Title Page
Contents
ABSTRACT 9
Chapter 1. Probiotic properties of Enterococcus faecium KU22001 isolated from human infant feces and its antioxidant and immune-enhancing activities of its heat-killed cells 11
1.1. Introduction 11
1.2. Material and Methods 14
1.2.1. Isolation and identification of LAB strains 14
1.2.2. Cell culture condition 14
1.2.3. Tolerance to artificial gastric juice and bile acid 15
1.2.4. Adhesion ability to HT-29 Cells 15
1.2.5. Antibiotic susceptibility 16
1.2.6. β-Glucuronidase activity 16
1.2.7. Hemolytic activity 17
1.2.8. D,L-Lactate production 17
1.2.9. Bile salt deconjugation 17
1.2.10. Indole production 18
1.2.11. Preparation of heat-killed LAB 18
1.2.12. DPPH radical scavenging assay 18
1.2.13. ABTS radical scavenging assay 19
1.2.14. β-Carotene bleaching inhibitory activity 19
1.2.15. Reducing power assay 20
1.2.16. Determination of ROS scavenging activity 20
1.2.17. Cell viability and NO production in RAW 264.7 cells 21
1.2.18. RNA extraction and semi-quantitative real-time RT-PCR 22
1.2.19. Western blotting 24
1.2.20. Statistical analysis 24
1.3. Results and discussion 25
1.3.1. Tolerance to artificial gastric juice and bile acid 25
1.3.2. Antibiotic susceptibility 27
1.3.3. β-Glucuronidase activity 29
1.3.4. Hemolytic activity 29
1.3.5. D,L-Lactate production 29
1.3.6. Bile salt deconjugation 30
1.3.7. Indole production 30
1.3.8. Antioxidant effects 32
1.3.9. ROS scavenging activity 34
1.3.10. Cell viability and NO production in RAW 264.7 cells 36
1.3.11. Expression of immune-associated genes 38
1.3.12. Protein expression of MAPK pathway components 40
1.4. Conclusion 42
Chapter 2. Heat-killed Enterococcus faecium KU22001 having effective anti-cancer effects on HeLa cell lines at a lower culture temperature 43
2.1. Introduction 43
2.2. Materials and Methods 46
2.2.1. Strains, culture media, and reagents 46
2.2.2. Culture conditions and sample preparation 46
2.2.3. Cell cultures 47
2.2.4. Anti-proliferative activity 47
2.2.5. EPS analysis using the phenol-sulfuric acid method 48
2.2.6. RNA extraction and semi-quantitative real-time PCR 48
2.2.7. Flow cytometry analysis of apoptosis 51
2.2.8. Flow cytometry analysis of cell cycle distribution 51
2.2.9. DAPI staining and fluorescence microscopy 52
2.2.10. Statistical analysis 52
2.3. Results and Discussion 53
2.3.1. Anti-proliferative activity 53
2.3.2. EPS production 57
2.3.3. RNA extraction and semi-quantitative real-time PCR 59
2.3.4. Apoptosis assay 64
2.3.5. Cell cycle analysis 66
2.3.6. Morphological changes using DAPI Staining 68
2.4. Conclusion 70
References 71
Abstract (in Korean) 82
Chapter 1 7
Table 1-1. List of primer sequences used for semi-quantitative RT-PCR 23
Table 1-2. Tolerance of LAB to artificial gastric conditions and its adhesion ability to the HT-29 cells 26
Table 1-3. Minimum inhibitory concentrations against LAB determined by microdilution 28
Table 1-4. Probiotics safety of LAB strains 31
Chapter 2 7
Table 2-1. List of primer sequences used for semi-quantitative RT-PCR 50
Table 2-2. Cell viability of MRC-5 cells treated with heat-killed cells of LAB strains detected using the MTT assay 54
Table 2-3. Cytotoxic effects of heat-killed cells of different LAB strains on cancer cell lines as assessed by the MTT assay 55
Table 2-4. Cytotoxic effects of 9 log CFU/mL of heat-killed LAB strains on HeLa cells assessed using the MTT assay 56
Table 2-5. Effect of temperature on EPS production by LGG and E. faecium grown at 25℃ and 37℃ 58
Chapter 1 8
Figure 1-1. Antioxidant activity of 9 log CFU/mL of heat-killed LAB strains as measured by (A) DPPH radical scavenging activity, (B) ABTS radical scavenging... 33
Figure 1-2. Production of ROS by 9 log CFU/mL of heat-killed LAB strains in RAW 264.7 cells stimulated with LPS. 35
Figure 1-3. Cell viability and NO production on RAW 264.7 cells of LAB strains. The cells were incubated with LPS (1 ng/mL) or heat-treated bacterial sample... 37
Figure 1-4. The expression of immune-associated genes induced by heat-killed LAB in RAW 264.7 cells. 39
Figure 1-5. The expression level of phosphorylated MAPKs induced by heat-killed E. faecium KU22001 in RAW 264.7 cells. 41
Chapter 2 8
Figure 2-1. The expression of apoptosis-associated genes in HeLa cells by heat-killed LAB strains. 61
Figure 2-2. Growth of LAB strains in MRS at 25℃ and 37℃. 63
Figure 2-3. Apoptosis of HeLa cells treated with heat-killed LAB strains grown at 25℃ and 37℃ determined using flow cytometry. 65
Figure 2-4. Cell cycle of HeLa cells treated with heat-killed LAB strains grown at 25℃ and 37℃ determined using flow cytometry. 67
Figure 2-5. Morphological changes in HeLa cells with heat-killed LAB strains grown at 25℃ detected using confocal imaging. 69