Title Page
Contents
ABSTRACT 12
Ⅰ. INTRODUCTION 15
Ⅱ. LITERATURE REVIEW 17
1. Lactic acid bacteria 17
1.1. Definition and characteristics 17
1.2. Stress adaptative response 18
2. Technologies for preserving lactic acid bacteria 21
2.1. Research trends 21
2.2. Freeze-drying 22
3. Strategy to enhance storage stability of freeze-dried bacteria 24
3.1. Cryoprotectants 24
3.2. Cross-protection 26
Ⅲ. MATERIALS AND METHODS 28
1. Freeze-drying of bacteria 28
1.1. Bacterial culture preparation 28
1.2. Preparation of the freeze-drying media 28
1.3. Freeze-drying process 29
1.4. Assessing the storage stability improvement effect of monosodium glutamate and optimal concentration 30
2. Identifying of impact of monosodium glutamate on cell membrane integrity 30
2.1. Scanning electron microscopy imaging 30
2.2. Cell membrane integrity assay 31
2.3. Quantification of intracellular amino acid contents 33
3. Transcriptomic analysis of related with cell membrane integrity 34
3.1. Gene expression analysis 34
3.2. Real-time fluorescence quantitative PCR 38
4. Statistical analysis 38
Ⅳ. RESULTS AND DISCUSSION 39
1. Storage stability 39
1.1. Storage stability improvement effect of monosodium glutamate 39
1.2. Optimization of concentration of monosodium glutamate 44
2. Effect of monosodium glutamate on reducing cell membrane integrity 46
2.1. Cell morphology observed by scanning electron microscopy 46
2.2. Cell membrane integrity 49
3. Mechanisms of maintaining cell membrane integrity by monosodium glutamate 60
3.1. Intracellular amino acid contents 60
3.2. Gene expression levels 63
Ⅴ. SUMMARY AND CONCLUSION 73
Ⅵ. REFERENCES 76
ABSTRACT IN KOREAN 89
Table 1. Primers used for the transcriptomic analysis 37
Table 2. Viable cell count (Log CFU/g) of freeze-dried bacteria during 8 weeks of storage at 40℃ 40
Table 3. Viable cell count (Log CFU/g) of bacteria during 8 weeks of storage at 40℃ 45
Table 4. Viable cell count (Log CFU/g) of bacteria during 4 weeks of storage at 40℃ 59
Table 4. Intracellular amino acid contents 62
Figure 1. Storage stability of freeze-dried bacteria with cryoprotectants during storage for 8 weeks at 40℃. 43
Figure 2. Scanning electron microscopy images of freeze-dried bacteria. 48
Figure 3. Acid resistance of freeze-dried bacteria. 52
Figure 4. NaCl resistance of freeze-dried bacteria. 53
Figure 5. Adhesion ability of freeze-dried bacteria. 56
Figure 6. Viable cell count of freeze-dried bacteria.[내용없음] 10
Figure 7. Relative gene expression level related to general stress chaperones genes in freeze-dried bacteria. 65
Figure 8. Relative gene expression level related to glutamate uptake genes in freeze-dried bacteria. 67
Figure 9. Relative gene expression level related to glutamate synthesis gene gltC in freeze-dried bacteria. 69
Figure 10. Relative gene expression level related to peptidoglycan synthesis genes in freeze-dried bacteria. 72