Handbook of Probiotics and Prebiotics

YUAN KUN LEE, PHD, is an Associate Professor in the Department of Microbiology at the National University of Singapore, where he researches fermentation and microbe-host interactions and probiotics. Dr. Lee has written ninety journal papers, sixty conference proceedings, twenty-eight book chapters, and three books. He is also the holder of three patents. SEPPO SALMINEN, PHD, is a Professor in the Department of Biochemistry and Food Chemistry at the University of Turku in Finland. He is also a Visiting Professor of Food Toxicology at RMIT University in Australia. Prior to his academic career, Dr. Salminen worked as a nutrition specialist, project manager, and toxicologist for private industry and government agencies. Dr. Salminen has written more than 240 refereed publications, several book chapters, and five books.

"Overall this is a useful handbook. Its primary aim, i.e. to put together the newest information and technology required to develop a successful pro- or prebiotic product from the laboratory to the market, is accomplished in my opinion." (Beneficial Microbes, September 2010) "This is a timely and highly informative production that will serve as a valuable resource for students, newcomers and active researchers in gut microbiology, food microbiology and biotherapeutics.... Highly recommended." (Microbiology Today, May 2009)

• Preface xvContributors xviiPart I Probiotics 11 Probiotic Microorganisms 31.1 Definitions 31.2 Screening, Identification, and Characterization of Lactobacillus and Bifidobacterium Strains 41.2.1 Sources of Screening for Probiotic Strains 51.2.2 Identification, Classification, and Typing of Bifidobacterium Strains 71.2.2.1 Taxonomy 71.2.2.2 Identification and Typing 81.2.3 Identification, Classification, and Typing of Lactobacillus Strains 141.2.3.1 Taxonomy 141.2.3.2 Identification and Typing 151.2.4 Characterization of Probiotic Properties in Bifidobacterium and Lactobacillus Strains 191.2.4.1 Survival to GIT Stressing Conditions 191.2.4.2 Adhesion/Colonization to/of GIT 231.2.4.3 Antimicrobial Activity 241.2.4.4 Other Probiotic Properties 241.2.5 Conclusion 241.3 Detection and Enumeration of Gastrointestinal Microorganisms 251.3.1 Methods for Intestinal Microbiota Assessment 251.3.1.1 Culture-Dependent Methods 251.3.1.2 Culture-Independent Methods 301.3.2 Detection and Enumeration in Dairy Products 371.3.3 Detection and Enumeration of Specific Probiotics in the Gut 381.3.4 The Problem of the Viability and Physiological State of Intestinal Bacteria 411.3.5 Conclusions 421.4 Enteric Microbial Community Profiling in Gastrointestinal Tract by Terminal-Restriction Fragment Length Polymorphism (T-RFLP) 431.4.1 T-RFLP 431.4.2 Universal and Group-Specific Primers 441.4.3 Fluorescent Dyes 441.4.4 DNA Extraction 461.4.5 PCR Amplification 461.4.6 Generation of Terminal Restriction Fragments (TRF) by Digestion of Amplicons with Restriction Enzymes 461.4.7 Software and Data Processing 471.4.8 Microbial Diversity in Different Intestinal Compartments of Pigs 471.4.9 Tracking the Fate of Orally Delivered Probiotics in Feces 481.4.10 Conclusion 511.5 Effective Dosage for Probiotic Effects 521.5.1 Acute (Rotavirus) Diarrhea in Children 531.5.2 Antibiotic-Associated Diarrhea 541.5.2.1 Combination of L. acidophilus + bifidobacteria or Streptococcus thermophilus 541.5.2.2 L. rhamnosus GG or Saccharomyces boulardii Applied Singly 571.5.3 Helicobacter pyroli 581.6 Incorporating Probiotics into Foods 581.6.1 Probiotic Ingredients 601.6.2 Factors Affecting the Viability of Probiotics in Foods 601.6.2.1 Choice of Probiotic Organism/Food Combinations 611.6.2.2 Physiologic State of the Probiotic 631.6.2.3 Temperature 631.6.2.4 pH 641.6.2.5 Water Activity 641.6.2.6 Oxygen 651.6.2.7 Toxicity of Ingredients 661.6.2.8 Growth Factors, Protective, and Synergistic Ingredients 671.6.2.9 Freeze–Thawing 671.6.2.10 Sheer Forces 671.6.3 Synbiotics 671.6.4 Delivery Systems 681.6.4.1 Microencapsulation 681.6.4.2 Delivery Devices 691.6.5 Probiotic Foods 691.6.6 Conclusions 691.7 Safety of Probiotic Organisms 751.7.1 Current Proposals for Probiotic Safety 771.7.2 Taxonomic Identification 791.7.3 Pathogenicity 811.7.4 Antibiotic Resistance and Susceptibility 831.7.5 Immune Modulation 871.7.6 Clinical Studies 901.7.7 Postmarket Surveillance 921.7.8 GMO Probiotics 931.7.9 Conclusion 941.8 Legal Status and Regulatory Issues 951.8.1 Human Probiotics 951.8.1.1 Asia 951.8.1.2 Europe 1061.8.1.3 The United States of America 1111.8.2 Animal Probiotics 1231.8.2.1 United States 1231.8.2.2 European Union 1231.8.2.3 China 1251.8.2.4 Japan 1251.8.2.5 Korea 1251.8.2.6 Thailand 1251.8.2.7 Australia 1251.8.2.8 New Zealand 1351.8.2.9 Indonesia, Malaysia, Philippines, and Vietnam 139References 1392 Selection and Maintenance of Probiotic Microorganisms 1772.1 Isolation of Probiotic Microorganisms 1772.2 Selection of Probiotic Microorganisms 1782.2.1 Manufacturing Criteria (General Criteria) 1792.2.2 Shelf Life and Gut Transit (General Criteria) 1792.2.2.1 Shelf Life of Viable Probiotics Under Different Storage Conditions 1792.2.2.2 Tolerance to Digestive Juices 1802.2.2.3 Adhesion and Colonization onto Specific Site of Body Surface 1812.2.3 Health Properties (Specific Criteria) 1812.2.4 Safety 1822.2.5 Identification 1822.3 Maintenance of Probiotic Microorganisms 184References 1873 Genetic Modification of Probiotic Microorganisms 1893.1 Mutants Obtained from Probiotic Microorganisms by Random Mutagenesis 1893.2 Plasmids 2023.3 Vectors for Lactobacilli and Bifidobacteria 2123.4 Genetic Recombination 222References 2294 Role of Probiotics in Health and Diseases 2574.1 Cell Line Models in Research 2594.2 Laboratory Animal Models in Research 2634.3 Effects on Human Health and Diseases 2674.3.1 Nutritional Effects 2674.3.1.1 Lactose Maldigestion 2684.3.1.2 b-Galactosidase in Fermented Milk Products 2694.3.2 Prevention and Treatment of Oral Infection and Dental Caries 2704.3.3 Prevention and Treatment of Diarrhea 2724.3.3.1 Acute (Rotavirus) Diarrhea in Children 2724.3.3.2 Antibiotic-Associated Diarrhea 2764.3.3.3 Clostridium difficile Associated Diarrhea 2794.3.3.4 Radiation-Induced Diarrhea 2794.3.3.5 Traveler’s Diarrhea 2804.3.3.6 Diarrhea in Tube-Fed Patients 2814.3.4 Treatment of Irritable Bowel Syndrome 2824.3.5 Prevention and Treatment of Inflammatory Bowel Diseases 2874.3.6 Treatment of H. pylori Infection 2924.3.7 Prevention of Postoperative Infections 2954.3.8 Prevention and Treatment of Respiratory Tract Infections 2994.3.9 Prevention and Treatment of Allergic Diseases 3024.3.10 Antitumor Effects 3104.3.11 Reduction of Serum Cholesterol 3134.3.12 Enhancement of Vaccine Responses 3184.4 Effects on Farm Animals 3214.4.1 Poultry 3224.4.2 Swine 3234.4.3 Ruminants 3314.4.4 Rabbits 3394.4.5 Pets 339References 3505 Mechanisms of Probiotics 3775.1 Adhesion to Intestinal Mucus and Epithelium by Probiotics 3775.1.1 Adhesion to Gastrointestinal Epithelial Cell Lines 3785.1.2 Adhesion to Intestinal Mucus 3785.1.3 Colonization of Probiotics in Human Intestine as Assessed by Biopsies 3795.1.4 Comparisons Between In Vitro and In Vivo Results 3795.1.5 Adhesins 3795.1.6 Factors Affecting the Adhesion Properties of Probiotics 3795.1.7 Adhesive and Inhibitory Properties of Nonviable Probiotics 3805.1.8 Role of Age and Diseases on Adhesion 3835.2 Combined Probiotics and Pathogen Adhesion and Aggregation 3845.2.1 Aggregation 3855.2.2 Adhesion 3855.2.3 Assay for Adhesion 3865.2.4 Assay for Aggregation 3865.2.5 Factors that Determine Adhesion 3895.2.6 In Vitro Models 3895.2.7 Probiotics in Combination 3905.2.8 Conclusion 3915.3 Production of Antimicrobial Substances 3915.3.1 Organic Acids 3925.3.2 Hydrogen Peroxide 3925.3.3 Carbon Dioxide 3935.3.4 Bacteriocins 3935.3.5 Low Molecular Weight Antimicrobial Compounds 3945.3.6 Other Antimicrobial Agents 3945.4 Immune Effects of Probiotic Bacteria 3955.4.1 The Neonatal Intestinal Microbiota 3955.4.2 The Importance of the Intestinal Microbiota in Immune Development 3955.4.3 Interaction of Commensal and Pathogenic Bacteria with the Intestinal Immune System 3965.4.4 Probiotic Effects on Immune Responses 3965.4.5 Probiotic Effects on Epithelial Cells 3975.4.6 Probiotic Effects on DCs 3975.4.7 Probiotic Effects on Adaptive Immune Responses: T Helper Cells and T Regulatory Cells 3975.4.8 Delivery of Probiotic Bacteria 3985.4.9 The Specificity of Probiotic Effects 3995.4.10 Summary 3995.5 Alteration of Microecology in Human Intestine 3995.5.1 Impact on Human Health: in Infants and the Elderly 3995.5.1.1 Stepwise Establishment of Microbiota 4005.5.1.2 Methodological Improvements in Microbiota Assessment 4015.5.1.3 Microbiota After Infancy 4035.5.1.4 Host–Microbe Cross Talk 4035.5.1.5 Microbiota in the Elderly 4045.5.1.6 Maintenance of Healthy Microbiota 4055.5.1.7 Conclusion 4055.5.2 Impact on Animal Health: Designer Probiotics for the Management of Intestinal Health and Colibacillosis in Weaner Pigs 4065.5.2.1 The Farrowing Environment 4065.5.2.2 The Weaning Environment 4065.5.2.3 Colibacillosis in Pigs 4075.5.2.4 Control of Colibacillosis 4085.5.2.5 Mechanism of Action 4085.5.2.6 Pathogenic and Commensal E. coli—the Concept of Gene Signatures 4095.5.2.7 Mosaicism and Genome Plasticity in Porcine E. coli (Clone Gene Signatures) 4105.5.2.8 Population Gene Signatures in Epidemiological Study 4125.5.2.9 Designer Lactic Acid Bacteria as Probiotics 4155.5.2.10 Population Gene Signatures as a Measure of Probiotic Bioefficacy 4175.5.2.11 Creation of Enteric Microbial Communities for Sustainable Intestinal Health (Probiosis) 419References 4216 Commercially Available Human Probiotic Microorganisms 4416.1 Lactobacillus acidophilus, LA-5® 4416.1.1 Gastrointestinal Effects 4416.1.1.1 Intestinal Microbial Balance 4416.1.1.2 Diarrhea 4426.1.1.3 Other Gastrointestinal Effects 4426.1.2 Immunomodulatory Effects 4436.1.2.1 Nonspecific Immune Responses 4436.1.2.2 Specific Immune Responses 4436.1.3 Other Health Effects 4436.1.4 Safety 4446.2 Lactobacillus acidophilus NCDO 1748 4446.2.1 Origin and Safety 4456.2.2 In Vitro and Animal Studies 4456.2.3 Human Studies 4466.3 Lactobacillus acidophilus NCFM® 4476.3.1 L. acidophilus NFCM Basic Properties 4476.3.2 Survival of Intestinal Transit and Change in Intestinal Microbiota Composition and Activity 4476.3.3 Lactose Intolerance 4486.3.4 Relief of Intestinal Pain 4486.3.5 Prevention of Common Respiratory Infections and Effects on Immunity 4496.3.6 Application 4496.3.7 Conclusion 4496.4 Lactobacillus casei Shirota 4496.4.1 Effects on Intestinal Environment 4506.4.2 Adhesive Property 4506.4.3 Intestinal Physiology 4516.4.4 Immunomodulation 4526.4.5 Effects on Cancer 4536.4.6 Prevention of Infectious Diseases 4546.4.7 Prevention of Life Style Diseases 4546.4.8 Clinical Application 4556.4.9 Safety Assessment 4566.5 Lactobacillus gasseri OLL2716 (LG21) 4576.5.1 Helicobacter pylori 4586.5.2 Selection of a Probiotic for H. pylori Infection 4586.5.3 Effects of LG21 on H. pylori Infection in Humans 4586.5.4 Mechanisms of Therapeutic Effects of LG21 on H. pylori Infection 4616.5.5 Conclusion 4626.6 Lactobacillus paracasei ssp. paracasei, F19® 4626.6.1 Identification and Safety 4626.6.2 In Vitro Studies 4636.6.3 Global Gene Expression 4636.6.4 Human Studies 4646.7 Lactobacillus paracasei ssp paracasei, L. casei 431® 4666.7.1 Adhesion and Survival Through the GI Tract 4666.7.2 Gastrointestinal Effects 4666.7.2.1 Intestinal Microbial Balance 4666.7.2.2 Diarrhea 4666.7.3 Immunomodulatory Effects 4686.7.4 Other Health Effects 4686.7.5 Safety 4686.8 Lactobacillus rhamnosus GG, LGG® 4696.8.1 Storage Stability 4696.8.2 Gastrointestinal Persistence and Colonization 4696.8.3 Health Benefits 4696.8.4 Source of LGG® 4706.9 Lactobacillus rhamnosus, GR-1® and Lactobacillus reuteri RC- 14® 4706.9.1 The Strains 4716.9.2 In Vitro Properties 4716.9.3 Animal Safety, Toxicity, and Effectiveness Studies 4716.9.4 Clinical Evidence 4726.9.4.1 Safety, Effectiveness, and Efficacy 4726.9.5 Summary 4736.10 Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 4736.10.1 Basic Properties of L. rhamnosus HN001 and B. lactis HN019 4736.10.2 Survival During the Intestinal Transit and Modulation of the Intestinal Microbiota 4746.10.3 Modulation of the Immune System 4746.10.4 Reduction of Disease Risk 4776.10.5 Application 4776.10.6 Conclusions 4776.11 LGG®Extra, A Multispecies Probiotic Combination 4776.11.1 Strain Selection for the Combination 4776.11.2 Adhesion and Gastrointestinal Survival 4786.11.3 Health Benefits 4786.11.4 Technological Characteristics 4796.11.5 Source of LGG® Extra 4806.12 Bifidobacterium animalis ssp. lactis, BB-12® 4806.12.1 Adhesion and Survival Through the GI Tract 4806.12.2 Gastrointestinal Effects 4806.12.2.1 Intestinal Microbial Balance 4806.12.2.2 Diarrheas 4816.12.2.3 Gastrointestinal Health of Infants 4826.12.2.4 Other Gastrointestinal Effects 4826.12.3 Immunomodulatory Effects 4836.12.3.1 Nonspecific Immune Responses 4836.12.3.2 Specific Immune Responses 4836.12.3.3 Other Immunomodulatory Effects 4846.12.4 Other Health Effects 4846.12.5 Safety 4856.13 Bifidobacterium breve Strain Yakult 4856.13.1 Effects on Intestinal Environment 4856.13.2 Intestinal Physiology 4856.13.3 Effects on Cancer 4866.13.4 Prevention of Infectious Diseases 4866.13.5 Prevention of Life Style Diseases 4866.13.6 Clinical Application 4876.14 Bifidobacterium longum BB536 4886.14.1 Evaluation of Safety of BB536 4886.14.2 Physiological Effects of BB536 4896.14.2.1 Improvement of Intestinal Environment 4896.14.2.2 Effects on Immunity and Cancer 4906.14.2.3 Antiallergic Activity 4906.14.3 Technologies in BB536 Applications 4916.15 Bifidobacterium longum Strains BL46 and BL2C—Probiotics for Adults and Ageing Consumers 4926.15.1 Safety of BL2C and BL46 4926.15.2 The Health Effects of BL2C and BL46 4936.15.2.1 BL2C and BL46 Stabilize the Gut Function in the Elderly 4936.15.2.2 Modulation of Gut Microbiota by BL2C and BL46 4936.15.2.3 BL46 is Effective Against Harmful Bacteria 4936.15.2.4 Effects of BL2C and BL46 on the Immune System and Infections 4936.15.2.5 BL2C and BL46 Can Bind Toxic Compounds 4936.15.3 Technical Properties and Sensory Qualities of BL2C and BL46 4946.15.4 Conclusions 494References 494Part II Prebiotics 5337 Prebiotics 5357.1 The Prebiotic Concept 5357.2 A Brief History of Prebiotics 5367.3 Advantages and Disadvantages of the Prebiotic Strategy 5367.4 Types of Prebiotics 5377.5 Production of Prebiotics 5407.6 Prebiotic Mechanisms 5467.7 Modulating the Intestinal Microbiota in Infants 5467.7.1 Breast Milk 5467.7.2 Infant Milk Formulas 5477.8 Modulating the Intestinal Microbiota in Adults 5487.8.1 Effects at the Genus Level 5487.8.2 Effects at the Species Level 5487.8.3 Altering the Physiology of the Microbiota 5497.9 Modifying the Intestinal Microbiota in the Elderly 5497.10 Health Effects and Applications of Prebiotics 5497.10.1 Laxatives 5507.10.2 Hepatic Encephalopathy 5507.10.3 Primary Prevention of Allergy in Infants 5517.10.4 Amelioration of Inflammatory Bowel Disease 5517.10.5 Prevention of Infections 5557.10.6 Mineral Absorption 5567.10.7 Prevention of Colorectal Cancer 5567.10.8 Reduction in Serum Lipid Concentrations 5597.10.9 Use in Weight Management and Improving Insulin Sensitivity 5597.11 Functional Foods for Animals 5597.12 Safety of Prebiotics 5607.13 Regulation of Prebiotics 5607.14 Conclusion 561References 562Author index 583Subject index 585