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Title Page

Contents

SUMMARY OF INDIVIDUAL EXPERIMENT 12

GENERAL INTRODUCTION 19

REVIEW OF LITERATURES 21

Feed Additives 21

Probiotics 21

Lactic acid bacteria 25

Bacteriophage 28

Gut Microbiota 30

Microbial Community Analysis 33

References 35

CHAPTER I. Effect of Fermenting Seaweed (Undaria pinnatifida) by-product with Lactic Acid Bacteria on Microbial Flora and Metabolites 39

Abstract 40

I. INTRODUCTION 43

II. Materials and Methods 46

1. Selective medium and isolation of lactic acid bacteria 46

2. Salt ranges for growth 47

3. Metabolite production 48

4. Antibacterial activities 48

5. Identification 49

6. Undaria pinnatifida by-product fermentation 50

7. Fermentation quality analysis 50

8. Predominant LAB 51

9. Barcoded Pyrosequencing, sequence processing and data analysis 52

10. Statistical methods 54

III. Results 55

1. Salt ranges for growth 55

2. Metabolite production 56

3. Antimicrobial activity 57

4. Identification 59

5. Fermentation quality 61

6. Predominant LAB 65

7. Bacterial and fungal diversity changes in SWBP feed during fermentation 67

8. Bacterial and fungal community changes in SWBP feed during fermentation 71

IV. Discussion 76

V. Conclusions 80

VI. References 81

CHAPTER II. Effect of Lactic Acid Bacteria Additives in Total Mixed Ration (TMR) on Fermentation Quality and in vitro Rumen Fermentation 85

Abstract 86

I. INTRODUCTION 88

II. Materials and Methods 90

1. Isolation, identification and metabolite production of microbes 90

2. Fermentation of TMR 91

3. Fermentation quality analysis 92

4. In vitro rumen fermentation 93

5. Parameters for analysis of in vitro rumen fermentation 95

6. Statistical methods 96

III. Results 97

1. Isolation and identification of LAB 97

2. Fermentation quality 98

3. Rumen fermentation in vitro parameters 100

IV. Discussion 104

V. Conclusion 107

VI. References 108

CHAPTER III. Effect of Bacteriophage Feed on Inhibition of Escherichia coli on in vitro Rumen Fermentation 111

Abstract 112

I. INTRODUCTION 114

II. Materials and Methods 116

1. Bacteriophage 116

2. Inhibition of bacterial growth by bacteriophage 117

3. In vitro rumen fermentation 117

3. Analysis of rumen fermentation in vitro parameters 119

4. Quantitative real-time PCR 121

5. Statistical methods 122

III. Results 123

1. Inhibition of bacterial growth by bacteriophage feed 123

2. Rumen fermentation in vitro parameters 124

3. Bacterial population 127

IV. Discussion 130

V. Conclusions 133

VI. References 134

CHAPTER IV. Effect of Dietary Fermented Sulfur Feed on Growth Performance and Gut Microbiota in Broiler Chickens 138

Abstract 139

I. INTRODUCTION 141

II. Materials and Methods 143

1. fermented Sulfur 143

2. Broilers Management and Experimental Design 144

3. Growth performance 144

4. Odorous compounds analysis 146

5. Blood parameters and immunoglobulin 146

6. Proximate composition, fatty acids composition of broier meat 147

7. Sampling and DNA extraction 148

8. Quantitative real-time PCR 148

9. Pyrosequencing analysis of gut microbiota 150

10. Statistical methods 152

III. Results 153

1. Growth performance 153

2. Concentration of odorous compounds in chicken building 155

3. Proximate composition and Fatty acid profiles of broiler meat 157

4. Blood profiles and immunoglobulin of chicken serum 159

5. Bacteria quantification in digesta of chicken 160

6. Bacterial diversity of the broiler gut microbiome 162

7. Bacterial community of the broiler gut microbiome 165

IV. Discussion 172

V. Conclusions 182

VI. References 183

국문초록 189

List of Tables

REVIEW OF LITERATURES 23

Table 1. Benefits of probiotics use in different type of animals 23

Table 2. DFM commonly used in animal feed 24

Table 3. The lactic acid bacteria fermentation type and products 26

Table 4. Bacteriophage type and life cycle 28

CHAPTER I. Effect of Fermenting Seaweed (Undaria pinnatifida) by-product with Lactic Acid Bacteria on Microbial Flora and Metabolites 47

Table 1. Culture media formula and preparation 47

Table 2. Organic acid concentration of isolated bacteria 57

Table 3. Inhibition of various gram positive, gram negative and yeast by 20-... 58

Table 4. Representative results of API 50 CHL system and 16S rRNA... 60

Table 5. Effects of additive treatment and time of fermentation on nutrient fermentation charcateristics of... 64

Table 6. The composition of dominant lactic acid bacteria during fermentation time of SWBP feed 66

Table 7. Summary of the barcoded pyrosequencing data sets and statistical analysis of the SWBP feed (bacteria) 68

Table 8. Summary of the barcoded pyrosequencing data sets and statistical analysis of the SWBP feed (fungi) 69

CHAPTER II. Effect of Lactic Acid Bacteria Additives in Total Mixed Ration (TMR) on Fermentation Quality and in vitro Rumen Fermentation 91

Table 1. Treatments of experiment 91

Table 2. Ingredient and chemical composition of total mixed rations without... 92

Table 3. 16S rRNA sequence analysis and organic acid concentration of... 97

Table 4. Fermentaion quality of TMR after 7 days of fermentation 99

Table 5. Total gas production, pH and ammonia-nitrogen production from... 102

Table 6. Volatile fatty acid production of in vitro fermentation by FTMR 103

CHAPTER III. Effect of Bacteriophage Feed on Inhibition of Escherichia coli on in vitro Rumen Fermentation 116

Table 1. Target of bacteriophage 116

Table 2. Experimental design for bacteriophage based theraphy in the... 119

Table 3. List of the real-time quantitative PCR primers and probes 122

Table 4. Total gas production, pH and NH₃-N concentration of in vitro... 125

Table 5. Volatile fatty acid (VFA) concentration of in vitro fermentation... 126

Table 6. Bacterial DNA copies from in vitro fermentation added with... 128

CHAPTER IV. Effect of Dietary Fermented Sulfur Feed on Growth Performance and Gut Microbiota in Broiler Chickens 143

Table 1. Ingredient and chemical composition of fermented sulfur 143

Table 2. Feed formula and chemical composition of experimental diets 145

Table 3. List of the real-time quantitative PCR primers and probes 150

Table 4. Body weight gain, feed intake, feed conversion rate and mortality... 154

Table 5. Summary of odorant concentration levels (ppb) measured from... 156

Table 6. Effects of dietary containing fermented sulfur on proximate... 158

Table 7. Effects of dietary containing fermented sulfur on blood profiles and... 159

Table 8. Effects of experimental diets on bacterial counts (log10 copies...(이미지참조) 161

Table 9. Summary of the barcoded pyrosequencing data set and statistical analysis of the broiler gut... 163

List of Figures

REVIEW OF LITERATURES 27

Figure 1. Major pathways active in lactic acid bacteria 27

Figure 2. Lytic or lysogenic cycles 29

Figure 3. Phylogenetic distribution of bacterial families in different GIT... 31

Figure 4. Abundance of glycoside hydrolase (GH) families in metagenomes... 32

Figure 5. Bacterial diversity is affected by ageing 34

CHAPTER I. Effect of Fermenting Seaweed (Undaria pinnatifida) by-product with Lactic Acid Bacteria on Microbial Flora and Metabolites 45

Figure 1. The pictures of brown algae 45

Figure 2. Growth response of isolated strains to different salt concentration 55

Figure 3. Changes in relative bacteria and fungi diversities shown through rarefaction analysis using gene... 70

Figure 4. Bacteria taxonomic compositions at the phylum level of SWBP feed during fermentation 73

Figure 5. Bacteria taxonomic compositions at the genus level of SWBP feed during fermentation 74

Figure 6. Fungal taxonomic compositions at the genus level of SWBP feed during fermentation 75

CHAPTER III. Effect of Bacteriophage Feed on Inhibition of Escherichia coli on in vitro Rumen Fermentation 123

Figure 1. Pathogenic microorganisms inhibition of bacteriophage feed 123

Figure 2. DNA copies of in vitro fermentation using rumen fluid inocula from bacteriophage feed 129

CHAPTER IV. Effect of Dietary Fermented Sulfur Feed on Growth Performance and Gut Microbiota in Broiler Chickens 164

Figure 1. Rarefaction analysis using bacteria 16S rRNA gene sequences... 164

Figure 2-1. Taxonomic composition at the phylum level of broiler gut microbiome 166

Figure 2-2. Taxonomic composition at the phylum level of broiler gut microbiome 167

Figure 3-1. Taxonomic composition at the family level of broiler gut microbiome 168

Figure 3-2. Taxonomic composition at the family level of broiler gut microbiome 169

Figure 4-1. Bacterial taxonomic composition of genus level in broiler gut microbiome 170

Figure 4-2. Bacterial taxonomic composition of genus level in broiler gut microbiome 171

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