Title Page
Summary
Contents
List of Abbreviations 18
Chapter 1. Review of Literature 21
1. Probiotics and eukaryotic probiotics 22
1-1. Probiotics 22
1-2. Eukaryotic Probiotics 24
1-3. Saccharomyces cerevisiae 30
2. IBD 33
2-1. IBD 33
2-2. Pathogenesis of IBD 36
3. Saccharomyces cerevisiae as Eukaryotic Probiotics in the Management of IBD 43
3-1. Medical Treatment for IBD 43
3-2. Probiotics for IBD treatment 45
3-3. Saccharomyces cerevisiae as eukaryotic probiotics in the management of IBD 48
Chapter 2. Probiotic potential of Saccharomyces cerevisiae GILA with alleviating intestinal inflammation in a dextran sulfate sodium induced colitis mouse model 50
1. Introduction 51
2. Materials and Methods 54
2-1. Isolation and culture conditions 54
2-2. Resistance to low pH and bile tolerance 56
2-3. Autoaggregation and coaggregation with pathogen 57
2-4. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging effect 58
2-5. Hydrophobicity 58
2-6. Hemolytic activity and biogenic amine production 59
2-7. ITS region sequencing and Phylogenetic analysis 61
2-8. In vitro gastrointestinal tract (GIT) models 62
2-9. Adhesion assay 64
2-10. Determination of nitric oxide production 65
2-11. ex vivo Experimental design 66
2-12. in vivo Experimental design 67
2-13. Colitis evaluation 68
2-14. RNA isolation and qRT-PCR 69
2-15. ELISA 70
2-16. Fermentative profiling 70
2-17. Cell wall β-glucan 71
2-18. Statistical analysis 71
3. Results 72
3-1. Isolation of yeasts 72
3-2. Resistance to low pH and bile tolerance 73
3-3. Auto aggregation and coaggregation with pathogens 75
3-4. Antioxidant ability by DPPH assay 78
3-5. Safety assessments 79
3-6. Identification and phylogenetic analysis of the ITS region 81
3-7. Survival rate in the GIT model 83
3-8. Adhesion to Caco-2 cell 84
3-9. Alleviating the inflammation in RAW 264.7 cells 85
3-10. Alleviating the intestinal inflammation in a dextran sulfate sodium-induced colitis mouse model 87
3-11. Fermentative profiles 93
3-12. Cell wall β-glucan content 94
4. Supplementary Figures 95
5. Discussion 98
Chapter 3. Cell wall component of Saccharomyces cerevisiae GILA 118 alleviates intestinal inflammation in a dextran sulfate sodium induced colitis mouse model 103
1. Introduction 104
2. Materials and Methods 107
2-1. The isolation of cell wall component 107
2-2. The protoplast isolation 109
2-3. in vitro RAW 264.7 cell assay 111
2-4. in vivo Experimental Design for Yeast cell components 112
2-5. qRT-PCR and ELISA 113
2-6. Histology 115
2-7-1. Isolation of Single cell 116
2-7-2. Phenotypic and Functional Examination of Immune Cells by Using Flow Cytometry 116
2-8-1. DNA extraction from the fecal sample 118
2-8-2. Gut microbiota analysis 118
2-9. Statistical analysis 119
3. Results 120
3-1. S. cerevisiae GILA 118 cell wall component administration reduced inflammatory biomarkers in murine macrophage RAW 264.7 cells 120
3-2. S. cerevisiae GILA 118 PZ administration alleviated intestinal inflammation in mice compared to PS and PP 122
3-3. S. cerevisiae GILA 118 PZ administration alleviated intestinal inflammation in mice compared to PS and PP by increasing IL-10 127
3-4. S. cerevisiae GILA 118 PZ shown more glucose residue than S. cerevisiae GILA 118 PS 131
3-5. S. cerevisiae GILA 118 PZ administration alleviated intestinal inflammation by regulating the Th17/Treg cell balance in spleen 133
3-6 Comparison of the influence to the gut microbiota on GILA 118 live cells and PZ in mouse feces 136
4. Supplementary Figures 147
5. Discussion 149
Chapter 4. Conclusion 155
Reference 158
Abstract in Korean 184
Chapter 1. Review of Literature 16
Table 1-1. Comparison between bacteria and yeast as probiotic implication. 23
Table 1-2. Commercial Eukaryotic Probiotic Product. 26
Table 1-3. Foods formulated with commercial Eukaryotic Probiotic. 27
Table 1-4. Medical Treatment for IBD. 44
Chapter 2. Probiotic potential of Saccharomyces cerevisiae GILA with alleviating intestinal inflammation in a dextran sulfate sodium induced colitis mouse model 16
Table 2-1. Component of decarboxylase medium. 60
Table 2-2. The solution, mixture, pH for in vitro gastrointestinal tract (GIT) models. 62
Table 2-3. Scoring of the disease activity index (DAI) 68
Table 2-4. Gene primer sequences. 69
Table 2-5. Coaggregation of yeast isolates with pathogens (n=33). 77
Table 2-6. Safety assessments of isolated yeast (n=7) 80
Supplementary Table 2-1. Hydrophobicity of yeast isolates 96
Chapter 3. Cell wall component of Saccharomyces cerevisiae GILA 118 alleviates intestinal inflammation in DSS-induced colitis mice model 16
Table 3-1. List of primer sequences 114
Table 3-2. Scoring scheme for DSS-induced colonic inflammation. 115
Chapter 1. Review of Literature 12
Figure 1-1. The molecular structure of soluble and insoluble yeast β-glucan. 31
Figure 1-2. S. cerevisiae Morphology, Cell wall structure and Life cycle. 32
Figure 1-3. The global prevalence of IBD in 2019. 35
Figure 1-4. IBD pathogenesis includes neutrophil, MPO infiltration, inflammatory cytokine, tight junction protein downregulation,... 42
Figure 1-5. Action of probiotics in therapy for IBD. 46
Chapter 2. Probiotic potential of Saccharomyces cerevisiae GILA with alleviating intestinal inflammation in a dextran sulfate sodium induced colitis mouse model 12
Figure 2-1. Scheme for isolation of yeast. 55
Figure 2-2. Overview of in vivo screening. 67
Figure 2-3. Gram positive yeasts are confirmed by a yeast smear's Gram staining. 72
Figure 2-4. Screening of yeast with over 90% acid tolerance (n=82) and bile tolerance (n=74). 74
Figure 2-5. Screening of yeast with over 80% auto aggregation (n=63). 76
Figure 2-6. Screening of yeast with over 85% DPPH scavenging activity (n=21). 78
Figure 2-7. Screening of yeast with growth characteristics comparable to S. boulardii CNCM I-745 (n=12). 79
Figure 2-8. The phylogenetic tree of S. cerevisiae GILA. Based on ITS region sequencing. 82
Figure 2-9. The survival rate of selected S. cerevisiae GILA strains at (a) mouth, (b) stomach and (c) intestine in the GIT model. 83
Figure 2-10. Adhesion percentages of S. cerevisiae strain on caco-2 cells. 84
Figure 2-11. Nitric oxide production of heat-killed S. boulardii and S. cerevisiae strains in LPS (1 µg/ml) induced RAW 264.7 murine... 86
Figure 2-12. in vivo Screening of S. cerevisiae GILA strain. 89
Figure 2-13. Alleviation of Intestinal Inflammation in Mice between Treatments of S. cerevisiae GILA. 92
Figure 2-14. Fermentative profiles of S. cerevisiae GILA. 93
Figure 2-15. Cell wall β-glucan content of S. cerevisiae GILA strains. 94
Supplementary Figure 2-1. Mouse IFN-γ expression by splenocyte from Balb/c mouse. 95
Supplementary Figure 2-2. The probiotic characteristics of seven S. cerevisiae GILA, isolated from Korean fermented beverage. 97
Chapter 3. Cell wall component of Saccharomyces cerevisiae GILA 118 alleviates intestinal inflammation in a dextran sulfate sodium induced colitis mouse model 13
Figure 3-1. Schematic diagram protoplast (PP), cell wall polysaccharide (PS), and cell wall polysaccharide treated with zymolyase (PZ) isolated... 110
Figure 3-2. The anti-inflammatory effect of S. cerevisiae GILA 118 cell wall component on RAW 264.7 cells. 121
Figure 3-3. S. cerevisiae GILA 118 PZ alleviated intestinal inflammation in mice compared to PP and PS. 123
Figure 3-4. S. cerevisiae GILA 118 PZ alleviated intestinal inflammation in distal colon in DSS-induced colitis mice 126
Figure 3-5. S. cerevisiae GILA 118 PZ alleviated the intestinal inflammation in mice and increased IL-10 in serum. 130
Figure 3-6. The structure of S. cerevisiae GILA 118 PS (A) and PZ (B) using FT-IR and H NMR. 132
Figure 3-7. S. cerevisiae GILA 118 PZ alleviated the intestinal inflammation by regulating Th17/Treg cell balance. 135
Figure 3-8-1. GILA 118 live cells and PZ-associated bacterial taxa in mouse feces 137
Figure 3-8-2. Relative abundance of specific genus modulated after live cells and PZ administration. 139
Figure 3-8-3. Alpha diversity after live cells and PZ administration. 141
Figure 3-8-4. Beta diversity of tested groups. 143
Figure 3-8-5. Relative abundance of specific Lactobacillus species modulated after live cells and cell wall components administration. 146
Supplementary Figure 3-1. Percentage of Treg, IL-17 producing cells in CD4⁺ T cells and IL-17 producing cells/Treg from (A) mLN and (B) PP. 147
Supplementary Figure 3-2. (A) Gating strategy for neutrophils (B) Percentage of neutrophils from spleen. 148
Chapter 4. Conclusion 15
Figure 4. Graphical summary. 157