표제지
국문초록
목차
I. 서론 17
II. 재료 및 방법 24
1. 재료 24
1) 사용균주 24
2) 배지 24
3) 시약 및 효소 25
2. 실험방법 28
1) 정량실험 28
2) 정성실험 29
3) 자외선 조사 32
4) 광펄스 조사 32
5) Chromosomal DNA 분리 33
6) RNA 분리 33
7) 효소처리 34
8) Agarose gel 전기영동 및 사진촬영 34
9) B. subtilis 모델균주의 포자형성 36
10) 생균수 측정 36
11) 투과전자현미경 37
12) 세포 내 단백질의 세포 외 유출도 측정 37
13) 통계적 분석 38
III. 결과 및 고찰 40
1. 수산식품에 존재하는 미생물의 정량분석과 분자유전학적 동정 40
1) 정량분석 40
2) 분자유전학적 동정 42
2. 수산식품에 존재하는 미생물의 정성분석 47
1) 선택배지에서의 미생물 분석 47
2) 그람염색 49
3) Latex test 51
4) Coagulase test 53
5) API test 55
3. 자외선 조사에 의한 E. coli 모델균주의 사멸기작 58
1) E. coli 모델균주의 사멸율 58
2) Double strand breaks 61
3) Single strand breaks 65
4) Cyclobutane pyrimidine dimers 65
5) RNA breaks 70
4. 자외선 조사에 의한 수산식품 유래 병원성 미생물의 사멸기작 73
1) 수산식품 유래 병원성 미생물의 사멸율 73
2) Double strand breaks 76
3) Single strand breaks 80
4) Cyclobutane pyrimidine dimers 80
5) RNA breaks 85
5. 광펄스 조사에 의한 E. coli 모델균주의 사멸기작 89
1) E. coli 모델균주의 사멸율 89
2) Double strand breaks 92
3) Single strand DNA breaks 95
4) Cyclobutane pyrimidine dimers 95
5) RNA breaks 98
6. 광펄스 조사에 의한 수산식품 유래 병원성 미생물의 사멸기작 101
1) 수산식품 유래 병원성 미생물의 사멸율 101
2) Double strand breaks 103
3) Single strand breaks 107
4) Cylobutane pyrimidine dimers 107
5) RNA breaks 111
7. 투과전자현미경에 의한 광펄스 조사의 생리학적 사멸기작 116
1) 투과전자현미경 116
2) 광펄스 조사에 의한 세포 내 단백질의 세포 외 유출도 확인 117
8. 광펄스 조사가 포자형성미생물의 세포발달과정에 미치는 영향 120
1) B. subtilis 모델균주의 사멸률 120
2) 투과전자현미경 121
IV. 결론 127
V. 참고문헌 132
ABSTRACT 138
Table 1. Bacterial strains 26
Table 2. Oligonucleotides used for PCR 39
Table 3. Viable cell numbers on the PCA and YMA media 41
Table.4. Identification of food-borne pathogenic bacteria using selection medium 48
Table 5. Gram staining 50
Table 6. Latex test for the identification of E. coli O157:H7 52
Table 7. Coagulase test for the identification of S. aureus 54
Table.8. API test for the identification of L. monocytogenes, B. cereus and S. Typhimurium 56
Table.9. Viable cell numbers and death rate of E. coli cells by UV irradiation 60
Table.10. Viable cell numbers and death rate of food-borne pathogenic bacterial cells by UV irradiation 75
Table.11. Viable cell numbers and death rate of E. coli cells by IPL irradiation 91
Table.12. Viable cell numbers and death rate of food-borne pathogenic bacterial cells by IPL irradiation 102
Table 13. Analysis of OD595 value for food-borne pathogenic bacterial cells(이미지참조) 119
Table 14. Viable cell numbers and death rate of B. subtilis cells by IPL irradiation 124
Fig. 1. Spectral distribution and intensity of light 22
Fig. 2. Schematic representation of a bench top experimental unit of pulsed light (Go´mez-lo´pez et al., 2005). 23
Fig. 3. Nucleotide sequence of 16S rRNA gene from isolated no. 1. 43
Fig. 4. Nucleotide sequence of 16S rRNA gene from isolated no. 2. 44
Fig. 5. Nucleotide sequence of 16S rRNA gene from isolated no. 3. 45
Fig. 6. Nucleotide sequence of 16S rRNA gene from isolated no. 4. 46
Fig. 7. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from E. coli AB1157 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric analysis... 63
Fig. 8. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from E. coli AB1886 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric analysis... 64
Fig. 9. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from E. coli AB1157 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis, (b) Densitometric scan... 68
Fig. 10. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from E. coli AB1886 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan... 69
Fig. 11. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from E. coli AB1157 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 71
Fig. 12. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from E. coli AB1886 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 72
Fig. 13. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from L. monocytogenes cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric analysis... 77
Fig. 14. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from E. coli O157:H7 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric analysis... 78
Fig. 15. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from P. aeruginosa cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) densitometric analysis... 79
Fig. 16. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from L. monocytogenes cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan... 82
Fig. 17. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from E. coli O157:H7 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan... 83
Fig. 18. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from P. aeruginosa cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan 84
Fig. 19. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from L. monocytogenes cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 86
Fig. 20. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from E. coli O157:H7 cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 87
Fig. 21. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from P. aeruginosa cells irradiated with artificial UV-C. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 88
Fig. 22. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from E. coli AB1157 cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric analysis... 93
Fig. 23. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from E. coli AB1886 cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric analysis... 94
Fig. 24. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from E. coli AB1157 cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan... 96
Fig. 25. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from E. coli AB1886 cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan... 97
Fig. 26. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from E. coli AB1157 cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 99
Fig. 27. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from E. coli AB1886 cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 100
Fig. 28. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from L. monocytogenes irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric analysis... 104
Fig. 29. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from E. coli O157:H7 irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric analysis Lane M, molecular weight marker; 1, control DNA (non-irradiated); 2,... 105
Fig. 30. Photograph of 0.8% neutral agarose gel electrophoresis of chromosomal DNAs extracted from P. aeruginosa irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric analysis Lane M, molecular weight marker; 1, control DNA (non-irradiated); 2,... 106
Fig. 31. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from L. monocytogenes cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan... 108
Fig. 32. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from E. coli O157:H7 cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan... 109
Fig. 33. Photograph of 0.8% alkaline agarose gel electrophoresis of chromosomal DNA extracted from P. aeruginosa cells irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan... 110
Fig. 34. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from L. monocytogenes irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 112
Fig. 35. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from E. coli O157:H7 irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 113
Fig. 36. Photograph of neutral 2% agarose gel electrophoresis of RNAs extracted from P. aeruginosa irradiated with IPL. (a) Photograph of electrophoresis (b) Densitometric scan Lane M, Molecular weight marker; 1, control RNA (non-irradiated); 2,... 114
Fig. 37. Evaluation of IPL-induced damages in microorganisms by TEM. a, control, b, UV-C treatment; c, intense pulsed light treatment; 1, L. monocytogenes; 2, E. coli O157:H7; 3, P. aeruginosa 118
Fig. 38. The life cycle of spore-forming bacteria (Errington, 2003). 123
Fig. 39. Evaluation of IPL-induced damages in microorganisms by TEM. 1, control, 2, intense pulsed light treatment; (a), B. subtilis 168; (b), B. subtilis PB3A; (c), B. subtilis Z3; (d), B. subtilis 94U; (e), B. subtilis P20; (f), B. subtilis 89; (g), B. subtilis 513 126