Diet, Microbiome and Health

Diet, Microbiome and Health

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Diet, Microbiome and Health Handbook of Food Bioengineering, Volume 11

Edited by

Alina Maria Holban Alexandru Mihai Grumezescu

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2018 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-811440-7 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisition Editor: Nina Bandeira Editorial Project Manager: Jaclyn Truesdell Production Project Manager: Punithavathy Govindaradjane Designer: Matthew Limbert Typeset by Thomson Digital

Contents List of Contributors..............................................................................................xv Foreword..........................................................................................................xvii Series Preface.....................................................................................................xix Preface for Volume 11: Diet, Microbiome and Health........................................... xxiii

Section 1: State of the Art and Applications........................................... 1 Chapter 1: Gut Microbes: The Miniscule Laborers in the Human Body........................3 Suma Sarojini 1 Introduction............................................................................................................ 3 2 Major Players of the Gut Microbiome................................................................... 4 3 Functions of the Gut Flora..................................................................................... 5 4 Infant Gut Flora.................................................................................................... 10 5 Evolution of Gut Microbial Flora........................................................................ 12 6 Diet in Shaping Composition of Gut Flora.......................................................... 13 7 Gut Microbiota and Diseases............................................................................... 16 7.1 Gut Bacteria and Obesity....................................................................................16 7.2 Gut Bacteria and Diabetes...................................................................................18 7.3 Gut Bacteria and Bowel Disorders......................................................................18 7.4 Gut Bacteria and Cancer.....................................................................................18 8 Gut Flora and Brain Functions............................................................................. 20 9 Effect of Antibiotics on the Gut Microbiome...................................................... 23 10 Fortifying Gut Flora............................................................................................. 25 11 Conclusions.......................................................................................................... 25 References.................................................................................................................. 26

Chapter 2: Role of Probiotics Toward the Improvement of Gut Health With Special Reference to Colorectal Cancer.................................................35 Mian K. Sharif, Sana Mahmood, Fasiha Ahsan 1 Probiotics............................................................................................................... 35 1.1 Introduction...........................................................................................................35 1.2 Types of Probiotics................................................................................................36 v

Contents 1.3 Role as Functional Food........................................................................................36 1.4 Probiotics as Food Ingredients..............................................................................37 1.5 Selection Criteria...................................................................................................37 1.6 Effective Dosage...................................................................................................38 1.7 Incorporation Criteria of Probiotics in Foods.......................................................39 2 Probiotics as Health Promoters.............................................................................. 39 2.1 Antimicrobial Activity...........................................................................................40 2.2 Immunomodulation...............................................................................................41 2.3 Gut Microbiota and Probiotics..............................................................................41 2.4 Diarrhea.................................................................................................................42 2.5 Hypercholesterolemia...........................................................................................42 2.6 Lactose Intolerance and Stomach Ulcers..............................................................42 2.7 Mineral Absorption...............................................................................................43 2.8 Inflammatory Bowel Disease................................................................................43 3 Probiotics and Colorectal Cancer........................................................................... 44 3.1 Pathophysiology of Colorectal Cancer..................................................................44 3.2 Colorectal Cancer and Diet...................................................................................45 3.3 Anticancer Activity of Probiotics..........................................................................45 4 Conclusions............................................................................................................ 48 References.................................................................................................................. 48

Section 2: Probiotics and Prebiotics..................................................... 51 Chapter 3: Therapeutic Aspects of Probiotics and Prebiotics....................................53 Asif Ahmad, Sumaira Khalid 1 Introduction............................................................................................................ 53 2 The Concept of Probiotics..................................................................................... 54 2.1 Taxonomy of Probiotic Microorganisms...............................................................54 2.2 Screening, Identification, and Characterization of Probiotic Microorganisms........55 3 Therapeutic Effects of Probiotics........................................................................... 57 3.1 Traveler’s Diarrhea..............................................................................................58 3.2 Acute Infectious Diarrhea...................................................................................59 3.3 Antibiotic-Associated Diarrhea...........................................................................59 3.4 Irritable Bowel Syndrome (IBS).........................................................................59 3.5 Crohn’s Disease...................................................................................................60 3.6 Ulcerative Colitis (UC).......................................................................................60 3.7 Lactose Intolerance.............................................................................................61 3.8 Heliobacter pylori Infections..............................................................................62 3.9 Hypercholesterolemia and Coronary Heart Diseases..........................................62 3.10 Diabetes...............................................................................................................63 3.11 Obesity................................................................................................................64 3.12 Colon Cancer.......................................................................................................65 4 Prebiotics and Synbiotics....................................................................................... 65 4.1 Types of Prebiotics................................................................................................65 4.2 Sources of Prebiotics.............................................................................................68 vi

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5 Therapeutic Effects of Prebiotics........................................................................... 69 5.1 Prebiotics as Dietary Fiber..................................................................................69 5.2 Effect on Influenza..............................................................................................70 5.3 Hyperglycemia/Diabetes.....................................................................................70 5.4 Obesity................................................................................................................71 5.5 Hepatic Encephalopathy......................................................................................71 5.6 Gastric and Colorectal Cancer............................................................................72 5.7 Ulcerative Colitis.................................................................................................73 5.8 Hypercholesterolemia.........................................................................................73 5.9 Immunomodulation.............................................................................................73 5.10 Bioavailability and Uptake of Minerals..............................................................74 5.11 Diarrhea...............................................................................................................74 5.12 Irritable Bowel Syndrome...................................................................................74 5.13 Prebiotics and Crohn’s Disease...........................................................................75 5.14 Atopic Dermatitis................................................................................................75 6 Synbiotics............................................................................................................... 75 7 Theuraptic Effects of Synbiotics............................................................................ 76 7.1 β-Glucan in Synbiotic Foods...............................................................................76 7.2 Effect on Immune System...................................................................................77 7.3 Diarrhea...............................................................................................................77 7.4 Mineral Absorption.............................................................................................78 7.5 Diarrheal Disorders.............................................................................................78 7.6 Hypercholesterolemia.........................................................................................79 7.7 Cancer.................................................................................................................79 7.8 Allergies..............................................................................................................80 7.9 Inflammatory Diseases........................................................................................80 7.10 Synbiotics and Obesity........................................................................................81 8 Conclusions............................................................................................................ 82 References.................................................................................................................. 82

Chapter 4: Lactic Acid Bacteria Beverage Contribution for Preventive Medicine and Nationwide Health Problems in Japan.............................................93 Akira Kanda, Masatoshi Hara 1 Introduction............................................................................................................ 93 1.1 Historical and General Remarks on Lactobacillus casei Strain Shirota�� 93 1.2 Structures and Functions of the Intestine..............................................................94 1.3 Pathogens in the Intestine......................................................................................95 1.4 Immunomodulatory Environment in Intestine......................................................96 2 Tolerance to Gastric Acid and Bile, and Viability in the Intestinal Tract��97 3 Modification of Gastrointestinal Function: Improvement of Diarrhea and Constipation��98 3.1 Constipation..........................................................................................................98 3.2 Diarrhea.................................................................................................................99 vii

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4 Metabolism: Changing Urinary Excretion of Nitrogen....................................... 99 5 Immunomodulation............................................................................................ 101 6 Prevention of Cancer.......................................................................................... 102 7 Effect on Inflammatory Bowel Disease............................................................. 102 8 Protection Against Infection.............................................................................. 103 9 Global Burden Diseases in Japan....................................................................... 105 10 Concluding Remarks.......................................................................................... 107 References................................................................................................................ 108

Chapter 5: Gut Microbiota Alterations in People With Obesity and Effect of Probiotics Treatment.....................................................111 Edwin E. Martínez Leo, Armando M. Martín Ortega, Maira R. Segura Campos 1 Obesity: A Multifactorial Disease........................................................................ 111 1.1 Obesity and Inflammatory Process......................................................................112 2 Human Microbiome............................................................................................. 116 2.1 Human Microbiome Composition.......................................................................117 2.2 Microbiota and Obesity.......................................................................................119 2.3 Probiotics’ Role in Obesity.................................................................................121 3 Conclusions.......................................................................................................... 125 References................................................................................................................ 125

Chapter 6: Safety of Probiotics...........................................................................131 Dorota Zielin´ska, Barbara Sionek, Danuta Kołoz˙yn-Krajewska 1 Introduction.......................................................................................................... 131 2 Regulatory Systems............................................................................................. 132 3 Most Frequent and Important Adverse Events of Probiotics............................... 137 3.1 Risk of Probiotic Infections................................................................................137 3.2 Safety of Probiotics in Healthy Populations.......................................................139 3.3 Safety of Probiotics in Ill and Immunocompromised Patients............................139 3.4 Deleterious Metabolic Activities.........................................................................142 3.5 Gene Transfer......................................................................................................146 4 Safety Assessment Studies................................................................................... 151 5 Proposed Evaluation of the Safety of Probiotics by FAO/WHO......................... 152 6 Conclusions.......................................................................................................... 156 References................................................................................................................ 157

Section 3: Nutritional Aspects.......................................................... 163 Chapter 7: Flavonoids in Foods and Their Role in Healthy Nutrition.......................165 Silvia Tsanova-Savova, Petko Denev, Fanny Ribarova 1 Introduction.......................................................................................................... 165 2 Chemistry and Classification of Flavonoids........................................................ 166 3 Antioxidant Activity of Flavonoids...................................................................... 168 3.1 Oxidants and Antioxidants in the Body..............................................................168 3.2 Mechanisms of the Antioxidant Activity of Flavonoids......................................169 viii

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4 Bioavailability of Flavonoids............................................................................... 171 5 Physiological Role and Pharmacological Activities of Flavonoids..................... 172 5.1 Flavonoids and Cardiovascular Diseases............................................................173 5.2 Flavonoids and Cancer........................................................................................174 5.3 Flavonoids and Obesity.......................................................................................176 5.4 Flavonoids and Type 2 Diabetes Mellitus...........................................................176 5.5 Flavonoids and Alzheimer’s Disease...................................................................177 6 Flavonoid Content and Antioxidant Activity of Selected Bulgarian Plant Foods��178 6.1 Flavonoid Composition and Antioxidant Activity of Bulgarian fruits�� 182 6.2 Flavonoid Composition and Antioxidant Activity of Bulgarian Vegetables�� 185 7 Conclusions.......................................................................................................... 192 References................................................................................................................ 193

Chapter 8: The Role of Milk Oligosaccharides in Host–Microbial Interactions and Their Defensive Function in the Gut............................199 Sinead T. Morrin, Jane A. Irwin, Rita M. Hickey 1 Introduction.......................................................................................................... 199 2 Effect of Oligosaccharides on Pathogen Colonization........................................ 201 3 Effects of Oligosaccharides on Commensal Colonization................................... 205 4 Immunomodulation by Oligosaccharides............................................................ 210 5 Mucin Expression, Defensive Function, and Indirect Effects of Oligosaccharides��214 6 Developing Areas................................................................................................. 217 6.1 Allergy Intervention by Oligosaccharides...........................................................217 6.2 Influence of Oligosaccharides on Brain Development........................................219 7 Conclusions and Future Perspectives................................................................... 222 References................................................................................................................ 223

Chapter 9: Nutritional Yeast Biomass: Characterization and Application................237 Monika E. Jach, Anna Serefko 1 Introduction.......................................................................................................... 237 2 Saccharomyces cerevisiae Preparations............................................................... 238 2.1 Saccharomyces cerevisiae β-Glucans.................................................................239 2.2 Se-Enriched Saccharomyces cerevisiae Yeasts...................................................243 2.3 Cr-Enriched Saccharomyces cerevisiae Yeasts....................................................246 2.4 Safety Issues Concerning Saccharomyces cerevisiae Preparations....................248 3 Saccharomyces boulardii as a Probiotic Yeast..................................................... 249 4 Yarrowia lipolytica as a Source of Bioactive Compounds................................... 252 5 Nutritional Benefits of Other Yeast Strains.......................................................... 257 6 Conclusions.......................................................................................................... 259 References................................................................................................................ 260 ix

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Section 4: Health, Disease, and Therapy........................................... 271 Chapter 10: Effect of Diet on Gut Microbiota as an Etiological Factor in Autism Spectrum Disorder.................................................273 Afaf El-Ansary, Hussain Al Dera, Rawan Aldahash 1 Introduction.......................................................................................................... 273 2 Factors Affecting Infant Gut Microbiota............................................................. 275 2.1 Microbial Characteristics in Amniotic Fluid, Placenta, and Colostrum..............275 2.2 Early Colonization and Homeostasis of Gut Microbiota....................................276 3 Gut Microbiota of Autistic Patients..................................................................... 278 4 Dietary Factors Affecting the Gut Microbiota..................................................... 282 4.1 Undernutrition and Gut Microbiota....................................................................282 4.2 Diet Composition and Gut Microbiota................................................................283 5 Manipulation of Imbalanced Gut Microbiota...................................................... 286 5.1 Role of Colostrum...............................................................................................286 6 Role of Probiotics, Prebiotics, and Symbiotics.................................................... 287 7 Conclusions.......................................................................................................... 291 References................................................................................................................ 291

Chapter 11: Dietary Fibers: A Way to a Healthy Microbiome................................299 Prerna Sharma, Chetna Bhandari, Sandeep Kumar, Bhoomika Sharma, Priyanka Bhadwal, Navneet Agnihotri 1 Introduction.......................................................................................................... 299 2 Gut Microbiota..................................................................................................... 300 2.1 Types of Human Gut Microbiota.........................................................................301 3 Dietary Fiber and Gut Microbiota........................................................................ 304 4 Types of Dietary Fiber......................................................................................... 308 4.1 Resistant Starch.................................................................................................308 4.2 Resistant Dextrins.............................................................................................308 4.3 Arabinoxylans...................................................................................................309 4.4 Fructans.............................................................................................................309 4.5 Cellulose............................................................................................................310 4.6 Hemicellulose....................................................................................................310 4.7 Pectins...............................................................................................................311 4.8 Gums.................................................................................................................311 4.9 β-Glucans..........................................................................................................312 4.10 Lignin................................................................................................................312 4.11 Chitin and Chitosan...........................................................................................313 5 Interplay Between Gut Microbiota and Host Metabolism................................... 313 5.1 Metabolism of Dietary Fiber...............................................................................313 5.2 Short-Chain Fatty Acid Metabolism...................................................................314 5.3 Utilization of Host-Derived Substrates...............................................................315 5.4 Hydrogen Disposal..............................................................................................316 5.5 Vitamin Synthesis................................................................................................317 x

Contents 5.6 Amino Acid Metabolism...................................................................................317 5.7 Regulation of Fatty Acid Metabolism...............................................................318 5.8 Regulation of Cholesterol Homeostasis by Bile Acid Metabolism...................319 5.9 Xenobiotic Metabolism.....................................................................................321 5.10 Host Immunity and Gut Microbiota..................................................................322 6 Role of Dietary Fiber in Disease Prevention....................................................... 325 6.1 Cardiovascular Disease.......................................................................................325 6.2 Inflammatory Bowel Diseases.............................................................................326 6.3 Cancer.................................................................................................................328 6.4 Obesity and Diabetes Mellitus............................................................................331 6.5 Nonalcoholic Fatty Liver Disease.......................................................................333 7 Conclusions.......................................................................................................... 334 References................................................................................................................ 336

Chapter 12: Effects of the Gut Microbiota on Autism Spectrum Disorder................347 Nalan H. Nog˘ay 1 Introduction.......................................................................................................... 347 2 Gut Microbiota..................................................................................................... 348 2.1 Gut Bacteria and the Immune System.................................................................349 2.2 Gut Bacteria Benefit the Host.............................................................................349 2.3 Dietary Intake and Gut Microbiota.....................................................................349 3 Gut–Brain Axis and the Microbiota..................................................................... 350 3.1 Effect of Microbiota on the Gastrointestinal System..........................................351 3.2 Effect of Gastrointestinal System Physiology on Microbiota.............................351 3.3 Effect of Brain on Microbiota.............................................................................352 3.4 Effect of Microbiota on Brain and Behavior.......................................................352 3.5 Intestinal Microbiota and Central Nervous System............................................353 3.6 Potential Mechanisms of Microbiota’s Effect on the Function of Central Nervous System�� 354 4 The Microbiota–Gut–Brain Axis and Autism Spectrum Disorder....................... 355 4.1 Nutritional Problems in Autism Spectrum Disorder...........................................355 4.2 Animal and Human Studies.................................................................................357 4.3 Disturbance in the Microbiota–Gut–Brain Axis Contributing to Autism�� 358 5 Treatments to Modify the Gut Microbiota in Order to Recover the Symptoms in Autism Spectrum Disorder��362 5.1 Antibiotics...........................................................................................................362 5.2 Antifungals..........................................................................................................363 5.3 Probiotics.............................................................................................................363 5.4 Digestive Enzymes..............................................................................................364 5.5 Vitamins...............................................................................................................364 5.6 Unprocessed and Fermented Nutrients...............................................................364 5.7 Diet Treatments...................................................................................................365 6 Summary.............................................................................................................. 366 References................................................................................................................ 366 xi

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Chapter 13: Diet, Microbiome, and Neuropsychiatric Disorders.............................369 Gabriel A. Javitt, Daniel C. Javitt 1 Introduction........................................................................................................ 369 1.1 Microbiome.......................................................................................................370 1.2 Dietary Effect on Microbiome..........................................................................372 2 Mechanisms in Which the Microbiome Effects the Brain and Central Nervous System��374 2.1 Direct Diffusion Through the BBB...................................................................374 2.2 Circumventricular Organs.................................................................................374 2.3 Enteric Nervous System (ENS).........................................................................377 2.4 Oxidative Stress.................................................................................................377 2.5 Immune Modulation..........................................................................................378 3 Major Depression Disorder (MDD)................................................................... 379 3.1 Micronutrients...................................................................................................381 3.2 Oxidative Stress.................................................................................................381 3.3 ENS Effects.......................................................................................................382 3.4 Immune System–Mediated Effects...................................................................382 4 Schizophrenia..................................................................................................... 383 4.1 Immune System–Mediated Effects...................................................................384 5 Bipolar Disorder................................................................................................. 386 5.1 Oxidative Stress.................................................................................................387 5.2 Immune System–Mediated Effects...................................................................387 6 Anxiety Disorders.............................................................................................. 388 6.1 Circumventricular Organs.................................................................................388 6.2 ENS Effects.......................................................................................................389 7 Obsessive-Compulsive Disorder (OCD)............................................................ 390 7.1 Circumventricular Organs.................................................................................390 7.2 Immune System–Mediated Effects...................................................................391 8 Autism Spectrum Disorders (ASD)................................................................... 391 8.1 Micronutrients...................................................................................................393 8.2 Oxidative Stress.................................................................................................394 8.3 Immune System–Mediated Response...............................................................395 9 Conclusions........................................................................................................ 396 10 Conflict of Interest............................................................................................. 396 References................................................................................................................ 397

Section 5: Function and Safety......................................................... 407 Chapter 14: Gastrointestinal Exposome for Food Functionality and Safety..............409 Yuseok Moon 1 Gross Structure of Food-Associated Gastrointestinal Network........................... 409 2 Mutual Interaction Between Food Components and Gut Microbiota.................. 410 3 Crosstalk Between Food Contaminants and the Gut Microbiota: Potent Implications in Environmental IBD Etiology��412 xii

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4 Crosstalk Between Foodborne Toxins and Gut Pathogens: Cases in Mycotoxicoses��413 4.1 Effects of Mycotoxin Exposure on Pathogenesis of Gut Pathogenic Bacteria�� 413 4.2 Effects of Foodborne Mycotoxins on Enteropathogenic Viral Infection�� 415 5 Food and Drug Metabolism in the Gastrointestinal Tract.................................... 415 5.1 Host Cell-Derived Food and Drug Metabolism..................................................415 5.2 Gut Microbiota-Derived Metabolism of Food Components...............................416 6 Neuroendocrine Regulation in the Gastrointestinal Exposome........................... 418 6.1 EEC-modulated Immune Regulation in Response to Food Components and Gut Microbes�� 419 6.2 Diet/Gut Microbiota-Modulated Neuronal Regulation, Nutrient Sensing, and Energy Balance�� 419 7 Impact of Foodborne Pathogens and Microbial Toxins on Gastrointestinal Immunity��420 7.1 Impact of Gut Pathogens and Microbial Toxicants on the Gastrointestinal Barrier and Related Cellular Signals�� 420 7.2 Impact of Foodborne Pathogens and Microbial Toxicants on Gastrointestinal Immunity�� 424 7.3 Gastrointestinal Tolerance to Proinflammation Stimulation by Microbiota or Foodborne Xenobiotics Via the Regulatory Factors�� 425 8 Integrated Management of Food-Linked Gastrointestinal Exposome Networks��427 References................................................................................................................ 428

Chapter 15: Risk From Viral Pathogens in Seafood..............................................439 Samanta S. Khora 1 Introduction.......................................................................................................... 439 2 Risks From Pathogenic Microbes in Seafood...................................................... 440 3 Risks From Pathogenic Viruses in Seafood......................................................... 441 4 Historical Perspectives......................................................................................... 442 5 Seafood as Vehicles for Viruses........................................................................... 446 6 Outbreaks and Prevalence.................................................................................... 447 7 Viral Pathogens in Seafood.................................................................................. 449 7.1 Hepatitis A Virus (HAV)...................................................................................454 7.2 Poliovirus (PV)..................................................................................................455 7.3 Coxackieviruses (CVs)......................................................................................456 7.4 Echoviruses.......................................................................................................457 7.5 Aichivirus A (AiV-1).........................................................................................458 7.6 Norovirus (NoV)...............................................................................................459 7.7 Sapoviruses (SaV).............................................................................................460 7.8 Hepatitis E Virus (HEV)....................................................................................462 7.9 Rotavirus (RV)..................................................................................................463 7.10 Astroviruses (AstVs).........................................................................................464 xiii

Contents 7.11 Parvovirus (ParV)............................................................................................465 7.12 Adenoviruses (AdVs)......................................................................................466 8 Risk Factors....................................................................................................... 467 9 Detection and Diagnosis.................................................................................... 468 10 Risk Management.............................................................................................. 469 11 Conclusions........................................................................................................ 470 References................................................................................................................ 471

Index...............................................................................................................483

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List of Contributors Navneet Agnihotri Panjab University, Chandigarh, India Asif Ahmad Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Punjab, Pakistan Fasiha Ahsan National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan Hussain Al Dera King Saud bin Abdulaziz University for Health Sciences; King Abdullah International Medical Research Center, Riyadh, Saudi Arabia Rawan Aldahash King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia Priyanka Bhadwal Panjab University, Chandigarh, India Chetna Bhandari Panjab University, Chandigarh, India Maira R. Segura Campos Autonomous University of Yucatan, Mérida, Yucatán, México Petko Denev Institute of Organic Chemistry with Center of Phytochemistry, Plovdiv, Bulgaria Afaf El-Ansary King Saud University; Autism Research and Treatment Center, Riyadh, Saudi Arabia Masatoshi Hara The Japan Dietetic Association, Tokyo, Japan Rita M. Hickey Teagasc Food Research Centre, Fermoy, Ireland Jane A. Irwin University College Dublin, Dublin, Ireland Monika E. Jach The John Paul II Catholic University of Lublin, Lublin, Poland Daniel C. Javitt Columbia University Medical Center, New York, NY, United States Gabriel A. Javitt Technion University, Haifa, Israel Akira Kanda HANA Nutrition College, Tokyo, Japan Sumaira Khalid Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi; Government College University, Faisalabad, Punjab, Pakistan Samanta S. Khora VIT University, Vellore, Tamil Nadu, India Danuta Kołożyn-Krajewska Warsaw University of Life Sciences, Warsaw, Poland Sandeep Kumar Panjab University, Chandigarh, India Edwin E. Martínez Leo Autonomous University of Yucatan, Mérida, Yucatán, México Sana Mahmood National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan Yuseok Moon Department of Biomedical Sciences, Pusan National University, Yangsan; Immunoregulatory Therapeutics Group in Brain Busan 21 Project, Busan, South Korea Sinead T. Morrin Teagasc Food Research Centre, Fermoy; University College Dublin, Dublin, Ireland

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List of Contributors Nalan H. Nog˘ay Erciyes University, Kayseri, Turkey Armando M. Martín Ortega Autonomous University of Yucatan, Mérida, Yucatán, México Fanny Ribarova Medical College J. Filaretova, Medical University–Sofia, Sofia, Bulgaria Suma Sarojini Department of Life Sciences, Christ University, Bangalore, Karnataka, India Anna Serefko Medical University of Lublin, Lublin, Poland Mian K. Sharif National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan Bhoomika Sharma Panjab University, Chandigarh, India Prerna Sharma Panjab University, Chandigarh, India Barbara Sionek Warsaw University of Life Sciences, Warsaw, Poland Silvia Tsanova-Savova Medical College J. Filaretova, Medical University–Sofia, Sofia, Bulgaria Dorota Zielińska Warsaw University of Life Sciences, Warsaw, Poland

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Foreword In the last 50 years an increasing number of modified and alternative foods have been developed using various tools of science, engineering, and biotechnology. The result is that today most of the available commercial food is somehow modified and improved, and made to look better, taste different, and be commercially attractive. These food products have entered in the domestic first and then the international markets, currently representing a great industry in most countries. Sometimes these products are considered as life-supporting alternatives, neither good nor bad, and sometimes they are just seen as luxury foods. In the context of a permanently growing population, changing climate, and strong anthropological influence, food resources became limited in large parts of the Earth. Obtaining a better and more resistant crop quickly and with improved nutritional value would represent the Holy Grail for the food industry. However, such a crop could pose negative effects on the environment and consumer health, as most of the current approaches involve the use of powerful and broad-spectrum pesticides, genetic engineered plants and animals, or bioelements with unknown and difficult-to-predict effects. Numerous questions have emerged with the introduction of engineered foods, many of them pertaining to their safe use for human consumption and ecosystems, long-term expectations, benefits, challenges associated with their use, and most important, their economic impact. The progress made in the food industry by the development of applicative engineering and biotechnologies is impressive and many of the advances are oriented to solve the world food crisis in a constantly increasing population: from genetic engineering to improved preservatives and advanced materials for innovative food quality control and packaging. In the present era, innovative technologies and state-of-the-art research progress has allowed the development of a new and rapidly changing food industry, able to bottom-up all known and accepted facts in the traditional food management. The huge amount of available information, many times is difficult to validate, and the variety of approaches, which could seem overwhelming and lead to misunderstandings, is yet a valuable resource of manipulation for the population as a whole. The series entitled Handbook of Food Bioengineering brings together a comprehensive collection of volumes to reveal the most current progress and perspectives in the field of food engineering. The editors have selected the most interesting and intriguing topics, and have dissected them in 20 thematic volumes, allowing readers to find the description of xvii

Foreword basic processes and also the up-to-date innovations in the field. Although the series is mainly dedicated to the engineering, research, and biotechnological sectors, a wide audience could benefit from this impressive and updated information on the food industry. This is because of the overall style of the book, outstanding authors of the chapters, numerous illustrations, images, and well-structured chapters, which are easy to understand. Nonetheless, the most novel approaches and technologies could be of a great relevance for researchers and engineers working in the field of bioengineering. Current approaches, regulations, safety issues, and the perspective of innovative applications are highlighted and thoroughly dissected in this series. This work comes as a useful tool to understand where we are and where we are heading to in the food industry, while being amazed by the great variety of approaches and innovations, which constantly changes the idea of the “food of the future.” Anton Ficai, PhD (Eng) Department Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, Bucharest, Romania

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Series Preface The food sector represents one of the most important industries in terms of extent, investment, and diversity. In a permanently changing society, dietary needs and preferences are widely variable. Along with offering a great technological support for innovative and appreciated products, the current food industry should also cover the basic needs of an ever-increasing population. In this context, engineering, research, and technology have been combined to offer sustainable solutions in the food industry for a healthy and satisfied population. Massive progress is constantly being made in this dynamic field, but most of the recent information remains poorly revealed to the large population. This series emerged out of our need, and that of many others, to bring together the most relevant and innovative available approaches in the intriguing field of food bioengineering. In this work we present relevant aspects in a pertinent and easy-to-understand sequence, beginning with the basic aspects of food production and concluding with the most novel technologies and approaches for processing, preservation, and packaging. Hot topics, such as genetically modified foods, food additives, and foodborne diseases, are thoroughly dissected in dedicated volumes, which reveal the newest trends, current products, and applicable regulations. While health and well-being are key drivers of the food industry, market forces strive for innovation throughout the complete food chain, including raw material/ingredient sourcing, food processing, quality control of finished products, and packaging. Scientists and industry stakeholders have already identified potential uses of new and highly investigated concepts, such as nanotechnology, in virtually every segment of the food industry, from agriculture (i.e., pesticide production and processing, fertilizer or vaccine delivery, animal and plant pathogen detection, and targeted genetic engineering) to food production and processing (i.e., encapsulation of flavor or odor enhancers, food textural or quality improvement, and new gelation- or viscosity-enhancing agents), food packaging (i.e., pathogen, physicochemical, and mechanical agents sensors; anticounterfeiting devices; UV protection; and the design of stronger, more impermeable polymer films), and nutrient supplements (i.e., nutraceuticals, higher stability and bioavailability of food bioactives, etc.).

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Series Preface The series entitled Handbook of Food Bioengineering comprises 20 thematic volumes; each volume presenting focused information on a particular topic discussed in 15 chapters each. The volumes and approached topics of this multivolume series are: Volume 1: Food Biosynthesis Volume 2: Food Bioconversion Volume 3: Soft Chemistry and Food Fermentation Volume 4: Ingredients Extraction by Physicochemical Methods in Food Volume 5: Microbial Production of Food Ingredients and Additives Volume 6: Genetically Engineered Foods Volume 7: Natural and Artificial Flavoring Agents and Food Dyes Volume 8: Therapeutic Foods Volume 9: Food Packaging and Preservation Volume 10: Microbial Contamination and Food Degradation Volume 11: Diet, Microbiome and Health Volume 12: Impact of Nanoscience in the Food Industry Volume 13: Food Quality: Balancing Health and Disease Volume 14: Advances in Biotechnology for Food Industry Volume 15: Foodborne Diseases Volume 16: Food Control and Biosecurity Volume 17: Alternative and Replacement Foods Volume 18: Food Processing for Increased Quality and Consumption Volume 19: Role of Material Science in Food Bioengineering Volume 20: Biopolymers for Food Design The series begins with a volume on Food Biosynthesis, which reveals the concept of food production through biological processes and also the main bioelements that could be involved in food production and processing. The second volume, Food Bioconversion, highlights aspects related to food modification in a biological manner. A key aspect of this volume is represented by waste bioconversion as a supportive approach in the current waste crisis and massive pollution of the planet Earth. In the third volume, Soft Chemistry and Food Fermentation, we xx

Series Preface aim to discuss several aspects regarding not only to the varieties and impacts of fermentative processes, but also the range of chemical processes that mimic some biological processes in the context of the current and future biofood industry. Volume 4, Ingredients Extraction by Physicochemical Methods in Food, brings the readers into the world of ingredients and the methods that can be applied for their extraction and purification. Both traditional and most of the modern techniques can be found in dedicated chapters of this volume. On the other hand, in volume 5, Microbial Production of Food Ingredients and Additives, biological methods of ingredient production, emphasizing microbial processes, are revealed and discussed. In volume 6, Genetically Engineered Foods, the delicate subject of genetically engineered plants and animals to develop modified foods is thoroughly dissected. Further, in volume 7, Natural and Artificial Flavoring Agents and Food Dyes, another hot topic in food industry— flavoring and dyes—is scientifically commented and valuable examples of natural and artificial compounds are generously offered. Volume 8, Therapeutic Foods, reveals the most utilized and investigated foods with therapeutic values. Moreover, basic and future approaches for traditional and alternative medicine, utilizing medicinal foods, are presented here. In volume 9, Food Packaging and Preservation, the most recent, innovative, and interesting technologies and advances in food packaging, novel preservatives, and preservation methods are presented. On the other hand, important aspects in the field of Microbial Contamination and Food Degradation are shown in volume 10. Highly debated topics in modern society: Diet, Microbiome and Health are significantly discussed in volume 11. Volume 12 highlights the Impact of Nanoscience in the Food Industry, presenting the most recent advances in the field of applicative nanotechnology with great impacts on the food industry. Additionally, volume 13 entitled Food Quality: Balancing Health and Disease reveals the current knowledge and concerns regarding the influence of food quality on the overall health of population and potential food-related diseases. In volume 14, Advances in Biotechnology for Food Industry, up-to-date information regarding the progress of biotechnology in the construction of the future food industry is revealed. Improved technologies, new concepts, and perspectives are highlighted in this work. The topic of Foodborne Diseases is also well documented within this series in volume 15. Moreover, Food Control and Biosecurity aspects, as well as current regulations and food safety concerns are discussed in the volume 16. In volume 17, Alternative and Replacement Foods, another broad-interest concept is reviewed. The use and research of traditional food alternatives currently gain increasing terrain and this quick emerging trend has a significant impact on the food industry. Another related hot topic, Food Processing for Increased Quality and Consumption, is considered in volume 18. The final two volumes rely on the massive progress made in material science and the great applicative impacts of this progress on the food industry. Volume 19, Role of Material Science in Food Bioengineering, offers a perspective and a scientific introduction in the science of engineered materials, with important applications in food research and technology. Finally, in volume 20, Biopolymers for Food Design, we discuss the advantages and challenges related to the development of improved and smart biopolymers for the food industry. xxi

Series Preface All 20 volumes of this comprehensive collection were carefully composed not only to offer basic knowledge for facilitating understanding of nonspecialist readers, but also to offer valuable information regarding the newest trends and advances in food engineering, which is useful for researchers and specialized readers. Each volume could be treated individually as a useful source of knowledge for a particular topic in the extensive field of food engineering or as a dedicated and explicit part of the whole series. This series is primarily dedicated to scientists, academicians, engineers, industrial representatives, innovative technology representatives, medical doctors, and also to any nonspecialist reader willing to learn about the recent innovations and future perspectives in the dynamic field of food bioengineering. Alina M. Holban University of Bucharest, Bucharest, Romania Alexandru M. Grumezescu Politehnica University of Bucharest, Bucharest, Romania

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Preface for Volume 11: Diet, Microbiome and Health To define the ecological community of commensal, symbiotic, and potentially pathogenic microorganisms normally living on our skin and mucosa, the term of “microbiota” was proposed. Gut microbiota represents the most diverse and numerous part of the whole microbiota in mammals, including humans. As the microorganisms that compose the normal microbiota are very diverse (i.e., bacteria, fungi, archaea, and viruses) and many of them are uncultivable under laboratory conditions and difficult to identify, researchers have proposed a new term to better reflect the great diversity and huge impact of this microbial community on the host. Thus, the microbiome comprises all the genetic material within a microbiota (the entire collection of microorganisms in a specific niche, such as the human gut). Due to its diversity and importance, the microbiome is considered by some researchers as the second genome, and it seems that for every one human gene we have, there are at least 100 associated genes within our microbiome. It is well known that our physical and mental well-being depends on our diet, and the gut microbiota plays a key role in processing and converting food into usable nutrients. Along with a major role in digestion, our microbiota is responsible for other important processes, such as immunity, detoxification, absorption, infection protection, and even in the occurrence of some diseases, such as metabolic disorders, obesity, diabetes, and some cancers. The composition of microbiome largely changes after a diet switch or a particular therapy, especially antibiotic therapies and this change somehow impacts on our general health and well-being. At present, the human microbiome project, and the elucidation of the functions of microbiome genes, represents one of the prioritized areas. Researchers believe that these studies would have a great impact on the elucidation of mechanisms and development of therapeutic approaches of numerous currently incurable diseases. This volume reveals the main roles of human microbiota and discusses how these inhabitants of the human body could influence the development or therapy of some diseases. The impact of diet and some particular dietary ingredients on the composition of microbiota, as well as potential risks associated with the utilization of some elements that could interfere with normal microbiota, such as probiotics, are also highlighted. xxiii

Preface for Volume 11: Diet, Microbiome and Health The volume contains 15 chapters prepared by outstanding authors from India, Japan, Ireland, Poland, Bulgaria, Mexico, USA, Saudi Arabia, Pakistan, Korea, and Turkey. The selected manuscripts are clearly illustrated and contain accessible information not only for a wide audience, especially food scientists, microbiologists, biotechnologists, biochemists, molecular biologists, geneticists, healthcare representatives, but also for any reader interested in learning about the most interesting and recent advances on the correlations among diet, microbiome and health. Chapter 1 of this volume was prepared by Sarojini and is entitled Gut Microbes: The Miniscule Laborers in the Human Body. The chapter reveals the role of the gut microbes in the proper functioning of the human body, emphasizing on the digestive and nervous systems and implications of microbiota in balancing health and disease. Sharif et al., in Chapter 2, Role of Probiotics Toward the Improvement of Gut Health With Special Reference to Colorectal Cancer, discuss various types of probiotics, their selection criteria, and functional roles in the improvement of overall health. A special reference toward the potential impact of probiotics on the progression of colorectal cancer is made. Ahmad and Khalid, in Chapter 3 entitled Therapeutic Aspects of Probiotics and Prebiotics, highlight the potential of various prebiotics in relation to microflora and key health benefits. Probiotics and prebiotics are gaining interest as therapeutic food ingredients in the current era of complementary medicine due to their supporting therapeutic roles against different ailments, such as decreasing symptoms of lactose intolerance, improving intestinal heath and bioavailability of nutrients, reducing the susceptibility to the prevalence of allergy and risk of certain cancers, and acting as a functional food in treatment of diarrhea-predominant irritable bowel syndrome. Chapter 4, Lactic Acid Bacteria Beverage Contribution for Preventive Medicine and Nationwide Health Problems in Japan, prepared by Kanda and Hara, discusses how lactic acid bacteria–containing beverages has contributed and still have the potential to contribute to health and a longer life span of Japanese people by maintaining a healthy intestinal tract. In Chapter 5, Gut Microbiota Alterations in People With Obesity and Effect of Probiotics Treatment, Martínez Leo et al. review the main microbiome modifications in patients with obesity, how they are a risk factor for obesity, and finally the role that probiotics play in the dietetic treatment for obesity, the weight management, and the microbiome balance reestablishment. Zielińska et al., in Chapter 6, Safety of Probiotics, discuss documented cases of side effects of some probiotics, such as infections caused by probiotics and epidemiological data, assessment of the risk connected with intake of food probiotic products, and other safety concerns of these “generally recognized as safe” products. xxiv

Preface for Volume 11: Diet, Microbiome and Health Chapter 7, Flavonoids in Foods and Their Role in Healthy Nutrition, written by TsanovaSavova et al., presents the current scientific knowledge on the importance of flavonoids for human health. It also presents original results on flavonoid content and composition in various fruits and vegetables, as well as data about their antioxidant activities. Morrin et al., in Chapter 8, The Role of Milk Oligosaccharides in Host–Microbial Interactions and Their Defensive Function in the Gut, describe the mechanisms by which oligosaccharides reduce infection, increase commensal microbiota numbers, induce mucin expression, progress neurodevelopment status, and decrease the likelihood of allergic manifestation. Elucidating the specific functions of individual oligosaccharides and determining their contribution to intestinal health are required to obtain a greater knowledge of their potential benefits to infant formula and subsequently infant health. Chapter 9, entitled Nutritional Yeast Biomass: Characterization and Application, prepared by Jach and Serefko, discusses preclinical and clinical studies that indicate that nutritional yeast biomass is important for prophylactic and/or therapeutic purposes. It is a rich source of amino acids, single-cell proteins, minerals (e.g., chromium, selenium, zinc, iron, magnesium, copper, and manganese), and vitamin B, which promote normal functioning of the immune system. El-Ansary et al., in Chapter 10, Effect of Diet on Gut Microbiota as an Etiological Factor in Autism Spectrum Disorder, show that understanding the mechanism of diet–microbiota interaction may help to avoid the increasing prevalence of autism. Strategies to decrease the overgrowth of pathogenic bacteria and thus improve the composition of gut microbiota of autistic patients through dietary intervention may help to ameliorate gastrointestinal disorders commonly seen in them. Chapter 11, Dietary Fibers: A Way to a Healthy Microbiome, prepared by Sharma et al., highlights the effect of dietary fiber consumption on the quality and diversity of gut microbiome and the interplay of these two in providing health and nutritional benefits to humans. In Chapter 12, Effects of the Gut Microbiota on Autism Spectrum Disorder, Nog˘ay explains that autism is responsive to the composition of gut microbiota, and understanding the early interaction between the intestinal microbiota and autism for nutritional interventions in a risk population would impact the development of this disorder. Javitt and Javitt, in Chapter 13, Diet, Microbiome, and Neuropsychiatric Disorders, review dietary/microbiomal contributions to neuropsychiatric illness, as well as emergent dietary-based prevention and management approaches. In addition, this chapter shows critical approaches by which the study of the microbiome can help elucidate mechanisms underlying severe and highly prevalent neuropsychiatric disorders, as well as mechanisms

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Preface for Volume 11: Diet, Microbiome and Health by which dietary and microbiomal interventions can replace or minimize the need for neuropharmacological intervention. Chapter 14, Gastrointestinal Exposome for Food Functionality and Safety, prepared by Moon, describes a gastrointestinal exposome-linked comprehension of food functionality, toxicity, and related biomarkers in terms of disease prevention or progression. The gastrointestinal exposome is the internal microenvironment that contains foodborne xenobiotics (dietary components and food contaminants) and microbiome, which cross talk with host sentinel components, including the immune and neuroendocrine systems. In the luminal parts of the mucosa, food functional components or contaminants are mixed with the gut microbiota and host-derived biomolecules, all of which cross talk, resulting in complex outcomes in the body. Khora, in Chapter 15, Risk From Viral Pathogens in Seafood, discusses the main pathogenic viruses that cause severe gut-related disorders and dysbiosis in consumers, also highlighting main contamination routes and associated risks. Alina M. Holban University of Bucharest, Bucharest, Romania Alexandru M. Grumezescu Politehnica University of Bucharest, Bucharest, Romania

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SECTION 1

State of the Art and Applications

1. Gut Microbes: The Miniscule Laborers in the Human Body 3 2. Role of Probiotics Toward the Improvement of Gut Health With Special Reference to Colorectal Cancer 35

1


CHAPTE R 1

Gut Microbes: The Miniscule Laborers in the Human Body Suma Sarojini Department of Life Sciences, Christ University, Bangalore, Karnataka, India

1 Introduction If there is anything that is of paramount and ultimate importance to mankind, then it is none other than health. The best way and how long one can lead a healthy life is the ultimate quest of human beings. Scientists have been looking into various routes to achieve this target for many centuries. Finally, they have come to a realization that the key for proper health lays not outside the body, but within. The miniscule living organisms that reside in our body— the gut bacteria—hold the key for a healthy state of the body and mind. The trillions of microbes living in association with the digestive tract of animals are collectively termed as gut microbiota. The past 2 decades saw many discoveries by scientists realizing the highly significant roles of these microbes in animal health. The chapter attempts to give an outlook into the evolution of these microbes, benefits offered to the host, problems caused when the gut microbial diversity is tampered with, and the potential benefits of replenishing the beneficial microflora. Joshua Lederberg had originally coined the term “microbiome” to define “the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space” (Lederberg and McCray, 2001). The study of all these bacteria that comprises the gut microbiome was a daunting task. The reason could be attributed to the uncultivable nature of about 20%–60% of the microbiome associated with the human beings (Bik et al., 2006; Pei et al., 2004). The culture of individual species of bacteria that was the major hurdle in this process (Walker et al., 2011) became insignificant with the advent of high end sequencing methods. This has led to an information explosion in this arena of biology. A lot of questions regarding the diversity and functions of these miniscule warriors in our body were considered during the human microbiome project (HMP). This project was funded by the NIH (Peterson et al., 2009).

Diet, Microbiome and Health http://dx.doi.org/10.1016/B978-0-12-811440-7.00001-6

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Copyright © 2018 Elsevier Inc. All rights reserved.

4 Chapter 1

2 Major Players of the Gut Microbiome Human cells comprise only one tenth of the number of human microflora cells. About 1014 bacteria reside in the human body (Berg, 1996). The number of human cells is estimated to be around 3.72 × 1013 (Bianconi, 2013). The collective mass of these microbes comprises about 2% of the body weight of a person. The microbes occupy almost all niches in our body ranging from the gut, skin, eyes, mouth and nose. The large intestine forms the greatest niche of these microbes (MacDougall, 2012). In fact, the genes of these tiny microbes encode about a 100-fold more genes than the human species (Ley et al., 2006). The human gastrointestinal tract does not have a uniform chemistry throughout. The differences at the physical and chemical level in turn give rise to a different population of diversity of microbiota in different regions of the gastrointestinal tract. Factors, such as age, illness, antibiotic intake, stress, diet, and lifestyle affect the composition of the gut microflora (Gerritsen et al., 2011; Woodmansey, 2007). It is estimated that the gut flora is composed of more than thousand different species. In the complex gut microflora, the most common ones comprise about 150–200 species and the less common ones come to about 1000 species. The genes encoded by the gut microbiota, known as the microbiome, are 100-fold more abundant than the genes of the human genome (Hamady and Knight, 2009). The gene pool of the gut bacteria contributes more than three million unique microbial genes in humans (Proctor, 2011). Most of the members of this community live in symbiotic association with the host and some of them may become opportunistic pathogens when there are changes in the host physiology. This intricate microbial system includes bacteria that live in a symbiotic relationship with their host and some microbes that have potentially pathogenic characteristics. The balance between the two ultimately decides whether the individual remains healthy or may be prone to diseases. In the past few years, a lot of information was generated on the gut microbiome primarily due to high throughput genome sequencing methods. The vast majority of the human gut microbiota is comprised of the four phyla: Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, with the former two being the most abundant in most of the normal healthy individuals (Belenguer et al., 2011). In a human being, at any given time, between 500 and 1000 different species of gut bacteria reside in the gastrointestinal tract. Firmicutes alone constitute more than 60% of the microbiota. Bacteroidetes account for more than 20% of the normal microbiota (Eckburg and Relman, 2007; Khor et al., 2011) The composition of the gut flora has been found to be different in individuals consuming plant- and animal-based food. Members of the genus Prevotella are seen mostly in people consuming a plant based diet whereas Bacteroides and Ruminococcus are predominant in people consuming animal based food (Damman et al., 2012). Results of the sequencing of about 22 faecal metagenomes of individuals from four different countries led to the classification of the human gut microbiome into three enterotypes.

Gut Microbes: The Miniscule Laborers in the Human Body 5 It was found that these three enterotypes were not dependent on the geography of the region. Bacteroides dominated type I whereas type II had a lesser number of the same. Type II showed a preponderance of Prevotella; and type III, of Ruminococcus (Armougom et al., 2011). In the past 3–4 years this domain of microbiology has been progressing at a rapid pace and each month more and more microbes are being discovered as potential players in the gut microbiome.

3 Functions of the Gut Flora The association between human beings and their gut flora can be categorized mostly into a symbiotic one, the latter getting shelter and food from humans and the former returning the favor in a bunch of ways, benefiting both the body and the mind of human beings. A plethora of functions are attributed to these warriors day by day, the most significant and the earliest discovered ones were the roles played in the breakdown of carbohydrates that is normally impossible by the human cell machinery. The immune system and these gut microbes are constantly looking out for each other. The main duty of the immune system is to control pathogens, hence they should keep a close watch on these microbiota and treat them as friends and not enemies of the host. The immune system also has a role in checking whether these individuals are becoming opportunistic pathogens too. The most important prerequisite to the exploration of the effects of the gut bacteria on the host physiology was the development of germ free animal models. One such system is the gnotobiotic mice. These are germ free mice into which we can introduce microbes individually or sequentially (Gordon and Pesti, 1971). These can be used as potential tools to study the effects of a single species of bacterium or a population of mixed species of microbes. The interplay of thousands of these gut bacteria could be studied using these tools. The interactions could be mutually beneficial or antagonistic ones. Critical analysis of these interactions on the hosts due to the introduction of a new member of gut bacterium can be carried out in this manner (Reigstad and Kashyap, 2013). Studies conducted by the members of the HMP suggest that the gut bacterial genes contributed more in the arena of human evolution and survival than the human genes. The human genome carries roughly 22,000 protein-coding genes whereas the human microbiome contributes some 8 million unique protein-coding genes. The number of genes is more than 300 times the number of human genes. These miniscule workers break down proteins, lipids, and carbohydrates in our diet into absorbable forms that can be easily taken up by the body. They also produce many compounds, such as vitamins and antiinflammatories, which the human genes cannot synthesize (MacDougall, 2012). The most abundant and diverse group of microbiota in the human colon are the bacteria that sum up to about 1014 citizens. A total of about 70 different genera of bacteria are depicted by the studies on their 16S rRNA genes to play a role in this (Xu and Gordon, 2003). Of

6 Chapter 1 the three predominant groups, Firmicutes and Actinobacteria are Gram-positive whereas Firmicutes are Gram-negative. The Bacteroidetes phylum mainly produces acetate and propionate, whereas the Firmicutes phylum has butyrate as its primary metabolic end product (Macfarlane and Macfarlane, 2003). Specific enzymes are required to digest the bulk of dietary fibers in human food. Consequently, these carbohydrates pass through the upper part of the gastrointestinal (GI) tract undigested. Later they are fermented in the cecum. Most of the enzymes required for this process are not synthesized by the human protein machinery and are therefore dependant on gut bacteria. The predominant products of this microbial digestion with the help of gut bacteria are the short-chain fatty acids (SCFAs) (Nicholson et al., 2012; Roy et al., 2006). The daily intake and type of food consumed by different people are reflected in the differential composition of the nondigestible carbohydrates that reach the large intestine. Plant cell-wall polysaccharides, oligosaccharides, and resistant starches comprise the primary components of fiber that pass the upper gastrointestinal tract (Flint et al., 2008). About 25 g of daily dietary fiber gets into the digestive tract by way of diet in Western societies (Bingham et al., 2003). Diets that predominantly constitute fruit and vegetables will have a fiber content of 60 g/day (Musso et al., 2011). Butyrate, a short-chain fatty acid, is a main end product of the intestinal microbial fermentation of dietary fibers. Butyrate is an important energy source for intestinal epithelial cells and plays a role in the maintenance of colonic homeostasis. The proximal colon is the venue of maximum bacterial diversity as the substrate availability is the highest in that zone. This nutrient richness becomes progressively lesser toward the distal end of the colon and hence one can find a lesser gut floral population there. These factors tend to make the proximal part of the colon the principal site of fermentation. The bacterial fermentation results in the production of SCFAs and also gases, such as hydrogen and carbon dioxide (Topping and Clifton, 2001). Short chain fatty acids, such as butyrate, propionate, acetate, and so on, have been shown in experimental models to have antineoplastic properties. Among these, butyrate was found to be the most potent (Waldecker et al., 2008). Butyrate is also a highly preferred energy source. This indeed is yet another advantage of the molecule in maintaining the proper health of the colon mucosa. A number of independent experiments have proven the inhibitory effect of butyrate on tumorigenesis. The best-hypothesized mechanisms for this inhibitory effect are its antiinflammatory and immunomodulatory effects. Various components of the host defense barrier could be boosted by this effect. Availability of butyrate has also been found to reduce oxidative stress and give a feeling of satiety (Greer and O’Keefe, 2011; Vipperla and O’Keefe, 2012). One of the most important butyrate producers is Faecalibacterim prausnitzii, which is a member of Clostridium cluster IV. This microbe has also been shown to have independent

Gut Microbes: The Miniscule Laborers in the Human Body 7 antiinflammatory properties related to secreted metabolites, which in turn was found to block nuclear factor kB activation and IL-8 production (Sokol et al., 2008). This is an important finding since it has correlations to the carcinogenesis events in the mammalian system. The antiinflammatory, antitumorigenic, and pathogen exclusion properties of the innumerable number of lactic acid bacteria in the gut benefit the host in multiple ways (Cummings et al., 1987; Marteau, 2013). Microbiota diversity of each individual is influenced by the genotype and physiology of the host, history of colonization, environmental factors, and diet and medicine intake (Zoetendal et al., 2001). The proper functioning of the metabolic pathways were not found to have any major deviations in a group of individuals under study as many biochemical pathways are redundant between alternative members of the microbiome. This was the finding of the Human Microbiome Consortium (Abubucker et al., 2012). A major chunk of fecal matter is made up of bacteria in the gastrointestinal tract. The number and composition of these different microbes decide the bowel health of an individual. Researchers have shown that a fiber-rich diet reduces the risk of colorectal cancer. This correlation is due to the dilution and elimination of toxins (Bingham et al., 2003) through fecal bulk, which in turn is decided by the fiber content of the dietary intake. High water holding capacity, action of fermentative bacteria, and the presence of water-holding fibers decide the efficiency of this process (Birkett et al., 1997; Cummings et al., 1992). Mate selection is another function that is directly shown to have correlation with gut bacteria. This finding was done using experiments conducted on insects. The fruit fly, Drosophila pseudoobscura, fed with starch, preferred to mate with other flies on a starch diet, rather than flies that were on a maltose sugar diet. The maltose-fed flies also preferred similar ones for mating. This research finding was proposed after continuous monitoring and data recording for about a decade (Dodd, 1989). Administration of antibiotics was shown to upset these preferences and mating was found to be at random in such cases. This suggested that the changes in gut microbes brought about by diet drove the mating behavioral pattern. As a continuation of the experiment, the researchers inoculated the antibiotic-treated flies’ food with Lactobacillus plantarum cultured from starch-raised flies. The mating preferences were then found to reverse the findings that were the case before antibiotic treatment. This experiment supports the hypothesis that gut bacteria can even affect sexual behavior. The researchers also hypothesize that this kind of selective mating is an early step toward splitting one species into two and hence the gut bacterial inhabitants are major contributors shaping not only behavior but also evolution (Rosenberg and Zilber-Rosenberg, 2011). In another study involving termites, it was found that antibiotic-treated termites showed a reduced diversity in their gut bacteria and produced fewer eggs. The antibiotic rifampicin was administered to two termite species Zootermopsis angusticollis and Reticulitermes flavipes. This was attributed to the change in the gut bacterial composition as a result of antibiotic

8 Chapter 1 Table 1.1: Gut microbial products and their potential functions. Microbial Products

Functions

References

Butyrates (SCFA)

Reduces inflammation and prevents ulcerative colitis Antiinflammatory Antineoplastic Binds to the vitamin D receptor and acts as detoxifying agent Causes chronic inflammation in obesity and diabetes Changes gut permeability, causes insulin resistance Promotes atherosclerosis Causes gallstone formation Activates the nuclear famesoid X receptor (FXR) and protects against muscle fat deposition Causes cell cycle arrest, induces apoptosis Prime T cell response

Machiels et al. (2013)

Conjugated linoleic acid Propionates, butyrates LCA LPS Endotoxin Trimethylamine DCA SBA

Isothiocyanates Capsular polysaccharides (PSA)

Delzenne and Cani (2011) Waldecker et al. (2008) Sun et al. (2008) Cani et al. (2007) Fei and Zhao (2013) Koeth et al. (2013) Thomas et al. (2000) Cipriani et al. (2010)

Myzak et al. (2006) Mazmanian et al. (2008)

DCA, Deoxycholic acid; LCA, lithocholic acid; LPS, lipopolysaccharides; PSA, polysaccharide A; SBA, secondary bile acids; SCFA, short-chain fatty acids.

administration. The nourishment status and fecundity were affected in such termites. These bacterial species were found to help in digestion and absorption of nutrients (Rosengaus et al., 2011). Thus, the gut microbiota are responsible for the smooth functioning of metabolic pathways involved in digestion and absorption of carbohydrates and lipids, providing signals for renewal of intestinal epithelium, preserving the gut integrity, production of vitamins, destruction of harmful xenobiotics, and development of a potent intestinal immune system. It is also responsible for the secretion of antimicrobial products, which has dual beneficial jobs, such as favoring the growth of beneficial microbes and preventing the growth of pathogenic bacteria by a mechanism of colonization resistance (Bearfield et al., 2002; Jiménez et al., 2008). Some of the products of the gut microbiota and their potential functions are depicted in Table 1.1. Physiological and phenotypic differences have been observed between germ-free and control animals in lab conditions with respect to their microbiome and thereby the host health (Turnbaugh et al., 2006). In germ-free animals, lymph nodes were found to be smaller in size and they had lesser body temperatures, absence of urease and β-glucuronidase activities, and lower organ weights when compared to the control animals (Berg, 1996; Tannock, 1999). Morphological, structural, and functional abnormalities were found to be present in germ-free animals. These included lower levels of digestive enzyme activity, reduced vascularity, thin

Gut Microbes: The Miniscule Laborers in the Human Body 9 muscle walls, lower rates of cytokine and serum immunoglobulin production, lesser number of intraepithelial lymphocytes, and Peyer’s patches (Compare et al., 2002). One of the first lines of defense against microbes in our body is the mucus layer overlying the intestinal epithelium. This consists primarily of glycoproteins secreted by goblet cells. Bacterial pathogens, such as Helicobacter pylori (Windle et al., 2000), Entamoeba histolytica (Moncada et al., 2000), and Pseudomonas aeruginosa (Aristoteli et al., 2003) have evolved cellular mechanisms that allow them to utilize the mucus associated nutrients. They do so by the reduction of mucin disulfide bonds or utilizing proteases. When pathogenic microbes do these kinds of damages, there is another set of bacteria, the commensals, which reinforce the barrier provided by the intestine. This job is done by both the resident gut microbiota and the ones that the host procures by way of having probiotics in the diet. Species of Lactobacillus present in probiotics have been shown to increase expression of mucin glycoproteins MUC2 and MUC3 in vitro in human intestinal cell lines (Mack et al., 2003; Mattar et al., 2002). However, convincing data from in vivo studies is lacking. Probiotics exert many beneficial effects on the health of the gastro intestinal tract, including increased mucus production, augmentation of secretory IgA in the mucus layer, synthesis of antimicrobial peptides, and tight junction integrity of intestinal epithelial cells (Ohland and Macnaughton, 2010). All these mechanisms put together will offer several advantages for the functioning of the gastrointestinal tract as an effective barrier against invasion by pathogenic microbes. The mammalian gut has an inbuilt mechanism to prevent colonization of pathogenic bacteria. This is accomplished by two methods—mechanical barriers and immunochemical barriers. A healthy gut bacterial population can further strengthen these barriers. This can be achieved by the consumption of probiotics. The beneficial gut microbiota competes with the pathogenic ones by putting forth competition for the attachment sites on the mucosal surface in the gut and also for nutrients (Stecher and Hardt, 2008). One of the species of gut flora, namely Oxalibacterium formigenes, helps to regulate the homeostasis of oxalic acid. This has a role to play in preventing the formation of kidney stones (Jalanka-Tuovinen et al., 2011). All these functions are of high physiological significance, because in the absence of gut bacteria, significant consequences, such as improper development of the gut immune system and the development of Clostridium difficile, antibiotic-associated colitis, can happen. One of the causes for this alteration in gut microbial diversity is the consumption of broad-spectrum antibiotics without replenishing the gut flora later by way of intake of vitamin B complex or probiotics. Probiotic microbes, such as Bacillus spp., Bifidobacterium spp., Lactobacillus spp., Lactococcus spp., Leuconostoc cremoris, Saccharomyces spp., or Streptococcus spp., alone or in combination, have shown a protective effect in preventing pediatric antibioticassociated diarrhea (Johnston et al., 2011). Gut microbiota help to protect the intestinal lining by allowing nutrients into the bloodstream and preventing disease-causing pathogens and toxins from passing through. The gut bacteria have carried out this function as a dynamic group. The members existing in the population

10 Chapter 1 would have been established through thousands of years of coevolution, elimination, and selection. Therefore, it can be argued that the imbalances found in the microbial composition of the gut may be catastrophic. It can result in emergence of multiple diseases and can make individuals less responsive to drugs. The hypothesis from the early days of research in this field was that the composition of the intestinal microbiota was relatively stable from early childhood. A phenomenon called gut dysbiosis has been pinpointed to induce carcinogenesis. Gut dysbiosis refers to the alteration of the composition of the gut flora and the consequent failure of the microbiome to sync up with the rest of the immune system. The inflammatory responses due to gut dysbiosis can trigger a chain of events in the different metabolic pathways of the body, which in turn will start affecting the different organ systems of the body. Mice lacking toll-like receptor 5, a transmembrane protein expressed in the intestinal mucosa that binds bacterial flagella, develop metabolic syndrome, and were found to exhibit gut dysbiosis and low-grade inflammation (Vijay-Kumar et al., 2010).

4 Infant Gut Flora The discovery that infants are born without gut microbiota and that infants get their share of the microflora rapidly after birth from the mother and the surrounding environment is a relatively new one. The composition of the microbiota was found to be constantly changing until about the infant reached 3 years of age. Later it becomes mature and will have a composition almost matching that of the adult one. Colonization of the gut by these microbes resulted in two potential benefits. The more important benefit is that these gut bacteria educate the immune system and increase the tolerance to microbial immunodeterminants. The second benefit comes by virtue of the microbiota acting as a big metabolic organ that can break down otherwise indigestible food components, degrade potentially toxic compounds in the diet, such as oxalate, and produce some amino acids and vitamins (Xu and Gordon, 2003). Several studies have shown that infants possess a signature microbiota by the end of the first year of life and between 2–5 years of age, the microbiota fully resembles that of an adult structurally and functionally (Backhed, 2011; Koenig et al., 2011; Palmer et al., 2007). This has led to a theory that the gut flora acquired in the first 3 years of life plays a crucial role in determining an individual’s health later in life. Even in the ayurvedic medical practices of ancient India, a major thrust was given to the diet of pregnant women, with emphasis on what to eat and what not to consume during the term of the pregnancy. With recent advances in medical science, now it has been proven that the mother’s diet and microflora have an impact on microbial colonization of the infant body. The child acquires his microbiome initially during the passage through the vaginal canal of the mother. In case of a cesarean delivery, this kind of vaginal contact is absent and hence the maternal vaginal flora has less of a role to play than the nonmaternally derived environmental bacteria in the infant’s intestinal colonization (Biasucci et al., 2008). It has been known from the mid-1980s that breaking the fecal and vaginal transmission route by Cesarean sections

Gut Microbes: The Miniscule Laborers in the Human Body 11 have a major impact on the infant gut microbiota (Bennet and Nord, 1987). The gut bacterial diversity has been on the lower side in C-section babies when compared to those delivered by the vaginal mode (Jakobsson et al., 2014). Meconium was also found to play a role in the acquisition of gut microbiota. It was earlier thought that meconium was sterile. In a recent experiment conducted on meconium samples of preterm babies, it was found that the meconium harbors a complex array of microbes (Moles et al., 2013). Meconium formation related events could play a major role in the infant gut colonization. This has a big role in shaping the immunity of the child (Jost et al., 2013). Research in the past few years has shown that gut bacteria can cross previously unsuspected barriers too. Pyrosequencing studies on the maternal feces, breast milk, and corresponding neonatal feces were carried out with a view to finding out the microflora composition in these three samples. It pointed to the fact that the mother and the neonate had a shared population of gut-associated obligate anaerobic genera (Bifidobacterium, Bacteroides, Parabacteroides), butyrate-producing members (Coprococcus, Faecalibacterium, Roseburia, and Subdoligranulum) and members of the Clostridia (Blautia, Clostridium, Collinsella, and Veillonella). A research group (Jost et al., 2014) put forth a theory of a novel way of communication between the newborn baby and the mother. They suggested that the mother’s gut flora reaches the breast milk via an enteromammary pathway. Such bacteria crossing these barriers were found to influence the neonatal gut colonization and immune system maturation. A multitude of evidences was obtained in the past few years that stresses the significance of breast-feeding on the initial neonatal gut colonization in the physical and mental health of an individual. Breast milk is known to provide a plethora of nutrients and bioactive immunological compounds. Apart from these it also supplies a substantial load of commensal bacteria, including the beneficial anaerobic bacteria, Bifidobacterium sp. It is with the goal of enhancing such commensal population that infant formulas are increasingly supplemented with probiotic bacteria. Though many attempts have been made to fortify infant formula by these various probiotic bacterial species, it can never compete with the richness offered by the breast milk. Earlier, doctors recommended Cesarean section delivery only in cases where some complication was expected. But in the past 20 years the rate of cesarean delivery (CD) has risen tremendously. In China, the country with the highest population, the rate of C-section is as high as 50%. In the case of Brazil, it is even higher, touching a whopping 80% (Lumbiganon et al., 2010). Though some of the C-section deliveries are preformed for obstetrical indications, a majority of them is done without the actual need. This increased rates of C-section deliveries has been found to be correlating with increasing incidences of autoimmune diseases, such as type 1 diabetes, Crohn’s disease, multiple sclerosis and allergic diseases, such as asthma, allergic rhinitis, dermatitis, and so on, especially in Western, industrialized countries (Bach, 2002; Okada et al., 2010).

12 Chapter 1 The study conducted by Dominguez-Bello et al. (2010) noted a difference in fetal colonization based on mode of delivery. A predominance of pathogenic bacteria typically found on the skin and in hospitals (such as Staphylococcus and Acinetobacter) was found in babies born by cesarean delivery, whereas a preponderance of Lactobacillus was found in babies born vaginally. But before reaching any conclusions, one has to think about another factor, too—the vaginally delivered patients in the study did not receive any antibiotics and the cesarean cases were administered antibiotics, such as cephalosporin prior to the delivery, which is not a standard norm in the United States. A conclusive statement can be made only after ruling out the possibility that the exposure to antibiotics could account for the difference in the gut colonization pattern. Probiotics provide an easy way of introducing and replenishing beneficial gut bacteria. Microbiota modulation by probiotics early in life is receiving great interest. During the first few months of life, where the microbial colonization and immune system maturation are still in progress, administration of probiotics can go a long way in shaping the gut microbiome. Taken together, the long-term health benefits for mothers and children may be conferred by balanced maternal nutrition during pregnancy, influencing the infant microbiota and immune system development, which have an impact on early and late health.

5 Evolution of Gut Microbial Flora Scientists study the evolution of the microbiome in two ways—the first is through coprolites, which are fossilized fecal matter. And the second method is through study of teeth enamel. Teeth preserve and fossilize very well, and the tooth surface bacteria are often calcified (called dental calculus). The evolution of diet, health, diseases, and overall genetics of primitive humans can be obtained from dental calculus (Weyrich et al., 2015). How and to what extent one species adjusts to a new diet is of paramount importance to the survival of that species on this planet Earth. If one looks at the history of life on this planet, examples of many such successful adaptations can be found—for example, the way the teeth and gut of the ruminants evolved in response to the significant amount of C4 plants (mainly grasses) available during the period of low CO2 in the atmosphere (Stevens and Hume, 2004). Over the course of evolution, human beings have struck a symbiotic relationship with the gut microbes. A constant source of nutrition is provided to these bacteria in the gut of humans. They return the favor in a multitude of ways by helping in the functioning of the human digestive and nervous systems (Chen et al., 2013; Geurts et al., 2013). Many human genes have their counterparts in bacteria. These are mainly derived by descent, but some by gene transfer from bacteria (McFall-Ngai et al., 2013). The cognitive performance of the human brain today owes a lot to the gut bacteria (Montiel-Castro et al., 2013). Coevolution has been hypothesized to occur in animal species whose parental care enables vertical transmission of whole gut communities, and where the properties of the community as a whole confer a fitness advantage to the host (Ley et al., 2006).

Gut Microbes: The Miniscule Laborers in the Human Body 13 A comparison between the genomes across different species of animals has revealed that 37% of the ∼23,000 human genes have homologs in bacteria and archaea and 28% originated in unicellular eukaryotes. Thus, most life forms share approximately one third of their genes, including those encoding central metabolic pathways (Domazet-Loso and Tautz, 2008). These genes, which could have been brought to the human genome by gut bacteria (Keeling and Palmer, 2008), might have accumulated mutations, a subset of which would have been lost from the genome and another subset would have persisted, as it would have conferred some selective advantage to the host. The products of some of these homologous genes would have provided the foundation for signaling between extant animals and bacteria (Hughes and Sperandio, 2008). When compared to the primitive human beings, the ever-increasing meat content in the hominin diets, beginning about 3 million years ago, has led to the progressive reduction in the gut length because they could extract more expeditious calories in shorter lengths. In a study conducted on wild African apes (chimpanzees, bonobos, and gorillas) and modern humans (Moeller et al., 2014), it was found that humans had lost some of their ancestral microbial diversity as compared to apes. This was attributed to the hominin’s animal-based diets. Environment also was found to play a major role in an individual’s gut flora diversity. The microbiomes of humans in America (USA) were slightly different from those of the African (Malawi) adults. The study found that the humans across three continents had significantly less microbiome diversity than extant wild apes. The authors have suggested that the meatbased diets could be the culprits.

6 Diet in Shaping Composition of Gut Flora Diet is the primary determinant of the composition of the gut floral associations in an individual. This dietary influence starts very early in an individual’s life—right from infancy. Infants dependent only on breast milk had higher levels of Bifidobacterium spp., whereas infants dependent on formula had higher levels of Bacteroides spp., C. coccoides, and Lactobacillus spp. (Brown et al., 2012; Fallani et al., 2010; Harmsen et al., 2000). Significantly greater levels of amino acid fermentation products and lower levels of carbohydrate fermentation products were observed in people having animal based diets when compared to those having plant based diets (David et al., 2014). This is reflected in the gut microfloral composition. In Thailand, a research study tried to enumerate the composition of gut bacteria in vegetarian and nonvegetarian subjects found that nonvegetarians had a much higher abundance of Bacteroides while vegetarian subjects had a preponderance of Prevotella genera (Ruengsomwong et al., 2014). An abundance of Bacteroides and Firmicutes enterotypes were observed in Western diet subjects. The African diet with high fiber content was dominated by the Prevotella enterotype. A reduction in the number of Firmicutes and increase in the number of Bacteroidetes were

14 Chapter 1 observed in these subjects. In terms of total microbial diversity, African diet was in the forefront. It also had a lesser load of pathogens belonging to Enterobacteriaceae (De Filippo et al., 2010). If the diet is rich in fats, there could be multiple changes in the gut population, such as the absence of gut barrier-protecting Bifidobacteria spp. A research found that dietary changes have more influence (57%) in the total structural variation in gut microbiota when compared to genetic aspects (12%) (Zhang et al., 2010). This is one more proof that the type of food that we consume has a significant role in shaping our gut microbiota. Altering key populations may tend to transform a healthy gut microbiota into a disease-inducing one. It has been discovered that the Western diet, high in sugar and fat, causes dysbiosis, which can affect the metabolic activities of the intestinal tract, which in turn can affect the immune system too (Sekirov et al., 2010). A lot of significance is being given to the vegan diet these days and a lot of people are slowly switching to this kind of diet. This might be attributed to the increased awareness of the dangers caused by prolonged consumption of red meat and animal fats and also an increased concern toward the sustainability of Earth as meat production extracts a lot of resources, such as water and also it indirectly contributes to emission of more green house gases. Vegan diets have gained acceptance as a dietary strategy for maintaining good health and managing disease conditions ranging from cardiovascular disease to cancer (Craig, 2009). A study involving vegetarians, vegans, and omnivorous subjects revealed a lower stool pH in the vegan and vegetarian people, although the total microbial count did not differ between the groups (Zimmer et al., 2012). Living food (LF) is an uncooked vegan diet and consists of berries, fruits, vegetables and roots, nuts, germinated seeds and sprouts. These are very rich sources of carotenoids, vitamin C, and vitamin E. Those people consuming vegan food were found to have increased levels of β- and α-carotenes, lycopene, lutein, vitamin C, and vitamin E. Due to the preponderance of fruits, primarily berries, a threefold increase in polyphenolic compounds, such as quercetin, myricetin, and kaempherol was found in subjects having a vegan diet. As the LF diet is rich in fiber, the urinary excretion of polyphenols, such as enterodiol, enterolactone, and secoisolaricirecinol were found to be on the higher side in people eating LF (Hänninen et al., 2000). A comparative study between vegans and omnivores Goff et al. (2005) found manifestations of lower levels of blood pressure, fasting triacylglycerol levels and glucose concentrations and a better biochemical profile that was cardioprotective and beta cell protective in the former group. Another study pointed out that the health status of long-term vegan diet subjects were comparable to that of endurance exercisers, with reduced BMI, lipoproteins, glucose, lipids, insulin, blood pressure, C-reactive protein, and so on (Fontana et al., 2007). A vegan diet leads to lesser inhabitation of the gut by pathogens of the family Enterobacteriacea (Zimmer et al., 2012), and a greater population of beneficial microbes,

Gut Microbes: The Miniscule Laborers in the Human Body 15 such as F. prausnitzii. One of the key mechanisms making vegan diet a beneficial one is the reduction in the levels of inflammation. l-Carnitine is a trimethyl amine found in red meat. Microbes can metabolize it to a compound called trimethylamine-N-oxide (TMAO), which has been shown to promote atherosclerosis (Koeth et al., 2013). Choline/phosphatidyl choline can also be metabolized by the gut flora to produce the intermediate compound trimethylamine (TMA). TMAO is produced by the oxidation of TMA. A direct link between TMAO levels and atherosclerotic heart lesions was observed in studies conducted in mice (Ussher et al., 2013). Vegan diet subjects have shown reduced levels of inflammation in their bodies. This could be a key aspect in conferring the health advantage to vegans. The fibers present in the vegan diet may have a significant role to play in this aspect. Inflammasomes, a group of protein complexes that recognize inflammation-inducing stimuli and obesity, metabolic syndrome, insulin signaling, and atherosclerosis have been well studied in the recent years. They have been positively correlated to a healthier gut microbiota and gut homeostasis, which in turn reflects on the health of the host’s immune system (Jin and Flavell, 2013; Nardo and Latz, 2011; Strowig et al., 2012). The part played by inflammasomes in regulating gut flora and thereby affecting the metabolic rhythm could be highly dependent on the composition of gut flora. This in turn is directly dependent on the diet of the individual. It is a well-established fact that the diet of the host has profound influence on the composition and number of bacteria present in the gut. The chemical compounds obtained via the diet— either digested or partly digested—will be subjected to gut bacterial metabolism. This can result in the production of both protective and harmful metabolites. Cancers are a result of genomic and epigenomic instability, which allows for the accumulation of genetic and epigenetic alterations. Such changes transform normal, healthy cells into cancerous ones. Microbial metabolites affect DNA by way of direct genotoxic effects on DNA, modulating DNA repair systems and by changing epigenetic mechanisms through histone acetylation or CpG Island methylation (Soreide, 2008). White blood cells are a significant part of the immune system of higher animals. One of the first groups of microbes to colonize the human intestine after birth is Escherichia coli. A study conducted in Portugal compared the guts of healthy mice with those of mice lacking leucocytes. This research work is the first of its kind that proves the hypothesis that the immune system influences the gut microbial evolution. The observations from this study concluded that the digestive and metabolic activities of the mice that were healthy were tuned or adapted quickly to changes in diet. Such changes were found to take a longer time in immune-deficient mice. The composition of the gut flora in the mice with functional lecuocytes were more or less uniform and aided digestive mechanism effectively during diet switches, whereas the mice lacking the white blood cells had a totally different gut flora composition. The fact that the immune system acts as a normalizer of the composition of gut microflora was revealed by this study. Also it concluded that the treatment

16 Chapter 1 strategies of intestinal diseases that occur as a result of an impaired immune system (e.g., inflammatory bowel disease) might require therapies based on personalized medicine; it is very important to find out the patient’s gut microbiota and pursue treatment accordingly (Batista et al., 2015). We often hear the association of probiotics, prebiotics, and gut bacteria. Live, nonpathogenic organisms used as food ingredients to benefit the health of an individual are known as probiotics. The list includes yeasts, such as Saccharomyces cerevisiae, lactic acid bacteria (LAB), and Bifidobacteria. Cancer and atopic dermatitis in children are diseases wherein probiotic bacteria play a role in the prevention (Kumar et al., 2010; Meneghin et al., 2012). Nondigestible food ingredients that beneficially affect the host are grouped as prebiotics. They do so by mechanisms, such as selective stimulation of growth of gut bacteria and enhancement of their activity resulting in improving the health of the host. Prebiotics include compounds, such as lactulose and resistant starch. In recent years, these are included in the food industry to modify the composition of the microbiota species with a view to improve the overall health of human beings (Gibson et al., 2004).

7 Gut Microbiota and Diseases Most of the gut flora would have been tuned to live in a friendly manner without causing any diseases or damages to the host. This situation can be altered when the environment is changed. In such situations, the usually commensal nature of association could be subjected to changes. In hospitals, surgery and antibiotic treatment were found to be related to the incidence of pseudomembranous colitis induced by the toxins of C. difficile. Similarly, sepsis caused by E. coli, Enterococcus faecalis, and so on, and intraabdominal abscesses caused by B. fragilis were also correlated to changes in gut flora composition (Wilcox, 2003). It is observed that a diet rich in highly processed foods causes more bloating. An unusually large number of microbes, such as Lachnospira pectinoschiza, Anaerotruncus colihominis, and Ruminococcus callidus were observed in individuals suffering from bloating. Members of the genus Streptococcus, especially S. alactolyticus were seen in reduced numbers during diarrhea (Hermann-Bank et al., 2013).

7.1 Gut Bacteria and Obesity Obesity is the result of the combination of factors, such as unhealthy diet, sedentary lifestyle, lack of physical exercise, and genetic factors. Studies conducted in the past 2 decades have shown the contribution of gut microflora composition and diversity in deciding the obesity factor of a person. The most dominant groups of bacteria in the gut of human beings and many other vertebrates are the Bacteroidetes and Firmicutes. The elucidation of the role of the gut bacteria in determining the obesity of individuals was obtained from studies comparing intestinal bacteria in obese and lean twins. Studies have shown that the gut flora community

Gut Microbes: The Miniscule Laborers in the Human Body 17 in lean people was, such as a rain forest filled with many diverse species whereas the gut bacterial composition was less diverse in obese people. A predominance of Bacteroidetes, a large tribe of microbes that specialize in breaking down bulky plant starches and fibers into shorter molecules that the body can use as a source of energy, was observed in lean people (Armougom et al., 2009). Studies involving humans, zebrafish, mice, and pythons have come out with factors regarding fat absorption and the number of Firmicutes in the gut microbiota. The number of Firmicutes was found to be inversely proportional to the number of Bacteroidetes. Also, Firmicutes have shown a rapid increase after a meal. The rate of weight loss in human beings was found to be directly proportional to the decrease in the population of the number of Firmicutes. The ratio of Firmicutes and Bacteriodetes was reflected in the obesity status of an organism. A study involving lean and obese mice showed an increased population of Firmicutes in obese ones and a decreased population in lean ones (Ley et al., 2006). Firmicutes have shown to be associated with efficient extraction of energy from food and increased breakdown of fibers. A study conducted in pythons revealed that the number of Bacteroidetes was high during fasting, replacing the Firmicutes (Costello et al., 2010). Gut bacteria can be attributed to increased weight gain as they help in increased uptake of monosaccharides, processing of polysaccharides and storage of triglycerides in the host cells (Backhed et al., 2004). An imbalanced gut flora correlated with obesity in animals. Bacteria belonging to Firmicutes were found in higher numbers in genetically engineered obese mice. It was discovered that the Firmicutes are very efficient at extracting energy from food, breaking down fiber, and even increasing the absorption of dietary fat. In this way, gut microbes could cause retention of body weight without the animal eating an extra morsel of food. Besides probiotics, other gut bacteria could also protect humans from obesity. Some members of the Bacteroidetes phylum were suggested to be mainly responsible for protection against increased adiposity. More complex bacterial interactions and associated metabolic disturbance were involved in protection against increased adiposity. One of the novel ways of treating obesity could be using gut bacteria as targets. Along with increased fatty acid absorption, more energy was also found to be efficiently obtained from diet in the obese mice compared to the lean mice, illustrating the connection between Firmicutes and improved efficiency in energy harvesting (Ley et al., 2005; Turnbaugh et al., 2006). The famous experiment conducted by Jeffrey Gordon’s lab, which was published in the journal Science, has revealed interesting facts correlating obesity and gut microflora. In the experiment, the team had used humanized, genetically identical baby rodents raised in a germ-free environment, whose guts were subsequently populated with intestinal microbes collected from obese women and their lean twin sisters. Though the mice were fed the equal amount of the same type of diet, it was found that the one that had the gut flora of the obese

18 Chapter 1 women gained more weight. In a continuation of the experiment, the two groups of mice were put in the same cage. Analysis of their subsequent body weight showed that the lean mice also gained weight due to consumption of fecal matter of the obese mice, which in turn changed their gut flora into the ones that are present in the obese mice (Riadura et al., 2015).

7.2 Gut Bacteria and Diabetes An overall increase in the onset of type 1 diabetes mellitus (type 1 DM) was also observed in the past 2 decades, which could be correlated to increase in C-section deliveries (Zivkovic et al., 2011). A study showed a 19% increase in type 1 DM in cesarean children when other factors were kept under controlled conditions (Fallani et al., 2010). A cause-effect relationship was found between endotoxin producers in the gut and obesity/ insulin resistance outcomes, which can be tracked by changes in gut permeability, serum endotoxin, and inflammatory biomarkers (Fei and Zhao, 2013). In yet another study, the Prevotella spp. (Bacteroidetes) to Eubacterium rectale ratio (Firmicutes) was positively correlated to plasma glucose levels (Larsen et al., 2010).

7.3 Gut Bacteria and Bowel Disorders One of the two major idiopathic IBDs is ulcerative colitis (UC) (Cummings et al., 2003). In patients suffering from UC, the disease is restricted to the colon. Scientists have found that the population of lactobacilli was significantly lower during the active phase of the disease. Experiments conducted using techniques, such as denaturing gradient gel electrophoresis showed the presence of bacteria, such as Pediococcus acidilactici, L. salivarus and L. manihotivorans were present in remission cases. However these microbes were seen during active inflammation (Bullock et al., 2004). Also, the complexity of the different types of gut bacterial species were found to be less in diseased mice, pointing to the lesser diversity of bacterial composition during acute inflammation. Members of the Clostridiales group dominated the samples from the inflamed colon. This is reflected in the increased accumulation of bacteria during colitis (Heimesaat et al., 2007).

7.4 Gut Bacteria and Cancer The energy production of cancer cells is different from most normal tissues in the body. These cells take up glucose and glutamine at a high rate for aerobic glycolysis. Some scientists had used this concept as a basis for reducing the growth of malignant cells by a program of calorie restriction. This was attributed to the fact that the growth of tumors seem to be mediated through the toxic effect of ketone bodies on cancer cells which have a defective mitochondrial function. The ketone bodies may not have this deleterious effect on normal cells as they can utilize these ketone bodies in a proper manner. Short-chain fatty acids, such

Gut Microbes: The Miniscule Laborers in the Human Body 19 as acetate, propionate, and butyrate have been found to suppress inflammation and cancer. Inflammation has been suspected to influence the development of colorectal cancer, but a new study suggests the potential role of the gut microbes in this disease development. Some other microbial metabolites, such as secondary bile acids, promote carcinogenesis. This increased production of secondary bile acids was attributed to the higher intake of high fat meat in Americans and to higher resistant starch intakes in Africans (O’Keefe et al., 2007). Cancer tissues are generally heterogeneous and each cancer is different in tissue origin and metabolic phenotype (Fantin et al., 2006). Each cancer cell in a cancer tissue might be different from its neighbor, and metabolic phenotypes in cancer are plastic. When compared to the normal cells, cancer tissues exhibit greater plasticity (Berridge et al., 2010). This could be attributed to several erroneous cellular functions that would have arisen due to the mutations in the DNA. An important research finding suggests that cancer cells may change their metabolic phenotypes to adapt to the changes at the microenvironment level. These changes help the survival of cancer cells under unfavorable conditions and give them a selective advantage (Chen et al., 2008; Marusyk and Polyak, 2010). Gut bacteria have been found to help in therapeutic aspects of cancer too. There are drugs called checkpoint inhibitors (e.g., Ipilimumab, pembrolizumab, and Nivolumab), which significantly reduce tumor growth. This is found in melanomas, lung cancer, head and neck cancers, and so on. Scientists were perplexed by the fact that this initiated a vigorous response in some patients, and not uniformly in all cases. In a research article published in the journal Science, the researchers give a clue for this, using mice as models. Mice bought from one laboratory mounted a better immune response to melanoma tumors than mice bought from a different lab, which could be attributed to the difference in their gut microbiota. Later, when the mice were cohoused in cages for 3 weeks, they found there were no more differences in the tumor growth. This could be due to the sharing of gut bacteria exchanged between the mice due to fecal feeding (Sivan et al., 2015). We all now know that abnormal cell division leads to cancers, but the triggers or primary causes of this malady are manifold. Some cancers have their underlying cause attributed to infectious agents. Cancers of the stomach, anus, cervix, penis, and liver, and some lymphomas, belong to this category (De Flora and Bonanni, 2011). About 20% of the total worldwide burden of cancer can be assigned to such infectious agents. This percentage is expected to rise over time (Blaser, 2008; Hausen, 2009). Viruses cause a significant number of known infection-associated cancers. This list includes the liver cancer caused by the hepatitis C and B viruses and the cervical cancer caused by the oncogenic human papillomavirus alpha types (Parkin, 2006). Though such viruses play roles as etiological agents of cancer, gut bacteria have also been implicated as carcinogenic agents (Mager, 2006). One of the bacteria associated with cancer is the enterotoxigenic B. fragilis, which is linked to colorectal cancer (Goodwin et al., 2011; Marchesi et al., 2011). Another potential bacteria

20 Chapter 1 Table 1.2: Gut microbiota and associated cancers. Type of Cancer

Associated Microbe

References

Breast cancer Colon cancer Colorectal cancer Cervical cancer Colorectal cancer MALT lymphoma Gall bladder cancer Gastric cancer Colorectal cancer Liver cancer

Staphylococus, Enterobacter Citrobacter rodentium F. nucleatum Human papillomavirus alpha type B. fragalis H. pylori S. typhi Helcobacter pylori Streptococcus bovis Hepatitis B virus, hepatitis C virus

Ubraniak et al. (2016) Newman et al. (2001) Castellarin et al. (2011) Bouvard et al. (2009) Goodwin et al. (2011) Cavanna et al. (2008) Lazcano-Ponce et al. (2001) Huang et al. (1998) Burnett-Hartman et al. (2008) Parkin (2006)

linked with colorectal cancer are Fusobacterium nucleatem (Castellarin et al., 2011) and H. pylori. This is grouped as a class I carcinogen by the IARC (International Agency for Research on Cancer). This correlates with gastric cancer and MALT lymphoma. These two account for more than 5% of the cancers worldwide (Huang et al., 1998; Parkin, 2006). Prolonged and multiple infections with the typhoid bacteria Salmonella typhi is thought to be linked with development of cancer in the gallbladder (Lazcano-Ponce et al., 2001). A list of gut microbes and associated cancers are given in Table 1.2. The global burden of various cancers is rising day by day. The causes could be manifold, but scientists world over are thinking of reasons as to why different patients respond differently to the drug regime, why certain cancers recur in some patients, why chemotherapy weakens some patients more than the disease itself, and so on. The clue for all these queries may lie in their gut microbiome. A lot of significance could be attributed to one’s gut microbiome in the field of personalized medicine.

8 Gut Flora and Brain Functions In the initial research studies regarding the functions of gut bacteria, scientists have been thinking of its role only with respect to the digestive system. As years went by and more information was obtained on the composition and diversity of the gut flora, scientists have come across previously unheard of or unsuspected functions. One such function is the connection between the gut bacteria and the brain. To carry out these functions, there has to be interplay between the different physiological systems in our body. The three main systems that could be thought of as the main players are the nervous system, digestive system, and the endocrine system. Under extreme stress or anxiety it is not uncommon to feel the need to empty your bowel. Scientists working on interactions of the brain and the gut have found that these feelings could be attributed to the different metabolic reactions carried out by the gut bacteria. Apart from helping in digestion and absorption of nutrients, these reactions also affect one’s

Gut Microbes: The Miniscule Laborers in the Human Body 21 thoughts, moods, and mental health. A part of the nervous system called the enteric nervous system (ENS) is very closely associated with the digestive system. The ENS is composed of two thin layers of more than 100 million nerve cells lining the gastrointestinal tract from the esophagus to the rectum. The gut brain axis refers to the biochemical signaling that takes place between CNS (central nervous system) and the GI tract. Scientists have been getting information on the interplay of gut and brain for the past few years, but the exact role of the gut flora in shaping the complicated gut brain axis was discovered very recently. In order to give due importance to the main players in shaping this pattern, the term was broadened as microbiome-gut-brain axis or gut-brain-microbiome axis (Wang and Kasper, 2014). The intestinal microbiota, along with the CNS, the neuroendocrine and neuroimmune systems, the sympathetic and parasympathetic nervous system, and the ENS plays a key role in deciding the efficiency of the BGM or brain-gut-microbiome axis (Mayer, 2011). The result of the interactions of all the aforementioned players is the formation of a complex reflex network with afferent fibers. The afferent fibers were found to project to the integrative CNS structures and efferent fibers to the smooth muscles (Grenham et al., 2011). This communication network is a bidirectional one. It was found that the gut influenced brain functions. These in turn could be reflected in the motor, sensory, and secretory functions (Montiel-Castro et al., 2013). The dominant players in this complex process were found to be the gut bacteria (Khanna and Tosh, 2014). The gut flora can produce a range of neuroactive molecules, such as acetylcholine, catecholamines, γ-aminobutyric acid (GABA), histamine, melatonin, and serotonin, which are essential for regulating peristalsis and sensation in the gut. One of the main inhibitory neurotransmitters in the human brain is GABA. Members of the genus Lactobacillus produce GABA. These are the bacteria that the babies born by vaginal delivery come across most likely for the first time (Dinan et al., 2015). It was found that the changes in the gut microbial composition due to medicines, diet, or any disease conditions were linked with the changes in levels of circulating cytokines. These chemicals were thought to affect brain function. The gut flora also release molecules that can directly activate the vagus nerve, which transmits information about the state of the intestines to the brain (Petra et al., 2015). The numerous essential neurochemicals produced with the help of the gut microbes decide the health of the human brain. In the absence of such chemicals, the brain’s serotonergic system does not develop properly. Hence, these gut bacteria are indispensible for the well being of the brain (Clarke et al., 2013). The emotional quotient of an individual is dependent on the serotonergic system activity and thus the gut microbiota are unconsciously regulating our behavior. Humans are social animals. In order to thrive and interact in this network, humans have evolved the trait of sociability, which in turn depends on our superior cognitive functioning. This has ultimately led to the emergence of humans as the superior species on the planet. Mice grown in the lab without any gut bacterial colonization showed behaviors that had autistic patterns (Desbonnet et al., 2014). It is suspected that the species of gut bacteria has

22 Chapter 1 coevolved with the humans. During several thousands of years, the best composition of gut flora could have been selected and stabilized in the human gastrointestinal tract. This effect of gut microbiota on the human cognitive functions would have facilitated human group living in a society and would have helped humans get the power for the environmental dominance (Lombardo, 2008). There is still debate among researchers as to whether stress is good or bad for human beings. According to the Yerkes-Dodson law, to a threshold point, stress is found to have positive impacts on an individual. Most people under mild stress have been found to be more competitive and can utilize their full potential and hence are more productive. But once the stress reaches a threshold but still continues at the same pace, it will have deleterious effects on the individual (Bregman and Mcallister, 1983). Individuals may have different pivot points and hence will have varying responses when subjected to the same amount of stress. This threshold level is decided by a multitude of factors, including the gut microbiota (Sudo et al., 2004). The process of formation of these new nerve cells is called neurogenesis. When the gut microbiome was switched off using a high dose of strong antibiotics, mice were found to have significantly fewer newly formed nerve cells in the hippocampus region of the brain when compared to the control mice. Neurogenesis is important for certain memory functions. Apart from impaired neurogenesis, the population of a specific immune cell in the brain—the Ly6Chi monocytes—decreased significantly when the microbiota was switched off. It was then hypothesized that these immune cells could be a previously unknown intermediary between the two organ systems. A study by Möhle et al. (2016), found that gut microbiome fortification could be significant in treating people with mental disorders, such as schizophrenia or depression, who have impaired neurogenesis. Antibiotics were first invented to kill bacteria and rescue patients affected by dreaded bacterial diseases. Over the years, people have started misusing antibiotics by way of incorrect dosage of drugs, failure to complete the antibiotic course and antibiotic additives in animal feed. The consumers of antibiotics range from millions of human beings and billions of cattle worldwide. It is an accepted fact that antibiotics have saved countless lives and reduced the pain and suffering of many more. A lesser-known fact in early days was the negative impact they had on the physiology and psychology of the patients (Bercik and Collins, 2014). Recent studies have reached a conclusion that this effect is due to the impact of antibiotics on the brain axis. Gut bacteria that make the intestinal lumen as their niche are thought to affect the host’s cognitive functions (Mayer, 2011). Autism is a neurodevelopmental disorder that affects more than 20 million people globally. It is characterized by impaired social interaction, verbal and nonverbal communication, and restricted and repetitive behavior. Usually the signs can be noticed in the first 2 years of a child’s life (Myers and Johnson, 2007). The range and severity of symptoms can vary widely. Common symptoms include difficulty in communication, difficulty with social interactions,

Gut Microbes: The Miniscule Laborers in the Human Body 23 obsessive interests, and repetitive behaviors. Early recognition, as well as behavioral, educational, and family therapies may reduce symptoms and support development and learning. Though initially it was believed that the cause of autism was genetic, it is now believed that both genetic and environmental factors play a role in the disease development. The complex nature of the disease could be attributed to the interactions among multiple genes—the environmental and epigenetic factors that do not change DNA sequence, but are heritable and influence gene expression (Rapin and Tuchman, 2008). The chromosome abnormalities that have been implicated in autism include deletions, duplications, and inversions (Beaudet, 2007). A significant role is played by the immune system in autism. Researchers have found significant levels of inflammation of both the peripheral and central immune systems in autistic children. The increased levels of proinflammatory cytokines and significant activation of microglia depict this (Onore et al., 2011). Recently, gut bacteria were also indicated to play a major role in disease manifestation. The gut microbes in a mouse model show the disease exhibits leaky gut, as well as core autismlike behaviors, such as repetitive actions and limited communication and socialization. When these mice were treated with the probiotic bacteria B. fragilis, the leaky gut and some other abnormal behaviors were corrected. Compared with control mice, the autistic mice had 46 times the normal amount of a molecule called 4-ethylphenyl sulfate (4-EPS), which is a predicted output of gut bacteria. This chemical is a close relative of p-cresol, a metabolite found in high concentrations in the urine of autistic patients. Scientists found that treatment with probiotics containing beneficial bacteria restored the normal levels of 4-EPS levels in mice. Also, when healthy mice were treated with 4-EPS they showed anxious behaviors, similar to those seen in the autistic ones. These studies have shown that people with autism have excess 4-EPS, or similar chemicals, present in their blood, which reaches the brain and affects their behavior (Hsiao et al., 2013).

9 Effect of Antibiotics on the Gut Microbiome Antibiotics are administered with a view to kill pathogenic bacteria. The correct timing and dosage of antibiotics have saved many lives. But this process has inadvertently done some harm also, along with the good, in that they can cause fluctuations in the native gut microbial composition. The native gut flora goes a long way in reducing the magnitude of gut colonization by noncommensal microbes. They do this by a variety of mechanisms varying from competition for attachment to niches, production of volatile fatty acids, and competition for food and nutrients. Some bacteria are negatively affected by the volatile fatty acids produced by anaerobic bacteria. Bacteriocins keep a check on the growth of some species of bacteria. Streptococci, enterobacteria, and some anaerobic bacteria produce these compounds (Nord et al., 1984).

24 Chapter 1 Fecal samples were the best-studied specimens in the domain of gut microbiome because access to the small intestine is a really challenging task. The microbial composition of the small intestine and the large intestine are quite distinct, and the way the gut flora responds to antibiotic administration is also quite different in these two loci as revealed by studies conducted in mice. Many broad-spectrum antibiotics affect the beneficial gut bacteria. The long-term health effects of antibiotic treatment especially in early life have been associated with the development of asthma, eczema, atopic dermatitis and other allergic sensitization, autoimmune encephalitis, candidiasis, cholera, and pathogen induced colitis (Willing et al., 2011). A multitude of studies have been conducted in different mammalian systems, predominantly mice, to understand the effects of antibiotic treatments on the gut microbial diversity. A few of them are listed in Fig. 1.1. Vancomycin, one of the last resorts for C. difficle induced diarrhea, exerts a harmful effect on gut commensals. Mice treated with oral vancomycin often became heavily colonized with bacteria belonging to the Enterobacteriaceae family, perhaps because this antibiotic has limited activity against Gram-negative bacteria. Treatment with Ampicillin, on the other hand, did not result in expansion of Enterobacteriaceae. Upon cessation of antibiotic treatment, bacterial density rapidly returned to pretreatment levels. On the other hand, studies have shown that the Thuricin CD, an antibacterial peptide or sactibiotic, can be used to treat C. difficile induced diarrhea without causing much harm to the gut commensals (Rea et al., 2011).

Figure 1.1: Effects of Antibiotic Treatments on the Gut Microbial Diversity.

Gut Microbes: The Miniscule Laborers in the Human Body 25

10 Fortifying Gut Flora Since every year we are getting newer and newer information about the beneficial aspects of the gut microbiota, scientists have been all the more interested in keeping the native gut bacterial population as natural as possible. Several strategies have been studied. One of them involved getting the caesarean born babies exposed to the mother’s vaginal bacterial population. A study was conducted in Puerto Rico, in which babies born by cesarean section are immediately swabbed with a gauze cloth laced with the mother’s vaginal fluids and resident microbes. Aspects, such as weight, disease resistance, and overall health of the infants in the study would be compared for a few years to get a concrete picture of the advantages conferred by this treatment in C-section babies when compared to those who did not receive the gauze treatment (Dominguez-Bello et al., 2016). There are some disease conditions of the gastrointestinal tract that could manifest due to unhealthy gut flora. Repeated doses of different antibiotic treatments also may not give 100% recovery in the case of such diseases. The technique of fecal microbiota transplant (FMT) could be of help in such cases. FMT, or bacteriotherapy, is the transplantation of fecal bacteria from a healthy individual into a recipient. FMT involves introducing healthy bacterial flora through infusion of stool, by enema, orogastric tube, or orally in the form of a capsule containing freeze-dried material obtained from a healthy donor. This technique has been used since 1980s for the treatment of anomalies of the digestive system. FMT has been reported as a treatment option in individuals with recurrent C. difficile infection (Bakken et al., 2011). While C. difficile infection could be treated with a single FMT infusion, multiple and recurrent infusions maybe required to treat diseases, such as ulcerative colitis (Borody and Campbell, 2011). Research is going on in labs across the world focused on the following two aspects: how best to make our gut flora healthy and also how to replenish the gut flora when there is an unhealthy composition. The coming years will definitely see a lot of research work in this booming field of microbiology.

11 Conclusions It has been established that each person has a signature microbiome in him or her that is unique to that person and is seldom seen exactly similar in another individual. From the time of their evolution, humans would have thrived with a plethora of microbes in their body, some of which would have done good and some would have done harm. This coevolution helped both the parties in the relationship. Microbes get shelter and nutrient rich food so that they can multiply and expand their race. The benefits obtained by humans far outnumber those got by the bacteria. Gut bacteria should be really a dynamic lot, as they have to adapt and evolve quickly in response to the ever-changing environment in the gastro intestinal tract. The shifts in

26 Chapter 1 the host’s diet puts pressure on them to have diversity in the gut flora composition. The exorbitant number of microbes residing in the human body paves way for a multitude of interactions among the human cells and the microbes and also between the microbes themselves. A lot of work has been going on in this field thanks to the high throughput sequencing methods available these days, which help scientists find out the microbiome of an individual at a very fast pace. Scientists are finding out potential functions carried out by these micro workers in our body. They are so numerous that almost every month or so, a couple of new functions are being attributed to the bacterial flora in the human body. The human race should be indebted to these microfriends of ours for blessing us with both good physical and mental health.

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Further Reading Al-Nassir, W.N., Sethi, A.K., Li, Y., et al., 2008. Both oral metronidazole and oral vancomycin promote persistent overgrowth of vancomycin-resistant enterococci during treatment of Clostridium difficile-associated disease. Antimicrob. Agents Chemother. 52, 2403–2406. Brismar, B., Edlund, C., Nord, C.E., 1993. Impact of cefpodoxime proxetil and amoxicillin on the normal oral and intestinal microflora. Eur. J. Clin. Microbiol. Infect. Dis. 12, 714–719. Dethlefsen, L., Relman, D.A., 2011. Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proc. Natl. Acad. Sci. USA 108 (Suppl. 1), 4554–4561.

Gut Microbes: The Miniscule Laborers in the Human Body 33 Lode, H., Höh, V., Ziege, S., et al., 2001. Ecological effects of linezolid versus amoxicillin/ clavulanic acid on the normal intestinal microflora. Scand. J. Infect. Dis. 33, 899–903. Maneval, M.L., Eckert, K.A., 2004. Effects of oxidative and alkylating damage on microsatellite instability in nontumorigenic human cells. RES 546, 29–38. Pletz, M.W., Rau, M., Bulitta, J., et al., 2004. Ertapenem pharmacokinetics and impact on intestinal microflora, in comparison to those of ceftriaxone, after multiple dosing in male and female volunteers. Antimicrob. Agents Chemother. 48, 3765–3772. Suchodolski, J.S., Dowd, S.E., Westermarck, E., et al., 2009. The effect of the macrolide antibiotic tylosin on microbial diversity in the canine small intestine as demonstrated by massive parallel 16S rRNA gene sequencing. BMC Microbiol. 9, 210.


CHAPTE R 2

Role of Probiotics Toward the Improvement of Gut Health With Special Reference to Colorectal Cancer Mian K. Sharif, Sana Mahmood, Fasiha Ahsan National Institute of Food Science & Technology, University of Agriculture, Faisalabad, Pakistan

1 Probiotics 1.1 Introduction The term “probiotics” originates from the Greek word probiotikos meaning “improving life.” These are live microorganisms that, when administered in sufficient quantities, improve the health of the host body (ISAPP, 2016). These were initially discovered by Lilly and Stillwell in 1965. Professor Elie Metchnikoff, known as the grandfather of probiotics, monitored the regular intake of fermented dairy products having lactic acid bacteria (LABs), such as yogurt, cheese, and kefir. In 1974, Parker elaborated probiotics as substances that assist the stability of gastrointestinal microbiota. In 1989, Fuller described probiotics as live microbes that upon ingestion impart health benefits in addition to their inherent ability to provide nutrition. The United States National Food Ingredient Association (USNFIA) considers bacteria, yeasts, and fungi as naturally existing probiotics. The most studied probiotics microbes are Lactobacillus spp., Bifidobacterium spp., and Saccharomyces boulardii (Dinleyici et al., 2014). Current public and market trends indicate a US $46.55 billion market by 2020. Currently, Europe is the leader regarding the use of probiotic substances. The major driving force is consumer demand for probiotics in various food products. Recent advances in the domain of food and nutrition have made it possible to manufacture commercial products containing probiotics prescribed by doctors for relieving different health ailments, such as constipation, cancer, and metabolic syndromes. The most important role of these companies is to develop new products by adding probiotics as ingredients in an array of food items as a supplementary approach. Furthermore, scientific advancements emphasizing potential benefits, consumer awareness, and advertisements through print and electronic media have further accelerated the global growth of probiotics.


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1.2 Types of Probiotics 1.2.1 Lactobacilli Lactobacillus is one of the major categories of LABs comprising more than 120 species and 20 subspecies, and the number is increasing annually. According to some reports, 13 novel strains of lactobacilli were introduced in 2005. On the basis of physical and morphological characteristics, lactobacilli are nonspore-forming anaerobes with harsh nutritional requirements (carbohydrates, amino acids, peptides, fatty acid esters, salts, nucleic acid derivatives, vitamins). These are cogently linked with a huge diversity of nutrient-enriched plants and animal-derived environments, and numerous stains are involved in the production and conservation of acidic foodstuffs (Charalampopoulos and Rastall, 2009). Furthermore, some species are generally used to attain food breakdown in acidic conditions, especially milk and milk products, such as yogurt, ice cream, and buttermilk, and other food products, including pickles, salami, sausages, and cereal-containing food supplies. These bacteria use glucose, lactose, and other sugar as sources of carbon to generate lactic acid through fermentation. Some strains of lactobacilli undergo fermentation in the same manner, whereas others have different fermentation perspectives and produce alcohol along with the formation of lactic acid from sugars. These bacteria are able to grow better at acidic pH and also block the progression of other organisms. These are present in different places, including fermented milk products, human and animal mucosal membranes, sewage, and various plants (Raman et al., 2015). 1.2.2 Bifidobacteria Bifidobacteria were primarily isolated by Henry Tissier from infants fed exclusively on breast milk. These leading gut microflora have proven beneficial for baby health by ameliorating diarrheal conditions and watery flow, which have deleterious effects on putrefactive bacteria, leading to the occurrence of diseases. On the basis of morphology, these are spore-forming, immobile, obligate aerobes existing in numerous shapes. Most of the strains are firmly anaerobic. Earlier these were considered group members of the genus Lactobacillus due to similar properties as exhibited by lactobacilli. Later on, they were assigned a separate category (Arboleya et al., 2016). The majority of the gut-lining microbiota belong to the genus Bifidobacterium. Basically, they become visible in the fecal material of babies within a few days of birth and then increase in number with the passage of time. The colon of an adult has about 10 colony-forming units per gram, but this amount tends to decrease with aging, mainly due to changes in dietary preferences and food choices (Charalampopoulos and Rastall, 2009).

1.3 Role as Functional Food Functional foods provide health benefits beyond normal nutrition. Dietary fibers, carotenoids, flavonoids, phenols, minerals, pre- and probiotics, fatty acids, plant stanols/sterols are considered prominent functional foods. Probiotics are considered as major stakeholder

Improvement of Gut Health 37 in the global market of functional foods due to numerous established health benefits and overall well-being of the consumer. In the human gut, these produce short-chain fatty acids as well as antibiotics. Likewise, these possess antagonistic effect suppressing the activities of putrefactive and pathogenic ultimately reducing the production of toxic substances. The majority of probiotics belong to Bifidobacterium, Lactobacillus, nonpathogenic Escherichia coli, and yeasts, such as S. boulardii. Antimicrobial activity of probiotics is linked with the synthesis of compounds, such as organic acids, ethanol, hydrogen peroxide, or proteincontaining components (e.g., bacteriocins). Medical and clinical researches have proven the curative abilities of probiotic bacteria in human beings as health promoters and in improving the overall quality of life (Balakrishnan and Floch, 2012).

1.4 Probiotics as Food Ingredients Probiotic microorganisms are principally bacteria chosen from the class of probiotics with well-established industrial and commercial applications for human well-being and prevention of various health ailments. There are numerous species in genera, and along with each species there are some strains having probiotic characteristics. Probiotic strains are usually selected to execute well technically, to remain alive in the intestinal passage, and to provide health benefits to consumers. Probiotics are provided to manufacturers either as dry powder or as refrigerated substitutes. As an ingredient in the final formulations, probiotics should not change either the organoleptic characteristics of the food products or their functional abilities. For dehydrated powders, cold storage and controlled humidity extend the shelf life of probiotics, and for refrigerated cultures it is essential to preserve a steady temperature and also keep them away from frequent freezing. In infant formulas, dry probiotics are blended with other ingredients, whereas in fluid or semiliquid products, such as fruit drinks or ice cream they are present as suspended particles. Likewise, in the case of acidic foods, such as yogurt and probiotic milks, they are cultured (Song et al., 2012).

1.5 Selection Criteria The Food and Agriculture Organization (FAO) of the United Nations and World Health Organization (WHO) have demonstrated rules for the assortment of safe probiotic strains (FAO/WHO, 2002). Probiotic strains for food applications should preferably be from human origin, be acid and bile tolerant, and have adhering properties with GIT linings, competition with pathogenic bacteria, and safe dosage for human use. 1.5.1 Human origin Infants’ stool is considered a rich source of healthy microbiota. Probiotics should preferably be derivatives of human beings. During product development, probiotics-containing products should be evaluated through animal and human efficacy trials to confirm the viability of selected strains in food commodities as well as the human body. Likewise, it should be

38 Chapter 2 ensured that supplementary probiotics did not alter the consumer acceptability of the food products, particularly the aroma, mouthfeel, and consistency of foods. Additionally, special attention should be given to possible changes in the shelf life of food products after the addition of probiotics as functional ingredients. 1.5.2 Acid and bile tolerance All the strains should exert their action in highly acidic conditions, as harmful intestinal microorganisms do not survive in low pH. This acidic pH is due to the high solubility and absorption of minerals. It is mandatory for probiotic strains to avoid and be protected from stomach gastric juices, and they must be capable of being nurtured in the presence of bile. Added probiotics have the ability to survive the harsh conditions of the stomach and bile. 1.5.3 Stickiness to human GIT linings The property of stickiness is necessary to enhance the bioavailability of added probiotics for improved health. Actually, infants are born without bacterial strains in the GIT; hence the foundation of the intestinal microbiota has not been entirely described. Probiotics must have the capacity to survive and stay active at the intended organ and to be effective and functional. The mucosal lining of the intestinal tract is designed in such a way that beneficial microorganisms after crossing impart health benefits. 1.5.4 Competition with harmful microbiota Probiotic species must fight against intestinal enzymes (e.g., lysozymes, nitroreductase) that are involved in the reproduction of carcinogenic compounds. After selection of probiotics lactobacilli and bifidobacteria, these are tailored to create and endure in appropriate ecological surroundings within the human large intestine. As in the upper GIT and the colon, the pH is usually close to neutral and the temperature is stable (37°C), and probiotics can easily survive to fight with harmful microbiota, provided that a constant nutrient (prebiotics) supply is available. 1.5.5 Safety Probiotics should be used according to safe doses suggested by various global agencies. In this context, safety assessment of probiotics and probiotics-containing products requires some research observations from human efficacy studies.

1.6 Effective Dosage To gain maximum benefits from probiotics, it is vital to incorporate them into food products as well as in the preferred gastrointestinal location in a large-enough amount. The viability of a strain is important for its functional role inside the body; hence, every probiotic is required

Improvement of Gut Health 39 to be tested correspondingly. The timing and period of consumption would principally depend on the requirements and form of the disease (e.g., treatment of diarrhea is short term whereas cancer protection is of longer duration). It should be kept in mind that probiotics species that are destroyed instantly by conditions in the alimentary canal should be provided in greater doses to counteract their disposal (Song et al., 2012). Overall, the functional role of probiotics depends on the probiotics strain (lactobacilli, bifidobacteria), daily dosage, regular amount of intake (1–4 times), time of dose intake (after or during meals), duration of use, method of provision (capsules, tablets, or powder), and feasibility.

1.7 Incorporation Criteria of Probiotics in Foods Conventionally, probiotic strains have been in use for decades through the consumption of acidic dairy products, such as yogurt, cheese, buttermilk, and more. However, the mechanisms and techniques to incorporate probiotics into fresh and frozen dairy commodities are quite new. With the advancement in research and clinical evidence regarding the chemotherapeutic benefits of probiotics, a global market of probiotics in the class of serviceable food products has emerged rapidly. Consequently, global food manufacturing industries and leading bodies are in continuous search of tools and ways for injecting these substances into a wide variety of foods and drinks. Incorporating live probiotic strains into foods and then developing ways for maintaining their activity until final consumption is a momentous task for food handlers and processors. Certainly, it is difficult and is opposite to the routine food-modification technologies where the main objective is to reduce the number of microbes to ensure food safety for consumers. Naturally there are many chances to decrease the activity of probiotics during handling and storage. Hence, a desirable feasible count of probiotics is usually attained by elevating the numbers of microbiota through production. Furthermore, the intake of probiotics products at larger doses is safe and does not cause any harm to the consumer. However, the following key points should be kept in mind while adding probiotics into foods: • • • • •

Choose a well-suited probiotic strain compatible with the end product. The food manufacturing environment should support probiotics endurance. In case of fermentation, make sure that the food medium will bear probiotics development. Choose a product material, wrapping, and environmental circumstances to confirm ample probiotics existence during the handling and transportation, and throughout life storage. Make sure incorporation of probiotics in the food products does not alter the mouthfeel and consistency of the food commodity.

2 Probiotics as Health Promoters The main therapeutic and health benefits of probiotics are described as follows.

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2.1 Antimicrobial Activity It is necessary for probiotics to stay alive in the body to help protect it from harmful pathogenic microorganisms. The abilities of lactobacilli to avoid and cure various urogenital ailments, including infections and oligouria, have been well documented. A number of methods have been documented for antimicrobial activity of probiotics mainly due to production of H2O2, bacteriocins, and induction of immune and signaling systems. Probiotics must be capable of providing intestinal and apical hemostasis (i.e., arresting of blood at specific site, particularly improving epithelial wall veracit). Friendly microorganisms are commercially used for the preparation of antimicrobial products, such as bacteriocins, lactic acid, hydrogen peroxide, and segregated bile acids to reduce pathogenic and cancer-causing microbes. Several probiotic bacteria can attach or fight with pathogens for nutritive molecules, stick on epithelial cells and break apart the attachment of pathogens, and compete with pathogens by formation of biofilms (Redman et al., 2014). Probiotics species are specialized to form short-chain fatty acids, such as acetates, propionates, butyrate, and lactic acids as final products, making the surrounding environment unfavorable for the pathogenic bacteria. The antimicrobial activity of acids is due to fall and blocking diversity of metabolic activities (Šuškovic´ et al., 2010). The ability of hydrogen peroxide to confer antimicrobial activity is credited to its physically powerful oxidation potential on bacterial cells and ability to obliterate the fundamental molecular morphology of the cell proteins. There is great capacity of hydrogen peroxide (H2O2) to protect the body from urinary tract diseases by destroying bacteria not only affecting the germicidal activity but also against spore formers. H2O2-producing lactobacilli may guard the vagina from the overgrowth of disease-causing native flora and externally by producing lactic acid, ultimately lowering the pH, which is not favorable for their growth (Liong, 2011). Advancements in molecular and genetic technologies have made possible the manufacture of bacteriocins as antimicrobial components chiefly from several Lactobacillus species classified with respect to shape, morphology, structure, method of action, antimicrobial effectiveness, defensive mechanisms, and desired cell receptors (Ohigashi et al., 2013). Currently, bacteriocins are categorized into lantibiotics, small heat-stable bacteriocins, and large heat-stable bacteriocins. Their role as clinical and therapeutic agents is well documented, and it is suggested that these be used as alternative agents to antibiotics due to their low damaging effects and the reality that they can be bioengineered (Cotter et al., 2013). Biosurfactants are surface-active components that are produced by bacteria and are considered helpful in the restoration and preservation of microorganism balance. These are synthesized by probiotics and act on nonsticking and antimicrobial constituents in the urogenital and intestinal tracts.

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2.2 Immunomodulation The intestinal microflora preserves the host’s health by enhancing the immunological resistance mechanism. The probiotic strains demonstrate great variation regarding immunomodulation involving antiinflammatory influences, enhancement of the gut mucosal obstruction homeostatic balance, valuable activity on gastrointestinal microbes, and a diminution of instinctive allergic reactions. Probiotics have the ability to stimulate the crossing of epithelial junctions, altering the provocative prospective of epithelial cells or unswervingly inducing the performance of immune cells (Caricilli et al., 2014). By stimulating immune system abilities, probiotics decrease sensitive, allergic, inflammatory, and self-immune ailments. At the same time, an improved immune comeback is desirable in various circumstances, especially illness and cancer. It is considered that immunomodulating activity of probiotics might depend not merely on the dosage of probiotics but also on the current immune system status of the host and the probiotic strain. In an investigation, the influence of orally consumed probiotics on lymphocyte propagation was observed ranging from inhibition of lymphocyte explosion to improved T lymphocytes and B cells (plasma cells) oncogenesis, depending on the strains used in the study (Hatakka et al., 2008).

2.3 Gut Microbiota and Probiotics It has been well known that an unbalanced microbiota population encourages settings for colorectal mutation. However, utilization of particular probiotic ingredients daily can boost human well-being, restoring the microbial balance and restraining gastrointestinal inhabitation by spoilage microorganisms (Lee et al., 2014). In a study, rats were fed on diets containing 1,2-dimethylhydrazine and strains of Lactobacillus rhamnosus to modulate colorectal cancer (CRCA). It was observed that the consumption of L. rhamnosus decreased the count of colony-forming bacteria and appreciably increased the number of lactobacilli (Bertkova et al., 2010). Likewise, in another animal study, the consumption of L. reuteri and Bifidobacterium bifidum considerably altered the stool microbial count by reducing enterobacteria found in the fecal mass and escalating the numbers of bifidobacteria and lactobacilli in the GIT. Similarly, in a clinical trial in which CRCA patients were orally administered various dosages of different probiotic strains, the amounts of Lactobacillus, Bifidobacterium, and Enterococcus were amplified besides reducing the counts of E. coli and Streptococcus species. Furthermore, a mixture of Lactobacillus and Bifidobacterium species, such as L. gasseri and B. lactis might have augmented Bifidobacterium and Lactobacillus in the stool microflora and lowered the number of disease-causing bacteria, including Clostridium perfringens. Much additional research is still needed to understand the mechanism of action and physiological properties of probiotics on pathogenic microorganisms in a well-described manner.

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2.4 Diarrhea Diarrhea is defined as a flow of watery material from the body, sometimes having blood in the feces, with reduced uniformity of stools. According to the World Health Organization (WHO), diarrhea reflects the health condition in which there are three or more watery stools for two or more succeeding days (Ma et al., 2010). The consumption of foods rich in probiotics may have an effect on the formation of gastrointestinal microflora. Comprehensive clinical trials have proved their effects on the anticipation and handling of short-term gastroenteritis (inflammation of the intestinal tract), antibiotic-associated diarrhea, and infant traveler’s diarrhea; however, their impact and benefits for pediatric ailments are still unclear. On the other hand, data favors the utilization of probiotics for the management of delicate viral gastroenteritis and for the preclusion of gastrointestinal diseases (Kanazawa et al., 2005).

2.5 Hypercholesterolemia Increased blood cholesterol is among the main hazardous factors for heart disease, hypertension, obesity, diabetes, and atherosclerosis around the globe. It is estimated that a 1% drop in blood cholesterol decreases the threat of coronary heart disease by 2%–3% (Amaretti et al., 2013). Numerous dietary and nutritional supplements are available in supermarkets to alleviate high serum cholesterol levels, and the eating of whole grain cereals, consumption of fruit and vegetables, use of olive oil, and avoiding trans fats, besides using moderate amounts of sugar and salt, are all advised. Decline of blood cholesterol level by probiotics has been well established in human and mice models (SadrzadehYeganeh et al., 2010). The ability of probiotics strains to lower serum cholesterol is chiefly attributed to the absorption of cholesterol by increasing cells, adhesion of cholesterol to the cell exterior side, amalgamation of cholesterol in the cell covering, breaking down of greenish-yellow bile through bile salt hydrolase, and denaturing of cholesterol, along with broken bile, attachment of bile by dietary fibers, and making of short-chain fatty materials by oligosaccharides. However, additional clinical and research studies are missing to support the suggestion (Raman et al., 2015).

2.6 Lactose Intolerance and Stomach Ulcers Lactose intolerance is a metabolic disorder arising from lack of the enzyme lactase that helps to digest lactose present in milk and milk products. An adequate action of lactase is required in the human digestive tract to prevent various ranges of peritoneal awkwardness. Probiotics involving Lactobacilli and Bifidobacteria construct β-dgalactosidase, which self-digests lactose and enhances lactose acceptance (Liong, 2011). Helicobacter pylori confers health damages to more than half of inhabitants globally, and in emergent countries up to 80% of middle-aged people may be contaminated with this

Improvement of Gut Health 43 microorganism. It is linked with inflammation of the GIT, stomach and duodenal soreness, and stomach cancers. No vaccination is available for the treatment of H. pylori, and medicinal conflict is also growing day by day. Outcomes resulting from in vitro studies and several clinical observations for utilizing probiotics against H. pylori have shown the viability of this development (Palm et al., 2014). A number of clinical studies have shown that provision of probiotics can lessen the undesirable attributes imparted by H. pylori (Lee et al., 2014).

2.7 Mineral Absorption Probiotics have the ability to enhance the assimilation of different mineral ions, especially Ca and Mg through intestinal mucosal barriers by the formation of short chain fatty acid, such as butyric acid, which lowers the pH of intestine and increasing the acidity. Consequently, solubility and absorption of micronutrients is elevated under highly acidic conditions. In this context, probiotics have proved to impart promising functions on the metabolism of bones and density of bone mass and is principally attributed to the improved mineral solubilization because of their ability to produce short-chain fatty acids, synthesis of enzyme phytase by the microflora to control the impact of mineral dejected by phytates found in cereals, grain products and green leafy vegetables, diminution of gastrointestinal inflammatory conditions ordered by boost up in the bone mass density and breakdown of glycoside linkage in the food whenever reaches to the intestines by gut microflora (Parvaneh et al., 2014).

2.8 Inflammatory Bowel Disease Inflammatory bowel disease (IBD) is a chronic abnormality of the GIT having irregular times of inflammation and reduction. It is divided into two subgroups, namely ulcerative colitis (UC) [i.e., inflammation of the large intestine (colon part) caused by damaged or highly acidic foods] and Crohn’s disease (CD) (i.e., a physiological disorder of the colon caused by a weak immune system). Both UC and CD vary depending upon the presence in the intestine and characteristics of the irritation. CD is frequently elucidated as a phenotype of T-helper lymphocytes 1-type ailment. It may also influence a part of the intestine in a segmentation manner and show a transmutable inflammation, moving along the whole intestinal wall. UC, demonstrated as a T-helper 2-mediated condition, is a widespread and constant inflammation limited to the mucosal lining of the colon (Koebnick et al., 2003). Minute injuries and trauma are observed on only the mucosa coating for UC, whereas the complete intestinal wall is influenced in CD. The etiology and pathogenesis of IBD depend on many factors and environmental conditions, such as low-fiber diet, reduced intake of water, increased intake of refined and processed food products, and sedentary lifestyle. Attributed to IBD may be modification in gastrointestinal mobility, intuitive allergic reactions, intestinal microbiota, reduction of epithelial cell barrier function, excess of appearance

44 Chapter 2 of proinflammatory stimulators in diverse effectors T lymphocyte sets, lacking defensive and authoritarian signals and/or anomalous antigen appearance, nonfunctioning of the gut–brain alliance, or some psychological and social factors. The utilization of probiotics and substances rich in probiotics suggests a substitute changing the intestinal microflora by spirited elimination, whereas probiotics struggle for the microbial pathogens for a restricted number of signals found on the exterior of the epithelium, immunomodulatory action and/ or encouragement of an immune response of gut-associated lymphoid and epithelial cells, antimicrobial activity and inhibition of pathogen development, upgrading of the gut barrier function, and initiation of T cell apoptosis (i.e., programmed cell death in the mucosal immune section) (Leblanc and De Leblanc, 2016).

3 Probiotics and Colorectal Cancer The word “cancer” is basically used for a disease in which abnormal cells in the human body start dividing and increasing without following the growth-controlling mechanism. These cells have capability to invade nearby tissues through lymphatic and blood systems, adopting a phenomenon known as metastasis (Kopetz et al., 2009). CRCA has emerged as a global issue and is a common malignancy of the GIT. The CRCA incidence rate in developed nations is 4 times that in developing countries (Haggar and Boushey, 2009). It is a type of cancer that initiates from the colon or rectum, which are parts of the large intestine in the GIT. The endurance rate of CRCA patients has been dramatically increased by current advancements in chemotherapy and biological agent–based therapies and by early detection techniques (Kopetz et al., 2009). Irrespective of advancements in its treatment, this cancer is still uncontrollable. In the United States, it is the third most commonly identifiable cancer both in women and in men. The threat of developing CRCA in one’s overall lifetime is about 1 out of 20 or 5%; however, mortality rates are frightening. According to the American Cancer Society, in 2013 there were 142,820 newly diagnosed cases of CRCA whereas about 50,830 patients died (Kankana and Kebin, 2013).

3.1 Pathophysiology of Colorectal Cancer Modifiable risk factors (nutrition, stress and bowel habits, alcohol drinking, and smoking) and nonmodifiable risk factors (age, heredity, environment, and previous colon disease) are involved in the pathophysiology of CRCA. Its pathophysiology involves the abnormal division of cells in the colon arising from the epithelial lining of the intestine and involved in the formation of polyps. If the polyps are not treated, cells continue to proliferate in the polyps and they start to increase in size and become the major reason for malignancy. Once this happens in the body, the proliferation rate of cells increases rapidly in the colon area, and colon cancer develops. Early detection is helpful to cure the body of this disease, but if this condition is left untreated, then death may occur (Anonymous, 2011).

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3.2 Colorectal Cancer and Diet Diet plays a fundamental role in the pathogenesis of CRCA. Epidemiological studies have shown that a diet rich in vegetables and fruit seems to be defensive against this cancer (Riboli and Norat, 2003), whereas risk increases with the ingestion of animal fat and red meat (Larsson and Wolk, 2006). There is strong evidence from various studies that supports the hypothesis that the relationship between CRCA and diet may be due to an inequality of the gastrointestinal microflora (Rafter et al., 2004). The GIT is first colonized by microbes at birth and serves as a home for diverse types of microorganisms throughout life. The normal microflora of the GIT contains many species of bacteria with genetic, morphological, and physiological features that help to grow, multiply, and make colonies under favorable environments at particular sites. These also help to inhibit the growth and colony-formation abilities of harmful or disease-causing bacteria. However, some factors, such as drugs and diet have the ability to change the configuration of the local microbiota, resulting in dysmicrobia and negative health consequences in individuals. The microflora of the intestine, especially that present in the colon, includes a huge number of anaerobic bacterial species, such as Fusobacterium, Clostridium, and Bacteroides species (Rastall, 2004). The consumption of probiotics-containing foods boosts the immune system, ultimately protecting from CRCA (Rafter et al., 2004).

3.3 Anticancer Activity of Probiotics No doubt, probiotics help prevent the development of CRCA; however, the mechanism is still unclear. It is believed that probiotics improve human health through numerous approaches, such as modification of the intestinal microflora, competition with pathogenic and putrefactive microbiota, regulating cell differentiation and apoptosis, pH modifications, and hindering the pathway of signaling tyrosine kinases (Mario et al., 2012). 3.3.1 Modification of intestinal microflora The liver is considered the house for various metabolic processes. Among these, the most important is glucuronide conjugation. The inactivation of harmful or cancer-causing compounds and the metabolization of hormones are very critical. When the substances or compounds bind with glucuronic acid, there is production of polar metabolites, which are powerfully released in the bile (Tephly and Burchell, 1990). There are several enzymes in humans involved in the formation of carcinogenic compounds, though mostly in inactive form. Once these are activated under favorable conditions, deconjugation of the substances from glucuronic acid occurs in the intestine (e.g., β-glucuronidase releases aglycones, which are basically carcinogenic or cancer-causing substances) (Reddy et al., 1997). Other harmful bacterial enzymes are nitroreductase and azoreductase, which catalyze the release of procarcinogenic materials in the intestine (Goldin and Gorbach, 1976; Hill, 1975). The

46 Chapter 2 probiotics basically alter the intestinal metabolism by modifying the action of these bacterial enzymes, and in this way probiotics decrease the danger of developing CRCA (Goldin and Gorbach, 1984). 3.3.2 Competition with pathogenic and putrefactive microbiota The colon part of the GIT is mainly occupied with bacteria, compared to the other parts of the GIT. Besides beneficial bacterial species in the GIT, numerous bacterial species are severely pathogenic and may participate in the development of short-term and longlasting complications and diseases, such as CRCA (Manning and Gibson, 2004). It is well recognized that a diet with a high ratio of animal fat stimulates the development of secondary bile salts, which can result in cell damage and cancer (Merchant et al., 2005; Nagengast et al., 1995). Likewise, consumption of products rich in red meat accelerates the progression of sulfate-decreasing bacteria, further creating hydrogen sulfide, which is experimentally recognized as genotoxic (Huycke and Gaskins, 2004; Kanazawa et al., 1996). Putrefactive gut microbiota, such as Clostridium and Bacteroides species are mostly involved in the initiation of CRCA (Sobhani et al., 2011). Higher intake of probiotics alone, as well as in combination with prebiotics, such as oligofructose-enriched inulin can increase the number of Lactobacillus and Bifidobacterium species in the fecal microflora of polyp and colon cancer patients, along with reducing the number of pathogenic bacteria, such as C. perfringens and coliforms (Rafter et al., 2007). 3.3.3 pH modifications Probiotics play a significant role in decreasing the pH of the gut by producing short-chain fatty acids, such as acetic, butyric, and lactic acid involved in the reduction of pathogenic microbes, and also assist in sustaining homeostasis by lowering the pH of the intestine (Roy et al., 2006). Probiotics also enhance the mineral absorption in the body by decreasing the absorption ability of ammonia and the solubility of bile acids. Among the short-chain fatty acids, butyrate is best associated with programmed cell death and cell differentiation (Donohoe et al., 2011). 3.3.4 Regulating the cell differentiation and apoptosis Programmed cell death or apoptosis is very important for the normal human body. It also plays an important role in the growth regulation of cells (Zhong et al., 2014). If there is damage to DNA or any disturbance occurs in the process of apoptosis, cells are unable to follow the normal growth control mechanism and continue to proliferate without any dividing signal, leading to the formation of cancer. However, regulating cell endurance and death at the molecular level in apoptosis could provide a way for chemoprevention and hold therapeutic potential. Probiotics are able to regulate the programmed cell death and cell differentiation and in this way can help prevent CRCA (Uccello et al., 2012).

Improvement of Gut Health 47 3.3.5 Hindering the pathway of signaling tyrosine kinase Probiotic strains also exert antimutagenic activity due to their potential to bind mutagenic substances on the cell surface (Raman et al., 2013). These also help in biotransformation and purification of procarcinogens and cancer-causing substances into less poisonous metabolites, and in this way the probiotics help prevent cancer formation (Pool-Zobel et al., 2005). This type of biotransformation of cancer-causing agents mostly occurs in the GIT with the help of the enzymes called phase I and phase II types. These enzymes basically control the harmful microorganisms causing mutation and neoplastic effects on environmentally friendly carcinogens. The role of phase I enzymes is to initiate bioactivation, and the function of phase II enzymes is to inactivate cancer-causing agents. The human probiotic strain L. rhamnosus 231 binds to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and MeIQx (2-amino3,8-dimethylimidazo quinoxaline), leading to their biotransformation and successive decontamination (Ambalam et al., 2011). Administration of feasible Lr 231 protects rats from MNNG-induced colon inflammation (Gosai et al., 2011) (Fig. 2.1).

Figure 2.1: Role of Probiotics in the Prevention of Colon Cancer. Adapted from Kumar, K.S., Sastry, N., Polaki, H., Mishra, V., 2015. Colon cancer prevention through probiotics: an overview. J. Cancer Sci. Ther. 7, 81–92.

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4 Conclusions The protection of gastrointestinal health is vital, as 70% of the human immune system is located in the intestine. Probiotics are live microorganisms that impart specific health benefits to humans when utilized in sufficient quantity. The chief probiotic bacterial species belong to the genera Lactobacillus and Bifidobacterium. These microorganisms help to maintain the integrity of the gut and other organs. The major health benefits imparted by the probiotic strains concern hypocholesterolemia, protection from irritable bowel syndrome, diarrhea, lactose intolerance, colon inflammation, CRCA, mutagenicity, and anti-H. pylori activity. Several in vitro and in vivo studies have welldocumented methodologies highlighting the functional role of probiotics in human gastrointestinal health, with special reference to CRCA. However, there is a dire need to undertake further studies to completely understand the mechanistic role of probiotics for sustainable health.

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50 Chapter 2 Ohigashi, S., Sudo, K., Kobayashi, D., Takahashi, O., Takahashi, T., Asahara, T., et al., 2013. Changes of the intestinal microbiota, short chain fatty acids, and fecal pH in patients with colorectal cancer. Digest. Dis. Sci. 58, 1717–1726. Palm, N.W., De Zoete, M.R., Cullen, T.W., Barry, N.A., Stefanowski, J., Hao, L., et al., 2014. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. Cell 158, 1000–1010. Parvaneh, K., Jamaluddin, R., Karimi, G., Erfani, R., 2014. Effect of probiotics supplementation on bone mineral content and bone mass density. Sci. World J 41, 1657–1660. Pool-Zobel, B., Veeriah, S., Bohmer, F.D., 2005. Modulation of xenobiotic metabolising enzymes by anticarcinogens—focus on glutathione S-transferases and their role as targets of dietary chemoprevention in colorectal carcinogenesis. Mutat. Res. 591, 74–92. Rafter, J., Bennett, M., Caderni, G., Clune, Y., Hughes, R., Karlsson, P.C., et al., 2004. Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients. Am. J. Clin. Nutr. 85, 488–496. Rafter, J., Bennett, M.G., Caderni, Y., Clune, R., Hughes, P.C., Karlsson, A., et al., 2007. Dietary synbiotics reduce cancer risk factors in polypectomized and colon cancer patients. Am. J. Clin. Nutr. 85, 488–496. Raman, M., Ambalam, P., Doble, M., 2015. Probiotics and Bioactive Carbohydrates in Colon Cancer Management. Springer, New Delhi, India. Raman, M., Ambalam, P., Kondepudi, K.K., Pithva, S., Kothari, C., Patel, A.T., et al., 2013. Potential of probiotics, prebiotics and synbiotics for management of colorectal cancer. Gut Microbes 4, 181–192. Rastall, R.A., 2004. Bacteria in the gut: friends and foes and how to alter the balance. J. Nutr. 134, 2022S–2026S. Reddy, B.S., Mangat, S., Weisburger, J.H., Wynder, E.L., 1997. Effect of high-risk diets for colon carcinogensis on intestinal mucosal and bacterial beta-glucuronidase activity in F344 rats. Cancer Res. 37, 3533–3536. Redman, M., Ward, E., Phillips, R., 2014. The efficacy and safety of probiotics in people with cancer: a systematic review. Ann. Oncol. 25, 1919–1929. Riboli, E., Norat, T., 2003. Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. Am. J. Clin. Nutr. 78, 559S–569S. Roy, C.C., Kien, C.L., Bouthillier, L., Levy, E., 2006. Shortchain fatty acids: ready for prime time. Nutr. Clin. Pract. 21, 351–366. Sadrzadeh-Yeganeh, H., Elmadfa, I., Djazayery, A., Jalali, M., Heshmat, R., Chamary, M., 2010. The effects of probiotic and conventional yoghurt on lipid profile in women. Br. J. Nutr. 103, 1778–1783. Sobhani, I., Tap, J., Roudot-Thoraval, F., Roperch, J.P., Letulle, S., Langella, P., Corthier, G., et al., 2011. Microbial dysbiosis in colorectal cancer (CRC) patients. PLoS One 6, e16393. Song, D., Hayek, S., Ibrahim, S., 2012. Recent application of probiotics in food and agricultural science. In: Rigobelo, E.C. (Ed.), Probiotics. InTech, Rijeka, Croatia. Šuškovic´, J., Kos, B., Beganovic´, J., Leboš Pavunc, A., Habjanicˇ, K., Matošic´, S., 2010. Antimicrobial activity— the most important property of probiotic and starter lactic acid bacteria. Food Technol. Biotech. 48, 296–307. Tephly, T.R., Burchell, B., 1990. UDP-glucuronosyltransferases: a family of detoxifying enzymes. Trends Pharmacol. Sci. 11, 276–279. Uccello, M., Malaguarnera, G., Basile, F., D’agata, V., Malaguarnera, M., Bertino, G., et al., 2012. Potential role of probiotics on colorectal cancer prevention. BioMed Cent. Surg. 12, S35. Zhong, L., Zhang, X., Covasa, M., 2014. Emerging roles of lactic acid bacteria in protection against colorectal cancer. World J. Gastroenterol. 20, 7878–7886.

PAR T S ection 2

Probiotics and Prebiotics

3. Therapeutic Aspects of Probiotics and Prebiotics 53 4. Lactic Acid Bacteria Beverage Contribution for Preventive Medicine and Nationwide Health Problems in Japan 93 5. Gut Microbiota Alterations in People With Obesity and Effect of Probiotics Treatment 111 6. Safety of Probiotics 131

51


CHAPTE R 3

Therapeutic Aspects of Probiotics and Prebiotics Asif Ahmad*, Sumaira Khalid*,** *Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Punjab, Pakistan; **Government College University, Faisalabad, Punjab, Pakistan

1 Introduction The use of probiotics, prebiotics, and synbiotics is based on the same goal; that is, to produce foodstuffs that supply healthy food microbes to the gut or intestines after ingestion, which can be achieved either by the addition of healthy microbes called “probiotics” or indigestible polysaccharides known as “prebiotics.” This addition of health-promoting ingredients to a food product’s basic qualities is the definition of functional food creation (Bottazzi, 1983). No doubt probiotics and prebiotics are food ingredients that fulfill all the requirements to produce functional foods, but they also fall under the umbrella of “beyond nutrition,” as they do not provide any nutrients (Gibson and Roberfroid, 1995). Fermented milk, which is a good source of probiotics, is one of the oldest functional foods, and has been consumed worldwide for thousands of years as a treatment for gastroenteritis problems (Ahmed et al., 2013a; Bottazzi, 1983). The basic purpose of adding probiotics to food is the prevention of pathogenic bacteria and their metabolites, the promotion of proper immune response against infection, and the maintenance of gastrointestinal functioning, while prebiotics are food constituents that promote microbes beneficial to health in the human gut and also improve the persistence of probiotic bacteria that are ingested orally at same time (Ahmed et al., 2013b). Recently it has been shown that the intake of antibiotics for several diseases is creating resistance in pathogens in a large segment of the population; this is prevalent on a large scale in underdeveloped countries. Probiotics can play a vital role in combatting drug-resistant bacteria and may be used as a prophylaxis against certain diseases and to improve general health conditions. Another benefit of probiotics is that they fulfill an environmental niche in the gut and promote growth of other beneficial microbes by modifying pH and other growth conditions necessary for beneficial microbes. Sometimes they completely or partially utilize those nutrients that pathogenic microorganisms require; thus, due to higher competition,

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54 Chapter 3 pathogenic microorganisms have a lower chance of survival. The positive effects of probiotics are not limited to intestinal health; they also activate the part of the enzyme system that may promote hydrolysis of certain undesirable protein and lipids having the capacity to cause allergic reactions. Patients receiving probiotics in food may face fewer allergic reactions (Tomasik and Tomasik, 2003). The following sections discuss in detail probiotics and prebiotics with respect to their therapeutic properties.

2 The Concept of Probiotics According to a recent definition from Germany, probiotics are defined as “Live microbes which can reach the intestine of the host in active form.” The term “probiotics” was first developed by Kollath in 1950, and the term was also used for live bacteria and spores in animal feed supplements by Lilly and Stillwell (1965). However, Fuller (1989) gave the first generally accepted definition: “Live microbes which can affect human health beneficially by improving intestinal balance.” This idea of suppressing the growth of harmful bacteria by oral administration of beneficial microorganisms was first presented by Carre (1887), while Metchnikoff (1908) created the concept of beneficial microorganisms that today we know as probiotics. With the passage of time similar properties of beneficial microorganisms, such as Bifidobacterium, was reported by Anukam and Reid (2007), who claimed that the oral consumption of Bifidobacterium reduced the growth of putrefactive disease-causing bacteria, while the intake of yogurt containing Lactobacillus caused the reduction of bacteria that caused toxins in the gut. Furthermore, probiotic microorganisms not only have a beneficial effect on the intestines, but also have an effect on other body organs by producing different regulatory and bioactive parameters. Hence the broader definition of probiotics, suggested by Schrezenmeir and de Vrese (2001): “Probiotics are live microbial feed supplements which beneficially influences the health of the host.” Probiotics were further defined by the FAO/WHO (2001) as “live microorganisms that when administrated in adequate amounts confer health benefit effects on the host.” Therefore, as summarized here, all of these definitions, irrespective of their differences, suggest that a probiotic microorganism should be alive and should have proven positive health effects.

2.1 Taxonomy of Probiotic Microorganisms Many genera of bacteria and some yeast have been suggested for use as probiotic cultures but the most common are Lactobacillus and Bifidobacterium; both are gram-positive, lactic acid–producing bacteria that constitute a major portion of intestinal microflora (Fujiya et al., 2014).

Therapeutic Aspects of Probiotics and Prebiotics 55 Table 3.1: Probiotic microorganisms. Microorganisms Used as Probiotics Bifidobacteria

Lactobacilli

Others

B. longum group B. pullorum group B. adolescentis group B. asteroids group B. boum group B. pseudolongum group

Lactobacillus lindneri L. sanfranciscensis L. rhamnosus L. kefiri L. plantarum L. pentosus L. fermentum L. panis L. casei L. delbrueckii subsp. lactis L. delbrueckii subsp. bulgaricus B. subtilis

Enterococcus faecalis S. thermophilus S. boulardii Lactococcus lactis Propionibacteria

Source: Fujiya, M., Ueno, N., Kohgo, Y., 2014. Probiotic treatments for induction and maintenance of remission in inflammatory bowel diseases: a meta-analysis of randomized controlled trials. Clin. J. Gastroenterol. 7(1), 1–13; De Vrese, M., 2007 Effects of probiotic bacteria on gastrointestinal symptoms, Helicobacter pylori activity and antibiotics-induced diarrhoea. Gastroenterology 124, A560; Preedy, V.R., Watson, R.R., 2016. Probiotics, prebiotics and synbiotics, second ed. Langford, London.

2.1.1 Lactobacilli Lactobacilli are gram-positive, rod-shaped, nonspore-forming bacteria with complex nutritional requirements. Mostly these bacteria are autofermentative, strictly anaerobic, and acidophilic (Senok et al., 2005). Carbohydrate-rich products from both plant and animal origins and fermented milk products are utilized as substrate by these bacteria (Schrezenmeir and de Vrese, 2001). Some of these microorganisms have the unique capacity to produce exopolysaccharides that possess functional as well as therapeutic properties. Recently, Lactobacillus kefiranofaciens (ZW3) was reported to have exopolysaccharide properties, and it can be used for coculturing in yogurt (Ahmed et al., 2013a,b). 2.1.2 Bifidobacteria Bifidobacteria comprise a major portion of gut microbiota; they appear in human stools some days after birth and increase in number thereafter. The normal range for these bacteria in the adult colon is 1010–10 11 CFU/g (Schrezenmeir and de Vrese, 2001). These bacteria are mostly rod shaped, gram positive, strict anaerobic, and nonspore forming. A brief list of these bacteria groups, along with other probiotics, is depicted in Table 3.1.

2.2 Screening, Identification, and Characterization of Probiotic Microorganisms According to FAO/WHO criteria, it is important to recognize bacteria at the species level because much of the evidence suggests that the beneficial health effects of probiotics are

56 Chapter 3

Figure 3.1: Screening Method for Probiotics.

strain specific. The recommendation is therefore to apply both phenotypic and genotypic techniques to carry out the identification, classification, and characterization of all strains (Osmanagaoglu et al., 2010). After identification, scientifically recognized names for nomenclature are applied to the bacteria, and it is further suggested to deposit these species in internationally recognized culture collections. These strains are further characterized by their probiotic aspects and safety–risk assessments. A brief description of the screening method is shown in Fig. 3.1. Furthermore, in vitro evaluation is useful to study the mechanism of action of these selected strains and to gain knowledge about them. Based on this knowledge and their long history as safe for consumption in many products, some of these strains have been accorded the status of “generally recognized as safe” (GRAS) by the US Food and Drug Administration (FDA) (FAO/WHO, 2001). There are two phases of trials that measure the safety of probiotics for human consumption. In the first phase, probiotics are validated in human models, while in the second phase these trials cover both the efficacy and safety aspects. However, the second phase should be randomized and placebo controlled so that the efficacy and possible adverse effects of these strains can be determined (BermudezBrito et al., 2012).

Therapeutic Aspects of Probiotics and Prebiotics 57

Figure 3.2: Probiotics Modes of Action.

3 Therapeutic Effects of Probiotics The maximum theraputic effects of probiotics are directly or indirectly dependent on their action in the gastrointestinal tract (GIT). This is not only because they are taken orally, but also due to the fact that probiotic microflora have efficacy because of their activity and interaction with the microflora of the host cell mucous membranes (Salminen et al., 2004). However, not all probiotic microorganisms are part of the human gut flora, so the benefical effects of one species may not pertain to other species. Apart from their benefical effects, the exact mechanism of the action of these microorgansism is still unknown, so many other mechanisms are described for them (Fig. 3.2). However, the basic pathway for their action is that when they are orally ingested, they pass through the stomach and mucous membranes and protect the epithelial membranes from pathogenic bacteria (Vibhute et al., 2011). Some of these bacteria, such as Bifidobacterium and Lactobacillus, also produce certain acids, such as lactic acid, propoinic acid, and acteic aicd, which lower the pH and prevent the growth of pathogenic bacteria (Chaucheyras and Durand, 2010). Another suggested mechanism for their action is their role in immunomodulation. A recent study from the University of Maryland school of medicine showed that ingesting LGG (Lactobacillus rhamnosus GG) can assist the ecosystem of the gut through the modification of the activities of other bacteria (Vibhute et al., 2011). Based on these mechanisms, probiotics have many preventive and curative effects against several maladies, such as diarrhea (Isolauri et al., 2002), inflammatory diseases (Majarmaa and Isolauri, 1997), irritable bowel syndrome (Duffy et al., 1993), cancer (Bengmark, 2012b; Mack et al., 1999), diabetes (Pronio et al., 2008), bacterial infections (Galdeano et al., 2007), and many others (Table 3.2).

58 Chapter 3 Table 3.2: Effects of probiotics on different diseases. Sr. No.

Diseases

Study Subjects Observed Effects

References

1

TD

Humans

Dupont et al. (2014), McFarland (2007)

2

Acute infectious diarrhea

Humans (children)

3

AAD

Humans (children)

4

IBS

Humans

Single probiotic at a low dose and with short treatment is more effective

5

CD

Humans (children)

6

UC

Humans

7

Lactose intolerance

Humans

8

H. pylori infection

Humans (children)

9

Rats

10

Hypercholesterolemia Cancer

11

Diabetes

Humans/rats

12

Obesity

Humans

Effect on PCDAI was to lower by 73% from baseline; CDAI fell from 217 to 150 Probiotic treatment was more effective than placebo in controlling UC Reduced effects of lactose intolerance; DDS-1 L. acidophilus strain was safe to use in acute lactose intolerance No strong evidence for eradication of H. pylori through probiotic supplementation Different probiotic strains have hypocholesterolemic effect on rats Reduced postoperative infectious complications in CRC; anticarcinogenic properties Effective for prevention and management of T1D and T2D; reduces serum CRP levels and increases GSH levels in T2D patients Regulates body weight and represents a viable treatment option for obesity; may facilitate alleviating metabolic syndrome

Humans/rats

Reduced relative risk and duration of diarrhea in travelers; useful in conjunction with rehydration Reduced risk and average duration of diarrhea; reduced risk of rotavirus infection Beneficial for treatment of AAD; reduction in diarrheal episodes and C. difficile infections

Luyer et al. (2005), Szajewska et al. (2011) Hickson (2011), McFarland (2007), Pillai and Nelson (2008) Wasilewski et al. (2015), Zhang et al. (2016) Gupta et al. (2000), McCarthy et al. (2003) Sanders (2003), Tsang et al. (2010) Vonk et al. (2012)

Anukam and Reid (2007), Pacifico et al. (2014) Anandharaj et al. (2014) Kumar et al. (2010), Liu et al. (2009), Maleki et al. (2016) de Oliveira et al. (2014), Ejtahed et al. (2011), Ghasemi-Niri et al. (2011) Sanz et al. (2013)

AAD, Antibiotic-associated diarrhea; CD, Crohn’s disease; CDAI, Clinical Disease Activity Index; GSH, glutathione; IBS, irritable bowel syndrome; PCDAI:, Pediatric Crohn’s Disease Activity Index; UC, ulcerative colitis.

3.1 Traveler’s Diarrhea Traveler’s diarrhea (TD) is a fecal-oral diffused disease due mainly to bacteria, and due less to viruses or protozoa. Escherichia coli is the most common bacteria to cause this disease, although in south Asia Campylobacter is also a leading cause (Shah, 2007). Using probiotics for treatment of this diarrhea has very unpredictable results (Dupont et al., 2014) and there

Therapeutic Aspects of Probiotics and Prebiotics 59 are insufficient studies that cover this purpose. However, according to a metaanalysis, probiotics had a significant effect in reducing TD (RR = 0.85, CI = 95%) in 12 study subjects (McFarland, 2007). Similarly, another study showed that Saccharomyces boulardii conferred dose-related protection against TD and that the L. rhamnosus strain also provided 12%–45% protection against TD (Dupont et al., 2014).

3.2 Acute Infectious Diarrhea Acute infectious diarrhea, either due to bacteria or viruses, is still a major health problem worldwide and a cause of death for children in underdeveloped countries. In developed countries people are affected by diarrhea resulting from foodborne illnesses. The effects of probiotics in reducing the risk and duration of diarrhea are the best-documented probiotic effects, based on detailed clinical studies that fulfill scientific requirements. These beneficial effects include a decrease in the number of infections; a decrease in the duration of stool episodes; and an elevation of the body’s immune system, producing antibodies against causative microbes, such as rotavirus and E. coli (Szajewska and Mrukowicz 2001).

3.3 Antibiotic-Associated Diarrhea Antibiotic-associated diarrhea (AAD) is a common clinical problem that usually occurs due to the excessive growth of Clostridium difficile, a harmless bacterium. The disturbance in the internal microbial balance due to antibiotics causes this excessive growth and leads to diarrhea (de Vrese and Marteau, 2007). The antibiotics can affect intestinal microflora directly by ceasing to resist intestinal pathogens, which facilitates the growth of C. difficile and other pathogens. Treatment of AAD through probiotics is usually a tool to test the effectiveness of probiotics and to validate of scientific health claims (Preedy and Watson, 2016). A metaanalysis by McFarland (2007) demonstrated that the use of probiotics significantly reduced diarrheal episodes in 25 randomized controlled trials. Similarly, another study showed that drinks with probiotics, including Lactobacillus bulgaricus, Lactobacillus casei, and Streptoccocus thermophilus, can reduce AAD to 21.6%, a significant decrease (Hickson, 2011). Based on the literature cited it can be concluded that probiotics can reduce the risk for AAD and can also be used for the treatment and prevention of diarrhea associated with C. difficile (Goldenberg et al., 2013).

3.4 Irritable Bowel Syndrome (IBS) Irritable bowel syndrome (IBS) is a functional gastroenterological disease mainly characterized by abdominal pain and changes in bowel habits (Lovell et al., 2010). The therapeutic options for this disease includes antibiotics, antidepressants, and spasmolytics,

60 Chapter 3 which can cause many health complications, so the new trend in the treatment of IBS includes the use of probiotics. To date, many clinical studies have proven the effect of probiotics on IBS. A metaanalysis demonstrated that the use of probiotics at a low dose with a short duration is the most effective therapy (Zhang et al., 2016). In another study, scientists stated that the exact mechanism by which probiotics work on IBS is still not understood; however, there are bacterial strains that have a positive effect on the GIT in IBS (Almansa et al., 2012; Wasilewski et al., 2015).

3.5 Crohn’s Disease Crohn’s disease (CD) is an inflammatory disease characterized by chronic inflammation within the gastrointestinal walls; the most effected part is the ileum. The exact pathogenesis of CD has not been determined; however, it might be caused by an abnormality in the intestinal microflora, a genetic predisposition and impaired immune system response that can be modified using prebiotics (Shanahan, 2002). In clinical trials to test the efficacy of probiotics on CD, Lactobacillus GG was administered to patients for one year after surgery at a dose of 1.2 × 1010 CFU and compared with a placebo treatment. The results showed that the reappearance of symptoms happened in 10% of the placebo-controlled patients and 16% of the probiotic-treated patients. In endoscopy results, in 60% of patients in remission pathological changes reappeared, in comparison to the control group in which there was reappearance in 35% of patients. This study suggests the failure of probiotic treatment for CD therapy (Prantera et al., 1992). However, in another study Lactobacillus GG was administrated to four children suffering from CD at a dose of 1010 CFU. The results showed significant progress in clinical activity after 1 week of probiotic administration, which was sustained throughout the study period. There was also a 73% reduction in CD activity index scores until the fourth week. The study suggests that Lactobacillus GG could improve gut barrier function and the clinical status of children suffering from CD (Gupta et al., 2000). The previous data suggest that the efficacy of probiotics against CD is still debatable and requires more study.

3.6 Ulcerative Colitis (UC) Ulcerative colitis (UC) is also an inflammatory disease, in this case of the mucous membranes in the rectum. It leads to ulceration, perforation, and necrosis of the intestines and mostly affects young people 20–40 years of age (Fedorak and Rioux, 2006). A metaanalysis was conducted to evaluate the effect of probiotics on UC. Thirteen randomized studies were performed that looked at remission and reoccurrence in comparison with a nonprobiotic group. Results showed that the probiotics group of UC patients had a remission rate of 1.35 (95% CI), while the nonprobiotic group had a 2% remission rate. The reoccurrence rate in patients who received a Bifidobacterium treatment was 0.25 (95% CI), while the nonprobiotic group had the same rate. This study suggested that probiotic treatment is more effective for

Therapeutic Aspects of Probiotics and Prebiotics 61 maintenance of remission in UC patients as compared with the placebo treatment (Tsang et al., 2010). However, important strategies based on clinical studies that consider different strains, dosages, and the heterogeneity of patients are needed to augment the use of probiotics for human health and disease.

3.7 Lactose Intolerance Lactose intolerance is a clinical syndrome in which a person is not capable of digesting milk sugar or lactose. It is characterized by symptoms such as abdominal pain, nausea, vomiting, watery diarrhea, flatulence, and bloating (Chitkara et al., 2005). Lactase, an intestinal enzyme located on the crypt-villus axis, hydrolyzes lactose into glucose and galactose. Lactose malabsorption is of two types: primary and secondary. Primary lactose intolerance is due to genetic linkage and occurs mostly in the people of Asia, Africa, North America, and the Mediterranean (Escher et al., 1992; Fajardo et al., 1994; Sahi, 1994; Troelsen, 2005), while secondary lactose malabsorption is due to different types of acquired diseases, such as celiac disease, allergic gastroenteritis, and viral gastroenteritis, which affect the lactase enzyme and the intestinal villi (Fig. 3.3). People with a low level of the lactase enzyme cannot absorb lactose in the small intestine, so it is transferred to the colon, where the microbial flora undergo fermentation and eventually develop short-chain fatty acids (SCFAs) and hydrogen gas. These SCFAs are absorbed by colon mucosa, thereby reducing the effect of lactose malabsorption. Probiotic bacteria (Lactobacillus and Bifidobacterium) enhance the production of the lactase enzyme, so they can be recommended for treating lactose malabsorption by improving the digestibility of lactose (Rastall, 2004). In a recent study, the effect of Lactobacillus acidophilus on lactose intolerance was studied in volunteers suffering from lactose malabsorption.

Figure 3.3: Mechanism of Probiotic Effect on Lactose Intolerance.

62 Chapter 3 They were treated with the DD-1 species of L. acidophilus for 4 weeks. Results showed that lactose intolerance symptoms such as abdominal pain, diarrhea, vomiting, and other all symptoms scored zero, without any side effects (De Vrese, 2007). Similarly, Vonk et al. (2012) stated that probiotics can reduce the symptoms of lactose malabsorption by increasing the hydrolysis of lactose in dairy products. However, early diagnosis of lactose intolerance is complex and the exact mechanism for its proper treatment requires more clinical studies. So new strategies should be developed for its treatment with probiotics, which could lead to an auspicious approach.

3.8 Heliobacter pylori Infections H. pylori is a gram-negative, flagellated organism that can survive in gastric mucosa. Infections from it affect approximately 50% of the world population. H. pylori is linked to gastric carcinoma, gastric ulcers, and many other gastric problems. Eradication of H. pylori has been a challenge over the last 30 years, but still an ideal treatment has not been identified. Beginning with the second decade of the 21st century, interest in microorganisms that can eradicate H. pylori, including probiotics, has increased (Bolton et al., 2015). Several studies have been conducted to evaluate the effect of probiotics on H. pylori. A recent study of schoolchildren suffering from H. pylori infections was conducted; one group was treated with probiotics and the other with standard antibiotics. The results showed that probiotics had no significant effect on eradicating H. pylori. Similar results were obtained from another study of human subjects; that is, the results showed probiotics had no significant effect on the eradication of H. pylori (Pacifico et al., 2014). Based on these studies, it is recommended that large, randomized, and controlled studies are needed to investigate the effect of probiotics on H. pylori eradication.

3.9 Hypercholesterolemia and Coronary Heart Diseases An elevated cholesterol level is thought by many to be the main risk element for the development of cardiovascular diseases (CVDs), including hypertension, atherosclerosis, and coronary heart diseases. According to the WHO, CVDs will remain the major cause of death through 2030 and affects 23.6 million people worldwide. It is also important to indicate that even a 1% reduction in cholesterol level can reduce the risk of CVDs to 2%–3% (Rasic et al., 1992). Nowadays many drugs are used to lower serum cholesterol levels, but based on long-term use these drugs have unwanted side effects. Therefore there is an increasing trend toward using dietary fiber and probiotics for lowering serum cholesterol levels (Ahmad et al., 2012). Many mechanisms have been suggested as the reason for the effect of probiotics on serum cholesterol levels, among which the most important is that probiotics bind bile salts to intestinal walls through an enzyme called the bile salt hydrolase enzyme (BSH). BSH activity breaks down deconjugated bile salts with


Figure 3.4: Mechanism of Probiotic Effect on Serum Cholesterol.

the help of probiotic microorganisms into free taurine, free cholic acids, and free amino acids, which are then reabsorbed by the liver and intestine or excreted from the body (Anandharaj et al., 2014) (Fig. 3.4). In a metaanalysis, the effect of probiotics on human subjects with high serum cholesterol levels was determined. The results showed a significant decrease in serum total cholesterol (TC) and LDL-c levels suggesting that probiotics supplementation can be used for prevention of hypercholesterolemia (Schrezenmeir and de Vrese, 2001). In the same way in another study, effect of probiotics (lactobacillus)was evaluated on lipid profile of hypercholesterolemic rats in 4 weeks’ study. Results showed a significant reduction in serum TC, low-density lipoprotein cholesterol, and triglyceride levels, suggesting that using Lactobacillus-based probiotic combinations can improve serum lipid profiles, thereby reducing the risk of CVDs (De Roos and Katan, 2000). Further cardiovascular studies have also suggested that probiotics can be used as dietary supplements for potential benefits. However, more analysis and trials are needed to develop the precise strains and specific dosages that can beneficially affect the cardiovascular system (Thushara et al., 2016).

3.10 Diabetes Diabetes is a multifactor-origin disease, caused by factors including genetics and the environment, which affects 10%–30% of the population worldwide. Some scientific studies have suggested that the reason behind the development of diabetes is increasing

64 Chapter 3 inflammatory stress, which causes insulin resistance and the interaction of intestinal microbes with environmental and genetic factors, leading to diabetes. However, improvement in the functioning of intestinal microbes through probiotics can help in managing this clinical condition. Probiotics maintain healthy gut microbes and can act as effective factors in insulin-resistant therapies (Ejtahed et al., 2011). A metaanalysis evaluated the effect of probiotics on the glycemic index and glucose level of diabetic patients. The results showed significant differences between probiotic and placebo-controlled subjects in terms of reduction in glucose, insulin, and HbA1c levels, suggesting that probiotics can be used as dietary supplements to reduce glucose levels and other glucose metabolism disorders linked to diabetes (Sun and Buys, 2016). Similarly, another study depicted the efficacy of probiotics on both type I diabetes (T1D) and type 2 diabetes (T2D) through many clinical trials, and determined that probiotics can be an effective means to prevent T1D and T2D through the modulation of intestinal microbes (de Oliveira et al., 2014).

3.11 Obesity The frequency of obesity is increasing day by day, especially among adults and children, and it is now becoming a worldwide health problem. The main causes of obesity are complex and include natural, genetic, and endocrine factors, while at the same time alimentary and physical habits are also contributing causes. Recently obesity has also been linked to structural changes in the gut microflora of animals and humans, suggesting a possible connection between these microbes’ genes and their functional disorders (Sanz et al., 2013). Moreover, animal studies evaluating these mechanisms have suggested that microbes have a big role in obesity through their contribution to nutrient digestion, absorption, and adsorption (Bäckhed et al., 2012; Semova et al., 2012). The metabolic activity of microbes can help the host to obtain energy from the diet, facilitating the breakdown of complex indigestible polysaccharides and providing approximately 10% of daily energy supply (McNeil, 1984). In addition, in most recent studies diet has been a primary factor influencing microbial structure in obese people, suggesting that diet and microorganisms have an important role in body-weight regulation (Sanz et al., 2013). However, in recent studies the effect of probiotic yogurt was compared with standard low-fat yogurt in obese people. The results showed insignificant differences in weight loss and anthropometric measurements between the two groups. Similarly, in another study the effect of the probiotic species Lactobacillus salivarius on biomarkers related to metabolic syndrome and inflammation in obese adolescents was examined. The results revealed that there were insignificant differences between the probiotic and the placebo groups for fasting glucose and insulin levels, lipoprotein profile, anthropometric values, and many other parameters relating to the metabolic syndrome baseline, suggesting that probiotic strains have no beneficial effect on biomarkers for metabolic syndrome in obese adolescents (Gobel et al., 2010).


3.12 Colon Cancer Cancer is defined as the growth and reproduction of abnormal cells and is the leading cause of deaths in many countries. It is an evolving and very common health problem, especially in developing countries. Complications in cancer are due to infections that cause death in cancer patients and are difficult to treat. Most cancer patients are in an immune-compromised situation, with different chemo- and radiotherapy treatments, bone-marrow suppression, and the infection itself, which causes the situation to deteriorate (Zhu et al., 2012). The main reason for these infections is internal gut microbes, mostly pathogenic bacteria, which can be transferred from bowel colonization through mucosa and other systems. Live microorganisms called probiotics can be used for treatment of these infections, which stimulate the gut immune system, compete for nutrition with pathogenic bacteria, and produce some bacteriocins and organic acids that can be fatal for pathogens. Probiotic species not only prevent infections but also act as antitumor agents (Maleki et al., 2016). A recent study suggests that probiotic metabolites have cancer-preventing abilities and interactions between these metabolites and molecular signals act as epigenetic agents for cancer prevention (Kumar et al., 2010). In another study, the effect of probiotic species against serum zonulin concentrations and their anticancer activity was evaluated in 150 patients. The results showed a reduction in zonulin concentrations, the period of postoperative pyrexia, and the time interval for antibiotic therapy. Above all, studies suggest that probiotic strains can reduce the risk of cancer and act as cancer-prevention agents (Maleki et al., 2016).

4 Prebiotics and Synbiotics Prebiotics were first defined by Gibson and Roberfroid (1995) as “undigestible food ingredients which can provide benefits to the host which can also motivate the development of discriminating probiotics in the colon and promote the health of the host.” Recently many ingredients have been recognized as preventing hydrolysis in the gut and small intestine, but now only four ingredients have been declared to meet the probiotic-stimulating criteria of prebiotics: fructooligosaccharides (FOS), inulin, galactooligosaccharides (GtOS), and soybean oligosaccharides (SOS) (Hayat et al., 2014; Rastall and Maitin, 2002). The general criteria for selecting a probiotic substrate include: (1) it should provide resistance for hydrolysis and absorption in the GIT, (2) it should act as a substrate for fermentation by probiotic organisms in the colon, (3) it should be useful in making the composition of gut microbiota healthier, and (4) it should impart a beneficial health effect to the host (Gibson and Roberfroid, 1995).

4.1 Types of Prebiotics Prebiotics are generally classified as low molecular weight oligosaccharides, but they also include polysaccharides, as they can be used as a rich source of carbon for gut microbiota.

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Figure 3.5: Monomers in Prebiotics.

After ingestion these compounds pass through the small intestine without being digested, while in the large intestine they will be utilized by gut microbes or probiotics as a substrate for fermentation and for the formation of SCFAs, which are fatal to pathogens (Rastall and Maitin, 2002). These oligosaccharides are naturally present as free sugars in plants, whereas in the colostrum of animals and in human milk they are also present in the form of glycol conjugates. There are two types of prebiotics: those naturally present in plants and those that are artificially synthesized from polysaccharides after enzyme digestion. 4.1.1 Natural prebiotics There are three main types of naturally existing prebiotics: GtOS, FOS, and SOS. 4.1.1.1 Galactooligosaccharides

GtOS are nondigestible carbohydrates that exist in cow’s milk, human milk, and yogurt in the form of glucose, galactose, or fructose monomers that have Glu-α-1-4{β-Gal-1-6} linkages (Fig. 3.5). They can pass through the gut but can be hydrolyzed in the large intestine by probiotic microorganisms and produce SCFAs, such as butyric acids, and propionic acids and gases, such as CO2, CH4, and H2. These microbes can also produce other metabolites by using GtOS, such as lactate, and vitamins that increase the growth of Lactobacilli and Bifidobacterium (Kolida et al., 2000). 4.1.1.2 Fructooligosaccharides

FOS are the most important and most well-known prebiotics because they were the first prebiotics to be made commercially available in the United States. FOS are short-to-medium chains of β-d-fructans containing fructosyl units linked through β-2-1 glycosidic linkages (Fig. 3.6). They are indigestible to gut microbes but can be digested by the microflora of the large intestine. They are insoluble in cold water but can be dissolved in hot water at approximately 80°C. They do not provide any sensory properties, except a little sweetness, and are quite stable; due to these properties they are added to many food products to improve the products’ physical properties.


Figure 3.6: FOS Structures.

4.1.1.3 Soybean oligosaccharides

The oligosaccharides present in soybeans are called SOS; namely, raffinose and stachyose (Gibson et al., 2004). These oligosaccharides can be digested in the stomach and small intestine and can also be hydrolyzed in the large intestine by microflora. They can also promote the growth of bifidobacteria in the large intestine (Hayakawa et al., 2012). 4.1.2 Synthetic prebiotics Synthetic prebiotics are created by using different enzymes to hydrolyze carbohydrates. The most common synthetic prebiotics are lactosucrose, lactulose, isomaltooligosaccharides (IMO), glucooligosaccharides, and xylooligosaccharides (XOS). Some artificial prebiotics and their production methods are listed in Table 3.3.

Table 3.3: Selected prebiotics and their production methods. Prebiotics

Synthesis Procedure

Inulin (FOS) GtOS XOS IMO

Extracted from chicory root through enzymatic hydrolysis Enzymatic of lactose Extracted from plant xylan through enzymatic hydrolysis Starch composed of isomaltooligosaccharides, slightly modified by isogalactosylated units Lactose isomerization

Lactulose

FOS, Fructooligosaccharides; GtOS, galactooligosaccharides; IMO, isomaltooligosaccharides; XOS, xylooligosaccharides.

68 Chapter 3 4.1.2.1 Lactosucrose

As the name indicates, lactosucrose is prepared by combining sucrose and lactose sugar using the β-fructofuranosidase enzyme. When lactosucrose was administrated orally to three volunteers to evaluate its efficacy, results showed that the amount of Bifidobacterium was increased 0.7 times while the pathogenic bacteria count was reduced 0.6 times (Bouhnik et al., 2004). 4.1.2.2 Lactulose

Lactulose is also prepared from lactose and has the structure Gal-β-1-4-Fru. It is soluble in water and insoluble in ether. Lactulose cannot be hydrolyzed by microbes in the gut, while bacteria in the large intestine can use it as a substrate for fermentation. Lactulose is also used as a functional component and is added to different food products, such as yogurt, chocolate, and cake, to improve their nutritional value. 4.1.2.3 Isomaltooligosaccharides

IMO are prepared from starch using different enzymes and is digestible in the small intestine. To evaluate its prebiotic effect, it was tested on subjects at 20 g/day; the findings showed that it can increase the content of Bifidobacterium and also produce butyrate through fermentation (Martín et al., 2003). 4.1.2.4 Glucooligosaccharides (GOS)

These prebiotics are synthesized by adding glucose units using the glycosylic transferase enzyme. GOS can be fermented by Bifidobacterium in the large intestine and can be hydrolyzed by bactericides. GOS are effective in stimulating the activity of Bifidobacterium breve and thereby reducing contamination by Salmonella in animal models, suggesting they are effective prebiotics (Marilyn et al., 2014). 4.1.2.5 Xylooligosaccharides

XOS are synthesized by joining xylose molecules through β-1-4 linkages. Both bifidobacteria and lactobacilli can hydrolyze it in the large intestine. XOS were found to be more effective than FOS, by enhancing the Bifidobacterium count and reducing the pathogenic bacteria count (Villamiel et al., 2014).

4.2 Sources of Prebiotics Naturally present prebiotics can be easily detected in many foods, such as chicory, asparagus, milk, wheat, and tomatoes, and in some components of human milk. Synthetic prebiotics are prepared from different simple and complex carbohydrates by using different enzymes as described previously. Sources of selected prebiotics are listed in Table 3.4.

Therapeutic Aspects of Probiotics and Prebiotics 69 Table 3.4: Sources of some prebiotics. Types of Prebiotics

Structures

Source(s)

Isomaltulose

—

FOS

α-d-Glu[-(1-2)-β-d-Fru]n (n = 2–4)

XOS

Xylo-OSs with zero, one, or multiple l-arabinofuranose residues attached by α-1,2 or/ and α-1,3 glycosidic linkages on the xylan backbone β-d-Gal-(1-4)-β-d-Fru α-d-Glu-(1-4)-β-d-Gal[-(1-6)-βd-Gal]n (n = 1–4) β-d-Gal-(1-4)-α-d-Glu-(1-2)β-d-Fru [α-d-Gal-(1-6)-]n-α-d-Glu-(1-2)β-d-Fru (n = 1–2) —

Sugarcane juice and Kohmoto et al. (1992) honey Chicory, sugar beets, Sangeetha et al. (2005) asparagus, onions, wheat, barley, honey, garlic, and sugarcane juice Bamboo shoots, Okazaki et al. (1990) vegetables and fruit, milk and honey

Lactulose GOS Lactosucrose SOS IMO

References

Milk sugar (lactose) Cow milk and human milk Lactose

Villamiel et al. (2014) Alander et al. (2001)

Soybean

Okazaki et al. (1990)

Starch

Yamada et al. (1993)

Fujita et al. (1991)

SOS, Soybean oligosaccharides.

5 Therapeutic Effects of Prebiotics The beneficial health effects of prebiotics demonstrated by various scientific studies through increasing the count of Bifidobacterium or reducing the number of pathogenic microbes begs the question as to whether these properties are sufficient for the promotion of human health (McAllister et al., 1994). Because of insufficient knowledge about the composition of probiotic microbes and their complex interactions it is difficult to reduce the effect of probiotic microorganisms even if the effect changes from organism to organism because they are generally regarded as safe. Therefore, definitive proof of the beneficial health effects of these organisms should be established through precise human interventions. The link between the strength of these microorganisms and the administration of prebiotics is fragile, because determining the increase in the number of probiotics after ingesting prebiotics depends on whether the exact number of probiotics in the host is known. However, some studies show that 4 g/day or less of inulin ingestion have relevant health effects. Some of the potential beneficial health effects of prebiotics are discussed in the sections that follow.

5.1 Prebiotics as Dietary Fiber In terms of nutrition physiology, dietary fiber includes bioactive substances defined as “the edible part of plants and carbohydrates which could not be absorbed and digested in humans and could be part of fermentation in the large intestine.” These substances include lignin,

70 Chapter 3 oligosaccharides, polysaccharides, and related plant substances (Jones and Holt, 2000). Based on its positive effects and upgrading definitions of dietary fiber, it has been classified into two types: soluble and insoluble dietary fiber (depending on its solubility in an aqueous solution). Usually dietary fiber, including β-glucan, enhances the bioavailability of nutrients, contributes to the transfer of nutrients in the GIT, and supports the growth of probiotic microorganisms in the gut. The beneficial health effects of these nondigestible food components occur in the human large intestine, as they cannot be digested in the small intestine, so they are transferred to the human colon, where they are totally or partially fermented by gut microbes. The effectiveness of fermentation in the colon is dependent upon the type of carbohydrates, their glycosidic linkages, and their structural arrangements. So, these different types of nondigestible carbohydrate with different structural arrangements enhance the growth of microflora in the human gut system (Ahmad et al., 2010). Based on this knowledge, dietary fiber is also termed a prebiotic, because the first prebiotic definition was based on the scientific study of dietary fiber’s beneficial health effects (Gibson and Roberfroid, 1995). The idea of prebiotics supports the concept of the effectiveness of selected nondigestible carbohydrates or dietary fiber in promoting human health.

5.2 Effect on Influenza Influenza is a virus infection causing approximately 250,000–500,000 deaths worldwide annually, and especially affects people age 65 and above. In developed countries influenza vaccines are suggested for older patients, however, older patients have compromised immune systems, which cause insufficient induction of antibodies. Many animal and clinical studies have shown the useful effects of prebiotics on influenza infections. The results of a recent study on effect of the prebiotics GOS and BGS showed that after ingestion of prebiotics for 8 weeks, there was a significant increase in body weight, number of probiotic microflora, and antibody (H1N1, H3N2, and B) count, suggesting that administrating different types of prebiotics simultaneously might facilitate the maintenance of enhanced antibody titers (Akatsu et al., 2016).

5.3 Hyperglycemia/Diabetes Nutrition has an important role in managing diabetes. Some nutrients can also reduce the postprandial glucose response. Foods such as fruits, cereals, legumes, and spices are important, containing active ingredients such as dietary fiber and polyphenols that can reduce the insulin immune response and glycemia levels. The glucose-reducing effect of these active ingredients depends upon the type of carbohydrates, their source, and dosage. Among all the nondigestible carbohydrates, inulin-type fructans (ITF) are important prebiotics that can modulate the gut’s microbial composition, thus conferring beneficial health effects on host physiology (Bindels et al., 2015). The mode of action of ITF is that they enhance the

Therapeutic Aspects of Probiotics and Prebiotics 71 count of endocrinal L cells in the colons of rodents and enhance the release of GLP-1 active forms, which reduce glycemic levels (Cani et al., 2007a,b). Similarly, another prebiotic called arabinoxylan (AX) has a beneficial effect on hyperglycemic levels. AX also undergoes colon fermentation through active microbes, and is most abundantly present in wheat, bran, and aleurone fractions (Andersson et al., 2013). A study based on a diabetic-induced mouse model shows the diabetes-lowering effect of AX through its increasing the count of probiotics in the colon, which enhances insulin resistance (Neyrinck et al., 2012). However, more studies are needed to determine the mechanism by which AX acts on gut microflora and its diabeteslowering effects.

5.4 Obesity The modulation of the composition of gut microbes occurs with obesity, which causes changes at the genetic level. For instance, people having a low count of Bifidobacterium at birth has been linked with developing obesity after childhood. Moreover, obese mothers are at risk for giving birth to children having low counts of Bifidobacterium, proving that obesity is an inheritable characteristic (Collado et al., 2009). Similarly, studies showed that elderly obese people have factors of increase in Bifidobacterium count lower than lean people, suggesting that Bifidobacterium has an important role in the human metabolic system and furthermore in the development of obesity (Madjd, 2016). Fruit and many other food products contain dietary fructans and oligosaccharides, which act as substrates to enhance the growth of Bifidobacterium in the human gut (Bomhof et al., 2014). Several studies have suggested significant increases in Bifidobacterium count in obese mice whose diets were supplemented with ITF (Cani and Delzenne, 2009; Cani et al., 2007a,b; Tilg and Moschen, 2009). Moreover, these studies also showed that the number of Bifidobacterium was inversely proportional to LPS and glucose levels and body fat mass (Cani et al., 2007a,b). Furthermore, prebiotics also have an important role in inhibition of the overexpression of those host genes that cause inflammation and adiposity by increasing the number of endocrinal L cells that produce GPL-1 and GPL-2 (Sarbini and Rastall, 2014). Studies on several experimental subjects have proven that GPL-1 actively participates in decreasing appetite, body fat, and hepatic insulin resistance (Tilg and Moschen, 2009). Meanwhile, GPL-2 participates in the reduction of intestinal-wall permeability and endotoxemia, which cause obesity (Sarbini and Rastall, 2014). Hence, overall studies suggest that ITF and dextran oligosaccharides actively participate in the prevention of genes that cause adiposity (Beserra et al., 2015; Sarbini and Rastall, 2014).

5.5 Hepatic Encephalopathy Hepatic encephalopathy (HE) is linked with the dreaded complications of liver disease defined as “Disorders in the central nervous system due to hepatic deficiency”

72 Chapter 3 (Blei and Cordoba, 2001). Even minimal HE can cause major health issues for patients suffering from it, due to its uncertain multifactor effects (Weissenborn et al., 2001). The suggested treatments for HE are prebiotic lactulose and poorly absorbable antibiotics. Lactulose is a nondigestible synthetic prebiotic having multiple beneficial effects on gut microflora by acting as a substrate for fermentation by bacteria species in the large intestine, resulting in the production of butyric, lactic, and acetic acid, and hydrogen gas. Production of these weak acids for colon bacteria with excessive hydrogen gas production in the large intestine causes flatulence in the colon area, which ultimately results in massive removal of colon bacteria, especially urease-positive bacteria and ammonia-producing bacteria. Accordingly, the main effect of lactulose is rapid stool transfer due to bacterial gas–mediated activation of intestinal peristalsis (Bongaerts and Severijnen, 2004).

5.6 Gastric and Colorectal Cancer Colorectal cancer (CRC) is the second most common reason for mortality and the fourth leading malignant neoplasm in the United States (Kumar et al., 2010). The rapidly increasing incidence of CRC is due to many reasons, such as changing lifestyles and dietary habits, including the consumption of more meat and fat, tobacco consumption, chronic alcoholism, and obesity (Zhu et al., 2011). CRC usually arises due to a sequence of precise histological fluctuations in parallel with activation of oncogenes due to mutations and mutagens and other carcinogenic chemicals damaging the heterozygosity of tumor suppressor genes, which occurs due to changes in the equilibrium between DNA repair and DNA damage, leading to cell progeny that causes a mismatch of unpaired DNA. This mutation starts from the internal cell lining of the intestines and then spreads to other muscle tissues and organs, which leads toward metastasis. Several studies suggest that prebiotics help to prevent or inhibit the risk of CRC. The anticarcinogenic effect of prebiotics is due to these characteristics: 1. 2. 3. 4. 5. 6.

Prebiotics stimulate the growth of beneficial gut bacteria They produce short chain fatty acids and other weak acids through fermentation Modulate the gene expression in colon, cecum and faeces Increases absorption of micronutrients in colon Promotes modification of xenobiotic metabolic enzymes Modifies immune response

Prebiotics change the gut microbiome positively and enhance the production of Lactobacillus and Bifidobacterium, which eliminate carcinogens from the host’s gut system. Many animal studies have shown the effect of prebiotics on the prevention of CRC. Feeding rodents ITF enhanced the Bifidobacterium count, lowered pH, and changed the immune response, and also decreased colon neoplasms induced by azoxymethane (AOM), and tumors in the colon and

Therapeutic Aspects of Probiotics and Prebiotics 73 small intestine (Verghese et al., 2007). However, the effects of prebiotics on humans were far less. Therefore more clinical studies with improved study designs are needed to build strong evidence for the effect of prebiotics on CRC (Clark et al., 2013).

5.7 Ulcerative Colitis There is much less evidence regarding the extensive use of prebiotics for UC as compared to probiotics. The available study data shows the remission and maintenance effect of prebiotics on UC. Different prebiotics were evaluated for remission effects in a small study of UC patients comparing plantago seeds with mesalamine. The results concluded that both agents were equally effective (MacFarlane et al., 2006). In short, well-planned clinical and animal studies on the effect of prebiotics on UC are limited at this time and more study trials are needed.

5.8 Hypercholesterolemia Prebiotics have been observed to increase hypocholesterolemic activities in many studies. Prebiotics, such as inulin and oligo-fructose can modify the hepatic lipid metabolism in animal models such that rats and hamsters supplemented with a western-style diet rich in fat and energy defecated dietary fiber. Postprandial serum cholesterol and triglyceride levels were decreased to 15% and 50%, respectively, whereas liver triglyceride accumulation was also inhibited. Similarly, in another study, the effect of inulin on the TC and triglyceride levels of hamsters was determined. The results showed a significant reduction in the levels of TC and TG through the consumption of dietary fiber, especially β-glucan (Ahmad et al., 2009b; Nguyen et al., 2007). In another study, the consumption of XOS resulted in a 27% reduction in total triglyceride level in 40 male Sprague-Dawley rats (Hsu et al., 2008).

5.9 Immunomodulation The exact mechanism of action of prebiotics on the immune system has not been determined yet, but several possibilities have been suggested, including: • • • • •

The production of SCFAs by prebiotics can regulate hepatic lipogenic enzymes SCFAs produced by prebiotic also modulate histone tail acetylation, resulting in the easy access of many enzymes to transcriptional factors Prebiotics modify the production of mucin Some prebiotics, such as FOS, are linked to enhancement of the number of lymphocytes in peripheral blood and GALT The increased secretion of IgA by GALT can also stimulate the phagocytic function of intraperitoneal macrophages (Liong, 2007)

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5.10 Bioavailability and Uptake of Minerals Micronutrients, such as Mg, Ca, K, and Fe are required for the proper functioning of the human body. Studies have revealed that prebiotics such as fructans can enhance Ca absorption. A study of 100 adolescents fed 8 g/day of short-chain ITF showed Ca absorption was increased and bone mineral density improved (Abrams et al., 2005). However, there was no effect on Ca absorption or retention in teenage girls, who consumed 9 g/day of FOS. It has also been suggested that fiber in the colon produces SCFAs through intestinal fermentation, which lowers pH. The unabsorbed and insoluble Ca converts into an ionic form in this acidic medium. SCFAs and low pH cause further hypertrophy of mucosal cells, which leads to enlargement of the intestine and increased Ca absorption. Mucin production, which helps to lower the incidence of bacterial transfers through gut barriers, also occurs due to prebiotic intake (Whisner et al., 2014).

5.11 Diarrhea According to the WHO, an incidence of three or more watery stools in a 24-h period is defined as diarrhea. The effect of prebiotics on different types of diarrhea has been investigated in many in vitro and in vivo studies done since the beginning of the 21st century (Narayanan, 2013). The beneficial effect of prebiotics on intestinal and gut microbiota requires the survival of microbes in the intestinal tract, where they have to face different acidic gastric conditions and lytic enzymes. Prebiotics develop a resistance to these difficult conditions. Hence, in these conditions prebiotics also lead to the protection of Lactobacillus and Bifidobacterium from acidic gastric conditions in vitro through not only improving bacterial strains but also providing energy to them when they reach the colon (Gorbach, 1984).

5.12 Irritable Bowel Syndrome The most common and functional gastrointestinal problem, usually characterized by abdominal pain and discomfort, flatulence, mucosal abnormality, and bloating, is known as IBS. Different pathophysical factors can induce IBS, such as (1) emotion and stress (physiological factors), (2) growth and support system (social factors), and (3) gut motility and sensitivity that cause different symptoms (biological factors). Nondigestible, soluble fibers (prebiotics) can be beneficial in reducing the inflammatory symptoms of IBS. In a recent study guar gum had a much better impact than wheat bran in minimizing abdominal pain and improving bowel habits, which can also enhance qualitative scores of inflammation and epithelial injury (Hardy et al., 2013).


5.13 Prebiotics and Crohn’s Disease CD is a type of IBS that can occur anywhere in the body from the mouth to the rectum but commonly affects the intestines. Prebiotics have an important useful effect in controlling CD. In one study, patients suffering from CD were administrated 24 g/day of inulin and showed a decrease in the number of bacteroides in their feces (Langen et al., 2009).

5.14 Atopic Dermatitis Atopic dermatitis (AD) is the most common and chronic inflammatory skin disease in infants. AD incidences have increased 20% in infants and young children over the last 30 years. The suggested treatments for AD are nutritional supplementation with nutrients such as pre- and probiotics, which can reduce the severity of the disease. Another study looked into the effect of FOS and oligosaccharides on infants suffering from AD at the ration of 9:1 with less risk. The results showed a significant decrease in the growth of AD in infants (Gruber et al., 2010).

6 Synbiotics Synbiotics are live microorganisms, which when administrated in adequate amounts can provide beneficial health effects. Along with defining the term “prebiotics” (Gibson and Roberfroid, 1995), Gibson also demonstrated that prebiotics mixed with probiotics can provide more beneficial health effects and called this combination “synbiotics” (Schrezenmeir and de Vrese, 2001). A synbiotic is a product that can provide beneficial health effects for the host by improving the implantation and survival of live microorganisms through stimulating the growth and metabolism of a selective number of live microorganisms in the GIT. The word “symbiotic” alludes to synergism, so it is a product in which prebiotics selectively favor probiotic organisms (de Vrese and Marteau, 2007). The reason for the development of synbiotics was to overcome the survival problem of probiotics. It is also important to note that the basis of synbiotics relies on all observations that show an improvement in the survival of probiotics in the GIT. Their stimulating effect on the growth of probiotics and other beneficial health bacteria maintains intestinal homeostasis and thereby body health (Pena-Soria et al., 2008). Moreover, different factors such as hydrogen peroxide concentration, pH, oxygen, and moisture stress are thought to affect probiotic growth and viability, especially in dairy products (Usami et al., 2011). The probiotic species that are used to formulate synbiotics include Bifidobacterium spp., lactobacilli, Bacillus coagulans, S. boulardii, and others, whereas common prebiotics involved in formulating synbiotics include inulin, FOS, GOS, XOS, and all the prebiotics that are extracted from natural plant sources.

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7 Theuraptic Effects of Synbiotics Synbiotics are claimed to have the following beneficial health effects on human body: 1. 2. 3. 4.

enhanced levels of Bifidobacterium and lactobacilli for balance with gut microflora enhancement in immunomodulation enhancement in liver functioning inhibition of bacterial transfer and decrease in infections of surgical patients (Steed et al., 2010; Zhang et al., 2014).

A summary of synbiotics’ mechanism of action is presented in Fig. 3.7. Selected details about synbiotics and their activity against various ailments are discussed in the following sections.

7.1 β-Glucan in Synbiotic Foods Synbiotics are an evolving type of functional foods that are introducing the new concept that humans can gain benefits from the health-promoting effects of ingesting pro- and prebiotics at the same time. Foods containing β-glucan are included in this theory because it acts as a substrate for the fermentation of probiotic organisms. Cereals such as barley and oats are major sources of β-glucan, and these cereals are thought to have several health benefits

Figure 3.7: Mechanism of Action of Synbiotics.

Therapeutic Aspects of Probiotics and Prebiotics 77 (Ahmad et al., 2008; Charalampopoulos and Rastall, 2012). β-Glucan can be extracted from these sources, purified, and used as supplements in different food products (Ahmad et al., 2009a; Brennan and Cleary, 2005). Moreover, the FDA has designated β-glucan from different sources as GRAS, based on different studies that looked at the addition of β-glucan to different products to evaluate its synbiotic effect. A synbiotic yogurt was produced with the addition of oat β-glucan, which prolonged the survival of Bifidobacterium longum at a concentration of 107 CFU, suggesting that β-glucan has a protective effect on the Bifidobacterium species (Rosburg et al., 2010). In another study, the effect of β-glucan in dairy and cereal-based products caused an increase in the viability of Lactobacillus paracasei at normal and cold storage temperatures. These studies suggest that β-glucan from different food sources can act as a symbiotic in food; however, more studies are needed to develop new food formulations.

7.2 Effect on Immune System The effect of synbiotics on immune response has been studied very little. A few recent reports have emphasized the evaluation of synbiotics in animal models. Synbiotics composed of Pediococcus acidilactici and short-chain FOS were administered to salmon species for 63 days. The results showed that salmon administered synbiotics expressed higher microbial diversity, higher villus length, and an upgrading of certain immune genes (Abid et al., 2013). Similarly, in another study using gilthead sea bream, the effect of synbiotics composed of Bacillus subtilis and inulin were evaluated for 2–4 weeks. The results showed an elevation in levels of peroxidase, phagocytosis, respiratory burst, and cytotoxic activities (Cerezuela et al., 2013). These studies support the role of synbiotics in improving the immune response; however, to our knowledge these effects of synbiotics are still less known, and studies should be planned to evaluate how intestinal microbial composition is linked with the peripheral immune response.

7.3 Diarrhea As discussed above, acute watery stools are a cause of mortality in children. The main complication is dehydration, which causes the death of 5–10 million children per year (King et al., 1998). The treatment suggested for acute diarrhea includes managing rehydration, preventing dehydration from reoccurring, and dietary treatment. The purpose of dietary treatment is to improve the microbial balance of the gut ecosystem (Rokhmawati et al., 2012). In this regard, synbiotics from different sources act as antibiotics, which have the potential to control gut infections (Bengmark, 2012a). They are also helpful in increasing the functioning of epithelial barriers and modulation of the bacterial ecosystem. Moreover, due to the synergistic effect of synbiotics, they enhance the growth of useful bacteria in the gut, which fight against infection-causing microbes there (Chuhan and Chorawala, 2012;

78 Chapter 3 Shafi et al., 2014). Furthermore, the effect of synbiotics has been evaluated with respect to the duration of diarrhea in hospitalized children suffering from acute diarrhea. The results showed that the synbiotic bacteria L. paracasei and FOS reduce the duration of the illness and episodes of stools within 24 h (Dinleyici et al., 2012). Studies suggest that synbiotics have a promising effect on acute infectious diarrhea and also reduce the consumption of other medications, such as antibiotics, antiemetics, and antipyretics.

7.4 Mineral Absorption An unintended effect of synbiotics on immune function may be attributed to the synbioticinduced increase in the recovery of some minerals, though studies are incomplete and center on calcium (Scholz-Ahrens et al., 2007). Calcium ions are divalent, which causes them to be considered a second messenger and to play a strong role in the immune system (Chaigne-Delalande and Lenardo, 2014). After being fed a mixture of L. acidophilus and FOS, ovariectomized rats showed an elevation in calcium absorption (Marten et al., 2004). Similarly, in another study, aged rats fed Lactobacillus GG, Bifidobacterium lactis, and FOS showed higher levels of plasma calcium concentration as compared to placebo-controlled rats (Naughton et al., 2011). Moreover, rats supplemented with B. longum and GOS had increased resistance to breaking bones. Increased mineral absorption involves different mechanisms, including enhanced mineral solubility due to the low pH developed because of the production of SCFAs and bacterial fermentation products causing expansion of the absorption surface, especially butyrate and Ca-binding proteins, which have increased expression (Flesch et al., 2014). However, further studies are essential to validate the mineral-absorbing effect of synbiotics on immune function.

7.5 Diarrheal Disorders AAD’s prevalence is 5%–30%. The risk increases with cephalosporins, aminopenicillin, and clindamycin therapies (Rayes et al., 2002). Although probiotics in combination with antibiotics have been studied as inhibition agents for AAD in both children and adults, there is very little evidence that supports synbiotic therapies. There is increasing interest in the potential use for infectious diarrhea, as different studies have determined that synbiotics can decrease pathogenic Gram (−ve) bacteria by increasing Bifidobacterium count and acetic acid concentrations (Hayakawa et al., 2012). Yet a subsequent randomized study of using synbiotics for the inhibition of TD has shown negative results (Virk et al., 2013), conflicting with the results of Dinleyici et al. (2012), who showed a significant decrease in TD; moreover, another study also showed a significant reduction in the duration of TD following the administration of synbiotics (Vandenplas and De Hert, 2012).


7.6 Hypercholesterolemia Synbiotics have demonstrated a promising role in controlling blood-lipid profiles, as evaluated in different studies in which hypercholesterolemia-induced male rats were fed fermented rice bran with L. acidophilus (Oberreuther-Moschner et al., 2004). In another study, 24 hypercholesterolemic male pigs were treated with a synbiotic formula of FOS, L. acidophilus, mannitol, and inulin for 8 weeks. The results showed the strong cholesterollowering effect of synbiotics (Liong, 2007).

7.7 Cancer Synbiotic treatment prevented AOM-induced suppression of the activity of NK cells in Peyer’s patches, an effect not observed in pro- and prebiotic treatments given individually (Saulnier et al., 2008). Dietary administration of B. longum, oligofructose, and inulin inhibited the formation of preneoplastic lesions. In addition, B. longum suppressed mammary and colon cancers (Gupta et al., 2000). Overall, studies of in vitro systems and in a wide range of animal models provide considerable evidence that probiotics, prebiotics, and synbiotics exert antineoplastic effects (Fotiadis et al., 2008). Many in vitro and in vivo studies have suggested the healthful effects of probiotics with respect to colon cancer, but the exact mechanism of their anticancer effect has still not been explained (Chong, 2014). However, some further studies have showed the effects of probiotics and prebiotics on tumors in patients. A randomized study was conducted on colon cancer to evaluate the effect of synbiotics (oligofructose-inulin, L. rhamnosus, and Bifidobacterium lactis) compared to a placebo-controlled study (Rafter et al., 2007). The results of fecal and blood samples and colorectal biopsies of 37 patients showed a significant elevation of Bifidobacterium and Lactobacillus strains, and a reduction in Clostridium perfringens strains, in small-intestine microbiota. Furthermore, synbiotics also significantly decreased colorectal cell proliferation and also showed improvement in epithelial barrier functions. Moreover, blood tests revealed that synbiotics prevented IL-2 production by PBMC in polypectomized patients and an elevation in the production of interferon gamma (IFN-γ) in cancer patients. Based on these results, it can be suggested that synbiotics can improve many parameters of CRC (Rafter et al., 2007). Similarly, in another study, a randomized, placebo-controlled trial was performed to analyze the effects of synbiotics on the intestinal immunity of 34 cancer patients and 40 polypectomized patients (Reddy et al., 2007). The group with the synbiotic treatment received inulinoligofructose and L. rhamnosus–B. lactis mixtures. The phagocytic and respiratory burst activity of neutrophilus monocytes showed lyctic activity in NK cells and production of IL-2, IL-10, and IL-12, as well as TNF-α and IFN-γ, after 6–12 weeks of investigation. It can be concluded that supplements of synbiotics have stimulatory effects on the systemic immune system.

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7.8 Allergies The effect of synbiotic combinations on the inhibition of food allergies has been evaluated by many scientists. In a recent study, a synbiotic combination containing L. rhamnosus GG and LC705, B. breve Bb99, and Propionibacterium freudenreichii ssp. shermanii, combined with GOS, was fed to infants for 6 months. The results showed that AD was decreased (Kukkonen et al., 2007) but prevention was limited to not more than 5 years (Kuitunen et al., 2009). In a double-blind randomized study, the effect of synbiotics (L. rhamnosus and metabolites) was observed in children 2 years of age suffering from AD. The results showed an improvement in reducing the symptoms of AD. The potential of synbiotics in the inhibition of allergic maladies was also observed in several clinical trials in both human and animal models. These studies showed the beneficial health effects of synbiotic products having a mixture of prebiotics (90% short-chain GOS and 10% long-chain FOS) and B. breve. Moreover, the authors determined that different synbiotic mixtures were effective in the treatment and prevention of allergic reactions; in Japanese cedar pollen season volunteers supplemented with L. casei and dextran showed no increase in cedar pollen–specific immunoglobulin E, IFN-γ levels as compared with the placebo-controlled group. Few studies have addressed the efficacy of synbiotics in healthy individuals.

7.9 Inflammatory Diseases Gut immunity is important for maintaining a balance between inflammatory and noninflammatory conditions at systemic and localized sites. Experimental studies and epidemiology suggest that changes in gut microbiota can be related to intestinal disorders such as IBS (Frank et al., 2007). Further clinical studies on human subjects reinforced synbiotic supplementation in inflammatory bowel disease. In a double-blinded randomized control trial, results showed that synbiotics had promising results for patients suffering from UC who were treated with a combination of B. longum and inulin-oligofructose (Furrie et al., 2005). Synbiotic treatments have significantly reduced the TNF-α and IL-1 α in mucosal tissues, and have also showed significant reductions in inflammatory cells and restoration of normal epithelium, whereas no effects were seen on the production of IgA, IgG, or IL-10, or on the functioning of neutrophil. It has also been determined, through observations of surgical infections in the gut area (Goris et al., 1986), that septic complications were advanced through inflammation, leading to the conclusion that the principle cause of severe sepsis is an autodestructive inflammatory response. Patients who suffer from severe septic problems may develop characteristics such as higher levels of proinflammatory cytokines (such as IL-6) and enhanced production of intracellular adhesion molecule-1 and proinflammatory platelet-activating factor, as well as augmented endothelial adhesion of polymorphonuclear cells (Bengmark, 2012a). Treatment with Synbiotic 2000 Forte, which consists of four different probiotics and four fibers, was effective in

Therapeutic Aspects of Probiotics and Prebiotics 81 reducing the risk of sepsis in patients with multiple injuries by mechanisms involving the immunomodulatory effect and prevention of bacterial translocation (Giamarellos-Bourboulis et al., 2009). A study that intended to assess the safety of synbiotic and probiotics in immunecompromised individuals suggested that adversative effects were less frequent in immunecompromised subjects receiving probiotics and/or synbiotics compared to the control group. However, the authors of this study did not reach a clear conclusion due to inconsistent and imprecise reporting, as well as variations in probiotic strains, dosages, and administration regimes (Van den Nieuwboer et al., 2015).

7.10 Synbiotics and Obesity The host’s relationship with microbiota is not limited to the gut, but also includes a series of metabolic reactions that connect with the brain, liver, gut, and muscles (Nicholson et al., 2012). Similarly, recent studies have showed that allergies and other chronic diseases are not only connected with immune dysfunction but also failure of gut-host communication, which leads to many other diseases, such as obesity, T2D, and other metabolic disorders. The WHO has concluded that the number of obese and overweight people is increasing continuously. Obesity is an ailment resulting from a complex combination of many factors, such as changes in lifestyle, diet, and genetic disorders, which is linked to many metabolic disorders, such as increased serum lipids levels, which cause CVDs and insulin resistance. Furthermore, gut microbiota’s role in the development of many chronic inflammatory diseases and obesity has been illuminated, which can be further characterized by increased levels of TNF-α, IL-18, IL-1β, and other cytokines that are associated with production of immune cells that infiltrate adipose tissues, the liver, and muscles (Gregor and Hotamisligil, 2011). Many studies of the gut microbiota of obese and lean people showed that there is a reduction in the diversity of microbiota of obese people, suggesting that the composition of gut microbiota can affect fat storage and metabolism (Cani and Delzenne, 2009). Moreover, the body-weight reduction mechanisms induced by probiotics are not clearly understood, yet studies have suggested that the consumption of probiotics could be an additional strategy in reducing the incidence of obesity and other metabolic diseases. The mechanism involved is that the gut activates genes linked to lipid transfer, catabolism of fatty acids, and the proper functioning of the immune system in response to a high-fat diet (de Wit et al., 2011). To highlight the role of microbiota in developing a better inflammatory status in obese models, a recent study was conducted to examine the role of inflammatory genes in both conventional and germ-free mice induced with a high-fat diet (Ding et al., 2010). The results showed an increased expression of TNF-α and Nf-kB in the conventional mice but not in the germfree mice, leading to the conclusion that the interaction between a high-fat diet and bacteria promotes inflammatory changes in the intestines, which can increase obesity and also has a strong link to the progression of obesity (Ding et al., 2010). Further studies explained that the composition of gut microbiota in both nondiabetic and diabetic lean and obese people

82 Chapter 3 is different (Diamant et al., 2011; Larsen et al., 2010), suggesting that the difference in composition affects energy storage and metabolism (Cani and Delzenne, 2009). Similarly, some other studies have shown the ability of prebiotics and probiotics to decrease the effect of inflammation in adipose tissues in models using obese animal and human beings (Kotzampassi et al., 2006; Miyoshi et al., 2014), and some studies have reported that some synbiotics have the capability to improve the inflammatory markers linked to overweight and obese patients. Kelishadi et al. (2014) reported that supplementing overweight children with a combination of probiotics and FOS for 8 weeks can induce a decrease in serum IL-6 and TNF-α while elevating adiponectin; however, synbiotic supplementation did not influence high-sensitivity C-reactive protein (CRP) results. So it can be suggested that consumption of synbiotics have a positive effect on different parameters, such as energy intake, body composition, human blood-lipid profiles, glucose, and homeostasis; however, these effects need to be confirmed through properly designed clinical trials (Yokoyama et al., 2014).

8 Conclusions This chapter discusses the overall effect of probiotics, prebiotics, and synbiotics on a host’s health, metabolism, and immune system with respect to systemic effects. Synbiotic selection is based on the exploitation of the prebiotic by the probiotics so that a good synergy between both can be maintained, enhancing their beneficial health effects. The design of functional foods for enhanced health effects can be improved by creating proper mechanisms of preand probiotics. The ability to control the conformation of gut microbes through a therapy based on a proper diet enriched with prebiotics is an exciting approach to controlling and treating some chronic diseases. Advanced technology has developed advance sequences and analysis of some unexpected microbes of the GIT, which can be helpful for the prevention of ailments and also beneficial for human health. There is sufficient scientific research on the use of probiotics, but more studies are needed on the effect of pro- and prebiotics on several diseases. Furthermore, the health benefits of pro-, pre-, and synbiotics should be validated by different properly designed large-scale clinical trials. In this regard, research on the effectiveness of using target microorganisms in the large intestine for specific, beneficial health purposes would be more valuable. At the same time, in developing new synbiotics it should be kept in mind that different bacterial species have different mechanisms for consuming carbohydrates.

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Further Reading Ahmed, Z., Wang, Y., Anjum, N., Ahmad, A., Khan, S.T., 2013. Characterization of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir—part II. Food Hydrocolloids 30, 342–350.

Therapeutic Aspects of Probiotics and Prebiotics 91 Collado, M.C., Meriluoto, J., Salminen, S., 2007. Measurement of aggregation properties between probiotics and pathogens: in vitro evaluation of different methods. J. Microbiol. Methods 71, 71–74. Erasmo, B.S.M., 2014. Meta-analyses, a systematic review and meta-analysis of the prebiotics and synbiotics effects on glycaemia, insulin concentrations and lipid parameters in adult patients with overweight or obesity. Clin. Nutr., 1–14. Goldin, B.R., Gualtieri, L.J., Moore, R.P., 1996. The effect of Lactobacillus GG on the initiation and promotion of DMH induced intestinal tumors in the rat. Nutr. Cancer 25, 197–204. Michael, N., Jay, K.U., Jhanna, P.M., Michael, S., 2016. The effects of the DDS-1 strain of Lactobacillus on symptomatic relief for lactose intolerance—a randomized, double-blind, placebo-controlled, crossover clinical trial. Nutr. J. 15, 56.


CHAPTE R 4

Lactic Acid Bacteria Beverage Contribution for Preventive Medicine and Nationwide Health Problems in Japan Akira Kanda*, Masatoshi Hara** *HANA Nutrition College, Tokyo, Japan; **The Japan Dietetic Association, Tokyo, Japan

1 Introduction 1.1 Historical and General Remarks on Lactobacillus casei Strain Shirota In 1921, Japan was not a healthy or wealthy country. Lack of hygiene control and poor nutritional status caused a prevalence of communicable diseases, such as cholera, dysentery, and tuberculosis. Dr. Minoru Shirota of the University of Kyoto struggled with this problem from the standpoint of preventive medicine. Then he found a strain of lactic acid bacteria that strongly restricted harmful bacteria living in the intestine. In 1930, he successfully selected the Lactobacillus casei strain Shirota, which resists gastric and biliary secretions that reach the intestine. Through this finding, a commercial lactic bacteria beverage was developed, called Yakult. On the basis of Dr. Shirota’s hypothesis, daily intake of a beverage containing L. casei strain Shirota can improve the internal environment, especially in the intestine, and then can prevent food poisoning and infectious diseases. Daily intake of such beverage also gave resident Japanese an understanding of behavioral modification for primary prevention of diseases. Since then Yakult has been consumed much more around the world for 80 years. Yakult and similar fermented milk products contribute to a healthy intestinal tract, good health, and longer life span of Japanese (Chorley, 2014). About 100 trillion bacteria derived from more than 1000 species are found to be colonized in the intestine of hosts (human beings), and then they form intestinal microbiota. Some intestinal bacteria can be harmful to the host when producing toxins or poisonous substances that cause infection after penetrating intestinal mucosa. Inversely, there are some intestinal bacteria that have beneficial effects on the host by producing essential nutrients, by inhibiting colonization of pathogens, by promoting integrity of the intestinal barrier, and by augmenting protective immunity in the intestine. Because intestinal microbiota form an ecosystem


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94 Chapter 4 composed of diverse microorganisms, an optimal balance between beneficial and harmful microorganisms is essential for maintaining homeostasis of the host. Such bacteria, called “probiotics” (Klaenhammer et al., 2012), living in microorganisms, play a beneficial role for the host’s health if administered in the appropriate amount into individuals. Although probiotics have been defined with various meanings during their history, what is basically understood is they promote the health of the host and reduce the risk of incidence of diseases. As probiotics are ingested orally, their characteristics should be considered well from the standpoint of food safety. Physiological mechanisms of these advantages in beneficial microbiomes have not been thoroughly clarified until now; however, some researchers have reported outstanding results (Yakult Central Institute for Microbiological Research, 2013). In Chapter 11, the authors primarily review recent findings of preventive effects of L. casei strain Shirota. Some of the references have been obtained from researchers of the Yakult Central Institute (Tokyo, Japan), and more were searched and listed in the References. Lactic acid bacteria beverages are widely available in Japan and also many other countries. When ingested, these types of beverages reach the intestine and then play a beneficial role for the host. They are frequently used as probiotics, which improve some biological functions in the host’s internal environment. Researchers have shown that probiotic bacteria have preventive effects against intestinal or bowel diseases. L. casei strain Shirota has been shown to react with both systemic and secretory immune responses via many complicated interactions among different components of intestinal circumstances, such as microflora, epithelial cells, and other immune cells (Perdigón et al., 2001). This chapter focuses particularly on the effect of L. casei strain Shirota as a kind of probiotic that is consumed as Yakult, a lactic acid fermented beverage.

1.2 Structures and Functions of the Intestine First we show the structures of intestinal mucosa and its role. This is essential to understand preventive effects of L. casei on the host and the interrelationship between the intestinal mucosa and symbiotic bacteria, such as L. casei strain Shirota. The intestinal mucosa is a kind of epithelial surface that has a large number of microorganisms called bacteria that form microbiota. Microbiota of the host are made of trillions of bacterial cells, most of which are found in the large intestine (Eckburg et al., 2005). The surface of the mucosa is the entrance for bacteria into gastrointestinal, respiratory, and urogenital tracts, where colonization of epithelial surfaces starts working; then a symbiotic state is formed that is beneficial for the host (Tlaskalová-Hogenová et al., 2011). Most exogenous pathogens also enter the host via surfaces of mucosa (Hill and Artis, 2010). These bacterial cells exist in tissues of the gastrointestinal tract and show the highest prevalence of immunogenic agents. The agents contain food and food components, such as fermented milk products (Hill and Artis, 2010). The function of the surfaces of the mucosa as a barrier in the intestine involves a number of complicated mechanisms that work on several levels. Interaction between the mucosa and

Lactic Acid Bacteria Beverage 95 bacteria forms a part of these natural mechanisms that provides protection against pathogenic microorganisms (Chung and Kasper, 2010; Turner, 2009). Such protection plays a role in the host’s resistance against pathogens. The protection interacts with pathogenic bacteria and by priming the host’s immune system. Then it results in mucosal tolerance (Foey, 2012; Turner, 2009). When L. casei strain Shirota starts composing, a series of beneficial events occur. L. casei strain Shirota prevents pathogenic or virulent microorganisms from attaching to these surfaces of intestinal mucosa. Then it prevents pathogenic or virulent microorganisms from multiplying on these surfaces of intestinal mucosa. This prevention inhibits the invasion of these microorganisms into epithelial cells and subsequent circulatory system in enterocytes of the host by struggling against pathogens (Chung and Kasper, 2010). The barrier exists between microvilli of the intestine and epithelial cells. This is a highly glycosylated macromolecule called mucin as a constituent of mucus. Mucin protects intestinal epithelial cells against direct contact with symbiotic bacteria and their components (Linden et al., 2008). Another defensive barrier is the secretion of mucus. The barrier traps pathogenic microorganisms. Secretion of mucus is from goblet cells that are interspersed with epithelial cells in the lining of the intestine (Foey, 2012). Tight junctions, adherence junctions, and desmosomes in the paracellular spaces between epithelial cells strengthen the epithelial layer of the intestinal mucosa (Chung and Kasper, 2010).

1.3 Pathogens in the Intestine However, most symbiotic bacteria are subject to cause diseases after being translocated through mucosa under specific conditions (e.g., in the case of immunodeficiency) (Tlaskalová-Hogenová et al., 2004). Another example of such disease is an inflammatory bowel disease, whose symptoms sometimes become chronic. These disorders include Crohn’s disease and ulcerative colitis; 0.2% of the population in the United States suffers from these diseases (Tlaskalová-Hogenová et al., 2011). This was a statistical research, so it was not clear about etiological and pathogenic characteristics of these diseases. Concomitant activation of three factors—an immune factor, an environmental factor, and a genetic factor— possibly induces inflammation and then development of mucosal lesions in both diseases (Xavier and Podolsky, 2007). Chronic intestinal inflammation possibly occurs by disruption of T-cell lymphocyte regulatory functions and abnormal mucosal immune responses to symbiotic bacteria (Bengmark, 2007). The results suggest that the intestinal mucosa may be a sensitive indicator of immune dysfunction. T-cell lymphocyte response against symbiotic bacteria in inflammatory bowel disease may explain the relationship between symbiotic bacteria and intestinal inflammation (Bengmark, 2007). Growing interest in the effects of intestinal microbiome on the host’s health resulted in an attempt to optimize the proportion of probiotics in microbiome (O’Flaherty et al., 2010). The most commonly used microorganisms in probiotic products are lactic acid bacteria. Thus various studies have found that these

96 Chapter 4 probiotic bacteria provide a beneficial effect on the function of the intestinal mucosa. Their potentiality possibly could work for the treatment or the alleviation of inflammatory bowel disease (O’Flaherty et al., 2010).

1.4 Immunomodulatory Environment in Intestine Probiotics have been shown to interact with a wide variety of cells, such as enterocytes, macrophages, dendritic cells, Th1, and Th2 in the intestine, and may modulate the immune response toward a proinflammatory or antiinflammatory response (Gourbeyre et al., 2011). In vitro cell experiments have shown that probiotics, such as L. casei strain Shirota promoted secretion of cytokines by intestinal antigen presenting cells. L. casei strain Shirota initiated an adaptable response to these cells (Gourbeyre et al., 2011). Studies on experiments using animals showed that L. casei strain Shirota strengthened the function of phogocytic cells. In 1983, Saito et al. (1983) described how L. casei strain Shirota experimentally stimulated the production of specific antibodies in mice against pseudomonas antigen by increasing the level of circulating IgM antibodies. Then in 1984, Kato et al. (1984) demonstrated using a murine model that intraperitoneally applied L. casei strain Shirota activated macrophages by increasing their phagocytic capacity and enzyme activity. Additionally, the method induced activation of natural killer cells, which plays some role in preventing tumor development. In 1985, Hashimoto et al. (1985) established, by using an in vitro assay, that L. casei strain Shirota activated Kupffer cells and immune cells associated with spleen, lung, and peritoneal macrophages. In mice, fermented milkcontaining L. casei strain Shirota was more effective in secreting lysosomal enzymes from macrophages than fermented milk containing other probiotic bacteria, such as L. acidophilus (Perdigón et al., 1986). After that, they showed that treatment with L. casei strain Shirota activated cells in the intestine even at low doses. This led to a significantly high production of secretory IgA in intestinal fluid, providing protection against infections, such as Salmonella (Perdigón et al., 1990, 1991). These results proposed that L. casei strain Shirota works possibly against intestinal infections. Thus, oral intake of L. casei strain Shirota could be functional as a supplement for preventing intestinal infections. L. casei strain Shirota has also been shown to increase the activity of natural killer cells (Dong et al., 2010; Takeda et al., 2006). In an in vitro study using human peripheral blood mononuclear cells, Dong et al. (2010) showed that incubating the cells with L. casei strain Shirota at 106 cfu/mL for 24 h increased the activity of natural killer cells. A similar study showed that heatkilled L. casei strain Shirota increased the activity of natural killer cells and induced the production of interleukin-12 (Takeda et al., 2006). These results suggest that L. casei strain Shirota possibly increases the activity of natural killer cells and then induces interleukin-12 production. Moreover, the researchers showed that antiinterleukin-12 monoclonal antibody reduced the enhancement of the activity of natural killer cells that were induced by L. casei strain Shirota. These results showed how L. casei strain Shirota can increase the activity

Lactic Acid Bacteria Beverage 97 of natural killer cells in vivo and in vitro in humans. This is possibly due to interleukin-12 (Takeda et al., 2006). An in vitro study demonstrated that heat-killed L. casei strain Shirota stimulated the production of interleukin-10, interleukin-12, tumor necrosis factor alpha, and interferon-gamma. L. casei strain Shirota also promoted natural killer cell activity and activated CD69 expression on NK cells (Shida et al., 2011). In an in vivo study by Takeda and Okumura (2007), voluntary subjects ingested fermented milk containing 4 × 1010 live cells of L. casei strain Shirota every day for 3 weeks. The activity of natural killer cells in subjects significantly increased. The result suggested that daily intake of L. casei strain Shirota increased the activity of positive natural killer cells. A similar study by Seifert et al. (2011) showed that L. casei strain Shirota did not modulate the activity of natural killer cells. The subjects who ingested a probiotic drink identical to the commercially available Yakult for 4 weeks showed no significant effect on natural killer cell numbers of function. This may be explained by the result that the probiotic drink used by Takeda and Okumura (2007) contained more cells than the probiotic drink used by Seifert et al. (2011). It should also be considered that, when probiotics were administered via a probiotic drink, many of the cells will not pass through stomach and small intestine and will not reach the large intestine as a destination. This may explain the positive results seen in the study by Takeda and Okumura (2007). They reported that higher numbers of cells in the probiotic drink would sufficiently increase the numbers of cells reaching the large intestine, where they could exert an effect on the natural killer cells.

2 Tolerance to Gastric Acid and Bile, and Viability in the Intestinal Tract Gastric secretory fluid is a mixture of gastric acid, pepsin, and mucus. Healthy adults secrete 2–3 L of gastric secretory fluid per day from the parietal cells in the stomach. The pH of gastric secretory fluid is about 1.5–2 when the stomach is empty, while the pH reaches about 3 after a light meal and has a range from pH 3 to slightly less than pH 6 after a regular meal. If 250 mL of skim milk or yogurt enters the empty stomach, gastric pH immediately increases to 5–6 and remains above pH 3 for 40–60 min (Conway et al., 1987). It has also been reported that gastric half emptying time after ingestion of yogurt is about 40 min (Berrada et al., 1991). Therefore, lactic acid bacteria were inoculated into artificial gastric secretory acid for 1 or 3 h to assess their tolerance to gastric acid. L. casei strain Shirota was more tolerant of this artificial gastric acid than other representative species of lactic acid bacteria (L. plantarum, L. acidophilus, Streptococcus thermophiles, and L. delbrueckii subsp. bulgaricus). The viable cell count of Shirota strain showed little change even after 3 h of incubation in artificial gastric acid. Upon exposure to severe stress, such as acid, bile, or oxygen, lactic acid bacteria can undergo spontaneous mutation to produce a new strain that is more stress tolerant than the parent strain. L. casei strain Shirota may have developed against such exposure to stress. Tolerance to gastric acid and bile by lactic acid bacteria is an issue for microbiological research, as well as for investigations targeting the commercial use of these

98 Chapter 4 bacteria. Many previous studies have addressed physiological and biochemical properties of lactic acid bacteria; however, studies have not yet fully elucidated the mechanisms involved in their high tolerance to stress.

3 Modification of Gastrointestinal Function: Improvement of Diarrhea and Constipation 3.1 Constipation Sakai et al. (2015) evaluated the effect of fermented milk containing L. casei strain Shirota on consistency and frequency of stool, constipation-related symptoms, and incidence of hemorrhoids during the puerperal period. This study was similar to an intervention study except there was no observation at baseline. Forty female subjects who had natural childbirth were divided randomly into two groups. One group consumed one bottle/day of fermented milk containing at least 6.5 × 109 cfu of L. casei strain Shirota, and the other group consumed a placebo for 6 weeks after childbirth. The subjects kept a diary on their bowel movement, stool consistency, and incidence of hemorrhoids. They also answered questionnaires on constipation-related symptoms and quality of life. After intervention, the L. casei strain Shirota group marked better scores on constipation-related symptoms (P = 0.013), subscales of the questionnaires; abdominal symptoms (P = 0.043), rectal symptoms (P = 0.031), and better scores on questionnaires on constipation-related quality of life, satisfaction subscale (P = 0.037) in comparison with the placebo group. In the L. casei strain Shirota group, two to four subjects suffered hemorrhoids during the first 3 weeks of intervention. The number of hemorrhoids decreased at the 4th week, and then at the 5th and 6th week almost all recovered from the hemorrhoids. These results support the possibility that continuous intake of fermented milk containing L. casei strain Shirota might soothe constipation-related symptoms. It also probably provides satisfactory bowel circumstances, resulting in earlier recovery from hemorrhoids in women during puerperium. Matsumoto et al. (2006) reported the effect of daily intake of 80 mL Yakult 400LT (it contains 4 × 1010 L. casei strain Shirota) for 2 weeks on the frequency of defecation and fecal properties in 40 healthy adults who were previously screened to detect mild constipation (7 men and 33 women with a mean age of 39.2 ± 10.3 years). In this randomized, placebo-controlled study, daily intake of Yakult 499KT significantly increased the frequency of defecation from 4.0 ± 1.3 per week at baseline to 4.7 ± 1.5 per week after 1 week of intake and to 4.8 ± 1.5 per week after 2 weeks. The number of days when defecation occurred also showed a significant increase after daily intake of Yakult 400LT for 1 week. Treatment with a placebo caused no significant changes in the frequency of defecation and the number of days, indicating that daily intake of Yakult 400LT improved constipation. Subgroup analysis based on the severity of constipation [baseline defecation frequency = 4.0 per week (n = 21) or 4.5 per week (n = 19)] more clearly showed that there was improvement of defecation with the daily intake of Yakult 400LT. In subjects with severe constipation, daily intake of Yakult 400LT significantly increased frequency of

Lactic Acid Bacteria Beverage 99 Table 4.1: Incidence of diarrhea in Indian children and preventive effect of L. casei strain Shirota. Number of subjects Subjects with diarrhea Incidence of diarrhea (number of diarrhea per subject-year)

Yakult

Placebo

1802 608 0.88a

1783 674 1.029

P radish > broccoli > celery root > spring onion > okra (gumbo) > green bean > dill > red pepper > green pepper > tomato > carrot. To visualise the results, a graphical form is presented in Tables 7.1 and 7.2, which allows for the rapid assessment of the richest food sources of total flavonoids and the raw materials with the highest ORAC values. Additionally, data for the total polyphenol content of fruits (Fig. 7.7) and vegetables (Fig. 7.8) is added. According to the results presented in Fig. 7.7, there was a direct relationship between the total phenolic content and ORAC antioxidant activity of fruits with correlation coefficient r = 0.95, and ORAC values correlate better with the total polyphenol content than with the flavonoid content. This could be explained by the presence of other polyphenol compounds in samples, as it is evident from the graph that flavonoids represent only a small portion of the polyphenols present in fruit samples. Fig. 7.8 presents the results for the overall content of flavonols and flavones in selected Bulgarian vegetables, as well as the results for their total polyphenol content and their ORAC antioxidant activity. The results in Fig. 7.8 show that parsley and celery leaves are the richest sources of flavonoids (flavones and flavonols) and possess the highest antioxidant activity among the investigated vegetables. Furthermore, the amount of total polyphenols in these leafy condiments is about fivefold higher than in the other vegetables. An interesting case here is dill, which despite having high amounts of flavonoids and total polyphenols revealed moderate antioxidant activity, comparable to that of green beans and celery roots. Similarly to results for fruits, it is evident that flavonoids represent just a small portion of the phenolic compounds present in vegetables. The present study is the first to present consolidated analytical data of flavonoid content of Bulgarian fruits and vegetables based on individual HPLC determination of catechins, flavonols, and flavones, along with original data for their total polyphenol content and ORAC antioxidant activity. The present set of data is of great importance, not only for nutritional science, but for the characterization of bioactive compounds in plant foods as well. The results presented in Figs. 7.7 and 7.8 clearly show the necessity of combining data from different sources, obtained via different analytical methods and procedures in order to better understand and interpret their integrity in the dietary recommendation for healthy nutrition.

190 Chapter 7

Figure 7.7: Sum of Flavonoids (Catechins and Flavonols), Total Polyphenol Content and ORAC Antioxidant Activity of Bulgarian Fruits.

Flavonoids in Foods and Their Role in Healthy Nutrition 191

Figure 7.8: Sum of Flavonoids (Flavonols and Flavones), Total Polyphenol Content and ORAC Antioxidant Activity of Bulgarian Vegetables.

192 Chapter 7

7 Conclusions Flavonoids are potent antioxidants, acting along with antioxidant vitamins, protecting the human body against reactive oxygen species. Their beneficial role for human health was confirmed during the last 20 years, since the first study of their potential protective effect against cardiovascular disease was described. Flavonoid consumption might also be associated with reduction of risk for the development of some forms of cancer, obesity, and diabetes, and has shown improvement of cognitive function in those with Alzheimer’s disease. The present results for the flavonoid content, ORAC antioxidant activity, and total polyphenols content show that leafy vegetables, such as parsley, dill, and celery leaves are among the best sources of polyphenols and antioxidants in the Bulgarian diet. The obtained results are a useful tool for medical professionals to promote the consumption of plant foods with high antioxidant activity as a part of a healthy lifestyle. Flavonoids are one of the top research areas in food and nutrition science concerning their bioactivity and potential to promote health and to reduce the incidences of coronary heart disease, diabetes, cancer, and other chronic diseases. Recent epidemiological studies have confirmed and enhanced the scope of diseases and pathophysiological conditions where flavonoids have displayed certain positive health effects, thus challenging science to clarify all aspects associated with their bioactivity. Although a great number of flavonoid representatives have been identified, studies continue searching for new bioactive components in the broad variety of plant species. Those data are of vital importance for the compilation of national/regional food composition databases that are used in global nutritional policy and information exchange. Besides that, they are important for the characterization and identification of existing biodiversity. The current stage of research implements unified sampling approaches and strives to use more and more perfect analytical equipment, providing high precision level and very good reproducibility, guaranteeing significant results. Large scientific potential is focused also on the assessment of bioaccessibility, bioavailability, and bioactivity of flavonoids that could be modulated on the basis of impact of various factors along the gastrointestinal tract with special emphasis on microbiota. The effect on the processes of digestion, absorption, and metabolism cause certain changes in flavonoid bioactivity. Numerous further studies are necessary in this aspect in order to establish the action specifics of the various factors and clarification of the biochemical mechanisms associated with the modifications of flavonoid bioefficacy. From a bioengineering point of view, it should be noted that the current knowledge on flavonoids also covers the development of new technologies using various nanoparticles and nanoemulsions for the protection of native flavonoid activity, preventing the impact of relevant gastrointestinal factors.

Flavonoids in Foods and Their Role in Healthy Nutrition 193 The content of this chapter unites current scientific evidence with our experience, knowledge, and data on flavonoids, from their identification in the different plant species, through their prevalence in nature, analytical approaches for their determination, their bioaccessibility and bioavailability, determination of their exact composition and content in various foods, description of their activity and the role of new technologies in the preservation of their native power. All those dimensions manifest the significance of the extensive flavonoids issue. The power, assigned by nature through plant food flavonoids is a challenge to nutrition science.

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Further Reading Katiyar, S.K., Brgamo, B.M., Vyalil, P.K., Elmets, C.A., 2001. Green tea polyphenols: DNA photodamage and photoimmunology. J. Photochem. Photobiol. B. 65 (2–3), 109–114.

CHAPTE R 8

The Role of Milk Oligosaccharides in Host–Microbial Interactions and Their Defensive Function in the Gut Sinead T. Morrin*,**, Jane A. Irwin**, Rita M. Hickey* *Teagasc Food Research Centre, Fermoy, Ireland; **University College Dublin, Dublin, Ireland

1 Introduction Milk oligosaccharides (OS) are unconjugated glycan/carbohydrates, which are found primarily in breast milk. In fact, within breast milk, human milk oligosaccharides (HMO) constitute the third most abundant element. OS structures are composed from the 5 main building blocks consisting of the monosaccharides glucose, fucose, sialic acid (N-acetylneuraminic acid), galactose, and N-acetylglucosamine. Most OS, if not all, have a lactose core at the reducing end, which is elongated with N-acetyllactosamine units decorated by fucosyl and sialic acid residues at terminal ends, contributing to each OS’s functional and structural diversity. Between 20% and 50% of OS contain sialic acid residues and 70% contain fucose residues, and their presence and abundance is governed by the lactation stage (Chaturvedi et al., 2001). OS are comprised of neutral and acidic OS whereby neutral OS contain uncharged carbohydrate residues whereas in contrast, acidic OS have negatively charged COOH residues, typically due to the monosaccharide sialic acid. The OS concentration and the acidic and neutral charge profile change considerably during the lactation period, with highest concentrations found within the first week of lactation, with a subsequent decrease in concentration by approximately 50% by month 3 of lactation. Independent of the initial presence of neutral and acidic oligosaccharides, the HMO profile shifts to a more blood group epitope specific profile at the 3-month mark (Albrecht et al., 2011; Froehlich et al., 2010; Thurl et al., 2010). At this stage, the HMO content of milk from different donors becomes divergent, varying in levels of fucosylation and sialylation and differing in concentrations. The abundant OS tend to stay abundant in each sample, but the minor OS display the most heterogeneity (Ninonuevo et al., 2006).


199


200 Chapter 8 OS content and composition vary greatly for each infant, a difference largely dependent on the mother’s secretor status (Se) and Le blood group, characterized by two genes; FUT2 (Se gene product) and FUT3 (Le gene product). Mothers with the presence of an active FUT2 gene are known as secretors and their milk contains α1-2-fucosylated HMOs. Mothers without the FUT2 gene are known as nonsecretors. Secretor/nonsecretor mothers with an active FUT3 gene are Le+ and produce α1-4-fucosylated OS, whereas secretors/nonsecretors without this FUT3 gene are Le− and do not contain α1-4-fucosylated HMOs in their milk (Xu et al., 1996). Based on the presence or absence of the two genes, four groups apply to breast-feeding mothers: FUT2+FUT3+(Se+Le+), FUT3−FUT2−(Se−Le−), FUT2−FUT3+(Se−Le+), and FUT2+FUT3−(Se+Le−). However, the production of fucosylated HMOs has been shown to be governed also by another mechanism of production outside of these conditions, as (Se−Le−) carriers have fucosylated HMOs within their milk and α1-2-fucosylated HMOs are found in the milk of (Se−Le+) groups (Kumazaki and Yoshida, 1984). Nonetheless, the majority of fucosylated HMOs are found in secretor mothers milk where concentrations of OS are higher than those found in nonsecretor milk (Gabrielli et al., 2011). Secretor women are also thought to give added protection to their infants due to the production of higher amounts of the core structures needed for oligosaccharide production, namely lacto-N-biose and GlcNAc (Nacetylglucosamine), when compared to nonsecretor women (Thurl et al., 2010). Breast milk has long been recognized as the gold standard in infant nutrition and over the years, the biological functions of oligosaccharides have gained interest due to their abundance in breast milk. These complex sugars have to date been shown to possess antiadhesive properties through competitive inhibition of pathogens, act as energy sources for commensal bacteria, and regulate the immune system, ultimately contributing to the overall health of the infant intestine and its gut microbiota (Coppa et al., 1993; Viverge et al., 1990). The configuration and maturation of the intestinal microbiota of an infant is reliant on a number of factors, including the mode of delivery, the time of birth (preterm vs. full-term), genetic factors, and also exposure to microbes. These factors can in turn affect the profile of OS and how well they are used and metabolized. Preterm infants have a higher susceptibility to infection due to a lower lactose content and a higher protein content in their mother’s milk when compared with full-term infants (Narang et al., 2006). The preterm milk HMO content was also shown to be quite variable, with the presence of OS, such as 2′-fucosyllactose being inconsistent throughout lactation. This immaturity in HMO production and regulation may be one of the reasons preterm infants acquire more infections when compared to full-term infants (De Leoz et al., 2012). When breast milk is ingested by the infant, lactose is digested along with other milk components, while HMO pass through the gastrointestinal tract intact until they reach the intestine and colon, whereby the intestinal microbiota ferment the OS for use as an energy source. The low molecular weight OS, such as 2′-fucosyllactose (2′-FL), lacto-N-neotetraose (LNnT), and 3′-fucosyllactose (3′-FL) have also been shown to be absorbed into the blood stream and have been found excreted in feces and urine still intact. For this reason, OS are

The Role of Milk Oligosaccharides 201 thought to have other systematic effects, as well as modulating the intestinal microbiota population (Goehring et al., 2014). The isolation and production of alternative OS that mimic HMO for potential supplementation in infant formula has great appeal. For instance, the combination of 9:1 galactooligosaccharides:fructooligosaccharides (GOS:FOS) at 8 g/L has been hugely studied and is regularly added to specific infant formulas (Fanaro et al., 2005). The benefits of this addition have been verified through various studies, showing increases in commensal bacteria colonization and a reduction in pathogen colonization in the infant intestine. For instance, formula-fed infants who received oligosaccharide (GOS:FOS) supplements had a reduced incidence of infection due to atopic dermatitis during the first 6 months when compared to formula-fed infants with no oligosaccharide supplementation (Arslanoglu et al., 2007; Moro et al., 2006). However, GOS/FOS does not represent the oligosaccharide fraction of human milk and the benefits and functions of the range of OS found in breast milk are not currently relayed in infant formula. Considering, sourcing functionally similar OS for use in infant formula is attracting the interest of a number of infant milk formula (IMF) companies. OS from other origins are now being investigated as potential substitutes for HMO, which cannot be produced in large quantities unless focusing on individual HMO structures. Bovine milk as a source of OS has long been studied as the main understudy of human milk but is still under investigation due to structural differences, which may affect its applicability. Such differences include the low concentration of fucosyl OS and the presence of more acidic OS, with the main sialyllactose in bovine milk being 3′-SL (3′-sialyllactose) in contrast to 6′-SL (6′-sialyllactose) which predominates in human milk and also the presence of other components which are not found in human milk, such as Neu5Gc and (α1-3) linked galactose (Albrecht et al., 2014; Neeser et al., 1991). Other animal sources under investigation include giraffe, camel, rabbit, monkey, sheep, goat, horse, and buffalo, with one study showing the efficiency of giraffe, monkey, buffalo, and camel milks in inhibiting lectin-dependent adhesion of Pseudomonas aeruginosa and Chromobacterium violaceum (Zinger-Yosovich et al., 2010). However, despite this, animal milk remains an attractive alternative source of OS and warrants further investigation. The effects of OS both from breast milk and other sources are not only observed in relation to the intestinal microflora but also in immune defense and prevention of pathogen infection, as well as cognitive development and allergy alleviation. This chapter reviews the influence of OS under each of the health pillars mentioned, as well as highlighting the potential of OS from sources other than human milk.

2 Effect of Oligosaccharides on Pathogen Colonization Pathogens have many ways to colonize and infect, but adherence is the first vital step, which must occur in order for the later steps to ensue. Pathogenic strains express a variety of binding proteins and adhesins for attachment to the glycan moieties found on intestinal

202 Chapter 8 epithelial cells (Glycocalyx) (McGuckin et al., 2011). Considering that milk OS share structural similarity with the glycan moieties of intestinal receptors, they can prevent adhesion through interaction with the pathogen by acting as decoys and preventing pathogens adhering to the host (Manthey et al., 2014). Inhibition of adherence of three pathogenic microbial species Escherichia coli, Cronobacter sakazakii, and Salmonella enterica to human epithelial type 2 (HEp-2) cells has been demonstrated with the use of protein reduced colostrum. Removal of lactose from the colostrum fraction resulted in a higher antiadhesion capability when compared to the nonlactose reduced colostrum demonstrating a higher OS concentration may be important and beneficial to the antiadherence effect (MaldonadoGomez et al., 2015). The ability of OS to prevent adherence of pathogens to the intestinal epithelium has been widely accepted and the different specificities of OS can be associated with their individual structures (Table 8.1). As mentioned previously, OS are quite similar to the receptors on human epithelial cells to which pathogens bind, and therefore can prevent pathogen attack by the pathogen attaching to the oligosaccharide instead of the receptor. Ruiz-Palacios et al. (2003) demonstrated the affinity of Campylobacter jejuni for α1,2-fucosylated glycan moieties and moreover these OS prevented C. jejuni from attaching to host carbohydrate moieties (Ruiz-Palacios et al., 2003). 3′-SL has been shown to prevent adherence of Helicobacter pylori, while fucosyloligosaccharides have been shown to reduce adhesion Table 8.1: Milk oligosaccharides that inhibit pathogen colonization. Oligosaccharides

Effects

References

3′-SL

Inhibition of adherence of H. pylori to epithelial cells Inhibition of H. pylori nonopsonic neutrophilactivating capacity Exposure of 3′-SL leads to altered glycosylated state on cell surface and subsequent reduction of EPEC adhesion Reduces pneumocyte invasion by pathogen P. aeruginosa Inhibition of adhesion of E. coli serotype O119, V. cholerae, and S. fyris to caco-2 cell line Inhibition of adhesion of influenza virus Inhibition of adhesion of P. aeruginosa Inhibit adhesion of P. aeruginosa to caco-2 and A459 cell line Norovirus inhibition Protective agent against enterotoxin of E. coli

Simon et al. (1997)

Inhibition of attachment of uropathogenic E. coli strains

Martín-Sosa et al. (2002)

3′-SL 3′-SL

6′-SL 3′- and 6′-SL 3′- and 6′-SL 3′- and 6′-SL 2′- and 3′-FL 2′- and 3′-FL Total fraction of neutral milk oligosaccharides Total fraction of acidic milk oligosaccharides

EPEC, Enteropathogenic E. coli; FL, fucosyllactose; SL, sialyllactose.

Teneberg et al. (2000) Angeloni et al. (2005)

Marotta et al. (2014) Coppa et al. (2006) Kunz and Rudloff (1993) Thomas and Brooks (2004) Weichert et al. (2013) Weichert et al. (2016) Newburg et al. (1990)

The Role of Milk Oligosaccharides 203 of C. jejuni to duodenal carcinoma HuTu-80 cells and caliciviruses in breast-fed infants (Morrow et al., 2004; Simon et al., 1997). 2′- and 3′-FL have displayed antiviral activity against noroviruses by blocking the virus-like particles of noroviruses from attaching to their known carbohydrate attachment sites on histo-blood group antigens (HBGAs) (Weichert et al., 2016). Bovine colostrum OS have also been shown to directly interact with C. jejuni, thereby preventing it from binding to intestinal mucin in vitro (Lane et al., 2012). The aforementioned studies illustrate the relationship between structure and function when focusing on antipathogenic effects of OS and show that individual compositions or genetically coded groups of OS offer better protection against particular infections reiterating the need for a variety of OS in infant formula to emulate the wide variety of OS found in human milk. Acidic milk OS are regarded as the dominant antipathogenic group, however, there are studies which have also shown that neutral milk OS display significant inhibition of attachment of pathogenic bacteria. For instance, Coppa et al. (2006) demonstrated that high molecular weight neutral OS were effective in inhibiting Vibrio cholerae and E. coli 0119 adhesion to caco-2 cells, while Hakkarainen et al. (2005) demonstrated that acidic bovine colostral oligosaccharides (BCO) and neutral HMO were able to reduce Neisseria meningitidis pili adhesion to bovine thyroglobulin by 50% (Coppa et al., 1993; Hakkarainen et al., 2005) (Fig. 8.1). Other OS extracted from natural sources, such as pectic OS and chitosan have also been shown to prevent pathogen invasion, (e.g., C. jejuni) thus preventing infection (Ganan et al., 2009, 2010). Individual OS of milk, such as fucosyllactose, tetroses, and sialyllactose, have been extensively studied but much research has also been devoted to exploring the

Figure 8.1: Oligosaccharides Act as Antiadhesive Agents Against Pathogenic Strains. (A) Due to oligosaccharides sharing structural similarity with glycan moieties on the cell surface, (B) these molecules can act as decoy receptors for pathogens, and (C) result in the pathogen interacting with the oligosaccharide instead of its target receptor allowing it to be flushed from the body.

204 Chapter 8 unknown functions of commercial OS (FOS, GOS, inulin, and lactulose), particularly those commonly added to infant formula. OS, such as GOS confer better resistance to Entamoeba histolytica attachment in vivo, as its terminal Gal is not decorated with α1-2 linked Fuc, a trait associated with nonsecretor women and not observed in secretor women (JantscherKrenn et al., 2012). Thus, in relation to this particular pathogen, nonsecretor women could confer better protection to their offspring when compared with secretor women. GOS also displayed the greatest inhibition of attachment of the enteropathogenic (EPEC) strain 0127:H6 to HEp-2 and caco-2 cells when compared with raffinose and fructan type OS. Shoaf et al. (2006) also demonstrated that this inhibition was only functional against bacteria not already adhered to the cells (Shoaf et al., 2006). This phenomenon was also observed when GOS and polydextrose (PDX) were compared for their ability to prevent attachment of C. sakazakii to the HEp-2 human cell line with GOS having the better antiadhesive ability (Quintero et al., 2011). As well as inhibiting attachment by acting as a physical barrier or through competitive inhibition, pathogens are now thought to be inhibited by OS through modulating their gene expression. This phenomenon may lead to an oligosaccharide-induced change in expression of the binding proteins and adhesins of the pathogen, lessening its ability to attach to the host. For instance, Vanmaele et al. (1999) demonstrated that the expression levels of intimin and bundle-forming pilus (BFP) of the EPEC strain E2348/69 were reduced when incubated with the carbohydrate moiety LacNAc-BSA, preventing the expression of two proteins required for the attachment of the pathogen (Vanmaele et al., 1999). Ebersbach et al. (2012) validated this hypothesis using xylo-OS (XOS), by demonstrating their ability to prevent the adhesion of L. monocytogenes 4446 and ScottA to intestinal cells in vitro by reducing the expression of proteins required for attachment including internalin A and listeria adhesion protein (LAP). However, XOS did not reduce the expression of proteins in another L. monocytogenes strain (7291) tested, possibly due to the inability of the strain to ferment XOS and internalize their end products. Hence, the ability of pathogens to ferment OS may make them more susceptible to transcriptional modulation by OS and their breakdown products (Ebersbach et al., 2012). Ortega-Gonzalez et al. (2014a) also demonstrated using 1EC18 cells, that FOS reduced the amount of exotoxin A produced, a major virulence factor of P. aeruginosa (Fig. 8.2). Another emerging area of interest is the ability of OS to modulate the intestinal surface and therefore modulate the response to pathogenic invasion. The addition of 3′-SL led to a change in the glycosylation pattern of caco-2 cells, with reductions in sialyltransferase gene expression of ST3-Gal associated genes and lactosamine expression. This ultimately led to reductions in α2-3- and α2-6-linked sialic acid residues and lactosamine glycan moieties on the cell surface and directly correlated with a 50% reduction in EPEC adhesion. This phenomenon may occur by either decreasing glycan expression and/or the expression of modifying enzymes (Angeloni et al., 2005).

The Role of Milk Oligosaccharides 205

Figure 8.2: Oligosaccharides may Influence Bacterial Transcription. (A) Pathogens express surface proteins and adhesins to interact with epithelial surface receptors, (B) Oligosaccharides can influence expression of these proteins, and (C) lead to reduced levels available for interaction with the host cell surface.

OS also display antiadhesive ability beyond the gastrointestinal tract. As an example, milk OS were shown to reduce adhesion of Burkholderia pseudomallei and Yersinia pestis to respiratory cell lines (Thomas and Brooks, 2004, 2006). This direct interaction was also observed by Marotta et al. (2014), whereby 6′-siallylactose was shown to directly interact with P. aeruginosa and prevent pneumocyte invasion in vitro. HMO have also been shown to reduce uropathogenic E. coli UPEC invasion of bladder epithelial cells (Lin et al., 2013). In addition, these milk sugars have also been shown to reduce invasion of the fungus Candida albicans by reducing the ability of hyphae, an important virulence factor, to invade premature intestinal cells at the initiation stage of hyphal development. HMO also influence gene expression, reducing the hyphal adhesin gene ALS3, thus preventing the fungal infection (Gonia et al., 2015). Further investigation is required to determine if OS could alleviate symptoms of adult diseases such cystic fibrosis. OS may prevent pathogen attachment and thus prevent the frequent use of antibiotics as is common with cystic fibrosis sufferers. Thus, the beneficial effects of milk OS for breast-fed infants may have applications beyond the gastrointestinal tract and have potential if developed for adult subjects also. Understanding how these OS interact with pathogens and what they attach to is the next step in understanding what receptors and carbohydrate moieties pathogens use for attachment and will provide a better understanding of which OS may provide decoy interaction activities for therapeutic purposes.

3 Effects of Oligosaccharides on Commensal Colonization Colonization of the intestine is thought to begin immediately after birth and it is at this stage that an environment capable of hosting a wide microbial community is established, facilitating the growth of bacteria, such as bifidobacteria, enterococci, clostridia,

206 Chapter 8 staphylococci and many other species. Existing studies now suggest that colonization may occur earlier, as the amniotic fluid, placenta, and infant meconium may harbor distinct populations of microbiota and may be already interacting with the “sterile” fetus intestine long before birth (Collado et al., 2016). Historically, studies have shown that infants who are breast-fed have a more diverse population of microbiota when compared with their formula fed peers. Comparing the two groups, breast-fed infants have a microbiota dominated by Bifidobacterium spp. with Bifidobacterium longum and Bifidobacterium bifidum being the most dominant, compared to lower numbers of these species found in the formula-fed infant. This difference in composition has been attributed in recent years to the fact that breast milk has an abundance of OS, which the commensal microbiota population metabolize as an energy source (Table 8.2). Differences in the mother’s secretor status leads to different bifidobacterial populations in the infant gut (Lewis et al., 2015) and additions of alternative OS to infant formulas have also been shown to increase counts of bifidobacteria (Boehm et al., 2002). Milk OS are used by commensal bacteria that express glycosidase enzymes, which cleave these structures into disaccharide and trisaccharide units, utilizing them as an energy source to survive and proliferate. Infant associated bifidobacteria have the ability to utilize milk OS as a sole carbon source, explaining their abundance in the gut microbiota of breast-fed infants (Fallani et al., 2010; Penders et al., 2006). B. infantis in particular is the predominant metabolizer of the HMO species, demonstrating more than a threefold higher cell density in growth when compared to other bifidobacteria strains including B. bifidum, B. breve, and B. longum (Ward et al., 2007). In fact, each type of bifidobacteria species have displayed specific preferences for which HMO structure(s) they consume, with B. breve preferring to metabolize monosaccharides, B. infantis preferring small HMO constituents Table 8.2: Milk oligosaccharides which influence commensal colonization. Oligosaccharides

Effects

References

2′-FL

Bifidobacteria colonization occurs earlier and more prevalent in infants of secretor mothers versus nonsecretor offspring Preferred prebiotic of infant-associated bifidobacteria LNnT mimics mucus glycans to confer advantage to mucin-adapted commensals, such as B. fragilis Increased growth of B. breve Induction of transcriptional response in B. longum subsp. infantis 16597; increased adhesion of strain to HT-29 colonic epithelial cells Bifidobacterium spp. and Bacteroides spp. display increased growth versus reduced pathogen numbers; prebiotic activity B. longum subsp. infantis expresses sialidases; ability to metabolize sialylated milk oligosaccharides

Lewis et al. (2015)

DS-LNT LNnT LNT and LNnT 3′- and 6′-SL

2′- and 3′-FL Total fraction of neutral milk oligosaccharides

LoCascio et al. (2007) Marcobal et al. (2011) Ruiz-Moyano et al. (2013) Kavanaugh et al. (2013)

Yu et al. (2013)

Sela et al. (2012)

DS-LNT, Disialyllactose; FL, fucosyllactose; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; SL, sialyllactose.

The Role of Milk Oligosaccharides 207 and B. bifidum having preference for lacto-N-biose (LNB) extracting this from intact HMO structures while not utilizing free sialic acid, fucose, and N-acetylglucosamine (LoCascio et al., 2007; Ward et al., 2007). The subsequent breakdown products of oligosaccharides, namely short-chain fatty acids (SCFA) are also beneficial to the host microbiota as they in turn can act as an energy source, as well as inhibiting pathogenic colonization (Den Besten et al., 2013; Fukuda et al., 2011). SCFA have also been correlated with multiple health benefits from antiinflammatory to antiproliferative (Blouin et al., 2011). OS have recently been associated with promoting colonization of the commensal bacteria population through influencing their colonization factors. Bifidobacteria demonstrate a strong adaption to the infant neonatal intestine. For example, B. bifidum encodes gene clusters associated with Tad and sortase-dependent pili, including BopA while B. breve UCC 2003 produces type IVb tight adherence pili and B. longum subsp. longum expresses a polymorphic fimbrial protein (BL0675) demonstrating an affinity for porcine colonic mucins. Such components are heavily involved in the ability of these strains to adhere to intestinal epithelial cells (Gleinser et al., 2012; Preising et al., 2010). Likewise, some adult strains, such as B. longum subsp. longum produce a polymorphic fimbrial protein with an affinity for porcine colonic mucins (Suzuki et al., 2016). OS may contribute in helping the specific adhesive ability of a wide variety of Bifidobacterium strains. The ability to increase adherence of B. longum subsp. infantis 15697 has also been demonstrated, with a 1:1 mixture of 3′ and 6′-SL resulting in an increase in adherence of B. infantis to HT-29 cells by nearly 9.8-fold compared to that of nontreated B. infantis. This also correlated with a change in gene expression with potential colonization factors, such as Dnak and groEL increasing in expression (Kavanaugh et al., 2013). Human milk and formula containing GOS and lcFOS have been shown to upregulate the expression of a type 2 glycoprotein binding FimA fimbriae and the sortase-like fimbrial associated protein of B. longum, both of which have previously been associated with adhesion and colonization (González et al., 2008). Chichlowski et al. (2012) also demonstrated that when HMO were used as the sole carbon source, they contributed to increased adherence of B. longum subsp. infantis ATCC 15697 to the HT-29 epithelial cell line. Milk OS therefore may be an important requirement for bifidobacteria in ensuring adequate colonization. Breast-fed babies with this more diverse microbial population have also displayed a better resistance to infection and lower susceptibility to disease later in life, which can be linked to three lines of defense from the host microflora, namely: (1) the production of antimicrobial substances, (2) competitive inhibition of pathogenic strains through colonization/steric hindrance, and (3) modulation of the intestinal cells’ immune repertoire to enhance pathogen inhibition (Bisgaard et al., 2011). The training of the innate and adaptive immune system to distinguish between the host microbial community and nonhost community is particularly important in maintaining a homeostatic balance. Commensal bacteria, such as bifidobacteria and lactobacilli have been shown to ellicit various modulating effects on the gastrointestinal immune system via

208 Chapter 8 receptors/cytokine production and antiinflammatory properties (Christensen et al., 2002; Turroni et al., 2014). Commensal bacteria influence the immune system through a wide variety of means, with some of bifidobacteria’s health-promoting benefits being linked to their ability to produce exopolysaccharides (EPS) enabling them to colonize more efficiently through modulation of the immune repertoire. Fanning et al. (2012) demonstrated that in mice with Citrobacter rodentium infection, the presence of a B. breve strain producing EPS resulted in lower bacterial counts of C. rodentium when compared to mice with a non-EPS producing B. breve strain and a stronger immune response was observed for B. breve-EPS inoculated mice when compared with B. breve + EPS mice. B. breve could remain immunologically silent through producing EPS (Fanning et al., 2012). Commensal strains also produce immunomodulins, which act as immune regulators in human monocyte cells. For instance, Lactobacillus reuteri 6475, caused a TNF-α reduction via modulation of expression of three genes; histidine/histamine antiporter (hdcP), histidine decarboxylase pyruvoyl type A (hdcA), and hdcB (thought to regulate hdcA) (hdcB), (Thomas et al., 2012). Ladda et al. (2015) found that the cell-free supernatants of two Lactobacillus strains, L. paracasei MSMC39-1, and L. casei MSMC39-3, reduced the amount of the proinflammatory cytokine TNF-α produced in monocytes (Ladda et al., 2015). The constant Toll-like receptor (TLR) stimulation at a low nondetectable level is thought to be beneficial for the intestine in maintaining homeostasis and helping to boost the immune response toward future pathogen attacks (Pinto et al., 2009). In fact, Jiang et al. (2016) demonstrated that L. plantarum NDC 75017 interacted with the TLR2 receptor causing subsequent signaling pathways NF-kβ and p38 MAPK to become activated and inducing IL-1β, TNF-α, and IL-6 expression in caco-2 cells. When S. typhimurium was then introduced to the HT-29 cells, more IL-8 was secreted, which recruited various cells of the innate response to fight off this pathogen (Jiang et al., 2016). The importance of modulating intestinal cells with commensal bacteria was also shown by Resta-Lenert and Barrett (2003), as only pretreatment of intestinal cells with S. thermophilus and L. acidophilus instigated a reduction in pathogen adhesion, increased the TEER (transepithelial resistance) reflecting epithelial barrier strength, increased epithelial cell permeability and reduced the serine phosphorylation of occludin and tyrosine phosphorylation of ZO-1 caused by pathogen infection (Resta-Lenert and Barrett, 2003). The commensal microbiota may also induce glycan expression to aid its own colonization stability. As demonstrated by Nanthakumar et al. (2005), glycosyltransferase gene expression was dependent on the colonizing adult microflora during the weaning period, with little initiation of induction of glycosyltransferase production in germ free subjects (Nanthakumar et al., 2005). Colonizing bacteria were also required for the traditional shift to heightened fucosylation and lowered sialylation in the mature intestine, with induction of FUT2 dependent on inoculation with adult microflora, such as Bacteriodes fragilis (Nanthakumar et al., 2013). Therefore, the colonizing microbiota may have the ability to induce transcriptional changes to provide a different glycan profile on the epithelial cell surface


Figure 8.3: The commensal microbiota population may (A) modulate the glycan moieties expressed on the cell surface, which in turn may (B) defer pathogen attachment and stabilize its own commensal colonization.

thereby increasing numbers of specific subsets of bacteria. This mutualistic relationship may also be a pathogenic defense mechanism, as altering the terminal glycan moieties of glycoconjugates may then render these attachment sites unattainable for pathogenic strains (Freitas et al., 2003; Naarding et al., 2005) (Fig. 8.3). As mentioned before, the majority of commensal strains can metabolize OS as an energy source and when selected OS are used, studies have shown expression of specific gene clusters which are not expressed with other carbon sources, such as lactose (Garrido et al., 2011). For instance, individual strains of bifidobacteria utilize 3′ and 6′-sialyllactose differently, with infant strains, such as B. infantis and B. bifidum utilizing 3′- and 6′-sialyllactose more efficiently when compared to adult-resident strains, such as B. adolescentis (Moon et al., 2016). B. longum subsp. infantis ATCC 15697 has been shown to express several fucosidase genes for metabolism of fucosylated HMO (Sela et al., 2012). The advantages of OS are further highlighted by the fact that several Enterobacteriaceae strains cannot utilize certain OS (2′-FL and 6′-SL) as carbon sources for their growth (Fig. 8.4). Careful consideration and further research is required to decide which OS are most suitable for supplementation in infant formula. Research groups are now focusing on other strains outside the popular genus of Bifidobacterium to assess their ability to grow on HMO, a trait which is known to be variable and strain specific (Krausova et al., 2015). OS generally only aid specific strains of commensal bacteria to persist (as observed for probiotics studied in recent times) (Arboleya et al., 2013). Many other OS commonly used in infant formula, such as GOS and XOS stimulate the attachment and growth of commensal bacteria, such as Lactobacillus and Bifidobacteria (Christensen et al., 2014; Giovannini et al., 2014). Mao et al. (2015) carried out a BLASTP homology search to investigate strains, encoding genes for glycoside hydrolazes and transporters potentially capable of metabolizing FOS.

210 Chapter 8

Figure 8.4: (A) Oligosaccharides are established prebiotics serving as an energy source for the commensal microbiota to metabolize. (B) The majority of pathogenic to the advantage of the commensal microbiota population.

These strains were then tested for their ability to metabolize FOS and those that produced proteins for homologous transporter and glycosidase hydrolases of FOS could metabolize FOS. Homology searches may be a quicker and more cost-effective method in understanding a bacterial strain preference for OS as carbon sources. However, these results may not translate in vitro due to the availability of other carbon sources but it is a step in the right direction (Mao et al., 2015). Some bifidobacterial strains encode genes for endoglycosidase activity thereby cleaving N-glycans from proteins, thus conferring them an advantage over other strains in acquiring substrates for growth (Garrido et al., 2012). The resulting free N-glycans serve as better growth substrates when compared to their conjugates with specific Bifidobacterium strains, such as B. infantis possessing this ability and being a key colonizer of the infant gut (Karav et al., 2016). Hence, OS attached to milk conjugates, such as lactoferrin, immunoglobulins, etc. may be just as important for infant strain growth as their free oligosaccharide peers.

4 Immunomodulation by Oligosaccharides OS have the ability to resist enzymatic degradation in the gastrointestinal tract until they reach the colon. As mentioned previously, it is now thought that OS can act systematically throughout the body and are most likely solid contributors to the infant immune response (Table 8.3) (Goehring et al., 2014; Rudloff et al., 2012). Various OS have been shown through many studies to interact with the immune system, whether it be by direct interaction with carbohydrate receptors or indirectly by helping to increase the numbers of commensal

The Role of Milk Oligosaccharides 211 Table 8.3: Milk oligosaccharides with immunomodulating properties. Oligosaccharides

Effects

References

2′-FL

Antiinflammatory

Matthies et al. (1996); Oliveros et al. (2016); Vazquez et al. (2015) Holscher et al. (2014)

LNnT

Influences maturation of intestinal cells, increases in transepithelial resistance Antiinflammatory activities LNFP I Immunomodulation of mononuclear cells, antiinflammatory LNFP III Activate macrophage cytokine secretion Signaling interaction with TLR4 receptor, influence dendritic cell maturation, proinflammatory Antiinflammatory activity 3′-SL Reduces pneumocyte invasion by pathogen 6′-SL P. aeruginosa Total fraction of acidic Reduce PNC formation, antiinflammatory milk oligosaccharides Effect on lymphocyte maturation Decrease sialyl Lewisx ligand binding to P-selectin Reduce leukocyte rolling and adhesion

Terrazas et al. (2001) Sotgiu et al. (2006) Atochina and Harn (2005) Velupillai and Harn (1994) Zenhom et al. (2011) Marotta et al. (2014) Bode et al. (2004b) Eiwegger et al. (2004) Schumacher et al. (2006) Bode et al. (2004a)

FL, Fucosyllactose; LNFP, lacto-N-fucopentaose; LNnT, lacto-N-neotetraose; PNC, platelet neutrophil complex; SL, sialyllactose; TLR, toll-like receptor.

bacteria. The varying effects of distinct OS are thought to relate to their structural diversity and length of the oligosaccharide chain (Johnson-Henry et al., 2014). Many carbohydrate receptors of the immune repertoire can interact with milk OS and at present, it is not known how many oligosaccharide-receptor interactions are possible. Dendritic cells are one of three types of antigen presenting cells which link the early innate immune response to the later adaptive immune response. Dendritic cells are involved in recognizing antigens, capturing, processing and presenting them to immune cells. The way in which dendritic cells recognize pathogens is through Pattern Recognition Receptors (PRRs) that recognize specific molecular structures associated with the pathogen and stimulate danger signals (signals of inflammatory stress), leading to activation of an immune response (Mellman, 2013). These PRRs are carbohydrate receptors and are believed to have the ability to interact with OS, which in turn may lead to immunomodulation. Among the PRR network, are toll-like receptors (TLRs), C-type lectins and siglecs. TLRs are important contributors to pathogen defense as even the absence of one of the ten TLRs, or it’s over expression can have detrimental consequences, such as a higher susceptibility to infection or chronic inflammation (Takeda et al., 2003). Upon recognition of specific pathogen structures, TLRs signal through specific molecules (TIR domain-containing adaptors, such as MyD88, TRIF, and TRAM) leading to a signaling cascade whereby transcription factors, such as NF-kβ

212 Chapter 8 are activated and proinflammatory genes are then expressed. In recent years, research has shown that OS may act as ligands for TLRs providing a regulatory and homeostatic role. This interaction between OS and TLR4 was demonstrated again by Kurakevich et al. (2013) whereby 3′-SL stimulation in mice produced CD11c+ MLN-derived dendritic cells, again through TLR4 signalling and induced expression of inflammatory cytokines leading to a Th1 and Th17 response (Kurakevich et al., 2013). 2′-FL also inhibits increased IL-8 expression as observed in response to type 1 pili E. coli infection by suppressing the CD14 coreceptor of TLR4 thereby preventing damage to the immature infant intestine (He et al., 2014). HMOs and GOS are also capable of modulating the inflammatory response by reducing TNF-α and Il-1β induced expression of IL-8, MIP-3α, and MCP1 thus inhibiting an excessive infiltration of inflammatory mediators of the immature infant intestine (Newburg et al., 2015). 3′-SL and other OS from breast milk may act as priming signals to coach the infant intestine to differentiate between its own glycan moieties and bacterial glycan moieties to initiate effective host recognition. These studies support the idea that some OS mount a proinflammatory response due to sharing homology with receptors with pathogens, for instance sialic acid, a common monosaccharide interacts with TLR4, which is known to be utilized by sialylated C. jejuni to attach to activated dendritic cells (Kuijf et al., 2010). TLR4-oligosaccharide interactions are the most studied, which is not surprising given that LPS, the main affinity ligand of TLR4, is a saccharide. Exposure to other sources of OS, such as FOS, GOS, goat milk OS (GMOS) and inulin led to an upregulation in the expression of the cytokines MCP1, GROα (IL-8) and MIP2 in intestinal cells. GOS and FOS were dependent on interaction with a TLR for upregulation of expression of these cytokines. Without the presence of TLR4, these cytokines did not display any upregulation in expression. The upregulation occurred at a more reduced level when compared to TLR4’s pathogen-alerting ligand LPS, possibly suggesting that a reduced activation of NF-kB may be beneficial to epithelial healing and integrity (Ortega-Gonzalez et al., 2014b). CapitanCanadas et al. (2014) also observed that the same mechanism was occurring with monocytes via induction with the OS FOS and inulin. Milk OS have also been shown to interact with various C-type lectins, such as selectins, which are involved in platelet neutrophil complex (PNC) formation and leukocyte recruitment. Acidic HMO possesses the ability to inhibit PNC formation through P-selectin inhibition, therefore reducing inflammation due to a reduced amount of (neutrophilproduced) reactive oxygen species (ROS) (Bode et al., 2004a,b; Schumacher et al., 2006). The regulation of PNC formation may prevent a subsequent excessive onset of neutrophil recruitment, a trait associated with onset of disease and this added regulation by acidic HMO may provide extra protection (Conese et al., 2003). The HMO, Lacto-N-fucopentaose, is also a ligand for P-selectin and has been shown to stimulate the proliferation of B-cells, which then leads to cytokine IL-10 and PGE2 production (Velupillai and Harn, 1994). DC-SIGN, another dendritic cell lectin, is known to be an attachment for pathogens and thus may also be stimulated by interaction with OS causing immunomodulation of leukocytes

The Role of Milk Oligosaccharides 213 (Koning et al., 2015). Also, DC-SIGN binds to high-mannose glycan moieties and has shown to have high affinity for Lex sugar epitopes commonly found on HMO. This affinity confers an advantage in the prevention of HIV transmission from mother to infant. The HIV-1 glycoprotein gp120 is suppressed from attaching to DC-SIGN by 80% with HMO supplementation (Hong et al., 2009; Naarding et al., 2005). Siglecs, yet other carbohydrate receptors found on dendritic cells, can recognize sialylated molecules and have displayed affinity for milk OS. Siglec-2 and Siglec-4 attach to 3′- and 6′-sialyllactose, suggesting that these OS may be modulating inflammation in which siglecs are involved (Chang et al., 2012; Koliwer-Brandl et al., 2011; Paulson et al., 2012). Galectins, a group of secreted proteins, are increasingly believed to be pattern recognition molecules, recognizing foreign microbes and also having the ability to modulate innate and adaptive immune responses (Nieminen et al., 2005; Rabinovich et al., 2002). Galectins share affinity for β-galactosides, molecules which are found at nonreducing ends of HMO in breast milk. As a result, it is thought OS may cause immune modulation through galectin binding. This hypothesis has been validated in airway epithelial cells whereby loss of sialic acid increased pneumococcal adhesion to galectins thus showing the necessity of incorporated sialic acid (Nita-Lazar et al., 2015). Recently, galectins have shown to be able to bind human milk glycans, which contain the monosaccharides galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (Neu5Ac), with each galectin showing differing specificities for differing glycans (Noll et al., 2016). OS may also interact with receptors to enhance immune homeostasis and prevent exaggerated immune responses in the very immature immune repertoire of the infant leading to fewer inflammatory conditions occurring in the breast-fed infant when compared with milk formula fed infants, such as atopic dermatitis (Moro et al., 2006). Sialyllactose and commercial OS have not just displayed interaction with TLRs, but also with other molecules, such as the PPARγ receptor, which led to the induction of the expression of PGlyRP3, causing suppression of the NF-kB pathway and a reduction in inflammatory cytokines. This indicates that OS, namely sialyllactose, may not just exert their modulatory effects through TLR receptors and lectins, but also through other recognition molecules found on the epithelial surface (Zenhom et al., 2011). Indeed, there may be a range of receptors/surface molecules modulated by OS, which have yet to be characterized. Kuntz et al. (2009) demonstrated the acidic oligosaccharide fraction of milk could induce cell cycle (G2/M) arrest via direct interaction with the EGF receptor, causing its phosphorylation and thus initiation of a signaling cascade through the Ras/Raf/ERK pathway (Kuntz et al., 2009). This indicates HMO has a direct influence on cell cycle proliferation and growth. The HMOs LNnT, 6′-SL and 2′-FL all led to a decrease in proliferation of HT29 and caco-2 Bbe cell lines (Holscher et al., 2014). Moreover, in cord blood T cells directly exposed to acidic milk OS, a significant increase in the number of IFN-γ producing CD4+CD8+ cells, as well as IL-4 and IL-13 producing CD3+CD4+ and CD3+CD8+ cells was observed. This study demonstrated uniquely the direct influence milk OS have on lymphocyte maturation, and how HMOs

214 Chapter 8 may help balance the Th1/Th2 cytokine profile during infant development (Eiwegger et al., 2004). OS have also been shown to directly interact with goblet cells, as observed with GOS supplementation of LS174T cells (goblet cell like cell line). Bhatia et al. (2015) demonstrated that stimulation of the cells with GOS led to an upregulation of the secreted products MUC2, RETNLB, CHST5 and TFF3, all involved in epithelial cell integrity and mucosal barrier protection. This effect, however, was not thought to be caused by stimulation of an inflammatory receptor, such as the NF-kB pathway, as IL-8, a key marker of this, was not affected by the addition of GOS (Bhatia et al., 2015). Kubota et al. (2014) demonstrated that FOS supplementation in pregnant and lactating women caused an increase in IL-27 production, which then through Th1 and Th17 responses lead to upregulation of IL-10 production (Kubota et al., 2014). This in turn could aid the colonization of the gut by commensal bacteria, as an absence of IL-10 leads to a heightened immune response against commensal bacteria (Ueda et al., 2010). Secretory IgA (SIgA) is hugely important for the newborn due to an inability to produce it, whereby they rely on receiving it passively from the mother. SIgA has been shown to have a wide variety of benefits, including a role in developing the gut microbiota and infection defence. It prevents adhesion through acting as a decoy receptor and attaching to the epitopes found on microbial pathogens. IgA also has the ability to modulate pathogen gene expression of surface epitopes used for adhesion (Peterson et al., 2007). Indeed, weaning mice deprived of SIgA passively have a higher number of bacteria and an increased risk of inflammation and associated disease (Rogier et al., 2014). Sangwan et al. (2015) demonstrated that supplementation of GOS gave mice a greater resistance to Listeria monocytogenes infection and increased levels of IgA and IgG, which are involved in repair of intestinal homeostasis, were observed (Sangwan et al., 2015). Similar results were observed by Hosono et al. (2003), whereby FOS supplementation increased intestinal IgA secretion in infant mice and reduced serum IgG1. The studies described above highlight the vital function OS in human milk play in the development of the neonate immune system (Hosono et al., 2003). Indirectly, as mentioned before, HMO may also act as an immunomodulator by allowing the commensal population flourish, which in turn benefits the host immunity. The full effects of OS on immune modulation may only become clear with further studies and human trials.

5 Mucin Expression, Defensive Function, and Indirect Effects of Oligosaccharides The gastrointestinal tract is protected by a constantly recycled mucus layer. This mucus layer consists of two main types of glycosylated material; glycoproteins secreted by either goblet or epithelial cells and glycolipids, also secreted by epithelial cells. These specialized goblet cells then secrete high molecular weight glycosylated mucin glycoproteins, which can be split into three individual groups; gel-forming secreted mucins, nongel forming secreted mucins and cell surface mucins. Mucins consist of a core protein rich in a number of serine,

The Role of Milk Oligosaccharides 215 threonine, and proline-linked residues and are saturated with a number of O and to a less extent N-linked OS, with many being sulfated or sialylated, through the monosaccharide N-acetylgalactosamine (GalNAc). Mucins, along with the proteins that associate with them, have an extremely important role in protecting the intestinal epithelium. Distinct mucins have diverse ways of preventing pathogen adhesion via creation of a physical barrier whereby pathogens and toxins cannot physically pass. Also secreted mucins form decoy receptors/ attachment sites for bacteria that result in bacteria being flushed away from the intestinal epithelium (Sando et al., 2009). Mucins mimic target receptors thereby preventing pathogens attaching to the intestinal surface (Mack et al., 2003). Considering this, mucins in many diverse ways limit disease and infection associated with pathogen colonization. All mucins are thought to actively take part in intestinal homeostasis, and have been shown upon stimulation by a pathogenic/inflammatory source to have higher rates of expression with differing functions of either antiinflammatory or proinflammatory responses (Enss et al., 2000; Sheng et al., 2013). Many mucins have been implicated with antiapoptotic/ proapoptotic functions (Elmore, 2007; Ishida et al., 2008; Raina et al., 2004). Studies have shown the strong link between mucin expression and TLR signaling in regulating the early response to infection. The upregulation of proinflammatory cytokines, such as TNF-α and IL-8 lead to secretion of certain mucins (Iwashita et al., 2003; Smirnova et al., 2000; Ueno et al., 2008). The ligands for TLRs have also been shown to stimulate goblet cells, which in turn result in mucin secretion in a concentration-dependent manner (Nishida et al., 2012; Schroeder et al., 2001). This highlights the importance of correct mucin expression in a controlled manner as deregulated mucin expression has been shown to be associated with many chronic diseases. Antiviral activity has also been associated with increased mucin content and mucins may provide a source of protection at varying levels to individual bacterial strains (Yolken et al., 1992). MUC2 has shown to be important in stimulating the production of β-defensin 2, an important antimicrobial peptide (Cobo et al., 2015). Mucins have also been shown to repress the expression of flagella in E. coli 0157:H7 preventing its colonization and an innate immune response due to the flagella signal. The core 2 O-glycan (building block of mucin) is known to be associated in preventing E. coli pathogenesis (Kim et al., 2012; Ye et al., 2015). MUC1 can prevent pathogens interacting with cells and receptors, such as DC-SIGN on dendritic cells, which in the neonate, appears extremely important and could also be a preventative in allergies (Koning et al., 2015). Liu et al. (2012) found that human milk MUC1 and MUC4 could prevent Salmonella invasion and prevent its pathogenesis in two different epithelial cell lines with MUC1 being the more potent inhibitor. Mucin content changes considerably during the course of infection with lower rates of cell surface mucins and higher rates of secreted mucins. Mucins are a primary target to overcome for efficient infection and C. rodentium directly interacts with goblet cells resulting in their destruction and thus preventing the recycling of the mucus layer. MUC1’s expression has

216 Chapter 8 shown to be upregulated in response to C. rodentium infection while other mucins were downregulated (Lindén et al., 2008). Acute H. pylori infection also caused a dramatic decrease in mucin proportions, which were then restored after infection (Cooke et al., 2009). The glycans attached to the external mucin surface play an important role in defending against pathogenic attachment. If gastric mucins are deglycosylated, the ability to repel pathogenic strain attachment, such as Staphylococcus aureus and Pseudomonas aeruginosa is reduced (Co et al., 2015). The O-glycans of mucins are also important in bacterial repulsion, protecting against pathogens, such as Staphylococcus aureus in the invasion of corneal epithelial cells (Ricciuto et al., 2008). Similarly, Salmonella is known to express glycosyl hydrolases (nanH and malS), which degrade the Glycocalyx layer in a bid to gain access to the epithelial cell membrane and also use these hydrolases to cause subsequent alteration of the glycans expressed by the host (Arabyan et al., 2016). Some pathogenic bacteria also produce virulence factors, which have responsive elements (short fragments of DNA) in mucin promoter regions and are capable of downregulating transcription and thus expression of certain genes and hence evade protection. Perrais et al. (2014) demonstrated that H. pylori had associated urease-responsive elements in the MUC5AC promoter region and induced a downregulation of MUC5AC at the transcriptional level. Also, C. jejuni has been shown to modulate its gene expression when exposed to MUC2, aiding pathogenesis and colonization (Tu et al., 2008) Probiotics, such as Bifidobacterium, L. bulgaricus, and Streptococcus thermophiles have been shown to increase MUC2 expression in caco-2 cells and avert oncoming E. coli infection (Yu et al., 2015). The commensal Ruminococcus gnavus has the ability to secrete soluble molecules, which altered the glycosylation of mucins by interacting with glyco-enzymes involved in glycan production, as well as influencing increased expression of specific mucins, MUC1 and MUC2 (Graziani et al., 2016). This increase in mucin expression is used beneficially by the strain as a carbon source for increased growth and attachment as observed with other strains in the Ruminococcus family, such as R. torques, which has the ability to degrade MUC2 as a carbon energy source. Dominant commensals, such as Bacteroides fragilis and B. longum subsp. infantis specifically attach to intestinal mucins and have the ability to digest intestinal mucins as a carbon source (Huang et al., 2011; Kim et al., 2013; Roberton and Stanley, 1982). Hence, mucins play a very important role in the colonization of the gastrointestinal tract by beneficial commensal microbes and are essential for their attachment and growth. Mucins inevitably influence the type of microbiota, which can colonize the intestine, with the glycan moieties attached to mucins catering for specific commensal strains in terms of attachment and also as a food source. In turn, the microbiota may be counterregulating mucin production and the overall mucus layer. Comelli et al. (2008) demonstrated that the presence of a microbiota-influenced mucin gene expression, including glycogene expression of N-glycans. The type of microbiota present in the infant intestine is reliant on the OS received

The Role of Milk Oligosaccharides 217 through breast milk. Certain nondigestible OS may stimulate mucosal intestinal epithelial cell to induce mucins, and their homeostatic/protective functions of the intestine, as has been observed in the presence of probiotics (Dykstra et al., 2011). GOS have been shown to modulate goblet cell function of LS174T cells increasing the expression of goblet secretory genes, such as MUC2 thus contributing to mucosal defense through direct interaction with goblet cells (Bhatia et al., 2015). In another study involving GOS, its addition instigated a substantial increase in O-linked glycoprotein mucin content in the mucosa of the intestine without affecting the mRNA levels of either MUC2 or MUC4 (Leforestier et al., 2009). Caprine milk OS have been demonstrated to downregulate and renormalize mucin gene expressions levels in a rat colitis model (dextran sodium sulfate–induced colitis) and addition of them to noninfected caco-2 cells has also shown them to increase mucin gene expression, specifically MUC5AC, MUC2, and MUC4. Hence, OS may influence slight changes in the glycan moieties of the glycocalyx and mucus layer, overall contributing to increased barrier function (Barnett et al., 2016; Lara-Villoslada et al., 2006). Thus, OS may also act to fuel-heightened mucin production, which aids better protection to the infant intestine from pathogenic agents. Given the diverse range of OS available in milk, it is possible that mucin production is heavily influenced by these molecules in early infant development.

6 Developing Areas 6.1 Allergy Intervention by Oligosaccharides The incidence of allergy has increased in infants in recent years and a means of preventing or alleviating symptoms early before complete manifestation/sensitization occurs is required. A lower incidence of allergy is observed in infants who are breast-fed when compared to those that are formula fed, and this may be due partly to the formula-fed infant not receiving the OS profile found in human milk. As reported in the immune section, OS have immune modulatory effects and have been shown to have direct interactions with the immune repertoire of receptors, transcription factors, cytokines, and so forth. Thus, OS may play an important role in the infant with respect to allergy prevention (Table 8.4). A Th2-weighted profile is necessary in pregnancy to reduce the risk of miscarriage and control Th1 responses. Therefore, newborn infants begin their immune life with a Table 8.4: Oligosaccharides involved in allergy prevention. Oligosaccharides

Effects

References

DS-LNT Total fraction of neutral milk oligosaccharides Total fraction of acidic milk oligosaccharides

Reduced necrotizing enterocolitis Reduction in incidence of allergy 1–5 years of life Allergy prevention

Jantscher-Krenn et al. (2012) Arslanoglu et al. (2012)

DS-LNT, Disialyllactose

Eiwegger et al. (2004, 2010)

218 Chapter 8 Th2-favored profile. The Th1/Th2 profile gradually becomes balanced over time but in infants that develop allergic manifestations, a reduced Th1 profile is observed and a more active Th2 profile is evident. Hence, OS that help balance the Th1/Th2 fluctuation in early infancy may provide an important step in the prevention of allergy development (Makhseed et al., 2001). Eiwegger et al. (2004) demonstrated that acidic HMO might in part function to balance the Th1/Th2 profile and prevent allergic manifestation through maturation of the immune response. In adult human subjects with allergies to peanuts, HMO supplementation induced a Th-0 shift and reduced IL-4 production and increased IFN-γ levels. Thus, the typical Th2 profile commonly observed in allergic phenotypes is suppressed in the infant through acidic OS found in breast milk (Eiwegger et al., 2004). This was also observed with 2′-FL and 6′-SL where their introduction to OVA-sensitized mice, alleviated allergic symptoms through stimulation with IL-10 producing T-regulatory cells and diminished mast cell initiation (Castillo-Courtade et al., 2015). Different synthetic oligosaccharide mixtures have differing effects on allergy alleviation with scGOS/lcFOS being the most studied in recent times due to its present use in infant formula. OS use has also been studied in influenza vaccination models with van’t Land et al. (2010) demonstrating the influenza vaccine immune response had been modulated by scGOS/lcFOS/ pAOS toward a Th1 response enabled by CD25+ Treg cells. GOS/FOS supplementation in a murine influenza vaccination model also directed the immune response again toward a Th1 phenotype (Vos et al., 2007b). Female dam mice that were sensitized to hen’s egg protein and supplemented with scGOS: lcFOS: pAOS (9:1:2) during pregnancy displayed decreased allergic responses and a reduced development of allergic symptoms in their female offspring, with expression of markers of a heightened Th1 response. Supplementation of OS to the mother during pregnancy may have long-standing immunomodulatory effects in the offspring (Hogenkamp et al., 2015a,b). This OS combination has also proven effective in alleviating asthma symptoms and reducing inflammation caused by chronic asthma (Sagar et al., 2014; Van der Aa et al., 2011). This mixture (scGOS/lcFOS) has also led to alleviation of cow’s milk allergy through a reduction in Th2 activation, whereas a combination of pectic (acidic) OS and scGOS/lcFOS, alleviated symptoms through strengthening the Th1 and Treg responses, which avoids the Th2 misbalance (Kerperien et al., 2014). This use of OS to promote a Th1 response may aid a variety of immune diseases, such as asthma, inflammatory airway conditions, atopic dermatitis among other allergies (Moosbrugger-Martinz et al., 2016; Vos et al., 2007a; Ying et al., 2005). scGOS/lcFOS was shown to reduce the incidence of atopic dermatitis in infants susceptible to the allergy and this correlated with reduced levels of kappa and lambda Ig-fLC levels in plasma, an immunoglobulin shown to be heightened in allergic disease (Schouten et al., 2011). Arslanoglu et al. (2008) demonstrated that an scGOS/lcFOS mixture given to infants of up to 6 months of age reduced the incidence of infection and appearance of allergies during the first 2 years of life (Arslanoglu et al., 2012). This scGOS/lcFOS mixture

The Role of Milk Oligosaccharides 219 has resulted in alleviation of allergic manifestation and a strengthening of immunity when supplemented in infant formula (Van Hoffen et al., 2009). Significant differences have been observed when infants have been fed scGOS/lcFOS, such as higher saliva counts of IgA, lower α-1-3 defensin counts, higher counts of bifidobacteria and lactobacilli in feces, lower incidence of infection, and a lower incidence of allergic reactions to food products and cow’s milk protein compared to control groups (Ivakhnenko and Nyankovskyy, 2013). The lower incidence of allergy could be attributed to the lower amount of immunoglobulins present, as well the immunomodulatory potential of OS. However, aside from synthetic OS, the various OS mixtures found in breast milk require further investigation, along with their routes of action for potential use in alleviating allergies. Nonetheless, studies do give an indication of the possible functions HMO may take part in. Differences in breast milk composition between individual mothers may also play a part in allergic manifestation considering that FUT2-dependent OS (only being received in secretor mothers and not nonsecretor mothers) influence the commensal microbiota profile, which establishes in the infant intestine. Infants born through C-section and having a high hereditary risk for allergies might have a lower risk to manifest IgE-associated eczema at 2 years, but not 5 years of age, when fed breast milk with FUT2-dependent milk oligosaccharides (Sprenger et al., 2016). The colonization of the neonate gut is thought to be of huge importance to allergy development. Studies have demonstrated that a reduced microbial diversity in early childhood is associated with allergic disease, such as rhinitis (Bisgaard et al., 2011). Indeed, maternal and infant use of antibiotics, which depletes microbial numbers, is associated with an increased risk of infants developing cow’s milk allergy (Metsälä et al., 2013; Russell et al., 2013). Gut microbial dysbiosis/disturbance within the first 100 days of life has been correlated with increased risk of allergic development and asthma. For instance, reduced content of strains from the genus Faecalibacterium, Lachnospira, Veillonella, and Rothia correlated with a heightened risk (Arrieta et al., 2015). Considering the strong links between commensal strain colonization of the intestine and allergy development, the types of OS received by the infant may influence allergy prevention or development. Synbiotic mixtures of FOS and commensal strains, such as B. breve, have been shown to be important in suppressing allergic symptoms in murine subjects (Castillo-Courtade et al., 2015; Sagar et al., 2014; Verheijden et al., 2016). In parallel with this, early colonization and infection by pathogenic strains has also been associated with allergic manifestation. Early infection by nontypeable Haemophilus influenza and S. pneumoniae correlates with increased acquirement of asthma (Bisgaard et al., 2007).

6.2 Influence of Oligosaccharides on Brain Development The infant brain undergoes vast changes within the first 3 years of life, doubling in size within the first year. Hence, nutritional intake is extremely important within this time period for

220 Chapter 8 Table 8.5: Brain development. Oligosaccharides

Effects

References

2′-FL

Enhanced memory and cognitive function, neurodevelopment Reduced stressor-induced anxietylike behavior Enhanced memory, cognitive function, neurodevelopment

He et al. (2014)

3′- and 6′-SL Total fraction of acidic milk oligosaccharides

Tarr et al. (2015) Ward et al. (2007); Wang (2012); Wang et al. (2003, 2007)

FL, Fucosyllactose; SL, sialyllactose.

efficient cognitive development of the infant (Table 8.5). There has long been an association between breast-feeding and cognitive development of the infant and this has been confirmed by many studies, which display a positive correlation between breast-feeding, IQ assessment and motor development, with increases in white matter growth observed in infants that are breast-fed compared to those that are not (Isaacs et al., 2010). This finding remains stable even when other variables, such as maternal education and socioeconomic status, are accounted for (Bernard et al., 2013; Kramer et al., 2008; Lucas et al., 1994; Quigley et al., 2012). The positive relationship between higher IQ status and breast-feeding has even been shown to positively correlate with improved educational qualification and subsequent higher income in the later adult years in a 30-year cohort study in Brazil (Victora et al., 2015). The ability of OS to affect the growth of bacteria and modulate various immune responses has been discussed but these findings have also led to further research into how OS may influence the gut–brain axis. Commensal bacteria, such as Bifidobacteriium and Lactobacillus, have already been shown to have a reducing effect on anxiety and serum cortisol levels in subjects (Krishna, 2015; Messaoudi et al., 2011). These bacteria, importantly, have the ability to break down OS for use as a carbon energy source and possess sialidases, which degrade sialylated OS and release free sialic acid. Free sialic acid itself has the ability to cross the blood brain barrier and travel to certain parts of the brain for incorporation into glycolipids and glycoproteins associated with cognitive function (Wang, 2012). Brain maturation and development can be correlated to an increase in gangliosides and sialoglycoproteins content. Sialylated OS are found in large amounts in human breast milk and their intake has been directly linked to elevated concentrations of ganglioside and protein bound sialic acid within the infant brain. Indeed, various studies have shown that infants that are breast fed when compared to formula fed have higher concentrations of ganglioside- and protein-bound sialic acid in the gray matter of the brain, which may contribute to higher brain development and higher cognitive function (Wang et al., 2003). Supplementation of sialylated casein glycomacropeptide to young piglets correlated with a heightened expression of genes associated with learning performance, memory capacity and levels of protein-bound sialic concentrations were also heightened in the brain when compared to nonsupplemented piglets (Wang et al., 2007). Thus, ingestion of sialic acid obtained through breast milk may define


Figure 8.5: Oligosaccharides, Such as Free Sialic Acid and 2′-FL, may be Able to Pass the Stringent Blood–Brain Barrier and be Incorporated Into Developing Structures in the Infant Brain. This ability has been positively correlated with higher cognitive function and development.

sialic acid as an essential nutrient and component of OS within infant development and health (Fig. 8.5). Indeed, OS have been shown to have an impact on lowering the salivary cortisol awakening response (marker of hypothalamic-pituitary-adrenal axis activity) in GOS supplemented subjects when compared to nontreated ones, thus showing their ability to indirectly affect brain activity (Schmidt et al., 2015). Supplementation of prebiotic GOS led to elevation of specific hippocampal proteins, namely brain-derived-neurotrophic factor (BDNF), GluN2A and synaptophysin all involved in neurotransmission and neural signaling, with levels remaining elevated into young adulthood (Williams et al., 2016). Other OS, such as 2′-FL have been detected in the blood and urine of infants and have the ability to interact with organs, such as the brain (Goehring et al., 2014). 2′-FL, was shown to enhance, when supplemented, long-term potentiation (memory and learning) of the hippocampus and to increase levels of BDNF, CaMKII, and PSD95 in the hippocampus and different brain areas, all markers of synapse function and ultimately brain development (Matthies et al., 1996; Vazquez et al., 2015). Cognitive skills spanning into adulthood have also been correlated to 2′-FL intake in early life, with supplemented animal subjects demonstrating better learning and memory skills in later life compared to their nonsupplemented counterparts (Oliveros et al., 2016). The effect the full array of OS has on brain development has yet to be deciphered. In particular, the lack of sialic acid in infant formula may disadvantage the newborn infant and its clear benefit in brain development may support for supplementation of a variety of OS in infant formula. 2′-FL also is only

222 Chapter 8 found in trace amounts in cow’s milk and with infant milk formulas mainly being generated from cow’s milk, bottle-fed infants have a lesser chance of being exposed to this beneficial oligosaccharide. Another group at risk of not receiving 2′-FL is the offspring of nonsecreting mothers, who cannot physically synthesize 2′-FL. The concentration of 2′-FL declines greatly in human milk from 3.93 g/L in colostrum to 0.06–4.85 g/L in mature milk. Hence, the function of 2′-FL may be extremely important in the early days of life. In this respect, formula producers have recently started to add 2'-FL to formula (Goehring et al., 2016). The gut–brain axis has been well characterized with studies demonstrating that a healthy gut microbiota is associated with stable regulation of neurodevelopment and behavior in murine subjects (Heijtz et al., 2011). A healthy gut microbiota has been shown through various studies to contribute to normal brain function. To create and maintain a normal gut microflora, OS may be an essential dietary component. The type of microbiota present in the gut will also affect the immune repertoire function as the commensal population of the gastrointestinal tract has shown to influence the immune response as discussed. This factor will also include brain function considering receptors for cytokines are present on neuron components of the brain (Cua et al., 2003; Verma et al., 2006). Hence, the development of a healthy intestinal microbiota population in infancy positively correlates with enhanced cognitive function and neurodevelopment. Dietary interventions, such as supplementation with OS to nurture this intestinal–brain axis environment may be key.

7 Conclusions and Future Perspectives Breast-fed infants have historically been shown to have a lower incidence of infection and a more efficient immune repertoire when compared with their formula-fed peers. Individual constituents in breast milk, such as OS, have been shown to largely influence this difference. OS reduce infection, increase commensal microbiota numbers, induce mucin expression, progress neurodevelopmental status, and lessen the likelihood of manifestation of allergies. Elucidating the specific functions of individual OS and how their addition contributes to intestinal health is required to obtain a greater knowledge on their potential benefit on infant health. OS not only modulate the intestinal microbiota and inhibit pathogenic strains but also modulate the intestinal surface. OS may provide a naturally sourced alternative to antibiotic use, which is important in the fast approaching antibiotic resistant epidemic faced by practitioners today. Sourcing OS in large quantities is the main difficulty in elucidating their biological functions more extensively. Methods for producing milk oligosaccharides at industrial scale are required to validate their activity in human trials. As discussed, the milk of domestic animals is being investigated at as a source of functionally active oligosaccharides given that these molecules share structural similarity with HMO. However, the abundance and concentration of oligosaccharides in animal milk is much lower in comparsion with the pool of HMO found in breast milk.

The Role of Milk Oligosaccharides 223 Chemical synthesis of OS has been employed using solid-phase synthesis (Seeberger and Werz, 2005). However, the many deprotection and protection steps associated with this method hinder the yield of OS produced and require the use of chemical solvents, which also makes the process less applicable to the food and infant formula industry. Enzymatic synthesis, using glycosyltransferases from genetically engineered microbial cells, has also been investigated. The coupled one-pot enzymatic reaction mechanism has been used for the production of sialyllactose, lactose-N-biose and LNT with high yields and small amounts of enzymes and substrate used (Koeller and Wong, 2000; Woo et al., 2014; Yao et al., 2015; Yu et al., 2010). The biological production of these OS is favorable also and progress has been made in this area using various bacterial strains. Two thermostable GH1 β-galactosidases (Ttβ-gly of Thermus thermophiles and CelB of Pyrococcus furiosus) and the GH42 βgalactosidase of Bacillus circulans were codon-optimized and cloned in E. coli. The strain was capable of producing LacNAc from GlcNAc and lactose and LNnT from LNT2 and lactose, while the GH42 β-galactosidase of B. circulans resulted in LacNAc and LNnT production (Zeuner et al., 2016). 2′-Fucosyllactose has also been produced by this method using engineered E. coli containing the GDP l-fucose synthesis pathway for the production of this HMO (Chin et al., 2016). Biological synthesis using microbial fermentation for largescale production of OS may be the safest, fastest and most cost-effective method available for authorized use in the food industry. Membrane filtration has also been employed to isolate OS from whey whereby OS are retained while lactose, salts, and other contanminants pass through the low molecular weight membrane (Mehra et al., 2014). Ultrafiltration and anion exchange chromatography methods are also being explored for enrichment of OS from various milk sources and dairy streams (Altmann et al., 2016; Mehra et al., 2014).

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234 Chapter 8 Wang, B., Yu, B., Karim, M., Hu, H.H., Sun, Y., McGreevy, P., Petocz, P., Held, S., Brand-Miller, J., 2007. Dietary sialic acid supplementation improves learning and memory in piglets. Am. J. Clin. Nutr. 85, 561–569. Ward, R.E., Niæonuevo, M., Mills, D.A., Lebrilla, C.B., German, J.B., 2007. In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteria. Mol. Nutr. Food Res. 51, 1398–1405. Weichert, S., Jennewein, S., Hüfner, E., Weiss, C., Borkowski, J., Putze, J., Schroten, H., 2013. Bioengineered 2′-fucosyllactose and 3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and enteric pathogens to human intestinal and respiratory cell lines. Nutr. Res. 33 (10), 831–838. Weichert, S., Koromyslova, A., Singh, B.K., Hansman, S., Jennewein, S., Schroten, H., Hansman, G.S., 2016. Structural basis for norovirus inhibition by human milk oligosaccharides. J. Virol. 90 (9), 4843–4848. Williams, S., Chen, L., Savignac, H.M., Tzortzis, G., Anthony, D.C., Burnet, P.W., 2016. Neonatal prebiotic (BGOS) supplementation increases the levels of synaptophysin, GluN2A-subunits and BDNF proteins in the adult rat hippocampus. Synapse 70, 121–124. Woo, J.S., Sohng, J.K., Kim, B.G., Kang, S.Y., Kim, D.H., Jang, K.S., Yang, J.Y., Jung, Y.S., Seo, W.M., Gil, T.G., 2014. N-acetylglucosamine-2-epimerase and method for producing CMP-neuraminic acid using the same. Google Pat. US 8,852,891 B2. Xu, Q.W., Gitti, R., Bush, C.A., 1996. Comparison of NMR and molecular modeling results for a rigid and a flexible oligosaccharide. Glycobiology 6, 281–288. Yao, W., Yan, J., Chen, X., Wang, F., Cao, H., 2015. Chemoenzymatic synthesis of lacto-N-tetrasaccharide and sialyl lacto-N-tetrasaccharides. Carbohydr. Res. 401, 5–10. Ye, J., Song, L.L., Liu, Y., Pan, Q., Zhong, X.L., Li, S.S., Shang, Y.Y., Tian, Y., He, Y.H., Chen, L., Chen, W.S., Peng, Z.H., Wang, R.Q., 2015. Core 2 mucin-type o-glycan is related to EPEC and EHEC O157:H7 adherence to human colon carcinoma HT-29 epithelial cells. Digest. Dis. Sci. 60, 1977–1990. Ying, S., O’Connor, B., Ratoff, J., Meng, Q., Mallett, K., Cousins, D., Robinson, D., Zhang, G., Zhao, J., Lee, T.H., 2005. Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity. J. Immunol. 174, 8183–8190. Yolken, R., Peterson, J., Vonderfecht, S., Fouts, E., Midthun, K., Newburg, D., 1992. Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. J. Clin. Invest. 90, 1984. Yu, H., Thon, V., Lau, K., Cai, L., Chen, Y., Mu, S., Li, Y., Wang, P.G., Chen, X., 2010. Highly efficient chemoenzymatic synthesis of β1-3-linked galactosides. Chem. Commun. 46, 7507–7509. Yu, Z.T., Chen, C., Newburg, D.S., 2013. Utilization of major fucosylated and sialylated human milk oligosaccharides by isolated human gut microbes. Glycobiology 23, 1281–1292, p.cwt065. Yu, J.Y., He, X.-L., Puthiyakunnon, S., Peng, L., Li, Y., Wu, L.-S., Peng, W.-L., Zhang, Y., Gao, J., Zhang, Y.-Y., 2015. Mucin2 is required for probiotic agents-mediated blocking effects on meningitic E. coli-induced pathogenicities. J. Microbiol. Biotechnol. 25, 1751–1760. Zenhom, M., Hyder, A., de Vrese, M., Heller, K.J., Roeder, T., Schrezenmeir, J., 2011. Prebiotic oligosaccharides reduce proinflammatory cytokines in intestinal caco-2 Cells via activation of PPAR gamma and peptidoglycan recognition protein 3. J. Nutr. 141, 971–977. Zeuner, B., Nyffenegger, C., Mikkelsen, J.D., Meyer, A.S., 2016. Thermostable β-galactosidases for the synthesis of human milk oligosaccharides. New Biotechnol. 33, 355–360. Zinger-Yosovich, K.D., Iluz, D., Sudakevitz, D., Gilboa-Garber, N., 2010. Blocking of Pseudomonas aeruginosa and Chromobacterium violaceum lectins by diverse mammalian milks. J. Dairy Sci. 93, 473–482.

Further Reading Bergstrom, K., Kissoon-Singh, V., Gibson, D.L., Ma, C., Montero, M., Sham, H.P., Ryz, N., Huang, T., Velcich, A., Finlay, B.B., 2010. Muc2 protects against lethal infectious colitis by disassociating pathogenic and commensal bacteria from the colonic mucosa. PLoS Pathog. 6, e1000902–e11000902. Croix, J.A., Carbonero, F., Nava, G.M., Russell, M., Greenberg, E., Gaskins, H.R., 2011. On the relationship between sialomucin and sulfomucin expression and hydrogenotrophic microbes in the human colonic mucosa. PLoS One 6, e24447.

The Role of Milk Oligosaccharides 235 Kerr, S.C., Fischer, G.J., Sinha, M., McCabe, O., Palmer, J.M., Choera, T., Lim, F.Y., Wimmerova, M., Carrington, S.D., Yuan, S., 2016. FleA expression in Aspergillus fumigatus is recognized by fucosylated structures on mucins and macrophages to prevent lung infection. PLoS Pathog. 12, e1005555. Rodríguez-Piñeiro, A.M., Bergström, J.H., Ermund, A., Gustafsson, J.K., Schütte, A., Johansson, M.E., Hansson, G.C., 2013. Studies of mucus in mouse stomach, small intestine, and colon. II. Gastrointestinal mucus proteome reveals Muc2 and Muc5ac accompanied by a set of core proteins. Am. J. Physiol. 305, G348–G356.


CHAPTE R 9

Nutritional Yeast Biomass: Characterization and Application Monika E. Jach*, Anna Serefko** *The John Paul II Catholic University of Lublin, Lublin, Poland; **Medical University of Lublin, Lublin, Poland

1 Introduction Yeasts are regarded as fungi with vegetative states that predominantly reproduce by budding or fission, growing mainly as single cells in the vegetative phase. They include ascomycetous and basidiomycetous yeasts. It has been well documented that yeasts have many applications in fermentation, food, feed, agricultural, biofuel, medical, and chemical industries, as well as environmental protection (Adedayo et al., 2011; Kurtzman and Fell, 2000). They have been used for production of fermented food for at least as long as since 7000 BC (Reed and Nagodawithana, 1988). Yeast biomass from so-called nutritional yeasts is widely used as a source of nutritional components, such as single-cell protein (SCP) (Ferreira et al., 2010; Gonçalves et al., 2014; Nayak, 2011). Moreover, yeast biomass contains fats, carbohydrates, nucleic acids, vitamins, and minerals. It is rich in certain essential amino acids, such as lysine and methionine, which are limited in most plant and animal foods (Adedayo et al., 2011; Suman et al., 2015; Uchakalwar and Chandak, 2014). Nutritional yeasts are heat deactivated for production of nutritional components. Yeast cells are killed and dried. They have a wide amino acid spectrum and a high protein:carbohydrate ratio. Yeast SCP is used as a highnutrient feed substitute (Burgents et al., 2004). The most popular yeast species are Saccharomyces and the typical oily yeast genera, such as Candida and Yarrowia (Jach et al., 2015; Suman et al., 2015). Saccharomyces cerevisiae biomass occurs on various fruit waste (Dhanasekaran et al., 2011; Khan et al., 2010; Mondal et al., 2012; Tanveer, 2010; Vaidya et al., 2014). Yeasts can utilize inexpensive raw materials, feedstock, and various wastes to produce biomass, protein, and/or amino acids concentrate. Both conventional substrates (e.g., starch, molasses, fruit, vegetable wastes) and unconventional ones (e.g., petroleum by-products, natural gas, ethanol, methanol, and lignocellulosic biomass) have been used for production of biomass and metabolites (Suman et al., 2015). Therefore, nutritional yeasts are environment friendly—they can Diet, Microbiome and Health http://dx.doi.org/10.1016/B978-0-12-811440-7.00009-0

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238 Chapter 9 grow on wastes and thus help in waste recycling. They grow faster than plants or animals and produce large quantities of biomass from a relatively small amount of substrates and in a relatively short time, irrespective of the season (Adedayo et al., 2011). The method of harvesting, drying, and processing has an effect on the nutritive value of the final product (Bhalla et al., 2007). Yeasts can be easily separated by centrifugation because of their large cells. Yeast protein biomass can be used as an addition to the main diet instead of sources known to be relatively expensive, such as soybean and fish (Uchakalwar and Chandak, 2014). Thus, the use of yeast biomass as an alternative nutrient supplement can solve the problem of food scarcity for the rapidly growing population, especially in the developing countries (Suman et al., 2015). According to the World Food and Agriculture Organization (FAO), 25% of the world population has protein deficiency (Uchakalwar and Chandak, 2014). In spite of all benefits, nutritional yeast production has not gained great importance because yeast biomass is not generally accepted as a protein supplement for people. Therefore, efforts should be made to find methods that can lead to acceptance of this valuable nutrient supplement on a global basis (Suman et al., 2015). Besides, yeast biomass does not always have desirable functional properties for incorporation thereof into food. The cell wall of yeasts may be nondigestible and may have unacceptable color and flavour, as the cells of the microorganism must be killed before consumption (Adedayo et al., 2011). However, the digestibility of nutritional yeasts can be significantly improved by drying at a high temperature under defined conditions (Nasseri et al., 2011). Drying yeast biomass enhances shelf life of food products (Ibarr and Barbosa-Cánovas, 2014). In the case of probiotic yeast biomass, the freeze-drying process is usually applied, since this type of drying saves the living cells. The moisture content of food powders is usually between 2% and 8%. At this level, powders are stable with an average shelf life of 12–24 months. Drying is implemented before or after grinding operations and plays a major role in the texturing and stabilization of food materials (Baudelaire, 2013). Yeast biomass fulfills all the above requirements of drying and moisture for inclusion thereof as a diet supplement for both humans and livestock (Adedayo et al., 2011), especially for vegans and vegetarians, in poor regions and the world at large. Yeast biomass products are usually found in the form of powder, flakes, tablets (capsules), or in a liquid form. Liquid yeasts contain enzymatically digested cells for better digestion, absorption, and utilization (Ferreira et al., 2010).

2 Saccharomyces cerevisiae Preparations According to the US Food and Drug Administration (FDA), S. cerevisiae cell wall and products of its fractionation are generally recognized as safe (GRAS) (FDA, 1997). The so-called brewer’s yeasts or baker’s yeasts are the special strains of S. cerevisiae that are

Nutritional Yeast Biomass: Characterization and Application 239 widely used for food processing (i.e., production of bread, beer, wine, etc.). Since they are known as a rich natural source of lipids, proteins (including enzymes), peptides, amino acids, B-complex vitamins (except for vitamin B12), and trace minerals, they occupy an important place in the pharmaceutical industry (Stewart and Russell, 1985). Brewer’s and baker’s yeasts biomass is naturally low in fat and sodium. Moreover, it is deprived of the components that are not tolerated and/or should not be consumed by some people (i.e., sugar, dairy products, gluten) (Pérez-Torrado et al., 2015). In human alimentary tracts, after disruption of the yeast cell wall and digestion of the yeast proteins, the cellular constituents (amino acids, peptides, carbohydrates, vitamins, minerals) are released and absorbed (EFSA, 2008). The food and dietary supplement markets are abundant in brewer’s and baker’s yeast products, which are particularly recommended for people with increased vitamin B requirements, that is, adolescents, convalescents, and persons with high physical activity (Gottschalk et al., 2016). Apart from that, the food market offers so-called brewer’s and baker’s yeast extracts containing amino acids, peptides, nucleotides, or other soluble components of broken yeast cells. Yeast extracts are usually used as flavor additives in soups, sauces, gravies, stews, snacks, and canned food (Chae et al., 2001). Podpora et al. (2015) suggest that the postfermentation brewing yeasts could be used for preparation of yeast autolysates and in this form applied in the functional food and dietary supplements. The authors demonstrated that the autolysates from postfermentation brewing yeasts are rich in amino acids and peptides, and possess a high antioxidant potential. On the other hand, Rayman (2004) pointed out the problems with reproducibility as a main negative issue concerning mineral-enriched yeasts that are used in food or dietary supplements. Depending on a mineral source added to a growth medium, the amount of a relevant microelement, as well as its form in the yeast biomass may be different. Moreover, the complications with 100% identification of a yeast species in mineral-enriched biomass may occur.

2.1 Saccharomyces cerevisiae β-Glucans The major components of S. cerevisiae biomass—that is, β-glucans, may induce a dual-way immunomodulatory activity: they enhance immune reaction (i.e., they exert a prophylactic effect against common cold infections), and on the other side—they may diminish inflammation (Table 9.1). The European Food Safety Authority (EFSA, 2011) assessed βglucans from S. cerevisiae as a safe component of both food supplements (up to 375 mg/day) and foods for particular nutritional uses (up to 600 mg/day). β-Glucans potentiate the innate immune response, since mammalian cells lack this component. They are recognized and bound by macrophages present in the cells lining the digestive track and transported to spleen, bone marrow, and lymph nodes (Chan et al., 2009). After disintegration into smaller, soluble β-glucan oligosaccharides, they are released into the bloodstream and there they are bound by circulating monocytes, macrophages, neutrophils, natural killers, and dendric cells with β-glucan recognizing receptors, including toll-like receptor 2 (Underhill et al., 1999), dectin-1

240 Chapter 9 Table 9.1: Clinical studies on the immunomodulatory effects of β-glucans preparations made from S. cerevisiae. Authors (Year)

Subjects

Auinger et al. (2013)

Healthy participants with 16 Weeks recurring infections (n = 167)

Babineau et al. (1994b)

High-risk patients undergoing major abdominal or thoracic surgery (n = 34) Patients who underwent a major surgical procedure (n = 67)

Babineau et al. (1994a)

Carpenter et al. (2013)

Recreationally active participants (n = 60)

Dellinger et al. (1999)

Patients scheduled for gastrointestinal procedure lasting 2–8 h (n = 1249)

Feldman et al. (2009)

Healthy community-dwelling participants (n = 40)

Fuller et al. (2012)

Healthy participants (n = 100)

Graubaum et al. Healthy participants with (2012) recurring infections (n = 100)

HargerDomitrovich et al. (2008) Kohl et al. (2009)

Moyad et al. (2008)

Wildland firefighters (n = 54)

Duration of Treatments

Products

Insoluble (1,3)–(1,6)-β-glucan preparation made from brewer’s yeasts, 900 mg/day (n = 81), or placebo (n = 81) Multiple sequential doses PGG-glucan preparation made by intravenous infusion from S. cerevisiae, 0.5 mg/kg before and after surgery (n = 17), or placebo (n = 13) Multiple sequential doses PGG-glucan preparation made by intravenous infusion from S. cerevisiae, 0.1, 0.5, or before and after surgery 1.0 mg/kg (n = 51), or placebo (n = 16) 10 Days before strenuous (1,3)–(1,6)-β-Glucan exercise session preparation made from brewer’s yeasts, 250 mg/day (n = 60), or placebo (n = 60) Four doses by PGG-glucan preparation made intravenous infusion from S. cerevisiae, 0.5 or 1.0 mg/ (once preoperatively and kg, or placebo 3 times postoperatively) 12 Weeks (1,3)–(1,6)-β-Glucan preparation made from brewer’s yeasts, 500 mg/day (n = 17), or placebo (n = 16) 90 Days (1,3)–(1,6)-β-Glucan preparation made from brewer’s yeasts, 250 mg/day (n = 48), or placebo (n = 49) 26 Weeks Insoluble (1,3)-(1,6)-β-glucan preparation made from brewer’s yeasts, 900 mg/day (n = 50), or placebo (n = 50) 14 Days Yeast-based (β-glucan) antioxidant supplement (n = 54) or placebo (n = 54) 4 Weeks (1,3)–(1,6)-β-Glucan preparation made from S. cerevisiae, 1500 mg/day (n = 12), or placebo (n = 12)

Overweight and obese participants with moderately and repeatedly elevated CRP levels, indicating subclinical inflammation (n = 12) Healthy participants with 12 Weeks up-to-date vaccination histories (n = 116)

S. cerevisiae–based dietary supplement, 500 mg/day, or placebo

Nutritional Yeast Biomass: Characterization and Application 241 Table 9.1: Clinical studies on the immunomodulatory effects of β-glucans preparations made from S. cerevisiae. (cont.) Authors (Year) Talbott and Talbott (2009)

Subjects Healthy, asymptomatic adults who were marathon participants (n = 75)

Duration of Treatments 4 Weeks postmarathon

Talbott and Talbott (2010)

Healthy moderate to highly stressed participants (n = 150)

4 Weeks

Talbott and Talbott (2012)

Healthy women with moderate levels of psychological stress (n = 77)

12 Weeks

Talbott et al. (2010)

Healthy participants with moderate levels of psychological stress (n = 122)

12 Weeks

Products (1,3)–(1,6)-β-Glucan preparation made from brewer’s yeasts, 250 or 500 mg/day, or placebo (1,3)–(1,6)-β-Glucan preparation made from brewer’s yeasts, 250 mg/day (n = 50), 500 mg/day (n = 50), or placebo (n = 50) (1,3)–(1,6)-β-Glucan preparation made from brewer’s yeasts, 250 mg/day (n = 39), or placebo (n = 38) (1,3)–(1,6)-β-Glucan preparation made from brewer’s yeasts, 250 mg/day, or placebo

CRP, C-reactive protein; PGG, poly-[1-6]-d-glucopyranosyl-[1-3]-d-glucopyranose glucan

(Brown et al., 2007), complement receptor 3 (CR-3) (Ross et al., 1987), and lactosyl ceramide receptor (Brown et al., 2007; Ross et al., 1987; Underhill et al., 1999). The nonspecific-innate immune response (i.e., phagocytosis, production of proinflammatory factors) is induced (Qi et al., 2011). The compounds with β-(1,6)-linked side chains are more effective. Solubility and the size seem to be important as well (Sonck et al., 2011). Marathon runners treated with either 250 or 500 mg of a β-(1,3)-(1,6)-glucan preparation from S. cerevisiae for 4 weeks postmarathon reported significantly fewer symptoms of infection along with better overall health and mood state than the group that was given placebo (Talbott and Talbott, 2009). Though in the clinical trial by Feldman et al. (2009), 90-day ingestion of β-glucan (500 mg/ day) derived from S. cerevisiae did not considerably change the incidence of symptomatic respiratory infections in comparison to the placebo-supplemented group, the patients from the treatment group had significantly lower fever scores and none of them missed work or school due to common cold. In another placebo-controlled, double-blind, randomized clinical trial with 100 healthy participants with recurring infections, 26-week administration of the insoluble β-(1,3)-(1,6)-glucan (900 mg/day) obtained from brewer’s yeasts exerted a prophylactic effect on the occurrence of common colds during seasons with increased common cold incidences. The symptoms were less pronounced and lasted for a shorter time period (Graubaum et al., 2012). After 16 weeks of treatment with the same preparation made from S. cerevisiae of 81 healthy subjects with recurring common cold episodes, the diminished number of infections, reduced severity of symptoms, as well as improved sleep were noted (Auinger et al., 2013). In spite of the fact that after 90-day supplementation with

242 Chapter 9 250 mg/day of β-glucan, the number of days with upper respiratory tract infection was not smaller in the tested subjects as compared to placebo group, some symptoms seemed to be less severe (particularly in terms of the “ability to breath”) (Fuller et al., 2012). In a 12-week randomized double-blind, placebo-controlled trial conducted during the acute period of the year for cold and flu seasonal symptoms, once-daily dose of the S. cerevisiae–based dietary supplement (500 mg) reduced incidence and duration of the common cold or influenzalike symptoms in healthy participants who had been given the influenza vaccine (Moyad et al., 2008). A lower incidence of upper respiratory tract infection symptoms and better overall health parameters were recorded for the wildland firefighters after consumption of β-glucan antioxidant preparation. However, no significant difference was noted between the tested groups in terms of days missed from work or the average daily activity measured with accelerometers (Harger-Domitrovich et al., 2008). Considerably fewer upper respiratory tract symptoms, as well as improved general physical and psychological well-being displayed the stressed subjects who had taken yeast β-glucan preparations for several weeks (Talbott and Talbott, 2010, 2012; Talbott et al., 2010). The outcomes suggested that supplementation with yeast extracts could provide an additional protection against negative consequences of everyday stress. Carpenter et al. (2013) found out that β-glucans (250 mg/day) from baker’s yeast taken for 10 days before strenuous physical exercise increased level of monocytes and cytokines production (particularly IL-4, IL-5, and IFN-γ), reducing postexercise suppression of immune system. A significantly lower number of postsurgical infections and need for the antiinfective medications, as well as a shorter stay in the intensive care unit and hospital were recorded in the case of patients treated intravenously with a highly purified β-glucan (PPG-glucan) obtained from S. cerevisiae. According to the literature data, PPG-glucan is deprived of the pyrogenic and inflammatory activity but possesses immunostimulatory potential—it easily binds to the respective receptors of human monocytes and neutrophils, improves their functioning, and potentiates the microbicidal effect of the phagocytic cells against a wide range of pathogens (Babineau et al., 1994a,b; Dellinger et al., 1999). In spite of administration by the parenteral route, PPG-glucan was well tolerated by both healthy subjects (Wakshull et al., 1992) and patients (Babineau et al., 1994a,b; Dellinger et al., 1999). On the other hand, the antiinflammatory action of (1,3)-(1,6)-β-glucan preparation was noticed by Kohl et al. (2009) after administration to overweight and obese patients. Fourweek supplementation increased mRNA expression, as well as the circulating level of the antiinflammatory cytokine IL-10, which is known to inhibit the production of TNF-α and IL6, that is, the proinflammatory cytokines. The authors underlined that any significant changes in circulating levels and mRNA expression of proinflammatory cytokines were detected after ingestion of β-glucan preparation. In other in vivo and in vitro studies it was found that the combined treatment with antitumor monoclonal antibodies and β-glucan was more effective and better tolerated than monotherapy with anticancer monoclonal antibodies (Liu et al., 2009; Salvador et al., 2008). Yeast β-glucan improved clinical outcomes in patients treated for breast, colorectal, colon, leukemia, lung, ovarian, and skin cancers. As an adjuvant

Nutritional Yeast Biomass: Characterization and Application 243 it was helpful in chemotherapy, radiotherapy, antireumatic drug therapy, antifungal therapy (Kwiatkowski and Kwiatkowski, 2012; Sener et al., 2006), treatment of hospital pneumonia, acute renal failure (Koc et al., 2011), healing of pressure ulcers, wounds, and burns caused by heat, UV, or X-ray radiation (Kwiatkowski and Kwiatkowski, 2012; Spruijt et al., 2010). However, both large doses and prolonged administration of β-glucan may lead to undesirable adverse effects, since the long-term proinflammatory action may induce the development of autoimmune diseases (Kwiatkowski and Kwiatkowski, 2012). A number of studies demonstrated the antioxidant activity of the intact S. cerevisiae cells, as well as cell extracts. The exact mechanism of action has not been discovered yet, but the authors suggest the contribution of the high content of β-glucans [particularly (1,3)-β-glucan] and the presence of superoxide dismutase, glutathione peroxidase, and catalase (Abbas, 2006; Chen et al., 2010; Jaehrig et al., 2007; Jensen et al., 2007). In the placebo-controlled preliminary studies of Jensen et al. (2008), after 5 weeks of daily consumption of 500 mg of the S. cerevisiae–based dry fermentate nutritional product, trends of the increased mucosal protection and reduced allergies were observed. Moreover, 12-week administration of the same preparation resulted in significantly fewer symptoms of cold/flu (Moyad et al., 2010), as well as the reduced mean severity of specific allergic rhinitis symptoms (Moyad et al., 2009) in comparison to the respective placebo-treated groups. The obtained outcomes were at least partially due to the changes in serum antioxidant status and cytokine levels. A single dose of S. cerevisiae–based fermentate nutritional product influenced the number of circulating T lymphocytes and NK cells, increasing their homing into tissue (Jensen et al., 2011).

2.2 Se-Enriched Saccharomyces cerevisiae Yeasts Selenized yeasts seem to be an attractive supplementary source of Se for people with poor eating habits. Due to high protein content, S. cerevisiae cultured in the presence of the inorganic sources of Se (i.e., sodium selenite) incorporate this microelement quite easily, mainly as selenomethionine—an organic well bioavailable compound. Levels of the inorganic forms of Se in the Se-enriched biomass usually do not exceed 1% (EFSA, 2008; Ponce de León et al., 2002). In humans, absorption of Se from the selenized yeasts is approximately 1.5–2 fold higher than that from the inorganic forms, and ranges between 50% and 90%, depending on the study (Dumont et al., 2006; Sloth et al., 2003). Se from selenomethionine is effortlessly built-in into human tissue proteins. The repeated supplementation with Seenriched yeasts increases Se concentration in plasma and tissue to a certain level; after 6–12 months, a steady state of Se is reached in a human body. The studies indicated that selenomethionine from yeast biomass is widely utilized and reutilized, with the half-life of c. 252 days. Selenized yeasts are available in the pharmaceutical market in a form of dietary supplements but they can also be used for baking bread instead of the conventional baker’s yeasts, as the incorporated selenium in yeast biomass is thermally stable. As absorption of a

244 Chapter 9 dietary Se is high (about 80%), healthy people whose daily diet is balanced and varied usually do not need any additional supplementation of this microelement. The recommended dietary allowance (RDA) for adults is 55 µg of Se/day (EFSA, 2008). Regular consumption of food containing more than 1 µg Se/g is risky and may result in toxic effects (Wada et al., 1993). Chronic selenosis (hair loss, brittle, thickened and stratified nails, garlic breath, skin lesions) is observed after mean daily intake of Se doses of 4990 µg/day. However, the toxicity of the organic forms of Se (i.e., Se-enriched yeast biomass) is significantly lower than that of inorganic selenite or selenate (EFSA, 2008; Whanger et al., 1996). Basing on the literature data (EFSA, 2008), daily consumption of the yeast biomass containing up to 200 µg of Se is safe and does not lead to exceeding the tolerable upper intake level established by the Scientific Committee on Food (i.e., 300 µg/day) or US Food and Nutrition Board (i.e., 400 µg/day). As an average dietary intake of Se by Europeans was estimated within the range of 27–70 µg/day, the total intake of Se from both daily diet and dietary supplements falls within the limits. However, the safety data concerning a long-term intake of Se-enriched yeasts by humans are not consistent. The clinical studies on the effects of the Se-enriched yeasts are presented in Table 9.2. Thirty-five subjects out of 1312 who received placebo or 200 µg of Se/day from the yeast biomass for a mean of 4.5 years, suffered from adverse events (mainly associated with gastrointestinal upset). Twenty-one of them were in the Se-treated group, while 14 from the placebo group (Clark et al., 1996). On the other hand, no adverse reactions were reported after daily administration of 200 µg Se in a form of Seenriched yeasts or placebo for 4 years. In this clinical trial performed by Yu et al. (1997), 226 hepatitis surface-antigen positive subjects took part. Similarly, no safety concerns were detected after administration of 400 µg of Se from yeasts/day for at least 3 years (Stratton et al., 2003). During the study of Reid et al. (2004), patients receiving 3200 µg Se-yeast/ day for an average period of almost 12 months complained more about garlic breath, brittle nails and hair, stomach upset, and dizziness than the subjects receiving daily 1600 µg of Se obtained from yeasts. Neither their blood chemistry nor hematology results were abnormal (Reid et al., 2004). Thirty-two patients out of 500 who were given placebo or 100, 200, or 300 µg/day of Se as Se-enriched yeast, reported adverse effects. Twenty-five of them were taking yeast biomass, while the other seven were the placebo-treated patients (Rayman, 2002). In a comparable clinical investigation, Larsen et al. (2004) did not notice any toxic effects after a long-term intake of yeast biomass enriched with 300 µg/day of Se. No diabetogenic effect was demonstrated by Rayman et al. (2012) after 6-month treatment of the elderly volunteers with selenized yeast (100, 200, or 300 µg/day). A significantly increased risk of type 2 diabetes was detected in the Nutritional Prevention of Cancer trial, in which the subjects were supplemented over an average period of 7.7 years with 200 µg of Se in the form of Se-enriched yeasts (Stranges et al., 2007). Although it is generally supposed that consumption of the selenized yeasts by lactating women may lead to an elevated level of Se in milk and excessive Se intake by infants, a maternal supplementation with 200 µg of Se/ day from the yeast biomass turned out to be safe for their children (Trafikowska et al., 1998).

Nutritional Yeast Biomass: Characterization and Application 245 Table 9.2: Clinical studies on the activity of the Se-enriched yeasts (S. cerevisiae). Duration of Treatments

Authors (Year)

Subjects

Algotar et al. (2013)

Men at high risk for 5 Years prostate cancer (n = 699)

Blot et al. (1993)

Adults from four Linxian communes, China (n = 29,584)

Clark et al. (1996)

Participants with history Mean (SD) of 4.5 of basal cell or squamous (2.8) years cell carcinomas of the skin (n = 1,312) Healthy adult males 9 Months (n = 52)

El-Bayoumy et al. (2002)

Peretz et al. (1991)

5 Years

Elderly institutionalized participants (n = 22) Rayman (2002); Elderly volunteers Rayman et al. (n = 501) (2006, 2008, 2011, 2012); Men with biopsy-proven Reid et al. (2004) prostate cancer (n = 24)

6 Months

Stranges et al. (2007)

Participants without type 2 diabetes at baseline (n = 1,312) Men considered at high risk for prostate cancer (n = 514) Lactating mothers (n = 67)

Average period of 7.7 years

Participants positive for HBsAg (n = 226)

4 Years

Stratton et al. (2003)

Trafikowska et al. (1998)

Yu et al. (1997)

Minimum 6 months 12 Months

Up to 5 years

3 Months

Products Se-enriched yeasts 200 µg/ day of Se (n = 234), or 400 µg/ day of Se (n = 233), or placebo (n = 232) β-Carotene, vitamin E, and Se-enriched yeasts; retinol and zinc; riboflavin and niacin; or vitamin C and molybdenum Se-enriched yeasts, 200 µg/day of Se, or placebo

Se-enriched yeasts, 247 µg/ day of Se (n = 26), or placebo (n = 26) Se-enriched yeasts, 100 µg/day of Se, or placebo Se-enriched yeasts, 100, 200, or 300 µg/day of Se or placebo Se-enriched yeasts, 1600 µg/ day of Se (n = 8), or 3200 µg/ day of Se (n = 16) Se-enriched yeasts, 247 µg/ day of Se (n = 653), or placebo (n = 659) Se-enriched yeasts, 200 or 400 µg/day of Se or placebo Se-enriched yeasts, 200 µg/ day of Se, or sodium selenite, 200 µg/day of Se Se-enriched yeasts, 200 µg/ day of Se (n = 113), or placebo (n = 113)

HBsAg, Hepatitis B surface antigen.

In the randomized clinical trial by Clark et al. (1996), daily consumption of the selenized S. cerevisiae yeasts (i.e., 200 µg/day of Se) reduced risk of several cancers (lung, colon, and prostate cancer), though it did not protect against development of basal or squamous cell carcinomas of the skin. In hepatitis B surface antigen-positive patients, continuous intake of Se in the form of selenized yeast protected subjects from the development of primary liver cancer. However, this effect was not maintained after cessation of treatment (Yu et al., 1997). Changes suggesting a decrease in oxidative stress along with a protective effect against prostate cancer, that is, reduction

246 Chapter 9 in prostate-specific antigen, increase in blood glutathione, and decrease in bound: free glutathione ratio after supplementation with Se-yeast, were also found by El-Bayoumy et al. (2002). However, the authors did not detect any changes in testosterone metabolism due to Se intake, and the decrease in prostate-specific antigen relatively quickly disappeared after discontinuation of the treatment. No protective effect on the incidence of prostate cancer in men at risk was demonstrated in studies by Algotar et al. (2013). Stomach-cancer mortality rate was diminished after consumption of Se-yeast, β-carotene, and vitamin E in one of Chinese clinical trials (Blot et al., 1993). Besides, the investigators recorded a link between low baseline serum level of Se and death from oesophageal squamous cell carcinoma or gastric cardia cancer (Wei et al., 2004). Based on the results obtained in the preclinical studies, selenized yeasts reduced the yield of breast cancer (Ip et al., 2000). Beneficial effects of Se-enriched yeast were also observed in studies performed by Peretz et al. (1992) and Aaseth et al. (1998) on subjects with rheumatoid arthritis. Chronic supplementation reduced joint involvement and alleviated pain with morning stiffness. Six-month therapy with Seenriched yeasts (100 µg of Se/day) significantly improved immune response to mitogen challenge in elderly people (Peretz et al., 1991). In another study conducted on elderly volunteers, consumption of the selenized yeast brought modestly beneficial effects on plasma lipid levels (Rayman et al., 2011), but it did not improve mood or quality of life (Rayman et al., 2006). It did not affect thyroid function, either (Rayman et al., 2008).

2.3 Cr-Enriched Saccharomyces cerevisiae Yeasts An adequate intake of chromium (Cr) for adults established by US Food and Nutrition Board (2001) is 25–35 µg/day. According to the literature data (Balk et al., 2007), Cr supplementation is not necessary in healthy, not Cr-deficient people. However, Cr preparations are widely used by diabetics, obese patients, and subjects with an increased appetite, since Cr is believed to be essential for normal glucose and lipid homeostasis. Severe Cr deficiency may lead to glucose intolerance, elevated circulating insulin, fasting hyperglycemia, reversible insulin resistance, and even diabetes. The results of clinical trials focused on efficacy of dietary supplements based on Cr-enriched brewer’s yeasts in regulation of carbohydrate and lipid metabolism are divergent (Table 9.3). Yin and Phung (2015), after conducting a systemic literature search of different medical databases, concluded that Cr supplementation with brewer’s yeasts might provide marginal benefits in lowering fasting plasma glucose in patients with type 2 diabetes mellitus; however, it did not affect the level of glycated hemoglobin. On the other hand, several preclinical and clinical studies demonstrated that supplementation of Cr-yeasts was beneficial to both diabetic humans and diabetic animals in relation to carbohydrate and lipid metabolism (Bahijiri et al., 2000; Balk et al., 2007; Lai, 2008; Lai et al., 2006). A significant decrease in the means of glucose (fasting and 2-h postglucose load), fructosamine, and triglycerides, as well as increased the means of HDL-cholesterol, and serum and urinary Cr values were

Nutritional Yeast Biomass: Characterization and Application 247 Table 9.3: Clinical studies on the activity of the Cr-enriched yeasts (S. cerevisiae). Duration of Treatments

Authors (Year)

Subjects

Bahijiri et al. (2000)

Type 2 diabetic participants (n = 78)

Hosseinzadeh et al. (2013) Hunt et al. (1985)

Type 2 diabetic 12 Weeks participants (n = 84) Diabetic and nondiabetic 90 Days participants (n = 78) Type 2 diabetic 8 Weeks participants (n = 20)

Król et al. (2011)

Lai (2008)

Type 2 diabetic participants (n = 30)

8 Weeks

6 Months

Offenbacher and Pi-Sunyer (1980)

Diabetic and nondiabetic 8 Weeks elderly participants (n = 24) Offenbacher et al. (1985) Healthy elderly volunteers 10 Weeks (n = 23) Rabinowitz et al. (1983)

Outpatient diabetic men (n = 43)

4 Months

Racek et al. (2006)

Type 2 diabetic participants (n = 36) Type 2 diabetic participants (n = 40)

12 Weeks

Sharma et al. (2011)

Uusitupa et al. (1983)

3 Months

Elderly participants 6 Months with persistent impaired glucose tolerance (n = 26)

Products Cr-enriched yeasts, 23.3 µg/day of Cr (n = 78), or chromium chloride, 200 µg/day of Cr (n = 78) Brewer’s yeasts, 1800 mg/day, or placebo Cr-enriched yeast, 68 µg/day of Cr, or placebo Cr-enriched brewer’s yeasts, 500 µg/ day of Cr (n = 20), or placebo (n = 20) Cr-enriched yeasts, 1000 µg/day of Cr (n = 10), Cr-enriched yeasts, 1000 µg/day of Cr + 1000 mg of vitamin C + 800 IU of vitamin E (n = 10), or placebo (n = 10) Cr-enriched brewer’s yeasts, 9 g/day, or placebo Cr-enriched brewer’s yeasts, 5 g/day, or chromium trichloride, 200 µg/ day of Cr Brewer’s yeast with Cr as GTF (n = 43), or brewer’s yeast extract without GTF (n = 43), or chromium trichloride (n = 43) Cr-enriched yeasts, 400 µg/day of Cr (n = 19), or placebo (n = 17) Cr-enriched brewer’s yeasts, 378 µg/ day of Cr (n = 20), or placebo (n = 20) Cr-enriched yeasts, 160 µg/day of Cr, or placebo

GTF, Glucose tolerance factor.

noted in a clinical trial conducted by Bahijiri et al. (2000). Cr-rich brewer’s yeasts improved glucose tolerance, insulin sensitivity, and total lipids in some elderly diabetic and nondiabetic people (Offenbacher and Pi-Sunyer, 1980). However, these parameters were not affected in healthy, well-nourished elderly volunteers taking part in another study performed by the same team (Offenbacher et al., 1985). Six-month treatment with Cr-yeasts alone or with vitamin C and E improved blood glucose, hemoglobin A1c, and insulin resistance index, as well as diminished oxidative stress. Sharma et al. (2011) demonstrated beneficial effect of similar supplementation for 3 months in terms of fasting blood glucose and hemoglobin

248 Chapter 9 A1c measurement. In a double-blind placebo-controlled study, 12-week consumption of Cr-yeasts (400 µg of Cr/day) significantly reduced serum glucose and insulin levels without influencing lipid indices (Racek et al., 2006). On the other hand, Król et al. (2011) did not observe considerable changes in blood glucose or lipid parameters in subjects with type 2 diabetes mellitus after 8-week intake of the same yeast preparation containing a higher dose of Cr (i.e., 500 µg/day). The participants presented only weak improvement in β cell function index. Consumption of Cr-enriched yeasts does not affect their body mass, blood parameters, or mineral status, except for Cr serum and hair levels. Similarly, Hunt et al. (1985) did not record any significant changes of the blood parameters in diabetic participants who received preparation of brewer’s yeast for 90 days. The same observations were made by Uusitupa et al. (1983), who noticed that neither glucose tolerance nor serum lipid levels were improved in elderly participants with stable impaired glucose tolerance. Based on the outcomes of Balk et al. (2007), Cr supplementation (including Cr formulas manufactured from brewer’s yeasts) had no significant effect on lipid or glucose metabolism in people without diabetes, but it improved glycemia in diabetic patients. Although brewer’s yeasts supplementation did not alter fasting plasma glucose and lipids levels or change the glucose response to meal or tolbutamide in the study by Rabinowitz et al. (1983), the preparation containing Cr as glucose tolerance factor induced a significant increase in postprandial insulin in the ketosis-resistant subgroups. Moreover, consumption of brewer’s yeast by diabetic patients may reduce systolic and diastolic blood pressures (Hosseinzadeh et al., 2013).

2.4 Safety Issues Concerning Saccharomyces cerevisiae Preparations Consumption of dietary supplements manufactured from yeast biomass cannot be recommended to the whole population, as there are people sensitive to yeast proteins. Though serious allergies are reported very rarely, patients who suffer from yeast infection or atopic dermatitis, as well as people exposed to yeasts by inhalations, belong to the increased risk group (EFSA, 2008). The results of the experiments carried out by Baldo and Baker (1988) demonstrated allergenic cross-reactivity between bakers’ yeasts, bakers’ yeasts enolase, and Candida albicans. Though S. cerevisiae is not considered as a pathogenic microorganism, there are some reports of opportunistic infections attributed to the pretreatment with preparations containing biomass of this yeast (for review, see Hempel et al., 2011). Several cases were detected in an intensive care unit (Lherm et al., 2002; Muñoz et al., 2005). Both baker’s yeasts and S. cerevisiae var. boulardii were isolated from patients with symptoms of infection (de Llanos et al., 2006b; Hennequin et al., 2000; Lherm et al., 2002; Riquelme et al., 2003). Worryingly, Piechno et al. (2007) described a case of fungemia in a cancer patient due to S. boulardii (cerevisiae) resistance to amphotericin B and possibly fluconazole. Infections related to S. cerevisiae ranged from not-severe cutaneous ones to systemic infections, depending on the general condition of a patient (de Llanos et al., 2011; Enache-Angoulvant and Hennequin, 2005; Muñoz et al., 2005). Although most of the

Nutritional Yeast Biomass: Characterization and Application 249 afflicted patients were immunocompromised, suffered from the underlying diseases and/or their normal bacterial flora was suppressed due to antibiotic therapy (Hempel et al., 2011), fungemia cases have been also described in healthy hosts (Debelian et al., 1997; Fung et al., 1996; Smith et al., 2002). Among the other adverse effects associated with use of S. cerevisiae products, gastrointestinal symptoms (i.e., abdominal distension or pain, constipation, meteorism, food protein-induced enterocolitis syndrome, emesis, diarrhea) are most common. Fever has been reported as well (for review, see Hempel et al., 2011). de Llanos et al. (2006a) found that both commercial baker’s yeasts and S. cerevisiae var. boulardii isolated from a product available in the pharmaceutical market presented similar pathogenicity-associated traits to clinical strains. The preclinical studies on animal model of systemic infection confirmed their remarkable dissemination capacity (de Llanos et al., 2006b, 2011). After inoculation into blood, some Saccharomyces isolates from dietary products were able to colonize, spread to different murine organs, trigger a systemic infection, and even cause death (Llopis et al., 2014). Because of these cases, several authors have started to regard S. cerevisiae as an emerging opportunistic pathogen (de Llanos et al., 2006a; Herbrecht and Nivoix, 2005; Pérez-Torrado and Querol, 2016). A special attention should be paid in the case of elderly people, children, and patients suffering from immunosuppression associated with HIV/AIDS, treatment with immunosuppressive drugs, or suffering from other conditions that compromise immune response. On the other hand, it should be underlined that not all S. cerevisiae strains are capable of developing infection in the favorable conditions (Byron et al., 1995; Clemons et al., 1994; de Llanos et al., 2011). Llopis et al. (2014) suggested that dietary supplements manufactured from Saccharomyces hybrids (i.e., a combination of different Saccharomyces species) could be safer than S. cerevisiae strains alone, as the hybrid nature most probably does not facilitate colonization, invasion, and dissemination during a systemic infection. Some safety concerns regarding consumption of the yeast products by pregnant women, children, elderly people, and immunocompromised patients were also raised by Gottschalk et al. (2016). The authors revealed that due to their interesting feature of being capable of adsorbing and degrading mycotoxins (Moslehi-Jenabian et al., 2010), the brewer’s yeasts that are commonly used in the dietary supplements are widely contaminated with ochratoxin A. Though the level of contamination was not assessed as high, it brings an additional load of ochratoxin A to the total intake of mycotoxins from food, and in the case of the sensitive population it should not be ignored (Gottschalk et al., 2016).

3 Saccharomyces boulardii as a Probiotic Yeast The best-known probiotic strain of yeast is S. cerevisiae var. boulardii which has been effective in double-blind clinical studies (McFarland, 2010; Szajewska et al., 2007; Szajewska and Kołodziej, 2015). Probiotics are defined as live, nonpathogenic microbial

250 Chapter 9 supplements that exert a positive influence on the health or physiology of the host (D’Souza et al., 2002; Marteau et al., 2001). S. boulardii is manufactured as freeze-dried and living biomass and used as a biotherapeutic agent for prevention and treatment of diarrhea induced by enteral and parenteral nutrition or by Clostridium difficile infection or several gastrointestinal diseases (Hennequin et al., 2000; Kelesidis and Pothoulakis, 2012). This viable yeast is used in intensive care adult patients at a dose of 1–2 g/day, for preventing diarrhea associated with antibiotics or enteral feeding (Lherm et al., 2002). S. boulardii resists the effects of gastric acid and bile and survives at 37°C. These features give it a unique advantage of being one of the few types of yeast that thrive best at human body temperatures (Graff et al., 2008; McFarland, 2010). S. boulardii mediates responses resembling the protective effects of the normal healthy gut flora (Kelesidis and Pothoulakis, 2012). The clinical activity of S. boulardii is especially relevant to antibiotic-associated diarrhea (AAD) and recurrent C. difficile intestinal infections. In Table 9.4, clinical trials with outcomes clearly demonstrating specific probiotic properties of S. boulardii are presented. That data has opened the door to a new therapeutic use of this yeast as an “immunobiotic” (Czerucka et al., 2007). The multidirectional mechanisms of S. boulardii action generally depend on antimicrobial and antitoxin activity, such as: (1) inhibition of growth of bacteria or parasites, (2) reduction of gut translocation of pathogens, (3) neutralization of bacterial virulence factors, (4) suppression of host cell adherence, which interferes with bacterial colonization, (5) inhibition of toxin receptor binding sites, (6) stimulation of antibody production against C. difficile toxin A, (7) direct proteolysis of infective toxins (e.g., production of serine protease, which cleaves C. difficile toxin A and B and diminishes the ability of toxins A and B to bind to the human colonic brush border membrane receptor), (8) secretion of enzymatic proteins against pathogens (e.g., 63 kDa phosphatase, which destroys the endotoxin of pathogenic Escherichia coli and a 120 kDa protein that reduces the effects of cholera toxin), (9) stimulation of effects on the intestinal mucosa, such as trophic effects on the brush border enzymes and immunostimulatory effects (Czerucka et al., 2007; Herbrecht and Nivoix, 2005; Vandenplas et al., 2009). Moreover, there is evidence that S. boulardii is beneficial for the treatment of acute gastroenteritis and prevention of antibiotic-associated diarrhea in pediatric populations (Szajewska and Kołodziej, 2015; Vandenplas et al., 2009). However, more data is needed to confirm other indications, such as travelers’ diarrhea, Helicobacter pylori eradication, and inflammatory bowel disease (Vandenplas et al., 2009). S. boulardii was found to be significantly efficacious and safe in 84% of the treatment arms. A metaanalysis of randomized controlled trials (RCTs) demonstrated that this yeast is efficient in preventing AAD both in children and adults treated with antibiotics for any reason. The daily dose of S. boulardii biomass ranged from 50 to 1000 mg. There was high variability in the type of antibiotics administered, which were given as a single medicinal product or in combinations. In this

Nutritional Yeast Biomass: Characterization and Application 251 Table 9.4: Randomized, controlled trials for various disease conditions using yeast probiotics during the last 25 years. Daily Dose Duration of (mg/day) Treatments

Indications

Treatment Groups

Prevention of AAD (antibiotics for infections) in children Prevention of AAD (antibiotics as a part of H. pylori eradication therapy) in children Prevention of AAD in adults

S. boulardii versus placebo

250–500


250


226–1000

Acute adult disease conditions associated with H. pylori (as a part of eradication therapy) Acute adult diarrhea

50–1000 S. boulardii versus placebo or versus Lactobacillus rhamnosus GG versus mix (L. acidophilus and Bifidobacterium lactis S. boulardii versus placebo 300–750

Adult traveler’s diarrhea


250–1000

Adult traveler’s diarrhea

S. cerevisiae Hansen (CBS 5926) versus a standard medicinal product contains S. boulardii S. boulardii versus placebo S. boulardii versus placebo S. boulardii versus placebo S. boulardii + mesalamine (2 g/d) versus mesalamine alone (3 g/d) S. cerevisiae versus placebo S. boulardii + metro (2.25 g/day) versus placebo + metro S. boulardii versus placebo

600

Prevention of CDAD in children C. difficile disease in adults Crohn’s disease in adults Crohn’s disease in adults Irritable bowel syndrome in adults Giardiasis in adults

HIV-related diarrhea in adults

500

Authors (Year)

During antibiotic therapy or 2 weeks 2 Weeks–1 year

Casem (2013); Erdeve et al. (2004); Kotowska et al. (2005); Shan et al. (2013) Bin et al. (2015); Namkin et al. (2016); Zhao et al. (2014)

6–14 Days or during antibiotic therapy + 3–14 days

Bravo et al. (2008); Can et al. (2006); Cindoruk et al. (2007); Cremonini et al. (2002); Duman et al. (2005); Lewis et al. (1998); McFarland et al. (1995); Pozzoni et al. (2012) Cindoruk et al. (2007); Cremonini et al. (2002); Kyriakos et al. (2013); Zojaji et al. (2013)

2 Weeks

8–10 Days 5 Days before trip and mean 21 days trip 5 Days

Mansour-Ghanaei et al. (2003) Kollaritsch et al. (1993)

Bruns and Raedsch (1995)

1000

During antibiotic Kotowska et al. (2005); therapy Shan et al. (2013) 4 Weeks McFarland et al. (1994); Surawicz et al. (2000) 7–52 Weeks Bourreille et al. (2013); Plein and Hotz (1993) 6 Months Guslandi et al. (2000)

500

8 Weeks

500

10 Days + 4 weeks Besirbellioglu et al. (2006) follow-up time

3000

7 Days

1000 750–1000

AAD, Antibiotic-associated diarrhea; CDAD, C. difficile–associated diarrhea.

De Chamburn et al. (2015)

Saint-Marc et al. (1995)

252 Chapter 9 metaanalysis, in children subjected to antibiotic therapy, S. boulardii reduced the risk of diarrhea from 20.9% (observed for the placebo-treated or no treatment group) to 8.8% (6 RCTs). In turn, in adults, this risk was diminished from 17.4% to 8.2% (15 RCTs). Furthermore, in both children and adults, a considerable reduction in the risk of AAD was noted, regardless of the reason for which the antibiotics were used (Szajewska and Kołodziej, 2015). By contrast, Pozzoni et al. (2012) observed that S. boulardii was not effective in preventing the development of AAD in elderly hospitalized patients. Similarly, the results from a recent large, clinical, randomized, placebo-controlled, multicenter trial did not support the efficacy of S. boulardii in prevention of AAD and C. difficile–associated diarrhea (CDAD) in hospitalized adult patients (Ehrhardt et al., 2016). On the other hand, randomized trials with adult subjects support the use of this yeast probiotic for prevention of enteral nutrition-related diarrhea and reduction of H. pylori treatment-related symptoms. S. boulardii has promising effects in prevention of C. difficile disease recurrence, and treatment of irritable bowel syndrome, acute adult diarrhea, Crohn’s disease (CD), giardiasis, and human immunodeficiency virus-related diarrhea. However, further studies that will give more supporting evidence are recommended for these indications (McFarland, 2010). As for CD, S. boulardii does not appear to have any beneficial effects for patients with CD in remission after steroid or salicylate therapies (Bourreille et al., 2013). S. boulardii preparations are also used for other inflammatory disorders, acute gastroenteritis in children, chronic diarrhea in patients with AIDS, and diarrhea caused by Vibrio cholerae and several Enterobacteriaceae (Czerucka et al., 2007). The use of S. boulardii as a therapeutic probiotic has been proved as safe and well tolerated (Bourreille et al., 2013; McFarland, 2010; Szajewska and Kołodziej, 2015). However, some rare cases of S. boulardii fungemia have been reported, although they are essentially restricted to immunocompromised patients or associated with patients contaminated through a central venous catheter (Foligné et al., 2010; Herbrecht and Nivoix, 2005). The hypotheses proposed for explaining these cases of S. boulardii fungemia include: (1) intestinal translocation of S. boulardii administered at a high dosage in severely ill patients; (2) contamination of the central venous catheter, especially in patients not pretreated with S. boulardii; and (3) massive colonization of critically ill patients by the yeast, as reported for Candida species or S. cerevisiae (Lherm et al., 2002; Riquelme et al., 2003). Though several trials reported no adverse effects related to S. boulardii, consumption thereof is not without risk in specific patient groups, such as immunocompromised subjects or in patients with other life-threatening illnesses managed in the intensive care unit (Szajewska and Kołodziej, 2015; Whelan and Myers, 2010).

4 Yarrowia lipolytica as a Source of Bioactive Compounds Among nutritional yeasts, Yarrowia lipolytica, capable of producing important metabolites and having intense secretory activity, is one of the most extensively studied “nonconventional” yeasts (Coelho et al., 2010). This yeast species belongs to the Ascomycota

Nutritional Yeast Biomass: Characterization and Application 253 fungal phylum, likewise the well-known baker’s and brewer’s yeasts (Madzak, 2015). However, it is phylogenetically distant from S. cerevisiae and exhibits many metabolic differences: it is strictly aerobic yeast, which can use normal hydrocarbons and various fats as a carbon source, it secretes diverse hydrolytic enzymes (proteases, lipases, RNases), and its peroxisome is constitutive (Loira et al., 2012). At first, this yeast was noticed for its capacity to grow on n-paraffins and to produce high amounts of organic acids (Groenewald et al., 2014). It was also found in a large range of ecosystems, such as soils, marine waters, mycorrhizae (symbiotic associations of fungi with roots of plants), and in a variety of foods, particularly in fermented dairy products (e.g., many cheeses, butter, cream, margarine, yogurt) and meat products (e.g., salami, Spanish fermented sausages) (Groenewald et al., 2014; Madzak, 2015). Among others, Y. lipolytica is very important for aroma formation in Muenster, Parmesan, and Cheddar cheeses (Bourdichon et al., 2012; Ferreira and Viljoen, 2003). It fulfills specific criteria to be regarded as a costarter for cheese making and as a good candidate for a ripening agent in cheese (Ferreira and Viljoen, 2003; Groenewald et al., 2014). The natural occurrence of this yeast in food is an additional argument in favour of its safety. The occasional occurrence of opportunistic infections of Y. lipolytica in immunocompromised and catheterized patients does not differ from that of other microorganisms with a history of safe use, such as S. cerevisiae (Groenewald et al., 2014). The recent research suggests that Y. lipolytica should be considered as normal human flora, especially of the adult respiratory tract. Given its prevalence in distal lung tissue, rather than more proximal or bronchoalveolar sputum specimens, the yeast may be present as a distal airway saprophyte with little clinical significance (Irby et al., 2014). Y. lipolytica is considered as nonpathogenic and several processes based on this organism have been classified by FDA as GRAS (Coelho et al., 2010; Groenewald et al., 2014; Kamzolova et al., 2012). Y. lipolytica is known as oleaginous yeast. This means that it can store at least 20% of its dry biomass as lipids (Loira et al., 2012). Lipids produced by oleaginous microorganisms are called single cell oils (SCOs), as well as microbial lipids (Liu et al., 2015). Y. lipolytica can accumulate lipids intracellularly to ≥40% of its cell dry weight (Beopoulos et al., 2011). Lipids from this yeast represent a high level of unsaturated fatty acids (about 90%), of which over 50% is isoleic acid and about 27 and 10% are linoleic acid (LA, C18:2, w-6) and linolenic acid, respectively (Musiał et al., 2003). The metabolism of microbial lipids includes fatty acid synthesis and triacylglycerol production. Generally, SCOs play a significant role in the delivery of major polyunsaturated fatty acids (PUFAs), which are important in nutrition and human health. Naturally, fatty acids (C14 and/or C16) can be synthesized via a de novo pathway from hydrophilic substrates or an ex novo pathway directly from a medium containing hydrophobic substrates (Liu et al., 2015; Zhang et al., 2012). LA is the major PUFA synthesized by wild-type Y. lipolytica, but genetic engineering can be used to generate recombinant strains overproducing several

254 Chapter 9 important SCOs, for example, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA, C20:4, w-6), and conjugated LA, or γ-linolenic acid (GLA,C18:3, w-6) (Chuang et al., 2010; Groenewald et al., 2014; Zhang et al., 2012). In general, PUFAs, including GLA, ARA, EPA, and DHA, are classified as either w-3 or w-6 fatty acids (Liu et al., 2015). Many clinical and animal studies have demonstrated that w-3 and w-6 fatty acids play an important role in neural development, aging, and neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. Long-chain PUFAs (LCPUFA) are important for neuronal membrane integrity and function, and contribute to prevention of brain hypoperfusion. Cerebral perfusion can be compromised as a result of obesity, cerebrovascular disease, hypertension, or diabetes mellitus type 2. Perinatal LCPUFA supplementation demonstrated beneficial effects in neural development in humans and rodents resulting in improved cognition and sensorimotor integration (Janssen and Kiliaan, 2014). Generally, the major sources of w-6 and w-3 fatty acids can be obtained from various foods. Mammals, including humans, cannot synthesize LA or α-linolenic acid (ALA, C18:3, w-3). Therefore, these functional PUFAs should be obtained from the diet directly or synthesized indirectly in the human body via fatty acid synthesis reaction with appropriate w-3 and w-6 precursors as substrates. Nevertheless, with the growing population and the increasing consumption of edible oils, the production of SCOs is becoming more important. Due to the similar composition of yeast lipids to that of plant oils and fats, SCOs are viewed as alternative edible oils with potential applications (Liu et al., 2015). It is worth mentioning that, in 2010, DuPont submitted a GRAS Notice for its EPA-enriched oil derived from Y. lipolytica biomass to the FDA (Groenewald et al., 2014). Y. lipolytica biomass is also characterized by a high content (40%–50%) of SCP (Kamzolova et al., 2012; Michalik et al., 2014; Musiał et al., 2003). Yarrowia SCP contains of a high quantity of essential amino acids, apart from methionine (Juszczyk et al., 2013; Kamzolova et al., 2012; Michalik et al., 2014; Musiał et al., 2003). The amount of essential amino acids is in agreement with the FAO standards for fodder yeasts (Kamzolova et al., 2012; Musiał et al., 2003). In the yeast protein amino acid profile the contents of lysine, leucine, threonine, valine, and phenylalanine are even higher than these required by FAO/WHO for human diet (Table 9.5). Furthermore, yeast SCP is characterized by a high biological value of about 80% (Musiał et al., 2003). It is interesting that the protein content of Y. lipolytica is similar to that of S. cerevisiae biomass (Table 9.5). The low share of sulfur amino acids of Y. lipolytica and S. cerevisiae limits the nutritional value of SCP of these yeasts. The biological value of proteins does not differ between the two yeast species. Y. lipolytica is also a rich source of highly digestible ether extracts (over 57%) (Michalik et al., 2014). Yarrowia biomass is enriched with minerals, particularly chromium and selenium, as well as phosphorus and calcium (Musiał et al., 2005; Michalik et al., 2014). Y. lipolytica has the ability to assimilate minerals from the medium and incorporate them in its cell structures. It contains β-glucans, that is a natural component of its cell wall (Esteban et al., 1999).

Nutritional Yeast Biomass: Characterization and Application 255 Table 9.5: Comparison composition of S. cerevisiae and Yarrowia lipolytica dried biomass. Contents in Dried Biomass, Mean Biomass Compounds

S. cerevisiae

Protein (SCP)

g/100-g dried biomass 48

Exogenous amino acids Threonine Valine Isoleucine Leucine Phenylalanine Lysine Methionine Tryptophan Lipids C18:1 C18:2 C18:3n−3 C16:0 C16:1 C18:0 SFAs MUFAs PUFAs Dry matter

FAO Requirements of Daily Intake for Adults

Y. lipolytica

50-g proteina 46

mg/g protein 46 49 37 64 33 65 14 10

42 47 40 71 39 62 14 15 % of total fatty acids

29 9 0.4 18 32 5 28 62 10

56 27 6 6 2 1.5 8 58 34

mg/g proteinb 40 42 42 48 28 42 22 — ga

20

g/kg dried biomass 925

Ether extract

952 g/kg dried biomass

7 Ash


62 Minerals


ga

Phosphorus

10

5

0.7

Calcium

1

2

0.8

Heavy metals

mg/kg dried biomass

mg/kgc

Cadmium

0.89

0.38

1.0

Mercury

0.04

0.02

0.1

Arsenic

0.28

0.04

—

MUFAs, Monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; SCP, single-cell protein; SFAs, saturated fatty acids. a Regulation (EU) No 1169/2011. b Adedayo et al. (2011). c Regulation (EC) No 1881/2006. Source: Michalik, B., Biel, W., Lubowicki, R., Jacyno, E., 2014. Chemical composition and biological value of proteins of the yeast Yarrowia lipolytica growing on industrial glycerol. Can. J. Anim. Sci. 94, 99–104.

256 Chapter 9 Furthermore, when cultivated under nutrient-limited conditions, Y. lipolyticais able to produce several organic acids, for example, citric acid from diverse carbon sources including sugars, plant oils, n-alkanes, ethanol, starch hydrolysates, and raw glycerol (Coelho et al., 2010; Liu et al., 2015). Y. lipolytica can be made to synthesize some practically important compounds, for example, α-ketoglutaric acid, succinic acid, diethyl succinate, and an unusually large amount of γ-aminobutyric acid under the conditions of thiamine deficiency (Kamzolova et al., 2012). Y. lipolytica has been used or is considered for multiple industrial applications as a high-quality protein and a source of other nutrient compounds source for livestock feeding (Groenewald et al., 2014) and human diet. Some valuable metabolites of Y. lipolytica, such as α-ketoglutaric acid, succinic acid, diethyl succinate, and biomass enriched with protein and essential amino acids, can be used in food and medical industries (Kamzolova et al., 2012; Madzak and Beckerich, 2013). The unique amino acid composition of the yeast makes it possible to use its biomass as a component of parenteral nutrition mixtures and a basis for neuroleptics (Kamzolova et al., 2012). Y. lipolytica grows on a variety of hydrophobic substrates, especially different fractions of petroleum, as well as streams from different industries (Groenewald et al., 2014). Such a large amount of biodiesel waste may be a problem; therefore, new methods are needed to use it in the production of more valued products. One of the most important solutions is to use glycerol as a source of carbon and energy in microbial production processes. Y. lipolytica and glycerol have been successfully applied to biotechnology for the production of citric acid, hydrogen, ethanol, dihydroxyacetone, erythritol, mannitol, and fodder yeast (Loira et al., 2012; Rywin´ska et al., 2010; Stasiak-Róz˙an´ska et al., 2010; Tomaszewska et al., 2012). It is easy to modify genetically, and presents many opportunities for metabolic engineering (Loira et al., 2012). In this view, Y. lipolytica has also been explored as a production host for therapeutic and industrial proteins and enzymes. For example, the Y. lipolytica LIP2 lipase may serve as a therapeutic agent for the treatment of exocrine pancreatic insufficiency (Turki et al., 2010). Another direction in the utilization of biofuel production waste is the biosynthesis of yeast protein biomass including essential amino acids and using it as a feed additive. This approach could become helpful due to the recent stricter regulations on the use and cultivation of genetically modified organisms in the European Union, in particular genetically modified soybean (Michalik et al., 2014). Very interesting is also the ability of Y. lipolytica strains to grow on olive mill wastewater–based medium and produce high-value compounds (Coelho et al., 2010). Crucial in determining the potential of Y. lipolytica for bioremediation might be its capacity to produce biosurfactants that may be applied to enhance oil recovery, as well as in crude oil drilling, food processing, and cosmetic formulation (Amaral et al., 2006; Groenewald et al., 2014; Trindade et al., 2008). Y. lipolytica can also be used for the production of aroma

Nutritional Yeast Biomass: Characterization and Application 257 chemicals, especially of γ-decalactone, which has a characteristic peach flavor (BieleckaFlorjanczyk et al., 2012; Garcia et al., 2014; Groenewald et al., 2014). Furthermore, Y. lipolytica is used as a production host for carotenoids, a class of natural coloring and stabilizing agents for food and feed. A GRAS self-affirmation has been prepared for βcarotene produced with Y. lipolytica (Groenewald et al., 2014). However, it is important that, in general, the characteristics of various Y. lipolytica strains isolated from different environments are strain dependent (Liu et al., 2015). Y. lipolytica is a “safe-to-use” organism whose dried biomass may be used as a rich source of both high-quality proteins (and amino acids) with minerals and polyunsaturated fatty acids (especially linoleic acid) for food and feed (Groenewald et al., 2014; Jach et al., 2015). Y. lipolytica strain HY4 has been considered as a potential probiotic, which may assimilate cholesterol in the human intestine. It assimilates about 40% of cholesterol after 72 h of incubation. Y. lipolytica strain HY4 has a high capability of adhesion to HT-29 cells. This strain survived at pH 3.0 and 5.0 after 5 days of incubation. It showed good bile resistance, even at a concentration of 0.5%. Survival at low pH and in bile is an important selection criterion that probiotic microorganism can pass along the human gastric environment (Chen et al., 2010; Nayak, 2011).

5 Nutritional Benefits of Other Yeast Strains It is known that yeast biomass and yeast-based products are typically rich in proteins and several important compounds, such as vitamins and minerals. Besides the yeast strains described earlier, scientist search for other new yeasts with similar features that could be widely used. Other yeasts are characterized by a broad range of natural habitats; they are commonly found in soil and salt water, as well as on plant leaves and flowers (Amata, 2012). The biomass of other yeasts, such as Candida spp., Rhodosporidium paludigenum, Phaffia rhodozyma, and Debaryomyces hansenii are beneficial to animal growth and performance when added to animal feed (Akiba et al., 2001; Bjerkeng et al., 2007; Nanjundaswamy and Vadlani, 2015; Sajeevan et al., 2006; Sarlin and Philip, 2011; Yang et al., 2010). Several other yeasts, such as Candida spp., Kluyveromyces, Hansenula, Pichia, Torulopsis, and Rhizopus oligosporus, as well as the typical oily yeast genera, including Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces produce enriched protein biomass (Adedayo et al., 2011; Amata, 2012; Babu et al., 2014; Kurbanoglu, 2001; Nasseri et al., 2011; Suman et al., 2015; Valentino et al., 2015; Yunus et al., 2015). Among them, the most popular are Candida yeast species, such as C. parapsilosis, C. guilliermondi, C. tropicalis, C. utilis (Torula yeast), C. novellas, and C. intermedia used to produce SCP (Adedayo et al., 2011; Kurbanoglu, 2001; Nasseri et al., 2011; Valentino et al., 2015; Yunus et al., 2015). Candida spp. contain about 30 and 33% of crude protein in their biomass using brewery (distillery sludge) or food production waste (rice bran) as a substrate, respectively

258 Chapter 9 (Valentino et al., 2015; Yunus et al., 2015). However, Candida spp. growing in n-alkans can produce 65% of crude protein. In turn, C. utilis utilizing ethanol and sulfite waste liquor contains 50%–55% SCP in biomass (Adedayo et al., 2011; Kurbanoglu, 2001). Its dried biomass also contains essential amino acids: threonine 34, cysteine 24, valine 40.5, methionine 15.5, isoleucine 32, leucine 44, phenylalanine 30, and lysine 76 mg/g of protein, 5.4% of lipids, and 9.7% ash (Kurbanoglu, 2001). Moreover, Kluyveromyces marxianus biomass contains 48% of crude protein and about 0.5% of fats (Babu et al., 2014). These amounts of exogenous amino acids are sufficient to supplement the human diet. Biomasses of Candida spp., Kluyveromyces, or Pichia spp. are also rich sources of organic forms of minerals, such as selenium, chromium, iron, or zinc (Paš et al., 2007; Roepcke et al., 2011). Additionally, yeast cell walls contain polysaccharides, such as β-d-glucans and α-d-mannans [mannan oligosaccharide (MOS)], which are beneficial to animal health (Nanjundaswamy and Vadlani, 2015). Several yeast strains, such as P. rhodozyma and Sporobolomyces roseus also produce carotenoids in their biomass, which are additives in broiler chicken, poultry, and aquaculture feeds (Akiba et al., 2001; Ananda and Vadlani, 2010, 2011; Johnson et al., 2010). According to the FDA, the dried and killed P. rhodozyma biomass is permitted as a salmonid feed colorant to provide up to 80 mg/kg of astaxanthin in the finished feed (Nanjundaswamy and Vadlani, 2015). Besides carotenoids, monocultures and mixed cultures of fermenting red yeasts P. rhodozyma and S. roseus cultivated on corn whole stillage are also rich in crude fat and PUFAs (up to 81%) and contain 77% of fiber (Ananda and Vadlani, 2010). However, high levels of carotenoids in S. roseus-fermented soy flour and P. rhodozyma–fermented rice bran are accompanied by reduced fiber, protein, and amino acids and respective enhancement or reduction of crude fat, almost complete elimination of the antinutritional factor trypsin inhibitor in soy flour, and enrichment of health-promoting yeast cell wall polysaccharides (Nanjundaswamy and Vadlani, 2015). Yeasts may be a new source of glucosamine. Glucosamine is a structural component of cartilage. It is often used as a nutraceutical for horses, pet animals, and humans to relieve osteoarthritic conditions although the health benefits largely remain inconclusive (Igarashi et al., 2011; Nanjundaswamy and Vadlani, 2015). The carotenoid-rich red yeasts P. rhodozyma and S. roseus fermenting soy flour and rice bran contain 0.3%–4.6% and 0.13%–0.42% glucosamine, respectively, and in both substrates S. roseus yielded the highest glucosamine levels (Nanjundaswamy and Vadlani, 2015). Other yeasts can use several both conventional and unconventional carbon sources such as nalkanes and waste to produce biomass and metabolites. For example, the following substrates can be utilised: glucose by C. tropicalis and C. utilis; maltose by C. tropicalis; lactose by C. intermedia, unconventional materials, such as methanol (methylotrophic yeasts) by several Candida species, Hansenula polimorpha, Pichia pastoris, and Torulopsis sonorensis;

Nutritional Yeast Biomass: Characterization and Application 259 ethanol by Amoco torula; n-alkanes by C. novellas; and different industrial wastes, such as distillery yeast sludge by C. guillermondii and C. parapsilosis or rice bran by R. oligosporus (Amata, 2012; Nasseri et al., 2011; Valentino et al., 2015). Some strains of other yeasts have been considered as probiotics. Several strains of Pichia, namely P. fermentans BY5, P. kudriavzevii BY10 and BY15, and P. guilliermondii BY31 may serve as potential probiotics to assimilate cholesterol in the human intestine. They assimilate 40%–45% of cholesterol after 72 h incubation. These strains grew in the presence of 0.3% bile and survived at pH 3.0 and 5.0. However, at pH 1.5 and 2.0, only P. kudriavzevii BY10 displayed viability. These potential probiotic strains can adhere to HT-29 cells very well (Chen et al., 2010). Obtaining biomasses from other yeasts is not expensive. They are a rich source of nutritional compounds for feed and, if their safety is proved, they can be used for human diet. The natural occurrence of some other yeast in food may be an additional argument in favour of its safety. For example, in addition to Y. lipolytica, Debaryomyces hansenii is also very important for aroma formation in Muenster, Parmesan, and Cheddar cheeses (Bourdichon et al., 2012; Ferreira and Viljoen, 2003). D. hansenii fulfills a number of criteria to be regarded as a costarter with Y. lipolytica for cheese-making. D. hansenii has proteolytic and lipolytic activity, as well as compatibility and stimulating action with lactic acid starter cultures when coinoculated, enhancing development of flavor during cheese maturation (Ferreira and Viljoen, 2003). However, it is crucial that consumption of other yeasts may not be without risk in specific patient groups, such as immunocompromised subjects or catheterized patients as is the case of microorganisms with a history of a safe use, such as S. cerevisiae, S. boulardii, or unconventional Y. lipolytica.

6 Conclusions In conclusion, yeast biomasses obtained from S. cerevisiae and Y. lipolytica and other yeasts show very attractive features as a nutrient supplement and additive for both humans and animals. They are a rich source of essential amino acids, proteins, and several bioavailable dietary minerals. Yeast biomass contains sufficient quantities of phenylalanine, valine, threonine, tryptophan, leucine, isoleucine, lysine, and histidine, but insufficient amounts of methionine. It also has a good balance of amino acids, especially indispensable amino acids. Nutritional yeast biomass can be used as food or dietary supplements, particularly recommended for a vegan or vegetarian diet and for people who avoid eating meat, as well as for young people during maturation. Due to the high amount of proteins (and essential amino acids), they can also be used as an aid for people who build muscle mass or convalescents, as well as in countries where meat availability is a problem. In turn, the multiple prophylactic and therapeutic effects of S. boulardii in inflammatory gastrointestinal diseases underline the efficacy of this probiotic yeast in enteric diseases.

260 Chapter 9

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PAR T S ection 4

Health, Disease, and Therapy

10. Effect of Diet on Gut Microbiota as an Etiological Factor in Autism Spectrum Disorder 273 11. Dietary Fibers: A Way to a Healthy Microbiome 299 12. Effects of the Gut Microbiota on Autism Spectrum Disorder 347 13. Diet, Microbiome, and Neuropsychiatric Disorders 369

271


CHAPTE R 10

Effect of Diet on Gut Microbiota as an Etiological Factor in Autism Spectrum Disorder Afaf El-Ansary*,**, Hussain Al Dera†,‡, Rawan Aldahash† *King Saud University, Riyadh, Saudi Arabia; **Autism Research and Treatment Center, Riyadh, Saudi Arabia; †King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; ‡ King Abdullah International Medical Research Center, Riyadh, Saudi Arabia

1 Introduction It is well accepted that neurodevelopmental disorders, such as intellectual disability, attentiondeficit/hyperactivity disorder (ADHD), and autism are due to the impairments of the growth and function of the central nervous system. These alterations are clinically presented as disabilities in regards to communication, learning, social interaction, movement, or behavior. It is well known that the developing brain is highly vulnerable to damage from environmental toxins, such as lead (Jusko et al., 2008; Schnaas et al., 2006), methyl mercury (Harada et al., 1999), and pesticides (Jacobson and Jacobson, 2003). It should be pointed out that fetal development is affected by the intrauterine environment and any disruptions in that environment may eventually lead to various learning, behavioral, and neurological disorders in childhood, as well as complex diseases later in life (Schuurmans and Kurrasch, 2013). Another factor that affects brain development and behavior is the gut microbiota, especially during microbial colonization of a newborn. Studies of the microbiota–gut–brain axis could provide a deeper understanding of the relationship between intestinal bacteria and their hosts, which could help suggest potential therapeutic strategies through affecting the composition of gut microbiota. Infants are completely sterile at birth, but being in contact with a huge amount of microbial communities through the fecal, vaginal, and skin microbiota of the mother usually leads to remarkable changes in gut flora. The composition of gut flora is usually affected with age, mode of delivery (vaginal vs. Cesarean section), mode of feeding (breast vs. bottle), state of immune maturation, and environmental factors. The stability of gut microbiota is attained


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274 Chapter 10 between 6 and 36 months of life, and during this period the composition of gut microbiota is highly sensitive to environmental stimuli (Round et al., 2010; Scaldaferri et al., 2012). The gut microbiota play a critical role in the maturation of the immune system: it initiates innate immunity in the early years of life, which helps in the maturation of the acquired immunity through stimulation of local and systemic immune responses (Nell et al., 2010). Moreover, it helps in the synthesis and metabolism of certain nutrients, hormones, vitamins, and clearance of drugs and toxins. Gut microbial products can impact our health either positively or negatively. These products are taken up by the gastrointestinal tissues, potentially reach circulation and other tissues, and are finally excreted in urine or breath. Beneficial bacteria, such as Bifidobacterium can produce many vitamins, such as K, B12, biotin, folate, and thiamine (Nicholson et al., 2012). Secondary bile acids, important components of lipid transport, are usually synthesized by many bacteria species, such as Lactobacillus, Bifidobacterium, and Bacteroides (Nicholson et al., 2012). On the other hand, lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria that can induce tissue inflammation (Trent et al., 2006). Moreover, enteropathogenic bacterial species, such as Escherichia coli can produce toxins. Under normal healthy conditions, nonpathogenic commensal bacteria with similar metabolic activities can compete and eliminate the overgrowth of the pathogenic species (Kamada et al., 2012). Bacteria, such as Bifidobacterium can also help prevent pathogenic infection through the production of acetate (Fukuda et al., 2011). There is a great amount of evidence that the intestinal microbiota influences the brain and greatly affect behaviors, especially those that are relevant to anxiety (Kang et al., 2013; O’Mahony et al., 2009). Based on this, recent studies suggest that manipulation of the gut microbiota with specific probiotics or with antibiotics (Relman, 2012; Sommer and Dantas, 2011) can influence depression-like behaviors (Moloney et al., 2014; O’Mahony et al., 2015). This can highlight or point out the importance of the gut–brain axis or more specifically the gut–microbiota–brain axis in the etiology of neurodevelopmental disorders among which are Autism Spectrum Disorders (ASD) (Dinan and Cryan, 2012). In a reciprocal relationship, the CNS and the gut microbiota are interacting. Many recent studies have now confirmed that anxiety as a phenotype can be related to altered gut microbiota (Desbonnet et al., 2014; Li et al., 2009). It is well documented that the signals generated by the stimulation of the gut have a strong effect on the nervous system presented as strong modulation of brain activity, immune response, emotional status, and many other homeostatic functions (Rhee et al., 2009). However, this influence is not unidirectional, but is a bidirectional communication pathway. The CNS is able to alter the composition of the gut microbiota and to break the equilibrium in the gut permeability, modulating motility and secretion through the activation of the hypothalamus pituitary–adrenal (HPA) axis, autonomic and neuroendocrine system with an

Effect of Diet on Gut Microbiota as an Etiological Factor in Autism Spectrum Disorder 275 immediate and direct impact on the microbiota (Cryan and Dinan, 2012; Rhee et al., 2009). Important molecules have been shown to play a critical role in the gut–brain axis, among which are vasoactive intestinal peptide (VIP) serotonin, melatonin, gamma-aminobutyric acid (GABA), catecholamines, histamine, and acetylcholine (Barrett et al., 2014; Forsythe et al., 2010; Lyte, 2011; O’Mahony et al., 2009; Velickovic et al., 2014), but still the mechanisms by which these molecules are acting is not fully understood. Autism is a neurodevelopmental disorder associated with multiple phenotypic features, including impairment of communication skills, social response impairment, and repetitive behaviors (Whitehouse and Stanley, 2013). The prevalence of autism has increased dramatically, and it is found to affect 1 in 68 children (DDMNS, 2014; Rutter, 2005). It is well documented that boys are 4 times more likely to have autistic features than girls. Numerous hypotheses have been proposed regarding the etiopathology of autism but still it remains poorly understood. Recent studies have correlated gut dysfunction with autism and suggested a possible role of the gut microbiota in symptomatology and/or severity of symptoms in autistic children highlighting the role of homeostatic imbalance in gut-to-brain connections (Kotagal and Broomall, 2012). Based on the impact of imbalanced gut microbiota on the CNS, new terms, such as “psychobiotics” and “psychomicrobiotics” are used to describe the effect of bad microbiota on feelings, attitudes, and whole mental states. Numerous studies indicate that gastrointestinal alterations of the microbial ecosystem promote gut permeability, causing leaky gut syndrome. This condition results in an escape of microbial products and cytokines into the bloodstream, causing neurodevelopmental disorders

2 Factors Affecting Infant Gut Microbiota 2.1 Microbial Characteristics in Amniotic Fluid, Placenta, and Colostrum The microbial populations in amniotic fluid and placenta are similar and highly consistent across individuals. Proteobacteria was the most prevalent phylum in amniotic fluid and placenta samples with a particularly high abundance of species belonging to Enterobacteriaceae, Enterobacter, and Escherichia/Shigella. These genera were also present in colostrum, meconium, and infant feces but in lower abundance (Collado et al., 2016). Propionibacterium was the second most predominant genus present in the amniotic fluid and placenta, and was also detectable in meconium. Streptococcus was present in low abundance in the amniotic fluid, placenta, and meconium (