Title Page
Contents
ABSTRACT 12
Chapter 1. Literature review 16
1.1. Introduction 17
1.2. Kefir 19
1.3. Types of kefir microorganisms 21
1.3.1. Kefir lactic acid bacteria 21
1.3.2. Kefir yeast 25
1.3.3. Kefir acetic acid bacteria 28
1.3.4. Symbiosis of kefir microorganisms 30
1.4. Functional properties of K. marxianus 32
1.4.1. Survivability, growth potential, and cell adhesion activity 32
1.4.2. Anti-microbial activity 33
1.4.3. Anti-inflammatory and antioxidant activities 35
1.4.4. Anti-carcinogenic activity 37
1.5. Conclusion 39
1.6. References 41
Chapter 2. Survivability of Kluyveromyces marxianus isolated from kefir in a simulated gastrointestinal environment 55
2.1. Introduction 57
2.2. Materials and Methods 60
2.2.1. Experimental design 60
2.2.2. Isolation of yeast strains from kefir 62
2.2.3. DNA extraction 62
2.2.4. Identification of yeast strains 63
2.2.5. Evaluation of survivability/growth potential in simulated gastrointestinal tract conditions 63
2.2.6. Biochemical analysis of yeast strains 66
2.2.7. Evaluation of factors affecting survival in the host gastrointestinal tract 66
2.2.8. Statistical analysis 68
2.3. Results 70
2.3.1. Yeast strain identification 70
2.3.2. Survival in artificial gastrointestinal tract fluid 70
2.3.3. Biochemical activities 72
2.3.4. Evaluation of factors affecting survival in the host gastrointestinal tract 77
2.4. Discussion 83
2.5. Conclusion 89
2.6. References 90
Chapter 3. A combined in vitro and in vivo assessment of the safety of the yeast strains Kluyveromyces marxianus A4 and A5 isolated from kefir 98
3.1. Introduction 100
3.2. Materials and Methods 103
3.2.1. Yeast and bacterial strains 103
3.2.2. Identification of yeast strains 103
3.2.3. Virulence attributes of yeast strains 104
3.2.4. Animals and experimental design 105
3.2.5. Blood analysis and visual observation of the liver and spleen 107
3.2.6. Statistical analysis 108
3.3. Results 109
3.3.1. Yeast identification 109
3.3.2. In vitro evaluation of virulence attributes of yeast strains 111
3.3.3. Visual observations, changes in body weight, and food and water intake of mice 113
3.3.4. Organ measurement 115
3.3.5. Blood parameters 118
3.4. Discussion 123
3.5. Conclusion 127
3.6. References 128
Chapter 4. Gut microbiota modulation via short-term administration of potential probiotic kefir yeast Kluyveromyces marxianus A4 and A5 in Balb/c mice 136
4.1. Introduction 138
4.2. Materials and Methods 140
4.2.1. Yeast strains 140
4.2.2. Cells and cell culture 140
4.2.3. Cell adhesion assay 140
4.2.4. Animals and experimental design 141
4.2.5. Gut microbiota analysis 145
4.2.6. Statistical analysis 145
4.3. Results 147
4.3.1. Adhesion ability of yeast strains to Caco-2 cells 147
4.3.2. Modulation of gut microbiota 149
4.4. Discussion 155
4.5. Conclusion 159
4.6. References 160
Chapter 5. Synergistic anti-inflammatory effects of a novel β-glucan rich kefir yeast Kluyveromyces marxianus A4 with sulfasalazine by modulating the gut microbiome in a mouse model of colitis 166
5.1. Introduction 169
5.2. Materials and Methods 172
5.2.1. Preparation of yeast strains 172
5.2.2. Measurement of β-glucan 172
5.2.3. Assessment of anti-inflammatory effects in mice with DSS-induced colitis 173
5.2.4. Hematological analysis 176
5.2.5. Histological evaluation 176
5.2.6. Reverse-transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) 179
5.2.7. Gut microbiota analysis using next-generation sequencing (NGS) 181
5.2.8. Statistical analysis 181
5.3. Results 183
5.3.1. β-glucan content of Km A4 and Sb MYA-796 183
5.3.2. General animal observations 185
5.3.3. Blood parameters 187
5.3.4. Histological findings and scoring of colon tissues 190
5.3.5. qPCR analysis of the gene expression levels of cytokine in the spleen 193
5.3.6. qPCR analysis of the gene expression levels of Cox-2, inos, Zo-1, Ocln, and Cldn-1 in the distal colon 196
5.3.7. Modulation of the gut microbiota 199
5.3.8. Correlation analysis among colitis biomarkers 204
5.4. Discussion 206
5.5. Conclusions 212
5.6. References 214
Abstract (in Korean) 221
〈Table 1-1〉 Types of kefir lactic acid bacteria. 22
〈Table 1-2〉 Kefir lactic acid bacteria and their functions. 23
〈Table 1-3〉 Types of kefir yeasts. 26
〈Table 1-4〉 Various kefir yeasts and their functions. 27
〈Table 1-5〉 Types of kefir acetic acid bacteria. 29
〈Table 1-6〉 Synergistic effects of kefir microorganisms. 31
〈Table 2-1〉 Composition of the artificial gastrointestinal fluids. 65
〈Table 2-2〉 Biochemical activities of the five Kluyveromyces marxianus strains isolated from kefir and Saccharomyces boulardii ATCC MYA-796 analyzed using the VITEK® 2 system.[이미지참조] 73
〈Table 2-3〉 Selected biochemical activities of the five Kluyveromyces marxianus strains isolated from kefir and Saccharomyces boulardii ATCC MYA-796 differing at the species or strain levels, as analyzed using the VITEK® 2 system.[이미지참조] 76
〈Table 2-4〉 Adhesion properties of Kluyveromyces marxianus strains isolated from kefir compared with that of Saccharomyces boulardii ATCC MYA-796. 78
〈Table 3-1〉 Physical parameters and food and water intakes of the negative control (NC) and experimental groups (NC_Saline, Km A4_H, Km A4_L, Km A5_H, Km A5_L, Sb MYA-796_H, and Sb MYA-796_L) administered 0.9% saline, Kluyveromyces marxianus... 114
〈Table 3-2〉 Weight and length of the spleen and liver of mice in the negative control (NC) and experimental groups (NC_Saline, Km A4_H, Km A4_L, Km A5_H, Km A5_L, Sb MYA-796_H, and Sb MYA-796_L) administered 0.9% saline, Kluyveromyces marxianus... 116
〈Table 3-3〉 Hematological parameters of the negative control (NC) and experimental groups (NC_Saline, Km A4_H, Km A4_L, Km A5_H, Km A5_L, Sb MYA-796_H, and Sb MYA-796_L) administered 0.9% saline, Kluyveromyces marxianus A4, K. marxianus A5,... 119
〈Table 3-4〉 Plasma biochemical parameters of the negative control (NC) and experimental groups (NC_Saline, Km A4_H, Km A4_L, Km A5_H, Km A5_L, Sb MYA-796_H, and Sb MYA-796_L) administered 0.9% saline, Kluyveromyces marxianus A4, K. marxianus... 121
〈Table 5-1〉 Scores for the histological evaluation of colitis. 178
〈Table 5-2〉 List of the primers used to evaluate gene expression. 180
〈Table 5-3〉 β-glucan content of Kluyveromyces marxianus A4 and Saccharomyces boulardii ATCC MYA-796. The content was determined using an enzymatic yeast β-... 184
〈Table 5-4〉 Hematological analysis of mice with 3% dextran sulfate sodium (DSS)-induced colitis administered sterilized saline, sulfasalazine, Kluyveromyces marxianus A4 (Km A4), Km A4 plus sulfasalazine, Saccharomyces boulardii ATCC MYA-796 (Sb... 188
〈Table 5-5〉 Histological score of inflammation severity, inflammation extent, and crypt damage in mice with dextran sulfate sodium (DSS)-induced colitis treated with... 192
〈Table 5-6〉 Significant relative taxonomy abundance ratio of the gut microbiota in mice with dextran sulfate sodium (DSS)-induced colitis at the phylum, class, order, family, and genus levels via 16S rDNA amplicon-based community analysis. 202
〈Figure 1-1〉 Physical appearance of a typical kefir milk and grains. (A) Kefir milk, (B) freeze dried kefir grain, and (C) fresh kefir grain. 20
〈Figure 1-2〉 Schematic representation of the functional properties of Kluyveromyces marxianus derived from kefir. 40
〈Figure 2-1〉 Flowchart of the experimental procedures in the present study. 61
〈Figure 2-2〉 Changes in the number of viable cells of the five Kluyveromyces marxianus strains isolated from kefir and Saccharomyces boulardii ATCC MYA-796... 71
〈Figure 2-3〉 (A) Biofilm formation and (B) auto-aggregation analysis of Kluyveromyces marxianus strains compared with those of Saccharomyces boulardii... 80
〈Figure 2-4〉 Effect of phenol concentration on the growth of the Kluyveromyces marxianus strains and Saccharomyces boulardii ATCC MYA-796. (A) The 0.2%... 82
〈Figure 3-1〉 Identification result of Kluyveromcyes marxianus A4 and K. marxianus A5 isolated from kefir using the VITEK® 2 system.[이미지참조] 110
〈Figure 3-2〉 Virulence attributes of Kluyveromyces marxianus A4 (Km A4) and K. marxianus A5 (Km A5). (A) Gelatin hydrolysis activity of Staphylococcus aureus... 112
〈Figure 3-3〉 Comparison of visual appearance in the spleen and liver of the negative control (NC) and experimental groups (NC_Saline, Km A4_H, Km A4_L, Km A5_H, Km A5_L, Sb MYA-796_H, and Sb MYA-796_L) administered 0.9% saline,... 117
〈Figure 3-4〉 Median fluorescence intensity (MFI) of plasma cytokines of the negative control (NC) and experimental groups (NC_Saline, Km A4_H, Km A4_L, Km A5_H, Km A5_L, Sb MYA-796_H, and Sb MYA-796_L) administered 0.9% saline,... 122
〈Figure 4-1〉 Schematic diagram of the animal experiment. 144
〈Figure 4-2〉 Adhesion ability of kefir yeast strains (Kluyveromyces marxianus A4 and K. marxianus A5) and Saccharomyces boulardii ATCC MYA-796 to colorectal... 148
〈Figure 4-3〉 Alpha diversity indices of the gut microbiota using Balb/c mouse fecal samples. There are no significant differences among the alpha diversity indices using... 151
〈Figure 4-4〉 Beta diversity of gut microbiota using Balb/c mouse fecal samples. Weighted UniFrac principal coordinate analysis (PCoA) results of (A) all groups and (B) the compared groups (negative control group and high concentrations of Kluyveromyces... 152
〈Figure 4-5〉 Effect of Kluyveromyces marxianus A4, K. marxianus A5 and Saccharomyces boulardii ATCC MYA-796 on the Balb/c mouse fecal microbiota. Relative taxonomy abundance of the gut microbiota at the (A) phylum, (B) order, and (C) genus level. 153
〈Figure 4-6〉 Heatmap of the significant differences in gut microbiota at the phylum, order, and genus levels. The heatmap represents the relative abundance of gut... 154
〈Figure 5-1〉 Experimental framework for evaluating the anti-inflammatory effects of combined treatment with Kluyveromyces marxianus A4 and sulfasalazine compared to that of Saccharomyces boulardii ATCC MYA-796 in mice with dextran sulfate sodium... 175
〈Figure 5-2〉 Effects of sterilized saline, sulfasalazine, Kluyveromyces marxianus A4 (Km A4), Km A4 plus sulfasalazine, Saccharomyces boulardii ATCC MYA-796 (Sb... 186
〈Figure 5-3〉 Hematoxylin and eosin staining in mice colon tissue with dextran sulfate sodium (DSS)-induced colitis treated with sterilized saline, sulfasalazine,... 191
〈Figure 5-4〉 Effects of sterilized saline, sulfasalazine, Kluyveromyces marxianus A4 (Km A4), Km A4 plus sulfasalazine, Saccharomyces boulardii ATCC MYA-796 (Sb MYA-796), and Sb MYA-796 plus sulfasalazine on the mRNA expression of (A) Il-... 195
〈Figure 5-5〉 Effect of sterilized saline, sulfasalazine, Kluyveromyces marxianus A4 (Km A4), Km A4 plus sulfasalazine, Saccharomyces boulardii ATCC MYA-796 (Sb MYA-796), and Sb MYA-796 plus sulfasalazine on the mRNA expression of (A) Cox-... 197
〈Figure 5-6〉 Effects of sterilized saline, sulfasalazine, Kluyveromyces marxianus A4 plus sulfasalazine, and Saccharomyces boulardii ATCC MYA-796 plus sulfasalazine on the beta diversity of the gut microbiota. (A) Weighted and (B) unweighted UniFrac principal... 201
〈Figure 5-7〉 Effects of sterilized saline, sulfasalazine, Kluyveromyces marxianus A4 sulfasalazine, and Saccharomyces boulardii ATCC MYA-796 plus sulfasalazine on the significantly different relative taxonomy abundances of the gut microbiota. (A) Heatmap... 203
〈Figure 5-8〉 Pearson correlation analysis of the significantly different physical parameters, mRNA gene expression levels of cytokines, inflammatory mediators,... 205
〈Figure 5-9〉 Proposed mechanism for the anti-inflammatory activity of Kluyveromyces marxianus A4 in dextran sulfate sodium-induced colitis mice. 213