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Page 1: Prebiotics and Probiotics Science and Technology978-0-387... · 2017-08-28 · prebiotics and probiotics field, covering their taxonomy, the potential beneficial effects to humans

Prebiotics and Probiotics Science and Technology

Page 2: Prebiotics and Probiotics Science and Technology978-0-387... · 2017-08-28 · prebiotics and probiotics field, covering their taxonomy, the potential beneficial effects to humans

Dimitris Charalampopoulos, Robert A. Rastall (Eds.)

Prebiotics and ProbioticsScience and Technology

With 67 figures and 97 tables

Page 3: Prebiotics and Probiotics Science and Technology978-0-387... · 2017-08-28 · prebiotics and probiotics field, covering their taxonomy, the potential beneficial effects to humans

Editors:Dimitris CharalampopoulosDepartment of Food BiosciencesUniversity of ReadingWhiteknights, [email protected]

Robert A. RastallDepartment of Food BiosciencesUniversity of ReadingWhiteknights, [email protected]

Library of Congress Control Number: 2009929416

ISBN: 978-0-387-79057-2

This publication is available also as:Electronic version under ISBN 978-0-387-79058-9Print and electronic bundle under ISBN 978-0-387-79059-6

� Springer Science+Business Media, LLC 2009

All rights reserved. This work may not be translated or copied in whole or in partwithout the written permission of the publisher (Springer Science+Business Media,LLC., 233 Spring Street, New York, NY 10013, USA), except for brief excerpts inconnection with reviews or scholarly analysis. Use in connection with any form ofinformation storage and retrieval, electronic adaptation, computer software, or bysimilar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similarterms, even if they are not identified as such, is not to be taken as an expression ofopinion as to whether or not they are subject to proprietary rights.

springer.com

Printed on acid‐free paper SPIN: 12084159 – 5 4 3 2 1 0

Page 4: Prebiotics and Probiotics Science and Technology978-0-387... · 2017-08-28 · prebiotics and probiotics field, covering their taxonomy, the potential beneficial effects to humans

To Elie Metchnikoff, Glenn Gibson and Marcel Roberfoid for originating

the concept of probiotics and prebiotics

Page 5: Prebiotics and Probiotics Science and Technology978-0-387... · 2017-08-28 · prebiotics and probiotics field, covering their taxonomy, the potential beneficial effects to humans

Preface

With growing consumer and industrial interest in self-care and integrative medi-

cine, our understanding of the relationship between health and diet has grown

stronger. As a result, the market for functional foods, dietary supplements and

nutraceuticals is continuing to expand rapidly. Among these products, probiotics

and prebiotics have carved their own special niche because of their scientifically-

supported health promoting properties, and have been in the forefront of re-

search over the past twenty years or so. This is driven by the realisation that the

gut microbiota can play a critical role in human health. Important functions of

the gut microbiota include the inhibition of the colonisation of the gut by

potentially pathogenic microorganisms, the microbial fermentation of substrates

yielding metabolic products which can serve as sources of energy for the gut cell

wall, and the modulation of the immune system. A substantial amount of

research has shown that the human gut microbiota can be modulated using

probiotics and prebiotics leading to various beneficial effects.

The prebiotics and probiotics area is a fast evolving field that attracts

significant interest by both the academic and industrial communities. As a

result, a substantial amount of research is generated every year. The use

of post-genomics, encompassing transcriptomics, proteomics, metabolomics

and meta-genomics has helped greatly in making significant advances in the

field, as they provide the means to carry out in-depth studies of the mechanisms

involved and of the beneficial effects likely to be exerted to the host. The target of

many probiotics and prebiotics is the prevention and treatment of disorders

associated with the gastrointestinal tract (GIT), including infectious, traveller’s,

and antibiotic-associated diarrhoeas, Clostridium difficile infections, and consti-

pation. They have also been suggested as therapeutic agents against irritable

bowel syndrome and inflammatory bowel diseases. An increasing amount of

evidence from in vitro and in vivo studies suggests that they are effective in

the prevention of atopic allergies and may have potential anti-carcinogenic

effects. In addition to the above, there has been considerable interest in

# Springer Science+Business Media LLC, 2009

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extra-intestinal application of probiotics and prebiotics, such as in urogenital

infections and oral health, and in their applications in animals.

This book will give a detailed and up to date account of the advances in the

prebiotics and probiotics field, covering their taxonomy, the potential beneficial

effects to humans and animals, the proposed mechanisms of actions, the molec-

ular techniques used, and the challenges faced in manufacturing such products.

The book consists of thirty one chapters. It starts with an introduction into the

gastrointestinal tract ecosystem and its interaction with prebiotics and probiotics

(Chapter 1), it then describes the molecular techniques used for analysing the

complex gut ecosystem (Chapters 2 and 3), and discusses the design of human

trials for evaluating their efficacy (Chapter 4). It then moves into the area of

prebiotics and starts off by discussing the mechanisms of prebiotic impact on

health (Chapter 5); it then provides a systematic overview of established as well as

potential prebiotic oligosaccharides (Chapters 6 to 11). Each of these chapters

covers the processes used to manufacture these compounds, and critically dis-

cusses their efficacy based on published data from in vitro and in vivo studies. The

rest of the chapters in the prebiotics part cover the application of prebiotics in

animals (Chapter 12), the analysis of prebiotic oligosaccharides (Chapter 13), and

their manufacture from biomass sources (Chapter 14). The book then focuses on

probiotics and begins with a taxonomic study of probiotic microorganisms

(Chapter 15). It then discusses their interaction in the human gut (Chapter 16),

provides an overview of functional genomic studies of probiotics (Chapter 17),

discusses the challenges in their manufacture (Chapter 18) and in their addition

to foods (Chapter 19), and presents the encapsulation methodologies used

to improve probiotic delivery (Chapter 20). Subsequent chapters cover the

potential beneficial effects of probiotics focusing on antibiotic-associated and

Clostridium difficile diarrhea (Chapter 21), infectious and traveller’s diarrhea

(Chapter 22), immune modulation (Chapter 23), chronic gastrointestinal infec-

tions (Chapter 24), allergic diseases (Chapter 25), potential anti-carcinogenic

effects (Chapter 26), urogenital applications (Chapter 27), oral health

(Chapter 28), and on the development of mucosal vaccines based on lactic acid

bacteria (Chapter 29). Finally, the remaining chapters cover the applications of

probiotics in livestock animals (Chapter 30) and safety aspects of probiotics

(Chapter 31).

viii Preface

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The aim of this book is to provide a comprehensive overview of the research

in the field of prebiotics and probiotics covering the achievements, challenges and

future needs. As such, we hope that this book will be a valuable reference to both

researchers and industrialists working in the field.

Dimitris Charalampopoulos

Bob Rastall

Preface ix

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Table of Contents

Volume 1

Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

1 Using Probiotics and Prebiotics to Manage the Gastrointestinal

Tract Ecosystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Randal Buddington

2 Molecular Tools for Investigating the Gut Microbiota . . . . . . . . . 33

Christophe Lay

3 Post-Genomics Approaches towards Monitoring Changes

within the Microbial Ecology of the Gut . . . . . . . . . . . . . . . . . . . . 79

Kieran M. Tuohy . Leticia Abecia . Eddie R. Deaville . Francesca Fava .

Annett Klinder . Qing Shen

4 Designing Trials for Testing the Efficacy of Pre- Pro- and

Synbiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Stephen Lewis . Charlotte Atkinson

5 Mechanisms of Prebiotic Impact on Health . . . . . . . . . . . . . . . . . 135

H. Steed . S. Macfarlane

6 Fructan Prebiotics Derived from Inulin . . . . . . . . . . . . . . . . . . . . 163

Douwina Bosscher

7 Galacto-Oligosaccharide Prebiotics . . . . . . . . . . . . . . . . . . . . . . . 207

George Tzortzis . Jelena Vulevic

# Springer ScienceþBusiness Media, LLC 2009

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8 Prebiotic Potential of Xylo-Oligosaccharides . . . . . . . . . . . . . . . . 245

H. Makelainen . M. Juntunen . O. Hasselwander

9 Resistant Starch and Starch-Derived Oligosaccharides as

Prebiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

A. Adam-Perrot . L. Gutton . L. Sanders . S. Bouvier . C. Combe .

R. Van Den Abbeele . S. Potter . A. W. C. Einerhand

10 Oligosaccharides Derived from Sucrose . . . . . . . . . . . . . . . . . . . . 293

Pierre F. Monsan . Francois Ouarne

11 Prebiotic Potential of Polydextrose . . . . . . . . . . . . . . . . . . . . . . . 337

Julian D. Stowell

12 Prebiotics in Companion and Livestock Animal Nutrition . . . . . 353

Kathleen A. Barry . Brittany M. Vester . George C. Fahey, Jr.

13 Analysis of Prebiotic Oligosaccharides . . . . . . . . . . . . . . . . . . . . . 465

M. L. Sanz . A. I. Ruiz-Matute . N. Corzo . I. Martınez-Castro

14 Manufacture of Prebiotics from Biomass Sources . . . . . . . . . . . . 535

Patricia Gullon . Beatriz Gullon . Andres Moure . Jose Luis Alonso .

Herminia Domınguez . Juan Carlos Parajo

15 Taxonomy of Probiotic Microorganisms . . . . . . . . . . . . . . . . . . . 593

Giovanna E. Felis . Franco Dellaglio . Sandra Torriani

Volume 2

Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

16 Ecological Interactions of Bacteria in the Human Gut . . . . . . . . 641

Gwen Falony . Luc De Vuyst

xii Table of Contents

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17 Genomics of Probiotic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 683

Sarah O’Flaherty . Yong Jun Goh . Todd R. Klaenhammer

18 Manufacture of Probiotic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . 727

J. A. Muller . R. P. Ross . G. F. Fitzgerald . C. Stanton

19 Some Technological Challenges in the Addition of Probiotic

Bacteria to Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763

Claude P. Champagne

20 Micro-Encapsulation of Probiotics . . . . . . . . . . . . . . . . . . . . . . . . 807

Jean-Antoine Meiners

21 Probiotics and Antibiotic-Associated Diarrhea and

Clostridium difficile Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827

Christina M. Surawicz

22 Probiotics for Infectious Diarrhea and Traveler’s

Diarrhea – What Do We Really Know? . . . . . . . . . . . . . . . . . . . . . 847

Patricia L. Hibberd

23 Immunological Effects of Probiotics and their Significance to

Human Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901

Harsharn S. Gill . Sunita Grover . Virender K. Batish . Preet Gill

24 Probiotics and Chronic Gastrointestinal Disease . . . . . . . . . . . . . 949

Francisco Guarner

25 Probiotics and Allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 977

Seppo Salminen . Erika Isolauri

26 Potential Protective Effects of Probiotics and Prebiotics

Against Colorectal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 997

Philip Allsopp . Ian Rowland

27 Urogenital Applications of Probiotic Bacteria . . . . . . . . . . . . . . 1049

Gregor Reid

Table of Contents xiii

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28 Prebiotics and Probiotics and Oral Health . . . . . . . . . . . . . . . . . 1067

J. H. Meurman

29 Development of Mucosal Vaccines Based on Lactic Acid

Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1099

Luis G. Bermudez-Humaran . Silvia Innocentin . Francois Lefevre .

Jean-Marc Chatel . Philippe Langella

30 Application of Prebiotics and Probiotics in Livestock . . . . . . . . 1123

James W. Collins . Roberto M. La Ragione . Martin J. Woodward .

Laura E. J. Searle

31 Safety Assessment of Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . 1193

Sampo J. Lahtinen . Robert J. Boyle . Abelardo Margolles . Rafael Frias .

Miguel Gueimonde

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237

xiv Table of Contents

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Editors

Dimitris Charalampopoulos

Department of Food Biosciences

University of Reading

Whiteknights, Reading

UK

[email protected]

Robert Rastall

Department of Food Biosciences

University of Reading

Whiteknights, Reading

UK

[email protected]

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List of Contributors

Leticia Abecia

Food Microbial Sciences

Department of Food Biosciences

University of Reading

Reading, UK

A. Adam-Perrot

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq

France

Philip Allsopp

Northern Ireland Centre for Food

and Health

Centre for Molecular Biosciences

University of Ulster

Coleraine, UK

Jose Luis Alonso

Department of Chemical Engineering

University of Vigo

As Lagoas, Ourense, Spain

Charlotte Atkinson

Lecturer in Nutrition

University of Bristol and United Bristol

Healthcare, NHS Trust

Bristol, Avon, UK

Kathleen A. Barry

Department of Animal Sciences

University of Illinois at

Urbana-Champaign

Champaign, IL, USA

Virender K. Batish

National Dairy Research Institute

Karnal (Haryana), India

Luis G. Bermudez-Humaran

Unite d’Ecologie et de

Physiologie du Systeme Digestif

INRA 0910, Jouy-en-Josas, France

Douwina Bosscher

Laboratory of Functional Food Science

and Nutrition

Department of Pharmaceutical Sciences

University of Antwerp

Wilrijk, Belgium

S. Bouvier

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq, France

Robert J. Boyle

Department of Paediatrics

Imperial College, London, UK

# Springer ScienceþBusiness Media, LLC 2009

Page 16: Prebiotics and Probiotics Science and Technology978-0-387... · 2017-08-28 · prebiotics and probiotics field, covering their taxonomy, the potential beneficial effects to humans

Randal Buddington

Health and Sports Sciences

University of Memphis

Memphis, Tennessee, USA

Claude P. Champagne

Food R & D Centre

Agriculture and Agri-Food Canada

Casavant, St. Hyacinthe

QC, Canada

Jean-Marc Chatel

Unite d’Ecologie et de

Physiologie du Systeme Digestif

INRA 0910, Jouy-en-Josas, France

James W. Collins

Department of Bacterial Diseases

VLA (Veterinary Laboratories Agency)

Woodham Lane

New Haw, UK

C. Combe

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq, France

N. Corzo

Instituto de Fermentaciones Industriales

(C.S.I.C.) Juan de la Cierva

Madrid, Spain

Luc De Vuyst

Research Group of Industrial

Microbiology and Food Biotechnology

Department of Applied Biological

Sciences and Engineering

Vrije Universiteit Brussel

Pleinlaan, Brussels, Belgium

Eddie R. Deaville

Food Microbial Sciences

Department of Food Biosciences

University of Reading

Reading, UK

Franco Dellaglio

Dipartimento Scientifico e Technologico

Facolta di Scienze MM. FF. NN.

Universita degli Studi di Verona

Verona, Italy

Herminia Domınguez

Department of Chemical Engineering

University of Vigo

As Lagoas, Ourense, Spain

A. W. C. Einerhand

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq, France

George C. Fahey, Jr.

Department of Animal Sciences

University of Illinois at

Urbana-Champaign

Champaign, IL, USA

Gwen Falony

Research Group of Industrial

Microbiology and Food Biotechnology

Department of Applied Biological

Sciences and Engineering

Vrije Universiteit Brussel

Pleinlaan, Brussels, Belgium

Francesca Fava

Food Microbial Sciences

Department of Food Biosciences

xviii List of Contributors

Page 17: Prebiotics and Probiotics Science and Technology978-0-387... · 2017-08-28 · prebiotics and probiotics field, covering their taxonomy, the potential beneficial effects to humans

University of Reading

Reading, UK

Giovanna E. Felis

Dipartimento di Scienze Tecnologie

e Mercati della Vite e del Vino

Facolta di Scienze MM. FF. NN.

Universita degli Studi di Verona

Verona, Italy

G. F. Fitzgerald

Department of Microbiology

University College Cork

Cork, Co. Cork, Ireland

Rafael Frias

Central Animal Laboratory

University of Turku

Finland

Harsharn S. Gill

Primary Industries Research Victoria

Department of Primary Industries

Australia and Victoria University

Werribee, Victoria, Australia

Preet Gill

School of Medicine

Griffith University

Southport, Australia

Yong Jun Goh

Department of Food

Bioprocessing and Nutrition Sciences

and

Southeast Dairy Foods

Research Center

North Carolina State University

Raleigh, NC, USA

Sunita Grover

National Dairy Research Institute

Karnal (Haryana), India

Francisco Guarner

Digestive System Research Unit

University Hospital Vall d’Hebron

Barcelona, Spain

Miguel Gueimonde

Department of Microbiology and

Biochemistry of Dairy Products

Instituto de Productos Lacteos de

Asturias. CSIC

Asturias, Spain

Beatriz Gullon

Department of Chemical Engineering

University of Vigo

As Lagoas, Ourense, Spain

Patricia Gullon

Department of Chemical Engineering

University of Vigo

As Lagoas, Ourense, Spain

L. Gutton

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq, France

O. Hasselwander

Technology & Business Development

Danisco (UK) Limited

Redhill, UK

List of Contributors xix

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Patricia L. Hibberd

Director, Center for Global Health

Research

and

Departments of Public Health and

Family Medicine,

Medicine, and Pediatrics

Tufts University School of Medicine

Boston, MA, USA

Silvia Innocentin

Unite d’Ecologie et de Physiologie du

Systeme Digestif INRA 0910

Jouy-en-Josas, France

andUnite de Virologie et Immunologie

Moleculaires

Domaine de Vilvert

INRA, UR892, Jouy-en-Josas, France

Erika Isolauri

Department of Paediatrics

University of Turku

and

Turku University Central Hospital

Finland

M. Juntunen

Danisco Finland Health & Nutrition

Kantvik, Finland

Todd R. Klaenhammer

Department of Food

Bioprocessing, and Nutrition Sciences;

and

Southeast Dairy Foods Research Center

North Carolina State University

Raleigh, NC, USA

Annett Klinder

Food Microbial Sciences

Department of Food Biosciences

University of Reading

Reading, UK

Roberto M. La Ragione

Dept. of Food & Environmental Safety

Veterinary Laboratories Agency (Defra)

Woodham Lane, KT15 3NB, Addlestone

Surrey, UK

Sampo J. Lahtinen

Danisco Finland Health & Nutrition

Kantvik, Finland

Philippe Langella

Unite d’Ecologie et de Physiologie du

Systeme Digestif

INRA 0910, Jouy-en-Josas, France

Christophe Lay

Department of Microbiology and

Immunology

University of Otago

Dunedin, New Zealand

Francois Lefevre

Unite de Virologie et Immunologie

Moleculaires

INRA, UR892, Domaine de Vilvert

Jouy-en-Josas, France

Stephen Lewis

Derriford Hospital

Plymouth, Devon, UK

xx List of Contributors

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S. Macfarlane

Microbiology and Gut Biology Group

Ninewells Hospital Medical School

Dundee, UK

Abelardo Margolles

Department of Microbiology and

Biochemistry of Dairy Products

Instituto de Productos Lacteos de

Asturias. CSIC

Asturias, Spain

I. Martınez-Castro

Instituto de Quımica Organica General

(C.S.I.C.)

Juan de la Cierva Madrid, Spain

Jean-Antoine Meiners

Meiners Commodity Consultants S.A

Colombier, Switzerland

J. H. Meurman

Institute of Dentistry of Helsinki

and

Department of Oral and Maxillofacial

Disease

Helsinki University Central Hospital

Finland

H. Makelainen

Danisco Finland Health & Nutrition

Kantvik, Finland

Pierre F. Monsan

LISBP-INSA, UMR CNRS 5504

UMR INRA, Toulouse cedex, France

Andres Moure

Department of Chemical Engineering

University of Vigo

As Lagoas, Ourense, Spain

J. A. Muller

Teagasc

Moorepark Food Research Centre

Fermoy, Co. Cork, Ireland

Sarah O’Flaherty

Department of Food

Bioprocessing and Nutrition Sciences;

and

Southeast Dairy Foods Research Center

North Carolina State University

Raleigh, NC, USA

Francois Ouarne

CRITT Bioindustries, INSA

Toulouse cedex, France

Juan Carlos Parajo

Department of Chemical Engineering

University of Vigo

As Lagoas, Ourense, Spain

S. Potter

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq, France

Gregor Reid

Canadian Research & Development

Centre for Probiotics

Lawson Health Research Institute

and

List of Contributors xxi

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Department of Microbiology &

Immunology

University of Western Ontario

ON, Canada

R. P. Ross

Teagasc

Moorepark Food Research Centre

Fermoy, Co. Cork, Ireland

Ian Rowland

Hugh Sinclair Unit for Human Nutrition

Department of Food Biosciences

University of Reading

Whiteknights, Reading, UK

A. I. Ruiz-Matute

Instituto de Quımica Organica General

(C.S.I.C.)

Juan de la Cierva Madrid, Spain

Seppo Salminen

Functional Foods Forum

University of Turku

20014 Turku, Finland

L. Sanders

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq, France

M. L. Sanz

Instituto de Quımica Organica General

(C.S.I.C.)

Juan de la Cierva Madrid, Spain

Laura E. J. Searle

Department of Bacterial Diseases

VLA (Veterinary Laboratories Agency)

Woodham Lane

New Haw, UK

Qing Shen

Food Microbial Sciences

Department of Food Biosciences

University of Reading

Whiteknights, Reading, UK

C. Stanton

Teagasc

Moorepark Food Research Centre

Fermoy, Co. Cork, Ireland

H. Steed

Microbiology and Gut Biology Group

Ninewells Hospital Medical School

Dundee, UK

Julian D. Stowell

Danisco Sweeteners

Danisco (UK) Ltd

Redhill, Surrey, UK

Christina M. Surawicz

Division of Gastroenterology

Department of Medicine

School of Medicine

University of Washington

Seattle, WA, USA

Sandra Torriani

Dipartimento di Scienze Technologie

e Mercati della Vite e del Vino

Universita degli Studi di Verona,

Verona, Italy

xxii List of Contributors

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Kieran M. Tuohy

Food Microbial Sciences

Department of Food Biosciences

Food Biosciences and Pharmacy

University of Reading

Reading, UK

George Tzortzis

Clasado Ltd

Wolverton Mill

Milton Keynes, UK

R. Van Den Abbeele

TATE & LYLE Innovation Centre

Parc Scientific de la Haute Borne

Villeneuve d’Ascq, France

Brittany M. Vester

Department of Animal Sciences

University of Illinois at

Urbana-Champaign

Champaign, IL, USA

Jelena Vulevic

Department of Food Biosciences

University of Reading

Whiteknights, Reading, UK

Martin J. Woodward

Department of Bacterial Diseases

VLA (Veterinary Laboratories Agency)

Woodham Lane

New Haw, UK

List of Contributors xxiii

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