Title Page
Contents
Abstract 14
Abbreviation 19
Chapter Ⅰ. Probiotic properties of Lacticaseibacillus rhamnosus HMP013 and HMP014 20
Ⅰ. Introduction 20
1. Beneficial effects of Lacticaseibacillus rhamnosus as probiotics 20
2. Purpose of this study 30
Ⅱ. Materials and methods 31
1. Screening and identification 31
2. Analysis of probiotic properties 38
3. Statistical analysis 43
Ⅲ. Results and discussion 44
1. Screening and identification 44
2. Probiotic properties 60
Ⅳ. Conclusion 76
Chapter Ⅱ. Protective effects of Lacticaseibacillus rhamnosus HMP013 and HMP014 on LPS-stimulated human colon epithelial cells 78
Ⅰ. Introduction 78
1. Protective effects of Lactobacillus strains in intestinal epithelial cells 78
2. Purpose of this study 81
Ⅱ. Materials and methods 83
1. Sample preparation 83
2. Cell culture 84
3. Analysis of protective effects in LPS-stimulated human colon epithelial cells 85
4. Statistical analysis 89
Ⅲ. Results and discussion 90
1. Protective effects in LPS-stimulated human colon epithelial cells 90
Ⅳ. Conclusion 108
Chapter Ⅲ. Anti-inflammatory and antimicrobial effects of s Lacticaseibacillus rhamnosus on bacterial vaginosis 110
Ⅰ. Introduction 110
1. The role of Lactobacillus strains in vaginal health 110
2. Purpose of this study 118
Ⅱ. Materials and methods 119
1. Bacterial strains culture condition 119
2. Analysis of antioxidant activity of cell-free supernatant 124
3. Analysis of anti-inflammatory effects in LPS-induced RAW 264.7 macrophages 126
4. Analysis of antimicrobial effects of cell-free supernatant 133
5. Statistical analysis 137
Ⅲ. Results and discussion 138
1. Antioxidant effects of Lacticaseibacillus rhamnosus HMP013 and HMP014 138
2. Anti-inflammatory effects in RAW 264.7 macrophages 144
3. Antimicrobial effects of s Lacticaseibacillus rhamnosus HMP013 and HMP014 157
Ⅳ. Conclusion 169
References 172
국문요지 200
Table 1. Recent studies on the efficacy of Lacticaseibacillus rhamnosus strains in both in vitro and in vivo animal models 25
Table 2. Media compositions 33
Table 3. List of indicators on antimicrobial activity 34
Table 4. Oligonucleotide primers for sequence analysis 37
Table 5. Screening of lactic acid bacteria with antimicrobial activities 46
Table 6. Screening of lactic acid bacteria with antimicrobial activities against multidrug-resistant bacteria 47
Table 7. Identification of lactic acid bacteria isolated from human breast milk with antimicrobial activities against multidrug-resistant... 56
Table 8. Carbon-source utilization of Lacticaseibacillus rhamnosus HMP013 and HMP014 by API 50 CHL 57
Table 9. Comparison of enzyme activity of Lacticaseibacillus rhamnosus HMP013 and HMP014 by API ZYM 58
Table 10. Antibiotic susceptibility of Lacticaseibacillus rhamnosus HMP013 and HMP014 74
Table 11. Primer sequences used in real-time qPCR 88
Table 12. Recent studies for inhibitory effects of various Lactobacillus strains on bacterial vaginosis 117
Table 13. Media compositions 120
Table 14. List of indicators on antimicrobial activity 121
Table 15. Primer sequences used in real-time qPCR 132
Table 16. Determination of the MIC and MBC of Lacticaseibacillus rhamnosus HMP013 and HMP014 163
Table 17. Determination of the MIC and MBC of Lacticaseibacillus rhamnosus HMP013 and HMP014 against multidrug-resistant bacteria 164
Figure 1. Gut-brain axis. 22
Figure 2. Neighbor-Joining phylogenetic tree of Lacticaseibacillus rhamnosus HMP013 and HMP014. 55
Figure 3. Growth profile of Lacticaseibacillus rhamnosus HMP013 and HMP014. 59
Figure 4. Survival rate in acidic condition and bile salts of Lacticaseibacillus rhamnosus HMP013 and HMP014. 62
Figure 5. Auto-aggregation of Lacticaseibacillus rhamnosus HMP013 and HMP014. 65
Figure 6. Hydrophobicity of Lacticaseibacillus rhamnosus HMP013 and HMP014. 66
Figure 7. Adhesion ability to HT-29 intestinal epithelial cells of Lacticaseibacillus rhamnosus HMP013 and HMP014. 69
Figure 8. Cell viability in HT-29 intestinal epithelial cells. 71
Figure 9. Hemolytic phenotypes of Lacticaseibacillus rhamnosus HMP013 and HMP014 compared with positive controls. 75
Figure 10. Cell viability in HT-29 intestinal epithelial cells. 91
Figure 11. mRNA expression levels of a pro-inflammatory cytokine gene (IL-8) in LPS-stimulated HT-29 intestinal epithelial cells. 94
Figure 12. mRNA expression levels of a pro-inflammatory cytokine gene (IFN-γ) in LPS-stimulated HT-29 intestinal epithelial cells. 95
Figure 13. mRNA expression levels of an anti-inflammatory cytokine gene (IL-10) in LPS-stimulated HT-29 intestinal epithelial cells. 98
Figure 14. mRNA expression levels of an anti-inflammatory cytokine gene (IFN-α) in LPS-stimulated HT-29 intestinal epithelial cells. 99
Figure 15. mRNA expression levels of COX-1 gene in LPS-stimulated HT-29 intestinal epithelial cells. 102
Figure 16. mRNA expression levels of COX-2 gene in LPS-stimulated HT-29 intestinal epithelial cells. 103
Figure 17. mRNA expression levels of HSP70 gene in LPS-stimulated HT-29 intestinal epithelial cells. 106
Figure 18. mRNA expression levels of MUC2 gene in LPS-stimulated HT-29 intestinal epithelial cells. 107
Figure 19. The role of live lactic acid bacteria in the genital tract against vaginitis. 114
Figure 20. DPPH and ABTS radical scavenging activity. 140
Figure 21. Superoxide dismutase-like activity. 143
Figure 22. Cell viability in LPS-induced RAW 264.7 macrophages. 145
Figure 23. Inhibition activity of NO production in LPS-induced RAW 264.7 macrophages. 148
Figure 24. mRNA expression levels of a pro-inflammatory cytokine gene (TNF-α) in LPS-induced RAW 264.7 cells. 152
Figure 25. mRNA expression levels of a pro-inflammatory cytokine gene (iNOS) in LPS-induced RAW 264.7 cells. 153
Figure 26. mRNA expression levels of a pro-inflammatory cytokine gene (COX-2) in LPS-induced RAW 264.7 cells. 154
Figure 27. mRNA expression levels of a pro-inflammatory cytokine gene (IL-6) in LPS-induced RAW 264.7 cells. 155
Figure 28. mRNA expression levels of a pro-inflammatory cytokine gene (IL-1β) in LPS-induced RAW 264.7 cells. 156
Figure 29. RP-HPLC chromatograms of lactic acid. 160
Figure 30. RP-HPLC chromatograms of bacteriocin-like substance. 161
Figure 31. Release of DNA. 166
Figure 32. Scanning electron microscope images. 168