Biosafety Assessment of Probiotic Potential

Foreword
Preface to the Series
Preface
Contents
Contributors
Part I: In Vitro Biosafety Assessment of Probiotics
	Chapter 1: Determination of Biogenic Amine Production
		1 Introduction
		2 Materials
			2.1 Media Composition
			2.2 Decarboxylase Media(DCM) Plate and Broth Method of Screening Biogenic Amines
			2.3 High-Performance Liquid Chromatography (HPLC)Method of Screening Biogenic Amines
			2.4 Isolation of Genomic DNA
			2.5 Polymerase Chain Reaction (PCR) of Biogenic Amine Specific Genes
			2.6 16S rRNA Gene Sequencing
			2.7 Genome Sequencing and Analysis
		3 Methods
			3.1 Culture of Probiotic Isolates
			3.2 Qualitative Screening of Biogenic Amine Production [6, 15  ]
			3.3 Quantitative Detection of Biogenic Amines Using Reversed Phase-High-Performance Liquid Chromatography-Diode Array Detector...
			3.4 Genomic DNA Isolation
			3.5 Detection of Biogenic Amine Producing Strains
			3.6 Molecular Identification of Microbial Strain Using 16S rRNA Gene Sequencing
			3.7 Whole Genome Sequencing
		4 Inference
		References
	Chapter 2: Determination of Gelatinases, Glycosidases, and Enolase Production
		1 Introduction
		2 Materials
			2.1 Chemical Composition of Culture Media
			2.2 Bacterial Genomic DNA Isolation
			2.3 Screening of Virulence Genes
			2.4 DNA Sequence Analysis
			2.5 Preparation for Gelatinase Assay
			2.6 Preparation for Glycosidase Assay
			2.7 Preparation for Enolase Assay
		3 Methods
			3.1 Culture of Probiotic Isolates
			3.2 Extraction of Genomic DNA
			3.3 Screening of Virulence Genes
			3.4 DNA Sequence Analysis
			3.5 Gelatinase Activity
				3.5.1 Protocol 1
				3.5.2 Protocol 2
				3.5.3 Protocol 3
			3.6 Glycosidase Activity
			3.7 Enolase Activity Assay
		References
	Chapter 3: Determination of β-Glucuronidase Production
		1 Introduction
		2 Materials
			2.1 Qualitative Method
			2.2 Quantitative Method
		3 Methods
			3.1 Qualitative Method
			3.2 Quantitative Method
		4 Notes
		References
	Chapter 4: Determination of Nitroreductase Production
		1 Introduction
		2 Materials
			2.1 Qualitative Methods
				2.1.1 Method 1
				2.1.2 Method 2
			2.2 Quantitative Methods
				2.2.1 Method 1
				2.2.2 Method 2
		3 Methods
			3.1 Qualitative Methods
				3.1.1 Method 1
				3.1.2 Method 2
			3.2 Quantitative Methods
				3.2.1 Method 1
				3.2.2 Method 2
		4 Notes
		References
	Chapter 5: Determination of Azoreductase Production
		1 Introduction
		2 Materials
			2.1 Qualitative Method
			2.2 Quantitative Method
		3 Methods
			3.1 Qualitative Method
			3.2 Quantitative Method
		References
	Chapter 6: Determination of Hemolytic Activity
		1 Introduction
			1.1 Alpha Hemolysis
			1.2 Beta Hemolysis
			1.3 Gamma Hemolysis
			1.4 Alpha Prime or Wide Zone Alpha Hemolysis
		2 Materials
		3 Methods
		References
	Chapter 7: Determination of DNAse Activity
		1 Introduction
		2 Materials
			2.1 Chemical Composition of Culture Media
		3 Methods
			3.1 Culture of Probiotic Isolates
			3.2 Determination of DNase Activity
				3.2.1 Protocol 1 (HCI-DNA Precipitation Method)
				3.2.2 Protocol 2 (DNAse Methyl Green Agar Method)
		References
	Chapter 8: Determination of Bile Salts Deconjugation
		1 Introduction
		2 Materials
		3 Methods
			3.1 Qualitative Determination of BSH Activity
			3.2 Quantitative Determination of BSH Activity
				3.2.1 Spectrophotometric Estimation BSH Activity
				3.2.2 HPLC Method
		4 Inference
		5 Notes
		References
	Chapter 9: Determination of D-Lactic Acid Production
		1 Introduction
		2 Materials
			2.1 Chemical Composition of Culture Media
			2.2 Preparation for D-Lactate Assay
			2.3 Bacterial Genomic DNA Isolation
			2.4 16S rDNA Gene Sequencing
			2.5 Genome Sequencing and Analysis
			2.6 In-Silico Tools for D-Lactate Gene Identification
		3 Methods
			3.1 Culture of Probiotic Isolates
			3.2 Enzymatic Measurement of D-Lactate
			3.3 Extraction of Genomic DNA
			3.4 16S rRNA Based Identification of the Isolate
			3.5 Whole Genome Sequencing and Analysis of the Isolate
			3.6 Identification of D-Lactate Related Genes
		4 Notes
		References
	Chapter 10: Determination of Antibiotic Resistance
		1 Introduction
		2 Materials
			2.1 Antimicrobial Susceptibility Test of Probiotic Bacteria (Disc Diffusion Method)
			2.2 Determination of Antibiotic Resistance Gene in Probiotic Bacteria by Mutiplex PCR
		3 Methods
			3.1 Antimicrobial Susceptibility Test of Probiotic Bacteria by Disc Diffusion Method
				3.1.1 Preparation of 0.5 McFarland Standard Probiotic Inoculum
				3.1.2 Inoculation of MHAM Plate
				3.1.3 Application of Antibiotic Discs
			3.2 Detection of Antibiotic Resistant Gene in Probiotic Bacteria by Mutiplex PCR
				3.2.1 Preparation of PCR Reaction Mix
				3.2.2 PCR Amplification Cycle
				3.2.3 Agarose Gel Electrophoresis
					Loading of the PCR Amplicon
					Electrophoresis
					Visualization
		4 Observation
			4.1 Antimicrobial Susceptibility of Probiotic Bacteria by Disc Diffusion Method
			4.2 Detection of Antibiotic Resistant Gene by Multiplex PCR
		References
	Chapter 11: Determination of Antibiotic Resistance Gene Transfer
		1 Introduction
		2 Materials
			2.1 Equipment and Accessories
			2.2 Media, Reagents and Recipes
				2.2.1 Brain Heart Infusion Broth (BHIB)
				2.2.2 MRS Broth (for Lactic Acid Bacteria -LAB)
				2.2.3 Peptone Physiological Saline (PPS)
				2.2.4 Tetracycline (Tet)
				2.2.5 Rifampicin (Rif)
				2.2.6 Physiological Saline
				2.2.7 Brain Heart Infusion Agar (BHIAM)
			2.3 Microbial Culture
		3 Method
		4 Interpretation
		5 Notes
		References
	Chapter 12: Determination of Toxin Production
		1 Introduction
			1.1 Bacillus Diarrheal Enterotoxin Visual Immunoassay (BDE VIA) Kit (3M Tecra)
				1.1.1 Principle
			1.2 BCET-RPLA Toxin Detection Kit
				1.2.1 Principle
			1.3 Duopath Cereus Enterotoxins (Merck)
				1.3.1 Principle
		2 Materials
			2.1 Enterotoxin Detection Kits
			2.2 Media for Bacterial Culture Growth
			2.3 Bacillus Diarrheal Enterotoxin Visual Immunoassay (BDE VIA) Kit (3M Tecra)
				2.3.1 Kit Components
				2.3.2 Materials Required but Not Provided with the  Kit
			2.4 B. Cereus Enterotoxin Reversed Passive Latex Agglutination (BCET-RPLA) Kit (Oxoid)
				2.4.1 Kit Components
				2.4.2 Materials Required but Not Provided with the  Kit
			2.5 Duopath Cereus Enterotoxins
				2.5.1 Materials Required but Not Provided with the  Kit
		3 Methods
			3.1 Sample Preparation
			3.2 Bacillus Diarrheal Enterotoxin Visual Immunoassay (BDE VIA) Kit (3M Tecra)
				3.2.1 Reagent Preparation
				3.2.2 Assay Procedure
				3.2.3 Observation of Result
			3.3 B. cereus Enterotoxin Reversed Passive Latex Agglutination (BCET-RPLA) Kit (Oxoid)
				3.3.1 Reagent Preparation
				3.3.2 Assay Procedure
				3.3.3 Observation of Result
			3.4 Duopath Cereus Enterotoxins (Merck)
				3.4.1 Assay Procedure
				3.4.2 Interpretation of Results
		4 Notes
		References
	Chapter 13: Detection of Toxin Genes by PCR Based Methods
		1 Introduction
		2 Materials
			2.1 Positive and Negative Enterotoxigenic Reference Strains
			2.2 Bacterial Culture Media for Maintaining Reference Strains
				2.2.1 DNA Isolation
					Instruments and Equipment
					Reagents
				2.2.2 PCR Amplification
				2.2.3 Agarose Gel Electrophoresis
					Preparation of 1% Agarose Gel
					Sample Preparation
		3 Methods
			3.1 Bacterial DNA Isolation
			3.2 Detection of Enterotoxin Genes by PCR Amplification
				3.2.1 PCR Amplification
					Primer Design
					PCR Master-Mix
					PCR Protocol
				3.2.2 Agarose Gel Electrophoresis
					Gel Preparation
					Loading of Samples
					Gel Electrophoresis
					Examining the Gel
					Observation
		4 Inference
		5 Notes
		References
	Chapter 14: Evaluation of Pathogenicity Potential by Phenotypic and Genotypic Methodologies
		1 Introduction
		2 Materials
			2.1 Phenotypic Approach
			2.2 Genotypic Approach
		3 Methods
			3.1 Phenotypic Approach
				3.1.1 Hemolysis
				3.1.2 Gelatinase Production
			3.2 Genotypic Approach
				3.2.1 Total DNA Isolation (Genomic and Plasmidic)
					Rapid DNA Isolation
					High-Quality DNA Isolation (Guanidium Thiocyanate Methodology, Adapted from
					Commercial Kits
				3.2.2 Screening Procedures
					Conventional PCR
					Real-Time PCR
					Whole Genome Sequencing (WGS)
		4 Conclusions
		5 Notes
		References
	Chapter 15: Determination of Toxicity Through Cytotoxicity Assays
		1 Introduction
			1.1 Principle of MTT Assay
		2 Materials
			2.1 Reference Bacterial Strains [8, 26 ]
			2.2 Bacterial Culture Media
			2.3 Cell Line and Cell Culture Media/Reagents
			2.4 Reagents for Cell Culture
			2.5 Reagents for MTT Assay
			2.6 Equipment/Apparatus
		3 Methods
			3.1 Preparation of Bacterial Toxin/Supernatant for Cytotoxicity Assay [8, 26 ]
			3.2 Plate Setup for Cytotoxicity Assay
			3.3 Treatment of Cells
			3.4 Cytotoxicity Assay
			3.5 Calculation and Inference
			3.6 HEp-2 Cell Vacuolation Assay
				3.6.1 Filtrate Preparation
				3.6.2 Tissue Culture Assay
				3.6.3 Observation
		4 Notes
		References
	Chapter 16: In Vitro Evaluation of the Nitric Oxide Pathway
		1 Introduction
		2 Materials
			2.1 NO Sensitivity
			2.2 Estimation of NO
		3 Methods
			3.1 Estimation of NO Sensitivity of Probiotic Species
			3.2 Estimation of NO in Probiotic Using Griess Reaction
			3.3 Estimation of NO Using Metmyoglobin Method
				3.3.1 Qualitative Method Using MRS Agar
				3.3.2 Quantitative Method Using MRS Broth
		4 Notes
		References
	Chapter 17: Assessment of Capsule Formation
		1 Introduction
		2 Materials
			2.1 Microscopy Methods
			2.2 PCR Based Method
		3 Methods
			3.1 Microscopy Methods
				3.1.1 Anthony´s Method [4]
				3.1.2 Hiss´s Method
				3.1.3 Maneval´s Method [6]
				3.1.4 M´Fadyean´s Method
				3.1.5 Duguid´s Method [8]
				3.1.6 India Ink Staining Method
				3.1.7 The Negative-Positive Method
			3.2 PCR Based Method
		References
	Chapter 18: Assessment of Platelet Aggregation
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Lactobacillus Culture
			3.2 Preparation of Blood Samples
			3.3 Platelet Aggregometry Assay
			3.4 Inhibition of Platelet Aggregation Assay
			3.5 Pronase Treatment
			3.6 Heat Treatment
			3.7 Extraction of Bacterial Surface Components
			3.8 SDS-PAGE
		4 Observation
		References
	Chapter 19: Assessment of Fibrinogen and Fibronectin Binding Activity
		1 Introduction
			1.1 Fibrinogen Binding Is an In Vitro Infectivity Measure
			1.2 Fibronectin Binding Is an In Vitro Probiotic Measure
		2 Materials
			2.1 Adhesion of Probiotic Bacteria with Fibronectin Coated Microtiter Plate
			2.2 Adhesion of Probiotic Bacteria with Fibrinogen Coated Microtiter Plate
		3 Methods
			3.1 Adhesion of Probiotic Bacteria with Fibronectin Coated Microtiter Plate
			3.2 Adhesion of Probiotic Bacteria with Fibrinogen Coated Microtiter Plate
		4 Inference
		References
	Chapter 20: Assessment of Probiotics Adhesion to Mammalian Cells
		1 Introduction
		2 Materials
			2.1 Assessment of Adhesion Ability
		3 Methods
			3.1 Assessment of Adhesion Ability
		4 Inference
		References
	Chapter 21: Assessment of Mutagenicity
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Media, Reagents, and Recipes
				2.2.1 Minimal Glucose Agar Medium
				2.2.2 Vogel-Bonner Salts (50x)
				2.2.3 Glucose Solution (40% w/v)
				2.2.4 Ampicillin Solution (10 mg/mL)
				2.2.5 Histidine-Biotin Solution 0.5 mM (S. typhimurium Strains)
				2.2.6 Tryptophan Solution 0.25 mM (E. coli Strains)
				2.2.7 Top Agar Supplemented with Histidine-Biotin or Tryptophan
				2.2.8 Nutrient Broth (HiMedia)
				2.2.9 Phosphate Buffer 0.2 M (pH 7.4)
				2.2.10 Positive Control Mutagen
				2.2.11 S9mix
			2.3 Microbial Cultures
		3 Methods
			3.1 Preparation of Probiotic Culture
			3.2 Preparation of Ames Test Strain
			3.3 Preparation of Probiotic Cell Suspension/Cell-Free Supernatant
			3.4 Preparation of Reaction Mixture
			3.5 Plate Incorporation
		4 Interpretation of Result
		5 Notes
		References
	Chapter 22: Assessment of Induction and Destruction of Thrombi
		1 Introduction
		2 Materials
			2.1 Determination of Thrombi Induction of Probiotic Bacteria by Flow Cytometry Analysis
			2.2 Determination of Thrombolytic Activity of Probiotic Bacteria (by In Vitro Clot Lysis Method)
		3 Methods
			3.1 Determination of Thrombi Induction of Probiotic Bacteria by Flow Cytometry Analysis
				3.1.1 Preparation of Probiotic Bacterial Cell Suspension
				3.1.2 Collection of Blood Samples
				3.1.3 Treatment of Samples
				3.1.4 Flow Cytometry Analysis
			3.2 Determination of Thrombolytic Activity of Probiotic Bacteria by In Vitro Clot Lysis Method
				3.2.1 Preparation of Probiotic Culture Supernatant
				3.2.2 Blood Collection
				3.2.3 In Vitro Clot Lysis
		4 Observation
			4.1 Determination of Thrombi Induction of Probiotic Bacteria by Flow Cytometry Analysis
			4.2 Determination of Thrombolytic Activity of Probiotic Bacteria by In Vitro Clot Lysis Method
		References
	Chapter 23: Assessment of Degradation of Mucin
		1 Introduction
		2 Materials
			2.1 Preparation of MM Media
			2.2 Preparation of B Media
			2.3 Preparation of MRS Broth
			2.4 Assessment of In Vitro Biosafety Aspects of Isolates for Mucin Degradation by Zone Clearance
			2.5 Genomic DNA Isolation
			2.6 16S rRNA Gene Profiling
			2.7 RNA Isolation
			2.8 Transcriptomics Analysis for Mucin Degrading Genes
			2.9 Extraction of Free Oligosaccharides
			2.10 Determination of O-glycans
		3 Methods
			3.1 Assessment of In Vitro Biosafety Aspects of Isolates for Mucin Degradation by Zone of Clearance
			3.2 Identification of Mucin Degrading Bacteria
				3.2.1 Genomic DNA Isolation
				3.2.2 16S rRNA Gene Profiling
			3.3 Identification of Mucin Degrading Genes
				3.3.1 RNA Isolation
				3.3.2 Transcriptomics Analysis for Mucin Degrading Genes
			3.4 Estimation of Mucin O-glycans Released by Mucin Degraders
				3.4.1 Extraction of Free Oligosaccharides
				3.4.2 Determination of O-glycans
				3.4.3 Analysis of Untargeted Mass Spectra
		References
Part II: In Vivo Biosafety Assessment of Probiotics: Monitoring In Vivo Toxicity of Probiotics
	Chapter 24: Evaluation of General Health Status of the Animals During the In-Life Phase
		1 Introduction
		2 Materials
			2.1 Administration of Probiotics in Animals
			2.2 Cylinder Test
			2.3 Irwin Test
			2.4 Wire Suspension Test
			2.5 Vertical Pole Test
		3 Methods
			3.1 Administration of Probiotics in Animals
			3.2 Cylinder Test
			3.3 Irwin Test
				3.3.1 Basic Protocol for Rats
				3.3.2 Preparation of Animals
			3.4 Wire Suspension Test
				3.4.1 Fall and Reaches Method
				3.4.2 Longer Suspension Method
			3.5 Vertical Pole Test
		4 Note
		References
	Chapter 25: Assessment of Bacterial Translocation Through Blood Cultures
		1 Introduction
		2 Materials
			2.1 Preparation of Probiotic and Animal Model
			2.2 Bacterial Translocation Assay
			2.3 Random Amplified Polymorphic DNA (RAPD)
		3 Methods
			3.1 Preparation of Probiotics
			3.2 Oral Administration of Probiotic in BALB/c Mice
			3.3 Bacterial Translocation Assay
			3.4 RAPD-PCR
		4 Note
		References
	Chapter 26: Determination of Splenic Weight Index and Weight-to-Length Ratio
		1 Introduction
		2 Materials
			2.1 Probiotics and Growth Conditions
			2.2 Animal Preparations
			2.3 Administration of Probiotics
			2.4 Specimen Collection
			2.5 Tissue Processing, Staining and Histopathological Evaluation
			2.6 Splenic Weight Index
			2.7 Weight-to-Length Ratio
		3 Methods
			3.1 Probiotics and Growth Conditions
			3.2 Animal Preparation
			3.3 Administration of Probiotics
			3.4 Specimen Collection
			3.5 Tissue Processing, Staining and Histopathological Evaluation
			3.6 Determination of Splenic Weight Index
				3.6.1 Splenic Weight Calculation Through Weighing Balance
				3.6.2 Splenic Weight Calculation Through Ultrasonography
			3.7 Determination of Weight-to-Length Ratio
			3.8 Inferences
		4 Notes
		References
	Chapter 27: Determination of Total Liver Glutathione and Plasma Malondialdehyde Concentrations
		1 Introduction
		2 Materials
			2.1 Probiotics and Growth Conditions
			2.2 Animal Preparations
			2.3 Administration of Probiotics
			2.4 Specimen Collection
			2.5 Preparation of Samples
			2.6 Estimation of Glutathione and Glutathione Disulfide Concentration Through DTNB and Glutathione Reductase Recycling Method
				2.6.1 Preparation of Assay Buffer Solution
				2.6.2 Preparation of DTNB Stock Solution
				2.6.3 Preparation of N-ethylmaleimide (NEM) Stock Solution
				2.6.4 Preparation of Glutathione Disulfide Standard Stock Solution
				2.6.5 Preparation of Glutathione Disulfide Working Standard
				2.6.6 Preparation of Reaction Mixture #1
				2.6.7 Preparation of Reaction Mixture #2
			2.7 Estimation of Glutathione and Glutathione Disulfide Concentration Through HPLC
				2.7.1 Preparation of Plasma Buffer Solution
				2.7.2 Preparation of Sample Buffer
				2.7.3 Preparation of Standards
			2.8 Estimation of Lipid Peroxidation (Spectrophotometrically)
			2.9 Estimation of Malondialdehyde Concentration Through HPLC
		3 Methods
			3.1 Probiotics and Growth Conditions
			3.2 Animal Preparation
			3.3 Administration of Probiotics
			3.4 Preparation of Samples
				3.4.1 Plasma Samples
				3.4.2 Cells Preparation
				3.4.3 Tissue Preparation
			3.5 Estimation of Glutathione and Glutathione Disulfide Concentration Through DTNB and Glutathione Reductase Recycling Method
				3.5.1 Glutathione Assay Procedure
					Glutathione Data Analysis
				3.5.2 Glutathione Disulfide Assay Procedure
			3.6 Estimation of Glutathione and Glutathione Disulfide Concentration Through HPLC
			3.7 Estimation of Lipid Peroxidation Using Spectrophotometer
			3.8 Estimation of Malondialdehyde Concentration Through HPLC
				3.8.1 Preparation of Malondialdehyde Standards
				3.8.2 Assay Procedure for Determination of Malondialdehyde Concentration Through HPLC
		4 Inferences
		5 Notes
		References
	Chapter 28: Determination of Serum Lactate and Fecal Calprotectin for Assessing the Intestinal Inflammation
		1 Introduction
		2 Materials
			2.1 Probiotics and Growth Conditions
			2.2 Animal Preparations
			2.3 Administration of Probiotics
			2.4 Preparation of Serum Samples
			2.5 Assessment of Serum Lactate Levels Through Spectrophotometer
			2.6 Chromatographic Measurement of Serum Lactate Through High-Performance Liquid Chromatography
			2.7 Preparation of Stool Sample
			2.8 Detection of Fecal Calprotectin Through ELISA
		3 Methods
			3.1 Probiotics and Growth Conditions
			3.2 Animal Preparation
			3.3 Administration of Probiotics
			3.4 Preparation of Serum Samples
			3.5 Assessment of Serum Lactate Levels Through Spectrophotometer
			3.6 Chromatographic Measurement of Serum Lactate Through High-Performance Liquid Chromatography (HPLC)
			3.7 Preparation of Stool Sample
			3.8 Detection of Fecal Calprotectin Through ELISA
		4 Inferences
		5 Notes
		References
Part III: In Vivo Biosafety Assessment of Probiotics: Measuring Infectivity in Animal Models
	Chapter 29: In Vivo Evaluation of Adhesion Properties of Probiotics
		1 Introduction
		2 Materials
			2.1 De Man, Rogosa and Sharpe (MRS) Broth
			2.2 Nematode Growth Medium (NGM)
			2.3 M9 Buffer
			2.4 Triton X-100
			2.5 Phosphate Buffer (PBS)
			2.6 Acridine Orange
		3 Method
			3.1 Determination of In vivo Adhesion Properties of Probiotics
		4 Inferences
		5 Notes
		References
	Chapter 30: Determination of Streptomyces Probiotics Oral Administration in Broiler Chicken
		1 Introduction
		2 Materials
			2.1 Preparation of Streptomyces Probiotics
			2.2 Procurement and Rearing of Broiler Chicks in Experimental House
			2.3 Formulation of Experimental Probiotic Diet
		3 Methods
			3.1 Preparation of Streptomyces Probiotics
			3.2 Procurement and Rearing of Broiler Chicks in Experimental House
			3.3 Formulation of an Experimental Probiotic Diet
			3.4 Experimental Design
			3.5 Probiotics Administration Using Feed
			3.6 Probiotics Administration Using Drinking Water
			3.7 Probiotics Administration Using Litter Application
			3.8 Probiotics Administration Using Oral Gavage
		4 Observation
			4.1 Sample Collection, Processing, and Probiotic Safety Assessment
		References
	Chapter 31: Determination of Infectivity of Probiotics Using Animal Model
		1 Introduction
		2 Materials
			2.1 Bacterial Isolation and Growth of Probiotics
			2.2 Animals Utilized and Safety Equipment to Be Used in Animal Colony Rooms/Farms
			2.3 Experimental Endocarditis in Rats to Determine Infectivity of Probiotics
			2.4 Infectivity of Probiotics in Rabbits
		3 Methods
			3.1 Experimental Endocarditis in Rats to Determine Infectivity of Probiotics
			3.2 Infectivity of Probiotics in Rabbits
		4 Notes
		References
	Chapter 32: Determination of Infectivity Using Immunosuppressed Hosts
		1 Introduction
		2 Materials
			2.1 Preparation of Immunodeficient Mouse
			2.2 Strains of Probiotics
			2.3 In Vitro Toxicity Tests
				2.3.1 Mucin Degradation Test
				2.3.2 Platelet Aggregation Test
				2.3.3 Antibiotic Susceptibility Testing
				2.3.4 Assessment of Cytotoxicity
			2.4 Alternative Method of Preparation of Immunodeficient Mouse and Assessment of Immunity
				2.4.1 Analysis of Parameters for Establishment of Immunosuppression
					Assay of Splenocyte Proliferation
					Assay of NK Cell Activity
					Determination of Pinocytosis of Peritoneal Macrophages
					Cytokine Quantification
				2.4.2 Assessment of Hematological Parameters (Blood Cell Count)
			2.5 Assessment of Potential of Probiotic Bacteria to Colonize and Infect Animal Model
				2.5.1 Preparation of Animal Model
				2.5.2 Inoculation of Probiotic Bacterial Culture
				2.5.3 Assay of Colonization of Probiotics Species in the Gastrointestinal Tracts of GF Mice
				2.5.4 Determination of Number of Viable Bacteria in the Internal Organs
				2.5.5 Histological Evaluations
				2.5.6 Determination of Immune Response to Probiotics
		3 Methods
			3.1 Preparation of Immunodeficient Mouse
			3.2 In Vitro Toxicity Tests
				3.2.1 Mucin Degradation Test
				3.2.2 Platelet Aggregation Test
				3.2.3 Antibiotic Susceptibility Testing
				3.2.4 Assessment of Cytotoxicity
			3.3 Alternative Method of Preparation of Immunodeficient Mouse and Assessment of Immunity
				3.3.1 Experimental Design
				3.3.2 Analysis of Parameters for Establishment of Immunosuppression
					Analysis of Body Weight
					Analysis of Immune Organ Index
					Assay of Splenocyte Proliferation Induced by T-Cell Mitogen conA
					Assay of NK Cell Activity
					Determination of Pinocytosis of Peritoneal Macrophages
					Cytokine Quantification
					Assessment of Hematological Parameters (Blood Cell Count)
						Sample Preparation
						Blood Cell Count Through Flow Cytometry
			3.4 Assessment of Potential of Probiotic Bacteria to Colonize and Infect Animal Model
				3.4.1 Preparation of Animal Model
				3.4.2 Inoculation of Probiotic Bacterial Culture
				3.4.3 Survival and Growth of Immunodeficient Mice Colonized with Probiotics Species
				3.4.4 Assay of Colonization of Probiotics Species in the Gastrointestinal Tracts of GF Mice
				3.4.5 Determination of Number of Viable Bacteria in the Internal Organs
				3.4.6 Histological Evaluations
				3.4.7 Determination of Immune Response to Probiotics
		4 Inference
		References
Part IV: In Vivo Biosafety Assessment of Probiotics: Measuring Reproductive and Developmental Toxicity in Animal Models
	Chapter 33: Assessment of Reproductive Toxicity
		1 Introduction
		2 Materials
			2.1 Probiotics and Its Culture
			2.2 Animal Preparations
			2.3 Administration of Probiotics
		3 Methods
			3.1 Probiotics and Growth Conditions
			3.2 Selection of Animal Species and Strain
			3.3 Age, Body Weight, and Inclusion Criteria
			3.4 Animal Preparation
			3.5 Administration of Probiotics
			3.6 Dosing Schedule and Administration of Doses
			3.7 Housing and Feeding Conditions
			3.8 Matting and Pregnancy
			3.9 Litter Size
				3.9.1 Assessment of Offspring Parameters
		4 Inferences
		5 Notes
		References
	Chapter 34: Assessment of Developmental Toxicity in Zebrafish Model
		1 Introduction
			1.1 Principle of the Method to Assess Developmental Toxicity
		2 Materials
		3 Methods
			3.1 Administration of Probiotics in Zebrafish Eggs
			3.2 Exposure Conditions
			3.3 Test Concentration
			3.4 Controls
			3.5 Initiation of Exposure and Duration of Test
			3.6 Eggs Distribution Over 24-Well Plates
			3.7 Validity of the Test
			3.8 Observation
				3.8.1 Embryo Coagulation
				3.8.2 Somite Formation
				3.8.3 Non-Detachment of Tails
				3.8.4 Lack of Heartbeat
				3.8.5 Hatching Rate
				3.8.6 Lethal Concentration 50 (LC50)
		4 Expected Results
		5 Inference
		References
Part V: In Vivo Biosafety Assessment of Probiotics: Measuring Immunological Parameters in Animal Models for Safety
	Chapter 35: Assessment of Inflammation in Animal Models (Macroscopic or Histological Inflammation in the Ileum or in the Colon)
		1 Introduction
		2 Materials
			2.1 Administration of Probiotic in Animal Model
			2.2 Dextran Sulfate Sodium (DSS) Induced Colitis Model
		3 Methods
			3.1 Determination of Inflammation Through Dextran Sulfate Sodium Induced Colitis Model
				3.1.1 Preparation of Animal
				3.1.2 Determination of Intestinal and Spleen Inflammation
				3.1.3 Myeloperoxidase (MPO) Assay
				3.1.4 RT-PCR Analysis
				3.1.5 Histological Staining
				3.1.6 Determination of Severity of Inflammation
		4 Inferences
		5 Notes
		References
	Chapter 36: Assessment of Inflammation in Animal Models (Quantification of TNFA, IFNG, IL4, and IL10 mRNAs by Real-Time PCR)
		1 Introduction
		2 Materials
			2.1 Collection and Storage of Animal Tissue Material
			2.2 Isolation of RNA
			2.3 Synthesis of cDNA from RNA by Reverse Transcription
			2.4 Reverse Transcriptase-Quantitative PCR (RT-qPCR)
			2.5 Selection of Housekeeping Genes
			2.6 Selection of Genes as Inflammation Markers
		3 Methods
			3.1 Isolation of RNA Using TRIzol or TRI Reagent
			3.2 Synthesis of cDNA from RNA by Reverse Transcription
			3.3 Reverse Transcriptase-Quantitative PCR (RT-qPCR) Approach for Quantification of Pro- and Anti-Inflammatory Cytokines
			3.4 Statistical Analysis of Relative Target Gene Expression
		4 Inference
		5 Notes
		References
	Chapter 37: Assessment of Inflammation in Animal Models (Quantification of TNF-α, IFN-γ, IL-4, and IL-10 Proteins by ELISA)
		1 Introduction
		2 Materials
			2.1 Preparation of Animal
			2.2 Preparation of Probiotic Strain
			2.3 Administration of Probiotic Bacteria
			2.4 Collection and Preparation of Animal Samples
			2.5 Materials and Equipment for ELISA Assay
		3 Methods
			3.1 Preparation of Animal Model
			3.2 Preparation of Probiotic Strain
			3.3 Administration of Probiotic Bacteria
			3.4 Collection and Preparation of Animal Samples
				3.4.1 Blood
				3.4.2 Tissue Homogenates
			3.5 Direct Sandwich ELISA
			3.6 Indirect Sandwich ELISA
			3.7 Preparation of Standard Curve Solution
			3.8 Quantification of Cytokines (TNF-α,IFN-γ, IL-4, and IL-10)
		4 Notes
		References
	Chapter 38: Detection of Myeloperoxidase Activity by Enzyme Linked Immunosorbent Assay
		Abbreviations
		1 Introduction
		2 Materials
		3 Methods
			3.1 Sample Extraction from the Animals Treated with the Probiotic Strains
			3.2 Determination of MPO Activity
				3.2.1 Extraction of Extracellular Protein
				3.2.2 Extraction of Intracellular Proteins and Determination of MPO Activity
			3.3 Positive Control
		4 Inference
		5 Notes
		References
	Chapter 39: Assessment of Bacterial Translocation Through Mesenteric Lymph Nodes [MLN] and Spleen Cultures
		1 Introduction
			1.1 Measures of Bacterial Translocation
		2 Materials
			2.1 Reagent Preparation
				2.1.1 Phosphate Buffered Saline (PBS) (1x, pH 7.4)
			2.2 Reagents for Gram Staining
				2.2.1 Gram Crystal Violet Solution
				2.2.2 Gram Iodine Solution
				2.2.3 Gram Decolorizer Solution
				2.2.4 Gram Safranin Solution
			2.3 Preparation of Alcohol Gradient (for 1 L)
			2.4 Bacteriological Media Preparation
			2.5 Preparation of Buffers for DNA Isolation (Using QIAamp DNA Mini Kit)
			2.6 Primer Designing
			2.7 Composition of PCR Reaction Mix
		3 Methods
			3.1 Animal Preparation
			3.2 Microbiological Analysis from Blood
			3.3 Microbiological Analysis from Tissue
			3.4 Histological Analysis
				3.4.1 Paraffin Embedding and Sectioning
			3.5 Detection and Identification of Bacterial DNA
				3.5.1 DNA Isolation
				3.5.2 DNA Amplification
				3.5.3 16S rRNA Sequencing and Data Analysis
		References
Index