표제지
요약
Abstract
목차
I. 서론 16
1. 프로바이오틱스(Probiotics) 16
2. Clostridium butyricum 20
3. 산 스트레스와 프로바이오틱스 22
4. 중금속 제거 23
4.1. 중금속 23
4.2. 미생물의 중금속 제거 및 내성 25
5. 연구 목적 27
II. 재료 및 방법 28
1. Lactic acid bacteria strains 28
2. Stimulation of C. butyricum growth by Lactobacillus spp. 30
2.1. Stimulation of C. butyricum growth by Lactic acid bacteria 30
2.2. Quantification of buk gene expression 31
3. Resistance of Lactobacillus in acidic conditions 33
3.1. Screening for acid resistant lactic acid bacteria 33
3.2. Survival of Lactobacillus in extreme acidic condition 34
3.3. Stress responses of Lactobacillus in other environmental condition 35
3.4. Expression of atpD gene in acidic condition 36
3.5. Addition of the substrate for improvement acid resistance 37
4. Heavy metal removal by Lactobacillus spp. 38
4.1. Confirmation of Heavy metal resistance and minimum inhibitory concentration (MIC) 38
4.2. Heavy metal removal assay 39
4.3. Antibiotic resistance assay 40
5. Characterization of properties for probiotics 41
5.1. Assessment of Lactobacillus strains as probiotics 41
5.2. Accelerated storage testing 42
5.3. Large-scale cultivation 42
III. 결과 및 고찰 43
1. Stimulation of C. butyricum growth by Lactobacillus spp. 43
1.1. Stimulation of C. butyricum growth by lactic acid bacteria 43
1.2. Stimulation of C. butyricum growth according to the initial dose ratio of C. butyricum and lactic acid bacteria 48
1.3. Expression of butyric acid producing gene of C. butyricum in co-culture with Lactobacillus spp. 51
1.4. Characterization for probiotics 54
1.5. Accelerated storage testing for freeze-dried Lactobacillus strains 58
1.6. Large-scale culture of L. parabuchneri MH44 60
2. Resistance of Lactobacillus in acidic conditions 62
2.1. Assessment of acid resistance to lactic acid bacteria 62
2.2. Survival of Lactobacillus in extreme acidic condition overtime 66
2.3. Morphology of Lactobacillus acidic condition 68
2.4. Expression of atpD gene in acidic condition 70
2.5. Addition of the substrate for improvement acid resistance 73
2.6. Stress responses of Lactobacillus in other environmental condition 76
2.7. Characterization for probiotics 79
2.8. Accelerated storage testing for freeze-dried Lactobacillus strains 82
2.9. Large-scale culture of L. johnsonii MH33 and gasseri KLB240 84
3. Heavy metal removal activities of Lactobacillus spp. 86
3.1. Heavy metals resistance of Lactic acid bacteria 86
3.2. Heavy metal removal by lactic acid bacteria 89
3.3. Antibiotic resistance pattern of lactic acid bacteria 92
3.4. Characterization for probiotics 95
3.5. Accelerated storage testing for freeze-dried Lactobacillus strains 98
IV. 결론 100
V. 참고문헌 102
Table 1. Pollution sources and adverse health effects of some heavy metals 24
Table 2. The list of lactic acid bacteria strains used in this study 29
Table 3. C. butyricum genes examined by qPCR in this study. 32
Table 4. The primers of Lactobacillus genes examined by RT-qPCR in this study 36
Table 5. Optical Density of C. butyricum by supernatant of lactic acid bacteria. 44
Table 6. Expected k (h-1) values and shelf-life of L. parabuchneri MH44 and...[이미지참조] 59
Table 7. Survival rate of lactic acid bacteria in acidic condition (pH 2, pH 2.5, pH 3) for 3 h 64
Table 8. Survival rate of L. johnsonii MH33, MH21 and L. gasseri KLB240 in... 67
Table 9. Expected k (h-1) values and shelflife of L. johnsonii MH33,...[이미지참조] 83
Table 10. The number of lactic acid bacteria resistant to four heavy... 88
Table 11. Minimum inhibitory concentration of various lactic acid bacteria 88
Table 12. Antibiotic resistance patten of heavy metal resistant lactic acid bacteria 94
Table 13. Expected k (h-1) values and shelflife of L. fermentum KLB79,...[이미지참조] 99
Fig. 1. Domestic and global probiotics market trends 16
Fig. 2. Properties for probiotics in the researches 17
Fig. 3. Damage and response of microorganisms by heavy metals 26
Fig. 4. PCR standard curve of spo0A gene 32
Fig. 5. Growth of C. butyricum with supernatant of lactic acid bacteria 45
Fig. 6. Growth of C. butyricum and lactic acid bacteria after coculture. Quantification of spo0A gene... 47
Fig. 7. Quantification of spo0A gene concentration in C. butyricum according to... 50
Fig. 8. Neighbor-joining tree with bootstrap values for the identifiction... 50
Fig. 9. Relative expression of buk gene in C. butyricum without (MRS) and... 53
Fig. 10. Probiotic properties of L. parabuchneri MH44 and L. brevis JL16; acid resistance (A); bile (0.3%,... 57
Fig. 11. Arrhenius plots of the logarithms of the specific rates of thermal... 59
Fig. 12. Growth of L. parabuchneri MH44 during 4L-scale culture 61
Fig. 13. Neighbor-joining tree with bootstrap values for the identification of... 65
Fig. 14. Survival rate of L. johnsonii MH33, MH21 and L. gasseri KLB240 in... 67
Fig. 15. SEM analysis of (A) Lactobacillus johnsonii MH33, (B) Lactobacillus... 69
Fig. 16. Relative expression of atpD gene in L. johnsonii MH33 and... 72
Fig. 17. Survival of L. gasseri KLB240 with arginine or glutamate in acid... 75
Fig. 18. Survival of L. johnsonii MH33, MH21 and L. gasseri KLB240 in environmental stresses; Heat... 78
Fig. 19. Probiotic properties of L. johnsonii MH33, MH21 and L. gasseri... 81
Fig. 20. Arrhenius plots of the logarithms of the specific rates of thermal... 83
Fig. 21. Growth of L. johnsonii MH33 (A) and L. gasseri KLB240 (B) during... 85
Fig. 22. Heavy metals removal efficiency of the seven lactic acid bacteria. 90
Fig. 23. Effect of initial lactic acid bacteria concentration on removal... 91
Fig. 24. Probiotic properties of L. fermentum KLB79, KLB100, and KLB203; acid resistance (A);... 97
Fig. 25. Arrhenius plots of the logarithms of the specific rates of thermal... 99