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| 研究生： |
黃柏晨 Huang, Po-Chen |
|---|---|
| 論文名稱： |
枯草芽孢桿菌分離株及其表面活性素對葡萄球菌的抗菌機制 Antibacterial effect of surfactin produced by Bacillus subtilis isolates on Staphylococcus |
| 指導教授： |
陳又嘉
Chen, Yo-Chia |
| 學位類別： |
碩士 Master |
| 系所名稱： |
農學院 - 生物科技系 Department of Biological Science and Technology |
| 畢業學年度： | 110 |
| 語文別： | 中文 |
| 論文頁數： | 73 |
| 中文關鍵詞： | 枯草芽孢桿菌 、細胞膜損傷 、表面活性素 、協同作用 |
| 外文關鍵詞： | Bacillus subtilis, cell membrane damage, surfactin, synergy |
| DOI URL： | http://doi.org/10.6346/NPUST202200062 |
| 相關次數： | 點閱：36 下載：0 |
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從土壤中所分離出的1-2、1-4、3-3菌株對金黃色葡萄球菌(Staphylococcus aureus)以及中間葡萄球菌 (Staphylococcus intermedius)表現出優秀的抗菌能力。再經過革蘭氏染色、內孢子染色、碳水化合物利用分析、16S rRNA核醣體基因鑑定比對後，判斷此三株分離株均為枯草芽孢桿菌(Bacillus subtilis)，枯草芽孢桿菌的抗菌效果主要歸功於表面活性素(surfactin)的生產。在本研究中透過將分離株的培養上清液沉澱純化後再經高效液相層析（high performance liquid chromatography，HPLC）、三(羥甲基)甲基甘氨酸-十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(Tricine sodium dodecyl sulphate–polyacrylamide gel electrophoresis，Tricine SDS PAGE)分析，確定了分離株1-2、1-4具有生產表面活性素的能力，並且在300mL的LB medium中培養48hr後總產量分別為23.77mg及17.21mg，並且二株分離株生產表面活性素的能力會因為在培養基當中額外添加L-谷胺酸 (L-Glutamate)而提升約1.6倍。值得一提的是雖然分離株1-2具有更高的表面活性素產量但是在相同濃度下分離株1-4所生產的表面活性素對金黃色葡萄球菌及中間葡萄球菌的抗菌作用更為明顯。而3-3菌株則僅能生產微量的表面活性素因此對金黃色葡萄球菌以及中間葡萄球菌的抗菌作用均不明顯。表面活性素的抗菌機制涉及破壞細胞膜結構從而使膜內外的滲透壓失衡進而發揮抗菌效果，在本研究中透過sytox green染色、胞外蛋白外流分析從而驗證該現象，在含有sytox green染劑的LB medium中經過1-2、1-4所生產的表面活性素處理的金黃色葡萄球菌以及中間葡萄球菌相較於控制組出現螢光單位大幅度上升的現象，而經過液相層析法-質譜聯用(Liquid chromatography–mass spectrometry，LC-MS/MS)的分析結果也顯示出經過表面活性素處理後的金黃色葡萄球菌及中間葡萄球菌的樣品有鑑定出胞內蛋白的現象。而分離株1-2與1-4所生產的表面活性素能夠與抗生素apramycin達到最佳的協同效果，兩者的共同處理可以在對抗金黃色葡萄球菌以及中間葡萄球菌時大幅度的降低apramycin的使用濃度(MIC/10)。綜合以上實驗結果顯示出B. subtilis分離株1-2、 1-4可以透過生產表面活性素造成金黃色葡萄球菌及中間葡萄球菌的細胞膜損傷從而發揮抗菌效果，並且具有與抗生素進行協同作用，進而降低抗生素使用濃度的潛力。
The 1-2, 1-4, 3-3 strains isolated from soil showed strong antibacterial activity against S.aureus and S.intermedius. After Gram staining, endospore staining, qualitative analysis of carbohydrates, and 16S rRNA ribosomal gene identification, it was confirmed the three isolates were Bacillus subtilis. The antibacterial effect of B.subtilis was mainly attributed to the production of surfactin. In this study, by precipitating and purifying the culture supernatant of the isolates and then analyzing by high performance liquid chromatography (HPLC)and Tricine sodium dodecyl sulphate–polyacrylamide gel electrophoresis(Tricine SDS PAGE), it was confirmed that isolates 1-2 and 1-4 had the ability to produce surfactin, and they were able to produce surfactin in 300 mL of the total yields were 23.77 mg and 17.21 mg after culturing in LB medium for 48 hours, and the ability of the two isolates to produce surfactin was increased by about 1.6 times due to the addition of L-Glutamate to the medium. It is worth mentioning that although isolates 1-2 had higher surfactin production, the surfactin produced by isolates 1-4 had better antibacterial effects on S.aureus and S.intermedius at the same concentration. However, isolates 3-3 can only produce a small amount of surfactin, so the antibacterial effect on S.aureus and S.intermedius is not obvious. The antibacterial mechanism of surfactin involves the destruction of the cell membrane structure to unbalance the osmotic pressure inside and outside the membrane to exert an antibacterial effect. In this study, this phenomenon was verified by sytox green staining and extracellular protein efflux analysis. Compared with the control group, the 1-2, 1-4 surfactin-treated group showed a significant increase in the fluorescence unit, and the analysis results of liquid chromatography-mass spectrometry (LC-MS/MS) also showed that the surfactin-treated of S.aureus and S.intermedius could identify intracellular proteins. The surfactin produced by isolates 1-2 and 1-4 can achieve the best synergistic effect with the apramycin, the co-treatment of them can greatly reduce the level of apramycin concentration(MIC/10) against S.aureus and S.intermedius. These experimental results show that B. subtilis isolates 1-2 and 1-4 can cause the cell membrane damage of S.aureus and S.intermedius through the production of surfactin, thereby exerting an antibacterial effect, And it has the potential to synergize with antibiotics, thereby reducing the concentration of antibiotics used.
中文摘要 I
Abstract III
謝誌 V
目錄 VI
圖目錄 X
表目錄 X
第一章、前言 1
第二章、文獻回顧 2
2.1葡萄球菌 2
2.1.1金黃色葡萄球菌 2
2.1.2中間葡萄球菌 4
2.1.3治療手段 5
2.2枯草芽孢桿菌 6
2.2.1枯草芽孢桿菌 6
2.2.2枯草芽孢桿菌的抗菌能力及其抗菌機制 6
(一)臨界微胞濃度對表面活性素的影響 8
(二) 表面活性素自身結構對其活性的影響 9
(三)細胞膜的組成對表面活性素的影響 10
2.2.3伊枯草菌素及豐原素 11
2.2.4表面活性素的應用前景 12
2.2.5表面活性素的發展瓶頸 13
2.2.6表面活性素的產量提升 14
2.3表面活性素與抗生素的協同作用 15
2.4研究目的 15
第三章、材料與方法 16
3.1實驗架構 16
3.2菌種 17
3.3 抗菌能力評估 17
3.4革蘭氏染色 17
3.5內孢子染色 18
3.6分離株的碳水化合物利用能力分析 18
3.7分離株之基因鑑定 18
3.7.1基因組DNA萃取 18
3.7.2 PCR擴增 19
3.7.3 PCR產物純化 20
3.7.4 載體與嵌入基因的黏合 20
3.7.5 轉型作用至E.coli DH5α 20
3.7.6 轉型株確認 21
3.7.7液態培養 21
3.7.8 質體萃取 21
3.7.9 限制酶截切確認 22
3.7.10 確認基因&序列 22
3.8分離株的16S rRNA親緣關係樹 22
3.9分離株的抗菌能力分析 22
3.9.1分離株的抗菌胜肽萃取 22
3.9.2 表面活性素的分子量估算 23
3.9.2.1 Tricine-SDS-PAGE Sample buffer配製及樣品前處理 23
3.9.2.2電泳膠體製作 23
3.9.2.3 SDS PAGE電泳 24
3.9.2.4 膠體染色與退染 24
3.9.3 分離株抗菌胜肽對金黃色葡萄球菌、中間葡萄球菌的抗菌能力分析 25
3.9.4 表面活性素濃度定量 25
3.9.5三株分離株之表面活性素產量提升 26
3.9.5.1 OD值、pH值與表面活性素產量之間的關係 26
3.10表面活性素對葡萄球菌的抗菌機制驗證 26
3.10.1表面活性素對葡萄球菌的最小抑菌濃度 26
3.10.2表面活性素對葡萄球菌的最小殺菌濃度 27
3.10.3細胞膜完整性染色 27
3.10.4經表面活性素處理後，金黃色葡萄球菌與中間葡萄球菌的胞內蛋白洩漏評估 28
3.10.5液相層析-串聯式質譜(LC-MS/MS)鑑定蛋白質身分 29
3.11表面活性素與抗生素的協同作用 30
第四章、結果 31
4.1具抗菌潛力菌株的篩選 31
4.2分離株的鑑定 31
4.2.1革蘭氏染色 31
4.2.2內孢子染色 32
4.2.3分離株的API test分析 33
4.2.4 DNA鑑定 34
4.2.4.1定序後經BLAST比對後結果 36
4.2.4.2三株分離株的16S rRNA親緣關係樹 37
4.3分離株抗菌胜肽的抗菌能力分析 37
4.4分離株抗菌胜肽的成分分析 38
4.4.1 Tricine SDS PAGE 38
4.4.2分離株抗菌胜肽的成分分析 39
4.4.3 表面活性素定量 40
4.4.4分離株的表面活性素產量提升分析 42
4.4.5分離株之表面活性素產量與OD值、pH值之關聯 43
4.5表面活性素對金黃色葡萄球菌及中間葡萄球菌的抗菌機制分析 47
4.5.1表面活性素對金黃色葡萄球菌及中間葡萄球菌的最小抑菌濃度 47
4.5.2表面活性素對金黃色葡萄球菌及中間葡萄球菌的最小殺菌濃度 49
4.5.3 Sytox green染色 51
4.5.4經表面活性素處理後，金黃色葡萄球菌及中間葡萄球菌的胞內蛋白質洩漏評估 53
4.5.5洩漏蛋白之身分鑑定 54
4.6表面活性素與抗生素的協同作用 58
第五章、討論 65
第六章、結論 67
第七章、參考文獻 68
作者簡介 74
圖目錄
圖1. 表面活性素的結構圖 7
圖2. 表面活性素破壞細胞膜的作用示意圖 8
圖3. 臨界微胞濃度示意圖 8
圖4. 表面活性素在不同濃度對由流動相DOPC和凝膠相DPPC所組成的雙層膜作用的示意圖 9
圖5. 細胞膜上的膽固醇對表面活性素的影響示意圖。 11
圖6. 伊枯草菌素的結構圖。 12
圖7. 豐原素的結構圖 12
圖8. 實驗架構 16
圖9. 具抗菌潛力菌株的篩選結果 31
圖10. 分離株與金黃色葡萄球菌及大腸桿菌革蘭氏染色結果 32
圖11. 內生孢子染色結果 33
圖12. 三株分離株的API test分析結果 34
圖13. DNA跑膠結果圖 35
圖14. 以16S rRNA核醣體基因建構的三株分離株親緣關係樹 37
圖15. 三株分離株所生產之抗菌胜肽的抗菌能力分析結果 38
圖16. 三株分離株所生產之抗菌胜肽的Tricine SDS PAGE分析結果 39
圖17. 抗菌胜肽的層析結果 40
圖18. 透過表面活性素標準品所繪製之表面活性素檢量線 41
圖19. 於300mL LB medium中加入不同含量之谷胺酸對三株分離株生產表面活性素的影響圖 42
圖20. 於300mL LB medium中加入不同含量之葡萄糖對三株分離株生產表面活性素的影響圖 43
圖 21. 添加葡萄糖對於培養分離株1-2之菌數、pH值之影響 44
圖 22. 添加葡萄糖對於培養分離株1-4之菌數、pH值之影響 45
圖 23. 添加谷胺酸對於培養分離株1-2之菌數、pH值之影響 46
圖 24. 添加谷胺酸對於培養分離株1-4之菌數、pH值之影響 47
圖25. 不同濃度之分離株1-2所產表面活性素在24hr內抑制金黃色葡萄球菌與中間葡萄球菌生長的結果 48
圖26. 不同濃度之分離株1-4所產表面活性素在24hr內抑制金黃色葡萄球菌與中間葡萄球菌生長的結果 49
圖27. 分離株1-2所產表面活性素殺死金黃色葡萄球菌與中間葡萄球菌所需的最小濃度 50
圖28. 分離株1-4所產表面活性素殺死金黃色葡萄球菌與中間葡萄球菌所需的最小濃度 51
圖29. 在MIC濃度的表面活性素處理之下的金黃色葡萄球菌及中間葡萄球菌螢光單位增長率曲線圖 52
圖30. 在MBC濃度的表面活性素處理之下的金黃色葡萄球菌及中間葡萄球菌螢光單位增長率曲線圖. 53
圖31. 經表面活性素、MeOH、TBS buffer以及滅菌處理後， 金黃色葡萄球菌及中間葡萄球菌的胞內蛋白分析結果圖 54
圖32. 各種抗生素與1-2所生產的表面活性素在相隔適當的距離下在LA平板上共同對抗金黃色葡萄球菌及中間葡萄球菌結果 58
圖 33. 各種抗生素與1-4所生產的表面活性素在相隔適當的距離下在LA平板上共同對抗金黃色葡萄球菌及中間葡萄球菌結果 59
圖34. 分離株1-2、1-4所生產之表面活性素與抗生素ampicillin於24hr內共同抑制金黃色葡萄球菌與中間葡萄球菌生長的結果 62
圖35. 分離株1-2、1-4所生產之表面活性素與抗生素daptomycin於24hr內共同抑制金黃色葡萄球菌與中間葡萄球菌生長的結果 62
圖36. 分離株1-2、1-4所生產之表面活性素與抗生素tetracycline於24hr內共同抑制金黃色葡萄球菌與中間葡萄球菌生長的結果 63
圖 37. 分離株1-2、1-4所生產之表面活性素與抗生素apramycin於48hr內共同抑制金黃色葡萄球菌與中間葡萄球菌生長的結果 64
表目錄
表 1.配置Ligation藥劑: 20
表 2. Tricine-SDS-PAGE sample buffer(pH6.8)配方 23
表 3. HPLC分析使用儀器 26
表 4. 2X SDS sample buffer(pH6.8)配方: 28
表 5. 1-2、1-4、3-3經比對後的相似菌株 36
表 6. 1-2表面活性素處理金黃色葡萄球菌的蛋白洩漏之蛋白身分鑑定 56
表 7. 1-2表面活性素處理中間葡萄球菌的蛋白洩漏之蛋白身分鑑定 56
表 8. 1-4表面活性素處理金黃色葡萄球菌的蛋白洩漏之蛋白身分鑑定 57
表 9. 1-4表面活性素處理中間葡萄球菌的蛋白洩漏之蛋白身分鑑定 57
表 10. 1-2所生產的表面活性素與六種抗生素共同對抗金黃色葡萄球菌的結果. 59
表 11. 1-2所生產的表面活性素與六種抗生素共同對抗中間葡萄球菌的結果 60
表 12. 1-4所生產的表面活性素與六種抗生素共同對抗金黃色葡萄球菌的結果 60
表 13. 1-4所生產的表面活性素與六種抗生素共同對抗中間葡萄球菌的結果 61
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校外公開2027/02/07