Title Page
Contents
ABSTRACT 9
Chapter 1. Antibacterial and anti-biofilm effects of Weissella cibaria strains against Streptococcus mutans 11
Ⅰ. Introduction 11
Ⅱ. Materials and methods 14
1.2.1. Bacterial strains and culture condition 14
1.2.2. Sample preparation 14
1.2.3. Evaluation of antimicrobial activity 15
1.2.4. Assessment of biofilm formation 15
1.2.5. Analysis of polysaccharides 16
1.2.6. Spectrophotometry aggregation analysis 16
1.2.7. Scanning electron microscopy (SEM) 17
1.2.8. Confocal laser scanning microscopy (CLSM) 18
1.2.9. Quantitative real-time PCR analysis (qRT-PCR) 18
1.2.10. Statistical analysis 19
Ⅲ. Results and discussion 21
1.3.1. Effect of W. cibaria on bacterial growth 21
1.3.2. Effect of W. cibaria on biofilms 25
1.3.3. Effect of W. cibaria on EPS production and aggregation 28
1.3.4. Morphology of biofilms 34
1.3.5. Gene expression of S. mutans 38
Ⅳ. Conclusion 41
Chapter 2. Antioxidant and anti-inflammatory effects of heat-killed Levilactobacillus brevis KU15151 in LPS-induced A549 cells 42
Ⅰ. Introduction 42
Ⅱ. Materials and methods 45
2.2.1. Bacterial strains and sample preparation 45
2.2.2. Cell culture condition 45
2.2.3. Cytotoxicity measurement 45
2.2.4. Antioxidant assay 46
2.2.5. Measurement of ROS production 47
2.2.6. Western blot analysis 48
2.2.7. Measurement of IL-6 and eotaxin 49
2.2.8. Quantitative RT-PCR 49
2.2.9. Statistical analysis 50
Ⅲ. Results and discussion 52
2.3.1. Cytotoxicity of heat-killed L. brevis KU15151 52
2.3.2. Antioxidant activity of heat-killed L. brevis KU15151 55
2.3.3. ROS scavenger effect of heat-killed L. brevis KU15151 57
2.3.4. Anti-inflammatory effect on MAPK signaling pathway 60
2.3.5. Anti-inflammatory effect on NF-κB signaling pathway 64
2.3.6. Inhibition of pro-inflammatory cytokine expression 67
Ⅳ. Conclusion 72
References 73
Abstract (in Korean) 83
Table 1-1. Primer sequences used in qRT-PCR 20
Table 1-2. Inhibition zone of S. mutans strains treated with live LAB cells 23
Table 1-3. The MIC values of LAB-derived CFS against S. mutans strains 24
Table 1-4. Water-insoluble EPS production rate of S. mutans treated with CFS 30
Table 1-5. Water-soluble EPS production rate of S. mutans treated with CFS 31
Table 1-6. Auto-aggregation rate of S. mutans treated with CFS 32
Table 1-7. Co-aggregation rate of S. mutans treated with live LAB cells 33
Table 2-1. Primer sequences used in qRT-PCR 51
Figure 1-1. Effects of LAB strains on biofilms. 27
Figure 1-2. Biofilms on glass cover surface visualized using FE-SEM (magnification: x 20,000). 36
Figure 1-3. Biofilms on confocal dish visualized using CLSM (magnification: x 100). 37
Figure 1-4. Expression of virulence genes associated with biofilm formation of S. mutans as determined using qRT-PCR. 40
Figure 2-1. Cytotoxicity of heat-killed LAB in A549 cells. 54
Figure 2-2. Antioxidant activities of heat-killed LAB. 56
Figure 2-3. Reduced ROS production in LPS-induced A549 cells. 59
Figure 2-4. Effect of heat-killed LAB on LPS-induced A549 cells in MAPK signaling pathway. 63
Figure 2-5. Effect of heat-killed LAB on LPS-induced A549 cells in NF-κB signaling pathway. 66
Figure 2-6. mRNA expression of cytokine/chemokine as measured by qRT-PCR. 70
Figure 2-7. Main cytokine and chemokine levels related to respiratory diseases as measured by ELISA. 71