Title Page
Contents
ABSTRACT 17
I. INTRODUCTION 19
1. Inflammation 19
2. Cytokines 22
3. Microbiome 26
4. Lactic acid-producing bacteria (LAB) 29
5. Probiotics 34
6. Limosilactobacillus and Lactocaseibacillus 42
7. Limosilactobacillus fermentum PL9988 45
8. Lacticaseibacillus rhamnosus PL60 46
II. Material and Methods 47
1. Preparation of LABs for experiments 47
1.1. Preparation of Limosilactobacillus fermentum PL9988 and Lacticaseibacillus rhamnosus PL60 47
1.2. Preparation of heat-killed Limosilactobacillus fermentum PL9988 and Lacticaseibacillus rhamnosus PL60 47
2. Minimal inhibitory concentration (MIC) assay 48
3. Hemolysis 51
4. Growth inhibition of intestinal pathogens 51
5. Self-aggregation check 52
6. Toxigenicity assay 53
7. Acid tolerance 54
8. Bile tolerance 54
9. Bile-salt conjugation 55
10. In vitro Binding Assay of LABs to HT-29 Cells 55
10.1. Observation of live or heat-killed LABs attached to HT-29 cells 55
10.2. Assay of live LABs attached to HT-29 cells 56
11. Optimum treatment time of LPS to HT-29 cells 57
12. Treatment of HT-29 Cells with L. fermentum PL9988 or L. rhamnosus PL60 for cytokine assay 58
13. Cytokine assay related to inflammation in in vitro experiment 58
14. Preparation of mouse for in vivo experiment 59
15. Examination of L. fermentum PL9988 and L. rhamnosus PL60 attached to mouse intestines 60
16. Assay of cytokines related to inflammation in in vivo experiment 61
17. Statistical analysis 61
III. Results 62
1. Minimal inhibitory concentration (MIC) assay 62
2. Hemolysis 64
3. Growth inhibition of intestinal pathogens 64
4. Self-aggregation 67
5. Toxigenicity assay 67
6. Acid and bile tolerance 69
7. Bile-salt conjugation 69
8. Optimum LPS treatment time 69
9. Binding of L. fermentum PL9988 and L. rhamnosus PL60 to HT-29 cells 71
10. Binding of L. fermentum PL9988 and L. rhamnosus PL60 to Mice intestines 74
11. IL-10 assay of the culture supernatant of HT-29 cells treated with L. fermentum PL9988 and L. rhamnosus PL60 77
12. TNF-α assay in the spent culture supernatant of HT-29 cells treated with Limosilactobacillus fermentum PL9988 and L. rhamnosus PL60 80
13. IL-10 assay in serum of mice treated with L. fermentum PL9988 and L. rhamnosus PL60 83
14. TNF-α Assay in Serum of Mice Treated with L. fermentum PL9988 and L. rhamnosus PL60 86
IV. Discussion 89
References 98
국문초록 123
Table 1. List of commonly assayed cytokines 25
Table 2. Lactic acid bacteria used in fermented food products 31
Table 3. List of genus/species for probiotic in Korean Food Standards Codex (KFDA) 38
Table 4. Bacterial cut-off values 50
Table 5. Minimal inhibitory concentration (MIC) of Limosilactobacillus fermentum PL9988 and Lacticaseibacillus rhamnosus PL60 63
Table 6. Growth inhibition of intestinal pathogens by Limosilactobacillus fermentum PL9988 and Lacticaseibacillus... 66
Table 7. The MTT assay of Limosilactobacillus fermentum PL9988 and Lacticaseibacillus rhamnosus PL60 with HT-29 cells 68
Table 8. Acid tolerance and Bile tolerance of Limosilactobacillus fermentum PL9988 and Lacticaseibacillus rham nosus PL60 (CFU) 70
Figure 1. Enriched taxa at various niches of the human body in diverse populations around the world. 28
Figure 2. Timeline of selected milestones in the history of probiotics and next-generation probiotics. 36
Figure 3. Hemolysis test with Limosilactobacillus fermentum PL9988 and Lacticaseibacillus rhamnosus PL60. 65
Figure 4. Limosilactobacillus fermentum PL9988 attached to HT-29 cells. 72
Figure 5. Lacticaseibacillus rhamnosus PL60 attached to HT-29 cells. 73
Figure 6. Limosilactobacillus fermentum PL9988 attached to mouse intestinal epithelial cells. 75
Figure 7. Lacticaseibacillus rhamnosus PL60 attached to mouse intestines. 76
Figure 8. Concentration of IL-10 in the culture supernatant of HT-29 cells after treated with LPS and L. fermentum PL9988. 78
Figure 9. IL-10 concentration in the culture supernatant of HT-29 cells after treated with LPS and L. rhamnosus PL60. 79
Figure 10. Concentration of TNF-α in the spent culture supernatant of HT-29 cell line after treated with LPS and L. fermentum PL9988. 81
Figure 11. TNF-α concentration in the spent culture supernatant of HT-29 cells after treated with LPS and L. rhamnosus PL60. 82
Figure 12. Concentration of IL-10 in mouse serum after L. fermentum PL9988 treatment. 84
Figure 13. IL-10 assay in mouse serum after treated with L. rhamnosus PL60. 85
Figure 14. Concentration of TNF-α in mouse serum after L. fermentum PL9988 treatment. 87
Figure 15. TNF-α assay in mouse serum after treated with L. rhamnosus PL60. 88