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Title Page
Abstract
Contents
1. Introduction 10
2. Materials and Methods 14
2.1. Bacterial strains and Growth cultures 14
2.2. Optimal working concentration of Berenil 15
2.3. Administration time of Berenil 15
2.4. The effect of Growth Temperature on the antibacterial activity of Berenil for E.coli 15
2.5. pH Effects and pH Converting 16
2.6. Reactive Oxidative Species Assay 16
2.7. Peroxidase Activity Assay 17
2.8. Extracellular Protease Assay 19
2.9. The effect of KCl on the surviability of KO157 20
2.9.1. Examination of cell morphology by SEM 20
3. Results and Discussion 21
3.1. Berenil Dosage 21
3.2. Administration Time 24
3.3. Working Temperature 27
3.4. Working pH 29
3.5. Enzyme Activities 31
3.6. The effects of KCl on the viability of E.coli O157:H7 40
4. Conclusion 42
5. References 43
Table 1. The effect of increasing concentrations of Berenil on the growth of... 24
Table 2. Temperatures' effects on anti-bacterial activity of Berenil for E.coli 28
Fig. 1. Chemical Structure of Berenil (DMZ). 11
Fig. 2. The Bacteriostatic effects of Berenil on the growth of E.coli. 22
Fig. 3. The effects of Berenil administrated at time (+6hr) on the growth... 26
Fig. 4. The effects of increasing concentrations Berenil administrated at mid... 27
Fig. 5. The effects of acidic pH (5.0) Berenil administrated at mid log phase... 29
Fig. 6. Berenil's effects when broth pH was converted at 8hr. 30
Fig. 7. Internal ROS activity of different strains of E. coli grown in LB-... 32
Fig. 8. The effects of Berenil on whole cell peroxidase activity. Standard... 33
Fig. 9. Whole cell peroxidase activity of K12, MO157 and KO157 after... 34
Fig. 10. Peroxidase activity of KO157 grown in LB broth with different... 36
Fig. 11. Whole cell protease activity assay screen for E.coli O157. 21 pNA... 37
Fig. 12. Protease activity assay and Berenil's effects on it. 4 pNA substrates... 39
Fig. 13. Effects of KCl (50mM) on E.coli O157: H7. 40
Fig. 14. SEM images of E.coli O157: H7 at different conditions (A) normal... 41
초록보기 더보기
This study investigated the bacteriostatic and bactericidal effects of Berenil against 5 strains of pathogenic bacteria and 2 strains of non-pathogenic bacteria. The results showed that under optimized conditions (temperature and pH), broth containing between 2-5 mg/L of Berenil effectively limited the growth of various strains of E.coli in the first 24 hours by as much as 77% compared to the controls. However the growth of Enterococcus faecalis, Listeria monocytogenes and Staphylococcus aureus was only significantly suppressed when the concentration of Berenil in the starting broth exceeded 50 mg/L. In the next part of this study we examined the effects of adding Berenil in mid-log phase on the growth of different strains of E.coli only. Low concentrations of Berenil (5mg/mL) did not induce any change in the growth pattern of K12 or E.coli 10005. However Berenil was a potent bactericide for both strains of E.coli O157:H7 inducing a collapse in both bacterial populations 6-8 hours later. Further investigation showed that the collapse in the population was dependent on the potassium ion concentration and the pH of the broth, and that the bactericidal action of Berenil was probably due the uncoupling of the membrane ion gradient in both strains of E.coli O157:H7. In the final part of this study we examined the effects of Berenil on the extracellular and intracellular peroxidase activity of E.coli O157:H7 as well as its influence on extracellular protease activity. It was found that the extracellular peroxidase activity of E.coli O157:H7 was reduced in a concentration dependent manner by Berenil. When compared to glucose, Berenil was 3-4 orders of magnitude more effective in reducing the extracellular peroxidase activity of E.coli O157:H7, however Berenil did not inhibit peroxidase activity directly only the secretion of peroxidases. Lastly a screen of 21 p-NA substrates was used to assess the extracellular protease/proteolytic activity of E.coli O157:H7. It was found that E.coli O157 and K12 in glucose (5%) assay media showed enhanced activity for 2 substrates, H-Asp-pNA and Ac-Asp-pNA. Activity was further enhanced (factor of 2-3) for H-Asp-pNA when Berenil was added to the medium.
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