas the DRI identify optimal nutrient intakes for different age and sex categories to prevent deficiencies and reduce the risk of developing chronic diseases.

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Box 7.5 Examples of FDA-approved qualified health claims related to cardiovascular disease ●

●

●

●

●

●

●

Scientific evidence suggests but does not prove that eating 1.5 ounces per day of most nuts [name of specific nut] as part of a diet low in saturated fat and cholesterol may reduce the risk of heart disease. Supportive but not conclusive research shows that eating 1.5 ounces per day of walnuts, as part of a low saturated fat and low cholesterol diet and not resulting in increased caloric intake, may reduce the risk of coronary heart disease. See nutrition information for fat [and calorie] content. Supportive but not conclusive research shows that consumption of EPA and DHA w-3 fatty acids may reduce the risk of coronary heart disease. One serving of [name of food] provides [x] g of EPA and DHA w-3 fatty acids. As part of a well-balanced diet that is low in saturated fat and cholesterol, folic acid, vitamin B6 and vitamin B12 may reduce the risk of vascular disease. FDA evaluated the above claim and found that while it is known that diets low in saturated fat and cholesterol reduce the risk of heart disease and other vascular diseases, the evidence in support of the above claim is inconclusive. Limited and inconclusive scientific evidence suggests that eating about 2 tablespoons (23 g) of olive oil daily may reduce the risk of coronary heart disease due to the monounsaturated fat in olive oil. To achieve this possible benefit, olive oil is to replace a similar amount of saturated fat and not increase the total number of calories you eat in a day. One serving of this product contains [x] g of olive oil. Limited and inconclusive scientific evidence suggests that eating about 1½ tablespoons (19 g) of canola oil daily may reduce the risk of coronary heart disease due to the unsaturated fat content in canola oil. To achieve this possible benefit, canola oil is to replace a similar amount of saturated fat and not increase the total number of calories you eat in a day. One serving of this product contains [x] g of canola oil. Very limited and preliminary scientific evidence suggests that eating about 1 tablespoon (16 g) of corn oil daily may reduce the risk of heart disease due to the unsaturated fat content in corn oil. FDA concludes that there is little scientific evidence supporting this claim. To achieve this possible benefit, corn oil is to replace a similar amount of saturated fat and not increase the total number of calories you eat in a day. One serving of this product contains [x] g of corn oil.

7.4.1

Dietary Guidelines for Americans

Consistent with federal law, the Dietary Guidelines for Americans (USDA/DHHS, 2005) are reviewed every 5 years by an advisory committee composed of nutrition experts, and if deemed appropriate on the basis of new scientific data are revised. Dietary Guidelines for Americans was first issued in 1980. Currently, the responsibility for reissuing the guidelines rests jointly with the DHHS and USDA. The law governing the Dietary Guidelines for Americans specifies that the publication shall contain nutritional and dietary information

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and guidelines for the general public and should be based on the most current scientific data. The committee tasked with reviewing and revising the Dietary Guidelines for Americans serves in an advisory capacity to the two federal agencies, making recommendations rather than having final authority. Since the first version in 1980, each committee thereafter has determined that some aspect of the guidelines should be revised. Prior to the 2000 Dietary Guidelines for Americans, the recommendations focused on limiting intake of dietary fat. Only minor changes were made to the original wording: “avoid too much fat” (1980), “avoid too much fat, saturated fat and cholesterol” (1985), and “choose a diet low in fat, saturated fat and cholesterol” (1990 and 1995). This message was changed in the 2000 Dietary Guidelines for Americans to “choose a diet that is low in saturated fat and cholesterol, and moderate in total fat”. The message was further refined in the 2005 Dietary Guidelines for Americans. Its recommendations told Americans to “consume less than 10 percent of energy from saturated fatty acids, less than 300 mg/day of cholesterol and keep trans fatty acid consumption as low as possible” and “keep total fat between 20 to 35 percent of energy, with most fats coming from sources of polyunsaturated fatty acids such as fish, nuts and vegetable oils”. Recent recommendations from the 2010 Dietary Guidelines Advisory Committee to DHHS and USDA is to limit the intake of “solid fats”, a term that encompasses both saturated and trans fatty acids. The basic shift in emphasis from quantity to quality of dietary fat was made in recognition of the scientific data that has evolved since 1980 (Lichtenstein, 2003). With respect to total dietary fat, the observation is that low-fat diets increase triglyceride concentrations and decrease highdensity cholesterol (HDL) concentrations (Lichtenstein et al., 1994; Schaefer et al., 1995), and that the fatty acid profile of the diet, rather than the total amount of fat, is the major determinant of lipoprotein profiles and subsequent cardiovascular disease risk (Lichtenstein, 2003). Additional “key messages” from the 2005 Dietary Guidelines for Americans include the following: when selecting and preparing meat, poultry, dry beans, and milk or milk products, make choices that are lean, low-fat, or fat-free, limit intake of fats and oils high in saturated and/or trans fatty acids, and choose products low in such fats and oils. In keeping with previous editions of the Dietary Guidelines for Americans, these recommendations were stated as food-based messages.

7.4.2

National Cholesterol Education Program

The National Heart, Lung and Blood Institute of the National Institutes of Health has convened panels of experts, the NCEP Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel or ATP) to develop guidelines for the treatment of individuals with high blood cholesterol concentrations. Three reports have been issued, in 1988, 1993 and 2001 (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 1988, 1993, 2001). The original dietary recommendations proposed for the treatment of cardiovascular disease included what were termed a Step 1 and Step 2 diet. The emphasis of the Step 1 diet was on obvious sources of saturated fatty acids and cholesterol in the diet and the elimination of excess energy, and was designed to be implemented by the physician or her/his immediate staff. If the lowdensity lipoprotein cholesterol goals were not achieved by 3 months, the patient was advised to adopt a Step 2 diet (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 1988). The Step 2 diet was a more aggressive form of the Step 1 diet and more likely to necessitate changes in the overall diet rather than targeting dietary fat and total energy. It was designed to be implemented by a registered dietitian

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(Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 1988). In both cases emphasis was on total fat (less than 30%) and type of fat. Criteria for the Step 1 diet included 30% of energy total fat, less than 10% of energy as saturated fat, up to 10% of energy as polyunsaturated fat, 10–15% of energy as monounsaturated fat and 300 mg cholesterol per day. Criteria for the Step 2 diet included less than 30% of energy total fat, less than 7% of energy as saturated fat, up to 10% of energy as polyunsaturated fat, 10–15% of energy as monounsaturated fat and 200 mg cholesterol per day. The second report from the NCEP, ATP II (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 1993), left the original recommendations unchanged. In response to changing dietary patterns of the general public in the United States the third report from NCEP, ATP III (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001), retired the Step 1 and Step 2 diets and shifted emphasis from diet alone to a multifaceted lifestyle approach for the treatment of hypercholesterolemia by introducing the Therapeutic Lifestyle Change Diet (TLC) (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001). By 2000 the general population had reduced saturated fat intake to a level approaching the Step 1 diet, eliminating the need for Step 1 and Step 2 diets. The components of the TLC diet, with regard to dietary fat are 25–35% of energy as total fat, less than 7% of energy as saturated fat, up to 10% of energy as polyunsaturated fat (PUFA), up to 20% of energy as monounsaturated fat and 200 mg cholesterol per day. The saturated fat recommendation was set at the level formerly associated with the Step 2 diet (less than 7% of energy) to help maintain population-wide efforts to reduce saturated fat intake. Noteworthy, in the biggest shift by NCEP, ATP III went from recommending a diet low in total fat (less than 30% of energy) to a diet moderate in total fat (25–35% of energy). This came about for two reasons: first, the recognition that when carbohydrate displaces fat from the diet, many individuals respond with carbohydrate-induced hypercholesterolemia (Lichtenstein et al., 1994); and second, a reduction in total fat intake does not necessarily translate to a reduction in saturated fat intake. Lowering fat by replacing traditional sources of unsaturated fat such as salad dressings with fat-free versions rather then replacing traditional sources of saturated fat such as dairy products with fat-free and reduced fat versions achieved the first dietary recommendation of reducing total fat intake but not the second of reducing saturated fat intake and maintaining unsaturated fat intake. The TLC diet guidelines placed renewed emphasis on balancing energy intake with expenditure.

7.4.3

Dietary Reference Intakes

The DRI values are the new standards for nutrient adequacy, replacing the former Recommended Dietary Allowances (RDA) values. They are established by panels of experts appointed by the Food and Nutrition Board (FNB) of the IOM and National Academy of Sciences. The first RDA values were developed during World War II to investigate issues of nutrition that might “affect national defense” (Nestle, 1993). The first RDA values were issued in 1941 and were intended to meet the nutrient needs of the majority of the healthy population (97.5%). Historically, the RDAs were revised every 5–10 years on the basis of new information. It was not uncommon to add recommendations for essential nutrients as they were identified and adequate information on which to base a recommendation emerged. In 1997 RDA values became one part of a broader set of

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dietary guidelines called the DRI values. The DRI values are set by joint expert panels composed of scientific expert members from the United States and Canada. As part of the new system of recommendations, the DRI values for clusters of nutrients were completed and released in sets rather than the prior approach of a single document for all essential nutrients (IOM Dietary Reference Intakes, 1997, 1998, 2000, 2001, 2005a,b, 2006; IOM Dietary Supplements, 2004). Dietary fat was a difficult topic for this panel to address because it encompasses a mixture of essential and non-essential nutrients. Whereas prior RDA/DRI panels had established reference values only for essential nutrients using well-established criteria to determine adequate intakes and avoid deficiency, this panel was mandated to establish recommendations not only for the essential nutrients (w-6 and w-3) but also for non-essential nutrients such as cholesterol, saturated fatty acids and trans fatty acids. In addition, it became apparent that in some cases there were ranges of nutrient intakes for macronutrients (fat, carbohydrate and protein) that were associated with optimal health outcomes, rather than a clear level that would prevent deficiency. To address this later challenge, the panel devised the Acceptable Macronutrient Distribution Range (AMDR) for carbohydrate, total fat, w-6 and w-3 PUFA and protein, as well as setting an Adequate Intake (AI) level for selected fatty acids (linoleic acid and a-linolenic acid). The AMDR is intended to represent a range of intakes high enough to ensure nutrient adequacy and energy balance, and low enough to minimize risk of chronic diseases, based on the available evidence. The 2005 DRI adult AMDR for dietary fat is 20–35% of energy, w-6 PUFA 5–10% of energy and w-3 PUFA 0.6–1.2% of energy. The 2005 DRI adult AI values for w-6 fatty acids is 11–12 g/day for women and 14–17 g/day for men, reflecting the difference in total energy intake. The AI for w-3 fatty acids is 1.1 g/day for women and 1.6 g/day for men, again reflecting the difference in total energy intake. The biological role of the very long chain w-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is thought to be unique from a-linolenic acid (Wang et al., 2006). Due to limited information for intakes of the individual w-3 fatty intakes, the panel recommended that approximately 10% of the w-3 AMDR be consumed as EPA and/or DHA. Saturated fatty acids, trans fatty acids, and cholesterol present a unique challenge for the DRI system. As indicated by the expert panel, “No required role for these nutrients other than energy sources was identified; the body can synthesize its needs for saturated fatty acids and cholesterol from other sources.” Nonetheless, zero intake could not be realistically recommended given the natural mix of these fatty acids in the food supply and essential nutrients associated with foods containing even low levels of these fatty acids. Therefore, for these three nutrients the panel recommended that intakes be “as low as possible while consuming a nutritionally adequate diet”.

7.4.4

American Heart Association

The American Heart Association Diet and Lifestyle Recommendations are developed with the mandate of reducing the risk of developing cardiovascular disease (Lichtenstein et al., 2006). They are revised approximately every 5 years. Their focus is prevention rather than treatment. The 2006 American Heart Association Diet and Lifestyle Recommendations are primarily food based and shown in Box 7.6. Those that pertain specifically to dietary fat include limiting intake of total fat to between 25 and 35%, saturated fats to less than 7% of energy, trans fat to less than 1% of energy (limit intake of partially hydrogenated fat) and cholesterol to less than 300 mg/day, and to consume fish, especially oily fish, at least twice a week.

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Box 7.6 American heart association diet and lifestyle recommendations ● ● ● ● ●

● ● ● ●

Balance calorie intake and physical activity to achieve or maintain a healthy body weight Consume a diet rich in vegetables and fruits Choose whole-grain, high-fiber foods Consume fish, especially oily fish, at least twice a week Limit your intake of saturated fat to less than 7% of energy, trans fat to less than 1% of energy, and cholesterol to less than 300 mg per day by: ° choosing lean meats and vegetable alternatives ° selecting fat-free (skim), 1% fat, and low-fat dairy products ° minimizing intake of partially hydrogenated fats Minimize your intake of beverages and foods with added sugars Choose and prepare foods with little or no salt If you consume alcohol, do so in moderation When you eat food prepared outside of the home, follow the AHA Diet and Lifestyle Recommendations

The saturated fat recommendation was reduced from less than 10% of energy in the 2000 recommendations (Krauss et al., 2000) to less than 7% of energy for similar reasons as stated for NCEP ATP IV (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001) in order to be consistent with current dietary practices and encourage evolution to optimal dietary patterns. The recommendation for a maximum percent of energy from trans fat is the first to specify a target number. The major emphasis of the recommendations was placed on the fatty acid profile of the diet rather than on total fat or dietary cholesterol. Partially hydrogenated fat was stressed because it is the major source of trans fatty acids in the American diet and that of most countries worldwide. The recommendation to consume fish reiterated the recommendations issued in the 2002 American Heart Association scientific statement on w-3 fatty acids (Kris-Etherton et al., 2002). These recommendations include the following: the general public should aim for two fish meals, preferably fatty fish, per week; individuals who have been diagnosed with coronary heart disease should consume the equivalent of 1 g of w-3 fatty acids per day, preferably from eating fish, but fish oil capsules may be used; and individuals with high triglycerides, under the care of a physician, may benefit from consuming 2–4 g of EPA plus DHA provided as capsules. Fish such as mackerel, lake trout, herring, sardines, albacore tuna, and salmon are high in the very long chain w-3 fatty acids EPA and DHA, which are most closely associated with decreased risk of cardiovascular disease. At about the same time that the American Heart Association recommendations were being developed, the American Diabetes Association and the American Cancer Society were also working on nutrition guidelines and recommendations, and each of their committees included representatives from the other organizations. These representatives were present to help facilitate the development of guidelines that were compatible among groups.

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7.4.5

American Diabetes Association

The American Diabetes Association guidelines (Bantle et al., 2008) are the most complex because they address issues related to both prevention and management. With regard to dietary fat, the focus of the American Diabetes Association guidelines is to maximize vascular health. While both obesity and weight gain are recognized risk factors for the development of type 2 diabetes, there is no evidence that high fat intakes per se increase risk for diabetes in humans. The American Diabetes Association's recommendations about fats (Bantle et al., 2008) are similar to those of other organizations (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 2001; IOM Dietary Reference Intakes, 2005b). They include limiting saturated fats to less than 7% of energy, minimize intake of trans fatty acids, limit dietary cholesterol to under 200 mg/day and eat two or more servings of fish per week, with the exception of fried fish fillets.

7.4.6

American Cancer Society

Creating dietary guidelines for cancer prevention is extremely challenging because the data for diet and cancer are much less definitive and extensive than those for heart disease and diabetes. Evidence linking high fat intakes and the development of cancer is mixed. Intervention studies have been limited by the lack of reliable intermediate markers for cancer development. The American Cancer Society (Kushi et al., 2006) based its recommendations on areas where the evidence between diet and cancer was strongest, emphasizing a plant-based diet. If adhered to, the American Cancer Society dietary recommendations will result in a diet low in saturated fat and cholesterol. They include choosing fish, poultry, or beans as an alternative to beef, pork, and lamb, when eating meat; selecting lean cuts; eating smaller portions; and preparing meat by baking, broiling, or poaching rather than by frying or charbroiling.

7.4.7

Case study: evolution of Dietary Guidelines for Americans

Periodic revision of any of these dietary guidelines is a necessary but painstaking and intensive process that involves many people and much time. The evolution of the US Dietary Guidelines provides an opportunity to temporally evaluate their evolution over time. The first Dietary Guidelines, published in 1980, were as follows: eat a variety of foods, maintain an ideal weight, avoid too much fat, saturated fat and cholesterol, eat foods with adequate starch and fiber, avoid too much sugar, avoid too much sodium, and if you drink alcohol, do so in moderation. In 1985 two changes were made to the guidelines: maintain a “desirable” weight instead of maintain an “ideal” weight, and “alcoholic beverages” instead of “alcohol”. In the 1990 revision, “desirable” weight evolved further to “healthy” weight, and there was a general shift from negative to positive wording. Instead of “avoid too much fat, saturated fat and cholesterol”, the guideline was reframed as a positive, actionable statement: “choose a diet low in fat, saturated fat and cholesterol”. Likewise, the guidelines for sugar and sodium were changed to positive recommendations: “use sugar only in moderation” and “use salt and sodium only in moderation”. Another important change in the 1990

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guidelines was a shift from starch and fiber to groups of foods that are sources of these nutrients: “choose a diet with plenty of vegetables, fruits, and grain products.” Five years later, in 1995, “maintain healthy weight” was modified to “balance the foods you eat with physical activity – maintain or improve your weight”. This was considered an important change at the time, because it removed descriptive adjectives and incorporated the idea of energy balance. In 1995 the guidelines were also reordered, with the fruit, vegetable, and grain guideline placed before the fat and cholesterol guideline. Theoretically, there is no particular order for the dietary guidelines, but there is an implied hierarchy and this change appeared to give greater priority to fruits, vegetables, and grains. The word “use” for sugar, salt and sodium was replaced with “choose”, placing more responsibility on the consumer. From 1995 to 2000 there were several notable changes. “Eat a variety of foods” was eliminated as a guideline because the committee felt there were few data to support a benefit from the recommendations and preliminary research had shown that with the exception of fruits and vegetables, people who ate a wider variety of foods ate more energy (McCrory et al., 1999). The guideline was replaced with “Let the Pyramid guide your food choices” in the hope that the visuals depicted in the Food Guide Pyramid would be a better way of communicating the concept of choosing a variety of foods among the different groups. Whether this change or any other word changes actually prompted an alteration in consumer behavior was never rigorously tested. The body weight and physical activity guideline was split into two parts: “Aim for a healthy weight” and “Be physically active every day”. The rationale for this change was twofold: first, the growing numbers of overweight and obese individuals in America made it clear that more emphasis was needed on body weight; and second, health benefits beyond weight control clearly result from regular physical activity. In 2000 the guideline “choose a diet with plenty of vegetables, fruits, and grains” was also divided into two guidelines: (1) “choose a variety of grains daily, especially whole grains” and (2) “choose a variety of fruits and vegetables daily”. At that time the intake of grains in the US population was generally meeting the guideline, but this was not the case with fruits and vegetables, so the committee thought that more emphasis would be placed on fruits and vegetables by singling them out in a separate guideline. In addition, focus group data suggested that some people interpreted the “5 a day” message to mean five servings from the entire group of grains, fruits, and vegetables. This demonstrated a need for greater clarity and consistency in dietary messages from all sources. In 2000 the guideline “choose a diet low in fat, saturated fat, and cholesterol” became “choose a diet moderate in total fat, and low in saturated fat and cholesterol” because the scientific data were sufficient to support the concept that type of fat, rather than amount of fat, is associated with better cardiovascular outcomes (Lichtenstein, 2006). Additional changes in the 2000 version of the Dietary Guidelines included adding the word “beverages” to the sugar guideline, with the intent of raising awareness of the increased intake of sugar-sweetened beverages in the United States; changing “choose a diet moderate in salt and sodium” to “choose and prepare foods with less salt”; and the first inclusion of a food safety guideline. The alcoholic beverage guideline itself did not change, but there was expansion of the accompanying text. Scientific data supported moderate alcohol intake for reduced risk of cardiovascular disease (Mukamal & Rimm, 2008), but other issues such as psychomotor changes, risk of breast cancer in women, fetal alcohol syndrome in pregnant women, traffic accidents, spousal abuse and child abuse could not be ignored.

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The 2005 committee took a very different approach for formulating and presenting the Dietary Guidelines. Instead of a list of guidelines, they developed a booklet with chapters corresponding to and elaborating on the topics of the previous guidelines considered of major public health importance. The information in these chapters was presented in the form of key recommendations and, when appropriate, key recommendations for specific populations.

7.5

CURRENT CHALLENGES

Research on consumer awareness and understanding as well as on implementation and behavior change would be useful for future committees as they continue to refine and improve dietary guidance documents. Are we communicating the message accurately? Where do consumers hear these messages: from the media, nutrition/medical community, or elsewhere? Who is listening and who is acting? How can we increase the proportion of the population who are following healthy diet and lifestyle behaviors? What are the barriers to implementing the guidelines? Are some messages more challenging to implement than others? For example, does the cost of fruit and vegetables represent a significant barrier, or does availability outside the home negatively impact on daily intake? Are some messages adhered to better than others? How can we learn from these successes?

REFERENCES Bantle JP, Wylie-Rosett J, Albright AL et al. (2008) Nutrition recommendations and interventions for diabetes: a position statement of the American Diabetes Association. Diabetes Care 31(Suppl 1): S61–S78. Capewell S, Ford ES, Croft JB, Critchley JA, Greenlund KJ, Labarthe DR (2010) Cardiovascular risk factor trends and potential for reducing coronary heart disease mortality in the United States of America. Bull WHO 88:120–30. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (1988) Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Arch Intern Med 148:36–69. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (1993) Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). JAMA 269:3015–3023. Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) (2001) Executive summary of the third report of the National Cholesterol Education Program (NCEP). JAMA 285:2486–2497. IOM Dietary Reference Intakes (1997) Calcium, Phosphorus, Magnesium, Vitamin D and Fluoride. Washington, DC: National Academy of Sciences. IOM Dietary Reference Intakes (1998) Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic acid, Biotin and Choline. Washington, DC: National Academy of Sciences. IOM Dietary Reference Intakes (2000) Vitamin C, Vitamin E, Selenium and Carotenoids. Washington, DC: National Academy of Sciences. IOM Dietary Reference Intakes (2001) Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium And Zinc. Washington, DC: National Academy of Sciences. IOM Dietary Reference Intakes (2005a) Water, Potassium, Sodium, Chloride and Sulfate. Washington, DC: National Academy of Sciences. IOM Dietary Reference Intakes (2005b) Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. Washington, DC: National Academy of Sciences.

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IOM Dietary Reference Intakes (2006) The Essential Guide to Nutrient Requirements. Washington, DC: National Academy of Sciences. IOM Dietary Supplements (2004) A Framework for Evaluating Safety. Committee on the Framework for Evaluating the Safety of Dietary Supplements. Washington, DC: National Academy of Sciences. Krauss RM, Eckel RH, Howard B et al. (2000) AHA Dietary Guidelines: revision 2000: A statement for healthcare professionals from the Nutrition Committee of the American Heart Association. Circulation 102:2284–2299. Kris-Etherton PM, Harris WS, Appel LJ (2002) Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Circulation 106:2747–2757. Kushi LH, Byers T, Doyle C et al. (2006) American Cancer Society guidelines on nutrition and physical activity for cancer prevention: reducing the risk of cancer with healthy food choices and physical activity. CA: Cancer J Clin 56:254–281. Lichtenstein AH (2003) Dietary fat and cardiovascular disease risk: quantity or quality? J Women's Health 12:109–114. Lichtenstein AH (2006) Thematic review series: patient-oriented research. Dietary fat, carbohydrate, and protein: effects on plasma lipoprotein patterns. J Lipid Res 47:1661–1667. Lichtenstein AH, Ausman LM, Carrasco W, Jenner JL, Ordovas JM, Schaefer EJ (1994) Short-term consumption of a low-fat diet beneficially affects plasma lipid concentrations only when accompanied by weight loss. Hypercholesterolemia, low-fat diet, and plasma lipids. Arteriosclerosis Thrombosis 14:1751–1760. Lichtenstein AH, Appel LJ, Brands M et al. (2006) Diet and lifestyle recommendations revision 2006: a scientific statement from the American Heart Association Nutrition Committee. Circulation 114:82–96. McCrory MA, Fuss PJ, McCallum JE et al. (1999) Dietary variety within food groups: association with energy intake and body fatness in men and women. Am J Clin Nutr 69:440–447. Mukamal KJ, Rimm EB (2008) Alcohol consumption: risks and benefits. Current Atherosclerosis Reports 10:536–543. Nestle M (1993) Dietary advice for the 1990s: the political history of the Food Guide Pyramid. Caduceus 9:136–153. Schaefer EJ, Lichtenstein AH, Lamon-Fava S et al. (1995) Body weight and low-density lipoprotein cholesterol changes after consumption of a low-fat ad libitum diet. JAMA 274:1450–1455. USDA/DHHS (2005) Dietary Guidelines for Americans. Available at www.healthierus.gov/ dietaryguidelines/ Wang C, Harris WS, Chung M et al. (2006) n-3 Fatty acids from fish or fish-oil supplements, but not alphalinolenic acid, benefit cardiovascular disease outcomes in primary- and secondary-prevention studies: a systematic review. Am J Clin Nutr 84:5–17.

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8

Probiotics and Health Claims: a Japanese Perspective

Fang He and Yoshimi Benno

8.1

INTRODUCTION

The definition of probiotics has been broadened through the years with the expansion of the knowledge on the symbiotic interaction between microbes and host animal (Salminen et al., 2000, 2006; Guarner et al., 2005). Today, probiotics are among the most researched and scientifically documented functional ingredients. In Japan, probiotics have a long history of cultural acceptance and safe use. The scientific studies conducted by Japanese scientists in the health-promoting effects of probiotic have a high reputation (Arai et al., 2001, 2002). However, in a manner similar to other developed countries, the Japanese Pharmaceutical Affairs Law strictly prohibits any statement that food products can exert efficacy like that of drugs to influence the structure or function of the human body. The commercialization of probiotics is historically regulated by the normal legislation on food with respect to packaging and labeling of fresh food products. In this legislation no claims related to health, prevention and cure are allowed to be used for food. There have been some dynamic changes in food regulation in Japan since the 1990s, and now food in the Japanese market can be divided into two categories. One allows foods to make health claims and is called “regulated foods”, while the other is “unregulated foods”, which actually represents all types of processed food, conventional foods including socalled “health foods”, and dietary supplements. Since they are not regulated, they are free to be sold as any processed food, and cannot carry any health claims (Arai et al., 2001, 2002; Fukushima & Iino, 2005; Amgase, 2008; Kitsukawa, 2008; Yamada et al., 2008). Most of the probiotic products in Japan still belong to this category, and are sold as general food without any health claim labeling. In the regulated foods category, called Foods with Health Claims, there are two health claim regulations. One is Foods for Specified Health Use (FOSHU). This regulation indicates that the authority approves the products and its health claims. The other is Foods with Nutrient Function Claims (FNFC), in which the government has set the range of nutrition and mineral content levels and labeling standards. Both regulations are governed under the Health Promotion Law, section of Foods with Health Claims Act of 2001. While the FOSHU system is a product-specific (not ingredient-specific) approval system, FNFC is a blanket approval system, meaning that any products that meet the standards may make standardized health claims without official approval. Some probiotic products in Japan have been approved as FOSHU and no probiotics can be called FNFC. 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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Table 8.1 Probiotic strains for FOSHU products. Probiotic strain

Company

Lactobcillus rhamnosus GG

Tanaknashi Milk Products Co. Ltd

Bifidobacterium longum BB536

Morinaga Milk Industry Co. Ltd

Lb. delbrueckii subsp. bulgaricus 2038

Meiji Dairies Corporation

Streptococcus salivarius subsp. thermophilus 1131

Meiji Dairies Corporation

Lb. casei Shirota YIT9029

Yakult Honsha

B. breve strain Yakult

Yakult Honsha

B. lactis FK120

Fukuchan milk

B. lactis LKM512

Kyodo Milk Industry Co. Ltd

Lb. acidophilus CK92

Matsutani Chemical Products Co. Ltd

Lb. helveticus CK60

Matsutani Chemical Products Co. Ltd

Lb. casei NY1301

Nissin York Co. Ltd

Lb. gasseri SP strain

Nippon Milk Community Co. Ltd

Bifidobacterium SP strain

Nippon Milk Community Co. Ltd

B. lactis BB-12

Koiwai Dairy Products Co. Ltd

B. lactis BB-12

Azumino Food Co. Ltd

B. lactis BB-12

Yotsub Co. Ltd

B. lactis BB-12

Ikaruga Milk Co. Ltd

Bifdobacterium Bb-12

Furuya Milk.Co.jp

Lb. johnsonii LC-1

Nestec Ltd

8.2

FOSHU HEALTH CLAIMS

FOSHU are foods containing food-originated components with specific health functions, contributing to maintenance or promotion of human health and consumed for the purpose of specific health use. It is only after the examination and approval of each product application by the Ministry of Health, Labor, and Welfare (MHLW) based on scientific evidence, including the results, that a food product can indicate the specific health use (Fukushima & Iino, 2005; Kitsukawa, 2008; Yamada et al., 2008). FOSHU was created in 1991 by MHLW to give foods a chance to claim health benefits. The creation of FOSHU helped consumers understand the relationship between food and health. The health claims printed on a label were vague and suggestive, but the health claim statements and FOSHU-approved logo gave the consumer better guidance when selecting products to benefit their health problems from store shelves. A total of 76 probiotic products, containing 16 specific probiotic strains, are listed among the 843 FOSHU products (Table 8.1). Under the current system, FOSHU approval includes standardized FOSHU, qualified FOSHU and reduction-of-disease FOSHU. At present, probiotics are still limited to standardized FOSHU, and there are no examples of qualified FOSHU and reduction of disease risk FOSHU for probiotics.

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Fig. 8.1 FOSHU logo.

8.2.1

History of FOSHU

In the 1970s and 1980s, modern technology brought many functional components into foods. These advances resulted in an expansion in the food industry and all types of socalled “functional foods” or “health foods” flooded the marketplace. Because there were no laws to regulate this growing industry, functional foods soon became a public health and safety issue, as well as causing confusion and mistrust among consumers. A specific study on the development and systematic analysis of functional food was initiated in Japan in 1984 with support from the Ministry of Education, Science and Culture (MESC). This study stated that food has three functions: (1) a nutrition function to maintain life and growth; (2) a taste function, with some components interacting with the sensory system; and (3) a body-enhancing function related to maintenance of health and the prevention of disease. This study proposed a new concept (i.e. functional food) focusing on the third function of food, and the term “functional foods” immediately took root internationally. In 1991, the FOSHU system was introduced in Japan as the world’s first approval system for health-claim labeling for food products, and the first FOSHU products were approved in 1993 (Figs 8.1 and 8.2). Since the Pharmaceutical Affairs Law stipulated that products aimed at influencing function or body structure should be categorized as drugs, the legal name of functional food was changed to FOSHU, and the concept Food With Functionality was subsequently modified to Food With Approved Label. The definition of “functional” was reduced to “expectation for the purpose of a specific health use”. The FOSHU system was revised in 1997 to eliminate the 2–4 year expiration, strengthen the regulation of quality control, and simplify the documentation for FOSHU application. In 2001, the type product was expanded to include tablets and capsules as FOSHU and the Food With Health Claim system was introduced under food sanitation law. FOSHU was

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Food FOSHU

1991

Medicine

Usual Food Food For Specified Health Uses

Food with Health Claims (FHC)

2001

Medicine

Usual Food

FNFC Food For Nutrient Function Claim

FOSHU

Food with Health Claims (FHC)

2005

Medicine

Usual Food

FOSHU

FNFC Standard FOSHU

Reduction of disese FOSHU

Qualified FOSHU

Fig. 8.2 Legal position for FOSHU regulation.

reclassified in the category Food With Health Claim as FNFC besides FOSHU. In 2005 MHLW developed a new rule for reduction-of-disease FOSHU and qualified FOSHU. As of March 2009, 847 items had been granted FOSHU status. The existing health claims on FOSHU can be classified into eight groups according to the health claims. These include gastrointestinal conditions, mineral absorption, blood pressure, blood cholesterol, bone health, dental health, and blood glucose levels. Probiotics are the most important functional ingredients in preparing FOSHU products for gastrointestinal health. There are 76 probiotic products approved as FOSHU with health claim. The first FOSHU probiotic drink was approved in May 1996. This yogurt drink, from Takanashi Milk Products Co., Ltd, contained Lactobacillus GG “to help increase intestinal Bifidobacteria and Lactobacilli and regulate GI condition”, scientifically proven in human intervention studies with Japanese subjects (Hosoda et al., 1994, 1998; Benno et al., 1996). This was followed in November 1996 by the simultaneous approval of two very popular yogurt brands that had been marketed for several years. Morinaga’s “Bifidus Plain Yoghurt” with Bifidobacterium longum BB536 and Meiji Milk Products’ (now Meiji Dairies) “Meiji Bulgaria Yoghurt LB 81” with Lactobacillus delbreukii subsp. bulgaricus 2038 and Streptococcus salivarius subsp. thermophilus 1131 were allowed to make similar gastrointestinal claims with reference to the specific strain(s) of bacteria used as the functional ingredient(s). This strainspecific approach has now become the standard for FOSHU approvals. The conversion of these popular yogurts to FOSHU status instantly added several hundred million dollars of sales to the already-growing FOSHU regulatory category. The conversion also helped increase consumer awareness of FOSHU. Major FOSHU sales growth also occurred in 1998 and 1999, when almost the entire line of Yakult brands, including their flagship “Yakult” beverage, converted to FOSHU status. All the Yakult products approved at that time were based on the Lactobacillus casei Shirota probiotic strain that had been developed nearly 70 years earlier.

8.2.2

Specifics of FOSHU health claims

The health claims allowed on FOSHU product labels are periphrastic. Although the FOSHU health claim has been legally approved, the effectiveness of food ingredients with regard to treatment, cure or prevention of disease conditions is not allowed to be expressed, because

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using such labels would be a violation of the Pharmaceutical Affairs Law. Therefore, the expression of FOSHU health claims is suggestive or recommendatory, even though the product is well tested and scientifically proven to be effective. For example, “Product ABC contains oligosaccharides to improve gastrointestinal conditions.” This statement does not say that consuming the product can improve a health condition but merely that the food contains a functional component to improve gastrointestinal conditions. Here are other examples of FOSHU health claim statements. ● ●

●

Gastrointestinal health: “This food contains fructo-oligosaccharides, which promote bifidobacteria in the colon to maintain a healthy gastrointestinal condition.” Diabetes: “This product contains guava-polyphenols that slow down the absorption of blood sugar. Therefore this product is suitable for people who are concerned about blood sugar levels.” Cardiovascular health: “This product contains isolated soy protein that may work to lower blood cholesterol levels; therefore, the product is suitable for people who have high cholesterol levels.”

FOSHU health claim statements differ from product to product, even though they are in the same health claim category with the same functional ingredient. In order for a product to be approved as FOSHU, companies need to go through an application process that takes about a year to complete. An application cost for MHLW is less than $2000 but scientific tests and other necessary studies may have a cost as high as $1 million or more. Furthermore, any changes in packaging design, content and production processing are prohibited before obtaining the permit from MHLW.

8.2.3

Procedure for obtainining permission for FOSHU

In summary, there are three essential requirements for FOSHU approval: (1) its effectiveness based on scientific evidence including clinical studies; (2) its safety as assessed from historical consumption pattern data and additional safety studies conducted in humans; and (3) analytical determination of the functional component responsible for the beneficial physiological action (Box 8.1). I Documentation of effectiveness should be prepared on the basis of substantiation not only by human clinical and animal studies but also by in vitro metabolism and biochemical data. Such data should demonstrate statistically significant differences between the control and intervention groups. Basically, human studies should be conducted for a reasonably long-term period (e.g. more than 3 months) using the food for which the claim is intended in a placebo-controlled, double-blind way. The results cannot be accepted until they are published in peer-reviewed scientific journals. The study should also be well designed (e.g. utilizing an appropriate biomarker and an appropriate sample size) in order to show statistically significant differences between sufficient numbers of subjects. Literature concerning relevant functional constituents, the food carrier, and the related function should be provided in the form of a review. II For documentation of safety, both in vivo and in vitro studies should be carried out to develop preliminary data on safety of intake in humans. Even if the effective component has been consumed in food by a reasonable number of people over a certain

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Box 8.1 Documentation required for application to ministry of health and labor and welfare for FOSHU status for a product ● ● ● ● ● ● ● ● ●

Sample of the entire package with labels and health claims Documentation that demonstrates clinical and nutritional proof for the product and/or its functional component for the maintenance of health Documentation that demonstrates clinical and nutritional proof of the intake amount of the product and/or its functional component Documentation concerning the safety of the product and its functional component, including additional human studies about the eating experience Documentation concerning the stability of the product and its functional component Documentation of the physical and biological characteristics of the product and the functional component Methods of qualitative/quantitative analytical determination of functional component, and analytical assay results of the component in the product Report on the analysis of the designated nutrient constituents and energy content of the product Statement of the production method, list of factory equipment, and an explanation of the quality control system

period, safety data regarding human consumption should be provided using at least three times the minimum effective dose. Literature concerning related functional components must be reviewed and provided. If the related literature or report implies an undesirable or adverse effect on health, it should be accompanied by a scientific explanation or a human study that confirms safety in humans. III Documentation of the methods of analysis of the functional components should be included in the claim submission. These analytical determinations typically precede clinical intervention studies in humans or preclinical animal studies or in vitro studies and stability testing. IV As additional documentation, evidence regarding the stability of related functional components should be provided. If a product is to be administered in the form of tablets or capsules, experiments should be conducted exploring the extent of disintegration or dissolution of the bioactive substance. In general, the evaluation process of both the benefit and the safety of FOSHU products differs from that of a medicine. FOSHU products are designed to target healthy people or people in a preliminary stage of a disease or a borderline condition of at-risk groups. Therefore, the effects observed in these people in FOSHU-related clinical intervention studies may be reduced compared with the effects seen for a medicine in patients. On the other hand, most FOSHU ingredients have been historically consumed by people and can thus be regarded as safer than innovative medicines.

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8.2.4

FOSHU health claim for probiotics: gastrointestinal conditions

As mentioned above, existing health claims on FOSHU can be classified into eight groups according to the health claims, such as gastrointestinal conditions, mineral absorption, blood pressure, or blood cholesterol. However, FOSHU health claims for probiotic products are still limited to “Improve GI condition”. The scientific evidence necessary for a probiotic FOSHU to use the health claim “Improve GI condition” should therefore consist of data showing improvement in intestinal microflora balance, increase in local bifidobacteria and ideally decrease in Clostridium perfringens and Escherichia coli, lower bacteria metabolite production, and increased defecation frequency in human studies. Approved FOSHU health claims for probiotics include the following. ● ● ● ● ● ● ●

Reaches the intestines alive. Increases the intestinal lactobacilli/bifidobacteria/beneficial bacteria. Promotes the maintenance of a good intestinal environment. Regulates/helps maintain good GI condition. Maintains the intestine in good health. Helps balance the intestinal flora. Reduces harmful bacteria.

FOSHU approval takes a strain-specific approach based on the concept that each probiotic strain can exhibit it own effect on the host animal. However, currently approved FOSHU health claims for the 76 probiotic products prepared with 16 strains are basically similar to each other, as demonstrated elsewhere. Therefore, the approved FOSHU health claim cannot characterize the function of each probiotic, although the scientific evidence for the health-promoting effects of probiotics are different qualitatively and quantitatively among specific strains. While reduction-of-disease claims have been approved for calcium (osteoporosis) and folic acid (neural tube defects), none as yet exist for probiotics.

8.3

NON-FOSHU HEALTH CLAIMS FOR PROBIOTICS IN JAPAN

There are many non-FOSHU probiotic products marketed in Japan with at least implied and/ or off-label claims that focus on important health issues based on some scientific evidence obtained from animal and human studies. These include cedar pollen allergy (Kawase et al., 2006, 2007a,b, 2009; Morita et al., 2006; Salminen et al., 2006; Hata et al., 2008; Iliev et al., 2008; Kubota et al., 2009), Helicobacter pylori colonization in the stomach, and immune function enhancement. Studies related to prevention and suppression of cancer with probiotics are being researched, with some encouraging developments, but related health claims are strictly prohibited. The accumulation of scientific evidence for safety and experience with efficacy in Japan is important in helping establish credibility for new probiotic health claims. MHLW has a stake in allowing such developments to proceed, but is expected to maintain a relatively cautious and conservative approach to new approvals. Although much time is still necessary, some new probiotic health claims could have potential in the future if Japanese scientists and industry combine in carrying out more clinical trials and related studies.

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REFERENCES Amgase H (2008) Current marketplace for probiotics: a Japanese perspective. Clin Infect Dis 46:S73–S75. Arai S, Osawa T, Ohigashi H et al. (2001) A mainstay of functional food science in Japan: history, present status, and future outlook. Biosci Biotechnol Biochem 65:1–13. Arai S, Moringa Y, Yoshikwa T et al. (2002) Recent trends in functional food science and the industry in Japan. Biosci Biotechnol Biochem 66:2017–2029. Benno Y, He F, Hosoda M et al. (1996) Effects of Lactobacillus GG yogurt on human intestinal microeclogy in Japanese subjects. Nutr Today Suppl 31:9–11. Fukushima Y, Iino H (2005) Probiotics in food safety and human health: current state of regulation on the use of probiotics in food in Japan. In: Goktepe I, Juneja VK, Mohamed Ahmedna M (eds) Probiotics in Food Safety and Human Health. New York: CRC Press, pp. 431–460. Guarner F, Perdigon G, Corthier G, Salminen S, Koletzko B, Morelli L (2005) Should yoghurt culture be considered probiotic? Br J Nutr 93:783–786. Hata J, Kobayakawa S, He F, Kawase M, Tochikubo T (2008) Effectiveness of combined treatment with Lactobacillus gasseri TMC0356 and Lactobacillus GG in a rat model of allergic conjunctivitis. J Med Soc Toho Univ 55:278–283. Hosoda M, He F, Hiramatsu T, Hashimoto H, Benno Y (1994) Effects of Lactobacillus GG strain intake on fecal microflora and defecation in healthy volunteers. Bifid (Journal of Japanese Bifidus Foundation) 8:21–28. Hosoda M, He F, Kojima T, Hashimoto H, Iino H (1998) Effects of fermented milk with Lactobacillus rhamnosus GG strain administration on defecation, putrefactive metabolites and fecal microflora of healthy volunteers. Health Nutr Food Res 11:1–9. Iliev ID, Tohno M, Kurosaki D et al. (2008) Immunostimulatory oligodeoxynucleotide containing TTTCGTTT motif from Lactobacillus rhamnosus GG DNA potentially suppresses OVA-specific IgE production in mice. Scand J Immunol 67:370–376. Kawase M, He F, Kubota A, Hata J, Kobayakawa S, Hiramatsu M (2006) Inhibitory effect of Lactobacillus gasseri TMC0356 and Lactobacillus GG on enhanced vascular permeability of nasal mucosa in experimental allergic rhinitis of rats. Biosci Biotechnol Biochem 70:3025–3030. Kawase M, He F, Kubota A, Harata G, Hiramatsu M (2007a) Orally administrated Lactobacillus gasseri TMC0356 and Lactobacillus GG alleviated nasal blockage of guinea pig with allergic rhinitis. Microbiol Immunol 51:1109–1114. Kawase M, He F, Harata G, Kubota A, Mizumachi K, Hiramatsu M (2007b) Characterization of inhibitory effects of lactobacilli against immunoglobulin E production in vitro and in vivo. Int J Probiotics Prebiotics 2:29–38. Kawase M, He F, Kubota A et al. (2009) Effects of fermented milk prepared with probiotic strains Lactobacillus gasseri TMC0356 and Lactobacillus GG on Japanese cedar pollinosis: a double-blind placebo-control clinical study. Int J Food Microbiol 128:429–434. Kitsukawa T (2008) Development of FOSHU focusing on its safety. Trace Nutr Res 25:36–40. Kubota A, He F, Kawase A et al. (2009) Lactobacillus strains stabilize intestinal microbiota of patients with Japanese cedar pollinosis. Microbiol Immunol 53:198–205. Morita H, He F, Kawase M et al. (2006) Preliminary human study for possible alteration of serum immunoglobulin E production in perennial allergic rhinitis with fermented milk prepared with Lactobacillus gasseri TMC0356. Microbiol Immunol 50:701–706. Salminen S, Ouwehand A, Benno Y, Lee YK (2000) Probiotics: how should they be defined? Trends Food Sci Technol 10:107–110. Salminen S, Benno Y, Winnok DV (2006) Intestinal colonisation, microbiota and future probiotics? Asia Pac J Clin Nutr 15:558–562. Yamada K, Sato-Mito N, Nagata J, Umegaki K (2008) Health claim evidence requirements in Japan. J Nutr 138:1192S–1198S.

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9

Regulation of Probiotics in China

Anu Lahteenmäki-Uutela

9.1

INTRODUCTION

Probiotic bacteria as food ingredients tend to come under the Chinese definition and regulation on novel foods. Final food products with health claims related to probiotics come under the Chinese definition and regulation on health foods. This chapter discusses Chinese regulations on health foods, and probiotics in particular, and on novel foods. The legal issues include product safety and product efficacy. Rules on efficacy translate into rules on claims. Novel foods cannot bear health claims, health foods can. No food can bear medicinal claims. I will also look at the distinction between foodstuffs and medicinal products. In China, national law is given by the National People’s Congress (NPC), the State Council, or authorities under the State Council. If the State has not formulated standards for a certain food, the people’s governments of the provinces, autonomous regions or municipalities directly under the Central Government may establish local standards. Local standards are plentiful, which has led to inconsistent standards and confusing licensing requirements (Li & Fung Research Centre, 2005). Existing principles of legislative competence are upheld by the 2009 Food Safety Law: under Article 24, local food laws can be developed in the absence of national laws. Besides discrepancies between national and local laws, other general issues of the Chinese legal system include the strong role of the Communist party, use of protectionist practices, and the weakness of courts. I will not discuss these general issues of the Chinese legal system here (see Lähteenmäki-Uutela, 2009.)

9.2

HEALTH FOOD OR MEDICINE?

Functional foods often comprise materials that are used in traditional Chinese medicine. The legal difference between health foods and medicines is that health foods have a special health function, but are not for curing a disease. The categorisation decision (medicine vesus non-medicine) is legally important. However, the Chinese classification rules are not clear and the State Food and Drug Administration (SFDA) has not yet published guidelines on the subject. Companies are consulting the SFDA on a case-by-case basis (Tsoi, 2007). In the Chinese Medicine Administration Law (2001), medicines are defined as ‘articles intended for use in the prevention, treatment or diagnosis of human diseases and intended 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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to affect human physiological functions, for which indications or major functions, usage and dosage are prescribed.’ Medicines include raw traditional Chinese medicinal materials, traditional medicines prepared in ready-to-use forms, and other prepared Chinese medicines, medicinal chemicals and their preparations, antibiotics, biochemical medicines, radioactive drugs, serums, vaccines, blood products, diagnostic aids, etc. (Medicine Administration Law, Article 102). The Chinese want to emphasise the equal status of modern and traditional medicines. Since June 2009, the basic piece of food law is the Food Safety Law. The Standing Committee of the National People’s Congress passed the new law on 28 February 2009, and it went into effect at the beginning of June 2009. Based on the new law and its implementing regulations, Chinese food law and particularly its implementation and control will be developed. The new law uses Western terminology in promoting safety ‘from farm to table’. The new law tries to lay out who is responsible for what, focusing on the critical points where there have been problems in the past. The law makes a distinction between foodstuffs and medicines in Article 50: ‘Medicines cannot be added to food, unless the added substance is traditionally considered both food and Chinese medicine’. This means that foods must not contain purely medicinal substances, but can contain materials that have traditionally served as both food and medicaments. The major implication of a product being classified as a medicine is that it imposes higher requirements for producers and sellers. For example, manufacturers of medicines must hold a Good Manufacturing Practice (GMP) certificate, and wholesale and retail enterprises must hold a Good Supply Practice (GSP) certificate. The implications of the categorisation decision on marketing are also important. Only medicines can bear medicinal claims, i.e. claims of preventing, treating or curing a disease. Only health foods can bear health claims. Article 11 of the Health Food Regulation separates health foods and medicines from each other: ‘Any medicinal product approved by the government should not apply for the Certificate of Approval on Health Food’. In this way the health food category separates health foods from regular foods at one end and medicines at the other. The function and the claim resolve the foodstuff versus medicine issue, not the raw material as such.

9.3

HEALTH FOOD REGULATIONS

On health foods, the basic national law is the Health Food Regulation by the Ministry of Health (1996). In addition, health foods are governed by over 20 other rules or notifications from the Ministry of Health or the SFDA. These include, for example, the Interim Regulations for the Registration of Health Foods, General Safety Requirements for Health Foods, Standards for Toxicological Assessment of Health Foods, Technical Standards for Testing and Assessment of Health Food, Provisions for Health Food Labelling and the Notification on Pre-Market Control of Health Food Advertisements. Health foods are subject to a pre-market control procedure by the SFDA. The Chinese category for health foods covers a wide range of different products and includes the European categories of food supplements, fortified foods and dietetic foods. According to Article 2 of the Health Food Regulation, health foods are foods that: ● ●

claim to have certain health functions or aim at supplementing vitamins and minerals; are used for certain groups of people with the aim of adjusting bodily functions;

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are not for therapetic purposes (for curing, treating or preventing diseases); will not cause any acute, subacute or chronic damage to human body.

The new Food Safety Law states the basic rules on health foods in its Article 51. It is first stated that ‘the state executes strict regulations on health foods’. Health foods may not pose acute, subacute or chronic hazard to the human body. Labels and instructions may not refer to disease treatment or prevention, all information and claims must be truthful, and the product must correspond to the information given. More precise rules on health foods are given by the Health Food Regulation of 1996 and its subsequent amendments. The Health Food Regulation was enacted to strengthen the administration and supervision of health foods, ensuring the quality of health foods (Article 1). The regulations focus on evaluation and approval of health foods, and are as such directed more at authorities than at entrepreneurs. However, there are also sections regulating production and marketing of health foods. Besides general hygiene rules, the safety of health foods is controlled by regulating the choice of raw materials. A separate Notification on Further Standardizing the Management of Raw Materials for Health Foods from 2002 governs the raw materials of health foods. First of all, the notification aims to clear the situation of overlapping pieces of legislation. If a health food has novel ingredients, the Novel Food Regulation shall be followed. Additive law applies to additives in health foods. This means that the health food regulation concerns final products with health claims, and that healthy raw materials may first need to be authorised according to the novel food regulation. The Raw Material Notification includes lists of plant and animal materials that can be used or which are prohibited in health foods. There are particular rules on fungal health foods, nucleic acid type of health foods, and probiotic health foods. The Probiotic Health Food Regulation applies since 2005. ‘Probiotic health food’ means a preparation that promotes ecological balance of the bacterial colonies in the intestine and is beneficial to human health (Article 2). The probiotic strains must belong to normal bacterial colonies in the human body. The probiotic health food must be safe and dependable, i.e. safe for eating with no adverse reactions. The strains used in production must have clear and stable biological characteristics, genetics and efficacy (Article 3). The use of live or dead bacteria and bacterial metabolites is allowed (Article 8). Only certain probiotic bacteria are allowed as health food raw materials. The SFDA promulgates a list of authorised probiotics (Article 4). If an entrepreneur wants a strain added to the list, an application to the SFDA must be made (Article 5). The 10 currently permitted beneficial bacteria are Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Lactobacillus delbrueckii subsp. bulgarigus, Lactobacillus acidophilus, Lactobacillus casei subsp. casei, Lactobacillus reuteri and Streptococcus thermophilus. Next, we turn to marketing of health foods. There is certain mandatory information that must be given to consumers via the labels or package leaflets on health foods: suitable and unsuitable user groups, dosage, storage life, storage method and precautions. The active ingredients and their content must also be given. Names of health foods should be accurate and scientific, and should not use names of people, names of places, names that are misleading or exaggerating, or names of minor effective components. There shall be no reference to therapeutic effects. Misleading advertising of health foods is prohibited. The content of the product description shall be accurate. The advertised functions and ingredients of the product shall be identical with the information given in the label and specification,

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and there shall be no false information. Claims that refer to prevention or treatment of disease (medicinal claims) are prohibited on health foods (Food Safety Law, Health Food Regulation) (Huang & Lapsley, 2005). It is important to notice that in China, only health foods can bear health claims. Regular foods cannot bear health claims. On the other hand, health foods cannot bear medicinal claims. This way the health food regulation separates normal foods from health foods, foodstuffs from medicines, and food advertising from medicine advertising. To make the separate functions clear, health foods must bear the advice ‘This product cannot substitute any medicine’ (Functional Ingredients, 2005). The verb ‘assist’ is now used in six health claims. For example, one cannot say that a product lowers hypertension; instead it is acceptable to say that it assists in hypertension alleviation. This approach is similar to the European rules on health claims: one has to remind the consumer of the lifestyle factors. The possible functions and claims for health foods include the following: ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

enhancing immune function; assisting in blood lipid reduction; assisting in blood sugar reduction; antioxidant (delay in ageing); assisting in memory improvement; reducing eye fatigue; facilitating lead excretion; thinning throat mucus (moistening of throat); assisting in hypertension (blood pressure) reduction; enhancing sleep; promoting lactation; alleviating physical fatigue; enhancing anoxia endurance; assisting protection against irradiation hazard; weight reduction; enhancing child growth and development; increasing bone density; alleviating nutritional anaemia; assisting in protection against liver chemical injury; alleviating acne; eliminating skin pigmentation; improving skin moisture; improving skin oil content; regulating gastrointestinal flora; facilitating digestion; alleviating constipation; assisting in protection against gastric mucosa injury.

The Chinese health claim categories cover many of the important claims that are interesting to functional food developers. The Ministry of Health revised the procedures for efficacy evaluation in 2003, when the current list of 27 functions/claims came into force. Gut health claims were separated into four different claims (Huang & Lapsley, 2005). Two important claim types are prohibited: claims related to cancer, and claims related to sexual

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functions. It is not possible to market an all-purpose health food: a health food with same recipe is not allowed to apply for more than two functions. As regards efficacy of health foods, necessary animal and/or human tests must have confirmed a clear and stable health effect, and formulation and dosage must be based on scientific evidence (Huang & Lapsley, 2005). According to the current rules on health food efficacy, animal trials are required for 22 of the 27 claims. Animal trials are naturally not required for cosmetic claims. They are also not required for alleviating eye fatigue. Animal trials are thus required for all gut health claims. Experimental requirements for animal trials include tests on mice or rats and that the length of test ranges from 7 to 45 days (Huang & Lapsley, 2005). Human trials are required for 20 claim categories. Requirements on test length range from 7 to 45 days. Human trials are not required for claims on enhancement of immune function, enhancement of sleep, alleviation of physical fatigue, enhancement of anoxia endurance, assisting in protection against irradiative hazard, increasing bone density, and assisting in protection against liver chemical injury (Huang & Lapsley, 2005). Human trials are thus required for all gut health claims: regulating gastrointestinal flora, facilitating digestion, alleviating constipation and assisting in protection against gastric mucosa injury. If both animal and human tests are required, human tests are performed after obtaining a positive result with animal tests. Before a human feeding trial can be started, it must receive approval from an ethical committee. The regulations do not give precise requirements on how effective a product must be. Similarly to the European rules on claims, the regulations only give the scientific criteria used in the evaluation of the application. Whether the product is effective enough is a question that the SFDA will have to evaluate on a caseby-case basis. For more information on the application process, see the Regulation. Choosing a distribution and marketing strategy is critical when selling health foods in China. Regulatory issues affect marketing strategy. According to Aunew Export Group, setting up chains of health food stores might be the most competitive alternative in China, and there is also potential with e-business. On health food advertisements, there is a premarket control system. Also, direct sale companies need to have a certificate (Aunew Export Group). In 2002, 90% of health foods were marketed by Chinese companies, while only 10% were imported (Huang & Lapsley, 2005). Many foreign food manufacturers, such as Danone, have set up Sino-foreign companies to produce locally in China (Li & Fung Research Centre, 2005). In 2002, 60% of authorised health foods were focused on three functions: immune regulation, blood pressure regulation, and anti-fatigue. Health foods were not often in regular food form; instead they were primarily in the form of oral liquid, capsule, tablet or powder. It is noteworthy that in about 90% of health foods, the active ingredients of the products were related to traditional Chinese medicine (Wang et al., 2003). Today, other types of health foods are also emerging, and probiotic products are among those gaining interest. According to Nutraingredients.com (2005), the probiotics market started to expand in 2005, when probiotics were included in more and more products. Most companies were simply adding probiotic bacteria to yogurts without communicating their benefits. The higher average income of urban Chinese consumers has increased the demand for meat, fresh seafood, organic vegetables and health foods. Consumers are broadening their diets to include poultry, eggs, dairy products, fish and refined vegetable oils. Quality food and branded food products are also gaining popularity, particularly baby foods and children’s foods. Luxury goods, gift-giving and entertainment are rising trends, as well as convenience/processed food in tidy packages. Stressful and quick-paced lifestyles have

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increased the demand for functional foods (Li & Fung Research Centre, 2005). Dairy products, especially milk and yogurt, are positioned as a source of quality protein and calcium (Li & Fung Research Centre, 2005). Urban households seem to be trading up from plain milk to higher value-added dairy products (Li & Fung Research Centre, 2006).

9.4

NOVEL FOOD REGULATION

A new Novel Food Regulation came into force in China in 2007. The regulation stipulates the definition, safety assessment, application and approval process, production management, and hygienic inspection of novel foods in China. The regulation does not cover genetically modified food, which is regulated separately. Production and import of novel foods requires pre-market approval by the Ministry of Health. Novel foods are divided into four groups: I II III IV

Animals, plants and microorganisms not traditionally consumed in China. Raw food materials derived from animals, plants and microorganisms and which are not traditionally consumed in China. New varieties of microorganisms used during food processing. Raw food materials, the original composition or structures of which are changed by the adoption of new techniques during production (Article 2 of the Regulation).

Novel foods shall comply with other food laws, and they shall not cause any acute, subacute, chronic or other latent health hazards (Article 5 of the Regulation). Safety assessment shall follow the principles of risk assessment and substantial equivalence (Article 6 of the Regulation). ‘Risk assessment’ refers to scientific evaluation on known or potential negative effects on human health, including four steps: hazard identification, hazard characterisation, exposure assessment and risk characterisation (Article 28 of the Regulation). Substantial equivalence is also explained in the Regulation: If raw materials or food ingredients of a novel food are substantially equivalent to traditional food or food ingredients or approved novel food in terms of species, source, biological characteristics, main ingredients, edible parts, dosage level, scope of application and group of application, and their processing techniques and quality standards adopted are basically identical, the novel food is considered equally safe as the traditional counterpart and has substantial equivalence (Article 28 of the Regulation).

The Assessment Committee responsible for the scientific evaluation shall base its decision on the following materials and data (Article 8 of the Regulation): source of novel food, traditional consumption history, processing techniques, quality standards, main ingredients and contents, estimated intake, usage and scope of application, toxicology, biological features, genetic stability, pathogenicity and toxicity of strains of microbiological products and other scientific data. If necessary, safety will be reassessed (Article 9 of the Regulation). The Ministry of Health shall publish a list of approved novel foods (Article 14 of the Regulation), and also a list of foods that are considered regular (non-novel) foods (Article 15). It is important to note that novel foods cannot bear health claims: ‘Enterprises manufacturing, operating and/or using novel foods shall not claim or imply the therapeutic effects and health functions of the novel food’ (Article 23 of the Regulation). This means

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that, as in the EU, novel food assessment is purely safety assessment. If health claims are required, the health food procedure must be followed. According to AP-Foodtechnology.com, the market for novel foods in China was still largely untapped in 2007, but was expected to grow with the wealth of the country. Novel foods are usually ingredients used in ready-to-consume products such as health drinks. The aim of the Chinese government was to tighten food safety while removing complex approval procedures and thus encouraging scientific research to add variety to the market. When the new Regulation came into force, there were some 340 novel foods on the market authorised according to the previous regulation. The sugar replacer Isomalt produced by the company Palatinit was in 2006 the first non-Chinese food to pass the existing novel food approval process (AP-Foodtechnology.com, 2007). In April 2009, GenMont Biotech gave a press release stating that its two probiotic strains, GM-080 and GMNL-33, had been approved as novel foods in China. According to the company, 15 probiotic strains had received approval under the novel food regulation in China, two of them being GenMont ingredients. In 2009, GenMont was negotiating with ‘some of China's leading food companies’ to have the ingredient added to health foods for sale on the Chinese market (Pharmalicensing.com, 2009).

REFERENCES AP-Foodtechnology.com (2007) Breaking News on Food and Beverage in Asia Pacific. China introduces new novel foods regulation. Article by George Reynolds, 24 July 2007. Available at www.ap-foodtechnology. com/Formulation/China-introduces-new-novel-foods-regulation Aunew Export Group (Australia New Zealand Healthcare Association). Available at www.aunew.org Functional Ingredients (2005) China News December 2005. Available at www.functionalingredientsmag.com Huang G, Lapsley K (2005) Chinese (health) functional food regulations. In: Hasler CM (ed.) Regulation of Functional Foods and Nutraceuticals. A Global Perspective. Malden, MA: Blackwell Publishing Ltd., pp. 263–292. Lähteenmäki-Uutela A (2009) Foodstuffs and medicines as legal categories in the EU and China. Functional Foods as a Borderline Case. Doctoral thesis, University of Turku. Available at https://oa.doria.fi/ handle/10024/52491 Li & Fung Research Centre (2005) Report on food consumption in China 2005. Available at www.idsgroup. com/profile/pdf/industry_series/industry_series3.pdf Nutraingredients.com (2005) Probiotics set for explosive growth in China. News headline by Dominique Patton, 15 December 2005. Available at www.nutraingredients.com Pharmalicensing.com (2009) GenMont probiotics products receive China market entry approval. Press release 16 April 2009. Available at www.pharmalicensing.com Tsoi A (2007) Pharmaceutical policies and regulations in China. Deacons law firm article. Available at www.deacons.com.hk/eng/knowledge/knowledge_290.htm Wang XC, Tao NP, Ni Y (2003) Current development of functional foods in China. Available at http://ift. confex.com/ift/2003/techprogram/paper_15155.htm

FURTHER READING China–Britain Business Council. Available at www.cbbc.org/ Hong Kong Trade Development Council. News on Chinese food regulations. Available at www.hktdc.com Li & Fung Research Centre (2006) Report on Chinese dairy market. Available at www.lifunggroup.com/ research/pdf/industry_series8.pdf USDA China. United States Department of Agriculture. Foreign Agricultural Service. China. Available at www.usdachina.org/ (includes English translations of Chinese food laws).

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REGULATIONS Food Safety Law of the People’s Republic of China. Entered into force 1 June 2009 (Food Safety Law). Ministry of Health. Administrative Measures on Novel Foods. Entered into force 1 December 2007 (Novel Food Regulation). Ministry of Health. Administrative Regulations for Health Foods. Entered into force 1 June 1996 (Health Food Regulation). State Food and Drug Administration. Regulation on Probiotic Health Foods. Entered into force 1 July 2005 (Probiotic Health Food Regulation). Medicine Administration Law of the People’s Republic of China. Entered into force 1 December 2001 (Medicine Administration Law).

GOVERNMENTAL WEB PAGES Official English-language web page of the Central People’s Government of the People’s Republic of China http://english.gov.cn/ State Food and Drug Administration English-language web page http://eng.sfda.gov.cn/eng/ Ministry of Health web page www.moh.gov.cn. (in Chinese only).

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10

Probiotics and Health Claims: an Indian Perspective

Jashbhai B. Prajapati and Nagendra P. Shah

10.1

THE BACKGROUND

India has recognized the role of diet in health and nutrition since ancient times, as evidenced by the treatises of Vedic culture. The tradition of using foods for healthpromoting or functional properties was influenced by the Ayurvedic system of medicine. These functional foods include herbal extracts, spices, fruits, and nutritionally improved foods or foods products with added functional ingredients and probiotics. An FAO (2004) report states that Indian people are known for often treating common ailments primarily with foods. Nine out of ten urban Indian consumers have been reported to generally choose foods based on health and wellness benefits (Ciocca, 2003). Fermented milk products like dahi (equivalent to western yogurt) and buttermilk are commonly recommended by elders in the family for control of diarrhea, dysentery and common intestinal ailments. A comprehensive historical overview of fermented foods and their application in health has been compiled by Prajapati and Nair (2003). Watson (2006) reported that, with its strong tradition of healthy eating, India ranks among the top 10 nations in buying functional foods, while Kotilainen et al. (2006) stated that the functional food industry in India is strong and growing, with the aim of becoming a major force in the international health foods market. Consumer spending has grown at an annual rate of more than 10% in the past decade in India. Robust growth is expected to continue in the functional foods industry in India, with productivity growing by over 60% during 2005 to 2010. Additionally, India's population is large and predominantly young, with 516 million people between the ages of 20 and 55; this number is expected to increase to 800 million within the next 40 years. As the younger generation moves toward middle age and disposable incomes increase, the need to maintain and/or establish a healthy diet will increase functional food consumption (Ismail, 2006).

10.2

THE STATUS

India is well aware of probiotics and their potential in human health. There is also a rich diversity of microorganisms, which are used as probiotics in Indian foods without sufficient scientific evidence of their clinical effects. However, increasing awareness in society, developing

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interests of the food industry, and scientific studies on use of probiotics in the Western world has also stimulated investigations in India. During the last two to three decades, a number of institutions have conducted scattered in vitro studies to establish the probiotic nature of selected microorganisms. However, there is paucity of systematic studies to prove the effects of microorganisms in animal and human models. There are only three or four clinical studies on well-known imported probiotic cultures in India. A limited number of reports is also available on small-scale studies in various animal models and human volunteers. However, the number of Phase I and II clinical studies on probiotics in India is on rise. The present chapter describes in vivo studies reported in animal models and clinical intervention studies in humans to give an idea about the Indian perspective on probiotics.

10.3

ANIMAL STUDIES

Most of the studies are in the form of feeding a dairy-based product containing probiotic culture to commonly used animal models like mice, calves and chicks.

10.3.1

Chicken

The immunomodulatory effect of probiotic Lactobacillus was studied in a chick model by Patidar and Prajapati (1999). Acidophilus milk prepared by using four different probiotic strains of Lactobacillus was fed orally at the rate of 1 mL (3.3–6.8 × 108 cells) per day to 3-day-old broiler chicks (N = 20 in each group) for 8 days and HI antibody titer was monitored as a measure of immune response for 5 weeks. All four chick groups fed fermented milk showed higher antibody titers (160–233) than the control group (148) fed only skim milk, which indicated the immunostimulating ability of the fermented milk. There were no significant differences in body weight gain, feed consumption rate, and fecal flora in chicks in all five groups. In a challenge study, three groups of 20 chicks each were fed with either skim milk or milk fermented by L. acidophilus C2 or L. acidophilus P for 16 consecutive days. All the chicks were then challenged with pathogenic Escherichia coli. Within 72 hours post challenge, 93% of birds in the control group died. However, in chicks fed acidophilus milk fermented by L. acidophilus C2 and P, the mortality was only 27% and 53%, respectively. This indicated that feeding acidophilus milk offered protective effect to the chicks against E. coli infection (Patidar & Prajapati, 1996). Katoch et al. (2003) studied the comparative dietary response of different strains of Lactobacillus, Lactococcus and yeast isolated from indigenous sources as probiotics in the commercial egg type chicken. Various treatments consisted of control (culture medium, T0), Lactobacillus plantarum isolated from carrot (T1), Lactococcus faecium isolated from onion (T2), Saccharomyces cerevisiae isolated from plum (T3), Lactobacillus bulgaricus standard L4 (T4), Lactococcus lactis standard S1 (T5), and Saccharomyces cerevisiae standard Y3 (T6). The results showed that weight gain and feed conversion ratio were better in treatment groups T1 and T4 compared with control group T0 during the starter phase. Growth was lower in all the treatment groups fed probiotics compared with control during the grower phase. Egg-laying performance during the first 4 months was better in the T1, T4, T5 and T6 treatment groups compared with that of the control group and showed significantly higher (P < 0.05) percent hen-day egg production.

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The dietary performance of the combination of L. acidophilus, Enterococcus faecalis and Saccharomyces carlsbergensis isolated from leopard (Panthera leo) excreta and the combination of their respective standard strains Lactobacillus bulgaricus L4, Lactococcus lactis S1 and Saccharomyces cerevisiae Y3 was studied by Katoch et al. (2000) in three strains of commercial broilers up to 6 weeks of age. During the starter (1–4 weeks), grower (5–6 weeks) and overall (1–6 weeks) phases, both the microbial combinations gave higher (P < 0.05) weight gain only in Vancob strain of broilers compared with the control treatment, while in Starbro and Kegbro strains the effects were not significant. The differences in feed conversion ratio and mortality were not significant in all situations. Ramesh et al. (2000) conducted a study on 160 1-day-old Salmonella-free chicks, which were subjected to various treatments, namely control (T1), L. acidophilus (108 CFU/bird) fed for 2 weeks (T2), L. acidophilus fed for 2 weeks followed by oral infection with Salmonella gallinarum (101 organisms per 0.1 mL bacterial suspension; T3), and the birds infected orally with S. gallinarum (T4). T4 birds manifested clinical signs of dullness, inappetence, reduced growth rate and diarrhea, while those pretreated with lactobacilli (T3) remained normal. Birds in the T1 and T2 groups were active and healthy. Birds in the T3 group showed viable counts of Salmonella only on day 1 and 3 post infection and which were significantly (P < 0.05) low compared with viable counts on respective days in T4 group birds. Birds fed Lactobacillus showed a lowered surface pH in the duodenum, jejunum, ileum and cecum. Maheswari and Kadirvel (2003) studied the interaction of fumaric acid (FA) with probiotic and antibiotic in day-old broiler chicks. Both antibiotic and probiotic significantly improved the growth rate of broilers, but FA supplementation in drinking water (10 g/L), with or without probiotics (100 mg/kg), antibiotics (virginiamycin 10 mg/kg) or both, depressed the growth rate significantly. FA supplementation increased the packed cell volume and probiotic reduced serum cholesterol levels. An experiment was conducted by Panda et al. (2000a) on 320 broiler chicks to evaluate the influence of dietary supplementation with probiotics on immunocompetence, response to E. coli, growth and carcass characteristics. The dietary treatments consisted of a basal diet (control) and three other diets with the same composition as the basal diet but supplemented with probiotic (Probiolac, 100, 150 or 200 mg/kg diet). Supplementation with 100 mg probiotic significantly improved body weight gain (0–4 weeks) but no difference was observed subsequently. Probiotic supplementation of the diet did not influence feed consumption or feed conversion ratio. There was significantly higher antibody production in the 100-mg probiotic supplementation group at 10 days and 5 days post inoculation in response to sheep red blood cell antigen when injected at 14 days and 21 days of age, respectively, as compared with the control. The birds fed probiotic were less susceptible to E. coli challenge than controls, although no difference was observed in the weight of bursa and spleen due to probiotic supplementation. The probiotic had no influence on dressing percentage or weight of internal organs such as liver, heart and gizzard. The effect of dietary supplementation with probiotics on growth, serum cholesterol and gut microflora of broilers was determined by Panda et al. (2001). It was observed that probiotic supplementation had no effect on liveweight gain at 6 weeks of age. A significant reduction in total serum cholesterol was observed, but no effect was found on high-density lipoprotein (HDL), very low density lipoprotein (VLDL), low-density lipoprotein (LDL) and triglycerides. A significant decrease in crop E. coli count was observed, although the reduction was not significant in the cecum.

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The efficacy of various Lactobacillus strains on broiler production was studied by Senani et al. (2000). Three strains of Lactobacillus, NCDC-3 (L. delbrueckii subsp. lactis), NCDC-8 (L. delbrueckii subsp. bulgaricus) and NCDC-14 (L. acidophilus), were given to three groups of 20 1-day-old chicks until they were 9 weeks of age. A fourth group served as the control. No difference in 6-week body weights was observed between treatments. Mortality (0–6 weeks) was highest in chicks given NCDC-14 and lowest in those on NCDC-3. However, 9-week body weights were higher in the NCDC-3 and NCDC-8 groups compared with control. Oral administration of the Lactobacillus strains NCDC-3 and NCDC-8 to broilers improved growth and feed conversion efficiency. Kumar et al. (2002) tested the efficacy of supplementing probiotics such as L. acidophilus, mannan oligosaccharide and native gut culture in the prevention of experimental Salmonella gallinarum infection in broiler chicks. Day-old commercial chicks were randomly divided into four groups with 36 chicks each. Birds infected with S. gallinarum but not pretreated with probiotics (group 1) manifested clinical signs of dullness, lack of appetite and diarrhea, whereas probiotic-supplemented birds did not show any clinical symptoms after inoculation of S. gallinarum. This study indicated that early establishment of L. acidophilus, mannan and native gut culture in the gastrointestinal tract helped increase the resistance to Salmonella colonization and may even have a possible role in competitive exclusion by elimination of Salmonella organisms from the gut. Satbir et al. (1999) investigated the influence of Lactobacillus sporogenes (note that this designation does not accord with current nomenclature) on mortality and economics of 480 broiler chicks. Day-old chicks of a commercial broiler strain were reared up to the age of 8 weeks on 2600, 2900 and 3200 kcal ME/kg diet, each with 0.00, 0.02, 0.03 and 0.04% probiotic (L. sporogenes). The addition of probiotic decreased mortality. Weight gain and feed efficiency were highest for diets containing 0.02% probiotic and lowest for diets with 0.04% probiotic. An experiment was conducted by Panda et al. (2000b) to examine the effect of probiotic supplementation on performance and immune response of White Leghorn layers aged 48–64 weeks. The birds in each group were supplied with one of three diets: basal diet, basal diet with 100 mg of probiotic (a commercial preparation containing L. acidophilus, L. casei, B. bifidum, Aspergillus oryzae, Enterococcus faecium and Torulopsis spp. with 27 × 109 CFU per 100 g), and basal diet with 200 mg of probiotic per kilogram diet. At 56 and 64 weeks of age, 12 birds per group were immunized with 0.1 mL of 0.5% sheep red blood cells (SRBC) and blood samples were collected after 5 days to measure antibody production. Supplementation of probiotic at 100 mg/kg improved (P < 0.05) hen-day egg production. Probiotic supplementation had no effect on daily feed intake, feed conversion, egg weight, or on concentration of albumin and yolk. However, there was an improvement (P < 0.05) in shell thickness by supplementation of with probiotic at 100 mg/kg. Antibody production in response to SRBC was also higher (P < 0.05) in the group supplemented with probiotic at 100 mg/kg.

10.3.2

Albino rats

Ruchi et al. (2006) conducted feeding trials on Swiss Albino rats (N = 10) with different strains of L. acidophilus and B. bifidum fed as free or immobilized cells via skim milk medium at the rate of 1 × 1010 cells per mouse per day. Examination of fecal flora revealed a greater persistence of probiotic strains in the intestinal contents of the groups fed on coimmobilized cells. Further, the feeding of probiotic cells resulted in lowering of fecal coliforms and b-glucuronidase activity.

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The preventive action of probiotic dahi containing L. acidophilus and L. casei was evaluated by Yadav et al. (2007) in a group of Wistar Albino rats that were induced to develop type 2 diabetes by 8 weeks of feeding 21% fructose in water. It was observed that the probiotic dahi-supplemented diet significantly delayed the onset of glucose intolerance, hyperglycemia, hyperinsulinemia, dyslipidemia and oxidative stresses, showing a lower risk of diabetes. The immunomodulatory effects of oral feeding of a dahi (stirred yogurt) containing L. casei was evaluated in mice for 8 days (Shalini et al., 2008). The animals (N = 15) fed probiotic dahi at the rate of 5 g per day per mouse (count of probiotic cells not indicated) showed a higher lactobacilli count in fecal flora and significant decreases in fecal b-galactosidase and b-glucuronidase activities. Further, the macrophages isolated from the peritoneal fluid showed increased phagocytic action as revealed by an in vitro yeast phagocytosis assay. Guhapriya et al. (2007) tested the effect of oral feeding for 4 days of the probiotics L. rhamnosus (1 × 107 CFU/mL) and L. acidophilus (1 × 107 CFU/mL) or the combination thereof in a rat model (N = 6 in each group) challenged by oral administration of Shigella dysenteriae 1 (12 × 108 CFU). Administration of S. dysenteriae 1 alone resulted in increased levels of myeloperoxidase, lipid peroxidation, alkaline phosphatase, and expression of MMP-2 and MMP-9 with concomitant decrease in antioxidant levels. Pretreatment with the combination of L. rhamnosus and L. acidophilus significantly attenuated these changes compared with the diseased group, indicating better protection. They further reported that L. rhamnosus and L. acidophilus synergistically reduced membrane damage during S. dysenteriae 1 infection by reducing membrane-bound ATPases and reduced expression of tight junction proteins (Guhapriya et al., 2009). Selvam et al. (2009) conducted a trial on 42 male Wistar Albino rats with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis to examine the therapeutic effect of a novel natural probiotic Bacillus subtilis PB6 (ATCC- PTA 6737). The animals treated orally with Bacillus subtilis PB6 at 1.5 × 108 CFU/kg thrice daily from 4 to 10 days significantly improved gross pathology of the colon with regain of colon weight to normal, compared with TNBS-induced positive control. Plasma levels of proinflammatory cytokines, i.e. tumor necrosis factor (TNF)-a, interleukin (IL)-1b, IL-6 and interferon (IFN)-g, were significantly lowered and anti-inflammatory cytokines, i.e. IL-10 and transforming growth factor (TGF)-b, significantly increased after the oral administration of PB6 on day 11. This proved the efficacy of probiotics in inflammatory bowel diseases. The effect of lyophilized lactic microbes on mouse macrophage-mediated immune functions was evaluated by Balasubramanya et al. (1999). The mice given lyophilized L. acidophilus showed a higher phagocytic index compared with L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

10.3.3

Pigs

A study was conducted on growing crossbred pigs by Ravi et al. (2000) to assess the effect of supplementation with yeast (Saccharomyces cerevisiae) or L. acidophilus or both to the diet on growth performance. A total 24 crossbred male pigs were randomly divided into four equal dietary treatment groups: control (R1), supplemented with live yeast 0.1% (R2), supplemented with 20 g L. acidophilus (R3), or supplemented with both live yeast 0.1% and 20 g L. acidophilus (R4). The study revealed that supplementation with L. acidophilus at 20 g/day was beneficial for growing pigs and economical during growth phase (13–35 kg liveweight).

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139

Sheep

The study by Rao et al. (2001) inferred that probiotic (L. acidophilus, Saccharomyces cerevisiae) supplementation to complete rations improved rumen bacterial count, total volatile fatty acid (TVFA) production and nutrient digestibility in sheep. The effect of supplementation with probiotics on growth performance in 20 Nellore brown male lambs was evaluated by Rao et al. (2003). A complete ration with 40 : 60 concentrate to roughage ratio supplemented with L. acidophilus 1 g, Saccharomyces cervisiae (Yea-Sacc1026) 10 g, or L. acidophilus 0.5 g plus (Yea-Sacc1026) 5 g was fed for 70 days. The study indicated that supplementation with probiotics to complete rations improved efficiency of feed utilization and daily weight gains in sheep.

10.3.5

Calves

Kalyankar and Khedkar (2007) conducted a study on 105 randomly selected buffalo calves to evaluate the health effects of feeding a low-cost probiotic preparation. The calves received a whey-based probiotic preparation containing 107 CFU/mL of L. delbrueckii subsp. bulgaricus BL44 and Saccharomyces cerevisiae BY13 at the rate of 500 mL per calf per day for 28 days. The generalized observation showed that, compared with a control group, the calves fed probiotics had a higher population of friendly microorganisms in the fecal matter and had a reduced incidence of diarrhea and lower rate of mortality. The effect of dietary inclusion of yeast cell suspension (Saccharomyces cerevisiae ITCCF 2094) on rumen fermentation was evaluated in 6-day-old crossbred calves (Panda et al. 1999). All calves were fed whole milk up to 9 weeks while calf starter and green berseem were offered ad libitum from week 2 onwards until 13 weeks of age. The yeast cell suspension was supplemented in the experimental group at 5 × 109 CFU per head per day. Ruminal pH, TVFA and NH3-N did not differ significantly between groups. Yeast supplementation had a significant effect on reducing the rumen lactate concentration (P < 0.01). Amylase and protease activities in the strained rumen fluid remained unaffected whereas CM cellulase activity was high at 50 and 90 days of age in the experimental group. The total protozoal count was also higher in the yeastsupplemented group than in the control group. In a similar study, Saha et al. (1999) evaluated the effect of oral feeding of yeast cell suspension (S. cerevisiae) on feed intake, growth performance and nutrient utilization in 15-week-old calves. Average daily liveweight gain and dry matter, crude protein and crude fiber digestibility were higher (P < 0.05) in the experimental group. Rumen bacterial and protozoan populations were also higher in the experimental group. The incidence of calf diarrhea was reduced due to feeding of S. cerevisiae in the experimental group compared with the control. In a study by Das et al. (2001), 32 crossbred female calves were divided into four groups of equal number and fed either grain-based concentrate with lactic acid bacteria (GC), grain-based concentrate without lactic culture (GC0), grainless concentrate with lactic culture (G0C) and grainless concentrate without lactic culture (G0C0). The lactic acid bacteria culture was given in the form of milk fermented with L. acidophilus, L. jugurti and L. casei. The feeding of lactic acid bacteria resulted in a reduced incidence of diarrhea and the number of days on which positive symptoms of diarrhea were observed. The digestibilities of organic matter, neutral detergent fiber and acid detergent fiber were significantly higher (P < 0.05) in animals fed on grainless concentrate with culture. Feed conversion efficiency

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was improved accompanied with lower intake of nutrients in probiotic-fed animals, but the differences were not significant. Kamra et al. (2002) studied the effect of dietary supplementation with yeast cells on liveweight gain, nutrient digestibilities, production of rumen metabolites and enzyme activities in the rumen of 16 crossbred calves. Calves in the experimental group were given a daily dose (10 mL) of yeast cell suspension containing 5 × 109 cells/mL Saccharomyces cerevisiae ITCCF 2094 for 159 days. There was no difference in liveweight gain, feed intake, feed conversion efficiency, digestibilities of nutrients and the nutritive value of diet between groups. The activities of rumen enzymes, namely carboxymethyl cellulase, amylase, xylanase, b-glucosidase, urease, and aspartate and alanine transaminases, were unaffected but protease was lower (P < 0.05) in yeast-fed animals. Production of TVFA remained unaffected but NH3-N and lactic acid decreased (P < 0.05) and pH increased (P < 0.05) in rumen liquor of the group given the yeast cell suspension.

10.3.6

Fish

A probiotic bacterium Bacillus circulans PB7 was used as a probiotic supplement in the feed for the fingerlings of Catla catla (Ham.) by Bandyopadhyay and Das Mohapatra (2009). Diets supplemented with three levels of B. circulans PB7 cells, i.e. 2 × 104, 2 × 105 and 2 × 106 per 100 g feed at 5% of body weight per day in two equal installments in triplicate treatments, was fed for 60 days. Fish fed with 2 × 105 cells displayed better growth, the highest RNA/DNA ratio, a lower feed conversion ratio, a higher protein efficiency ratio, highest carcass protein and lipid, highest protease and the highest TSP, albumin and globulin after the 60-day feeding trial. Phagocytic ratio, phagocytic index, and leucocrit values were highest in fish fed 2 × 105 cells. After the feeding trials, the fish were challenged for 10 days by bath exposure to Aeromonas hydrophila and the highest survival of 96.66% was observed in fish fed 2 × 105 cells of probiotics as compared with only 6.66% in the controls, indicating the effectiveness of B. circulans PB7 in reducing disease caused by A. hydrophila.

10.3.7

Post-larvae

The effect of formulated diets incorporated with different commercial probiotics on growth, feed utilization and survival of post-larvae of Macrobrachium rosenbergii was studied by Indulkar and Belsare (2003). Triplicate groups of 5-day-old post-larvae in an indoor nursery were fed for 15 days on one of the experimental diets. It was observed that the diet incorporated with probiotic (Gp-5) at the rate of 7.5 g/kg and consisting of combinations of L. sporogenes (45 × 109 CFU), L. acidophilus (45 × 109 CFU), B. subtilis (30 × 109 CFU), B. licheniformis (30 × 109 CFU), Saccharomyces cerevisiae (125 × 109 CFU) and seaweed extract (100 g/kg) resulted in better growth and better feed utilization of post-larvae of M. rosenbergii. In a similar study, Venkat et al. (2004) observed that probiotic strains had inhibitory effects against the Gram-negative bacterial flora present in the gut of post-larvae. Growth of the probiotic-fed groups was significantly higher (P < 0.05) than the control group. Significantly higher percent weight gain (132.5%), specific growth rate (1.41%), feed efficiency ratio (0.45), protein efficiency ratio (1.29) and protein gain (161.6%) were recorded in a group fed Artemia-bioencapsulated L. sporogenes over the control group. The growthpromoting effects of L. sporogenes were found to be higher than those of L. acidophillus.

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141

HUMAN STUDIES

Limited human clinical studies to test the efficacy and effectiveness of probiotics have been reported from India. Most of these studies are at Phase I level employing very limited numbers of volunteers/patients. A few studies in progress are employing commercial probiotic strains of non-Indian origin. Further, most of the studies concentrate on general well-being with less focus on the intestinal tract and intestinal ailments. Few studies also report data on the hypocholesterolemic and immunostimulating properties.

10.4.1

Probiotics in gut microbiology

In a very small and uncontrolled study, Prajapati et al. (1986) fed a probiotic L. acidophiluscontaining dried milk product at the rate of 10 g/day (8.7 × 108 cells/day) to six human volunteers with complaints of intestinal discomfort. During the 15 days of the feeding period and 15 days of follow-up, an increase in the lactobacilli population and a significant decrease in fecal coliforms in stool samples were recorded. Oral enquiry of the test subjects revealed that they felt relief from the gastrointestinal discomfort they were experiencing before the start of feeding. A random study of 11 human patients with various intestinal ailments was conducted in an institutional hospital by Khedkar et al. (1990a). All the patients were administered 200 mL of fermented milk containing 2 × 107 live cells/mL of the human strain of L. acidophilus V3 for 9 days, with a health check-up every third day. Patients were also receiving chemotherapy. It was observed that in all the cases a complete cure was possible within a period of 9 days. In another feeding trial, seven volunteers were each fed with 200 mL of milk fermented by L. acidophilus V3 or L. acidophilus I4 for a period of 7 days and the intestinal microbial population was monitored. Results showed a positive balance toward lactobacilli and a decrease in fecal coliforms (Khedkar et al. 1990b). Fresh bifidus milk containing 109 cells of Bifidobacterium adolescentis Hb1 was fed to a group of eight human volunteers at the rate of 100 mL/day for 1 week to study implantation ability in the intestine (Khedkar et al., 1994). Feeding of bifidus milk resulted in an increase (3–220 times) of bifidobacteria and decrease (3–30 times) in coliform counts in the feces of the volunteers. In an almost similar study, Patel et al. (1992) fed a consortia of three strains of Lactobacillus (5 × 107 cells/mL) in the form of acidophilus milk to human volunteers and noticed a positive balance of lactobacilli and reduction in coliform count in fecal samples. A study conducted by Khedkar et al. (2003) evaluated the implantation ability of a probiotic culture of L. acidophilus in the gastrointestinal tract of 135 tribal children aged 2–5 years. A control group received dahi containing 107 CFU/g of mixed lactic bacterial culture at the rate of 100 g/day per volunteer, a blank control group received 100 g buffalo milk per day per volunteer, and the test group volunteers received 100 g freshly prepared probiotic acidophilus milk (PAM) containing 107 CFU/g of a human isolate of L. acidophilus. Analysis of fecal flora was carried out before feeding, during feeding and after feeding to establish the effect on the gastrointestinal microflora. Feeding of PAM resulted in a manyfold increase in friendly types and a very sharp decline in harmful organisms after 15 days of commencing the feeding trial in test group children. This trend continued even after terminating the feeding, an indication of the positive implantation ability of the human

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strains in the gastrointestinal tracts of the subjects. There was no evidence of gastrointestinal ailments on the test group during and after 90 days of stopping the feeding trials. Fecal pH of all the test group subjects showed a steady decline during feeding. Residual lactase levels in fecal matter of the test group showed a significant increase over the control group. It was also observed that the fecal lactase level in the control group receiving dahi was comparable with that in test group volunteers. Agarwal et al. (2003) studied the effect of oral Lactobacillus GG (LGG) on enteric microflora in low-birth-weight neonates. A prospective randomized study was carried out on 71 preterm infants weighing less than 2000 g at birth. Infants weighing less than 1500 g (24 treated, 15 control) received 109 Lactobacillus GG orally twice daily for 21 days, while infants weighing 1500–1999 g (23 treated, 9 control) were treated for 8 days. Colonization with LGG occurred in 21% (in those less than 1500 g) and 47% (in larger infants). Larger infants had more bacterial species and higher log CFU compared with infants weighing less than 1500 g. Gram-positive mean log CFU showed a significant increase on day 21 (6.1 ± 0.9) as compared to day 0 (3.5 ± 0.9) (P < 0.05), with no significant change in species number and quantities in all infants. Furthermore, investigators concluded that neonatal response to the probiotic preparation depends on gestational and postnatal age and prior antibiotic exposure.

10.4.2

Probiotics in diarrheal diseases

A community-based, randomized, controlled, double-blind study was carried out by Aggarwal and Bhasin (2002) at the University College of Medical Sciences, Guru Teg Bahadur Hospital, Delhi to control acute diarrhea in children (N = 150) aged 6 months to 5 years. A total of 75 subjects in each hospital and slum cluster were randomized into three groups and administered with (1) the commercial fermented milk Actimel (108 organisms/g each of L. casei DN-11400, L. delbrueckii subsp. bulgaricus and S. thermophilus), (2) Indian dahi LF40 (108 organisms/g each of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Leuconostoc mesenteroides), and (3) ultra-heat-treated yogurt (without live bacterial cells) until the diarrhea was cured. Actimel showed superior effect (mean time to cure, 1.7 days) compared with Indian dahi (mean time to cure, 2.0 days) and UHT yogurt (mean time to cure, 2.25 days) in the control of acute diarrhea. Further, it was reported that families using Actimel as a starter for making household fermented milk and consuming it showed a reduction in diarrheal morbidity episodes by 40% of the children tested in the 3-month follow-up period. A hospital-based, randomized, placebo-controlled, double-blind study of 98 subjects with acute watery diarrhea aged 6 months to 12 years was carried out at the JN Medical College, Aligarh by Khanna et al. (2005). Treatment (N = 48) with a probiotic product containing 15 × 109 tyndalized L. acidophilus and with placebo (N = 50) with puffed rice powder were given until the patients recovered. The authors concluded that in the rotaviral diarrhea and in those subjects who had diarrhea of less than 60 hours, the difference did not reach statistical significance. The reasons could be small sample size or because these subjects had moderate to severe diarrhea or the period of supplementation was inadequate. Sur (2008) reported a study to evaluate the role of a probiotic milk-based beverage (Yakult) in prevention of acute diarrheal diseases in children aged 1–5 years in the urban slum area of Kolkata. The children were randomized into two groups: a probiotic arm (N = 1894) received 65 mL of probiotic beverage containing 15 × 109 live lactobacilli, while a second nutritional arm (N = 1864) received a placebo nutritional drink without lactobacilli. The baseline

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characteristics of the children in the two groups were comparable. The incidence of diarrhea was low in the probiotic group compared with the nutrient group (P < 0.05), and even the proportion of children suffering from diarrhea was significantly lower in the probiotic group compared with the nutrient group (33.7% vs. 37.8%; P < 0.05). Anthropometric indicators weight-for-age, Z-score and weight-for-age percent of median were compared between the groups and there was no significant difference between the two groups. A commercial probiotic preparation, Bifilac, containing bifidobacteria was evaluated in a randomized, double-blind, placebo-controlled study of 80 children aged 3 months to 3 years with acute viral diarrhea at Medical College Hospital, Mysore (Narayanappa, 2008). When compared with placebo, Bifilac showed clinical reduction in (1) number of episodes (frequency) of diarrhea in a day, (2) mean duration of diarrhea (4.35 days in Bifilac group, 5.45 days in placebo group), (3) degree of dehydration, (4) duration and volume of oral rehydration solution (ORS), (5) duration and volume of intravenous fluid therapy and (6) duration of rotaviral shedding. A hospital-based placebo-controlled study to evaluate the efficacy and tolerability of a commercial probiotic preparation, VSL#3 (CD Pharma India), in the treatment of acute rotavirus diarrhea in children was reported by Dubey et al. (2008). Oral treatment with the probiotic mixture or placebo in addition to usual care for diarrhea was carried out in 230 children with rotavirus-positive acute diarrhea for 4 days. Overall recovery rates were significantly better in the drug group compared with placebo. Use of the probiotic mixture resulted in earlier recovery and reduced frequency of ORS administration, reflecting decreased stool volume losses during diarrhea. No side effects were noted with the use of the probiotic mixture. Basu et al. (2009) tested the efficacy of L. rhamnosus GG in controlling acute watery diarrhea in Indian children in a randomized, controlled, hospital-based study over 1 year. The study comprised 559 patients, divided into group A (control, N = 185), group B (ORS + LGG powder containing 1010 cells, N = 188) and group C (ORS + LGG powder containing 1012 cells, N = 186), who were administered the probiotic twice daily for a minimum period of 7 days or until diarrhea stopped along with correction of dehydration. Frequency and duration of diarrhea, requirement for intravenous therapy, and hospital stay were significantly lower in both the intervention groups as compared with the controls. There was no significant difference between the two intervention groups. No complication was observed from the doses of LGG used. A randomized, controlled, hospital-based trial was carried out at the North Bengal Medical College and Hospital by Basu et al. (2007). A total of 235 subjects with persistent watery diarrhea were randomized to receive either 60 × 106 organisms of L. rhamnosus GG along with ORS (probiotic arm, N = 117) or ORS alone (control arm, N = 118) for 7 days or until diarrhea ceased. The results obtained with acute watery diarrhea indicated that the probiotic produced a significant decrease in the mean duration of diarrhea (5.3 vs. 9.2 days) as compared with the controls. Probiotic plus ORS could decrease the frequency and duration of diarrhea and vomiting and reduce hospital stay in children with persistent diarrhea.

10.4.3

Effects on lipid profile

A randomized feeding trial comprising 27 human volunteers with both normal lipid profile and hyperlipidemia was conducted by Ashar and Prajapati (2001). All the subjects were fed 200 mL of stirred acidophilus milk (containing 5 × 108 live lactobacilli per mL) for 20 days. Blood lipid parameters, namely total, LDL, HDL and VLDL cholesterol, triglycerides,

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LDL/HDL ratio and total/HDL ratio, were monitored for 20 days before feeding, 20 days during feeding and 20 days after feeding. The results showed wide variations among the volunteers. However, a significant reduction by 7.6% in total cholesterol and 15.7% in LDL cholesterol was noticed in the volunteers during the study. The volunteers were grouped according to sex, age, health status and initial cholesterol level. Feeding of acidophilus milk resulted in a reduction of total cholesterol by 11.7, 21.0, 12.4 and 16.4% in volunteer group A1 (40–60 years), C2 (200–220 mg/dL initial cholesterol), C3 (220–250 mg/dL initial cholesterol) and H1 (normal health), respectively. The feeding favorably affected total serum cholesterol and LDL/HDL or total/HDL ratios. However, its effect on triglycerides was neutral or negative in some cases. Overall, feeding was most beneficial to volunteers aged 40–60 years, who have the highest risk of heart attack. The feedback gathered from the volunteers was encouraging: apart from decreasing cholesterol level, probiotics also improved gastrointestinal health. Margaret and Roshanars (2007) conducted feeding trials with bioghurt on hypercholesterolemic women. A total of 50 postmenopausal women were recruited for the study through medical camps. Each volunteer received a bioghurt prepared with Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus containing 108 CFU/mL. The authors reported a significant decrease in total cholesterol, HDL cholesterol and serum triglycerides in the bioghurt-fed group as compared to placebo, thereby concluding that bioghurt is an effective hypocholesterolemic supplement.

10.4.4

Morbidity and nutritional status

A randomized, double-blind, placebo-controlled, preventive efficacy study was carried out on a semi-urban population in New Delhi by Sarkar et al. (2004). Children between 1 and 3 years of age (N = 634) with fever, diarrhea, ear infections, iron-deficiency anemia and malnutrition were randomized to receive either a milk fortified with probiotic [Bifidobacterium lactis HN019 (DR10)™, 9.6 × 106 CFU/day] and prebiotic (galacto-oligosaccharides 2.4 g/day) or unfortified milk for 12 months of study. The administration of probiotic and prebiotic resulted in a reduced incidence of dysentery [odds ratio (OR) 0.78, 95% confidence interval (CI) 0.61–1.00] and its prevalence (OR 0.85, 95% CI 0.71–1.01). Further, it also resulted in a non-significant reduction in the incidence of diarrhea (10%), a significant reduction in the prevalence of severe illness days (OR 0.84, 95% CI 0.74–0.95; P < 0.001), days with fever (OR 0.68, 95% CI 0.54–0.84), and prevalence of severe ear infections (OR 0.93, 95% CI 0.87–1.00). When both groups received isocaloric diets with the same iron content, there was a 35% reduction in the incidence of iron-deficiency anemia in the PP milk-supplemented group. Measles episodes were reduced by 53%. A study was conducted at a tertiary care hospital in east Delhi during September 2003 to August 2004 to assess the impact of supplementation with curd (dahi) and micronutrientrich leaf protein concentrate (LPC) on nutritional status and immunity as assessed by anthropometry, hemoglobin, ferritin levels, T-cell subpopulations and C-reactive protein (CRP) in children suffering from protein energy malnutrition (PEM) (Dewan et al., 2007). A total of 80 moderate to severely malnourished children (1–5 years) were randomized to receive either curd (group A, N = 32) or LPC (group B, N = 36) in addition to the WHO recommended two-step diet over 15 days. The results indicated that the change in weight, hemoglobin level and CD4/CD8 T-cell subpopulation were significantly higher in both the groups after supplementation. The response of CRP was blunted in PEM. Serum ferritin decreased significantly after supplementation in both groups.

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10.5

145

AN INDIAN PERSPECTIVE ON REGULATION OF PROBIOTICS

The regulation on probiotics in India is still not clear. There is a Prevention of Food Adulteration Act for foods and Food and Drug Authority Regulations for pharmaceutical products in India. The Food Safety and Standards Act 2006 has been formed with a view to consolidating the laws relating to food and to establish the Food Safety and Standards Authority of India for laying down science-based standards for articles of food and to regulate their manufacture, storage, distribution, sale and import. In order to propose guidelines for regulating the use of probotic microorganisms as ingredients, food supplements or probotic foods in India, a committee has been constituted by the Indian Council of Medical Research and Department of Biotechnology, Government of India. The proposed guidelines are on the website and comments from various stakeholders have been invited. Most of the issues are consistent with FAO/WHO guidelines, except that a special clause for use of indigenous strains of probiotics is given. It states that: Commercial probiotic cultures currently being used in India are of foreign origin. Inherent differences in gut flora of Indian population are known to occur, hence it is imperative to carry out efficacy studies in Indian population prior to their use in India. Further, there is an urgent need for development of indigenous probiotic strains for expressing optimal functionality.

Regarding the use of proven probiotics in our country, the guidelines suggest that, if the probiotic food has a record of documented long and safe use outside the country, the data regarding this may be reviewed and deemed as sufficient to allow its marketing within the country. However, labeling of health benefits may require to be evaluated in a different manner. While taking into account studies done abroad, efficacy studies of probiotics (which are of proven benefit in Western population) should also be conducted on Indian population. It is recommended that such ‘bridging’ human trials be repeated at more than one centre for verification of health claim(s).

Indian research is at present targeted at sponsored clinical trials by the manufacturers of probiotic cultures or foods at a limited number of hospitals and only one or two other places. Most of the clinical studies are still targeted to general health benefits or especially on gastrointestinal tract disorders. There is a wide gap between medical faculty and science faculty and hence coordination between the two needs to be strengthened in order to conduct meaningful clinical studies. The Indian Council of Agricultural Research has launched a project to isolate, preserve and maintain the germplasm of microbes native to the rich biodiversity of India. It seems that in the coming 5 years, India will have more systematic multicenter clinical trials on foreign strains of probiotics as well as on indigenous probotic strains.

ACKNOWLEDGEMENT The authors thank Aarti Patel, Momin Jafar and Ami Patel for their help in compiling reports and preparing the manuscript.

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REFERENCES Agarwal R, Sharma N, Chaudhry R et al. (2003) Effect of oral Lactobacillus GG on enteric microflora in low birth weight neonates. J Pediatr Gasteroenterol Nutr 36:397–402. Aggarwal KN, Bhasin SK (2002) Feasibility studies to control acute diarrhea in children by feeding fermented milk preparations Actimel and Indian dahi. Eur J Clin Nutr 56:556–559. Ashar MN, Prajapati JB (2001) Serum cholesterol level in humans fed with acidophilus milk. Indian J Microbiol 41:257–263. Balasubramanya NN, Lokesh BR, Krishnakantha TP (1999) Effect of oral administration of lyophilised lactic microbes on macrophage function in mice. Indian J Dairy Biosci 10:117–120. Bandyopadhyay P, Das Mohapatra PK (2009) Effect of a probiotic bacterium Bacillus circulans PB7 in the formulated diets: on growth, nutritional quality and immunity of Catla catla (Ham.). Fish Physiol Biochem 35:467–478. Basu S, Chatterjee M, Ganguly S, Chandra PK (2007) Efficacy of Lactobacillus rhamnosus GG on persistent diarrhea in Indian children: a randomized controlled trial. J Clin Gasterenterol 41:756–776. Basu S, Paul DK, Ganguly S, Chatterjee M, Chandra PK (2009) Efficacy of high-dose Lactobacillus rhamnosus GG in controlling acute watery diarrhea in Indian children: a randomized controlled trial. J Clin Gasterenterol 43:208–213. Ciocca L (2003) Key European and Asian consumer data unveiled. Functional Ingredients Magazine, October 2003. Das KC, Kamra DN, Pathak NN (2001) Effect of lactic acid bacteria in the diet of female crossbred cattle calves fed on grain based or grainless concentrate mixtures. Anim Nutr Feed Technol 1:69–77. Dewan P, Kaur I, Chattopadhya D, Faridi MMA, Agarwal KN (2007) A pilot study on the effects of curd (dahi) and leaf protein concentrate in children with protein energy malnutrition (PEM). Indian J Med Res 126:199–203. Dubey AP, Rajeshwari K, Chakravarty A, Famularo G (2008) Use of VSL#3 in the treatment of rotavirus diarrhea in children: preliminary results. J Clin Gastroenterol 42(Suppl 3):S126–S129. FAO (2004) Report on the Regional Expert Consultation of the Asia-Pacific Network for Food and Nutrition on Functional Foods and their Implications in the Daily Diet. Regional Office for Asia and the Pacific. RAP Publication 2004/33, Bangkok. Guhapriya M, Raman Murali M, Niranjali Devaraj S (2007) Protective role of lactobacilli in Shigella dysenteriae 1-induced diarrhea in rats. Nutrition 23:424–433. Guhapriya M, Raman Murali M, Niranjali Devaraj S (2009) Lactobacilli facilitate maintenance of intestinal membrane integrity during Shigella dysenteriae 1 infection in rats. Nutrition 25:350–358. Indulkar ST, Belsare SG (2003) Effect of probiotics on growth and survival of post larvae of Macrobrachium rosenbergii. J Aquaculture Tropics 18:287–297. Ismail A (2006) India: The Land of Opportunity. Functional Ingredients Magazine, January 2006. Kalyankar SD, Khedkar CD (2007) Effects of feeding a low cost probiotic preparation on the performance of buffalo calves. In: Proceedings of the National Symposium on Synbiotic Dairy and Food Products in Human Health and Nutrition organized by Tamilnadu Veterinary and Animal Sciences University and SASNET-Fermented Foods at Chennai, 19–20 January 2007, pp. 182–183. Kamra DN, Chaudhary LC, Agarwal, N, Singh R, Pathak NN (2002) Growth performance, nutrient utilization, rumen fermentation and enzyme activities in calves fed on Saccharomyces cerevisiae supplemented diet. Indian J Anim Sci 72:472–475. Katoch S, Kaistha M, Sharma KS, Kumari M, Katoch BS (2000) Effect of different strains of microbes isolated from the leopard (Panthra leo) excreta on the performance of chicks of different strains. Indian J Poultry Sci 35:57–61. Katoch S, Kaistha M, Sharma KS, Kumari M, Katoch BS (2003) Biological performance of chickens fed newly isolated probiotics. Indian J Anim Sci 73:1271–1273. Khanna V, Alam, S, Malik A, Malik A (2005) Efficacy of tyndalized Lactobacillus acidophilus in acute diarrhea. Indian J Pediatr 72:935–938. Khedkar CD, Dave JM, Sannabhadti SS, Megha RV (1990a) Use of acidophilus milk in treatment of human gastrointestinal disorders. Indian Dairyman XLII:233–236. Khedkar CD, Dave JM, Sannabhadti SS (1990b) Effect of feeding acidophilus milk on faecal lactobacilli and coliform counts in human volunteers. Indian Dairyman XLII:237–241. Khedkar CD, Patil MR, Gyananath G, Bajad DN, Sarode AR (2003) Studies on implantation ability of a probiotic culture of Lactobacillus acidophilus in gastrointestinal tract of tribal children. In: Proceedings

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of the International Seminar and Workshop on Fermented Foods, Health Status and Social Well being, organized by Gujarat Agricultural University, SASNET Fermented Foods and Department of Applied Nutrition Lund University, Sweden at Anand, India, 13–14 November 2003, pp. 62–63. Khedkar JN, Sannabhadti SS, Dave JM (1994) Inhibitory effect of Bifidobacterium adolescentis (Hb1) on faecal coliform counts. J Dairying Foods and Home Sci 13:187–191. Kotilainen L, Rikka R, Catherine R, Eija P (2006) Health enhancing foods: opportunities for strengthening the sector in developing countries. Agriculture and Rural Development Discussion Paper 30. Washington, DC: World Bank, pp. 1–95. Kumar BS, Vijayasarathi SK, Gowda RNS, Satyanarayana ML (2002) Probiotics for the prevention of experimental fowl typhoid in broilers: a pathomorphological study. Indian J Anim Sci 72:528–531. Maheswari D, Kadirvel R (2003) Interaction among fumaric acid, probiotic and antibiotic in broilers. Indian J Poultry Sci 38:285–287. Margaret M, Roshanars J (2007) Effects of supplementation of a bioyoghurt on the serum lipid profile of hypercholesterolemic women. In: Proceeding of the National Symposium on Synbiotic Dairy and Food Products in Human Health and Nutrition organized by Tamilnadu Veterinary and Animal Sciences University and SASNET Fermented Foods at Chennai, 19–20 January 2007, pp. 137–147. Narayanappa D (2008) Randomized double blinded controlled trial to evaluate the efficacy and safety of Bifilac in patients with acute viral diarrhea. Indian J Pediatr 75:709–13. Panda AK, Rameshwar Singh, Pathak NN (1999) Effect of dietary inclusion of Saccharomyces cerevisiae on rumen fermentation in crossbred calves. Indian J Anim Nutr 16:291–294. Panda AK, Reddy MR, Rao SVR, Raju MVLN, Praharaj NK (2000a) Growth, carcass characteristics, immunocompetence and response to Escherichia coli of broilers fed diets with various levels of probiotic. Archiv Geflugelkunde 64:152–156. Panda AK, Reddy MR, Ramarao SV, Praharaj NK (2000b) Effect of dietary supplementation of probiotic on performance and immune response of layers in the decline phase of production. Indian J Poultry Sci 35:102–104. Panda AK, Reddy MR, Praharaj NK (2001) Dietary supplementation of probiotic on growth, serum cholesterol and gut microflora of broilers. Indian J Anim Sci 71:488–490. Patel JR, Dave JM, Dave RI, Sannabhadti SS (1992) Effect of feeding milk fermented with mixed culture of human strains of lactobacilli on faecal lactobacilli and coliform counts in human test subjects. Indian J Dairy Sci 45:379–382. Patidar SK, Prajapati JB (1996) Effect of feeding milk fermented with Lactobacillus acidophilus on chicks infected with pathogenic E. coli. In: Proceedings of the 37th Annual Conference of the Association of Microbiologists of India, 4–6 December 1996, IIT, Madras, pp. 20. Patidar SK, Prajapati JB (1999) Effect of feeding lactobacilli on serum antibody titer and fecal microflora in chicks. Microbiol Aliment Nutr 17:145–154. Prajapati JB, Nair BM (2003) History of fermented foods. In: Farnworth ER (ed.) Handbook of Fermented Functional Foods. Boca Raton, FL: CRC Press, pp. 1–25. Prajapati JB, Shah RK, Dave JM (1986) Nutritional and therapeutic benefits of a blended-spray dried acidophilus preparation. Cult Dairy Prod J 21:16–21. Ramesh BK, Satynarayana ML, Gowda RNS, Vijayasarathi SK, Rao S (2000) Effect of Lactobacillus acidophilus on gut pH and viable bacterial count in experimental fowl typhoid in broilers. Indian Vet J 77:544–546. Rao TN, Rao ZP, Prasad JR (2001) Effect of probiotic supplementation to complete rations on the nutrient digestibility and rumen environment in sheep. Indian J Anim Nutr 18:133–137. Rao TN, Rao ZP, Prasad JR, Prasad PE (2003) Supplementation of probiotics on growth performance in sheep. Indian J Anim Nutr 20:224–226. Ravi A, Suresh J, Reddy IS, Rao DS (2000) Effect of feeding probiotics on growth performance of crossbred (LWY X DESI) pigs. Cheiron 29:158–159. Ruchi K, Anand SK, Chander H (2006) In vivo demonstration of enhanced probiotic effect of co- immobilized Lactobacillus acidophilus and Bifidobacterium bifidum. Int J Dairy Technol 59: 265–271. Saha SK, Senani S, Padhi MK, Shome BR, Shome R, Ahlawat SPS (1999) Microbial manipulation of rumen fermentation using Saccharomyces cerevisiae as probiotics. Curr Sci 77:696–697. Sarkar A, Sazawal S, Dhingra U et al. (2004) Effect of fortification on milk with probiotic Bifidobacterium lactis HN019 (DR-10)™ and prebiotic galacto-oligosaccharides on anemia, growth and development in children aged 1–4 years: a double blind masked randomized trial. In: Pediatric Gastroenterology, Hepatology and Nutrion, 2nd World Congress, Paris, France, 3–7 July 2004, pp. 59–63.

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Satbir S, Sharma VP, Panwar VS (1999) Effect of different levels of probiotic and microbial populations in broiler chicks. Indian Vet J 76:1026–1028. Selvam R, Maheswari P, Kavitha P, Ravichandran M, Sas B, Ramchand CN (2009) Effect of Bacillus subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in inflammatory bowel disease. Indian J Biochem Biophys 46:79–85. Senani S, Saha SK, Padhi MK, Rai RB (2000) Efficacy of various Lactobacillus strains on broiler production. Indian J Anim Sci 70:845–846. Shalini Jain, Yadav H, Sinha PR (2008) Stimulation of innate immunity by oral administration of dahi containing probiotic Lactobacillus casei in mice. J Med Food 11:652–656. Sur D (2008) Role of probiotics in prevention of acute diarrheal diseases in children [Abstract]. In: Second India Probiotics Symposium: Evidence-based Health Benefits of Probiotics, 7–8 November 2008, New Delhi, p. 16. Venkat HK, Sahu NP, Jain KK (2004) Effect of feeding Lactobacillus-based probiotics on the gut microflora, growth and survival of postlarvae of Macrobrachium rosenbergii (de Man). Aquaculture Res 35:501–507. Watson J (2006) Middle class India joins global organic food wave. Terra daily (UPI) 28 February. Yadav H, Jain S, Sinha PR (2007) Antidiabetic effect of probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei in high fructose fed rats. Nutrition 23:62–68.

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11

The Role of Meta-analysis in the Evaluation of Clinical Trials on Probiotics

Hania Szajewska

11.1

INTRODUCTION

Systematic reviews with or without a meta-analysis are a key element of evidence-based medicine. They are now a well-established means of reviewing existing evidence and integrating findings from various studies. Since the number of meta-analyses is increasing rapidly, it is essential that the strengths and limitations of this approach are well understood. This chapter provides an overview of the basic principles of systematic review and meta-analysis of randomized controlled trials (RCTs). A summary of published meta-analyses on the effects of probiotics in the prevention and/or treatment of various conditions is presented. This chapter also discusses whether a meta-analytical approach is appropriate for evaluating the effectiveness of probiotics. The problems and limitations of using a meta-analytical approach in the context of probiotics are discussed.

11.2

WHAT IS A SYSTEMATIC REVIEW? WHAT IS A META-ANALYSIS?

Although the terms “systematic review” and “meta-analysis” are commonly used interchangeably, there is a distinction between the two. According to one of the most commonly used definitions developed by the Cochrane Collaboration, a systematic review is a review of a clearly formulated question that uses systematic and explicit methods to identify, select and critically appraise relevant research, and to collect and analyze data from studies that are included in the review. Statistical methods may or may not be used to analyze and summarize the results of the included trials (Higgins & Green, 2008).

The strategies used in systematic reviews limit bias, which is likely to occur in traditional review articles (narrative reviews). For example, the studies selected for review articles may not represent all of the available evidence due to a flawed search strategy. A meta-analysis is the name given to any review article when statistical techniques are used in a systematic review to combine the results of included trials to produce a single 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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estimate of the effect of a particular intervention (i.e. a number or a graph) (Higgins & Green, 2008). At least two primary studies are needed to perform a meta-analysis.

11.3

HOW TO CONDUCT A SYSTEMATIC REVIEW

Guidelines for conducting a systematic review are provided by several organizations. As a detailed description is beyond the scope of this chapter, readers interested in conducting a systematic review should consult existing guidelines, preferably those developed by the Cochrane Collaboration (www.cochrane.org) (Higgins & Green, 2008). In brief, the following steps are needed to conduct a systematic review.

11.3.1

Formulation of the review question (the problem)

The key components of a research question about the effectiveness of an intervention should address the types of participants, interventions, comparisons, and outcomes of interest. The use of the acronym PICO (participants, intervention, comparison, outcome(s)) is helpful. Clear formulation of the research question is essential and helps to identify study designs that would most adequately answer it. For questions about the effectiveness of an intervention, the best study design, which reduces the risk of bias if performed correctly, would be a randomized controlled trial.

11.3.2

Searching

Searching is the locating and selecting of controlled trials based on predefined inclusion and exclusion criteria. The searches should be as extensive as possible using both free-text and subject headings. The more data sources searched (not just the most popular source, i.e. Medline), the more likely it is that none of the important trials will be missed. It is advisable to search at least Medline, EMBASE, and the Cochrane Library. Searching one database is never enough. If possible, no restrictions on language should be applied. The inclusion of only studies in specific languages (usually English only) might result in the omission of relevant studies. In order to minimize bias and error during study searching and selection, two or more reviewers should be involved. Inclusion of unpublished trials, also controversial as discussed below, may help to reduce the risk of publication bias. Unfortunately, there is no good strategy for identifying unpublished data no matter how many databases are searched.

11.3.3

Selecting studies and collecting data

Typically, studies are included if the title and abstract are clearly relevant. If the abstract contains insufficient information to warrant exclusion, full papers should be obtained. Once all relevant papers are identified, data are extracted based on the general characteristics of the studies (e.g. authors, year, location, language of publication, and source of funding), clinical issues (population, intervention, comparison, and outcomes assessed), methodological characteristics of the trials, and results. When important data are not reported or are unclear, an effort should be made to contact the corresponding authors of the primary studies for clarification. For dichotomous outcomes (e.g. the presence of diarrhea), the total number of participants and the number of

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participants who experienced the event are extracted. For continuous outcomes (e.g. the duration of diarrhea), the total number of participants, means, and standard deviations are extracted. As both the selection process of the trials and data extraction are subjective, it is desirable if more than one reviewer is involved. The strategy of how to handle disagreement between reviewers (or interpretations) should be defined. In practice, if differences in opinion exist between reviewers, they are usually resolved by discussion.

11.3.4

Assessment of methodological quality (i.e. the risk of bias in included trials)

Because of the limited ability to validate any scoring system, there is no gold standard for assessing the “true” validity of a trial. Usually, the following criteria generally associated with good-quality studies are evaluated: generation of allocation sequences and allocation concealment; blinding of investigators, participants, outcome assessors, and data analysts; intention-to-treat analysis; and comprehensive follow-up (80% or more). Preferably, two or more reviewers should be independently involved in the assessment.

11.3.5

Analysing the data and presenting the results

One of the most important decisions to be made by the reviewers is to decide whether or not to pool the results of individual trials. The Cochrane Handbook (Higgins & Green, 2008) recommends that data only be pooled if the data summarized are homogeneous, i.e. the participants, intervention, comparison, and outcomes must be similar (homogeneous) or at least comparable. The essential message is that it is always appropriate to perform a systematic review, and every meta-analysis should be preceded by a systematic review. However, not every systematic review should be finalized with a meta-analysis; in fact, it is sometimes erroneous and even misleading to perform a meta-analysis (Higgins & Green, 2008). If a meta-analysis is carried out, the measures of the treatment effect should be specified. To present the difference in dichotomous outcomes (e.g. presence of diarrhea versus no diarrhea), pooled statistics are reported as the risk ratio (RR) between the experimental and control groups, the odds ratio (OR), or the risk difference (RD) with 95% confidence interval (CI). To represent the difference in continuous outcomes (e.g. the duration or severity of diarrhea), the weighted mean difference (WMD) or less often the standardized mean difference (SMD) between the treatment and control groups is usually selected with 95% CI. To pool the data, either a fixed effects or random effects model approach is used according to the degree of heterogeneity in outcomes across the studies, or both. The fixed effects model assumes that the treatment effect is the same across trials and aims to estimate it with more precision. Conversely, the random effects model assumes that the treatment effect is not the same across studies and aims to determine the average effect (Higgins & Green, 2008).

11.4

WHY PERFORM A META-ANALYSIS?

There are a number of reasons why a meta-analysis is performed within a systematic review (Higgins & Green, 2008):

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To increase power, i.e. the chance to reliably detect a clinically important difference if one actually exists. The problem with many individual studies is that they are too small to detect small effects, which can only be detected if the data from several trials are combined. To improve precision in estimating effects, i.e. narrow the confidence interval around the effects. To answer questions not raised by individual studies. To resolve controversies arising from studies with conflicting results or to generate new hypotheses for future studies.

11.5

HETEROGENEITY

In the context of a meta-analysis, heterogeneity refers to any kind of variability (diversity) among studies. It is called “clinical heterogeneity” if it is due to clinical differences such as differences in the participants, interventions, comparisons and/or outcomes. Heterogeneity due to variability in study design is referred to as “methodological heterogeneity”. One tool to display heterogeneity is the forest plot, the interpretation of which is described in section 11.6. Heterogeneity can also be due to the use of different statistical methods. Heterogeneity is often quantified by c2 and I 2. The latter can be interpreted as the percentage of the total variation between studies that is attributable to heterogeneity rather than to chance. A value of 0% indicates no observed heterogeneity, and larger values show increasing heterogeneity. A value over 50% indicates substantial heterogeneity. If significant heterogeneity exists, the reviewers should attempt to identify and explain its potential sources.

11.6

HOW TO INTERPRET A FOREST PLOT

A forest plot is a graphical display of results from individual studies together with the combined result. Figure 11.1 shows a forest plot from a hypothetical meta-analysis comparing the effect of a new infant formula supplemented with probiotics with a standard infant formula for the prevention of gastrointestinal infections in children. The results of seven trials are presented. The study identifications are listed in a column on the left-hand side. The black square in the middle of each horizontal line represents the point estimate of the difference between the groups (the best single estimate of the benefit) presented as the relative risk (RR). The width of the horizontal line represents the 95% CI for this estimate. The vertical line is called the “line of no effect” (RR = 1.0). If visually the horizontal line representing the results of an individual trial does not cross the line of no effect (i.e. if the confidence interval for RR does not include 1), there is 95% chance that there is a real difference between the groups. On the other hand, if the horizontal line does cross the line of no effect (i.e. if the confidence interval for RR does include 1), there is no significant difference between the treatments and/or the sample size was too small to allow one to be confident about where the true result lies. The area of the black squares reflects the weight each study contributes to the meta-analysis. The center of the diamond below all of the horizontal lines represents the pooled treatment effect (RR 0.36). The horizontal tips of the diamond again represent 95% CI (in our example, 0.2–0.65). The diamond does not cross the line of no

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The Role of Meta-analysis in the Evaluation of Clinical Trials on Probiotics New infant formula Study or Subgroup Study A Study B Study C Study D Study E Study F Study G Total (95% CI)

Risk Ratio

Standard formula

Events

Total

Events

0 4 5 1 2 1 2

45 295 51 72 180 39 21

0 8 6 10 10 3 1

703

Total Weight M-H, Fixed, 95% CI

20.8% 18.1% 25.5% 25.2% 7.5% 2.8%

Not estimable 0.49 [0.15, 1.61] 0.59 [0.19, 1.78] 0.10 [0.01, 0.77] 0.21 [0.05, 0.94] 0.35 [0.04, 3.23] 1.62 [0.16, 16.37]

690 100.0%

0.36 [0.20, 0.65]

46 290 36 73 187 41 17

38 15 Total events Heterogeneity: Chi² = 4.66, df = 5 (P = 0.46); I² = 0% Test for overall effect: Z = 3.37 (P = 0.0008)

153

Risk Ratio M-H, Fixed, 95% CI

0.002 0.1 1 10 500 Favours new formula Favours control

Fig. 11.1 Forest plot from a hypothetical meta-analysis comparing the effect of a new infant formula containing probiotics with a standard infant formula on the risk of diarrhea. The relative risk of 0.36 suggests that compared with use of the standard formula, the use of the new infant formula reduces the risk of diarrhea in an infant (64% reduction). CI, confidence interval.

effect. Thus, this meta-analysis shows that the use of a new formula supplemented with probiotics compared with the use of standard formula reduces the risk of gastrointestinal infections (reduction of 64%). The forest plot also allows, at a glance, the opportunity to assess heterogeneity (the amount of variation among studies). If the confidence intervals (represented by the horizontal lines) all overlap to some extent, the trials are homogeneous. On the other hand, if some confidence intervals (horizontal lines) do not overlap at all, there is high heterogeneity. In our hypothetical example, all confidence intervals do overlap so the trials are considered to be homogeneous.

11.7

CRITICAL APPRAISAL OF A SYSTEMATIC REVIEW

As with any kind of research, not all systematic reviews and meta-analyses are equal. The quality of systematic reviews and meta-analyses varies. The main purpose of appraising a systematic review is to determine the extent to which it is protected against bias. Critical appraisal of a systematic review of intervention trials includes answering the following questions defined by Oxman et al. (2002). Are the results valid? ● Did the review question address a clearly formulated question with a clearly defined population, intervention, comparison, and outcome(s)? ● What was the search strategy? ● Were the included studies critically appraised? ● Were assessments of studies reproducible? What are the results? ● Were the results similar from study to study? ● What are the overall results of the review? ● How precise were the results?

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How can I apply the results to patient care? ● How can I best interpret the results to apply them to the care of patients in my practice? ● Were all clinically relevant outcomes considered? ● Are the benefits worth the costs and potential risks?

11.8

PUBLISHED META-ANALYSES ON THE EFFECTS OF PROBIOTICS

A PubMed search in May 2009 of the words “meta-analysis” or “systematic review” and “probiotics” yielded a substantial number of papers, many of which are Cochrane reviews. Most of the reviews addressed well-defined questions in terms of the study design, participants, intervention, and outcomes. The reviewers usually searched several relevant databases, and many made efforts to find further evidence by reviewing reference lists of retrieved articles. In contrast, searches for unpublished trials were uncommon. Most reviewers assessed the methodological quality of the included trials. Interestingly, some systematic reviews addressing the same clinical question, which were performed at almost the same time by reviewers with the same access to the literature, reached incompatible or even contradictory conclusions. The main conclusions from selected published reviews on the effects of probiotics on various conditions are summarized here.

11.8.1

Acute gastroenteritis

The rationale for the use of probiotics to treat and prevent diarrheal diseases is based on the assumption that they modify the composition of the colonic microflora and act against enteric pathogens. Evidence from several meta-analyses of RCTs (Szajewska & Mrukowicz, 2001; Huang et al., 2002; Van Niel et al., 2002; Allen et al., 2004) has consistently shown a statistically significant effect and moderate clinical benefit of some probiotic strains in the treatment of acute watery diarrhea, mainly rotaviral, in infants and young children mainly. Only two meta-analyses focused exclusively on the efficacy of only single probiotic microorganisms and found beneficial effects of Lactobacillus GG (Szajewska et al., 2007a) and Saccharomyces boulardii (Szajewska et al., 2007b, 2009) Overall, the beneficial effects of probiotics in the management of acute infectious diarrhea seem to be (1) moderate, (2) strain dependent, (3) dose dependent (greater for doses above 1010–1011 CFU), (4) significant in cases of watery diarrhea and viral gastroenteritis but not in cases of invasive bacterial diarrhea, (5) more evident when treatment with probiotics is initiated early in the course of the disease, and (6) more evident in patients living in developed countries. Given the available evidence, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition and the European Society of Paediatric Infectious Diseases Expert Working Group (Guarino et al., 2008) recently stated that selected probiotics with proven clinical efficacy (e.g. Lactobacillus GG, S. boulardii) that are administered in appropriate dosages, according to the strain and the patient population, may be used as an adjunct to rehydration therapy for the management of acute gastroenteritis in children. Other probiotic strains may also be used provided their efficacy is documented in highquality RCTs.

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11.8.2

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Antibiotic-associated diarrhea

A common side effect of antibiotic treatment is antibiotic-associated diarrhea (AAD), defined as otherwise unexplained diarrhea that occurs in association with the administration of antibiotics (Bartlett, 2002). In the pediatric population, AAD occurs in approximately 11–40% of children between the initiation of therapy and up to 2 months after cessation of treatment (Elstner et al., 1983; Turck et al., 2003). Several systematic reviews with or without a meta-analysis have shown most of the tested probiotics to be effective in reducing the risk of AAD in the general (mainly adult) population (Cremonini et al., 2002; D’Souza et al., 2002; Hawrelak et al., 2005; Szajewska & Mrukowicz, 2005). Evidence from recent systematic reviews of RCTs conducted in children (Johnston et al., 2006, 2007; Szajewska et al., 2006) is also encouraging. In the review by Szajewska et al. (search date December 2005) identified six RCTs involving 766 children. The review found that treatment with probiotics compared with placebo reduced the risk of AAD from 28.5% to 11.9% (RR 0.44, 95% CI 0.25–0.77, random effect model). Preplanned subgroup analysis showed that the reduction in the risk of AAD was associated with the use of Lactobacillus GG (two RCTs, 307 participants, RR 0.3, 95% CI 0.15–0.6), S. boulardii (one RCT, 246 participants, RR 0.2, 95% CI 0.07–0.6), or B. lactis and Streptococcus thermophilus (one RCT, 157 participants, RR 0.5, 95% CI 0.3–0.95). It was concluded that the use of probiotics reduces the risk of AAD in children. For every seven patients who would develop diarrhea while being treated with antibiotics, one fewer will develop AAD if also receiving probiotics. Two other reviews of the effects of probiotic therapy on pediatric AAD were performed by the same authors (Johnston et al., 2006, 2007). They found that the combined results, analysed with a per-protocol method that reported on the incidence of diarrhea during antibiotic treatment, showed a significant benefit for the use of probiotics compared with placebo (RR 0.43, 95% CI 0.25–0.75) (Johnston et al., 2006). In contrast, the results from the intention-to-treat analysis were non-significant overall (RR 1.01, 95% CI 0.64–1.61) (Johnston et al., 2006). However, as indicated by the authors of this review, the validity of the intention-to-treat analysis in this review can be questioned due to high losses to follow-up (Johnston et al., 2006).

11.8.3

Clostridium difficile-associated diarrhea

The bacterial agent commonly associated with AAD, particularly in the most severe episodes (pseudomembranous colitis), is Clostridium difficile (Bartlett et al., 1978). Evidence suggesting that there is an increasing incidence of C. difficile-associated diarrhea (CDAD) and emergence of the BI/NAP1/027 strain has renewed interest in the development of effective therapeutic and prophylactic measures (Parkes et al., 2009). One meta-analysis (McFarland, 2006) and two systematic reviews (Dendukuri et al., 2005; Pillai & Nelson, 2008) related to CDAD were identified with conflicting conclusions. The meta-analysis (search date 2005) concluded that, overall, there was sufficient evidence to support the use of probiotics in preventing recurrent CDAD (six RCTs, RR 0.59, 95% CI 0.4–0.85). However, there was criticism of the methods used (Dendukuri & Brophy, 2007). When S. boulardii was used alone, the meta-analysis showed a benefit of this treatment compared with placebo in the prevention of recurrent CDAD. However, a similar benefit was not observed with the use of L. rhamnosus GG or L. plantarum 299v. A systematic review (Dendukuri et al., 2005) (search date 2005) of probiotic therapy for the prevention and treatment of CDAD identified eight studies, four of which had

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prevention (N = 1) or treatment (N = 3) as a primary outcome. Overall, heterogeneity in the choice and dose of probiotic as well as in the criteria for diagnosing CDAD did not allow the authors to synthesize the data. The authors concluded that existing data provide insufficient evidence to support the routine clinical use of probiotics to prevent or treat CDAD. A Cochrane systematic review (Pillai & Nelson, 2008) (search date 2007) of probiotics used in the treatment of CDAD in adults included only four trials. The authors did not pool the results because of variability in the probiotics and antibiotics used. They also concluded that there is insufficient evidence to recommend the use of probiotics in the treatment of CDAD and insufficient evidence to comment on their use for secondary prophylaxis.

11.8.4

Traveler’s diarrhea

Traveler’s diarrhea is a common problem, occurring in 5–50% of travelers. It is usually due to bacterial enteropathogens, particularly enterotoxigenic Escherichia coli. There have been two meta-analyses of the role of probiotics in the prevention of traveler’s diarrhea with conflicting conclusions. The first (McFarland, 2007) (search date 2005) identified 12 studies with a total of 4709 subjects. The pooled relative risk indicates that probiotics significantly prevent traveler’s diarrhea (RR 0.85, 95% CI 0.79–0.91). The authors concluded that several probiotics, such as S. boulardii and a mixture of L. acidophilus and B. bifidum, had significant efficacy and that probiotics may offer a safe and effective method of preventing traveler’s diarrhea. However, opposite conclusions were reached by the authors of the second meta-analysis (Takahashi et al., 2007) (search date 2005). The pooled results of five RCTs of varying methodological quality involving 3326 subjects showed no significant difference between the probiotic and the control groups in the risk of traveler’s diarrhea (RR 0.93, 95% CI 0.85–1.01). The authors concluded that probiotics are not effective for the prevention of traveler’s diarrhea.

11.8.5

Necrotizing enterocolitis

The rationale for probiotic supplementation is based on data demonstrating differences in the establishment of the intestinal microbiota in preterm infants (Blakey et al., 1982; Gewolb et al., 1999; Magne et al., 2005). While the possible consequences to health are not known, it has been speculated that abnormal patterns of colonization in preterm infants may contribute to the pathogenesis of necrotizing enterocolitis (NEC) and to the increased susceptibility to infections. It has also been suggested that enteral administration of probiotics to preterm newborns could prevent infections, prevent NEC, and reduce the use of antibiotics (Caplan & Jilling, 2000). A number of systematic reviews, with or without a meta-analysis, have reviewed data on the effects of the enteral administration of probiotics on the risks of NEC and mortality in preterm infants (Deshpande et al., 2007, 2010, Alfaleh & Bassler, 2008, Barclay et al., 2007). Among them, the most recent is the updated meta-analysis by Deshpande et al. 2010 (search date March 2009), which identified 11 RCTs, including 4 recent trials, and involved 2176 preterm infants. Compared with the control group, preterm neonates in the probiotic group had a reduced risk of NEC (relative risk [RR] 0.35, 95% confidence interval [CI] 0.23 to 0.55) and all-cause mortality (RR 0.42, 95% CI 0.29 to 0.62), but there was no difference between groups in the risk of sepsis (RR 0.98, 95% CI 0.81 to 1.18). Heterogeneity between trials was low (I^2 = 0%), suggesting that the benefit appears to be a true class effect despite known differences between individual probiotic microorganisms.

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Helicobacter pylori infection

The rationale for the use of probiotics as adjunctive treatment for H. pylori infection is based on the results of studies which have shown that various lactobacilli (e.g. Lactobacillus johnsonii La1, L. acidophilus CRL 639, L. casei), or their metabolic products, can inhibit or kill H. pylori in vitro (Bhatia et al., 1989; Bernet et al., 1994). One meta-analysis (Tong et al., 2007) (search date October 2006) evaluated the effects of probiotics on H. pylori eradication rates and side effects of anti-H. pylori treatment. Fourteen RCTs of varying methodological quality involving 1671 patients were identified for inclusion. In patients with H. pylori infection, probiotic supplementation improved eradication rates. In two RCTs that evaluated patients with eradication failure, probiotic supplementation also improved eradication rates. Probiotic use reduced therapy-related side effects overall and individual symptoms of diarrhea, epigastric pain, nausea, and taste disturbance.

11.8.7

Functional gastrointestinal disorders

Functional gastrointestinal disorders are defined as a variable combination of chronic or recurrent gastrointestinal symptoms not explained by structural or biochemical abnormalities. In 1999, the Rome II diagnostic criteria for functional gastrointestinal disorders were formulated, primarily to assist research (Drossman, 1999). In 2006, they were replaced by updated Rome III criteria separate for infants and toddlers (Hyman et al., 2006), for children and adolescents (Rasquin et al., 2006), and for adults (Drossman, 2006). Functional gastrointestinal disorders account for a substantial number of referrals to gastroenterology clinics. Management remains difficult, prompting interest in new and safe treatment options. A Cochrane systematic review (Huertas-Ceballos et al., 2009) (search date December 2006) of dietary interventions for recurrent abdominal pain (currently usually classified according to the Rome II criteria) and irritable bowel syndrome in childhood included only three RCTs related to probiotics; only two provided data suitable for analysis. The authors concluded that there is no evidence that Lactobacillus supplementation is effective in the management of children with recurrent abdominal pain.

11.8.8

Irritable bowel syndrome

Irritable bowel syndrome (IBS) encompasses a group of functional bowel disorders in which abdominal discomfort and pain is often associated with an altered bowel habit and bloating for which there is no evidence of detectable organic disease. Surveys of Western populations have revealed IBS in 15–20% of adolescents and adults, with a higher prevalence among women (Drossman, 1999). In one pediatric study, children with IBS represented 25–50% of visits to a gastroenterologist’s clinic (El-Matary et al., 2004). IBS exemplifies a significant therapeutic challenge. At best, currently available therapies provide symptomatic relief, but there are none that can influence the natural course of the disorder. There have been four meta-analyses and one systematic review on the effects of probiotics for the treatment of IBS, with slightly different conclusions. The first (McFarland & Dublin, 2008) (search date 2007) identified 20 blinded RCTs that met the inclusion criteria involving 1404 adults and children. The probiotic species studied were Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., VSL#3, Bacillus subtilis, Lactococcus lactis, E. coli, Propionibacterium freudenreichii, and S. boulardii. The authors found significant

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variations in the strains of probiotics used, treatment doses and durations, clinical outcomes, and clinical follow-up. Only three of the included trials used intention-to-treat analysis, and thus most trials have methodological limitations. Compared with placebo, treatment with probiotics reduced IBS symptoms (14 treatment arms from 12 RCTs, RR 0.77, 95% CI 0.62–0.94) and reduced abdominal pain (12 treatment arms from 10 RCTs, RR 0.78, 95% CI 0.69–0.88). For both outcomes, tests for heterogeneity revealed statistically significant heterogeneity. It was concluded that the use of probiotics may be associated with improvement in IBS symptoms. Given the methodological limitations of the included studies, the authors called for caution in interpreting the data. The second meta-analysis (Nikfar et al., 2008) (search date September 2007) identified eight RCTs involving a total of 1011 subjects (adults and children) with IBS. The probiotics studied were Lactobacillus spp., Bifidobacterium spp., VSL#3, and P. freudenreichii. Pooling of the data from the eight RCTs for the outcome of clinical improvement yielded a significant RR of 1.22 (95% CI 1.07–1.4). Thus the authors concluded that probiotic therapy may improve IBS symptoms. The third meta-analysis (Hoveyda et al., 2009) (search date 2007) that assessed the role of probiotics in the treatment of IBS identified 14 RCTs involving both adults and children. All the studies were assessed for methodological quality in four specific areas: randomization method, allocation concealment, blinding, and use of intention-to-treat analysis. Total scores were given for the methodological quality of each included study, with a maximum score of 4 points.The methodological quality of the RCTs varied, with a maximum score of 4 points for only four trials, a score of 2 or less points for seven trials, and a score of 1 point each for three trials. This meta-analysis demonstrated improvement in overall symptoms after several weeks of probiotic treatment both for dichotomous data (seven RCTs, OR 1.6, 95% CI 1.2–2.2) and continuous data (six RCTs, SMD 0.23, 95% CI 0.07–0.38). A systematic review by Brenner et al. (2009) (search date June 2008) to assess the efficacy, safety, and tolerability of probiotics in the treatment of IBS identified 16 RCTs. Only studies in adults with IBS were considered for inclusion. As many as 11 of the 16 RCTs showed methodological limitations such as inadequate blinding, inadequate trial length, inadequate sample size, and/or lack of intention-to-treat analysis. In contrast to previous reviews, given the significant heterogeneity in the various study characteristics, the reviewers did not pool the results. Based on the results of two RCTs, only the use of B. infantis 35624 compared with placebo was associated with improvement in IBS symptoms, as assessed by the composite score for abdominal pain/discomfort, bloating/distention, and/or bowel movement difficulty. It was concluded that the overall data are inadequate to comment on the efficacy of other probiotics. The most recent meta-analysis (Moayyedi et al., 2010) (search date June 2008) to examine the role of probiotics in therapy for IBS identified 19 trials that included 1668 participants with IBS diagnosed according to the Rome or Manning criteria. Nine trials used an adequate method of randomization and six used adequate allocation concealment. Ten RCTs (N = 918) reported IBS symptoms as a dichotomous outcome, and it was found that probiotics compared with placebo significantly reduced IBS symptoms (RR 0.71, 95% CI 0.57–0.87, NNT 4, 95% CI 3–12.5). Compared with the lower-quality trials, the higher-quality trials reported a more modest treatment effect. No difference in the effects of the different types of probiotics used (Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., combination of probiotics) was found, with all showing a trend toward benefit. Fifteen RCTs (N = 1351) reported IBS symptoms as a continuous outcome. Again, compared with placebo, probiotics significantly reduced IBS symptoms (SMD −0.34,

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95% CI −0.6 to −0.07). However, there were differences in the effects of the different types of probiotics used, with Lactobacillus spp. (four RCTs, N = 200) showing no effect on IBS symptoms, combinations of probiotics (nine RCTs, N = 772) showing significant improvement in IBS symptoms, and Bifidobacterium spp. (two RCTs, N = 379) showing a trend toward improvement in IBS symptoms. Based on this meta-analysis, published earlier in an abstract form only (Moayyedi et al., 2008), the American College of Gastroenterology formulated the following recommendation regarding the effectiveness of probiotics in the management of IBS: “In single organism studies, lactobacilli do not appear effective; bifidobacteria and certain combinations of probiotics demonstrate some efficacy.”

11.8.9

Inflammatory bowel disease

The etiology of inflammatory bowel disease (IBD), which consists mainly of two distinct disorders, Crohn’s disease and ulcerative colitis, remains elusive. However, there is increasing evidence that gut microbiota play a role in the pathogenesis of IBD by both initiating and maintaining inflammation (Ogura et al., 2001; Hisamatsu et al., 2003). 11.8.9.1

Induction of remission in Crohn’s disease

A Cochrane review (search date 2007) identified only one small RCT with 11 participants that assessed the efficacy of using probiotics for the induction of remission in patients with Crohn’s disease. The authors stated that there is insufficient evidence to make any conclusions regarding the efficacy of such treatment for this purpose (Butterworth et al., 2008). 11.8.9.2

Maintenance of remission in Crohn’s disease

A Cochrane review (search date 2005) to assess the effectiveness of using probiotics for the maintenance of remission in patients with Crohn’s disease identified seven small RCTs. No formal meta-analysis was performed due to heterogeneity of the studies. The authors concluded that there is no evidence to suggest that probiotics are beneficial for the maintenance of remission in Crohn’s disease. All the included studies enrolled small numbers of patients and may have lacked sufficient statistical power to show differences should they exist. Larger trials are required to determine if probiotics are of benefit in the management of Crohn’s disease (Rolfe et al., 2006). Also, the authors of a second meta-analysis (Rahimi et al., 2008) (search date May 2007) concluded that probiotics confer no benefit over placebo in maintaining remission and preventing clinical or endoscopic relapses in patients with Crohn’s disease. The pooled trial results revealed no significant difference in the clinical relapse rate (seven RCTs, OR 0.92, 95% CI 0.52–1.62) or the endoscopic relapse rate (three RCTs, OR 0.97, 95% CI 0.54–1.78) between the probiotic and placebo groups. 11.8.9.3

Remission in ulcerative colitis

A Cochrane review (search date 2006) assessed the efficacy of probiotics compared with placebo or standard medical treatment for the induction of remission in patients with active ulcerative colitis. Four RCTs were identified. Given the differences in the probiotics studied, outcomes and methodological issues, no formal meta-analysis was performed. The authors concluded that conventional therapy combined with a probiotic does not

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improve overall remission rates in patients with mild to moderate ulcerative colitis. However, limited evidence suggests that probiotics added to standard therapy may provide modest benefits in terms of reducing disease activity in patients with mild to moderately severe ulcerative colitis (Mallon et al., 2007). 11.8.9.4

Pouchitis

Pouchitis is a non-specific idiopathic inflammation of the ileal reservoir and is the most common complication of pouch surgery for ulcerative colitis. A Cochrane review (search date 1997) concluded, based on the results of four RCTs, that oral probiotic therapy with VSL#3 appears to be an effective therapy for maintaining remission in patients with chronic pouchitis in remission (Sandborn et al., 2000). A more recent meta-analysis of five RCTs revealed that compared with placebo, probiotics reduce the risk of pouchitis defined by a pouchitis disease activity index (≥ 7) (OR 0.04, 95% CI 0.01–0.14). The most promising agent was VSL#3 (Elahi et al., 2008).

11.8.10

Functional constipation

Constipation is a common condition affecting children and adults (Higgins & Johanson, 2004; Van den Berg et al., 2006). In most cases, no underlying organic cause is found and functional constipation is diagnosed. Functional constipation is defined as infrequent evacuation of hard stools associated with retentive posturing, pain at defecation, and/or soiling (Rasquin et al., 2006; www.romecriteria.org/pdfs/AdultFunctGIQ.pdf). One rationale for using probiotics to treat constipation is data demonstrating differences in the intestinal microbiota between healthy individuals and patients with chronic constipation (Salminen & Salminen, 1997; Zoppi et al., 1998). Second, studies in which Bifidobacterium animalis DN-173 010 was administered have shown improved colonic transit times in both a healthy population (Picard et al., 2005) and constipated patients (Agrawal et al., 2008). Finally, probiotics lower the pH in the colon, which enhances peristalsis (Salminen & Salminen, 1997) and, subsequently, may decrease colonic transit time. One systematic review (Chmielewska & Szajewska, 2010; search date May 2009) to assess the efficacy and safety of probiotic supplementation for the treatment of constipation identified five RCTs with a total of 377 subjects (194 in the experimental group and 183 in the control group). The participants were adults (three RCTs, N = 266) and children (two RCTs, N = 111). In adults, data suggest a favorable effect of treatment with B. lactis DN 173 010, L. casei Shirota, and E. coli Nissle 1917 on defecation frequency and stool consistency. In children, only L. casei rhamnosus Lcr35, but not Lactobacillus GG, produced a beneficial effect. The pooled results of two pediatric RCTs revealed no difference in the rate of treatment success, defined similarly as three or more spontaneous defecations per week with no episodes of fecal soiling, between the probiotic and control groups (RR 2.3, 95% CI 0.26–20.5, random effect model). The authors concluded that until more data are available, the use of probiotics for the treatment of constipation should be considered investigational.

11.8.11

Allergy prevention

The rationale for using probiotics in the prevention of allergic disorders is based on several concepts. First, it has been suggested that improved hygiene and the reduced exposure of the immune system to the microbial stimulus early in childhood contribute to the

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rising number of allergic disorders worldwide (Prescot, 2003). Second, there are differences in the neonatal gut microflora that may precede or coincide with the early development of atopy. Atopic subjects have more clostridia and tend to have fewer bifidobacteria than non-atopic subjects (Kalliomaki et al., 2001a). Finally, there is evidence suggesting a crucial role for a balanced commensal gut microflora in the maturation of the early immune system. Two meta-analyses assessing the effects of probiotics in the prevention of allergic disorders were found (Osborn & Sinn, 2007; Lee et al., 2008). In the first meta-analysis (search date February 2007), 12 studies were eligible for inclusion. Of these, only six RCTs assessed allergic disease and/or food hypersensitivity outcomes. Outcomes were reported for only 1549 of the 2080 infants enrolled in these six RCTs. Although the studies generally had adequate randomization, allocation concealment, and blinding of treatment, many trials had excess losses in patient follow-up (17–61%). A meta-analysis of five RCTs, involving 1477 infants, revealed a significant reduction in infant eczema with probiotic supplementation. However, there was significant heterogeneity among the studies. One study evaluating the effect of LGG use demonstrated that the difference in the prevalence of eczema between the treatment and control groups persisted until 4 years of age (a further RCT, not included in the meta-analysis, showed an effect also at 7 years). However, the findings were no longer significant when the analysis was limited to studies reporting the prevalence of atopic eczema (confirmed by the skin-prick test or specific IgE). There were no reports of other benefits for any other allergic disease or food hypersensitivity outcome with the administration of probiotics and/or prebiotics. The authors concluded that there is insufficient evidence to recommend the addition of probiotics to infant feeds for the prevention of allergic disease or food reactions, as supported by the data reviewed. The second meta-analysis (Lee et al., 2008) was restricted to trials of probiotics for the prevention and treatment of pediatric atopic dermatitis. PubMed and Cochrane databases (up to July 2007) as well as reference lists were searched. Six prevention RCTs that compared treatment with different Lactobacillus species and placebo were included (including five RCTs identified in the previously mentioned meta-analysis). All the prevention studies were considered to be of high quality. The prenatal and/or postnatal administration of probiotics to pregnant woman and their infants resulted in a significant reduction in the incidence of atopic dermatitis in the infants. Exclusion of one RCT involving only postnatal administration of probiotics resulted in a more pronounced effect, suggesting the importance of prenatal administration. The third review identified three RCTs that evaluated the effect of probiotic use on the prevention of atopic dermatitis. The authors concluded that probiotics, especially Lactobacillus GG, seem to be effective for the prevention of this condition (Betsi et al., 2008). 11.8.11.1

Treatment of eczema/atopic dermatitis

There have been three meta-analyses with conflicting conclusions regarding the role of probiotics in the treatment of eczema. The first meta-analysis (Lee et al., 2008) (search date May 2007) assessed the effect of probiotics for the prevention and treatment of pediatric atopic dermatitis. For the latter outcome, four RCTs (N = 299) were identified. The reviewers stated that the clinical significance of the treatment trial findings of the intergroup reduction of Scoring of Atopic Dermatitis Severity Index by −6.64 points (−9.78, −3.49) and −8.56 (−18.39, 1.28), as well as the intragroup change of −1.06

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(−3.86, 1.73) and −1.37 (−4.81, 2.07), is questionable. In their view, current evidence is not convincing for a role of probiotics in the treatment of pediatric atopic dermatitis. A Cochrane review (Boyle et al., 2008) (search date April 2008) that identified 12 RCTs involving 781 participants (children only) also concluded that the evidence suggests that probiotics are not an effective treatment for eczema and that probiotic treatment carries a small risk of adverse events. Opposite conclusions were reached by the authors of the third meta-analysis (Michail et al., 2008) (search date January 2008) that assessed the efficacy of probiotics in the treatment of pediatric atopic dermatitis. The authors identified 11 RCTs, and data from 10 studies (N = 678) were available for analysis. Overall, there was a statistically significant difference favoring probiotics compared with placebo in reducing the Scoring of Atopic Dermatitis Severity Index score. Children with moderately severe disease were more likely to benefit. The outcome was not affected by the duration of probiotic administration, patient age, or type of probiotic used. According to the authors, evidence suggests a modest role for probiotics in the treatment of pediatric atopic dermatitis, with effects observed in patients with moderately severe rather than mild disease. Betsi et al. (2008) reviewed 10 RCTs that evaluated probiotics as a treatment for atopic dermatitis. Probiotics were found to reduce the severity of atopic dermatitis in approximately half of the RCTs evaluated.

11.8.12

Respiratory tract infections

The successful prevention of respiratory tract infections (RTIs), which are responsible for a significant number of consultations particularly in young children and the elderly, could be useful for patients, families, and society in general. One systematic review (Vouloumanou et al., 2009, search date February 2008) without a formal meta-analysis evaluated the effect of probiotics for the prevention or amelioration of RTIs and identified 14 RCTs (12 involving healthy subjects and two involving patients with RTIs). With regard to the incidence of RTIs, no significant difference was found between the probiotic and control groups in 10 RCTs, while the use of probiotics was beneficial in the remaining four RCTs. In five of six RCTs that provided relevant data, there was a reduction in the severity of symptoms related to RTIs in the probiotic group compared with the control group. In three of nine RCTs, the clinical course of RTIs was shorter in the probiotic group compared with the control group, whereas no intergroup difference was found in the remaining six RCTs. It was concluded that probiotics may have a beneficial effect on the severity and duration of symptoms of RTI but do not appear to reduce the incidence of RTIs (Michail et al., 2008).

11.9

IS A META-ANALYTICAL APPROACH APPROPRIATE FOR ASSESSING THE EFFICACY OF PROBIOTICS?

Analysis of the results of published meta-analyses reveals that, with few exceptions (Szajewska & Mrukowicz, 2005; Szajewska et al., 2007a,b, 2009), probiotics administered for treatment of a specific disease or condition are all evaluated together. The question

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remains: Is it appropriate to pool data on different probiotic microorganisms? It is tempting for reviewers to produce a single estimate of the treatment effect (presented as a diamond at the bottom of a forest plot). However, the results of a meta-analysis of all probiotics, regardless of the microorganisms used, may be misleading if appropriate consideration is not given to interpretation of the pooled results. Below, some arguments for and against the pooling of data on different probiotics, as well as some suggestions for solutions, are discussed.

11.9.1

Arguments for pooling data

The value of performing a meta-analysis is that by combining trials, the sample size is increased and thus the power. Pooled data on different probiotics allow one to (Higgins & Green, 2008) (1) establish whether there is evidence of an effect, (2) determine the direction of the effect, (3) determine the size of the effect (and the 95% CI around the effect), (4) assess the consistency of the effect across studies, and (5) identify the most promising probiotic(s). If there are many trials involving the administration of different probiotics to different participants with similar results consistently being seen in the various trials, the effect of the probiotic(s) has some generalizability. In addition, pooled data on different probiotics are important for demonstrating whether further research on these probiotics is substantiated. If so, this pooled data potentially may help to identify the most promising microorganisms as well as the research questions to be addressed in future studies.

11.9.2

Arguments against pooling data

There are a number of arguments against pooling data. First, there is evidence that the beneficial effects of probiotics, particularly the immunomodulatory effects of individual probiotics observed in the host, differ greatly and are strain specific. For example, O’Mahony et al. (2005) demonstrated that treatment with B. infantis 35624, but not L. salivarius UCC4331, resulted in normalization of the ratio of an anti-inflammatory to a proinflammatory cytokine. Second, probiotics vary by organism. In addition to the most commonly used lactic acid bacteria (e.g. lactobacilli, bifidobacteria), the yeast S. boulardii is often used. All these probiotics have different properties and antipathogenic mechanisms. Consequently, their efficacy may vary. For example, limited evidence suggests that S. boulardii, but not lactobacilli, is effective in preventing recurrent CDAD. Third, the dose of probiotics may be important. A study by Whorwell et al. (2006) involving 362 women with IBS found that treatment with B. infantis 35624 at a dose of 1 × 108 CFU was significantly superior compared with placebo and B. infantis at a dose of 1 × 106 or 1 × 1010 CFU for the primary efficacy variable of abdominal pain. In addition, treatment with B. infantis 35624 at a dose of 1 × 108 CFU compared with placebo resulted in significant changes from baseline in the composite Likert scale score and individual scores for bloating, bowel habit satisfaction, sense of incomplete evacuation, straining, and the passage of gas at the end of the 4-week study. Similar results were not observed with other dosages of B. infantis 35624. While the inefficacy of the dose of 1 × 1010 CFU was associated with significant formulation problems, no such problems were observed with the lower dose, suggesting dose–response issues. Finally, the results of some studies do suggest a different response to probiotics in various populations. For example, whereas coadministration of Lactobacillus GG (with antimicrobials) reduced both the incidence and duration of associated diarrhea in two RCTs

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involving children treated as outpatients (Arvola et al., 1999; Vanderhoof et al., 1999), it failed to prevent diarrhea in a large study of hospitalized adults (Thomas et al., 2001). Thus, the results observed in one population or setting cannot be simply extrapolated to the other. Collectively, these data suggest that is hard to consider probiotic supplementation as a homogeneous intervention. Pooling data from different genera, species, strains, and doses of probiotics obtained in different populations, presumably with variations in their native intestinal microbiota, may result in misleading conclusions. The risk is that the results could be erroneously extrapolated to other probiotics or other patient groups.

11.10

WHAT COULD BE THE SOLUTION?

Given these concerns, the best approach would be to perform a meta-analysis evaluating the effect of administering a clearly defined, single-organism probiotic preparation or an equally well-defined combination of probiotic microorganisms for treatment of a specific disease or condition. However, a lack of data often makes this infeasible. With few exceptions, only seldom are there data from more than single studies on given probiotic microorganism(s). There are various factors that discourage simple repetition (duplication) of trials that could clarify the effect of a given probiotic. These factors include a lack of scientific novelty and/or a lack of interest by potential sponsors in cases involving the administration of a commercially available probiotic product that has been proven effective in a single study. However, results from a study by Kopp et al. (2008) that assessed the effect of Lactobacillus GG use for the primary prevention of atopic dermatitis, which was carried out based on a protocol almost identical to that used in a study by Kalliomaki et al. (2001b), demonstrates the importance of such repeat studies. Whereas Kopp et al. (2008) observed no effect of Lactobacillus GG use on the prevention of atopic dermatitis, Kalliomaki et al. (2001b) reported that Lactobacillus GG use exerts a preventive effect on the development of atopic eczema. Another approach could be, and often is, to perform a review of all probiotics and then to perform subgroup analyses based on factors considered a priori that could potentially influence the magnitude of the treatment response (Szajewska & Mrukowicz, 2001; Allen et al., 2004; Moayyedi et al., 2010). Examples of such factors are (1) the type of probiotic administered, (2) whether the probiotic was live versus dead, (3) the medium, and (4) the study population (children, adults). It may be that the reader of a meta-analysis does not agree with pooling data on different probiotics. Still, one will have a comprehensive summary of the evidence and may learn from such analysis by assessing data from individual trials and ignoring the pooled results.

11.11

UNPUBLISHED DATA

Few, if any, meta-analyses on probiotics include unpublished data. This is because trial identification is the most difficult and demanding part of any systematic review, even if only published data are included. Identification of unpublished data is even more challenging, and there is no systematic process for finding controlled trials that were conducted but not published.

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However, does it matter if unpublished data are included in systematic reviews? This is a controversial issue (Cook et al., 1993). There are a number of arguments in favor of the inclusion of unpublished data. For example, it is well documented that unpublished studies differ systematically from those that have been published (Dickersin & Rennie, 2003; Whittington et al., 2004). Often, albeit definitely not always, because of methodological weaknesses, but more likely because of smaller or no treatment effects revealed in these trials. In addition, studies are often industry sponsored. There are concerns regarding sponsors who may withhold unfavorable trial data or will not make full trial reports available and/or the inadequate reporting of adverse events (Whittington et al., 2004). Nonpublication of a trial can lead to false assumptions regarding the efficacy of the treatment. Thus, the inclusion of unpublished data reduces the risk of publication bias, defined as the failure to report results of a negative trial. Initiatives such as the global registration of trials could contribute to better access to the results of relevant research, regardless of the results (Evans et al., 2004). However, the inclusion of unpublished data is not without challenges and drawbacks, which have been fully reviewed elsewhere (Higgins & Green, 2008). In brief, the first challenge is gaining access to the studies. The reviewers may contact all the companies who manufacture probiotics requesting unpublished data, but this approach may be unsuccessful. Regulations that all trial data, whether published or unpublished, should be fully accessible could help solve the problem. Even if unpublished trials are identified, obtaining unpublished information from the investigators may be difficult. The second challenge is obtaining sufficient information to evaluate the methodological quality of the research. While it has not been universally confirmed (MacLean et al., 2003), unpublished data may be of lower methodological quality than data in published trials. Expertise in the critical appraisal of studies would be needed to evaluate unpublished trials, but most reviewers do possess the necessary skills. The third challenge is that the located trial data may be an unrepresentative sample of data from all unpublished studies, and thus introduce further bias. Finally, it has been postulated that the absence of peer review of unpublished data poses a problem. On the other hand, some have expressed the view that the peer-review process does not guarantee the validity of published data. The most valid synthesis of available information will result when meta-analysts subject published and unpublished material to the same rigorous methodological evaluation and present results obtained with and without unpublished sources of data to provide a complete picture (MacLean et al., 2003).

11.12

QUALITY OF INCLUDED TRIALS

Any meta-analysis is only as good as the constituent studies, often summarized as “garbage in, garbage out” (Egger et al., 2003). Similar to any other research, often some of the trials included in the analysis have a number of methodological limitations (i.e. unclear or inadequate allocation concealment, no blinding, no intention-to-treat analysis). Study limitations also include small sample size in some trials. To obtain the most valid synthesis of available research data, the reviewers should subject available evidence to a thorough methodological evaluation and present a sensitivity analysis. The latter is defined in the Cochrane Handbook as “an analysis used to determine how sensitive the results of a study or systematic review are to changes in how it was done” (Higgins & Green, 2008). Sensitivity analyses are performed to assess the robustness of the results given the uncertain decisions or assumptions about the data and the methods that

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were used to obtain them. A review by Allen et al. (2004) on the efficacy of probiotics for the treatment of infectious diarrhea is an example of a meta-analysis in which sensitivity analyses according to each of the four parameters of trial methodological quality were performed.

11.13

INCONCLUSIVE SYSTEMATIC REVIEWS AND META-ANALYSES

One of the criticisms regarding systematic reviews or meta-analyses is that they are not useful because of often inconclusive results, with the inclusion of frustrating statements such as “no clear evidence”, “some evidence of a trend”, etc. Some examples of such inconclusive results were provided in the section describing the results of published systematic reviews and meta-analyses. However, the demonstration of clinical uncertainty about any therapeutic or preventive issue is an important finding. As pointed out by Alderson and Roberts (2000), clinical uncertainty is a prerequisite for the large-scale RCTs needed to evaluate the influence of such interventions; it also helps to clarify available treatment options and stimulate new and better research. In addition, such systematic reviews may allow more accurate calculation of the sample sizes required in future trials.

11.14

OPPOSITE CONCLUSIONS

Systematic reviews addressing the same clinical question and performed at almost the same time by reviewers with the same access to relevant databases may reach discordant conclusions. For example, after reviewing data from RCTs on the efficacy of probiotics for the prevention of traveler’s diarrhea, McFarland concluded that “several probiotics (S. boulardii and a mixture of Lactobacillus acidophilus and Bifidobacterium bifidum) had significant efficacy”. Other reviewers, however, have stated that probiotics are not effective in preventing traveler’s diarrhea (Takahashi et al., 2007). A number of factors may contribute to discordance among systematic reviews, which have been reviewed in detail elsewhere (Jadad et al., 1997). In brief, these include differences in the review question (e.g. participants being adults and children or adults only), search strategy (e.g. inclusion or exclusion of unpublished data), data extraction, assessment of study quality (e.g. inclusion of both high and low quality studies), and statistical methods used for data synthesis. Such discordance in the conclusions of reviews is frustrating and contributes to confusion instead of reducing uncertainty. For decision-making, an algorithm for interpreting discordant reviews has been developed (Jadad et al., 1997).

11.15 ● ●

SUMMARY AND KEY MESSAGES

Systematic reviews with or without a meta-analysis are now a well-established means of reviewing existing evidence and integrating findings from various studies. A distinction should be made between a systematic review and a meta-analysis. It is always appropriate to perform a systematic review (i.e. a review of a clearly formulated question using systematic and explicit methods), but it may be inappropriate or

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●

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misleading to perform a meta-analysis (i.e. to use statistical techniques to combine the results of included trials to produce a single estimate of the effect). An understanding of the strengths and limitations of the meta-analytical approach as well as the critical appraisal of the results of a meta-analysis is needed by everyone involved in decision-making regarding the use of probiotics. Meta-analyses on probiotics do provide valid information. However, caution should be exercised so one does not over-interpret the results of a meta-analysis when all probiotics have been evaluated together. Considering that the effects of probiotics are strain specific as well as population specific, they cannot be generalized.

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Kalliomaki M, Kirjavainen P, Eerola E, Kero P, Salminen S, Isolauri E (2001a) Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. J Allergy Clin Immunol 107:129–134. Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E (2001b) Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial. Lancet 357:1076–1079. Kopp MV, Hennemuth I, Heinzmann A, Urbanek R (2008) Randomized, double-blind, placebo-controlled trial of probiotics for primary prevention: no clinical effects of Lactobacillus GG supplementation. Pediatrics 121:e850–e856. Lee J, Seto D, Bielory L (2008) Meta-analysis of clinical trials of probiotics for prevention and treatment of pediatric atopic dermatitis. J Allergy Clin Immunol 121:116–121. McFarland LV (2006) Meta-analysis of probiotics for the prevention of antibiotic associated diarrhea and the treatment of Clostridium difficile disease. Am J Gastroenterol 101:812–822. McFarland LV (2007) Meta-analysis of probiotics for the prevention of traveler’s diarrhea. Travel Med Infect Dis 5:97–105. McFarland LV, Dublin S (2008) Meta-analysis of probiotics for the treatment of irritable bowel syndrome. World J Gastroenterol 14:2650–2661. MacLean CH, Morton SC, Ofman JJ, Roth EA, Shekelle PG (2003) Southern California Evidence-Based Practice Center. How useful are unpublished data from the Food and Drug Administration in metaanalysis? J Clin Epidemiol 56:44–51. Magne F, Suau A, Pochart P, Desjeux JF (2005) Fecal microbial community in preterm infants. J Pediatr Gastroenterol Nutr 41:386–392. Mallon P, McKay D, Kirk S, Gardiner K (2007) Probiotics for induction of remission in ulcerative colitis. Cochrane Database Syst Rev 4:CD005573. Michail SK, Stolfi A, Johnson T, Onady GM (2008) Efficacy of probiotics in the treatment of pediatric atopic dermatitis: a meta-analysis of randomized controlled trials. Ann Allergy Asthma Immunol 101:508–516. Moayyedi P, Ford AC, Brandt L et al. (2008) Efficacy of probiotics in the therapy of irritable bowel syndrome (IBS): a systematic review [Abstract]. Am J Gastroenterol 103(Suppl 1):S481. Moayyedi P, Ford AC, Talley NJ et al. (2010) The efficacy of probiotics in the therapy of irritable bowel syndrome: a systematic review. Gut 59:325–332. Nikfar S, Rahimi R, Rahimi F, Derakhshani S, Abdollahi M (2008) Efficacy of probiotics in irritable bowel syndrome: a meta-analysis of randomized, controlled trials. Dis Colon Rectum 51:1775–1780. Ogura Y, Bonen DK, Inohara N et al. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 411:603–606. O’Mahony L, McCarthy J, Kelly P et al. (2005) Lactobacillus and Bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 128:541–551. Osborn DA, Sinn JK (2007) Probiotics in infants for prevention of allergic disease and food hypersensitivity. Cochrane Database Syst Rev 4:CD006475. Oxman A, Guyatt G, Cook D, Montori V (2002) Summarizing the evidence. In: Guyatt G, Rennie D (eds) Users’ Guides to the Medical Literature. A Manual for Evidence-based Medicine. Chicago: AMA Press, pp. 155–173. Parkes GC, Sanderson JD, Whelan K (2009) The mechanisms and efficacy of probiotics in the prevention of Clostridium difficile-associated diarrhoea. Lancet Infect Dis 9:237–244. Picard C, Fioramonti J, Francois A, Robinson T, Neant F, Matuchansky C (2005) Review article: bifidobacteria as probiotic agents: physiological effects and clinical benefits. Aliment Pharmacol Ther 22:495–512. Pillai A, Nelson R (2008) Probiotics for treatment of Clostridium difficile-associated colitis in adults. Cochrane Database Syst Rev 1:CD004611. Prescot SL (2003) Allergy: the price we pay for cleaner living? Ann Allergy Asthma Immunol 90:64–70. Rahimi R, Nikfar S, Rahimi F et al. (2008) A meta-analysis on the efficacy of probiotics for maintenance of remission and prevention of clinical and endoscopic relapse in Crohn’s disease. Dig Dis Sci 53:2524–2531. Rasquin A, Di Lorenzo C, Forbes D et al. (2006) Childhood functional gastrointestinal disorders: child/ adolescent. Gastroenterology 130:1527–1537. Rolfe VE, Fortun PJ, Hawkey CJ, Bath-Hextall F (2006) Probiotics for maintenance of remission in Crohn’s disease. Cochrane Database Syst Rev 4:CD004826. Salminen S, Salminen E (1997) Lactulose, lactic acid bacteria, intestinal microecology and mucosal protection. Scand J Gastroenterol Suppl 222:45–48.

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Sandborn W, McLeod R, Jewell D (2000) Pharmacotherapy for inducing and maintaining remission in pouchitis. Cochrane Database Syst Rev 2:CD001176. Szajewska H, Mrukowicz J (2001) Probiotics in the treatment and prevention of acute infectious diarrhea in infants and children:a systematic review of published randomized, double-blind, placebo controlled trials. J Pediatr Gastroenterol Nutr 33:S17–S25. Szajewska H, Mrukowicz J (2005) Meta-analysis: non-pathogenic yeast Saccharomyces boulardii in the prevention of antibiotic-associated diarrhea. Aliment Pharmacol Ther 22:365–372. Szajewska H, Ruszczynski M, Radzikowski A (2006) Probiotics in the prevention of antibiotic-associated diarrhea in children: a meta-analysis of randomized controlled trials. J Pediatr 149:367–372. Szajewska H, Skórka A, Ruszczyñski M, Gieruszczak-Białek D (2007a) Meta-analysis: Lactobacillus GG for treating acute diarrhoea in children. Aliment Pharmacol Ther 25:871–881. Szajewska H, Skórka A, Dyląg M (2007b) Meta-analysis: Saccharomyces boulardii for treating acute diarrhoea in children. Aliment Pharmacol Ther 25:257–264. Szajewska H, Sko´rka A (2009) Saccharomyces boulardii for treating acute gastroenteritis in children: updated meta-analysis of randomized controlled trials. Aliment Pharmacol Ther 30:960–961. Takahashi O, Noguchi Y, Omata F, Tokuda Y, Fukui T (2007) Probiotics in the prevention of traveler’s diarrhea: meta-analysis. J Clin Gastroenterol 41:336–337. Thomas MR, Litin SC, Osmon DR, Corr AP, Weaver AL, Lohse CM (2001) Lack of effect of Lactobacillus GG on antibiotic-associated diarrhea: a randomized, placebo controlled trial. Mayo Clinic Proc 76:883–889. Tong JL, Ran ZH, Shen J et al. (2007) Meta-analysis: the effect of supplementation with probiotics on eradication rates and side effects during Helicobacter pylori eradication therapy. Aliment Pharmacol Ther 25:155–168. Turck D, Bernet JP, Marx J et al. (2003) Incidence and risk factors of oral antibiotic-associated diarrhea in an outpatient pediatric population. J Pediatr Gastroenterol Nutr 37:22–26. Van den Berg MM, Benninga MA, Di Lorenzo C (2006) Epidemiology of childhood constipation: a systematic review. Am J Gastroenterol 101:2401–2409. Vanderhoof JA, Whitney DB, Antonson DL, Hanner TL, Lupo JV, Young RJ (1999) Lactobacillus GG in the prevention of antibiotic-associated diarrhea in children. J Pediatr 135:564–568. Van Niel C, Feudtner C, Garrison MM, Christakis DA (2002) Lactobacillus therapy for acute infectious diarrhea in children: a meta-analysis. Pediatrics 109:678–684. Vouloumanou EK, Makris GC, Karageorgopoulos DE, Falagas ME (2009) Probiotics for the prevention of respiratory tract infections: a systematic review. Int J Antimicrob Agents 34:197.e1–e10. Whittington CJ, Kendall T, Fonagy P, Cottrell D, Cotgrove A, Boddington E (2004) Selective serotonin reuptake inhibitors in childhood depression: systematic review of published versus unpublished data. Lancet 363:1341–1345. Whorwell PJ, Altringer L, Morel J et al. (2006) Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable bowel syndrome. Am J Gastroenterol 101:1581–1590. Zoppi G, Cinquetti M, Luciano A, Benini A, Muner A, Bertazzoni Minelli E (1998) The intestinal ecosystem in chronic functional constipation. Acta Paediatr 87:836–841.

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Applied Studies with Probiotics: Fundamentals for Meeting the Health Claims

Hannu Mykkänen, Silvia W. Gratz and Hani El-Nezami

12.1

INTRODUCTION

Substantiation of a health claim is always based on human data, collected primarily from intervention studies. Although the final evidence is produced in randomized controlled trials, information on the mechanisms of effects often requires animal experimentation and in vitro studies. With probiotics it is especially important to generate data on plausible mechanisms of their beneficial health effects, since probiotics have been shown to produce a large variety of strain-specific responses in human physiology resulting in a wide variety of diseases and clinical conditions (Goldin & Gorbach 2008), and each of these requires the use of their characteristic biomarker in evaluation of the health effects. Using our experience from studies on the probiotic–aflatoxin interaction, we will demonstrate the problems associated with compiling evidence in support of health claims for probiotics. This chapter describes a series of studies showing the path from a laboratory-based finding in vitro and animal experiments to confirmation of the in vitro findings in vivo and to field studies in human subjects (Fig. 12.1). The relevance of the data collected in various stages is discussed, and a proof-of-concept study on the interaction of well-defined probiotic bacteria with a harmful dietary contaminant in an exposed population group is presented.

12.2

MYCOTOXIN PROBLEM

Toxin-producing fungi are ubiquitous in our environment and can invade our crops and produce toxic secondary metabolites known as mycotoxins. To avoid destroying edible crops because of fungal growth and spoilage, technologies have been developed to be used during harvest, processing and storing of crops. Despite this, mycotoxin exposure can occur either as low-level chronic exposure causing serious health problems (e.g. stunted growth, impaired immune function, liver cancer) or as an acute disease that may result in death especially in areas where populations depend on a highly contaminated single staple food commodity. Aspergillus, Penicillium, Fusarium and Alternaria are the major mycotoxin-producing fungi, and their toxins are found in corn and groundnuts (aflatoxins), corn, wheat and oats (trichothecenes, ochratoxin A), and in apple juice (patulin) (SCOOP, 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 Selection of bacteria with probiotic properties (available commercially or isolated from microbiota of healthy subjects) and good safety record

In vitro binding assays

Mechanisms of binding (to determine the chemical and structural factors)

Kinetic studies on binding and release of toxins (dose-response)

In vitro toxicity studies (to examine if the binding will detoxify the toxin)

Selection of bacteria with good binding properties and ability to deactivate food toxins

In vivo binding of single toxins and mixtures

Feedind studies (using animal model)

Ex vivo ligated loop in chicks

Stabillity of complex Effect on absorption and bioavailability

Clinical trials in populations exposed to toxins (body burden and biomarkers)

Fig. 12.1 Studies on the probiotic–aflatoxin interaction showing the path from a laboratory-based finding in vitro and animal experiments to field studies in human subjects.

2003). Since consumption of mycotoxin-contaminated food commodities cannot be fully avoided, especially in the developing countries where there is a shortage of food, reduction in mycotoxin bioavailability from foods may offer a useful way to prevent the harmful health effects exerted by mycotoxins. A series of studies started by Hani El-Nezami in his dissertation (El-Nezami, 1998) identified the probiotic lactic acid bacteria as a safe means of reducing availability of aflatoxins in vitro. These bacteria are characterized by a number of other beneficial health effects that make them even more suitable additives to food and feed. This finding was thought to be of great potential importance since aflatoxin exposure and its adverse health effects is well known (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2002) and aflatoxin B1, the most prevalent and potent form of aflatoxin, has been classified as a class 1A human carcinogen by the IARC (2002). Moreover, both acute and chronic aflatoxicosis are most prevalent in developing countries with very limited healthcare resources in prevention and treatment.

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12.3

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LACTOBACILLUS RHAMNOSUS STRAIN EFFECTIVELY BINDS AFLATOXIN: IN VITRO FINDINGS

El-Nezami found in his dissertation work that a Lactobacillus rhamnosus strain effectively binds aflatoxin in a test tube (El-Nezami et al., 1998a). After the original finding, more than 250 strains of lactic acid bacteria isolated from either dairy products or healthy human microbiota have been tested in vitro for this effect, but only some species and strains of the tested probiotics were shown to be capable of removing aflatoxin from solution (El-Nezami et al., 1998a; Pieridis et al., 2000). Even within a given species, not all strains are equivalent in terms of toxin binding. Gram-positive bacteria (several lactobacilli and propionibacteria) appear to be more efficient in removing aflatoxin from liquid medium than Gram-negative Escherichia coli (El-Nezami et al., 1998a). Lactobacillus rhamnosus strain GG (LGG) and strain LC705 appeared to be the most efficient binders of aflatoxin B1, removing approximately 80% of the toxin from liquid media within minutes of incubation. This fast removal has been confirmed by other investigators (Bueno et al., 2007; Khanafari et al., 2007). Peltonen et al. (2001) has also reported remarkable differences in aflatoxin B1-binding ability within a range of closely related strains of lactobacilli and bifidobacteria. Thus the efficacy of aflatoxin detoxification by probiotics is highly variable and depends on the genus and strain of bacteria, and identification of the effective species or subspecies is essential for substantion of the appropriate health claims. Lactobacillus rhamnosus GG, one of the most efficient aflatoxin binders, is used in various dairy products in many countries and hence is a good candidate for further studies aiming to confirm this in vitro finding in vivo and thereby to substantiate the possible health claim based on the interaction of this probiotic bacteria with harmful compounds in the gastrointestinal tract. The conditions under which lactobacilli bind aflatoxins and other mycotoxins in vitro have also been studied in detail (El-Nezami et al., 1998a,b, 2002a,b). Bacterial metabolism does not seem to play a significant role since killing the bacteria by boiling for 1 hour or by acid treatment increased their ability to remove aflatoxin B1 (El-Nezami et al., 1998b). Similarly, Thyagaraja and Hosono (1994) have reported that autoclaved L. casei removes significantly more aflatoxin from phosphate-buffered medium as compared to live bacteria. In addition, Peltonen et al. (2001) found increased aflatoxin binding by bifidobacteria after heat-treatment, while Lankaputhra and Shah (1998) have reported the opposite effects in their studies on binding of dietary mutagens by heat-treated versus viable bacteria. Since the binding does not require the presence of live bacteria, this phenomenon is clearly easier to apply to a variety of food commodities. Binding of mycotoxins appears to occur predominantly with carbohydrate and protein components at the surface of the bacteria. The mechanism involves hydrophobic interactions, bacterial cell wall polysaccharide peptidoglycans being the most likely binding sites for aflatoxin B1 to the surface of L. rhamnosus GG and L. rhamnosus LC705 (Haskard et al., 2000, 2001; Lahtinen et al., 2004). It was suggested that denaturation of cell wall proteins and breakage of the glycosidic linkages in polysaccharides and amine linkages in peptides and proteins and consequent increase in the pore size of the peptidoglycan layer of the bacterial surface may explain the increased binding by non-viable bacteria (Haskard et al., 2001). Revealing the mechanism of binding helps to explain the observed differences between strains of bacteria in their ability to sequester aflatoxins, and this information might be used to develop more efficient binders.

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The concentration of the bacteria also influences the binding. A minimum probiotic concentration for removing 50% of aflatoxin B1 from solution was found to be 2 × 109 CFU/mL and higher binding occurs at the level of 1010 CFU/mL (El-Nezami et al., 1998a). The information that a single bacterium is able to bind in excess of 107 aflatoxin B1 molecules (Haskard et al., 2000) may be useful in defining the dose of probiotic needed to have a beneficial effect in vivo. Interestingly, intestinal mucus has also been shown to compete with aflatoxin-binding sites at the bacterial surface (Gratz et al., 2004, 2005). Binding first to mucus significantly reduced aflatoxin binding by L. rhamnosus LC705 but not by LGG. Using a Caco-2 cell model, Kankanpää et al. (2000) demonstrated that adhesion of the complex formed by LGG and aflatoxin B1 is reduced compared with adhesion of LGG alone. After binding aflatoxin the bacteria appear to lose their ability to bind to the intestinal mucosa, thus increasing the potential removal of the toxin by the fecal route. Interactions of bacteria and mycotoxins with intestinal mucus may also partly explain the differences observed between the in vitro and in vivo results (Gratz et al., 2004, 2005) and should be taken into consideration when choosing a probiotic for binding.

12.4

ANIMAL MODELS FOR STUDYING THE AFLATOXIN–PROBIOTIC INTERACTION

The in vitro findings are useful in the screening of potential agents from a larger number of candidates, but the findings need to be confirmed in animal experiments to prove their efficacy and safety in vivo prior to collecting the final evidence in human studies. The reduced availability of aflatoxin B1 by probiotic bacteria was confirmed in two animal models. The ability of Lactobacillus strains to bind aflatoxin B1 was first tested using the in vivo ligated duodenal loop of chick. All tested probiotics (LGG, L. rhamnosus LC705 and Propionibacterium freudenreichii subsp. shermanii JS), when injected intraduodenally together with aflatoxin B1, proved capable of reducing aflatoxin in the soluble fraction of the luminal content of 1-week old chickens (El-Nezami et al., 2000a). The uptake of aflatoxin B1 by the intestinal mucosa was also reduced, indicating that these probiotics have potential to reduce the transport of aflatoxin across the intestinal mucosa. Following this finding, rats were dosed orally with aflatoxin B1 (1.5 mg/kg body weight) and probiotics (5 × 1010 CFU/rat) and fecal excretion of aflatoxin B1 was determined (Gratz et al., 2006). Probiotic dosing significantly increased the fecal excretion of aflatoxin B1 within 24 hours after dosing, indicating reduced absorption from the intestinal lumen. Furthermore, hepatotoxicity and weight loss induced by aflatoxin were alleviated in rats dosed with probiotics, giving further support to the original idea that binding by probiotics can be used to reduce the body burden of these toxicants (Gratz et al., 2006). It is evident from the above in vitro and in vivo findings that interaction between probiotics and aflatoxins does not require the presence of live bacteria. Thus there is no need to show that the probiotic bacteria have remained viable during their passage through the gastrointestinal tract. Moreover, use of non-viable bacteria is clearly less demanding under the conditions prevailing in the developing countries. On the other hand, the beneficial effect requires that the bacteria are present in the gastrointestinal lumen at the minimum effective concentration simultaneously with the harmful agent. Since the aflatoxin content

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of daily foods is highly variable, reflecting poor harvest conditions and improper home storage of foods, it is difficult to reduce exposure using probiotics unless daily consumption is adopted.

12.5

FIELD STUDIES WITH LACTOBACILLUS RHAMNOSUS STRAIN IN AFLATOXIN-EXPOSED POPULATIONS

To establish these beneficial effects of probiotic administration in humans, field studies were conducted in people exposed to aflatoxins via diet. In a pilot trial, 20 healthy Egyptian volunteers (age 20–40 years, 12 males and 8 females) from a rural area close to Alexandria were recruited, and 10 of them were given a probiotic supplement, two capsules per day (one each morning and evening) for 2 weeks (El-Nezami et al., 2000b). The capsules contained lyophilized L. rhamnosus strain LC705 and P. freudenreichii subsp. shermanii JS at a dose level of 2–5 × 1010 CFU/day. Each test subject had a control subject residing in the same household and receiving two placebo capsules per day containing only cellulose. Aflatoxin B1 was detected in the feces of five subjects from the test group and in six subjects from the control group at the baseline prior to the intervention period. The level of fecal aflatoxin was significantly reduced in five subjects receiving the probiotic supplement, and the number of subjects testing positive decreased to two, while the level of aflatoxin in the feces of the six control subjects remained unchanged. According to the in vitro and in vivo results, an increase in fecal excretion of aflatoxin was expected in the group receiving the probiotic bacteria. This contradictory finding could be explained by the use of fecal aflatoxin as a biomarker of aflatoxin exposure and by the timing of fecal collection (a spot sample 1 week after starting the supplementation). The fecal level reflects both the current dietary exposure and the aflatoxin in the body secreted via bile, and therefore it is not an optimal biomarker for dietary exposure. Interaction between the probiotic and aflatoxin probably removed most of the luminal aflatoxin immediately after starting the bacterial supplementation. After 1 week the fecal level in the test group reflects mainly the current dietary exposure while that in the control goup represents both the aflatoxin secreted via bile and current dietary exposure. This study clearly emphasizes the importance of timing of sample collection and selection of the relevant biomarkers in collecting evidence from human trials supporting health claims. In the other trial, healthy Chinese male students (N = 300) from southern China were screened for presence of the hydroxylated metabolite of aflatoxin B1, aflatoxin M1, in a spot urine sample (Mykkänen et al., 2005). Of those with detectable levels of aflatoxin, 90 were selected for an intervention aimed at investigating the effect of probiotic supplementation on urinary aflatoxin B1-N7-guanine, a well-established biomarker for dietary aflatoxin exposure (Groopman et al., 1992). The probiotics used were L. rhamnosus strain LC705 and P. freudenreichii subsp. shermanii JS at a dose level of 2–5 × 1010 CFU/day. The subjects were randomized into two groups, 45 subjects receiving a probiotic capsule and 45 subjects a placebo capsule twice a day for 5 weeks (El-Nezami et al., 2006). No difference in the percentage of detectable aflatoxin B1-N7-guanine levels were seen at baseline, but the percentage of negative values tended to be higher in the probiotic group during the 5-week intervention period, and the difference between the groups was not significant at the post-intervention visit 5 weeks after the cessation of the intervention.

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Urinary AFB1-N7-guanine (ng/mL)

1.2 Probiotic 1.0

Placebo

0.8 0.6 0.4 0.2 0.0 Baseline

Week 3

Week 5

Post intervention

Fig. 12.2 Urinary aflatoxin B1-N7-guanine (geometric means) in healthy male students in Guangzhou, southern China during a 5-week probiotic intervention and at post intervention.

Probiotic administration led to a significant decrease in the mean level of urinary aflatoxin B1-N7-guanine (Fig. 12.2). Thus this study showed that it is possible to reduce the biologically effective dose of aflatoxin by giving subjects with detectable aflatoxin exposure a dose of probiotics regularly on a daily basis. This result supports the earlier data collected from in vitro studies and animal experiments, and encourages additional field studies with different target populations. We are currently evaluating the data collected from a probiotic intervention in Egypt to assess the possibility of preventing aflatoxin exposure of breast-feeding infants by dietary probiotics given to their mothers. It is generally accepted that randomized controlled human trials are the cornerstone in compiling evidence for the substantiation of health claims. However, the present case report on the probiotic–aflatoxin interaction clearly illustrates that there is a long path of research activities preceding human trials. With regard to probiotics, this is especially important, since health claims for probiotics should be strain specific not generic, and therefore the amount and type of evidence can vary widely.

REFERENCES Bueno DJ, Casale CH, Pizzilitto RP, Salvano MA, Oliver G (2007) Physical adsorption of aflatoxin B1 by lactic acid bacteria and Saccharomyces cerevisiae: a theoretical model. J Food Prot 70:2148–2154. El-Nezami H (1998) Probiotic bacteria, an approach to detoxify aflatoxins. PhD thesis, RMIT-University, Melbourne, Australia. El-Nezami H, Kankaanpää PE, Salminen S, Ahokas JT (1998a) Ability of dairy strains of lactic acid bacteria to bind food carcinogens. Food Chem Toxicol 36:321–326. El-Nezami H, Kankaanpää P, Salminen S, Ahokas J (1998b) Physico-chemical alterations enhance the ability of dairy strains of lactic acid bacteria to remove aflatoxins from contaminated media. J Food Prot 61:466–468. El-Nezami H, Mykkänen H, Kankaanpää P, Salminen S, Ahokas J (2000a) Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B1 from the chicken duodenum. J Food Protect 63:549–552.

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El-Nezami H, Mykkänen H, Kankaanpää P, Suomalainen T, Salminen S, Ahokas J (2000b) The ability of a mixture of Lactobacillus and Propionibacterium to influence the faecal aflatoxin content in healthy Egyptian volunteers: a pilot clinical study. Biosci Microflora 19:41–45. El-Nezami HS, Chrevatidis A, Auriola S, Salminen S, Mykkänen H (2002a) Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium. Food Addit Contam 19:680–686. El-Nezami H, Polychronaki N, Salminen S, Mykkänen H (2002b) Binding rather than metabolism may explain the interaction of two food grade Lactobacillus strains with zearalenone and its derivative zearalenol. Appl Environ Microbiol 68:3545–3549. El-Nezami HS, Polychronaki NN, Ma J et al. (2006) Probiotic supplementation reduces a biomarker for increased risk of liver cancer in young men from Southern China. Am J Clin Nutr 83:1199–1203. Goldin BR, Gorbach SL (2008) Clinical indications for probiotics: an overview. Clin Infect Dis 46(Suppl 2):S96–S100. Gratz S, Mykkänen H, Ouwehand AC, Juvonen R, Salminen S, El-Nezami H (2004) Intestinal mucus alters the ability of probiotic bacteria to bind aflatoxin B1 in vitro. Appl Environ Microbiol 70:6306–6308. Gratz S, Mykkänen H, El-Nezami H (2005) AFB1 binding by a mixture of Lactobacillus rhamnosus strain LC-705 and Propionibacterium freudenreichii ssp. JS in vitro and ex vivo. J Food Protect 68:2470–2474. Gratz S, Täubel M, Juvonen R et al. (2006) Lactobacillus rhamnosus strain GG modulates intestinal absorption of aflatoxin B1 and its fecal excretion and toxicity in rats. Appl Environ Microbiol 72:7398–7400. Groopman JD, Jiaqi Z, Donahue PR et al. (1992) Molecular dosimetry of urinary aflatoxin–DNA adducts in people living in Guangxi Autonomous region, People’s Republic of China. Cancer Res 52:45–52. Haskard C, Binnion C, Ahokas J (2000) Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG. Chem Biol Interact 128:39–49. Haskard CA, El-Nezami HS, Kankaanpää PE, Salminen S, Ahokas JT (2001) Surface binding of aflatoxin B1 by lactic acid bacteria. Appl Environ Microbiol 67:3086–3091. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans (2002) Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. IARC Monogr Eval Carcinog Risks Hum 82, p. 171. Khanafari A, Soudi H, Miraboulfathi M, Osboo RK (2007) An in vitro investigation of aflatoxin B1 biological control by Lactobacillus plantarum. Pak J Biol Sci 10:2553–2556. Lahtinen SJ, Haskard CA, Ouwehand AC, Salminen SJ, Ahokas JT (2004) Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG. Food Addit Contam 21:158–164. Lankaputhra WE, Shah NP (1998) Antimutagenic properties of probiotic bacteria and of organic acids. Mutat Res 397:169–182. Mykkänen H, Zhu HL, Salminen E et al. (2005) Fecal and urinary excretion of aflatoxin B1 metabolites (AFQ1, AFM1, and AFB1-N-7-guanine) in young Chinese males. Int J Cancer 115:879–884. 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. Pieridis M, El-Nezami H, Peltonen K, Salminen S, Ahokas J (2000) Ability of dairy strains of lactic acid bacteria to bind aflatoxin M1 in a food model. J Food Prot 63:645–650. SCOOP (2003) Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states. Available at ec.europa.eu/food/fs/scoop/task3210.pdf Thyagaraja N, Hosono A (1994) Binding properties of lactic acid bacteria from “Idly” towards food-borne mutagens. Food Chem Toxicol 32:805–809.

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Probiotics Research: the Pediatric Perspective

Karl Zwiauer

13.1

INTRODUCTION

During the last two decades, probiotics, visibly and partly unperceived, have found their way into infant and childhood nutrition. At least in the European Union (EU), most of the infant milk formulas are enriched with probiotics and moreover hundreds of other food products available in normal food markets are enriched with different probiotic strains. Hardly any other food supplement has gained such great interest as probiotics and it seems almost obligatory to enrich milk products with probiotic strains in order to do something good for our or our children’s health. In parallel with the marketing cry that probiotics are good for infants and children, one would like to have clear evidence for the benefits of these ubiquitous food products. Probiotic functional foods in infancy are claimed to provide health benefits beyond the provision of essential nutrients. The introduction of these probiotic products has been promoted mainly by the food industry and only partly accompanied by scientific research. However, during the last decade, scientific interest has grown, and numerous studies have tried to answer the main questions. How do probiotics work? What are the beneficial effects and what are the indications for using probiotics? And last but not least, can probiotics do harm? This chapter provides an overview of the state of the art regarding the scientific basis of the use of probiotics in different diseases in infants and children and addresses the significance and value of probiotics in nutrition in infancy and childhood.

13.2

DEVELOPMENT OF THE GASTROINTESTINAL FLORA POSTNATALLY

The gastrointestinal tract of the unborn baby is sterile because the amniotic fluid is sterile. During delivery, the process of colonization of the mucosal membranes of the newborn commences rapidly and continues during the first postnatal days of life (Bettelheim et al., 1974; Brook et al., 1979; Bengmark, 1998; Mackie et al., 1999). Colonization of the newborn is dependent on the immediate environment and differs considerably between individuals. The maternal birth canal, fecal compounds, mother’s skin, and presence of birthing assistants determines the primary bacterial mixture the newborn is exposed to.

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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Different environmental factors influence the bacterial composition of the skin and membranes of the newborn, the most important of which are listed below: ● ● ● ● ● ●

maternal diet (Peltonen et al., 1992; Schultz et al., 2004); gestational age (Penders et al., 2006; Westerbeek et al., 2006); type of delivery (Penders et al., 2006); hospitalization (Bjorksten, 2004); infant nutrition (Harmsen et al., 2000; Penders et al., 2006); infant antibiotic use (Teitelbaum & Walker, 2002; Penders et al., 2006).

Maternal nutrition plays an important role in colonization of the gastrointestinal tract of the newborn. Penders and coworkers could demonstrate that infants whose mothers consumed an organic diet (foods produced without the use of synthetic fertilizers or pesticides, veterinary drugs, genetically modified seeds and breeds, preservatives, additives, and irradiation) or a biodynamic diet have significantly lower numbers of Escherichia coli in their stools than infants whose mothers consumed a regular diet (Penders et al., 2006). One of the reasons for this could be the fact that organically produced foods include spontaneously fermented vegetables containing lactobacilli (Alm et al., 2002). On the other hand, mothers who consume organic diets often breast-fed their infants exclusively, compared with mothers who consumed a regular diet (Penders et al., 2006). It seems biologically plausible that it is possible to influence colonization of newborns via the microbiota of the mother. It is not unlikely that transfer of probiotic bacteria from mother to child takes place, particularly in infants born vaginally, who are exposed to maternal feces during vaginal delivery. Therefore the concept of treating the mother with probiotics in order to (positively) affect the microbiota of the newborn infant seems fascinating. Moreover, by colonizing the naive gut of the newborn with specific probiotics, it might be possible to permanently influence the gut of the newborn with probiotics known to generate positive health effects. In contrast, in adults it is not possible to sustainably colonize the gut with probiotic bacteria, but this might be different for newborns and neonates, who do not yet have an established gut microbiota. Schultz et al. (2004) demonstrated that probiotics ingested by the mother may be transmitted to the infant during birth. They confirmed that a specific probiotic strain (Lactobacillus rhamnosus strain GG) orally administered to mothers was transferred to the newborns and was present in infants at the ages of 1, 6 and 12 months and in unexplained circumstances for as long as 24 months. Because of the oxidation–reduction potential of the intestinal environment of neonates, the gastrointestinal tract is first colonized by aerobes. The consumption of oxygen by these bacteria gradually changes the intestinal flora to one that is more reducing and, as a consequence, strict anaerobes subsequently start growing. One of the major determinants of colonization of the gut flora of newborns is the mode of delivery. Vaginally born newborns are colonized with the fecal and vaginal bacteria of the mother. Children born by Caesarean section are primarily exposed to bacteria originating from the environment of the hospital, e.g. healthcare workers. Moreover, children born by Caesarean section often show clusters of risk factors for unfavorable microbiota: they stay more often, and for longer, in hospital and they or their mothers often receive antibiotics. Thus it is hard to differentiate which of the factors is the major determinant. Vaginal delivery and breast-feeding of the child are associated with primary colonization predominantly with beneficial Bacteroides and bifidobacteria (Huurre et al., 2008). On the

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Probiotics and Health Claims 120 x 108 100 x 108

Vaginal delivery Caesarean section

80 x 108 60 x 108 40 x 108 20 x 108

2 x 106 1 x 106 0 x 106

Bifidobacterium

Total bacterial cell number

Fig. 13.1 Total gut bacterial cell counts and number of bifidobacteria in feces at 1 month after vaginal or Caesarean delivery. (Modified from Huurre et al. 2008.)

other hand, Caesarean section is associated with a decrease in these anaerobic microorganisms and an increase in Clostridium spp. (Grönlund et al., 1999). Several studies have demonstrated that colonization rates and counts of the Bacteroides fragilis group differ most markedly between vaginally delivered infants and infants born via Caesarean section (Bennet & Nord, 1987; Penders et al., 2006). Bacteroides fragilis group counts were considerably reduced as a result of Caesarean section. Furthermore, bifidobacterial counts were also lower and colonization rates of Clostridium difficile were higher in infants born via Caesarean section compared with infants born vaginally (Fig. 13.1). Penders et al. (2006) clearly demonstrated that the hospital environment itself is the major determinant for these differences in microbiota; after adjustment for potential confounding factors, the hospital environment was found to be the reason for colonization with spore-forming anaerobic microorganism. However, the origin for C. difficile colonization of the gastrointestinal tract of the newborn delivered by Caesarean section was found to be the hands and stools of healthy hospital personnel, particularly in the neonatal intensive care unit (Kim et al., 1981). The highest rates of carriage of C. difficile (64%) are found among premature infants (Larson et al., 1982). Prematurity is strongly associated with hospitalization but also with other factors influencing bacterial colonization, such as immature gastrointestinal tract or delayed oral feeding. Feeding type influences infant gut colonization. Human milk and breast-feeding are thought to create an environment favorable for the growth of bifidobacteria. In breast-fed infants the microbiota is dominated by bifidobacteria. Colonization rates with E. coli, C. difficile, B. fragilis group and lactobacilli are significantly lower than those found in formula-fed infants (Stark & Lee, 1982; Yoshioka et al., 1983; Benno et al., 1984). Exclusively and partially formula-fed infants were more often colonized with E. coli, C. difficile, B. fragilis group and lactobacilli than were their exclusively breast-fed counterparts. Infants fed with a formula supplemented with galacto-oligosaccharides and fructo-oligosaccharides have been shown to have counts of bifidobacteria and lactobacilli

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in their stools comparable with those of breast-fed infants (Stark & Lee, 1982; Yoshioka et al., 1983). Several factors have been proposed to cause the specific effects on the fecal flora of breast-fed infant: lower content of proteins in human milk, different composition of human milk proteins, lower phosphorus content of human milk, and various humoral and cellular mediators of immunological function in breast milk (Kunz et al., 1999). No influence of maternal antibiotics during pregnancy or of prolonged rupture of amniotic membranes before delivery has been described. However, the use of oral antibiotics (amoxicillin) in infants during the first month of life resulted in decreased numbers of bifidobacteria and B. fragilis group members. Similar trends for bifidobacteria were observed after oral use of antimycotics (miconazole). However, infants who experienced fever but who were not treated with antibiotics during the first month of life did not have a different gut microbiota composition compared with infants without fever.

13.3

PROBIOTICS IN INFANT NUTRITION

During the last decade in the EU several infant formulas have been enriched with probiotics that aim to mimic some of the beneficial effects of breast milk. These formulas, containing different probiotic strains, e.g. Streptococcus thermophilus, Bifidobacterium lactis (BB-12) and Lactobacillus reuteri, are used in many European countries as regular formulas in healthy newborns and infants. The microorganisms used in these infant formulas have been extensively studied in humans with so far no adverse effects observed in healthy subjects (Ishibashi & Yamazaki, 2001; Isolauri et al., 2002). Several expert committees have reported on the use of probiotics in infant nutrition and have urged strict control of their use. The Scientific Committee on Food of the European Commission commented on the use of probiotic bacteria in food products for infants and recommended that infant formulas with probiotic microorganisms should be marketed only if their benefit and safety have been evaluated. Moreover, the committee stated that only bacterial strains with identity and genetic stability demonstrated by cultural and molecular methods should be used and the identity of the probiotic strain should be described by molecular methods in a dossier and be available to the food control authorities (Scientific Committee on Food of the European Commission, 2004).

13.3.1

Growth of healthy infants

One of the most important safety factors with regard to infant formulas is evaluation of the impact of the breast milk substitute on growth. Several double-blind placebocontrolled studies that addressed this question have demonstrated that infant formulas containing different strains had no negative effects on stool habit, tolerance and, particularly, infant growth (Saavedra et al., 1994; Langhendries et al., 1995; Weizman et al., 2005). Vendt et al. (2006) studied the effect of probiotic supplementation on growth during the first 6 months of life. Infants up to age 2 months were randomized to receive either a formula enriched with Lactobacillus GG or a standard formula in a double-blind fashion. At the age of 6 months children in the LGG group showed significantly greater height and weight gain than the group with the standard formula. Weizman and Alsheikh (2006) reported similar data in their study.

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Several studies have demonstrated that the numbers of fecal lactobacilli or bifidobacteria could be increased in infants fed formulas enriched with probiotics, while fecal pH values were lower than in infants fed conventional formulas. Infant formulas supplemented with different probiotic microorganisms did not adversely affect growth (weight, length, head circumference), feeding (regurgitation, vomiting), stool habit (diarrhea, constipation), or infant behavior. However, there are some limitations to the data available. ● ● ● ●

The overall number of infants included in these studies was very limited: the total number of infants treated did not exceed 350 healthy full-term infants. The duration of treatment was rather short (2–6 months). Data regarding weight and growth during treatment and follow-up were not presented, while data for the intervention group were not compared with the control group. Only partial information on how conclusions were tested was available.

In conclusion, there are limited data from well-designed controlled studies on the growth of infants fed formulas supplemented with probiotics. However, there are no indications from the available data that formulas with probiotics have any adverse effects on growth, Moreover, the everyday use and experience with these widely used formulas do not indicate a negative influence on infant growth. A clinically relevant advantage of infant formulas enriched with probiotics, as compared with non-supplemented formulas, in healthy infants has not yet been unequivocally demonstrated.

13.3.2

Probiotics in preterm infants

Preterm infants are at risk for increased morbidity and mortality from sepsis and necrotizing enterocolitis (NEC). NEC is the most common serious disease of the intestine in preterm infants and is characterized by perforation and/or necrosis of the bowel. The pathogenesis of NEC is not fully understood and most likely represents interaction of different factors causing mucosal injury. One of the major pathogenic factors is colonization of the intestine by pathogenic bacteria (Kosloske, 1984). Intestinal colonization and microbiota of preterm and low birth weight infants are different from those of healthy full-term newborns: very low birth weight infants have delayed onset and abnormal fecal bacterial colonization with fewer normal enteric bacterial species (Goldmann et al., 1978; Gewolb et al., 1999). Administration of probiotic bacteria to premature infants is therefore aimed at modifying intestinal microbiota and thereby preventing infection and NEC. The mechanisms of action of probiotics in providing protection against NEC in the preterm neonate include an increased barrier to migration of bacteria across the mucosa, competitive exclusion of potential pathogens, augmentation of IgA mucosal responses, modification of immunological host response to microbial products with upregulation of immune responses, and improvement of enteral nutrition that inhibits pathogen growth (Link-Amster et al., 1994; Orrhage & Nord, 1999; Duffy, 2000; Mattar et al., 2001; Reid et al., 2001). Several reports on the ability of different probiotics to colonize the intestine of preterms have shown that different probiotics have colonizing capacities that vary from strain to strain (Millar et al., 1993; Grönlund et al., 1997; Agarwal et al., 2003). A recent meta-analysis performed in accordance with the Cochrane Neonatal Review Group reviewed nine randomized trials including 1425 preterm infants (Alfaleh & Bassler, 2008; Alfaleh et al., 2009). The included trials varied highly with regard to enrolment criteria, feeding regimens and probiotics supplemented; however, in the meta-analysis the

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Relative Risk (fixed) 95% Confidence Interval

A (7 studies)

B (5 studies)

0,36 [0,20 – 0,65]

0,32 [0,17 – 0,60]

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 1,1 Fig. 13.2 Effect of probiotics on the incidence of necrotizing eneterocolitis of stage 2 or higher. (Data from Deshpande et al. 2007[A]; Alfaleh & Bassler, 2008[B].)

prophylactic supplementation of enteral probiotics significantly reduced the severity of NEC [risk ratio (RR) 0.32, 95% CI 0.17–0.60] and mortality (RR 0.43, 95% CI 0.25–0.75). A total of five deaths were attributed to NEC in the control group, while no NEC-related deaths occurred in the probiotic-treated group. Five studies included in the meta-analysis reported on sepsis. There was no significant difference between both groups in the rate of culture-proven sepsis and no evidence was found for a significant reduction of nosocomial sepsis or duration on total parenteral nutrition. The review showed no significant effects of probiotics on hospital days as outcome, number of days on total parenteral nutrition, apnea or weight gain. Very similar results were found by a systematic review of 12 randomized controlled trials (RCTs) by Deshpande et al. (2007) (Fig. 13.2). The authors conclude that enteral supplementation with probiotics reduces the risk of severe NEC and mortality in preterm infants. They support a change in practice in premature infants weighing above 1000 g at birth, but emphasize the importance of large RCTs for investigating both the benefits and safety profile of probiotics in preterm infants. Obviously, more large controlled trials are required to address important questions, such as mechanisms of action, strain of probiotics, dosage of probiotic strains, mode of supplementation and duration. However, data on the safety and efficacy of administration of probiotics to the highest risk group for NEC, namely very low birth weight infants (< 1000 g at birth), are urgently needed.

13.3.3

Safety concerns

In general, probiotics are considered to be safe in children and adolescents. However, there have been some concerns that this conclusion requires additional evaluation, as there are only limited data on the safety of probiotic supplements or supplements in infant formulas. There are only a few reports of significant complications and adverse effects of probiotics in the clinical literature. Most of the reported adverse effects involve mild abdominal discomfort and flatulence. However, there are some concerns about the risk of septicemia and bacteremia. There are anecdotal case reports in the medical literature of systemic infections, bacteremia, and fungemia caused by probiotic organisms for infants, children and adults. Recently, De Groote et al. (2005) reported a multimorbid preterm infant with short-gut syndrome, indwelling catheter and gastrostomy tube who suffered from bacteremia with Lactobacillus GG at the age of 11 months. Land et al. (2005)

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described a 6-week-old infant with severe underlying medical problems who developed sepsis due to lactobacilli after ingesting these bacteria, and two other case reports describe borderline premature neonates with short-gut syndrome and chronic intestinal inflammation who developed sepsis due to Lactobacillus after treatment with Lactobacillus GG (Kunz et al., 2004). Most of the reported cases were children with pre-existing and predisposing diseases, for example immunocompromise or immunosuppression, structural heart defects, short bowel syndrome, central venous catheters (Salminen et al., 2004; Berger, 2005; Cannon et al., 2005; De Groote et al., 2005; Boyle et al., 2006; Cukovic-Cavka et al., 2006; Ledoux et al., 2006; Salvana & Frank, 2006; Burns et al., 2007; Lin et al., 2008). On the other hand, none of the studies in preterm children reported any cases of bacteremia or septicemia when using probiotic organisms. On the basis of existing data, probiotics have been declared safe, even in immunocompromised populations such as premature neonates. The European Society of Paediatric Gastroenterology, Hepatology and Nutrition Committee on Nutrition (2004) recently summarized its approach to probiotics as follows: “probiotics so far used in clinical trials can be generally considered as safe. However, surveillance for possible side effects, such as infection in high-risk groups, is lacking and is needed”. Nevertheless, due to this uncertainty about safety issues in particular age groups (extremely and very low birth weight infants) and in infants and children with underlying diseases, probiotics currently should not be used in cases where mothers are unable or not willing to breast-feed or the mother’s milk is unavailable or insufficient, in infants with (congenital) heart disease, with a compromised immune system or immunosuppressive treatment, and in critically ill infants and children. Preterm infants who cannot receive fortified human milk should receive a preterm formula without probiotics (Ernährungskommissionen DACH, 2009). However, in healthy children the risk of bacteremia is almost non-existent.

13.4

CLINICAL EFFECT OF PROBIOTICS IN CHILDREN

During the last decades a wide range of probiotic strains, primarily lactobacilli, bifidobacteria, E. coli, Saccharomyces boulardii, and streptococci, have been investigated in clinical trials for various indications as single agents or as combination therapies. Many promising studies have not been reproduced or confirmed and further studies are necessary to increase understanding of the mechanism of action and effect on the host.

13.4.1

Prevention of allergic disease: food hypersensitivity

The prevalence of allergic diseases has been continuously increasing over the last few decades. The cumulative prevalence of allergies in childhood in Western developed countries is high, with up to 7–8% developing food allergy, 15–20% atopic eczema, and 30–35% asthma or recurrent wheezing (Halken, 2004). Current research suggests that the “hygiene hypothesis”, proposed by Strachan (1989), explains the increasing prevalence rates as a result of the decrease in exposure to common infections during early life and reduced immunological interaction with microbial stimuli. In particular, the gastrointestinal microflora modulates mucosal physiology, barrier function, and systemic immunological and inflammatory responses. Another factor, altered microbial exposure in the gastrointestinal tract, may be partly responsible for the increase in allergic disease in

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populations with a Western lifestyle (Holt et al., 1997). Differences in the intestinal microflora can be found not only between infants delivered by Caesarean section and those delivered vaginally but also between infants with atopic disease and those without allergies. The primary prevention of allergic diseases by modulating the intestinal microflora seems to be fascinating and plausible. Prenatal and/or postnatal probiotic supplementation for primary and secondary intervention of atopic disease has been studied in randomized clinical trials, but results have been mixed. Assessment of the clinical efficacy of probiotics in preventing atopic disease is hindered by diverse heterogeneity of protocols, different strains studied, low sample sizes, different study populations with infants at risk or not at risk for allergies, various endpoints, and different outcomes. At the moment there is insufficient evidence to recommend the use of probiotics prenatally by mothers or the addition of probiotics to infant feeds for the prevention of allergic diseases in general (Osborn & Sinn, 2007).

13.4.2

Atopic dermatitis

13.4.2.1

Prevention

The first studies addressing the possibility of preventing atopic dermatitis by administration of probiotics was a Finnish RCT in infants at high risk for atopic disease based on family history (Kalliomäki et al., 2001, 2003, 2007). Mothers were given Lactobacillus GG for 4 weeks prenatally and 6 months postnatally while breast-feeding. Atopic eczema was diagnosed at age 2 years in 31 of 68 infants in the placebo group but in only 15 of 64 infants in the probiotics group and at age 4 years in 25 of 54 in the placebo versus 14 of 53 in the probiotics group (RR 0.51, 95% CI 0.32–0.84 at the age of 2 years; RR 0.57, 95% CI 0.33–0.97 at the age of 4 years). No difference was observed in the number of sensitized children between the groups. Several meta-analyses have assessed the use of probiotics for preventing atopic dermatitis. The first meta-analysis included five RCTs and, after limiting the analysis to studies dealing with atopic dermatitis, revealed no significant reduction in pediatric eczema with probiotics. Another meta-analysis included six RCTs that compared treatment with different Lactobacillus species with placebo (Lee et al., 2008). A recent review on the prevention of atopic dermatitis concludes that probiotics, especially L. rhamnosus GG, seem to be effective in preventing atopic dermatitis and seem to reduce the severity of atopic eczema in approximately half of the RCTs evaluated (Betsi et al., 2008). Although they were not found to significantly change most of the inflammatory markers measured in the majority of the RCTs evaluated, probiotics seem to have beneficial effects and seem useful for the prevention of atopic dermatitis. However, the promising results of the early studies could not be confirmed by other studies (Brouwer et al., 2006; Abrahamsson et al., 2007). A recent trial conducted by the study group of Susan Prescott found no difference in the development of atopic dermatitis but observed increased sensitization to allergens in neonates supplemented with a different strain (Taylor et al., 2007). In this study newborns at high risk for allergy received Lactobacillus acidophilus (LA VRI-A1) 109 CFU three times a day plus maltodextrin or maltodextrin alone from birth to 6 months. At the age of 6 months and 12 months no difference in the incidence of atopic eczema was found between the two groups: 34 (43.2%) of 88 children in the probiotic group compared with 34 (39.1%) of 87 children in the

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placebo group developed atopic dermatitis. In contrast to the Finish study, this report did not include a prenatal period of probiotic administration. Kopp et al. (2008) used a study concept almost identical to that of the Finnish study, with prenatal administration of probiotics. Supplementation with Lactobacillus GG started during pregnancy and early infancy and lasted for 3 months after birth. However, neither a reduced incidence of atopic dermatitis nor an influence on the severity of atopic dermatitis in affected children was found at the age of 2 years in the population of infants at high risk for allergic disease. Instead, there was a significantly increased rate of recurrent episodes of wheezing bronchitis during the first 2 years in the LGG-treated group compared with placebo. However, two recently published trials indicate a preventive effect of newly tested probiotic strains. In the PandA double-blind study, a blend of the probiotics Bifidobacterium bifidum W23, Bifidobacterium lactis W52, and Lactococcus lactis W58 (or a placebo) was administered to 156 women during the last 6 weeks of pregnancy and to their newborns for 12 months (Niers et al., 2009). In all cases, the families had a history of allergies. From birth to 3 months, the parents reported six cases of eczema out of the 50 children treated compared with 15 of 52 from the control group who received the placebo (P = 0.035). At the age of 3 months the reduction in relative risk was 58%, at 1 year 26% [odds ratio (OR) 0.495, P = 0.086], and at 2 years 22% (OR 0.518, P = 0.117). Total occurrences at the age of 1 and 2 years respectively were 23 of 50 (tested) versus 30 of 48 (control) (P < 0.05) and 27 of 50 (tested) versus 33 of 48 (control) (P < 0.05). This clinical study using a blend of probiotics (B. bifidum W23, B. lactis W52 and Lactococcus lactis W58) indicates that the early consumption of a combination of probiotics can prevent eczema in children at high risk at least up until the age of 2 years. The second RCT tested Lactobacillus paracasei F19 in children during the weaning period (West et al., 2009); 179 children were studied from age of 4 months to 13 months. During this period they received a milk drink with cereals containing the probiotics (N = 90) or no probiotics (N = 89). At the age of 13 months, the incidence of eczema was significantly lower in the group receiving the probiotic compared with the control group (11% vs. 22%, P < 0.05). Evaluation of the Th1/Th2 ratio based on measurement of the ratio of interferon (IFN)-g to interleukin (IL)-4 showed that this was significantly higher in the tested subjects (P < 0.05). No differences were found between the two groups with regards to serum IgE levels. This clinical study demonstrated that L. paracasei F19 administered to children during the weaning period reduces the total number of incidences of eczema and that this is coupled with modulation of the immune response controlled by T lymphocytes. In conclusion, there are several trials showing an allergy-preventive effect of different probiotics, although so far these effects have not been consistently established. At the moment no single probiotic or blend of probiotic bacteria can generally be recommended for the primary prevention of atopic dermatitis. 13.4.2.2

Treatment

Besides recent efforts to prevent atopic eczema with probiotics, interest has developed in treating atopic dermatitis in young infants and children. One of the first reports on the positive effects of Lactobacillus GG on the clinical score of atopic dermatitis comes from a Finnish study group (Majamaa & Isolauri, 1997). They evaluated the clinical and immunological effect of cows’ milk elimination with and without the addition of

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Lactobacillus GG in infants with atopic eczema and cows’ milk allergy. During the 1-month study period the clinical score of atopic dermatitis improved significantly in infants treated with Lactobacillus GG. In parallel, immunological factors improved, too, thus indicating a positive effect on intestinal inflammation. Several other clinical RCTs have been published. Rosenfeldt et al. (2003) performed a double-blind, placebocontrolled, crossover study with 41 children with moderate to severe atopic dermatitis. After administration of Lactobacillus rhamnosus and L. reuteri for 6 weeks, a significant decrease in the severity of the atopic eczema was observed. The improvement in atopic dermatitis correlated with decreased intestinal permeability. Other studies suggest that probiotics may be effective only in children with food sensitization and in children who have documented IgE hypersensitivity (Viljanen et al., 2005; Sistek et al., 2006). The data from several other trials were included in three meta-analyses, although the conclusions are inconclusive and controversial. Two reviewers came to the conclusion that the clinical significance of treatment with probiotics is questionable and that the current evidence is not convincing for a clinically relevant role of probiotics in the treatment of atopic eczema (Boyle et al., 2008; Lee et al., 2008). Moreover, the Cochrane review points out a small but not excludable risk of adverse events during treatment with probiotics. The authors of the third review suggest a modest role for probiotics in moderately severe rather than mild pediatric atopic dermatitis (Michail et al., 2008).

13.4.3

Prevention of antibiotic-associated diarrhea

Antibiotics are the most frequently prescribed medicines in children, frequently for a variety of minor infections of the urinary and respiratory tracts. Almost all antibiotics, particularly broad-spectrum antibiotics that act on aerobes, can cause diarrhea. The risk is highest for aminopenicillins, the combination of aminopenicillins and clavulanate, cephalosporins, and clindamycin and ranges from 11 to 31% (Turck et al., 2003; Correa et al., 2005). Antibiotic-associated diarrhea (AAD) can occur within a day of starting antibiotics or up to 8 weeks later. The severity of AAD may range from mild to severe diarrhea with serious complications, including electrolyte disturbance, dehydration, pseudomembranous colitis, and toxic megacolon (Bartlett, 1992). Antibiotic treatment disturbs the balance of the gastrointestinal microflora. Disruption of the microbial balance often results in overgrowth of pathogenic organisms and may disturb the metabolism of carbohydrates. Clostridium difficile has emerged as the major enteropathogen of AAD, responsible for 10–25% of cases and for almost all cases of pseudomembranous colitis (Bartlett et al., 1978). Numerous probiotics, such as Lactobacillus acidophilus, L. casei GG, L. bulgaricus, Bifidobacterium bifidum, B. longum, Enterococcus faecium, Strep. thermophilus, or Saccharomyces boulardii, have been tested for the treatment and prevention of AAD. However, many of the studies were small and suffered considerable methodological problems and most were conducted in adults. A recent meta-analysis and a Cochrane review summarized the pediatric data on probiotics in the treatment and prevention of AAD (Szajewska et al., 2006; Johnston et al., 2007). The meta-analysis by Szajewska et al. (2006) included six RCTs with a total of 766 children, 376 in the experimental group and 390 in the control group. Among the trials there was considerable clinical heterogeneity regarding sample size, settings, age of

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children, duration and dose of treatment, and type of antibiotics administered. All trials had short follow-ups. Treatment with probiotics reduced the risk of AAD in children from 28.5% to 11.9% (RR 0.44, 95% CI 0.25–0.77) compared with placebo. The risk reductions were similar for all types of probiotics used (Lactobacillus GG, S. boulardii, or Bifidobacterium lactis plus Strep. thermophilus). For every seven patients who would develop diarrhea while being treated with antibiotics, one fewer would develop AAD if he or she also received probiotics (number needed to treat 7, 95% CI 5–10). The meta-analysis did not allow final conclusions regarding the efficacy of probiotics for the prevention of C. difficile diarrhea in children because C. difficile diarrhea was not a primary endpoint in the trials included. However, despite all the systemic limitations of meta-analysis, the final conclusion of the authors was that probiotics reduce the risk of AAD in children. A second meta-analysis by Johnston et al. (2007) examined six RCTs that included 707 children. The combined results, although analysed with a per-protocol method, showed benefits for probiotics over placebo (RR 0.43, 95% CI 0.25–0.75). However, the more sensitive intention-to-treat analysis did not show a significant benefit for probiotics in reducing the incidence of AAD. The reason for the lack of significant effects could be attributed to excessive drop-out rate of the follow-up in several of the trials. Nevertheless, in a subgroup analysis, the investigators found a significant benefit (RR 0.36, 95% CI 0.25–0.53) in four studies that used at least 5 × 109 CFU daily of Lactobacillus GG, L. sporogenes, or S. boulardii. This leads to the conclusion that inadequate dosing could be an important factor in the trials that do not show an effect. The results of the two most recent meta-analyses therefore support the use of probiotics in the prevention of AAD in children. Doses of 5–10 × 109 CFU should be used, and various probiotic strains appear to be equally effective. However, further well-conducted clinical trials must evaluate the most effective probiotic strains, the best dosing scheme and cost-effectiveness before probiotics can be routinely recommended for the prevention of AAD.

13.4.4

Acute gastroenteritis and community-acquired diarrhea

13.4.4.1

Acute gastroenteritis

The most and best studied potentially beneficial effect of probiotics is mild in moderate infectious diarrhea. In contrast to other issues regarding the effects and benefits of probiotics, the data on acute gastroenteritis are rather consistent and positive. The rationale for using probiotics in infectious diarrhea is that they act against enteric pathogens by competing for available nutrients and binding sites, making the gut contents acid, producing a variety of chemicals, and increasing specific and non-specific immune responses. Szajewska and Mrukowicz (2001) have published the first systematic review of probiotics in acute diarrhea. This review included eight published randomized, placebo-controlled, double-blind studies of acute diarrhea lasting 3 days or more in infants and children. The effects of all probiotics and of individual strains were analysed. The risk of diarrhea lasting 3 days or more was significantly reduced in the probiotic-treated group compared with the placebo group (RR 0.40, 95% CI 0.28–0.57). The use of probiotics reduced the duration of

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diarrhea by 18.2 hours (95% CI 9.5–26.9 hours). The authors concluded that there is evidence of a clinically significant benefit of probiotics in the treatment of acute infectious diarrhea in infants and children, particularly in rotaviral gastroenteritis and particularly for Lactobacillus GG, which showed the most consistent effect. A meta-analysis undertaken by Van Niel et al. (2002) was restricted to adequately randomized and blinded studies of several strains of lactobacilli in children. Children who had received recent antibiotics were excluded. Probiotics reduced the duration of diarrhea by approximately two-thirds of a day (0.7 days, 95% CI 0.3–1.2 days) and diarrhea frequency on day 2 by 1.6 stools (95% CI 0.7–2.6). The results of the meta-analysis suggest that Lactobacillus is safe and effective as a treatment for children with acute diarrhea. A Cochrane review included data from unpublished studies and non-blinded (open) studies (Allen et al., 2003). This comprised a total of 23 studies which met the inclusion criteria, with a total of 1917 participants. Probiotics reduced the risk of diarrhea at 3 days (RR 0.66, 95% CI 0.55–0.77) and the mean duration of diarrhea by 30.48 hours (95% CI 18.51–42.46 hours). The authors of the review concluded that probiotics appear to be a useful adjunct to rehydration therapy in treating acute infectious diarrhea in adults and children, although they stated that more research is needed to inform the use of particular probiotic regimens in specific patient groups. A variety of different probiotics were used in these trials and causes of diarrhea were quite heterogeneous as outcomes, which were measured by parents rather than investigators. Finally, Szajewska et al. (2007) examined the treatment of acute gastroenteritis with Saccharomyces boulardii in children. They included five RCTs with 619 participants. Combined data from four RCTs showed that S. boulardii significantly reduced the duration of diarrhea compared with control by 1.1 days (95% CI 1.3–0.8) in children taking S. boulardii compared with placebo. Saccharomyces boulardii also significantly reduced the risk of diarrhea on days 3, 6 and 7 and the risk of diarrhea lasting more than 7 days (RR 0.25, 95% CI 0.08–0.83). However, the investigators restricted their conclusions by stating that their results should be interpreted with caution due to the methodological limitations of the included studies. Overall, however, there is good evidence from RCTs and meta-analyses that there is a significant effect and moderate clinical benefit of some probiotic strains in the treatment of acute diarrhea, particularly rotavirus-associated diarrhea. The benefits of treatment with Lactobacillus GG and with S. boulardii seem to be moderate and more pronounced in children living in developing countries. Treatment with probiotics seems to be more effective if started early and with greater doses (>1010 CFU). A joint statement of the European Society of Paediatric Infectious Diseases and the European Society for Paediatric Gastroenterology, Hepatology and Nutrition states that probiotics may be an effective adjunct in the management of diarrhea (Guarino et al., 2008). However, because there is no evidence of efficacy for many preparations, the committees suggest the use of probiotic strains with proven efficacy [Lactobacillus GG (IA) and Saccharomyces boulardii (IIB)] and in appropriate doses for the management of children with acute gastroenteritis as an adjunct to rehydration therapy. 13.4.4.2

Community-acquired diarrhea

Several studies have examined the ability of probiotics to prevent acute diarrheal episodes in the community setting. Community-acquired diarrhea still remains a major cause of death among children in the developing world; on the other hand, probiotic-fortified

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formulas can be manufactured easily and cheaply. If probotic-fortified formulas could be demonstrated to prevent community-acquired diarrhea, this would be of particular importance for the developing world. In a study of malnourished children aged 1 month to 2 years in Peru, 204 children were randomly assigned to receive either a formula supplemented with Lactobacillus GG or placebo for 15 months (Oberhelman et al., 1999). Children in the Lactobacillus GG group had significantly fewer diarrheal episodes (5.21 per child per year versus 6.02 in the placebo group, P < 0.03). Similar results have been found in studies done in developed countries. Chouraqui et al. (2004) examined 90 healthy children younger than 8 months living in residential or foster care settings in France. One group of children was treated with an acidified milk formula containing Bifidobacterium lactis BB12, the other group with a conventional formula. The authors found a significant difference in the daily probability of diarrhea in the treated group (0.84) compared with the placebo group (2.3). The difference was not statistically significant, but there was a significant difference between the groups in terms of the overall prevalence of diarrhea during the study period. Thibault et al. (2004) studied the prevalence of diarrhea in a sample of over 900 healthy infants in childcare settings. The infants were given a formula enriched with Bifidobacterium brevis plus Strep. thermophilus 065 or a standard formula. There was no difference in incidence or duration of diarrheal episodes or hospital admissions but the probiotic group had less diarrheal episodes, significantly fewer cases of dehydration, fewer prescriptions for oral rehydration solutions, and fewer medical consultations. Weizman et al. (2005) studied 201 infants aged 4–8 months in 14 childcare centers in Israel over a 3-month period. Children were randomized to receive a formula with Bifidobacteriunm lactis BB12 or with Lactobacillus reuteri, or a formula without probiotics. Infants on both probiotic formulas had a significantly lower frequency of diarrheal episodes and shorter duration of diarrhea than the untreated controls. The beneficial effects were more prominent in the group of children receiving L. reuteri. Figure 13.3 shows the prophylactic effect of four studies with B. lactis on the prevalence of community-acquired diarrhea in different settings. The available data from RCTs therefore suggest a modest protective effect of probiotics against community-acquired diarrhea.

13.4.5

Irritable bowel syndrome and constipation

Irritable bowel syndrome comprises a group of functional gastrointestinal disorders characterized by chronic abdominal pain and distension, discomfort, alterations in bowel habit, bloating, diarrhea, and constipation in the absence of a detectable organic cause. Between 4 and 25% of school-age children complain of recurrent abdominal pain of sufficient severity to interfere with daily activities (Williams et al., 1996; Drossman, 1999). The etiology of the syndrome remains unclear, although a number of hypotheses have been proposed. The Rome criteria commonly used for diagnosis of irritable bowel syndrome in adults have been modified into the Pediatric Rome Criteria in an attempt to improve diagnosis, study and treatment of children with recurrent abdominal pain (Schurman et al., 2005). Probiotics are commonly used in the treatment of irritable bowel syndrome, although so far only a few clinical trials have been performed. In an abstract, Young (1997) described a placebo-controlled, randomized, crossover trial using Lactobacillus plantarum 299V

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0

Percentage reduction (approximate values)

–10 –20 –30

? C

–40 –50

B?

–60

? D

–70 –80 –90

A? C

D

Saavedra et al. (1994)

Ziegler et al. (2003)

Chouraqui et al. (2004)

Weizmann et al. (2005)

Patients

Hospitalized chronicly ill children (N = 55)

Newborns (N = 122)

Infants in foster care (N = 90)

Infants in child care center (N = 201)

Age

5–24 months

Newborns

< 8 months

4–10 months

Ø approximately 80 days

4 months

12 months

3 months

P < 0.001

NS

P < 0.001

A

Observation period Probiotic vs. placebo

P = 0.035

B

Fig. 13.3 Prophylactic effect of probiotics on community-acquired diarrhea in different settings as shown in four studies.

(LP299V) versus placebo for 4 weeks in children with chronic recurrent abdominal pain. Preliminary data in 8 of 12 patients who had completed the first phase of the study are reported to show that 25% of the placebo group and 50% of the group receiving L. plantarum 299V experienced decrease in their abdominal pain, but no further details were provided. Bausserman and Michail (2005) performed an RCT in which they enrolled 64 children from a pediatric gastroenterology clinic with a diagnosis of irritable bowel syndrome made according to the Rome II criteria. Participants were randomized to receive a capsule containing either Lactobacillus GG or a placebo twice daily for a period of 6 weeks. After treatment with Lactobacillus GG or placebo for 6 weeks, they found no significant benefit on the abdominal pain scale (44% response in the treatment group vs. 40% response in placebo). However, there was a significant effect on abdominal distension scores. Gawronska et al. (2007) performed another RCT on 104 children referred to a pediatric gastroenterology clinic who fulfilled the Rome II criteria (functional dyspepsia, irritable bowel syndrome, or functional abdominal pain). The children were randomly allocated (stratified by diagnosis) to the intervention group where they received Lactobacillus GC capsules for 4 weeks, or to the control group where they received placebo capsules for the same length of time. Outcome measures were self-reported pain including severity and frequency, use of medication, and school absenteeism. After the 4-week intervention no significant benefit in the abdominal pain scale was found. Response rates in the treatment group were 44% compared with 40% response in the placebo group. All three trials were included in a Cochrane systematic review (Huertas-Ceballos et al., 2009), although data from only two trials were suitable for analysis. The authors concluded that there is a lack of high-quality evidence on the effectiveness of dietary interventions and

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that there is no evidence that Lactobacillus supplementation is effective in the management of children with recurrent abdominal pain. Chronic constipation is one of the most frequent complaints in childhood. There is good evidence that the gastrointestinal flora are an important factor in gut motility, even though there is little evidence that the gut flora is abnormal in children with constipation. In normal individuals lactobacilli and bifidobacteria can increase stool frequency and decrease stool consistency. According to several reviews, the evidence of probiotics for efficacy in constipation is limited. Lactose-free diets, fiber supplements, and Lactobacillus supplementation are effective in the management of children with recurrent abdominal pain and irritable bowel syndrome. Several studies with Lactobacillus GG in children showed negative results in children with chronic constipation. In contrast, Bifidobacterium animalis DN-173 010 has been shown to be effective in adults with constipation-predominant irritable bowel syndrome. However, these studies have not been performed in well-designed, large, placebo-controlled trials in children with constipation.

13.4.6

Infantile colic

Infantile colic is a common problem within the first 3 months of life and affects as many as 3–28% of newborn children. During the last century the role of the intestinal microflora steadily increased in importance: as in atopic infants, lower counts of lactobacilli were observed in colicky infants (Savino et al., 2004). The intestinal microflora may therefore play a role in the development and treatment of infantile colic. Recently, Savino et al. (2007) tested the hypothesis that oral administration of Lactobacillus reuteri would improve the symptoms of infantile colic. They compared the use of L. reuteri with simethicone, a widely used treatment of choice. A total of 90 breast-fed colicky infants who met the clinical criteria for colic were randomly assigned to receive either 108 live bacteria per day or 60 mg of simethicone per day. The beneficial effects of L. reuteri started in the probiotic group within 1 week, and by 28 days the average crying time in the probiotic group was decreased by 65%. Additionally, 95% of all infants receiving probiotics responded positively compared with only 7% responders in the simethicone group. The results suggest a potential role for L. reuteri as a therapeutic agent in infantile colic. So far, however, these results have not been confirmed by similar trials.

13.4.7

Inflammatory bowel disease

One of the prevailing theories on the etiology of inflammatory bowel disease (IBD) is that the adaptive immune system is hyperresponsive to the commensal intestinal microflora in genetically susceptible individuals. A healthy bowel flora therefore plays an important role in this development, for example through effects on cytokines. Probiotics can also influence the Toll receptors and dendritic cells involved in healthy gut immunity (Ezendam & van Loveren, 2006). Other possible therapeutic effects include correction of abnormal intestinal permeability (“leaky gut”), improved mucus production and production of short-chain fatty acids (Campieri & Gionchetti, 2001). Clinical trials of probiotics for IBD have predominantly involved adults and resulted in positive effects particularly for ulcerative colitis and pouchitis, less so for Crohn’s disease. Bousvaros et al. (2005) studied the effect of Lactobacillus GG (LGG) in addition to a standard maintenance therapy for children with Crohn’s diseases. A total of 57 children and

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young adults aged 5–21 years in remission were randomized to either LGG or placebo and followed for up to 2 years. The authors could find no differences in the median time to relapse (9.8 months in the LGG group and 11.0 months in the placebo group; P = 0.24). Of 39 patients in the LGG-treated group, 12 (31%) developed a relapse compared with 6 of 36 (17%) in the placebo group (P = 0.18). The time to relapse and proportion of patients relapsing was essentially identical in both LGG and placebo groups. The study suggests that LGG does not prolong time to relapse in children with Crohn’s disease when given as an adjunct to standard therapy.

13.4.8

Oral health effects: caries

Lactobacillus GG (LGG) has been shown to produce a substance with potential inhibitory activity against different bacterial species including cariogenic Streptococus spp. Näse et al. (2001) have shown that long-term consumption of milk containing LGG caused a significant reduction in caries in daycare centers. A total of 500 children aged 1–6 years old from 18 municipal daycare centers were randomized and received either milk containing LGG or normal milk. The children received the different milks 5 days a week in the daycare centers for 7 months. Oral health was recorded at baseline and at the end, using WHO criteria. At the end of the 7-month trial there was less dental caries and lower Streptococcus mutans counts in the LGG-treated group. LGG was found to reduce the risk of caries significantly (OR 0.56, P = 0.01; controlled for age and gender, OR 0.51, P = 0.004). The effect was particularly clear in the 3–4 year olds. The authors conclude that milk containing the probiotic LGG bacteria may have beneficial effects on children’s dental health. All these studies and preliminary results require further study before treatment recommendations can be made.

13.4.9

Other clinical conditions

The role of probiotics in the treatment of hepatic encephalopathy has been examined in a few pilot studies. Therapy with probiotics resulted in improvement in hepatic encephalopathy and lower blood ammonia levels (Loguercio et al., 1987). This effect may be related to colonization of the intestine with acid-resistant, non-urease-producing bacteria. Probiotics are generally not effective in eradicating Helicobacter pylori infection, but they probably can reduce the side effects of recommended antimicrobial therapy. It has been shown in vitro that certain probiotics are capable of suppressing the multiplication of H. pylori. The clinical results, obtained mainly in adults, remain controversial with regard to the benefits of consumption of probiotics. Some indicate a reduction in the pathogen load while others do not; some indicate a reduction in the side effects associated with conventional medication and others do not. A recent clinical, double-blind, placebo-controlled study was carried out on 83 children infected with H. pylori (Szajewska et al., 2009). For 7 days they were simultaneously subjected to a triple therapy (two antibiotics plus one proton-pump inhibitor), in addition to the probiotic Lactobacillus rhamnosus GG or a placebo. Eradication was observed in 69% of the children in the test group and 68% of the control group, indicating that there was no significant difference between the two groups (RR 0.98, 95% CI 0.7–1.4). The same was true of the side effects. This study concludes that, in children, the consumption of L. rhamnosus GG did not lead to a better rate of eradication of the pathogenic agent nor ease the side effects caused by the triple therapy.

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13.5 ●

● ●

● ● ●

SUMMARY AND KEY MESSAGES

The intestinal flora plays a key role in the development of normal immunological and digestive function in humans. Understanding the importance of the microflora of the intestine and facilitating the development of a healthy gastrointestinal flora by encouraging breast-feeding or minimizing the use of antibiotics is an important challenge for everyone caring for infants and children. Currently used probiotics are generally recognized as safe for healthy children. Significant complications due to probiotics are extremely rare. There is good evidence for a clinical benefit of probiotics in treating acute gastroenteritis, with a reduction in the severity and duration of acute diarrhea, particularly rotavirus diarrhea. Probiotics are effective in reducing the risk of antibiotic-associated disease. Probiotics may be effective in preventing the risk of necrotizing enterocolitis and community-acquired diarrhea. Probiotics may have a potential in the prevention and treatment of atopic dermatitis.

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Guarino A, Albino F, Ashkenazi S, Gendrel D, Hoekstra JH, Shamir R, Szajewska H (2008) European Society for Paediatric Gastroenterology, Hepatology, and Nutrition/European Society for Paediatric Infectious Diseases evidence-based guidelines for the management of acute gastroenteritis in children in Europe. J Pediatr Gastroenterol Nutr 46(Suppl 2):S81–S122. Halken S (2004) Prevention of allergic disease in childhood: clinical and epidemiological aspects of primary and secondary allergy prevention. Pediatr Allergy Immunol 15(Suppl 16):4–5. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendrop AA, Klijn N, Bindels JG, Welling GW (2000) Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 30:61–67. Holt PG, Sly PD, Björkstén B (1997) Atopic versus infectious diseases in childhood: a question of balance? Pediatr Allergy Immunol 8:53–58. Huertas-Ceballos AA, Logan S, Bennett C, Macarthur C (2009) Dietary interventions for recurrent abdominal pain (RAP) and irritable bowel syndrome (IBS) in childhood. Cochrane Database Syst Rev 1:CD003019. Huurre A, Kalliomäki M, Rautava S, Rinne M, Salminen S, Isolauri E (2008) Mode of delivery: effects on gut microbiota and humoral immunity. Neonatology 93:236–240. Ishibashi N, Yamazaki S (2001) Probiotics and safety. Am J Clin Nutr 73:465S–470S. Isolauri E, Ribeiro HC, Gibson G, Saavedra J, Salminen S, Vanderhoof J, Varavithya W (2002) Functional foods and probiotics: Working group report of the first World Congress of Pediatric Gastroenterology, Hepatology and Nutrition. J Pediatr Gastroenterol Nutr 35:S106–S109. Johnston BC, Supina AL, Ospina M, Vohra S (2007) Probiotics for the prevention of pediatric antibioticassociated diarrhea. Cochrane Database Syst Rev 2:CD004827. Kalliomäki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E (2001) Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 357:1076–1079. Kalliomäki M, Salminen S, Poussa T, Arvilommi H, Isolauri E (2003) Probiotics and prevention of atopic disease: 4-year follow-up of a randomised placebo-controlled trial. Lancet 361:1869–1871. Kalliomäki M, Salminen S, Poussa T, Isolauri E (2007) Probiotics during the first 7 years of life: a cumulative risk reduction of eczema in a randomized, placebo-controlled trial. J Allergy Clin Immunol 119:1019–1021. Kim KH, Fekety R, Batts DH, Cudmore M, Silva J Jr, Waters D (1981) Isolation of Clostridium difficile from the environment and contacts of patients with antibiotic-associated colitis. J Infect Dis 143:42–50. Kopp MV, Hennemuth I, Heinzmann A, Urbanek R (2008) Randomized, double-blind, placebo-controlled trial of probiotics for primary prevention: no clinical effects of Lactobacillus GG supplementation. Pediatrics 121:e850–e856. Kosloske AM (1984) Pathogenesis and prevention of necrotizing enterocolitis: a hypothesis based on personal observation and a review of the literature. Pediatrics 74:1086–1092. Kunz AN, James NM, Fairchok MP (2004) Two cases of Lactobacillus bacteremia during probiotic treatment of short gut syndrome. J Pediatr Gastroenterol Nutr 38:457–458. Kunz C, Rodriguez-Palmero M, Koletzko B, Jensen R (1999) Nutritional and biochemical properties of human milk. Part I: General aspects, proteins, and carbohydrates. Clin Perinatol 26:307–333. Land MH, Rouster-Stevens K, Woods CR, Cannon ML, Cnota J, Shetty AK (2005) Lactobacillus sepsis associated with probiotic therapy. Pediatrics 115:178–181. Langhendries JP, Detry J, Van Hees J, Lamboray JM, Darimont J, Mozin MJ, Secretin MC, Senterre J (1995) Effect of a fermented infant formula containing viable bifidobacteria on the fecal flora composition and pH of healthy full-term infants. J Pediatr Gastroenterol Nutr 21:177–181. Larson HE, Barclay FE, Honour P, Hill ID (1982) Epidemiology of Clostridium difficile in infants. J Infect Dis 146:727–733. Ledoux D, Labombardi VJ, Karter D (2006) Lactobacillus acidophilus bacteraemia after use of a probiotic in a patient with AIDS and Hodgkin’s disease. Int J STD AIDS 17:280–282. Lee J, Seto D, Bielory L (2008) Meta-analysis of clinical trials of probiotics for prevention and treatment of pediatric atopic dermatitis. J Allergy Clin Immunol 121:116–121. Lin HC, Hsu CH, Chen HL, Chung MY, Hsu JF, Lien RI, Tsao LY, Su BH (2008) Oral probiotics prevent necrotizing enterocolitis in very low birth weight preterm infants: a multicenter, randomized, controlled trial. Pediatrics 122:693–700. Link-Amster H, Rochat F, Saudan KY, Mignot O, Aeschlimann JM (1994) Modulation of a specific humoral immune response and changes in intestinal flora mediated through fermented milk intake. FEMS Immunol Med Microbiol 10:55–63.

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Loguercio C, Del Vecchio Blanco C, Coltorti M (1987) Enterococcus lactic acid bacteria strain SF68 and lactulose in hepatic encephalopathy: a controlled study. J Int Med Res 15:335–343. Mackie RI, Sghir A, Gaskins HR (1999) Developmental microbial ecology of the neonatal gastrointestinal tract. Am J Clin Nutr 69:1035S–1045S. Majamaa H, Isolauri E (1997) Probiotics: a novel approach in the management of food allergy. J Allergy Clin Immunol 99:179–185. Mattar AF, Drongowski RA, Coran AG, Harmon CM (2001) Effect of probiotics on enterocyte bacterial translocation in vitro. Pediatr Surg Int 17:265–268. Michail SK, Stolfi A, Johnson T, Onady GM (2008) Efficacy of probiotics in the treatment of pediatric atopic dermatitis: a meta-analysis of randomized controlled trials. Ann Allergy Asthma Immunol 101:508–516. Millar MR, Bacon C, Smith SL, Walker V, Hall MA (1993) Enteral feeding of premature infants with Lactobacillus GG. Arch Dis Child 69:483–487. Näse L, Hatakka K, Savilahti E, Saxelin M, Pönkä A, Poussa T, Korpela R, Meurman JH (2001) Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental caries and caries risk in children. Caries Res 35:412–420. Niers L, Martín R, Rijkers G, Sengers F, Timmerman H, van Uden N, Smidt H, Kimpen J, Hoekstra M (2009) The effects of selected probiotic strains on the development of eczema. Allergy 64:1349–1358. Oberhelman RA, Gilman RH, Sheen P, Taylor DN, Black RE, Cabrera L, Lescano AG, Madico G (1999) A placebo-controlled trial of Lactobacillus GG to prevent diarrhea in undernourished Peruvian children. J Pediatr 134:15–20. Orrhage K, Nord CE (1999) Factors controlling the bacterial colonization of the intestine in breastfed infants. Acta Paediatr Suppl 88:47–57. Osborn DA, Sinn JK (2007) Probiotics in infants for prevention of allergic disease and food hypersensitivity. Cochrane Database Syst Rev 4:CD006475. Peltonen R, Ling WH, Hanninen O, Eerola E (1992) An uncooked vegan diet shifts the profile of human fecal microflora: computerized analysis of direct stool sample gas-liquid chromatography profiles of bacterial cellular fatty acids. Appl Environ Microbiol 58:3660–3666. Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, van den Brandt PA, Stobberingh EE (2006) Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 118:511–521. Reid G, Howard J, Gan BS (2001) Can bacterial interference prevent infection? Trends Microbiol 9:424–428. Rosenfeldt V, Benfeldt E, Nielsen SD, Michaelsen KF, Jeppesen DL, Valerius NH, Paerregaard A (2003) Effect of probiotic Lactobacillus strains in children with atopic dermatitis. J Allergy Clin Immunol 111:389–395. Saavedra J, Bauman NA, Oung I, Perman JA, Jolken RH (1994) Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhea and shedding of rotavirus. Lancet 344:1046–1049. Salminen MK, Rautelin H, Tynkkynen S, Poussa T, Saxelin M, Valtonen V, Järvinen A (2004) Lactobacillus bacteremia, clinical significance and patient outcome, with special focus on probiotic L. rhamnosus GG. Clin Infect Dis 38:62–69. Salvana EM, Frank M (2006) Lactobacillus endocarditis: case report and review of cases reported since 1992. J Infect 53:e5–e10. Savino F, Cresi F, Pautasso S, Palumeri E, Tullio V, Roana J, Silvestro L, Oggero R (2004) Intestinal microflora in breastfed colicky and non-colicky infants. Acta Paediatr 93:825–829. Savino F, Pelle E, Palumeri E, Oggero R, Miniero R (2007) Lactobacillus reuteri (American Type Culture Collection Strain 55730) versus simethicone in the treatment of infantile colic: a prospective randomized study. Pediatrics 119:e124–e130. Schultz M, Göttl C, Young RJ, Iwen P, Vanderhoof JA (2004) Administration of oral probiotic bacteria to pregnant women causes temporary infantile colonization. J Pediatr Gastroenterol Nutr 38:293–297. Schurman JV, Friesen CA, Danda CE, Andre E, Welchert E, Lavenbarg T, Cocjin JT, Hyman PE (2005) Diagnosing functional abdominal pain with the Rome II criteria: parent, child and clinician agreement. J Pediatr Gastroenterol Nutr 41:291–295. Scientific Committee on Food of the European Commission (2004) Report of the Scientific Committee on Food on the Revision of Essential Requirements of Infant Formulae and Follow-on Formulae (adopted on 4 April 2003) Available at http://ec.europa.eu/food/food/labellingnutrition/children/formulae_en.htm (July 2010).

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Probiotics and Health Claims Related to OTC Products and Pharmaceutical Preparations

Frank M. Unger and Helmut Viernstein

14.1

INTRODUCTION

This chapter reviews the clinical indications, clinical studies and meta-analyses of probiotics for which health benefits have been claimed. In the first section, an overview is given of the production, processing and formulation of probiotic cultures in dosage forms suitable for pharmaceutical applications. In subsequent sections, the clinical indications are grouped into three areas: gastroenterology, gynecology and dentistry/stomatology. By far the most health claims concern the area of gastroenterology, so this section has been arbitrarily subdivided into two groups, namely frequently occurring diarrheas and inflammatory bowel disease (IBD). In the section on frequently occurring diarrheas, the clinical indications discussed include antibioticassociated diarrhea including Clostridium difficile disease and diarrheas occurring during treatment of Helicobacter infections, traveler's diarrhea, and diarrheas due to lactose malabsorption (lactose intolerance). Childhood diarrheas, such as those due to rotavirus infection, or clinical indications resulting from deficiencies in the gut flora of premature babies are discussed in Chapter 13. In the section on IBD, the clinical indications discussed include irritable bowel syndrome, ulcerative colitis, pouchitis and Crohn's disease. For each clinical indication, a short introduction comprising natural history, epidemiology and standard treatment of the condition is provided. Subsequently, selected clinical studies with probiotics are discussed. In the section Evaluation and Outlook, each of the above clinical indications is discussed in terms of health claims made in conjunction with the use of probiotics as pharmaceutical agents, as adjunct therapeutics, or as nutritional supplements with prophylactic activity. Where applicable, meta-analyses or reviews of clinical applications are cited. Also included is a brief discussion on recently discovered properties of probiotics that may lead to expanded therapeutic opportunities in the future.

14.2

PRODUCTION, PROCESSING AND FORMULATION OF PROBIOTIC CULTURES FOR PHARMACEUTICAL PURPOSES

Production of probiotic preparations useful for pharmaceutical applications requires several considerations, such as the origin of the strains, genetic stability, fermentability and fermentation yields, viability following processing of cultures on an industrial scale, viability upon

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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drying, and shelf-lives as pharmaceutical dosage forms (Viernstein et al., 2005). Recently, probiotic strains of human origin are preferred over dairy strains. Following their selection, samples are deposited in one of the recognized culture collections. Following large-scale culture, bacterial biomass is harvested by filtration or centrifugation. One classical modality of bacterial culture distribution is in the form of fresh frozen or chilled liquid cultures. For these preparations, loss of viability is usually small, but storage times are limited (comparable to milk products). At present, most of the pharmaceutically applied probiotics are supplied in pharmaceutical dosage forms such as powders (sachets), capsules or tablets. On a technical scale, drying of cultures may be performed by spray-drying, but freeze-drying (lyophilization) is the state-of-the-art technology for the production of dry but viable microbial cells. Some probiotic products with excellent stability may be stored for up to 2 years. Ideal storage conditions are a temperature of 5–10°C and an aw (water activity) of 0.1–0.25. Both higher and lower aw values usually result in more rapid losses of viability. Even under ideal storage conditions, products may lose 90% of viable bacteria after 1 year of storage.

14.3

CLINICAL STUDIES

In the following sections we review selected clinical studies undertaken to substantiate health claims made in conjunction with probiotics administered according to clinical indications.

14.3.1

Gastroenterology

14.3.1.1

Commonly occurring diarrheas

Discussion in this section focuses on antibiotic-associated diarrhea (AAD) including C. difficile infection, diarrheas occurring during treatment of Helicobacter infections, traveler's diarrhea, and diarrhea due to lactose intolerance. Infantile diarrheas such as those caused by rotavirus infection are considered in Chapter 13. Antibiotic-associated diarrhea and Clostridium difficile disease The use of antibiotics, especially in hospital settings, frequently results in AAD. These are a consequence of disruption of the function of the physiological gut flora by antibiotic action. AAD mostly occurs 2–3 weeks after treatment with antibiotics. Elimination of most of the physiological flora compromises the barrier function that usually prevents colonization by opportunistic pathogens. Therefore, infection by C. difficile and other pathogens is a common cause of AAD (Kelly & LaMont, 2008). Clostridium difficile disease Clostridium difficile was first described as Bacillus difficilis by Hall and O'Toole (1935). Presently, C. difficile is a frequent cause of AAD, especially following antibiotic regimens that involve amoxicillin, cephalosporins, clindamycin and fluoroquinolones (Kelly & LaMont, 2008). Clostridium difficile disease (CDD) is most frequent among elderly patients in hospital settings. Key pathogenicity factors of C. difficile are toxins A and B, which penetrate into epithelial cells, leading eventually to destruction of the cytoskeleton and cell death. Some aspects of the genetics and pathogenicity of toxins A and B are described in Kelly and LaMont (2008) and Dèneve et al. (2009). Many strains of C. difficile do not express toxins A or B, and carriage of C. difficile is asymptomatic in such cases. However,

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the presence of toxin-producing strains can be manifested in a wide range of conditions, from relatively harmless AAD to pseudomembranous colitis, which can be life-threatening. In recent years, several outbreaks of CDD have occurred in North America and Europe. Of the strains responsible, C. difficile NAP-1/027 strains contain a mutation in the gene tcdC, which is a putative negative regulator of toxin transcription. As a result, toxin transcription occurs earlier in the bacterial life cycle, resulting in enhanced pathogenicity of these strains. Whereas previously CDD mainly occurred in elderly or frail hospital or nursing home patients, recent epidemiology increasingly involves young and previously healthy individuals who would not have been considered at risk a few years ago. A probiotic drink against AAD and CDD in geriatric patients In a randomized, double-blind, placebo-controlled study (Hickson et al., 2007), 135 hospitalized geriatric patients were included to assess the efficacy of a probiotic drink containing Lactobacillus casei, Lactobacillus bulgaricus, and Streptococcus thermophilus for prevention of AAD, especially as caused by C. difficile. The verum group (N = 57) consumed the probiotic drink twice daily during a course of antibiotics and for 1 week thereafter. The placebo group (N = 56) consumed a longlife sterile milkshake. Primary outcome was the occurrence of diarrhea; secondary outcome was diarrhea plus the presence of C. difficile toxins A or B or both. Of the verum group, 7 of 57 participants (12%) developed diarrhea and no one had diarrhea caused by C. difficile. Of the placebo group, 19 of 56 participants (34%) had diarrhea (P = 0.007) and 9 of 53 (17%) had diarrhea caused by C. difficile (P = 0.001). The authors concluded that the consumption of a probiotic drink containing L. casei, L. bulgaricus and Strep. thermophilus can reduce the occurrence of AAD and CDD. Lack of effect of Lactobacillus GG on AAD In a prospective, randomized, double-blind, placebo-controlled study (Thomas et al., 2001), 302 patients treated with antibiotics were randomized to receive Lactobacillus GG (2 × 1010 CFU/day) or placebo for 2 weeks. Primary outcome was the number of patients developing diarrhea within the first 3 weeks of antibiotic treatment. In the verum group 29.3% (39 of 133) and in the placebo group 29.9% (40 of 134) developed diarrhea. The authors concluded that Lactobacillus GG in the dosage administered would not reduce the rate of occurrence of diarrhea in a group of patients taking antibiotics in a hospital setting. Prevention of AAD by Saccharomyces boulardii Surawicz et al. (1989) performed a prospective, double-blind, placebo-controlled study to evaluate the effect of Saccharomyces boulardii in AAD. In the verum group, 9.5% of patients experienced diarrhea compared with 22% in the placebo group. Interestingly, the authors found no significant association between AAD and C. difficile or cytotoxin: about 33% of patients without diarrhea had at least one C. difficile-positive stool, and nearly 50% of these patients had detectable cytotoxin. There was a trend toward fewer C. difficilepositive patients developing diarrhea in the verum group than those on placebo, but this trend did not reach statistical significance. The authors concluded that administration of S. boulardii reduces the incidence of AAD in hospitalized patients. Traveler's diarrhea Traveler's diarrhea is by far the most frequently occurring health problem of people traveling to subtropical and tropical countries in Africa, the Middle East, southern Asia and Latin America (DuPont & Ericsson, 1993). Between 20 and 50% of travelers contract

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this condition, commonly defined as the passage of three to ten liquid stools in 24 hours and which may be accompanied by abdominal pain or cramps, nausea and tenesmus (constantly feeling the need to empty the bowels, along with pain, cramping and straining; see www.nlm.nih.gov/medlineplus/ency/article/003131.htm for further information). The most prevalent infectious agents causing traveler's diarrhea are enterotoxigenic Escherichia coli (ETEC), shigella species, and Campylobacter jejuni (DuPont & Ericsson, 1993). However, depending on geographical location, Aeromonas and Salmonella species and a variety of other organisms may also play a role. In most cases, symptomatic therapy of traveler's diarrhea is sufficient (DuPont & Ericsson, 1993). Fluids such as broth or tea and salted crackers can be taken as long as stools are watery. If electrolyte replacement products are available, these should be taken together with sufficient fluids. In more severe cases of persisting fever or bloody diarrhea, medical attention should be sought and antibiotic therapy may be required. Probiotic bacteria can temporarily persist in the intestine where they metabolize carbohydrates to lactic acid and other organic acids. In this manner, the intraluminal pH is decreased and the growth of enteropathogens may be inhibited (DuPont & Ericsson, 1993). Therefore, a number of studies have been undertaken to assess the efficacy of probiotic preparations in preventing traveler's diarrhea. Effect of a lactobacilli preparation on traveler's diarrhea In a randomized double-blind trial (De Dios Pozo-Olano et al., 1978) of a commercial preparation, Lactinex, 50 volunteer travelers from the US to Mexico were given either the Lactobacillus preparation (N = 26) or placebo (N = 24). During 4 weeks of observation, the incidence and duration of diarrhea were similar between the two groups (placebo 29%, verum 35%). The authors concluded that ingestion of the Lactinex preparation for 1 week does not reduce the occurrence or duration of traveler's diarrhea during the ingestion period or during the 3 weeks thereafter. Lactobacillus GG in prevention of traveler's diarrhea In a placebo-controlled double-blind study (Oksanen et al., 1990), 820 participants traveling from Finland to two destinations in southern Turkey were randomized into two groups to receive identical-looking sachets of either Lactobacillus GG or placebo. During the return flight, each of the participants received a questionnaire to report the incidence of diarrhea and related symptoms. Of the 756 participants who completed the study, 331 (43.8%) reported having had diarrhea. In the placebo group, the incidence of diarrhea was 46.5% and in the probiotic group 41.0% giving a protection rate of 11.8%. Protection rates were different between the two destinations, the maximum being 39.5%. No side effects were reported. The authors concluded that Lactobacillus GG was able to reduce the incidence of diarrhea, at least in one of the two destinations. Efficacy of Saccharomyces boulardii in preventing traveler's diarrhea Prophylactic administration of 250 or 1000 mg daily of S. boulardii (in sachets) to Austrian travelers to different destinations (North and East Africa, Middle East, India, Far East, Central and South America) led to a significant reduction in the incidence of diarrhea (40% in the placebo group, 34% in those who received 250 mg/day of probiotic, and 29% in those who received 1000 mg/day of probiotic). The prophylactic effects of the probiotic were considerably more pronounced for travelers to North Africa and the Near East than in those traveling to other regions of the globe (Kollaritsch et al., 1993).

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Fig. 14.1 Sections of the human stomach.

In a prospective, double-blind, controlled study (Black et al., 1989), 94 Danish tourists participating in a 2-week round trip to Egypt were randomized into two groups. The probiotic group received one capsule three times daily, containing a mixture of about 3 × 109 live lyophilized organisms of the four species Lactobacillus acidophilus, Bifidobacterium animalis, Lactobacillus bulgaricus and Strep. thermophilus. Administration of capsules started 2 days prior to departure, ending on the last day of travel. The frequency of diarrhea was significantly reduced in the probiotic-treated group (from 71% in the placebo group to 43%; P = 0.019), corresponding to a protection rate of 39.4%. The authors concluded that the probiotic preparation can be recommended to travelers except for those belonging to the high-risk groups where the more effective antimicrobial chemotherapeutics should be used. Diarrheas occurring during treatment of Helicobacter infections Helicobacter pylori is found in 25–50% of the population in developed countries, and to a higher extent in developing countries (70–90%) (Go, 2002; Hamilton-Miller, 2002). Helicobacter pylori was first cultured in 1982 by J.R. Warren and B. Marshall who recognized its significance in the etiology of gastritis and ulcer disease (Warren & Marshall, 1983; Marshall & Warren, 1984). As the vast majority of people with H. pylori never develop symptoms, it has been suggested that presence of the organism be termed “carriage” rather than infection. The clinical course of an actual infection depends on both microbial and host factors (Suerbaum & Michetti, 2002). The type and localization of gastritis is strongly associated with the potential sequelae of gastric or duodenal ulcers, mucosal atrophy, gastric carcinoma or gastric lymphoma. The most common form of H. pylori gastritis is the antral-predominant form (Fig. 14.1), which predisposes to duodenal ulcers, whereas corpus-predominant gastritis and multifocal atrophy predispose to gastric ulcers, atrophy, metaplasia and gastric carcinoma. Since the discoveries of Warren and Marshall, peptic ulcer disease is treated as an infectious disease. Indeed, complete eradication of H. pylori is considered a cure for the disease. Present first-line therapies, so-called triple therapies, are based on combinations of proton-pump inhibitors (e.g. omeprazole) with two antibiotics (e.g. clarithromycin plus amoxicillin). For second-line therapy, quadruple therapy with a proton pump

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inhibitor or a histamine H2 receptor antagonist added to a bismuth-based triple regimen with high-dose metronidazole has been recommended (Hojo et al., 2001). A detailed discussion of Helicobacter infection and the respective therapeutic regimens is provided by Suerbaum and Michetti (2002). In recent years, probiotics have been increasingly studied either as stand-alone regimens for eradication of H. pylori or as adjunct treatments to triple therapies. Adjunct therapy with Saccharomyces boulardii In a prospective, randomized, placebo-controlled, double-blind study (Cindoruk et al., 2007), 124 patients with Helicobacter infection underwent triple therapy (antibiotics clarithromycin and amoxicillin, lansoprazole as proton pump inhibitor) for 2 weeks. Male to female ratio was 44 : 80. Participants were randomly attributed to one of two groups, the probiotics group receiving 1 g/day of S. boulardii (made available in sachet form) and the control group receiving identical-looking sachets with a placebo powder. Helicobacter pylori eradication rates in the treatment and placebo groups were similar (44/62 vs. 37/62; 71% vs. 60%); although the trend was in favor of the treatment group, the difference was not statistically significant. Nine patients (14.5%) in the treatment group experienced diarrhea compared with 19 (30.6%) in the placebo group; 27 participants (43.5%) in the control group complained of epigastric discomfort compared with only 9 (14.5%) in the probiotictreated group. Symptoms that were similar in both groups included abdominal gas, diffuse abdominal pain, taste disturbance, nausea and urticaria. Before initiation of therapy and at the end of the follow-up period (6 weeks), symptoms were recorded with the use of a modified Glasgow Dyspepsia Questionnaire (GDQ). For the treatment group, GDQ scores were significantly better than for the placebo group: 1.38 ± 1.25 (range 0–5) versus 2.22 ± 1.44 (0–6) (P < 0.01). The authors concluded that AAD, epigastric discomfort and treatment tolerability were improved in the S. boulardii-treated group, while the probiotic had no significant effect on H. pylori eradication rates. Positive effects of a combination of Lactobacillus and Bifidobacterium administered during standard triple or quadruple therapy of H. pylori infection De Vrese (2003) reported on a reduction of the metabolic activity of H. pylori in the stomachs of patients when a combination of Lactobacillus acidophilus La5 and Bifidobacterium animalis BB12 was administered in conjunction with triple or quadruple therapies of H. pylori-infected patients. At the same time, H. pylori eradication rates were augmented and antibiotic-associated side effects (such as AAD) reduced. Diarrheas due to lactose malabsorption Lactase-catalysed hydrolysis of lactose: natural history and nutritional significance For the nutritional requirements of infants during the first year of life, the disaccharide lactose 4-O-(b-d-galactopyranosyl)-d-glucose contained in breast milk is the most important source of energy (Vesa et al., 2000). Only the constituent sugars, glucose and galactose, but not intact lactose, can be absorbed in the small intestine. Therefore, lactose must be hydrolytically cleaved by the action of lactase, an enzyme located in the tips of intestinal villi. After weaning, the lactose ceases to be important as a source of energy and has no special nutritional significance for adults. Concomitantly, the activity of intestinal lactase decreases to 10% or less of the level found in infants. The diminished activity of lactase in adults is termed adult-type lactase deficiency, hypolactasia or, more appropriately, lactase non-persistence. Lactase non-persistence seems to be genetically determined (Sahi, 1994; Sahi & Launiala, 1977).

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Persistence of lactase activity into adulthood Persistence of the ability to digest lactose is more common in European whites than in most populations worldwide. Mutation of the genes encoding lactase non-persistence has been identified as the cause of this phenomenon. Indeed, a hypothesis has been formulated that in populations dependent on dairy farming, individuals with persistent high lactase activity would have had an advantage (Simoons, 1981). Whereas in Europe the prevalence of lactose maldigestion varies from 2% in Scandinavia to 70% in Sicily, it is above 50% in South America, Africa and Asia (reaching almost 100% in some Asian regions). In the United States, numbers depend on the origin of the respective population, amounting to 80% among blacks, 53% among Mexican-Americans and 15% among whites. In Australia and New Zealand, only 6 and 9% respectively of the adult population are lactose maldigestors (Scimshaw & Murray, 1988). Diagnosis of lactose intolerance Routine tests are available for the diagnosis of lactose malabsorption. For example, using breath hydrogen analysis, the amount of hydrogen gas formed from lactose through anaerobic metabolism in the large intestine may be estimated. A rise in hydrogen of 20 ppm over baseline after the ingestion of lactose is considered to indicate lactose malabsorption. Conversely, the levels of glucose in serum are expected to rise if lactose is cleaved in the small intestine with absorption of glucose and galactose: the absence of change (or a rise in glucose levels of less than 20 mg/dL) indicates lactose malabsorption (Swagerty et al., 2002). Biopsy of small bowel epithelia and the ex vivo/in vitro measurement of enzyme-catalysed lactose hydrolysis is another method but is invasive and cumbersome and hence rarely used. Bacterial lactase activity and longer gut transit time of yogurt In 1984, Kolars et al. reported that ingestion of 18 g of lactose in yogurt resulted in about one-third of the breath hydrogen excretion as the same amount of lactose in milk (Kolars et al., 1984). At the same time, fewer instances of diarrhea or flatulence were observed when lactose was ingested in yogurt than when milk or water were the vehicle (Savaiano et al., 1984). These results were confirmed in a study by Marteau et al. (1990), who performed hydrogen breath tests with eight lactase-deficient volunteers following ingestion of 18 g of lactose contained in milk, yogurt or pasteurized yogurt. Excess hydrogen excretion for the milk vehicle was about four times higher than that for yogurt and about twice that for pasteurized yogurt (P < 0.001). Gut transit time (measured from mouth to cecum) for lactose in yogurt was abut 165 minutes, for pasteurized yogurt about 206 minutes, and for milk about 103 minutes. After consumption of lactose contained in yogurt, significantly less lactose (about 1740 mg) reached the terminal ileum than when pasteurized yogurt was the vehicle (about 2825 mg; P < 0.05). The authors conclude that, in lactase-deficient individuals, more than 90% of the lactose contained in yogurt is digested in the small intestine. This is explained by the bacterial lactase activity and the slower gut transit of the yogurt vehicle. Similarly, Rizkalla et al. (2000) found that chronic consumption of fresh yogurt by men with lactose malabsorption resulted in lower breath hydrogen excretion. Varela-Moreiras et al. (1992) reported that 36% in a group of healthy institutionalized elderly individuals had lactose malabsorption which could be ameliorated by consumption of fresh but not pasteurized yogurt. Improved absorption of lactose from yogurt does not lead to adaptation of digestive capabilities in lactase-deficient adults In a study with 16 lactase-deficient individuals, Lerebours et al. (1989) confirmed that yogurt but not fermented-then-pasteurized milk (FPM) enhances the digestion of lactose.

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However, the results of hydrogen breath tests were not improved after 8 days of yogurt consumption in comparison with 24-hour ingestion, nor were mucosal lactase or b-galactosidase activities enhanced. The authors conclude that, in lactase-deficient individuals, no adaptation takes place on consumption of yogurt or FPM, and that beneficial effects of yogurt ingestion must be based on intraluminal processes. Failure of a Lactobacillus strain to change breath hydrogen levels following lactose ingestion In a randomized trial, Saltzman et al. (1999) attempted to determine whether oral ingestion, for 1 week, of Lactobacillus acidophilus BG2FO4, a strain with a high b-galactosidase activity and marked intestinal adherence, would lead to increased tolerance of lactose by lactose maldigestors. Of 42 participants with self-reported lactose intolerance, only 24 (57%) actually had lactose maldigestion as verified by breath hydrogen analysis. Of these, 18 individuals were given L. acidophilus BG2FO4 for 7 days. Breath hydrogen tests were performed, stool samples collected and symptom scores were recorded at baseline and at the termination of the study. Overall hydrogen production and symptom scores following Lactobacillus ingestion were not significantly different from the values recorded at baseline. The authors concluded that lactose maldigestion is overreported and that ingestion of L. acidophilus BG2FO4 does not significantly change breath hydrogen excretion after lactose ingestion. Overreporting of lactose malabsorption was confirmed by the study of Suarez et al. (1995) who studied 30 individuals who had claimed to be severely lactose intolerant. Of these, only 21 were identified as lactose malabsorbers. Participants were given either 240 mL of lactose-hydrolysed milk containing 2% fat or 240 mL of milk containing 2% fat plus aspartame to simulate the taste of lactose-hydrolysed milk. Evaluation of the mean symptom-severity scores for bloating, abdominal pain, diarrhea and flatus showed no statistically significant differences between the four gastrointestinal symptoms. The authors observed that of 30 individuals, nine were able to hydrolyse and absorb lactose; the other 21 were lactose malabsorbers but tolerated 240 mL of milk per day through a 1-week period with minimal if any symptoms. 14.3.1.2

Inflammatory bowel diseases

Clinical studies with probiotic preparations have been undertaken in the areas of irritable bowel syndrome, ulcerative colitis, pouchitis and Crohn's disease. Irritable bowel syndrome Irritable bowel syndrome (IBS) comprises a variety of conditions causing discomfort in the gastrointestinal tract. IBS is a functional bowel disorder and may be characterized by chronic abdominal pain, bloating, diarrhea, or constipation. Depending on the preponderant symptom, IBS may be categorized into IBS with diarrhea (IBS-D, more frequent in men), IBS with constipation (IBS-C, more frequent in women), and IBS with mixed bowel habits (IBS-A); each of the three categories accounts for approximately one-third of all cases (Mayer, 2008). IBS-PI (for post infection) is a further designation applied if IBS is known to start after an infection. Comprehensive overviews of aspects of IBS including differential diagnosis, etiology, role of diet, psychosomatic involvement, available treatment modalities, epidemiology, and numerous recent references can be found in Mayer (2008). Presently, there is no generally accepted cure for IBS. A variety of treatments are available for relief of symptoms. These include psychological interventions, dietary

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adjustments, laxatives, antidiarrheals, antibiotics, probiotics, serotonin agonists (for relief of constipation) and serotonin antagonists (for relief of diarrhea). Most of the available treatments have limited efficacy. Activity of a Bifidobacterium infantis strain in IBS The purpose of this study was a comparison of the symptoms and cytokine ratios in IBS with ingestion of probiotic preparations containing a Lactobacillus or a Bifidobacterium strain (O'Mahony et al., 2005). A total of 77 patients with IBS were randomized into three groups, one receiving 1010 CFU of Lactobacillus salivarius UCC 4331, another 1010 CFU of Bifidobacterium infantis 35624, and a third group receiving placebo for 8 weeks. The cardinal symptoms of IBS were recorded daily and were assessed each week. At baseline and at the end of the treatment phase, quality of life, stool microbiology, and release of the cytokines interleukin (IL)-10 and IL-12 were assessed. In the group receiving B. infantis 35624, scores for pain, bloating and bowel movement difficulty were significantly lower than scores in the placebo group for most weeks of the treatment phase. The abnormal ratio of IL-10 to IL-12, indicative of a proinflammatory immune status in patients with IBS, was normalized by feeding of B. infantis 35624. Clinical studies of the use of probiotics as therapeutic agents in IBS have been reviewed by Krammer et al. (2005). In a number of studies (Halpern et al., 1996; Nobaek et al., 2000; O'Sullivan & O'Morain, 2000; Koebnick et al., 2003) significant improvements of one or more parameters relevant to IBS have been demonstrated. For example, the symptom of obstipation was improved in a randomized, double-blind, placebo-controlled study after 4 weeks' treatment with Lactobacillus casei Shirota (Koebnick et al., 2003). Similarly, 6 weeks' treatment with Lactobacillus acidophilus improved the symptoms of pain, flatulence and bowel frequency (Halpern et al., 1996). In a 4-week double-blind, placebocontrolled study with Lactobacillus plantarum, flatulence was significantly (P < 0.001) reduced but reduction in pain did not reach statistical significance. Even after 12 months, general gastrointestinal functions of those in the probiotic-treated group were improved relative to participants in the placebo group (Nobaek et al., 2000). Treatment of IBS patients with Lactobacillus rhamnosus GG over 8 weeks (O'Sullivan & O'Morain, 2000) improved the symptom of diarrhea (P = 0.0045). Ulcerative colitis Ulcerative colitis (UC) is a form of IBD, specifically of the large intestine (colon) (Podolsky, 2002). UC is an intermittent condition with periods of exacerbated symptoms and relative symptom-free periods. Depending on the areas affected, UC occurs in the form of proctitis, proctosigmoiditis, left-sided colitis, or pancolitis (Fig. 14.2a, b, c and d). The principal symptom of active UC is constant diarrhea with bloody stools. Conventional treatment of UC is with anti-inflammatory and immunosuppressive drugs and biological agents targeting specific immune mediators. In severe cases, partial or total surgical removal of the colon is required and is considered a cure of UC (Kornbluth & Sachar, 2004). Improvement in chronic inflammation by a synbiotic therapy regimen In a double-blind, randomized, controlled pilot study, Furrie et al. (2005) studied the influence of a synbiotic preparation (Bifidobacterium longum combined with Synergy 1, an inulin–oligofructose nutrient of the bacterial strain) over 1 month on parameters of active UC in 18 patients (N = 9 in the synbiotic group, N = 9 in the placebo group). Parameters measured included sigmoidoscopy scores, mRNA levels for human b-defensins

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Fig. 14.2 Extent of gut involvement in ulcerative colitis: (a) proctitus; (b) proctosigmoiditis; (c) left-sided colitis; (d) pancolitis.

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2, 3 and 4, tumor necrosis factor (TNF)-a, IL-1a and inflammatory signs on rectal epithelial tissue histology. On a scale from 0 to 6, sigmoidoscopy scores were lower in the verum group compared with placebo. Similarly reduced were mRNA levels of human b-defensins 2, 3 and 4 (strongly upregulated in UC) and the inflammatory cytokines TNF-a and IL-1a. Gut tissue biopsies performed on the verum group showed a lesser degree of inflammation and some regeneration of epithelial tissue. The authors concluded that synbiotic treatment of active UC led to improvement of the appearance of chronic inflammation in these patients. Maintaining remission of UC by administration of a probiotic E. coli strain The efficacy was compared of the probiotic E. coli Nissle 1917 in maintaining remission of UC in comparison with mesalazine, the gold standard treatment for this indication (Kruis et al., 2004). A total of 327 patients were assigned to two groups. The probiotic-treated group (N = 162) received 200 mg E. coli Nissle 1917 once daily over 1 year while the control group (N = 165) received mesalazine 500 mg three times daily over 1 year. Analysis per protocol showed the occurrence of 40/110 relapses in the probiotic-treated group compared with 38/112 in the mesalazine-treated group, demonstrating statistical equivalence (P = 0.003). No differences were detected between the groups in terms of localization or duration of disease, or due to differences in treatment prior to the study. Tolerability and safety of the treatments were very good for both groups. The authors concluded that the probiotic E. coli Nissle 1917 shows efficacy and safety in maintaining remission equivalent to the standard mesalazine in patients with UC. Pouchitis The term “pouchitis” (Madden et al., 1990) describes inflammation of an ileal pouch (ileo-anal pouch, ileo-anal pouch anastomosis). An ileo-anal pouch is a surgical construct designed for patients who have their large intestine removed. Conditions requiring surgical removal of the large intestine include UC (see preceding section), familial adenomatous polyposis (an inherited condition wherein large numbers of colonic polyps grow, predisposing for colon cancer), colon cancer per se, and toxic megacolon. Toxic megacolon is a life-threatening complication of IBD; manifestations include a dilated colon (megacolon) and abdominal distension (bloating). Additional symptoms may include fever, abdominal pain and shock. Toxic megacolon may result in perforation of the colon wall, septicemia, and death (see www.answers.com/topic/toxic-megacolon for further information). The construction of an ileo-anal pouch for patients with Crohn's disease is controversial. The ileo-anal pouch is an internal reservoir that is located where the rectum would normally be. Its construction is performed by folding loops of ileum (the distal section of the small intestine) and suturing these together; finally, a reservoir is formed by removal of the internal walls. The pouch is then sutured into the perineum at the original location of the rectum. Typical symptoms of pouchitis are bloody diarrhea and urgency in passing stools. Frequent stools and loss of blood may lead to dehydration and nausea. For treatment, the Mayo Clinic recommends a short course (1–2 weeks) of antibiotics such as metronidazole and ciprofloxacin. If antibiotics should fail, potential treatments include mesalamine (5-aminosalicylic acid, a gastrointestinal anti-inflammatory agent) as a suppository or enema, oral and topical corticosteroids, immunosuppressive agents such as infliximab, and probiotics. When pouchitis persists and is unresponsive to medications, removal of the pouch may be required.

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Applications of probiotic preparations in patients with pouchitis In a double-blind placebo-controlled study, 40 patients with ileal pouch-anal anastomosis for UC were randomized into two groups of 20 patients each (Gionchetti et al., 2003). The verum group received the highly concentrated multistrain preparation VSL#3, 9 × 1011 CFU/day for 1 year (VSL#3 is composed of eight different bacterial species: Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Streptococcus salivarius subsp. thermophilus). The control group received an identical-looking placebo. Patients were examined clinically, endoscopically and histologically after 1, 3, 6, 9, and 12 months. Their quality of life was assessed with the aid of an IBD Questionnaire. Episodes of acute pouchitis occurred in two patients in the probiotictreated group (10%) and in eight patients of the placebo group (40%) (P < 0.05). IBD Questionnaire scores for the verum group, but not those for the placebo group, showed significant improvement. The authors concluded that treatment with VSL#3 is effective in the prevention of onset of acute pouchitis and improves the quality of life for patients with an ileo-anal pouch. Crohn's disease Crohn's disease (CD), also referred to as regional enteritis, is a chronic inflammatory disease of the digestive tract. CD progresses intermittently and can involve all sections of the alimentary canal from mouth to anus. In 50% of cases, lesions are found in the ileum and colon, whereas in 25% of cases the ileum or colon alone are affected (Fig. 14.3a, b and c). Lesions in the esophagus or stomach are rare (1–4%). Several characteristic features distinguish CD from UC. Whereas in UC, continuous shallow mucosal areas of inflammation are observed, CD lesions are patchy and may be transmural, reaching deep into tissues. In CD, recurrences are often seen after surgical removal of affected parts; on the other hand, UC is usually cured by the removal of colon but may be followed by pouchitis (see preceding section). Symptoms of CD include diarrhea (in 90% of cases, rarely bloody), pain (90%), weight loss (60–75%) and fever (33–70%). Attempted prevention of recurrence of CD following surgical resection In a randomized, double-blind, placebo-controlled trial (Marteau et al., 2006), patients were included if they had undergone bowel surgery of less than 1 m within the past 21 days. Participants were randomized to receive two sachets per day of Lactobacillus johnsonii LA 1 (2 × 109 CFU) or placebo for 6 months. Primary endpoint was endoscopic recurrence at 6 months of grade > 1 according to Rutgeert's classification (or an adapted classification for colonic lesions). Of 98 patients enrolled, 48 had been attributed to the probiotic-treated group. At 6 months, 30 of 47 patients in the placebo group and 21 of 43 in the verum group had endoscopic recurrences (64% vs. 49%, P = 0.15). Four clinical recurrences in the verum group were recorded compared with three in the placebo group. The authors concluded that the probiotic regimen involving Lactobacillus johnsonii LA 1 did not have sufficient efficacy in preventing the endoscopic recurrence of CD following surgical resection. In a randomized placebo-controlled study (Prantera et al., 2002) with orally administered Lactobacillus GG or identical placebo for the duration of 1 year, endpoints were the prevention of recurrent lesions after surgery or reduction of their severity. Endoscopic recurrence was defined as grade 2 or higher according to Rutgeert's classification (Rutgeert et al., 1990). Patients suffering from CD who had undergone surgery to completely remove the diseased

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Rectum

(c)

Stomach Large intestine (Colon)

Sigmoid colon

Small intestine Anus

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Rectum

Fig. 14.3 Most common sites of gut involvement in Crohn’s disease: (a) ileal; (b) ileocolic; (c) colonic.

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portion of the gut were attributed to receive either 1.2 × 1010 CFU or identical placebo for 1 year. Three patients in the Lactobacillus group (16.6%) and two patients in the placebo group (10.5%) had clinical recurrences. Of 15 patients in clinical remission on Lactobacillus, 9 (60%) had endoscopic recurrence compared with 6 of 17 (35.3%) on placebo (P = 0.297). No significant differences in the degree of severity of symptoms were observed between the two groups. The authors concluded that Lactobacillus GG seems neither to prevent endoscopic recurrence at 1 year post surgery nor reduce the severity of recurrent lesions.

14.3.2

Gynecology

In the field of gynecology, clinical studies have been performed to assess the benefit of probiotics in the prevention or treatment of infections of the genitourinary tract. Bacterial vaginosis Bacterial vaginosis (Spiegel, 1991) results from an imbalance in the vaginal flora. Generally, the numbers of lactobacilli decrease while the counts of Gardnerella (Gardner & Dukes, 1955), anaerobes and Mobiluncus (Spiegel et al., 1983) increase. Diagnostic criteria for bacterial vaginosis Amsel et al. (1983) have defined four criteria for bacterial vaginosis: a pH of the vaginal fluid higher than 4.5; a thin, homogeneous, milky vaginal discharge; clue cells (more than 20% of vaginal epithelial cells in a smear display adherent bacteria); and a fishy odor that is detected when vaginal fluid is mixed with 10% potassium hydroxide. A diagnosis of bacterial vaginosis is established when three of these four criteria are met. Alternatively, bacterial vaginosis is diagnosed according to the Nugent score (Nugent et al., 1991). Grades from zero (healthy flora) to 10 are assigned on the basis of the microscopically identified ratio of Gram-positive rods and lactobacilli to various Gram-negative organisms constituting the typical bacterial vaginosis flora. An alternative system of grades has been proposed by Ison and Hay (2002), whereby grade 1 corresponds to a normal healthy flora with lactobacilli predominating, grade 2 is intermediate (some lactobacilli, but also some Gardnerella or Mobiluncus), and grade 3 constitutes bacterial vaginosis with Gardnerella/Mobiluncus predominating, lactobacilli being few or none. Sequelae of bacterial vaginosis While bacterial vaginosis is frequently asymptomatic, serious sequelae such as infection of the amniotic fluid and miscarriage may arise from this condition. In a randomized placebo-controlled trial with 64 healthy women (Reid et al., 2003), a combination of strains L. rhamnosus GR-1 and L. reuteri RC-14 was examined to determine its safety and the changes it might effect in the quality of the vaginal flora. There were no side effects reported in conjunction with the antibiotic therapy. On day 0, all participants reported no vaginal symptoms, but 16 of 64 (25%) had asymptomatic bacterial vaginosis according to the Nugent criteria. Of 25 women who did not have vaginosis on day 0 and who received placebo, 6 (24%) had developed vaginosis by day 35 and 4 (16%) by day 56. In contrast, none of the women in the probiotic-treated group (N = 23) developed bacterial vaginosis (P < 0.05). By Nugent scoring, 94% of participants in the probiotic-treated group had lactobacilli present on day 35, and 97% on day 56, significantly more than in the placebo group (day 35, P = 0.08; day 56, P = 0.05). More participants in the probiotic-treated

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group relative to the placebo group developed a vaginal flora with lactobacilli present (P < 0.01). More participants in the placebo group than in the probiotic-treated group had fewer or no lactobacilli compared with the swabs taken initially (P < 0.01). According to culture findings, oral administration of lactobacilli resulted in a significant increase in vaginal lactobacilli compared with placebo in 4 weeks (P = 0.01). Concomitantly, there were decreases in yeasts (P = 0.01) and coliform bacteria (P = 0.001). By day 90 (30 days after cessation of probiotic intake), coliform counts in the verum group were still significantly lower (P < 0.01) than those in the placebo group. Probiotic combination adjunct to metronidazole in treatment of bacterial vaginosis In a randomized, double-blind, placebo-controlled study (Anukam et al., 2006), 125 African women diagnosed with bacterial vaginosis received two 500-mg capsules of metronidazole daily plus either oral Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 (1 × 109 CFU each) or placebo capsules twice daily for 30 days starting on day 1 of the metronidazole treatment. Primary outcome of the study was cure of bacterial vaginosis as diagnosed by Nugent score, absence of clue cells, negative sialidase test, and no symptoms or signs (discharge or odor) of bacterial vaginosis at day 30. At the termination of the study, 43 of 49 participants in the probiotic-treated group (88%) had normal Nugent scores and were sialidase negative, compared with 23 of 57 patients (40%) in the antibiotics-only group (P < 0.001). Clinically, all participants in the probiotic-treated group were no longer diagnosed with bacterial vaginosis, whereas in the placebo group 30% were positive despite metronidazole treatment. In a subsequently reported study, Martinez et al. (2009) demonstrated that the probiotic combination Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 provides significant benefit together with the antimicrobial chemotherapeutic tinidazole in an adjunct setting. According to a randomized double-blind protocol, 64 women who had been diagnosed with bacterial vaginosis were assigned to receive tinidazole (2 g) supplemented with either two capsules of L. rhamnosus GR-1 and L. reuteri RC-14 or two placebo capsules every morning for 28 days. Subsequently, the cure rate of the probiotic-treated group was 87.5%, whereas in the placebo group only 50% of women had been cured of bacterial vaginosis (P = 0.001). In the probiotic-treated group, 75% of women had Nugent scores corresponding to a “normal” vaginal flora, whereas that percentage was only 34.4% in the placebo group (P = 0.011). Quality of the vaginal flora of postmenopausal women In another randomized, double-blind, placebo-controlled study, Petricevic et al. (2008) evaluated the influence of the orally administered probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the quality of the vaginal flora in postmenopausal women, as assessed by Nugent scoring. Postmenopausal women with Nugent scores between 4 and 6 were randomized into two groups. The probiotic-treated group received capsules containing 2.5 × 109 CFU each of L. rhamnosus GR-1 and L. reuteri RC-14. Women in the control group received an oral placebo once daily, both groups for 2 weeks. On the day following the last administration, vaginal swabs were taken. The primary outcome variable was change in Nugent score of at least two grades. Of the 72 participants, 35 were assigned to the probiotic-treated group and 37 to the placebo group; 21 of 35 women (60%) in the probiotic-treated group and 6 of 37 women in the placebo group showed a reduction in Nugent score by at least two grades. The difference in the number of those improved was highly significant (P = 0.0001). The median difference in Nugent scores

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between day 1 and the end of the study was 3 in the probiotic-treated group and 0 in the placebo group (P = 0.0001). The results were taken by the authors to indicate an alternative modality for the restoration of the normal vaginal flora using orally administered specific probiotic strains.

14.3.3

Dentistry/stomatology

Clinical studies have been performed with probiotic strains to examine benefit regarding improvement in gingivitis, reduction of plaque, suppression of Streptococcus mutans, and alleviation of gum bleeding and other inflammatory signs related to imbalance of the bacterial flora of the oral cavity. A total of 59 participants with moderate to severe gingivitis were included in a randomized, placebo-controlled, double-blind study (Krasse et al., 2005) designed to assess if the probiotic Lactobacillus reuteri could be effective in the treatment of gingivitis. Further outcomes were the influence of the probiotic on plaque and the lactobacilli population in the saliva. Participants were given one of two different L. reuteri formulations (designated LR-1 or LR-2) at a dose of 2 × 108 CFU per day or a corresponding placebo over 2 weeks; 20 participants were randomized to LR-1, 21 to LR-2 and 18 to placebo. On day 0, gingival index and plaque index were measured on two surfaces, and saliva for lactobacilli determination was collected. The participants were instructed how to brush and floss efficiently. On day 14, gingivitis and plaque were assessed and the count of lactobacilli determined. The gingival index fell significantly in all three groups (P < 0.0001). LR-1, but not LR-2, improved more than placebo (P < 0.0001). Plaque index fell significantly in LR-1 (P < 0.05) and in LR-2 (P < 0.01) with no significant change in the placebo cohort. At the end of the study, 65% of the participants in LR-1 and 95% of the participants in LR-2 were colonized with L. reuteri. The authors concluded that L. reuteri was effective in reducing both gingivitis and plaque in patients with moderate to severe gingivitis. Nikawa et al. (2004) studied the effect of Lactobacillus reuteri against Streptococcus mutans, one of the more important cariogenic organisms. In a double-blind, placebocontrolled, crossover study, participants in the first group were given 95 g of placebo yogurt at lunchtime daily for a period of 2 weeks, and subsequently 95 g of L. reuteri yogurt at lunchtime daily for another 2 weeks. Participants in the second group were given L. reuteri yogurt at lunchtime daily for 2 weeks, and subsequently placebo yogurt at lunchtime daily for another 2 weeks. Levels of oral carriage of Strep. mutans were determined before and after consumption of each variety of yogurt. The eating of L. reuteri yogurt significantly reduced the oral carriage of Strep. mutans in each group (P < 0.05 for group 1; P < 0.01 for group 2). The effect was not observed when placebo yogurt was consumed (P > 0.05). The authors interpreted the data to indicate that fermented milk containing L. reuteri should be of value in the reduction of caries risk. In a randomized, double-blind, placebo-controlled study (Caglar et al., 2008), the effect was investigated of the probiotic Lactobacillus reuteri, delivered in the form of lozenges, on the levels of salivary mutans streptococci and lactobacilli in young women with high Strep. mutans counts. Participants in the probiotic group (N = 10) sucked the probiotic lozenges containing L. reuteri ATCC 55730 plus L. reuteri ATCC PTA 5289 (1.1 × 108 CFU) once daily for 10 days whereas the control group received placebo lozenges without bacteria. Streptococcus mutans and L. reuteri were enumerated with the aid of chair-side kits on day 0 and on the day after the final ingestion. In the probiotic

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group, salivary Strep. mutans were significantly reduced (P < 0.05) whereas no difference was observed with the placebo group. There was no significant change in salivary lactobacilli counts in either group.

14.4

EVALUATION AND OUTLOOK

In this section, an attempt is made to evaluate the current state of clinical evidence so as to determine which of the probiotic-related health claims are justified with regard to registered pharmaceutical specialties and nutritional supplements. However, the reader should be aware of the limitations inherent in the known clinical studies, reviews and meta-analyses regarding therapeutic activities of probiotics. On the one hand, the number of participants is small in most studies; on the other, in meta-analyses, probiotics are frequently treated as a homogeneous “class of drug”, although it is well established that certain probiotics are active and useful in one indication but may not be so in another.

14.4.1

Antibiotic-associated diarrhea and Clostridium difficile disease

14.4.1.1

Meta-analysis: probiotics for prevention of AAD and treatment of CDD

Following a rigorous selection of 31 of 180 studies examined, McFarland (2006) concluded that administration of certain probiotics significantly reduced the relative risk (RR) of AAD to 0.43 (95% CI 0.31–0.58; P < 0.001). Similarly, six randomized studies were analysed and indicated significant efficacy against C. difficile disease (RR 0.59, 95% CI 0.41–0.85; P = 0.005). In the meta-analysis of McFarland, three types of probiotics showed promise as effective therapeutic agents against AAD, namely Saccharomyces boulardii, Lactobacillus rhamnosus GG, and a probiotic mixture of Lactobacillus acidophilus and Bifidobacterium bifidum. Benefit against C. difficile disease was demonstrable only for S. boulardii. According to a recent study by Hickson et al. (2007), consumption of a probiotic drink containing Lactobacillus casei, Lactobacillus bulgaricus and Streptococcus thermophilus, can reduce the occurrence of AAD and CDD. 14.4.1.2

Conclusion and recommendation

Several probiotic preparations show promise of benefit against AAD, among them Saccharomyces boulardii, Lactobacillus rhamnosus GG, a mixture of Lactobacillus acidophilus and Bifidobacterium bifidum, and a probiotic drink containing Lactobacillus casei, Lactobacillus bulgaricus and Streptococcus thermophilus. The probiotic drink and Saccharomyces boulardii also show promise of benefit against CDD.

14.4.2

Traveler's diarrhea

In a meta-analysis of probiotics for the prevention of traveler's diarrhea, McFarland (2007) analysed 12 different studies and reached the conclusion that two probiotics, Saccharomyces boulardii and a mixture of Lactobacillus acidophilus and Bifidobacterium bifidum, had significant activity. A combination of Lactobacillus acidophilus, Bifidobacterium animalis,

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Lactobacillus bulgaricus and Streptococcus thermophilus is registered in a number of countries for the prevention of traveler’s diarrhea and has been successfully used in North Africa and the Middle East (Black et al., 1989). However, rigorous studies (“good clinical practice”) are difficult to perform with groups of travelers: causative organisms of traveler’s diarrhea may vary from one region to the other; hand hygiene and hygienic discipline may vary widely amoung participants; and, last but not least, individual susceptibility to opportunistic infections is bound to depend on individual gut flora composition and therefore to be different among participants. Finally, it must be noted that the maximum protection rates that can be achieved by prophylactic administration of probiotics are usually around 40%. 14.4.2.1

Conclusion and recommendation

The risk of experiencing traveler's diarrhea can be significantly lowered by taking prophylactic doses of probiotics. Clinical evidence exists for the efficacy of Lactobacillus GG, of Saccharomyces boulardii, and of a combination of Lactobacillus acidophilus, Bifidobacterium animalis, Lactobacillus bulgaricus and Streptococcus thermophilus.

14.4.3

Helicobacter pylori infection

14.4.3.1

Meta-analysis and systematic review of probiotic supplementation during H. pylori eradication therapy

Tong et al. (2007) reported on a meta-analysis of 14 randomized studies (N = 1671) comparing probiotic supplementation to placebo or no treatment during regimens directed against H. pylori infection. Pooled eradication rates were 83.6% for verum and 74.8% for placebo. Cumulative occurrence of side effects was 24.7% for verum and 38.5% for placebo. The authors concluded that probiotics could be of value in increasing eradication rates and in reducing the occurrence of side effects. Similar conclusions were drawn by Gotteland et al. (2006) in their systematic review of the subject. 14.4.3.2

Conclusion and recommendation

The Maastricht III Consensus Report (Malfertheiner et al., 2007) has recognized probiotics as a future tool in the adjuvant therapy of H. pylori infection.

14.4.4

Lactose intolerance

14.4.4.1

Helpful effects of probiotics are uncertain in lactose intolerance

In a systematic review, Levri et al. (2005) conclude that evidence is lacking for probiotic supplementation alleviating symptoms of lactose intolerance in adults. However, certain strains and preparations appear to be effective, suggesting further clinical trials. 14.4.4.2

Yogurts facilitate digestion of lactose

In their review, De Vrese et al. (2001) emphasize that yogurts facilitate digestion of lactose in the small intestine more efficiently than do probiotic bacteria, which naturally target the colon. Indeed, a number of research groups (Kolars et al., 1984; Savaiano et al., 1984;

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Marteau et al., 1990; Varela-Moreiras, 1992; Rizkalla et al., 2000) report that lactose administered in yogurt is digested by lactose maldigestors better than lactose administered in milk or in pasteurized yogurt. The improved digestion of lactose in yogurt is thought to be due to the intraluminal action of bacterial lactase enzymes, to a protracted gut transit of the yogurt-containing preparations, to positive effects on intestinal function and gut flora, and to decreased sensitivity to symptoms (De Vrese et al., 2001). Interestingly, while administration in pasteurized yogurt does not improve the digestion of lactose, the severity of symptoms of lactose maldigestion such as diarrhea and bloating is reduced when lactose is consumed in pasteurized yogurt (Kolars et al., 1984; Marteau et al., 1990). 14.4.4.3

Conclusion and recommendation

Clinical evidence indicates that the yogurt strains Lactobacillus bulgaricus and Streptococcus thermophilus when administered in live form in the context of yogurt products can assist lactose maldigestors in the digestion of lactose and ameliorate such symptoms of lactose intolerance as bloating and diarrhea. As the digestion of lactose takes place in the small intestine, probiotics proper, which target the colon, appear to be less generally useful in this context. Thus, a cup of yogurt twice daily might help to overcome lactase non-persistence and enable the digestion of the small “hidden” amounts of lactose present in many prepared foods of a normal Western diet. Similarly, a probiotic product composed of Lactobacillus acidophilus, Bifidobacterium animalis, Lactobacillus bulgaricus and Streptococcus thermophilus is available in capsule form. This product contains the two yogurt strains in addition to probiotic bacteria and maybe capable of ameliorating lactose intolerance. However, rigorous clinical evidence for such benefit is missing.

14.4.5

Irritable bowel syndrome

Brenner et al. (2009) reported on a systematic review of the use of probiotics in the treatment of IBS. The purpose of this review was to analyse available RCTs evaluating efficacy, safety and tolerability of probiotics in the treatment of IBS. A total of 16 RCTs met the selection criteria, of which 11 studies suffer from inadequate study design using Rome II criteria. According to this review, only Bifidobacterium infantis 35624 showed significant improvement in the composite score for abdominal pain/discomfort, bloating and/or bowel movement difficulty compared with placebo (P < 0.05) (O'Mahony et al., 2005). A probiotic preparation based on B. infantis 35624 is commercially available under the trade name Bifantis®. 14.4.5.1

Conclusion and recommendation

As indicated in the review of Krammer et al. (2005), a number of probiotic preparations are capable of alleviating one or more of the symptoms of IBS such as chronic abdominal pain, bloating, diarrhea or constipation. However, in the studies reviewed, none of the commonly available probiotics showed wider-ranging activity covering the whole spectrum of symptoms.

14.4.6

Ulcerative colitis

14.4.6.1

Conclusion and recommendation

The probiotic E. coli Nissle 1917 has been shown to be equivalent to mesalazine in maintaining remission in UC. Results with a synbiotic combination of Bifidobacterium longum

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and an inulin preparation Synergy 1 augur well for the development of clinically applicable probiotics and synbiotics for the treatment of active UC.

14.4.7

Pouchitis

14.4.7.1

Conclusion and recommendation

In their review, Gionchetti et al. (2003) state that “the administration of highly concentrated probiotic preparations represents a valid approach both for the prevention of pouchitis onset and relapses”. Positive clinical evidence exists for the preparation VSL#3, a multistrain product composed of Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Streptococcus salivarius subsp. thermophilus). Attempts at probiotic therapy of active pouchitis have so far been unsuccessful.

14.4.8

Crohn's disease

In a Cochrane review, Rolfe et al. (2006) concluded that insufficient evidence was available from studies concluded at the time for the efficacy of probiotics in inducing remission of Crohn's disease or preventing relapse following surgery (cited in Schulze et al., 2008).

14.4.9

Bacterial vaginosis

14.4.9.1

Conclusion and recommendation

The probiotic combination Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 provides significant benefit, in an adjuvant setting, during antibiotic therapy of bacterial vaginosis. Similarly, the intermediate quality of the vaginal flora in a cohort of postmenopausal women was significantly improved by a 2-week course of that probiotic combination. However, active bacterial vaginosis was refractory to probiotic treatment using the combination of L. rhamnosus GR-1 and L. reuteri RC-14.

14.4.10

Gingivitis, reduction of plaque and alleviation of gum bleeding

14.4.10.1

Conclusion and recommendation

Strains of Lactobacillus reuteri termed Prodentis have been demonstrated to improve oral/ dental symptoms such as gingivitis, gum bleeding and carriage of Strep. mutans. These products are available as probiotic lozenges and chewing gums and are presumed to establish a natural equilibrium of the bacterial flora inhabiting the oral cavity.

14.4.11

Selected experimental approaches to probiotic products with new properties and in new indications

Increasing numbers of studies have been performed demonstrating biological activities of probiotics or their cell-free supernatants on cellular pathways related to inflammation, cancer development and cancer progression (Karin & Greten, 2005). For example,

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specific strains of the commensal-derived Lactobacillus reuteri have been shown to inhibit IL-8 expression induced by TNF-a (Ma et al., 2004), alleviate Helicobacter hepaticus-induced inflammatory bowel disease in IL-10 deficient mice (Pena et al., 2005), and induce IL-10-producing regulatory T cells in vitro by modulating dendritic cell function (Smits et al., 2005). In a recent communication, the group of Versalovic reported on molecular mechanisms of pro-apoptotic effects exerted by supernatants of the human-derived Lactobacillus reuteri strain ATCC PTA 6475 on human chronic myeloid leukemia-derived KBM-5 cells (Iyer et al., 2008). Supernatants were shown, by fluorophore staining and by trypan blue methods, to enhance TNF-induced cytotoxicity by 3–38%. As demonstrated by means of a terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) assay, the proportion of apoptotic leukemia cells increased from 3 to 37%. The TUNEL assay measures DNA strand breaks as an indication of apoptosis (Gavrieli et al., 1992).

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Marteau P, Lémann M, Seksik P et al. (2006) Ineffectiveness of Lactobacillus johnsonii LA 1 for prophylaxis of postoperative recurrence in Crohn's disease: a randomised, double blind, placebo controlled GETAID trial. Gut 55:842–847. Martinez RCR, Franceschini SA, Patta MC et al. (2009) Improved cure of bacterial vaginosis with single dose of tinidazole (2 g), Lactobacillus rhamnosus GR-1, and Lactobacillus reuteri RC-14: a randomized, double-blind, placebo-controlled trial. Can J Microbiol 55:133–138. Mayer EA (2008) Clinical practice. Irritable bowel syndrome. N Engl J Med 358:1692–1699. Nikawa H, Makihira S, Fukushima H et al. (2004) Lactobacillus reuteri in bovine milk fermented decreases the oral carriage of mutans streptococci. Int J Food Microbiol 95:219–223. Nobaek S, Johansson ML, Molin G, Ahrné S, Jeppsson B (2000) Alteration of intestinal microflora is associated with reduction in abdominal bloating and pain in patients with irritable bowel syndrome. Am J Gastroenterology 95:1231–1238. Nugent RP, Krohn MA, Hillier SL (1991) Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. J Clin Microbiol 29:297–301. Oksanen PJ, Salminen S, Saxelin M et al. (1990) Prevention of traveller's diarrhoea by Lactobacillus GG. Ann Med 22:53–56. O'Mahony L, McCarthy J, Kelly P et al. (2005) Lactobacillus and Bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 128:541–551. O'Sullivan MA, O'Morain CA (2000) Bacterial supplementation in the irritable bowel syndrome. A randomized, double-blind, placebo controlled crossover study. Dig Liver Dis 32:294–301. Pena JA, Rogers AB, Ge Z et al. (2005) Probiotic Lactobacillus spp. diminish Helicobacter hepaticus induced inflammatory bowel disease in interleukin-10 deficient mice. Infect Immun 73:912–920. Petricevic L, Unger FM, Viernstein H, Kiss H (2008) Randomized, double-blind, placebo controlled study of oral lactobacilli to improve the vaginal flora of postmenopausal women. Eur J Obstet Gynecol Reprod Biol 141:54–57. Podolsky DK (2002) Inflammatory bowel disease. N Engl J Med 347:417–429. Prantera C, Scribano ML, Falasco G, Andreoli A, Luzi C (2002) Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn's disease: a randomized controlled trial with Lactobacillus GG. Gut 51:405–409. Reid G, Charbonneau D, Erb J et al. (2003) Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol Med Microbiol 35:131–134. Rizkalla SW, Luo J, Kabir M, Chevalier A, Pacher N, Slama G (2000) Chronic consumption of fresh but not heated yogurt improves breath-hydrogen status and short-chain fatty acid profiles: a controlled study in healthy men with or without lactose maldigestion. Am J Clin Nutr 72:1474–1479. Rolfe VE, Fortun PJ, Hawkey VJ, Bath-Hextall F (2006) Probiotics for maintenance of remission in Crohn's disease. Cochrane Database Syst Rev 4:CD004826. Rutgeert P, Geboes K, Vantrappen G, Beyls J, Kerremans R, Hiele M (1990) Predictability of the postoperative course of Crohn's disease. Gastroenterology 99:956–963. Sahi T (1994) Genetics and epidemiology of adult-type hypolactasia. Scand J Gastroenterology 29(Suppl. 202):7–20. Sahi T, Launiala K (1977) More evidence for the recessive inheritance of selective adult type lactose malabsorption. Gastroenterology 73:231–232. Saltzman JR, Russell RM, Golner B, Barakat S, Dallal GE, Goldin BR (1999) A randomized trial of Lactobacillus acidophilus BG2FO4 to treat lactose intolerance. Am J Clin Nutr 69:140–146. Savaiano DA, AbouElAnouar A, Smith DE, Levitt MD (1984) Lactose malabsorption from yogurt, pasteurized yogurt, sweet acidophilus milk, and cultured milk in lactase-deficient individuals. Am J Clin Nutr 40:1219–1223. Schulze J, Sonnenborn U, Ölschläger T, Kruis W (2008) Probiotika. Stuttgart: Hippokrates Verlag. Scrimshaw NS, Murray EB (1988) Prevalence of lactose maldigestion. Am J Clin Nutr 48:1086–1098. Simoons FJ (1981) Geographic patterns of primary adult lactose malabsorption. In: Paige DM, Bayless TM (eds) Lactose Digestion: Clinical and Nutritional Implications. Baltimore: Johns Hopkins University Press, pp. 23–48. Smits HH, Engering A, Van der Kleij D et al. (2005) Selective probiotic bacteria induce IL-10 producing regulatory T cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin. J Allergy Clin Immunol 115:1260–1267. Spiegel CA (1991) Bacterial vaginosis. Clin Microbiol Rev 4:485–502.

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Spiegel CA, Eschenbach DA, Amsel R, Holmes KK (1983) Curved anaerobic bacteria in bacterial (nonspecific) vaginosis and their response to antimicrobial therapy. J Infect Dis 148:817–822. Suarez FL, Savaiano DA, Levitt MD (1995) A comparison of symptoms after the consumption of milk or lactose-hydrolyzed milk by people with self-reported severe lactose intolerance. N Engl J Med 333:1–4. Suerbaum S, Michetti P (2002) Helicobacter pylori infection. N Engl J Med 347:1175–1186. Surawicz CM, Elmer GW, Speelman P, McFarland LV, Chinn J, Van Belle G (1989) Prevention of antibioticassociated diarrhea by Saccharomyces boulardii: a prospective study. Gastroenterology 96:981–988. Swagerty DL Jr, Walling AD, Klein RM (2002) Lactose intolerance. Am Fam Physician 65:1845–1850, 1855–1856. Thomas MR, Litin SC, Osmon DR, Corr AP, Weaver AL, Lohse CM (2001) Lack of effect of Lactobacillus GG on antibiotic-associated diarrhea: a randomized, placebo-controlled trial. Mayo Clin Proc 76:883–889. Tong JL, Ran ZH, Shen J, Zhang XC, Xiao SD (2007) Meta-analysis: effect of supplementation with probiotics on eradication rates and adverse events during Helicobacter pylori eradication therapy. Aliment Pharmacol Ther 25:155–168. Varela-Moreiras G, Antoine JM, Ruiz-Roso B, Varela G (1992) Effects of yogurt and fermented-thenpasteurized milk on lactose absorption in an institutionalized elderly group. J Am Coll Nutr 11:168–171. Vesa TH, Marteau P, Korpela R (2000) Lactose intolerance. J Am Coll Nutr 19:165S–175S. Viernstein H, Raffalt J, Polheim D (2005) Stabilization of probiotic microorganisms. In: Nedovic V, Willaert R (eds) Applications of Cell Immobilization Biotechnology. Berlin: Springer, pp. 439–453. Warren JR, Marshall B (1983) Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1:1273–1275.

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Probiotics and Health Claims: the Perspective of the Feed Industry

Anja Meieregger, Elisabeth Mayrhuber and Hans Peter Lettner

15.1

INTRODUCTION AND HISTORY

In human nutrition, probiotics have been used for centuries but were not accurately defined. It was very hard to prove their effects and therefore the whole probiotic concept was long regarded as scientifically unproven. The concept of using probiotics as feed supplements has been developing since the 1970s. In the beginning the focus was on improving the animal’s health and resistance to disease (Fuller, 1989). As the effect of probiotics is linked to the gastrointestinal tract, it was assumed that this led to a greater ability to overcome gut infections like diarrhoea, leading to better animal performance. The term ‘probiotic’ was used in 1974 by Parker to describe animal feed supplements that had a beneficial effect on the host animal by affecting its gut flora (Fuller, 1989). Gradually, the food industry has also shown interest in probiotics and redefined it accordingly. In the last decades, scientific progress has been tremendous, and nowadays there are reliable methods for selection and characterisation of useful microorganisms. The history of probiotics was largely driven by economic reasons. The beginning of the story, with use of probiotics in the feed industry, is closely linked to the development of, and cutback in, the use of antibiotics as growth promoters. The implementation of antibiotics as growth stimulants for farm animals was common in the United States from 1949 onwards, in Britain from 1953 (Fuller, 1989; Meikle & Brown, 1999), and immediately enjoyed heavy use. A number of antibiotics were incorporated in the feed as low-level growth promoters. These low-dose drugs were believed to negate bacteria in the animal’s intestines that would hinder the absorption of nutrients. The decision to reject this principle was based on the assumption that antibiotics not only inhibited undesirable but also desirable microorganisms. In the UK in 1967, 168 tonnes of antibiotics were fed or injected into animals compared with 240 tonnes in humans. Only three decades later in 1997, it was reported that over 750 tonnes of drugs were used in animals. In comparison, use of antibiotics in humans may only have risen to 560 tonnes (Meikle & Brown, 1999; Wise, 2007). There was growing concern that the widespread and uncontrolled use of antibiotics as growth promoters could result in the development of resistant bacterial populations. In 1969, the Swann Committee was asked to report on antimicrobial use in both human and veterinary practice. The report (Swann et al., 1969) documented that there was a significant problem with increasing resistance to antibiotics and several recommendations

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were made. One suggestion was that the antibiotics used in animal feed should not be the same as those used in human medicine as this could affect efficacy in humans. Accordingly, discussions about restricting the use of antibiotics in feedstuff started. It was quite a long time before the first growth promoters were banned by the European Union (EU) because they were very similar to drugs used in human medicine. This concept was continued and resulted in EU legislation: from the beginning of 2006 the use of antibiotics as growth promoters was banned. This decision has stimulated discussion about the use of probiotics as alternative growth promoters in farm animal feed. One of the big advantages of probiotics is that their principal function is based on benefit for the animal. At the moment more than 20 probiotic preparations are authorised as feed additives in the EU, most of them containing only a single strain while some are mixtures. The most common microorganisms are different strains of bacilli, enterococci, lactobacilli or yeast. The majority have authorisation for use in more than one animal category. Only a very small number of probiotics are already authorised under Regulation (EC) No. 1831/2003 (see section 15.5). Therefore a real market change may be expected after the end of the transition period, in 2011. The following paragraphs highlight some examples of market entrance of probiotic feed additives in different countries. These examples are based on reports obtained from three long-term insiders in the animal feed business. In the 1970s high prices for feed proteins led to the worldwide soy protein crisis, which resulted in a struggle for feed in animal production. This branch of the agriculture suffered from the high prices which resulted in a battle for financial survival. An intense search for alternative nitrogen sources began. In the case of feeding ruminants, it was known that special bacteria in the rumen can utilise the nitrogen of ammonia cleaved from urea to metabolise bacterial protein, which can be further utilised by the host animal. When feeding urea to pigs, the cleaved ammonia killed the animals quickly. Because of this, it was concluded that nitrogen in the form of urea could act as an adequate nitrogen source for ruminants but not for monogastric animals. However, the use of urea resulted in a significant reduction in plant protein needs. This raised the question of how it would be possible to use this plant protein saving effect found in ruminant animals for monogastric animals. In an interesting experiment, urea was fed in combination with ruminant bacteria to pigs for fattening and poultry. In fact this was one of the first attempts to use directly fed microbials to achieve positive effects with animals. However, neither scientifically based reports nor detailed information are available on these trials. From an experienced American specialist who has been working in the feed industry since the 1970s, we learned that the US feed industry started to use beneficial bacteria in the late 1970s. The history of feed probiotics in the United States is rather short. At this time the key company in the feed business in the United States was Nulab. This company produced a sort of feed additive enriched with lactic acid bacteria, which was produced via a wet mash fermentation process, similar to an ensiling process, using a complex mixture of plant material as substrate. In 1977 Nulab was merged with one of the big players in the agricultural business, who extended its microbiological activities. The development of better drying technologies gave more opportunities to reduce the mass of the material and to transport the material for greater distances. The bacterial strain first claimed to be an active ingredient was a Lactobacillus acidophilus, which may have been identified at the start of the process but could not be enriched during production. Practically, Enterococcus faecium was one of the dominant strains and was also claimed to be responsible for exerting beneficial effects. This development can be seen as the birth of the first commercially available probiotic feed in

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the United States. Along with the gradual improvements in drying technology, and especially the technology of lyophilisation, the first products were exported to Europe in the late 1970s. Another example concerns endospore-based probiotics in animal nutrition. In Japan, the use of microbial fermentation processes has a long tradition, for example sake, a traditional Japanese alcoholic beverage, is fermented from rice. Numerous bacterial species have been isolated and identified and their specific properties and potential benefits investigated in many Japanese studies. At a time when antibiotics were very popular ‘performance enhancers’ in Western Europe and the United States, Asian countries had already discovered the advantages of feeding products containing live microorganisms. In general, it may be assumed that the probiotic concept was always much more common in these countries. In 1978, data from a research project which intended to systematically screen for endospore-forming Bacillus strains from different samples of Japanese soil were published by Kozasa (1978). The isolates were analysed for their efficiency and safety in different types of animals. This search finally resulted in a product based on spores from a specific strain of Bacillus toyoi. This product has been successfully sold on the Asian market for several decades. Japanese scientists focused specifically on endospore-forming species, on the one hand being aware that microorganisms used as feed additives must be efficient and harmless and, on the other, stable during feed processing. The spore has a low water content and is naturally protected by a very resistant coating. In its resting phase there is no metabolic activity within the cell, which does not change as long as no moisture has access. As a result of this natural protective mechanism, spore-forming microorganisms are suitable for use in mixed feed. In the processes commonly used to produce mixed feed, like blending and pelletising, different physicochemical factors like heat, shearing force, moisture, pressure and soluble salts might all have a negative impact on the microorganisms. When selecting a microorganism as a probiotic, it is essential that the microorganism survives in the feed until finally reaching the intestine. Only living organisms are able to show a positive effect on the animal’s health and performance. Spores are persistent forms of Bacillus species. When fed to the animal, the spore has access to water and it starts to germinate in the mash. The vegetative cell, like other probiotic strains, is then a transient part of the gut flora. The probiotic microorganisms contribute to the balance between the different strains in the gut. This helps to improve the stability of the gut flora and avoids the development undesired microbes. The product described above, based on spores of B. toyoi, was brought to the mid-European market in the 1985/86. At this time, there was no uniform European legislation for probiotics: the different countries within Europe had different laws for national authorisation of such products. Although many things have changed since then, the B. toyoi product is still on the market, nowadays across the EU and no longer restricted to single countries. The authorisation process for probiotics as feed additives and what requirements have to be fulfilled is discussed later in this chapter.

15.2

FEED PROBIOTICS VERSUS FOOD PROBIOTICS

An increasing number of probiotic foods and drinks for human consumption have become available in the last few years. Because of the growing popularity of such products and the still insufficient scientific body of knowledge regarding efficacy and safety, the World

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Health Organization (WHO) and the Food and Agriculture Organization (FAO) started cooperation in 2001 in order to develop guidelines for the evaluation of probiotics in food. The Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food including Powder Milk and Live Lactic Acid Bacteria provides a list of the beneficial uses of probiotics in human nutrition. Recent advertisements constantly try to encourage healthy people to use probiotic products to remain healthy and to potentially reduce the long-term risk of some diseases. However, there are some problems with this assumption; in particular, the definition of ‘healthy’ is difficult, and thus more clinical studies with healthy subjects need to be performed. In the feed industry, probiotics are mainly marketed as feed additives under the category ‘zootechnical additive’ in the functional group of gut flora stabilisers. For producers it is seldom possible to base an authorisation according to Regulation (EC) No. 1831/2003 solely on health claims, although ‘improved well-being’ of the animals can be found as one criterion for authorisation. The problem is how to assess the well-being of animals; for authorisation, significant and measurable benefits are needed. It is therefore common practice to measure performance parameters as the methods of measurement and therefore argumentation of improvement are broadly accepted. For probiotics and health claims in the food industry the situation is different, as it is based on the Regulation (EC) No. 1924/2006. Safety is one of the major concerns of the European authorities with regard to products containing or consisting of living microorganisms. Thus great efforts have to be made to show the safety of the product for feed as well as for food probiotics. For feed probiotics, a tolerance trial with the target animal category has to be performed. Normally, for microorganisms a 100-fold level of the proposed maximum dose is applied; if only a 10-fold level is used, additional parameters have to be measured. If the strains used do not have ‘qualified presumption of safety’ (QPS) status, a full safety assessment is required, which requires producers to perform toxicity studies that are the same, or at least very similar, to those used for pharmaceutical products. Toxicity studies include tests for: ● ● ● ● ●

acute toxicity; genotoxicity; subchronic oral toxicity; chronic oral toxicity/carcinogenicity; reproductive toxicity including teratogenicity.

If these studies are not sufficient, other additional studies could be requested. The tests have to conform to OECD principles and standards of good laboratory practice, implying that only a limited number of laboratories in Europe can perform the tests. The QPS system is the counterpart to the US system of ‘generally recognised as safe’ (GRAS). However, there are some differences concerning single species between the two systems. The GRAS system applies to food additives in general, and microorganisms and other food ingredients that are derived from microorganisms are also included. To obtain a GRAS status, either a history of safe use in food prior to 1958 must be presented or scientific procedures have to be passed, which is quite similar to obtaining authorisation as a food additive. The general recognition of safety must be based on the views of experts qualified

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in safety evaluation. In the United States not only the Food and Drug Administration (FDA) can grant GRAS status but also independent experts. The first attempt to establish the European QPS system was started in 2002/2003 in order to simplify the safety assessment of microorganisms used in feed and/or food. The QPS approach was introduced by the European Food Safety Authority (EFSA) in November 2007 after long and careful cooperation with many other stakeholders. In 2008 the QPS list of strains was reviewed. These reviews are planned regularly in order to account for new scientific developments that might lead to the inclusion of further species. According to the EFSA opinions on QPS (EFSA-Q-2005-293 and EFSA-Q-2008-006), microorganisms used in feed and food can be divided into four categories, although the last category is not used as a probiotic. These are described in the following four sections.

15.2.1

Gram-positive non-sporulating bacteria

The following genera, all belonging to the phylum Firmicutes, have been considered for QPS status by the EFSA: Bifidobacterium, Corynebacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Propionibacterium and Streptococcus. The individual species granted QPS status by the EFSA are shown in Box 15.1. Nevertheless, the absence of antibiotic resistance has to be demonstrated for the single strains, unless the cells are not present in the final product. This is not applicable for probiotics.

15.2.2

Bacillus species

The following species have QPS status: Bacillus amyloliquefaciens, B. atrophaeus, B. clausii, B. coagulans, B. fusiformis, B. lentus, B. licheniformis, B. megaterium, B. mojavensis, B. pumilus, B. subtilis, B. vallismortis and Geobacillus stearothermophillus. Although

Box 15.1 Gram-positive non-sporulating bacteria that have been granted QPS status by the EFSA ● ● ●

● ● ● ● ●

Bifidobacterium: adolescentis, animalis, bifidum, breve, longum Corynebacterium: glutamicum (for production process only) Lactobacillus: acidophilus, amylolyticus, amylovorans, alimentarius, aviaries, brevis, buchneri, casei, crispatus, curvatus, delbrueckii, farciminis, fermentum, gallinarum, gasseri, helveticus, hilgardii, johnsonii, kefiranofaciens, kefiri, mucosae, panis, paracasei, paraplantarum, pentosaceus, plantarum, pontis, reuteri, rhamnosus, sakei, salivarius, sanfranciscensis, zeae Lactococcus lactis Leuconostoc: citreum, lactis, mesenteroides Pediococcus: acidilactici, dextrinicus, pentosaceus Propionibacterium freudenreichii Streptococcus thermophilus

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those species have QPS status, the absence of food poisoning toxins with surfactant activity and the absence of enterotoxic activity needs to be demonstrated for the single strain.

15.2.3

Yeasts

In addition to Saccharomyces cerevisiae, S. bayanus and S. pastorianus, EFSA regards various species of Candida, Hanseniaspora, Issatchenkia, Kluyveromyces, Metschnikowia, Pichia and Schizosaccharomyces as positive contributors in the manufacture of fermented foods, dairy products, meats, cereals, coffee and sauces. The most frequently encountered and important species in dairy products are Debaryomyces hansenii, Yarrowia lipolytica, Kluyveromyces marxianus, S. cerevisiae, Galactomyces geotrichum, Candida celanoides and various Pichia species. In the case of the fermentation of meat for sausages and maturation of hams, various species of Debaryomyces, Yarrowia lipolytica and various Candida species are involved. Saccharomyces cerevisiae, S. exiguous, C. humicola, C. milleri, C. kruseii, C. orientalis, Torulaspora delbrueckii and various Pichia species are used in the fermentation of cereal products. The growth and activities of a diversity of Hanseniaspora, Candida, Pichia, Issatchenkia, Kluyveromyces and Saccharomyces species have been reported in the fermentation of coffee beans and cocoa beans. Zygosaccharomyces rouxii, C. versatilis and C. etchellsii are important osmotolerant species that play a key role in soy sauce fermentation. Moreover, baker’s and brewer’s yeasts (S. cerevisiae) have been available for many years as dietary supplements because of their high content of B-group vitamins, proteins, peptides, amino acids and trace minerals. In summary, some species of the genera Candida, Debaryomyces, Hanseniaspora, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces and Xanthophyllomyces are included in the QPS list as the most relevant and commonly encountered yeasts

15.2.4

Filamentous fungi

Microorganisms of this group are very diverse and used for quite different purposes. None is used as a feed or food probiotic. No member of the group fulfils all the requirements for inclusion on the QPS list.

15.3

EFFICACY

In human nutrition the focus of probiotic products available in supermarkets in the form of probiotic yoghurts, drinks or other products is better health and well-being. These everyday products should not be mixed with probiotics available only in pharmacies, for example in the treatment of diarrhoea. The problem with these everyday probiotics is assessment of their efficacy: it is hard to define measurable endpoints for ‘well-being’. In proving the efficacy of food probiotics, the requirements grant producers some degree of freedom. On the other hand, feed probiotics are very strictly regulated and specific efficacy parameters are defined, although the main requirements are related to safety of the product. The problem of defining ‘well-being’ is one reason why the feed industry concentrates on performance parameters like weight gain, which can be easily measured and which are the most relevant factors for farmers. The requirements for proving the efficacy of feed products are exactly defined.

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FEED PROBIOTICS Fundamentals

With farm animals and pets, probiotic microorganisms are used for prophylaxis, as growth enhancers or for therapy. Improvements in the intestinal microbial balance or of the properties of the indigenous probiotic microflora should lead to beneficial effects for the host animal (Simon et al., 2003). Probiotics used as feed additives are defined as microorganisms that have a positive influence on the performance of healthy animals. This means that treatment of a disease is not within the scope of a feed additive, but such products are otherwise regulated. For feed purposes, the living microorganisms are applied as additives to mineral feed, protein concentrates, milk replacers, etc. These components are mixed or prepared to final feed, which is then directly fed to the animal; thus they are also called ‘direct fed microbials’ (DFM). This term is used favourably in the US market but is not so common within Europe. In general, DFM are used as prophylaxis, mainly for stabilising the microbial communities within the digestive system of the animal. As a result, stable eupepsia, a condition of good digestion with efficient degradation of feed, leads to enhanced performance. Knowledge about the exact mode of action of probiotics in farm animals is still very limited and much research work must be done. Simon et al. (2003) assess that it is inappropriate to extrapolate studies about probiotics in human nutrition to animal nutrition. Because of the regulatory system of the EU, the use of probiotics as feed additives is defined for each animal species and category (i.e. pigs, poultry, bovines and others). Data from one animal category cannot, with few exceptions, be used for another category. With farm animals, the use of probiotics is focused mainly on pigs, poultry and cattle. An upcoming number of probiotics is expected to be used in aquaculture for shrimps and fish and the first requests for the application of probiotics in these categories have been submitted to the European authorities in 2008. Probiotic microorganisms are usually used at concentrations of 108–109 CFU/kg feed (Simon et al., 2003). Dosing of the microorganisms is calculated by mixing the correct amount of probiotic material into the feedstuff. Depending on the animal’s weight, a certain amount of feed is usually eaten by the animal. Calculation of the correct concentration of probiotic microorganisms for a single animal is based on the amount of feed consumed. This approach differs from the dosing of pharmaceuticals, where the calculation is based on a defined value per kilogram body weight of the animal. Depending on the category and age of the animal, the application rate can range widely, sometimes up to 1010 CFU/kg feed. These ranges imply that the concentration of the probiotic needs to be higher during production, so that the additives can be incorporated into the final feed in a feasible and realistic range. Depending on the production process, up to 1010–1012 CFU/g can be found in concentrates. In extreme cases, even amounts below 100 mg could be sufficient for 1 tonne of final probiotic feed. As a result, pre-mixtures (e.g. in form of mineral feed, supplemental feed or protein concentrates) have to be produced. These products are provided to the farmer to ensure that only homogeneous feed is prepared. In modern farming the major objective is to achieve maximum utilisation of the farm animal. Therefore, the animal’s full genetic potential for energy conversion of feed is necessary. This can only be attained by excellent livestock husbandry, optimum farm and feed management, and management of the diet. The idea behind using probiotics as natural

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growth promoters is to support the animal’s gut microflora balance and enhance the immune response. Appropriate animal health is the basis for optimum feed conversion and high performance of farm animals. The majority of the scientific experiments in the literature have been performed with pigs and piglets. Especially in growing weaned piglets, there is high demand for improvment in growth performance and reduction in the incidence of diarrhoea. Because the conversion of feed in the bovine intestine is generally more complex than in monogastric animals or in poultry, the use of probiotic products also differs. The special digestive system of cattle is the main reason why probiotics, primarily yeasts, are used in these animals. Their mode of action is principally via rumen fermentation. Studies have shown that probiotic yeasts are metabolically active in the rumen and small intestine after ingestion but their numbers decrease in the lower sections of the intestine. One very important activity of live yeasts is their ability to consume oxygen. This causes a reduction of oxygen in the rumen, which can positively influence its ecosystem and so enhance the growth of relevant bacteria. The main effect of yeasts might be an increase in general bacterial numbers in the rumen and result in better degradation of fibre (FEFANA, 2005). The situation is different when feeding pre-ruminant calves, where the gastrointestinal tract is similar to that of monogastric animals. Here bacterial probiotics are used. Probiotic additives are known to have a generally positive influence on the intestinal flora in animals. However, according to EU regulations intended to protect consumers from products with no proven benefit, the efficacy of probiotic products has to be demonstrated by conducting feeding trials. The prime interest for the farmer, as user of the probiotics, is the impact on the performance of farm animals. The aim of the probiotics is to produce better performing animals through improved well-being. Measurement of well-being is not easy. The term is generally defined as a state of happiness, good health and/or prosperity or welfare. Swanson (1995) suggested three types of definition of animal welfare: legal, public and technical. ● ● ●

Legal definitions are influenced by legislators, who look to establish minimum standards that are accepted by society and which are a useful tool for lawyers. Public definitions are the result of the public’s knowledge, empathy and activism toward animals. Scientists focus on the technical definitions, since they are based on measures. A slightly different approach distinguishing other broad criteria to indicate animal well-being was described by Fraser (1993). One of these criteria is defined as ‘a high level of biological function’, like daily weight gain, feed uptake and feed conversion.

Animal scientists are most familiar and comfortable with this physiological approach. Biological measures have formed the basis of many studies of animal well-being by using models that detect changes in productivity (Curtis & Stricklin, 1991). Generally speaking, it is necessary to show significant differences between animals fed with probiotics and untreated animals. The most commonly used measurement is daily weight gain, which describes the increase in liveweight per day and is expressed in kilograms per day per animal. It is used mainly in the fattening of different categories of animal. The efficient growth of animals is indicated when a higher daily weight gain is measured in the group treated with probiotics than in the control group. Of course, not only weight gain but also meat quality needs to be monitored, because only a gain in high-quality meat provides an advantage for the user of probiotics.

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It is undesirable to obtain only a higher weight gain because the meat then has a higher water content. Seelig (2007) defined an increase in weight gain as a secondary effect caused by stabilisation of the intestine microbial eubiosis. Nevertheless, most companies monitor weight gain itself since it is much easier to measure than stabilisation of the eubiosis. A second important parameter is the daily feed intake, expressed in kilograms feed per day per animal. In healthy fattening animals, a higher daily feed intake usually results in a higher daily weight gain. In animals used for breeding the situation is different as the target is improvement in reproductive parameters. In order to quantify the relation between these two parameters, the feed conversion ratio is calculated. It is a measure of the efficiency in converting feed mass into body mass. Therefore it is a relative dimensionless parameter, calculated in kilograms feed per kilograms body weight. A good feed conversion ratio indicates that there is less feed needed to yield the same amount of meat. The feed conversion ratio differs between categories of animals. The range for pigs and poultry is 1 : 1.5–4 and for cattle 1 : 6–7. High-performance animals show a low feed conversion ratio, indicating that less feed is needed to produce the same amount of meat. These three parameters are the most commonly measured ones and the values are of great economic importance for farmers. Realistic improvements have been reported by different authors (Gunther, 1994; Kumprecht & Zobac, 1998; Simon et al., 2003). For piglets, the increase in daily weight gain ranged from 2 to 10%, with an average value of 7%, and the feed conversion ratio could be reduced by 0–8% (average 2.4%) in most trials. A quite similar conclusion was shown in experiments with chickens for fattening. The improvement in daily weight gain ranged from 0.1 to 3.8% (average 1.5%), with a reduction in the feed conversion ratio of 0.7–3.5% (average 2.0%). Depending on the animal category, endpoints other than the ones described above can be used to demonstrate the efficacy of a probiotic additive. Especially in young animals like piglets or calves, a reduction in diarrhoea leads to an improvement in animal health and results in reduction in animal losses. The effects of probiotics in laying hens might be a higher number of eggs or better egg quality, such as egg size, thickness of shell and weight of yolk. With regard to the details of the intestinal flora of animals, the number of bacteria in the intestine or the faeces of animals can be compared. It is important to remember that probiotic organisms should not permanently colonise the intestine. The microbes should adhere to the gut mucosa for a certain time but should generally be only transient. On the one hand, an increase in ‘beneficial’ microbes, such as introduced probiotic strains or all lactic acid bacteria, can be detected; on the other, a decrease in potentially pathogenic microorganisms, for example enterobacteria, can be a positive effect. Bacterial counts in the faeces can be assessed rather easily but there is some discussion as to the extent to which this number reflects the situation in the intestinal tract. Analysing samples directly drawn from the gut of animals requires invasive methods. If the endpoint of a trial is slaughter of the animal, it is possible to measure microbial counts in the intestine, but there are also other feeding trials where animals are not slaughtered afterwards (e.g. feeding trials with sows). Parameters investigated during feeding trials with sows include the beneficial effects on the reproductive parameters of the sow (e.g. less weight loss after birth, number and weight of newborn piglets) and the beneficial effects on the piglets (e.g. daily weight gain or health of weaning piglets). For pets and companion animals, the situation is rather different compared with farm animals. People nowadays have a very close relationship to their cats and dogs, and pets are often seen more as ‘family members’ than just animals. The reason for feeding probiotics

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to pets is therefore different from feeding probiotics to farm animals. The farmer basically wants to save money (lower feed costs for the same number of animals or amount of produced meat, healthy animals without high veterinary costs, etc.), whereas individuals are willing to spend a lot of money on their pets. For farm animals, performance parameters are most important. For pets, improved well-being and health status is the desired consequence. It has already been mentioned that performance parameters are the easiest measurement, but this is usually not useful for pets. Nobody buys a product for a dog where the main message of the advertisement is ‘better and higher weight gain’. However, with regard to the number of pets that are obese, the message might be better reversed. These ideas might give an indication why only very few products are authorised as feed additives for pets. It should be mentioned that probiotic products cannot only be sold as feed additives, but also in other forms (e.g. as a pharmaceutical product). So if performance parameters are not an option, how should the efficacy of probiotics be measured in pets? One possible way is to measure the immunomodulatory properties of probiotics. The immune system is highly complex and it is outside the scope of this chapter to provide an overview of this fascinating topic. What can be summarised here is that the determination of characteristics of the immune system is much more difficult than simple weighing of the animal. Usually blood samples are needed, causing some stress and inconvenience to the animal. Advanced laboratory techniques and special equipment are needed to differentiate between subpopulations of cells of the immune system or to measure the level of immunoglobulins. Despite those problems, it has already been proven in different studies that probiotic microorganisms can influence one or several components of an immune response (humoral, cellular and non-specific immunity). The main components of the specific immune system are T cells and B cells. B cells produce immunoglobulins (IgM, IgG, IgA and IgE) that mediate the body’s defence against antigens (e.g. viruses, bacteria) and which belong to the humoral immune response. With regard to the cellular component of the immune response, a higher activity of macrophages could be measured after feeding of probiotics (Altherr, 2006). In another study it could be shown that probiotic bacteria, here a strain of Enterococcus faecium, stimulated immune function in young dogs (Benyacoub et al., 2003). Faecal IgA levels and canine distemper virus vaccine-specific circulating IgG and IgA were higher in the group receiving the probiotic than in controls. Recently, other trials with dogs came to the conclusion that probiotic feed supplementation (another strain of Enterococcus faecium) leads to higher stimulation indices of lymphocyte proliferation with three different mitogens (Koiou & Zentek, 2010; Kröger & Zentek, unpublished trial data, 2009). Furthermore, the antibody titre was measured after vaccination and significantly higher titres against rabies and also against canine distempter virus were measured in animals receiving probiotics. Bacterial counts in the intestine or faeces of animals can also be compared in pets, as already described for farm animals. The regulatory system for feed additives is more focused on farm animals, although authorities seem to have recognised these developments. In a recent EFSA opinion (EFSA, 2008) the issue of possible new categories of feed additives is discussed, one of them being ‘immunomodulators’. This might be an opportunity to use probiotics not only for pets but also to extend the definition from the present ‘gut flora stabiliser’, with which most probiotics are categorised at the moment. However, this EFSA proposal might also be controversial because the border between probiotics and pharmaceutical products would become blurred. Products registered as feed additives in the EU and therefore included on the Community Register of Feed additives are listed in Table 15.1, as well as the animal categories for which an authorisation exists.

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Table 15.1 Probiotics registered as feed additives in the EU (list is not exhaustive). Product

Microorganism

EU Numbera

Animal categories

Toyocerin®

Bacillus cereus var. toyoi

1 E1701

Piglets, pigs for fattening, sows, chickens for fattening, laying hens, turkey for fattening, cattle for fattening, rabbits

Biosaf®

Saccharomyces cerevisiae

3 E1702 4b1702

Sows, piglets, dairy cows, cattle for fattening, horses, dairy goats, dairy sheep, lambs for fattening, rabbits for fattening

Yea Sacc®

Saccharomyces cerevisiae

5 E1704 4a1704

Calves, cattle for fattening, dairy cows, horses

Levucell SB®

Saccharomyces cerevisiae

6 E1703

Sows, piglets

Levucell SC®

Saccharomyces cerevisiae

7 E1711 4b1711 4d1711

Dairy cows, cattle for fattening, horses

Probios PDFM®

Enterococcus faecium

8 E1709

Chickens for fattening

Fermaid PA®

Pediococcus acidilactici

9

Chickens for fattening, pigs for fattening Salmonids, shrimps

E1712 4d1712 Cylactin®, LBC®

Enterococcus faecium

10 E1705

Chickens for fattening, piglets, pigs for fattening, sows, calves, cats, dogs

Biacton®

Lactobacillus farciminis

12 E1714

Piglets, chickens for fattening, laying hens, turkeys for fattening

Oralin®

Enterococcus faecium

13 E1707

Piglets, calves, chickens for fattening, turkey for fattening, dogs

Biosprint®

Saccharomyces cerevisiae

14 E1710

Dairy cows, cattle for fatteing, piglets

Lactiferm®

Enterococcus faecium

15 E1708

Piglets, calves, chickens for fattening

Provita LE®

Lactobacillus rhamnosus, Enterococcus faecium

E1706

Piglets, calves

Fecinor Plus®

Enterococcus faecium

18 E1713

Piglets, chickens for fattening

Bio Plus 2B®

Bacillus subtilis, Bacillus licheniformis

20 E1700

Piglets, sows, pigs for fattening, turkey for fattening, calves

Biomin IMB 52®

Enterococcus faecium

4b1850

Chickens for fattening

Bonvital®

Enterococcus faecium

22 4b1841

Piglets, pigs for fattening, sows, chickens for fattening

L. acidophilus D2/CSL

Lactobacillus acidophilus

23

Laying hens (cont’d)

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Table 15.1 (cont’d) Product

Microorganism

EU Numbera

Animal categories

MLB®

Lactobacillus acidophilus

25

Cats, dogs

Turval B0399

Kluyveromyces marxianus-fragilis

26

Piglets

Calsporin®

Bacillus subtilis

4b1820

Chickens for fattening

Bacillus subtilis O 35

Bacillus subtilis

4b1821

Chickens for fattening

Ecobiol®, Ecobiol Plus®

Bacillus amyloliquefaciens

4b1822

Chickens for fattening

Sorbiflore®

Lactobacillus rhamnosus and Lactobacillus farciminis

4d2

Piglets

a There are different numbering systems due to changes in the regulatory system. The current numbering scheme indicates the category and functional group of the additive, e.g. ‘4b’ for ‘zootechnical additive’ (4) and ‘gut flora stabiliser’ (b). Some products based on living microorganisms are not, or not exclusively, categorised as ‘Gut flora stabilisers’: two products are authorised as ‘Digestability enhancers’ (4a) and two others as ‘Other zootechnical additives’ (4d). Other numbers like ‘22’ or ‘E1706’ are an indication for authorisation according to former regulations. Many additives have not yet obtained an authorisation according to the latest EU regulation (EC) No. 1831/2003.

As the authorisation process is time-consuming and expensive, the number of products is not changing very fast, although there will be more changes after 2011. Changes in the animal categories are much more frequent, because it is easier to prove efficacy and tolerance in another species than obtaining authorisation for a completely new product. For an extension to another category, much existing data (e.g. identity of the strain, stability of the product) can be extrapolated from the previous authorisation. Table 15.1 shows that most probiotic products contain only one single strain of microorganism. This is partly due to the increased efforts for multistrain products requested by the current regulations. Table 15.1 also shows that different types of microorganisms are used for probiotic products. Basically, they can be divided into three groups, lactic acid bacteria, Bacillus spores and yeasts, all of which have advantages and disadvantages (FEFANA, 2005). The different characteristics of the diverse organisms are compared in Table 15.2 and described in detail above.

Table 15.2 Categories of microorganisms used as probiotics. Lactic acid bacteria

Bacillus spores

Yeast

Natural habitat

Gastrointestinal tract Plants

Soil

Fruits

Formation of biofilm

Yes

No

No

Metabolic product mainly produced

Lactate, acetate

CO2/H2O

CO2/H2O Ethanol

Resistance to environmental condition

Weak

Strong

Weak

Immunomodulation

Yes

Yes

Yes

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235

Lactic acid bacteria

Lactic acid bacteria are a diverse group of Gram-positive acid-tolerant rods or cocci that have specific metabolic and physiological characteristics. They are generally non-sporulating and therefore not as heat-tolerant as the spores of the Bacillus group. One natural origin of lactic acid bacteria like enterococci, streptococci or lactobacilli is the gastrointestinal tract of different animals. Strains used in products on the market are mainly isolated from this natural habitat. This has a huge advantage for establishment of the microorganism in the intestinal tract, since a strain isolated from this environment can cope with the conditions in the intestinal tract quite well. It should become active in an environment very similar to where it was originally isolated from. All lactic acid bacteria used as probiotics must show active growth in the gut to be a useful product. In vitro experiments have revealed several mechanisms of action in lactic acid bacteria, some of which are mentioned here. As the name states, lactic acid bacteria can produce acids, mainly lactic acid, by fermentation using certain types of sugars. These acids, and in certain cases also additional metabolic products, can act as antimicrobial substances against antagonistic microorganisms in the intestine. Another characteristic is the formation of a biofilm that protects the intestinal mucous membrane and leads to the exclusion of potentially pathogenic microorganisms. When pathogenic microorganisms are restricted in growth, their production of toxins may also be suppressed. Another beneficial effect of the lactic acid bacteria on the host animal is the strengthening of non-specific immunity. Stimulation of the local immune system in the intestine helps to improve the general health of the animal. Some influence of lactic acid bacteria on the physicochemical conditions in the intestine, for instance pH and redox potential, also helps to limit the growth of undesirable microorganisms. 15.4.1.2

Bacillus spores

The group of bacilli comprises a wide range of Gram-positive rod-shaped bacteria living under strict or facultative aerobic conditions (Schlegel, 1990). Bacilli are naturally found in soil, some are implicated in food poisoning, while others show strong enzymatic activities. Some species within this heterogeneous group have been found to have positive effects when fed to animals. All Bacillus species have the ability to form highly resistant endospores. They are therefore used as spore preparations, showing good stability against unfavourable environmental conditions like heat or chemicals. Probiotic microorganisms without heat stability are a problem especially in the production of poultry feed. In most countries poultry feed is pelleted at temperatures up to 100°C in order to reduce pathogens like Salmonella spp. (Simon et al., 2003). The good heat resistance of spores is advantageous; conversely, it is difficult to eliminate all spores from equipment, surfaces and machinery by common cleaning procedures. 15.4.1.3

Yeasts

Yeasts of the genus Saccharomyces normally grow on plant material but do not naturally occur in the digestive tract. Selected yeast strains have been used for food production for centuries, such as bakers yeast or for making alcoholic beverages. Probiotic products comprising yeasts are preferably used for ruminants. One of the beneficial effects is that yeasts consume oxygen in the rumen and therefore create more favourable conditions for

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the growth and activity of rumen microorganisms, which are strictly anaerobic. Traditionally, yeasts are also regarded as suppliers of vitamins.

15.4.2

Industrial production

Before a probiotic strain can be beneficial for farm animals, it needs to be manufactured under industrial conditions and retain viability during storage. Finally, the bacteria must survive the gastrointestinal system in sufficient numbers to be able to retain functionality in the intestine (Mattila-Sandholm et al., 2002; Saarela et al., 2000). All the different factors affecting viability need to be considered in the selection process and during the whole production process.

15.4.2.1

Selection process

Basic properties for the selection of probiotic microorganisms include efficacy and safety as well as functional and technological aspects. Even if a microorganism possesses the required characteristics for safety and functionality, the aspects related to production and processing are of high importance (Saarela et al., 2000). Specifications concerning safety aspects for microorganisms used as probiotics in feed are defined in EU regulations and guidelines are described in detail later in the text. In general, probiotic strains for animal feed are preferably of animal or human origin. This should be taken into account when starting the selection process. The isolation of potential probiotic strains from gastrointestinal material and the health status of the animals need to be documented. The selected species should have a history of being non-pathogenic and not associated with diseases like infective endocarditis or gastrointestinal disorders and should not carry transmissible antibiotic-resistance genes (Saarela et al., 2000). The functional requirements mainly describe the properties of the bacteria necessary to enable establishment in the host organism. This includes tolerance to acid, gastric juice and bile. Also important is the ability to adhere to gut mucosa for limited persistent establishment in the host organism, as well as the ability to be washed out of the gut. Additionally, functionality describes actual probiotic efficacy, which may include, for example, immunostimulation or antagonistic activity against pathogens. Within the selection process these characteristics are usually investigated by the use of in vitro methods. Other major features are several technological properties. For each bacterial strain, the production process needs to be optimised in order to enhance good growth and viability. Good stability of the bacteria in the product during storage is mandatory. This requirement results in significant challenges for the production of probiotics, especially for those organisms which have an intestinal origin. Some scientists (Lacroix & Yildirim, 2007) state that the selection of commercial strains is largely based on technological properties.

15.4.2.2

Production process

Economically competitive production of probiotics for the feed industry is carried out on a large scale. The efficiency of production depends on the yield of viable cells at the end of a multistep process. Cells are susceptible to environmental stresses such as acidity, heat, oxygen and shearing forces during fermentation and downstream processes. Optimisation of the process has to meet the requirements of the different strains. Depending on the manufactured microorganism (lactic acid bacteria, yeasts, bacilli),

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Table 15.3 Production steps and overview of the factors affecting viability of probiotics. Fermentation Composition of growth medium Toxic byproducts (organic acid, H2O2) Dissolved oxygen Final cell mass Downstream process Mechanical stress Composition of freezing and drying media Extreme temperature (spray-drying, freeze-drying) Oxygen stress Cell dehydration (intracellular osmotic) Storage Acidity of carrier feed Oxygen stress Temperature Moisture content Mineral content in product Gastrointestinal tract Acidic conditions in stomach Enzymatic activities Environment (e.g. fermentable sugars) Bile salt in the small intestine Source: Modified from Lacroix and Yildirim 2007.

there are some differences in the process. In general, the production process can be divided into the following steps. ● ● ● ●

Strain maintenance and strain control: genetic stability. Fermentation: pre-cultures to main culture. Downstream processing: concentration, drying, coating. Finishing process: milling, blending, storage, formulation.

Several factors affect the viability of probiotic bacteria until they reach the target site in the host. The most important ones are listed in Table 15.3. Cell bank: maintenance and control of strains For companies, the maintenance of their unique microbial strains is of the utmost importance, as these strains are the fundamental basis of the production process. A common system is the use of a master cell bank and a working cell bank. It is common practice to keep the strains either deep-frozen in liquid nitrogen at −179°C or in a freezer at −80°C. The master cell bank is made directly from a proven sample containing the genuine strain and state-of-the-art effort is made to prove identity. The master cell bank is designated for long-term storage of a production strain and usually contains sufficient units to serve as seed for the production of working cell banks for decades. Only a few proliferation steps are required between the master cell bank and the working cell bank. The working cell bank in general holds enough units for at least 1 year of manufacturing. For each new

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Primer 1

Primer 2

Primer 3 P2

P1

W

M

Marker

P2

P1

M

W

Marker

P2

P1

M

W

Fig. 15.1 Example of an RAPD-PCR profile.

production batch, one unit of the working cell bank is used as inoculum. This ensures that production is started with genetically identical material. One step in quality control is comparison of the identity of master cell bank and working cell bank. However, it is necessary to confirm the identity and genetic stability of the strain from the master cell bank to the end product. Usually, genetic stability is controlled by using suitable molecular biological methods like RAPD-PCR (randomly amplified polymorphic DNA) or PFGE (pulsed field gel electrophoresis), both methods being suitable for detecting differences between single strains as well as for showing the equality of samples. Figure 15.1 shows an example of an RAPD-PCR profile. It can be seen that different primers lead to different bands in the gel because the bacterial DNA is cut in other regions and therefore the size of the DNA fragments is not equal. No matter which primer is used, the pattern is the same for the master cell bank (M), the working cell bank (W) and the sample obtained after the production step (P). This is the evidence for genetic stability. The PFGE pattern shown in Figure 15.2 is an example of how to detect differences between different strains belonging to the same species. Additionally, commercially used microorganisms in the feed industry need to be deposited at an official strain collection. Strain collections (ATCC, BCCM/LMG, CBS, DSMZ, NCAIM, NCIMB, etc.) usually store the obtained cultures in the form of a freeze-dried powder. Only the depositor has access to the deposited strains.

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E.faecium DSM 7134

239

E.faeciumT Marker DSM 20477

[kb]

339,5 291,0 242,5 194,0

145,5

97,0

48,5 23,1

9,42

6,55

Fig. 15.2 Pulsed field gel electrophoresis of two different strains of Enterococcus faecium.

Fermentation: from pre-culture to main culture The biomass production process of a probiotic strain starts with proliferation of the microorganism in a fermentation process. The main factors affecting the viability of probiotics in this production stage include the composition of the growth medium, the final cell mass, dissolved oxygen or toxic byproducts (Lacroix & Yildirim, 2007). At present, industrial processes for probiotic culture production generally use strictly controlled conventional batch fermentation with suspended cells. Figure 15.3 gives a general schematic overview

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Bacterial strain working cell bank

Growth media

Inoculum

Fermentation

Concentration (separation, filtration)

Concentrated biomass

Addition of protectants, carriers

Drying (lyophilisation, spray drying, fluidised bed drying

Finishing processes (grinding, coating, blending)

Probiotic product

Fig. 15.3 Overview of the production process of a probiotic used in feed.

over the whole production process of probiotic microorganisms. All process steps in the fermentation are conducted under aseptic conditions. The equipment as well as the growth media need to be sterilised. A new fermentation starts with one unit of the working cell bank as inoculum for the seed stage carried out in flasks up to 5 L in capacity. The active culture of the seeding stage

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is transferred under aseptic conditions to a pre-culture fermenter. After propagation, the content of the pre-culture fermenter is transferred further to the final production fermenter. The fermentation is continued until a defined endpoint depending on the specific strain, is reached. During the fermentation, temperatures and pH values are controlled and adjusted to strain-dependent values. Temperatures may range from 25 to 42°C, and pH values between 3.5 and 8.0 are adjusted using caustic solutions like ammonia or sodium hydroxide. Oxygen-sensitive strains may need gassing with nitrogen. Downstream process: concentration, drying Probiotic feeds are usually administered as dry products. Therefore the cells have to be separated from the complex aqueous fermentation broths by centrifugation or filtration. Depending on the microorganism, the resulting concentrate is dried by lyophilisation, fluidised bed-drying or spray-drying. In order to minimise costs and maximise efficiency, fermentation and downstream processing should be regarded as an integrated process. The most efficient fermentation may not necessarily yield the optimum overall process if the required separation and drying steps are not adapted. Depending on the chosen downstream procedure, the mechanical and osmotic stress can have a highly negative influence on the recovery of the microorganism. To improve viability protectants may be added to the cell suspension before drying. They help to prevent, or at least reduce, cell injury while drying and subsequent storage. Some commonly used protectants at industrial scale are lactose, sucrose, monosodium glutamate and ascorbate (Lacroix & Yildirim, 2007). All protectants must be in accordance with the positive lists of European and national feed laws. The most commonly used drying procedures are freeze-drying, spray-drying and fluidised bed-drying. Freeze-drying (lyophilisation) Lyophilisation is a very gentle drying process, typically used to preserve perishable material or very sensitive products like proteins or living microorganisms. The principle of lyophilisation is to freeze the material and to evaporate the resulting ice at a defined vacuum in a process called sublimation. The water vapour is transported from the frozen bacterial concentrate and trapped on the ice condensers that are cooled to a lower temperature than the product. The driving forces for the sublimation process are the supply of controlled quantities of heat to the product and the temperature difference between the product and the ice condensers. The lyophilisation process leads to a substantially dry, porous product which has the same size and shape as the original frozen mass and is of good solubility. Spray-drying Spray-drying is a commonly used method of drying liquid solutions or pumpable suspensions through a hot gas and is highly suited for the continuous production of solids. Spraydrying involves the atomisation of a liquid feeding stock into a spray of droplets and exposing the droplets to hot air in a drying chamber. The sprays can be produced by either rotary or nozzle atomisers. Evaporation of moisture from the droplets and the formation of dry particles proceed under controlled temperature and airflow conditions. The hot drying gas can be passed as a co-current or counter-current flow to the direction of the particles. Despite the fact that spray-drying is more economical than freeze-drying, especially on a large scale, sensitive bacteria cannot tolerate the exposure to the process conditions involving relatively high temperatures.

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Fluidised bed-drying Fluidised bed-drying is an ideal process for the drying, agglomeration, granulation and coating of a wide range of products. For the drying process, a liquid microorganism concentrate is sprayed onto a carrier in the fluidised bed. This allows the formation of a product with a defined particle size. The fluidised state is created by a gas (usually air) that is passed through the product layer under controlled velocity, temperature and humidity conditions. For the coating process, in a second step a liquid coating material is sprayed onto the dried product under strictly controlled conditions to form a protective film. Fluidised bed coating can increase the stability of different probiotic products for use in animal feed. Storage and formulation For use in the feed industry, the dried probiotic products may have to be standardised to a particular concentration according to the authorisation. Depending on the drying method, the products therefore have to be milled and blended with carrier materials. The viability of probiotics in different feedstuffs depends on various factors like storage temperature, water activity, pH and the presence of inhibitors (e.g. mineral salts). The stability of sensitive strains can be improved by using microencapsulation and coating technologies. The ready mixed product is usually packed airtight and stored at a controlled, if necessary cool, temperature. Correct storage conditions are important for the quality and viability of the probiotic product.

15.5

AUTHORISATION PROCESSES

Before the 1980s probiotics were not regulated across the EU but rather on a national basis. For example, in Austrian law in 1984 the category of ‘microbial growth enhancer’ was included within the regulations for feedstuffs for the first time (Hammerer, 1990). Therein it allowed microorganisms to be used alone as a growth enhancer or in combination with antibiotic growth enhancers. By 1989, seven products were registered for the Austrian feed market, including one biological concentrate of living and stabilised microorganism from rumen, four products with Streptococcus faecium, spray-dried or lyophilised, one mixture of lactic acid bacteria, and one product including Bacillus toyoi. In order to place a probiotic on the European market today it is necessary to obtain an authorisation, where in principle the quality, safety and efficacy of a probiotic has to be proven. This requires many different scientific studies which make the authorisation process of a probiotic time-consuming and expensive. Depending on the animal categories and the probiotic microorganism itself (see QPS status, section 15.2), the costs for the whole authorisation process may exceed several hundred thousand Euros. In the EU, probiotics used as feed additives, in the sense of microorganisms that are not genetically modified, are regulated under Regulation (EC) No. 1831/2003 on additives for use in animal nutrition. This regulation lists the following categories and respective functional groups of feed additives. ●

● ●

Technological additives: preservatives, antioxidants, emulsifiers, stabilisers, thickeners, gelling agents, binders, substances for control of radionuclide contamination, anticaking agents, acidity regulators, silage additives, denaturants. Sensory additives: colourants, flavouring compounds. Nutritional additives: vitamins, compounds of trace elements, amino acids and derivatives, urea and its derivatives.

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Zootechnical additives: digestibility enhancers, gut flora stabilisers, substances which favourably affect the environment, other zootechnical additives.

●

Probiotics belong to the category ‘zootechnical additives’ (4) and therein to the functional group ‘gut flora stabilisers’ (b), definded as ‘microorganisms or other chemically defined substances, which, when fed to animals, have a positive effect on the gut flora’. Regulation 1831/2003 only provides a very general framework but no details on how to prepare the documents for the authorisation of a probiotic in the EU. In April 2008, a new regulation (EC) No. 429/2008 was published. This document was very much anticipated by all probiotic producers as it contains ‘detailed rules for the implementation of Regulation (EC) No. 1831/2003 of the European Parliament and of the Council as regards the presentation of applications and the assessment and the authorisation of feed additives’. Before 2003, probiotics needed an authorisation to be offered on the market. With the former directive 70/524/EEC it was possible to obtain an ‘authorisation without time limit’. With the coming into force of Regulation 1831/2003, this term ‘without time limit’ is no longer valid as there are higher requirements on the probiotic compared with former regulations. Thus these products need to be re-authorised according to Regulation 1831/2003 and documents must be submitted. This might be a challenge, as a huge amount of applications will be submitted to the EFSA in November 2010. All probiotics legally on the market are listed on the Community Register of Feed Additives, either as ‘Microorganisms’ (for all products still having an authorisation according to 70/524/EEC) or under category 4, group b (for all products already authorised according to Regulation 1831/2003). This register is regularly updated and available for the public online. From an industry point of view, there are three different institutions involved in the authorisation process (Fig. 15.4). I

II

III

European Food Safety Agency: this authority is tasked with the risk assessment of food and feed products. It is a relatively new authority founded in 2002, which followed the former Scientific Committee for Animal Nutrition (SCAN). The EFSA receives the whole technical dossier and scientific documentation from the applicant and publishes an independent scientific opinion. Currently, there are 11 scientific panels within EFSA. Feed additives are the responsibility of the FEEDAP Panel (Panel for Additives and Products or Substances Used in Animal Feed). European Commission: the applicant sends a request for authorisation to the EC, which in turn gives an official mandate to evaluate the product to the EFSA. After the EFSA opinion is published, the Commission prepares a draft regulation to grant authorisation, following the procedure involving member states within the SCFCAH (Standing Committee on the Food Chain and Animal Health) within the subgroup for Animal Nutrition. Community Reference Laboratory for Additives for Use in Animal Nutrition (CRL for Feed Additives): this institution assists the EFSA in the scientific evaluation of the technical dossier. The Joint Research Centre (JRC) as the CRL for Feed Additives and its Institute for Reference Materials and Measurements (IRMM) are, besides other tasks, responsible for evaluation of the analytical methods submitted in connection with an application. Details can be found in Commission Regulation (EC) No. 378/2005, last modified by Commission Regulation (EC) No. 850/2007. From an industry point of view, the CRL is important because it is not possible to complete the

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Applicant

Application

European Commission

Technical dossier + CRL statement

Reference samples + fee

EFSA

CRL

Officially forward the application

Acknowledgement of receipt (15 days) Completeness check of dossier (30 weekdays)

Dossier complete?

Yes

Publication of summary

No

Scientific evaluation of dossier (6 months)

Supplementary information necessary?

Preparation and adoption of the EFSA-opinion

Communication to European Commission

Request missing information from applicant

No

Yes

Report (concerning analytical method)

Request information from applicant → extension of 6 months

Publication of EFSA-opinion

Draft of EUregulation for authorisation

Translation of regulation into all languages

Publication of EU-regulation

Entry into Community Register of Feed Additives

Fig.15.4 Overview of the authorisation process for feed additives in the EU.

technical dossier for the EFSA before having a statement from the CRL that it received samples of the feed additive in sufficient quantity, which depends on type of additive, and that the fee is paid. Because the authorisation process is sophisticated, there are some organisations that support their members in regulatory issues. One example of such an organisation is FEFANA Asbl, the EU association of feed additives and premixtures operators. Founded in 2004, FEFANA is the successor to the Feed Additives Producers Association, which was founded in 1963. In summer 2008, nearly 100 companies were members of the organisation, which presents itself as the interface between industry and the regulatory authorities in the EU. It takes a lot of time for a probiotic to be authorised and be placed on the EU market. Some steps in the authorisation process have a time limit, for example the EFSA has, in principle, 6 months to publish their scientific opinion. However, because as soon as there is

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a question to the applicant, the clock is stopped, meaning that the time the applicant needs to answer the question is not included in this 6-month period. As some of the questions require further trials to be submitted, it can take years until EFSA finally comes to an opinion. The foundation of the EFSA as the risk assessor is advantageous for applicants because the questions are normally submitted all at once. In the former system, all the different member states of the EU could request additional information from the applicant, sometimes causing more delay, and it was more difficult to bundle the necessary information. Other steps in the authorisation process do not have a specified time limit, and often these are the steps resulting in delays that can hardly be foreseen by the applicant. Examples include the time the EU Commission needs to forward the application to the EFSA at the beginning of the authorisation process and the time between publication of the scientic opinion of EFSA and publication of the corresponding EU regulation, which is the basic requirement to launch the product on the market. In conclusion, it can be stated that the authorisation process typically takes several years. As soon as a microorganism is genetically modified, other additional regulations need to be followed, but currently no genetically modified organism is used as a probiotic feed additive in the EU and included on the Community Register of Feed Additives. In order to harmonise the safety assessment of microorganisms for applicants, the QPS concept was developed. A microorganism with QPS status is freed from the need for further safety assessment, whereas those not QPS classified are subject to a full safety assessment. Some safety studies, like resistance to antibiotics, must be provided for every strain, no matter if it has QPS status or not. The need to prove efficacy is not influenced by QPS status. Efficacy has to be shown on the strain level and for every animal sub-category for which an authorisation is sought, with some exceptions for minor species. This implies a lot of work for probiotic producers as there are eight major animal categories that are further divided in up to six sub-categories. ● ● ● ● ● ● ● ●

Pigs: piglets (suckling), piglets (weaned), piglets (suckling and weaned piglets), pigs for fattening, sows for reproduction, sows in order to have the benefits in piglets. Poultry: chickens for fattening, chickens reared for laying, laying hens, turkeys for fattening, turkeys for breeding purposes, turkeys reared for breeding. Bovines: calves for rearing, calves for fattening, cattle for fattening, dairy cows for milk production, cows for reproduction. Sheep: lambs for rearing, lambs for fattening, dairy sheep (for milk production), ewes for reproduction. Goats: kids for rearing, kids for fattening, dairy goats (for milk production), goats for reproduction. Fish: salmon and trout, salmon and trout (brood stock). Rabbits: rabbits suckling and weaned, rabbits for fattening, breeding does (for reproduction), breeding does (in order to have the benefits in young rabbits). Horses.

At least three trials with significant improvement at the level of P < 0.05 have to be presented for each sub-category, meaning that if the efficacy of a product is shown for piglets and pigs for fattening for example, the authorities do not accept the conclusion that there is a positive effect for sows; this has to be demonstrated in further trials. Many feed probiotic producers conduct these trials in cooperation with universities or other independent research centres because the realisation of a scientific trial on a typical

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farm is hardly ever possible, as the set-up of stables and feeding units are not suitable for scientific trials in most cases. Additionally, extra personnel have to be employed for all the necessary measurements.

15.6

PROBIOTICS AS PERFORMANCE ENHANCERS: CONCLUSIONS

Predicting future markets is never an easy task. For feed probiotics this may be even vaguer, because products on the market are not standardised and vary over orders of magnitude in concentrations of microorganisms, recommended quantity in compound feed, and price. Feed and feed additive industries are the drivers for the probiotic market as they purchase probiotics and incorporate them into feed. In 2006, the feed industry in the 27 countries of the EU produced 145 million tonnes of compound feed, corresponding to a turnover of €36 billion. In the same year, worldwide compound feed production was as high as 667 million tonnes, which may correspond to turnover of €166 billion. Probiotic products on the market for the feed industry have an average concentration of 1 × 1013 CFU/kg. Assuming that all compound feeds for cattle, pigs, poultry and other species could be supplemented with a typical probiotic concentration of 5 × 108 CFU/kg, 33,350 tonnes of probiotics would be needed. If the price of the probiotic is assumed to be €40,000 per tonne, global turnover could theoretically reach €1.33 billion. In reality it is not likely that the feed industry can pay €2 per tonne compound feed for a probiotic additive in standard feeds for fattening for example. Only special feed mixtures for specified purposes allow higher goods and materials employed. Because of the keen price situation on the feed market, the acceptable limit for costs of probiotic additives is only a fraction of the €2 per tonne compound feed mentioned above. Therefore the realistic turnover may lie far below the theoretical figure calculated above. However, according to a report of Global Industry Analysts, Inc., USA from May 2008, the whole feed additive market will have reached $US15.4 billion by 2010. Only a few percent of the feed additive market, which consists mainly of antibiotics, amino acids and vitamins, are probiotics. Another source reported that the global market for probiotics, summarizing food and feed probiotics, was valued at $US684 million in 2006. Feed probiotics had a slightly smaller market share than food probiotics. In both fields the European market was larger than the US market. A feed industry insider stated in an interview in 2009 that he believes the feed probiotic market will double in the coming 3–5 years. Although the estimations and assumptions about the history and development of the feed industries, feed additives and feed probiotics vary, the authors see a further chance for the probiotics market to continue to grow. A higher growth rate may be assumed for Europe due to the ban on antimicrobial growth promoters. Besides the development of new applications, proven by the significant number of open registrations for probiotics, the development of further targeted probiotic therapies for specific conditions and the uncertainties and fears about antibiotics as growth promoters are good reasons to believe in a further growing probiotics market. There is a continuing trend to healthier products, and people are more often concerned about the fact that farm animals often receive high levels of antibiotics before they end up as their lunch or dinner. Probiotic feed additives may be one option to reduce the application of antibiotics. When the first probiotic products became available as performance enhancers, customer expectations about efficacy were very high. People were looking for real alternatives to antibiotics, which produced, due to uncontrolled distribution, more and more resistant

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bacterial populations. As a consequence, the use of antibiotics as growth promoters in feed has already been restricted in many countries. At this time, there was not enough scientific work on probiotics, neither concerning details of the mode of action nor the concentration of microorganisms necessary to have an influence on animal health and performance. So, on the one hand, consumers wanted those “new and natural products”, on the other the scientific base was missing. Because of the lack of knowledge, not only about the bacteria but also about the production process and technical feasibility, some of the probiotic products could not meet expectations. Some products did not contain enough bacteria to show an effect, others were not as stable as promised. Additionally, the legal status of feed additives was unclear and the authorisation process was different in each country. All together this led to disappointed customers and the reputation of probiotics was tremendously harmed. Many years have passed since those starting points. There has been huge progress in the general scientific knowledge. Knowledge of the exact mode of action of probiotics is still often only fragmentary because it is too complex and not all the beneficial effects could be proven until now. Production processes have been developed and improved, so that stability is no longer a problem. It is really important that, with the current EU regulations, customers can be sure that an authorised probiotic feed additive has absolutely proven safety and efficacy. No product with poor stability or no effect on animals can be marketed as a feed additive in Europe. Therefore we hope that the once-damaged image and reputation of probiotics can be restored, and that customers see probiotics as what they are: not a miracle but a valuable contribution to the health and performance not only of humans but also our animals.

REFERENCES Altherr BJH (2006) Studies of the influence of Bacillus cereus var. toyoi on the cellular immunity of swine. Dissertation. Department of Veterinary Medicine, Free University of Berlin. Benyacoub J, Czarnecki-Maulden GL, Cavadini C et al. (2003) Supplementation of food with Enterococcus faecium (SF68) stimulates immune functions in young dogs. J Nutr 133:1158–1162. Curtis SE, Stricklin WR (1991) The importance of animal cognition in agricultural animal production systems: an overview. J Anim Sci 69:5001–5007. European Commission (2005) Commission Regulation (EC) No. 378/2005 of 4 March 2005 on detailed rules for the implementation of Regulation (EC) No. 1831/2003 of the European Parliament and of the Council as regards the duties and tasks of the Community Reference Laboratory concerning applications for authorisations of feed additives. Official Journal of the European Union OJ L59, Corrigendum OJ L 275:211–218. European Commission (2007) Commission Regulation (EC) No. 850/2007 of 19 July 2007 amending Regulation (EC) No. 378/2005 on detailed rules for the implementation of Regulation (EC) No. 1831/2003 of the European Parliament and of the Council as regards the duties and tasks of the Community Reference Laboratory concerning applications for authorisations of feed additives. Official Journal of the European Union OJ L188. European Commission (2008) Commission Regulation (EC) No. 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No. 1831/2003 of the European Parliament and of the Council as regards the preparation and the presentation of applications and the assessment and the authorisation of feed additives. Official Journal of the European Union OJ L133/1. European Commission, Health and Consumer Protection Directorate General, Directorate D – Animal Health and Welfare, Unit D2 Feed. Community Register of Feed additives pursuant to Regulation (EC) No. 1831/2003, 44th edn, published 3 April 2009. Latest version available online at http://ec.europa.eu/ food/food/animalnutrition/feedadditives/comm_register_feed_additives_1831-03.pdf European Council (1970) Council Directive 70/524/EEC of 23 November 1970 concerning additives in feeding-stuffs. Official Journal of the European Union OJ L 270:1–17. European Food Safety Authority (2008) Scientific opinion EFSA-Q-2007-173 of 11.12.2008 on Functional groups of additives as described in Annex 1 of Regulation (EC) No. 1831/2003. EFSA Journal 920:1–19.

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European Food Safety Authority, Scientific Committee (2007) Introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA (EFSA-Q-2005-293), adopted on 19.11. 2007. EFSA Journal 587:1–16. European Food Safety Authority, Panel on biological hazards (2008) Scientific opinion on the maintenance of the list of QPS microorganisms intentionally added to food or feed (EFSA-Q-2008-006), adopted on 10.12.2008. EFSA Journal 923:1–48. European Food Safety Authority, Scientific Panel on Dietetic Products, Nutrition and Allergies (2007) Scientific and technical guidance (EFSA-Q-2007-066) for the preparation and presentation of the application for authorisation of a health claim, adopted on 06.07.2007. EFSA Journal 530:1–44. European Parliament and Council (2003). Regulation (EC) No. 1831/2003 of the European Parliament and Council of of 22 September 2003 on additives for use in animal nutrition. Official Journal of the European Union OJ L268/29. European Parliament and Council (2006). Regulation (EC) No. 1924/2006 of the European Parliament and Council of 20 December 2006 on nutrition and health claims made on foods. Official Journal of the European Union OJ L 404, 30.12.2006. Corrigendum OJ L 12:3–18. FAO (2006) Food and Nutrition Paper No. 85. FEFANA (2005) Probiotics in Animal Nutrition. Goussainville, France: Editgraph. Fraser D (1993) Assessing animal well-being: common sense, uncommon science. In: Food Animal WellBeing Conference Proceedings and Deliberations, 13–15 April 1993, Indianapolis. West Lafayette, IN: Purdue University, Office of Agricultural Research Programs, pp. 37–54. Fuller R (1989) A review: probiotics in man and animals. J Appl Bacteriol 66:365–378. Gunther KD (1994) The role of probiotics as feed additives in animal nutrition. In: Piva G (ed.) Proceedings of the IV International Feed Production Conference. Piacenza, Italy: Piacenza Facolta Di Agraria, pp. 97–114. Hammerer J (1990) Über den Einsatz des mirkrobiellen Leistungsförderers Microferm bei laktierenden Sauen. Dissertation, Universität für Bodenkultur, Institut für Nutztierwissenschaften, Vienna. Koiou L, Zentek J (2010) Influence of Enterococcus faecium DSM 7134 on the immune response in vaccinated dogs. Dissertation. Department for Veterinary Medicine, Free University of Berlin. Kozasa M (1978) Feed additives of new type: viable bacterial spore preparation; Toyocerin’ powder. J Feed Feed Industry 18:45–48. Kumprecht I, Zobac P (1998) Study of the effect of a combined preparation containing Enterococcus faecium M-74 and mannan-oligosaccharides in diets for weanling piglets. Czech J Anim Sci 43:477–481. Lacroix C, Yildirim S (2007) Fermentation technologies for the production of probiotics with high viability and functionality. Curr Opin Biotechnol 18:176–183. Mattila-Sandholm T, Myllärinen P, Crittenden R, Morgensen G, Fonden R, Saarela M (2002) Technological challenges for future probiotic foods. Int Dairy J 12:173–182. Meikle J, Brown P (1999) Alarms rang 50 years ago. The Guardian Tuesday 7 September 1999. Available at guardian.co.uk Saarela M, Mogensen G, Fonden R, Maettoe J, Mattila-Sandholm T (2000) Probiotic bacteria: safety, functional and technological properties. J Biotechnol 84:197–215. Schlegel HG (1990) General Microbiology, 6th edn. Cambridge: Cambridge University Press. Seelig B (2007) Influence of the probiotics Enterococcus faecium SF 68 (NCIMB 10415) and Bacillus cereus var. toyoi on acidic and alkaline phosphates reactivity as well as on the endocrine cells of the intestinal mucous membrane in piglets. Dissertation. Department for Veterinary Medicine, Free University of Berlin. Simon O, Vahjen W, Scharek L (2003) Micro-organisms as feed additives: probiotics. In: Ball RO (ed.) 9th International Symposium on Digestive Physiology in Pigs, Banff, Alberta, Canada. Edmonton, Canada: University of Alberta, Vol 1, pp. 295–315. Swann MM, Baxter KL, Field HI et al. (1969) Report of the Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine. London: HMSO. Swanson JC (1995) Farm animal well-being and intensive production systems. J Anim Sci 73:2744–2751. Wise R (2007) An overview of the Specialist Advisory Committee on Antimicrobial Resistance (SACAR). J Antimicrob Chemother 60(Suppl 1):i5–i7.

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16

Developing LGG®Extra, a Probiotic Multispecies Combination

Maija Saxelin, Eveliina Myllyluoma and Riitta Korpela

16.1

INTRODUCTION

Most probiotic products are based on a single strain culture that is used alone or in combination with traditional dairy starter cultures. This has made research easier and facilitated studies to evaluate the mechanisms behind the probiotic effects. Lactobacillus rhamnosus GG is one of the best-known probiotic strains (for review see Doron et al., 2005). The role of Lactobacillus GG in effectively reducing the risk of intestinal infections and in shortening the duration of symptoms such as acute diarrhoea (Szajewska et al., 2007) and its potential use in relieving symptoms and reducing the risk of atopic dermatitis (Prescott & Bjorksten, 2007), as well as its demonstrated enhancement of immune response (Kaila et al., 1992; Vaarala, 2003; De Vrese et al., 2005), have been widely documented. However, a single strain probiotic may not be an optimal solution for use in all conditions (Karimi & Pena, 2003) as is illustrated by the case of irritable bowel syndrome (IBS) where Lactobacillus GG alone gave only minor advantages for those with diarrhoea-prone or abdominal distension symptoms (O'Sullivan & O'Morain, 2000; Bausserman & Michail, 2005). Several preparations containing two or more bacterial strains can be found on the probiotic market, especially in the food supplement sector, though in many cases without any documentation on the mutual effect of the combined strains or the efficacy of the combination. Timmerman et al. (2004) showed very nicely that the inclusion of additional strains does not necessarily improve the functionality of the combination; instead, antagonistic effects may also occur. Moreover, efficacy must be shown in human intervention trials as the definition of probiotics states that probiotics ‘confer a health benefit on the host’ (FAO/WHO, 2001). This chapter describes the rationale and the studies behind one probiotic multispecies combination (LGG®Extra, Valio Ltd, Helsinki, Finland) which is primarily targeted for alleviation of non-infectious, functional intestinal discomfort. The chapter introduces characteristics of the single strains used in the combination, summarises in vitro and in vivo data of their combinational use, considers the safety, and finally describes the results obtained by the combination in clinical trials.

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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16.2

STRAIN SELECTION

LGG®Extra, a multistrain probiotic combination, is based on Lactobacillus rhamnosus GG (Lactobacillus GG, ATCC 53103) combined with three other strains, L. rhamnosus LC705 (DSM 7061), Propionibacterium freudenreichii subsp. shermanii JS (PJS, DSM 7065), and Bifidobacterium animalis subsp. lactis Bb12 (DSM 15954). The strains LC705 and PJS were earlier shown to have inhibitory effects against yeast and moulds in dairy-based foods and fermented bread (Suomalainen & Mäyrä-Mäkinen, 1999). Bifidobacteria belong to the normal intestinal microbiota and are generally associated with health benefits in the human intestine, many strains being able to persist in the gut. They may also be able to beneficially modulate gut microbiota, showing immunological activity for example (Saxelin et al., 2005; Riedel et al., 2006). Bifidobacteria grow synergistically with propionic acid bacteria, which secrete metabolites that stimulate the growth of bifidobacteria (Kouya et al., 2007). Since the purpose was to develop a probiotic combination that can have influence in both small bowel and colon, the Bifidobacterium animalis subsp. lactis Bb12 strain was included in the multispecies combination. The B. breve 99 strain (Bb99, DSM 13692) was also tested for this probiotic multispecies combination. Since both of the bifidobacteria strains seemed to work as well in the combination, the Bb12 strain was selected for the final combination due to its excellent technological characteristics and abundant documentation.

16.3

PROBIOTIC CHARACTERISTICS OF THE STRAINS

16.3.1

Gastrointestinal persistence and colonisation

For the functionality and efficacy of probiotic bacteria, adhesion to the intestinal cell lining (intestinal mucus and epithelial cells) is supposed to play an important role, especially in the inhibition of pathogen adhesion and immunological effects. The strains in the present combination differ in their adhesion capacities. Lactobacillus GG is very adhesive on intestinal mucus and Caco-2 cell layer, but LC705 only poorly adhesive (Jacobsen et al., 1999; Ouwehand et al., 2000; Tuomola et al., 2000). Genome sequencing of these two strains revealed that specific pili structures containing a human mucus-binding protein protrude from the cell surface of GG but not of LC705 (Kankainen et al., 2009). Also Bifidobacterium strain Bb12 is quite adhesive, but adhesion of the PJS strain on intestinal mucus varied depending on the study (Ouwehand et al., 2000, 2002a). The adhesion capacity completely explained the difference in the persistence of the strains in the human gastrointestinal tract. After ceasing oral consumption of the strain combination, their median recovery times were 17 days for GG, 7 days for Bb12 and PJS, and 5 days for LC705 (Saxelin et al., 2009, submitted). Also, the adhesive properties of the strains were reflected in biopsy samples taken from the human colon: Lactobacillus GG was recovered in all biopsy samples taken from volunteers during colonoscopy on the following day and 1 week after cessation of its consumption (Alander et al., 1997, 1999a), but the strains LC705 and PJS were not recovered in colon biopsy samples on the following day or later after cessation of their consumption (Alander et al., 1999b). However, survival through the gastrointestinal tract was excellent for all the strains of the combination, when consumed in capsules, yoghurt or cheese (Saxelin et al., 2009, submitted). Traditional plate culture methods are laborious and do not fulfil the modern criteria of being able to accurately differentiate and identify administered strains

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from among the thousands of bacterial species and strains in the gastrointestinal tract. Recently, real-time strain-specific quantitative PCR (qPCR) methods to quantify the strains directly from the DNA isolated from faecal samples have been developed for Lactobacillus GG (Ahlroos & Tynkkynen, 2009), LC705 and PJS (Mikkola et al., 2006).

16.3.2

Influence on human intestinal microbiota

Probiotics are supposed to improve the balance of intestinal microbiota. The present multispecies combination did not have a remarkable effect on the composition of intestinal microbiota in healthy volunteers who had high counts of autochthonous bifidobacteria and lactobacilli, but the level of lactobacilli increased when the baseline counts were low (Tiihonen et al., 2008). However, the methods used to analyse the totality of intestinal microbiota may not be sensitive enough to detect the changes. In a recent study, the total microbiota of IBS patients was analysed with a microarray-based method, the Human Intestinal Tract Chip (HIT Chip), containing approximately 5500 oligonucleotide probes able to identify 1250 intestinal microbial phylotypes (Zoetendal et al., 2008). A similarity index, expressed as a percentage indicating the degree of preservation of the microbiota composition between two time points, assesses the stability of the microbiota. The results of the analyses of faecal samples of IBS patients showed that the microbiota similarity index increased with the 5-month probiotic supplementation, while it decreased in the placebo group, indicating that the administration of the probiotic combination stabilised the composition of faecal microbiota (Kajander et al., 2008). In another clinical study to treat symptoms of IBS with this multispecies combination, the composition of intestinal microbiota was analysed by real-time qPCR, and colon metabolism was evaluated by measuring the activity of the faecal enzymes β-glucosidase and β-glucuronidase and short-chain fatty acids. No changes in short-chain fatty acids occurred, but a decrease in β-glucuronidase activity was detected more frequently in subjects in the probiotic group compared with those in the placebo group (Kajander et al., 2007). When the multispecies combination was administered together with Helicobacter pylori eradication therapy, the level of total microbial counts was lowered during the antibiotic treatment in both the probiotic and the placebo groups and the probiotics could not prohibit the changes caused by the antibiotics. The levels of bifidobacteria and lactobacilli/enterococci were low, and the probiotic treatment could only slightly, though statistically significantly, counteract the decrease in total aerobic bacteria and the lactobacilli/enterococci group (Myllyluoma et al., 2007a). Thus, the influence of the present probiotic combination on human microbiota may be stabilising in conditions where there is certain instability. However, it is crucial to use methods of analysis that evaluate the totality of the microbiota.

16.3.3

Immunological effects in vitro

Immunological in vitro effects of the Lactobacillus GG and Bifidobacterium Bb12 strains have been widely studied, but in this connection only studies made with all the combination strains are referred to. The stimulation of cytokine production in human peripheral blood mononuclear cells (PBMC) with each single strain and the final combination has been studied (Kekkonen et al., 2008). Bifidobacteria (B. animalis subsp. lactis Bb12, B. breve 99) and propionic acid bacteria (P. freudenreichii subsp. shermanii JS) induced higher formation of anti-inflammatory cytokine interleukin (IL)-10 than the lactobacilli strains and a

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low level of proinflammatory cytokine IL-12. All the strains of the present composition (including GG, LC705, Bb12, PJS) induced tumour necrosis factor (TNF)-α formation in PBMC. The strength of responses induced by the different strain combinations was closer to an average of responses induced by the individual strains than of any individual strain. In monocyte-derived dendritic cells, bifidobacteria (Bb12 and Bb99) induced high levels of proinflammatory and anti-inflammatory cytokines, but L. rhamnosus GG or LC705 were poor inducers (Veckman et al., 2004; Latvala et al., 2008). Altogether, based on these in vitro studies, the present multispecies combination may be more potent for immune suppression than immune stimulation. Although in vitro cell tests are used to pre-screen potential immunological effects, the host's health status has its own influence on the outcome in vivo and it is important to study immunological and clinical effects case by case.

16.3.4

Potential for reducing dietary toxins

One of the important activities of the microbiota is to degrade harmful compounds, such as drugs, toxins and other carcinogenic and procarcinogenic compounds, to less harmful ones (Goldin, 1998). The strains of the present multispecies combination did not produce potentially harmful enzyme activities (β-glucuronidase, nitroreductase, azoreductase and trypsin activity), and their administration in human intervention studies resulted in a reduction of one or more of these enzyme activities in faecal samples (Goldin et al., 1992; Ling et al., 1992, 1994; Ouwehand et al., 2002b; Suomalainen et al., 2008). Aflatoxin B1 (AFB1) is produced in contaminated food and feed by Aspergillus flavus mould in warm and humid conditions. A few strains of probiotic bacteria, especially GG, LC705 and PJS, have been intensively studied for their ability to bind dietary toxins (El-Nezami et al., 1998, 2002a,b). Lactobacillus GG cells were the most effective in removing AFB1 in liquid medium (about 80%), but LC705 and PJS were also effective (El-Nezami et al., 1998; Haskard et al., 2001). The results indicated that the effect is not mediated via metabolic activity, since both heat and acid treatment of the bacteria enhanced the binding capacity (Peltonen et al., 2001). The most probable binding sites for the toxin were in the cell-wall polysaccharides and peptidoglycans (Haskard et al., 2000, 2001; Lahtinen et al., 2004). Interestingly, intestinal mucus has been shown to reduce the binding of AFB1 by Lactobacillus GG and especially by the combination of LC705 and PJS (Gratz et al., 2004), which may partly explain why the toxin binding of the LC705 and PJS combination was higher in vitro in liquid medium than the ex vivo binding in the duodenal loop of chicken (Gratz et al., 2005). In the intestinal lumen of chicken the combination of LC705 and PJS retained 39% of the administered dose of AFB1 in the lumen (Gratz et al., 2005), being the average of the amount retained by the single strains (El-Nezami et al., 2000). The amount retained in the lumen decreased over time but, in any case, the amount of AFB1 taken up by duodenal tissue was 74%, 37%, 63% and 40% lower compared with control absorption, with simultaneous administration of GG, LC705, PJS or the LC705 and PJS combination, respectively. The mechanism of action in toxin removal was studied using the Lactobacillus GG strain. In cell culture studies heat-inactivated Lactobacillus GG reduced the transport of AFB1 through Caco-2 monolayer, while the formation of a hydroxylated metabolite, AFM1, increased significantly, and the formation of aflatoxicol was clearly reduced (Gratz et al., 2007). This may indicate that a probiotic strain also has an active role in enhancing the metabolism of aflatoxin in epithelial cells. Inactivated Lactobacillus GG also attenuated the aberration in the cell-layer integrity caused by AFB1 and inhibited DNA damage in

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epithelial cells (Gratz et al., 2007). In rats a single high dose of AFB1 reduced body weight gain and there was a tendency to increase the activity of alanine transaminase, which can be connected to the occurrence of liver injury. The administration of Lactobacillus GG increased the excretion of faecal AFB1 and AFM1 during the first 24 hours after the toxin dose, and the level of AFB1 albumin adducts in plasma tended to be lower compared with the control group (Gratz et al., 2006). Aflatoxin is metabolised in the liver, and the metabolites (e.g. AFQ1, AFM1 and AFB1N(7)-guanine) can bind to guanine in DNA, resulting in DNA mutations and increased risk of tumour formation (El-Nezami et al., 2006). Aflatoxins are high-risk factors for hepatocellular carcinoma in humans, especially in persons infected with hepatitis B virus. AFB1 metabolites are excreted in milk, urine and faeces. The most common metabolite excreted by young Chinese males was AFQ1 (Mykkanen et al., 2005). Chinese men exposed to AFB1 were treated with the combination of LC705 and PJS versus placebo in a 5-week intervention and the excretion of AFB1 metabolites was studied (El-Nezami et al., 2006). The researchers expected that the probiotic binding of AFB1 would lead to reduced excretion of AFB1-N(7)-guanine in urine. The results showed that the proportion of samples negative for AFB1-N(7)-guanine increased significantly due to the probiotic intervention and a significant decline in the levels of urinary AFB1-N(7)-guanine (El-Nezami et al., 2006). These results indicate that the toxic effects of aflatoxin exposure in humans may be reduced by the administration of probiotic strains that effectively bind the toxin and may also have an influence in the metabolism of the toxin in human tissues. However, more studies are needed to evaluate the entire situation and the mechanisms behind the effects.

16.3.5

Safety aspects

By using the qualified presumption of safety (QPS) concept, the European Food Safety Authority (EFSA, 2007) has already approved the QPS status for several microbial species used in food processing. Among the QPS-approved microorganisms, all the species used in the LGG®Extra combination are mentioned. Furthermore, L. rhamnosus GG and B. animalis subsp. lactis Bb12 were regarded as safe to be used in infant foods by the US Food and Drug Administration. In addition to their probiotic use, L. rhamnosus and P. freudenreichii subsp. shermanii species are commonly isolated in ripened cheeses where they can reach levels of 107–108 CFU/g (Antonsson et al., 2003; Briggiler-Marco et al., 2007) and may be either intentionally added or native non-starter bacteria. All the strains included in the probiotic multispecies combination that is the subject of this review have a long history of safe use in dairy foods. PJS has been used as an adjunct culture in cheese production in Finland since 1979, LC705 also in cheese production since 1995, Lactobacillus GG in fresh dairy products since 1990, and Bb12 since the early 1980s. The LGG®Extra combination (GG, LC705, PJS, and Bb12, 107 CFU/mL, each strain, respectively) was introduced in Finland in January 2008, when a daily dose drink fermented with this particular combination was launched. A rough estimation for the annual consumption of GG, LC705 and PJS in Finland is 1010–1011 CFU per person. A large epidemiological study showed that isolates similar to Lactobacillus GG have been occasionally isolated at low frequencies in clinical blood cultures (Salminen et al., 2002), but isolates similar to LC705 have not been found among the L. rhamnosus isolates (S. Tynkkynen, Valio Ltd, Finland, personal communication). Differences in the ability of these strains to adhere to intestinal mucus and epithelial cells may explain why one type is able to translocate to blood circulation and the other is not. However, it was clearly shown that the exponentially increased consumption of Lactobacillus

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GG products did not increase the incidence of Lactobacillus blood isolates or the proportion of Lactobacillus GG-like isolates among them (Salminen et al., 2002). Important information to confirm the safe use of the present probiotic combination can be found in the results of all human intervention studies in infants, adults and the elderly, which have been completed without adverse effects. Probiotic bacteria should not carry transferable antibiotic-resistance genes. A standardised method for the determination of minimal inhibitory concentration (MIC) for lactobacilli and bifidobacteria was described a few years ago (Klare et al., 2005; Mättö et al., 2006). No high MIC values other than vancomycin (>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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Kaila M, Isolauri E, Soppi E, Virtanen E, Laine S, Arvilommi H (1992) Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Pediatr Res 32:141–144. Kajander K, Hatakka K, Poussa T, Farkkila M, Korpela R (2005) A probiotic mixture alleviates symptoms in irritable bowel syndrome patients: a controlled 6-month intervention. Aliment Pharmacol Ther 22:387–394. Kajander K, Krogius-Kurikka L, Rinttila T, Karjalainen H, Palva A, Korpela R (2007) Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome. Aliment Pharmacol Ther 26:463–473. Kajander K, Myllyluoma E, Rajilic-Stojanovic M et al. (2008) Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome and stabilizes intestinal microbiota. Aliment Pharmacol Ther 27:48–57. Kankainen M, Paulin L, Tynkkynen S et al. (2009) Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human-mucus binding protein. Proc Natl Acad Sci USA 106:17193–17198. Karimi O, Pena AS (2003) Probiotics: isolated bacteria strain or mixtures of different strains? Two different approaches in the use of probiotics as therapeutics. Drugs Today (Barc) 39:565–597. Kassinen A, Krogius-Kurikka L, Makivuokko H et al. (2007) The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects. Gastroenterology 133:24–33. Kekkonen RA, Kajasto E, Miettinen M, Veckman V, Korpela R, Julkunen I (2008) Probiotic Leuconostoc mesenteroides ssp. cremoris and Streptococcus thermophilus induce IL-12 and IFN-gamma production. World J Gastroenterol 14:1192–1203. Klare I, Konstabel C, Muller-Bertling S et al. (2005) Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria. Appl Environ Microbiol 71:8982–8986. Klein G, Hallmann C, Casas IA, Abad J, Louwers J, Reuter G (2000) Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods. J Appl Microbiol 89:815–824. Kouya T, Misawa K, Horiuchi M et al. (2007) Production of extracellular bifidogenic growth stimulator by anaerobic and aerobic cultivations of several propionibacterial strains. J Biosci Bioeng 103:464–471. Kuitunen M, Kukkonen K, Juntunen-Backman K et al. (2009) Probiotics prevent IgE-associated allergy until age 5 years in cesarean-delivered children but not in the total cohort. J Allergy Clin Immunol 123:335–341. Kukkonen K, Nieminen T, Poussa T, Savilahti E, Kuitunen M (2006) Effect of probiotics on vaccine antibody responses in infancy: a randomized placebo-controlled double-blind trial. Pediatr Allergy Immunol 17:416–421. Kukkonen K, Savilahti E, Haahtela T et al. (2007) Probiotics and prebiotic galacto-oligosaccharides in the prevention of allergic diseases: a randomized, double-blind, placebo-controlled trial. J Allergy Clin Immunol 119:192–198. Kukkonen K, Savilahti E, Haahtela T et al. (2008) Long-term safety and impact on infection rates of postnatal probiotic and prebiotic (synbiotic) treatment: randomized, double-blind, placebo-controlled trial. Pediatrics 122:8–12. Kukkonen K, Kuitunen M, Haahtela T, Korpela R, Poussa T, Savilahti E (2010) High intestinal IgA associates with reduced risk of IgE-associated allergic diseases. Pediatr Allergy Immunol 21:67–73 Lahtinen SJ, Haskard CA, Ouwehand AC, Salminen SJ, Ahokas JT (2004) Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG. Food Addit Contam 21:158–164. Latvala S, Pietila TE, Veckman V et al. (2008) Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells. World J Gastroenterol 14:5570–5583; discussion 5581–5582. Ling WH, Hanninen O, Mykkanen H, Heikura M, Salminen S, Von Wright A (1992) Colonization and fecal enzyme activities after oral Lactobacillus GG administration in elderly nursing home residents. Ann Nutr Metab 36:162–166. Ling WH, Korpela R, Mykkanen H, Salminen S, Hanninen O (1994) Lactobacillus strain GG supplementation decreases colonic hydrolytic and reductive enzyme activities in healthy female adults. J Nutr 124:18–23. Malinen E, Rinttila T, Kajander K et al. (2005) Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy controls with real-time PCR. Am J Gastroenterol 100:373–382.

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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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17

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

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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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

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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

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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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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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’.

REFERENCES Anal AK, Singh H (2007) Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends Food Sci Technol 18:240–251. Ananta E, Birkeland SE, Corcoran BM, Fitzgerald G (2004) Processing effects on the nutritional advancement of probiotics and prebiotics. Microbial Ecol Health Dis 16:113–124. Ananta E, Volkert M, Knorr D (2005) Cellular injuries and storage stability of spray-dried Lactobacillus rhamnosus GG. Int Dairy J 15:399–409. Ben Amor K, Vaughan EE, de Vos WM (2007) Advanced molecular tools for the identification of lactic acid bacteria. J Nutr 137:741–747. Biavati B, Mattiarelli P (2006) The Family Bifidobacteriaceae. In: Dworkin (ed.) The Prokaryotes, 3rd edn, Vol 4. New York: Springer, pp. 322–382. Bogovic-Matijasic B, Obermaier T, Rogelj I (2010) Quantification of Lactobacillus gasseri, Enterococcus faecium and Bifidobacterium infantis in a probiotic OTC drug by real-time PCR. Food Control 21:419–425. Boylston TD, Vinderola CG, Ghodussi HB, Reinheimer JA (2004) Incorporation of bifidobacteria into cheeses: challenges and rewards. Int Dairy J 14:375–387. Bujalance C, Jimenez-Valera M, Moreno E, Ruiz-Bravo A (2006) A selective medium for Lactobacillus plantarum. J Microbiol Method 66:572–575. Champagne CP, Raymond Y, Gonthier J, Audet P (2009) Enumeration of the contaminating bacterial microbiota in unfermented pasteurized milks enriched with probiotic bacteria. Can J Microbiol 55:410–418. Chavez BE, Ledeboer AM (2007) Drying of probiotics: optimization of formulation and process to enhance storage survival. Drying Technology 25:1193–1201. Dave RI, Shah NP (1996) Evaluation of media for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and bifidobacteria. J Dairy Sci 79:1529–1536. De Vuyst L, Falony G, Leroy F (2008) Probiotics in fermented sausage. Meat Science 80:75–78. EFSA (2004) European Food Safety Authority EFSA: Scientific Colloquium Summary Report. QPS Qualified Presumption of Safety of Microorganisms in Food and Feed, 13–14 December 2004. EFSA (2007) European Food Safety Authority Scientific Committee (EFSA) public consultation on the Qualified Presumption of Safety (QPS) approach for the safety assessment of microorganisms deliberately added to food and feed. Annex 3: Assessment of Gram-positive non-sporulating bacteria with respect to a qualified presumption of safety. Available at www.efsa.europa.eu/en/science/sc_commitee/ sc_consultations/sc_consultation_qps.html Euzeby JP (1997) List of bacterial names with standing in nomenclature: a folder available on the internet. Int J Syst Bacteriol 47:590–592 (List of Prokaryotic Names with Standing in Nomenclature: December 22, 2009.) Available at www.bacterio.net Felis GE, Dellaglio F (2007) Taxonomy of lactobacilli and bifidobacteria. Curr Issues Intest Microbiol 8:44–61. Furet JP, Quenee P, Tailliez P (2004) Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR. Int J Food Microbiol 97:197–2004. Garcia-Cayuela T, Tabasco R, Pelaez C, Requena, T (2009) Simultaneous detection of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR. Int Dairy J 19:405–409. Gueimonde M, Delgado S, Mayo B, Ruas-Madiedo P, Margolles A, de los Reyes-Gavilan CG (2004a) Viability and diversity of probiotic Lactobacillus and Bifidobacterium populations included in commercial fermented milk. Food Res Int 37:839–850.

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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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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. ●

●

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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REFERENCES Agence Française de Sécurité Sanitaire des Aliments/French Agency for Food Safety (AFSSA) (2005) Effects of probiotics and prebiotics on flora and immunity in adults. Available at http://www.afssa.fr/ Documents/NUT-Ra-PreprobiotiqEN.pdf Aggett PJ, Antoine JM, Asp NG et al. (2005) PASSCLAIM: consensus on criteria. Eur J Nutr 44(Suppl 1):i5–i30. Altermann E, Russell WM, Azcarate-Peril MA et al. (2005) Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM. Proc Natl Acad Sci USA 102:3906–3912. Asp NG, Bryngelsson S (2008) Health claims in Europe: new legislation and PASSCLAIM for substantiation. J Nutr 138:1210S–1215S. Aureli P, Fiore A, Scalfaro C, Franciosa G (2008) Metodi microbiologici tradizionali e metodi molecolari per l’analisi degli integratori alimentari a base di o con probiotici per uso umano. Rapporti ISTISAN 08/36. Barrangou R, Altermann E, Hutkins R, Cano R, Klaenhammer TR (2003) Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus. Proc Natl Acad Sci USA 100:8957–8962. Ben AK, Vaughan EE, de Vos WM (2007) Advanced molecular tools for the identification of lactic acid bacteria. J Nutr 137:741S–747S. Bertazzoni-Minelli E, Benini A, Marzotto M, De Dea P, Morelli L, Dellaglio F (2002) Commercial probiotic products: evaluation of microbial content and nomenclature. Microecol Ther 29:135–143. Besselink MG, van Santvoort HC, Buskens E et al. (2008) Dutch Acute Pancreatitis Study Group. Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial. Lancet 371:651–659. Bron PA, Grangette C, Mercenier A, de Vos WM, Kleerebezem M (2004) Identification of Lactobacillus plantarum genes that are induced in the gastrointestinal tract of mice. J Bacteriol 86:5721–5729. Castagliuolo I, Galeazzi F, Ferrari S et al. (2005) Beneficial effect of auto-aggregating Lactobacillus crispatus on experimentally induced colitis in mice. FEMS Immunol Med Microbiol 43:197–204. Castellazzi AM, Valsecchi C, Montagna L et al. (2007) In vitro activation of mononuclear cells by two probiotics: Lactobacillus paracasei I 1688, Lactobacillus salivarius I 1794 and their mixture (PSMIX). Immunol Invest 36:413–421. Cesena C, Morelli L, Alander M et al. (2001) Lactobacillus crispatus and its nonaggregating mutant in human colonization trials. J Dairy Sci 84:1001–1010. Contor L, Asp NG (2004) Process for the assessment of scientific support for claims on foods (PASSCLAIM) phase two: moving forward. Eur J Nutr 43(Suppl 2):II3–II6. Drago L, De V E, Nicola L, Colombo A, Gismondo MR (2004) Microbiological evaluation of commercial probiotic products available in Italy. J Chemother 16:463–467. Duncan SH, Lobley GE, Holtrop G et al. (2008) Human colonic microbiota associated with diet, obesity and weight loss. Int J Obes (Lond) 32:1720–1724. Elliot E, Teversham K (2004) An evaluation of nine probiotics available in South Africa, August 2003. S Afr Med J 94:121–124. EMEA-CPMP (2001) Note for guidance on the investigation of bioavailability and bioequivalence. CPMP/ EWP/QWP/1401/98. European Food Safety Authority. EFSA Journal (2008) 782:1–9; 824:2–12; 859:2–9; 860:2–8; 861:7–9; 862:2–8; 863:2–8; EFSA Journal (2009) 999:2–9. FAO/WHO Expert Consultation (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Available at www.fao.org FAO/WHO (2002) Guidelines for the evaluation of probiotics in food. Available at www.fao.org Fasoli S, Marzotto M, Rizzotti L, Rossi F, Dellaglio F, Torriani S (2003) Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Int J Food Microbiol 82:59–70. Food and Drug Administration (2003) Bioavailability and bioequivalence studies for orally administered drug products: general considerations. Rockville, MD: FDA. Goh YJ, Azcarate-Peril AM, O’Flaherty S et al. (2009) Development and application of a upp-based counterselective gene replacement system for the study of the S-Layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol 75:3093–3105. Hamilton-Miller JM, Shah S (2002) Deficiencies in microbiological quality and labelling of probiotic supplements. Int J Food Microbiol 72:175–176.

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Hamilton-Miller JM, Shah S, Winkler JT (1999) Public health issues arising from microbiological and labelling quality of foods and supplements containing probiotic microorganisms. Public Health Nutr 2:223–229. International Standards Organization (2006) Enumeration of presumptive Lactobacillus acidophilus on a selective medium: colony-count technique at 37°C. 20128:2006 (IDF 192: 2006). Kirjavainen PV, Ouwehand AC, Isolauri E, Salminen SJ (1998) The ability of probiotic bacteria to bind to human intestinal mucus. FEMS Microbiol Lett 167:185–189. Klijn A, Mercenier A, Arigoni F (2005) Lessons from the genomes of bifidobacteria. FEMS Microbiol Rev 29:491–509. Konstantinov SR, Smidt H, de Vos WM et al. (2008) S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions. Proc Natl Acad Sci USA 105:10475–10479. Ley RE, Bäckhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI (2005) Obesity alters gut microbial ecology. Proc Natl Acad Sci USA 102:11070–11075. Ley RE, Turnbaugh PJ, Klein S, Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444:1022–1023. Mai V, Draganov PV (2009) Recent advances and remaining gaps in our knowledge of associations between gut microbiota and human health. World J Gastroenterol 15:81–85. Martin FP, Wang Y, Sprenger N et al. (2008) Top-down systems biology integration of conditional prebiotic modulated transgenomic interactions in a humanized microbiome mouse model. Mol Syst Biol 4:7 (article n205). Mater D (2007) Market supply of probiotic dairy products and scientific publications show parallel increase. Probiotics Reference 33:1–3. Mercenier A, Lenoir-Wijnkoop I, Sander ME (2008) Physiological and functional properties of probiotics. Bull Int Dairy Fed No. 429. Metchnikoff E (1907) Lactic acid as inhibiting intestinal putrefaction. In: The Prolongation of Life: Optimistic Studies. London: W. Heinemann, pp.161–183. Midtvedt T (1986) Effects of antimicrobial agents upon the functional part of the intestinal flora. Scand J Infect Dis Suppl 49:85–88. Ministero della Salute (2005) Linee Guida Nutrizione/Probiotici. Available at www.ministerosalute.it/alimenti/nutrizione/linee.jsp Morelli L (2000) In vitro selection of probiotic lactobacilli: a critical appraisal. Curr Issues Intest Microbiol 1:59–67. Morelli L, Garbagna N, Rizzello F, Zonenschain D, Grossi E (2006) In vivo association to human colon of Lactobacillus paracasei B21060: map from biopsies. Dig Liver Dis 38:894–898. Official Journal of the European Union (2003) Regulation (EC) No. 1831/2003 L 268/29. Pridmore RD, Berger B, Desiere F et al. (2004) The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci USA101:2512–2517. Reid G (2008) Probiotic lactobacilli for urogenital health in women. J Clin Gastroenterol 42(Suppl 3 Pt 2):S234–S236. Rosander A, Connolly E, Roos S (2008) Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938. Appl Environ Microbiol 74:6032–6040. Schell MA, Karmirantzou M, Snel B et al. (2002) The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc Natl Acad Sci USA 99:14422–14427. Sha BE, Chen HY, Wang QJ, Zariffard MR, Cohen MH, Spear GT (2005) Utility of Amsel criteria, Nugent score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women. J Clin Microbiol 43:4607–4612. Sha BE, Gawel SH, Hershow RC et al. (2007) Analysis of standard methods for diagnosing vaginitis: HIV infection does not complicate the diagnosis of vaginitis. J Low Genit Tract Dis 11:240–50. Sheu SJ, Hwang WZ, Chen HC, Chiang YC, Tsen HY (2009) Development and use of tuf gene-based primers for the multiplex PCR detection of Lactobacillus acidophilus, Lactobacillus casei group, Lactobacillus delbrueckii, and Bifidobacterium longum in commercial dairy products. J Food Prot 72:93–100. Sokol H, Pigneur B, Watterlot L et al. (2008) Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci USA 105:16731–16736 Sul SY, Kim HJ, Kim TW, Kim HY (2007) Rapid identification of Lactobacillus and Bifidobacterium in probiotic products using multiplex PCR. J Microbiol Biotechnol 17:490–495.

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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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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

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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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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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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●

●

● ●

●

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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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Inada T, Kimata K, Aiba H (1996) Mechanism responsible for glucose-lactose diauxie in Escherichia coli: challenge to the cAMP model. Genes Cells 1:293–301. Kalliomäki M, Collado MC, Salminen S, Isolauri E (2008) Early differences in fecal microbiota composition in children may predict overweight. Am J Clin Nutr 87:534–538. Kassinen A, Krogius-Kurikka L, Mäkivuokko H et al. (2007) The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects. Gastroenterology 133:24–33. Kirjavainen PV, Arvola T, Salminen SJ, Isolauri E (2002) Aberrant composition of gut microbiota of allergic infants: a target of bifidobacterial therapy at weaning? Gut 51:51–55. Kolars JC, Levitt MD, Aouji M, Savaiano DA (1984) Yogurt, an autodigesting source of lactose. N Engl J Med 310:1–3. Kringelum BW, Soerensen NM, Garrigues C, Pedersen MB, Groen S (2006) Use of compounds involved in biosynthesis of nucleic acids to increase yield of bacterial cultures. Patent WO2006072257, Denmark. Ley RE (2010) Obesity and the human microbiome. Curr Opin Gastroenterol 26:5–11. Luesink EJ, van Herpen RE, Grossiord BP, Kuipers OP, de Vos WM (1998) Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol Microbiol 30:789–798. McCauley R, Heel KA, Barker PR, Hall J (1996) The effect of branched-chain amino acid-enriched parenteral nutrition on gut permeability. Nutrition 12:176–179. Martinez C, Antolin M, Santos J et al. (2008) Unstable composition of the fecal microbiota in ulcerative colitis during clinical remission. Am J Gastroenterol 103:643–648. Martini MC, Lerebours EC, Wei-Jin L et al. (1991) Strains and species of lactic acid bacteria in fermented milks (yogurts): effect on in vivo lactose digestion. Am J Clin Nutr 54:1041–1046. Merrell DS, Camilli A (2000) Detection and analysis of gene expression during infection by in vivo expression technology. Philos Trans R Soc Lond B Biol Sci 355:587–599. Metchnikoff II (1907) The Prolongation of Life: Optimistic Studies (reprinted edition 2004). New York: Springer. Morishita T, Tamura N, Makino T, Kudo S (1999) Production of menaquinones by lactic acid bacteria. J Dairy Sci 82:1897–1903. Nadal I, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y (2007) Imbalance in the composition of the duodenal microbiota of children with coeliac disease. J Med Microbiol 56:1669–1674. Nissle A (1916) Ueber die Grundlagen einer neuen ursaechlichen Bekaempfung der pathologischen Darmflora. Deutsch Med Wochenschr 42:1181–1184. Perrot F, Hebraud M, Charlionet R, Junter GA, Jouenne T (2001) Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock. Electrophoresis 10:2110–2119. Rediers H, Rainey PB, Vanderleyden J, De Mot R (2005) Unraveling the secret lives of bacteria: use of in vivo expression technology and differential fluorescence induction promoter traps as tools for exploring niche-specific gene expression. Microbiol Mol Biol Rev 69:217–261. Savaiano DA, Aboueknouar A, Smith DE, Levitt MD (1984) Lactose malabsorption from yogurt, pasteurized yogurt, sweet acidophilus milk, and cultured milk in lactase-deficient individuals. Am J Clin Nutr 40:1219–1223. Seksik P, Rigottier-Gois L, Gramet G et al. (2003) Alterations of the dominant faecal bacterial groups in patients with Crohn’s disease of the colon. Gut 52:237–242. Siezen RJ, Bachmann H (2008) Genomics of dairy fermentations. Micr Biotech 1:435–442. Smeianov VV, Wechter P, Broadbent JR et al. (2007) Comparative high-density microarray analysis of gene expression during growth of Lactobacillus helveticus in milk versus rich culture medium. Appl Environ Microbiol 73:2661–2672. Sokol H, Seksik P, Rigottier-Gois L et al. (2006) Specificities of the fecal microbiota in inflammatory bowel disease. Inflamm Bowel Dis 12:106–111. Sokol H, Lay C, Seksik P, Tannock GW (2008a) Analysis of bacterial bowel communities of IBD patients: what has it revealed? Inflamm Bowel Dis 14:858–867. Sokol H, Pigneur B, Watterlot L et al. (2008b) Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci USA 105:16731–16736. Song Y, Liu C, Finegold SM (2004) Real-time PCR quantitation of clostridia in feces of autistic children. Appl Environ Microbiol 70:6459–6465.

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Sybesma W, Burgess C, Starrenburg M, van Sinderen D, Hugenholtz J (2004) Multivitamin production in Lactococcus lactis using metabolic engineering. Metab Eng 6:109–115. Tavan E, Cayuela C, Antoine JM, Cassand P (2002) Antimutagenic activities of various lactic acid bacteria against food mutagens: heterocyclic amines. J Dairy Res 69:335–341. Tissier H (1900) Recherches sur la flore intestinale normale et pathologique du nourrisson. Thèse, Paris. Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI (2009) The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci Transl Med 1:6–14. Valdivia RH, Falkow S (1996) Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol Microbiol 22:367–378. Van Tongeren SP, Slaets JPJ, Harmsen HJM, Welling GW (2005) Fecal microbiota composition and frailty. Appl Environ Microbiol 71:6438–6442. Weber P (2001) Vitamin K and bone health. Nutrition 10:880–887. Wen L, Ley RE, Volchkov PY et al. (2008) Innate immunity and intestinal microbiota in the development of type 1 diabetes. Nature 455:1109–1113.

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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

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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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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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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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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329

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

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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

9781405194914_6_index.indd 333

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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Index

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

9781405194914_6_index.indd 335

335

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

9781405194914_6_index.indd 336

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

11/1/2010 6:33:31 PM

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

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