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研究生:宮紹凱
研究生(外文):Shao-Kai Kung
論文名稱:不同品系金黃色葡萄球菌與大腸桿菌對低分子量幾丁聚醣敏感差異性及抗菌機制之初步研究
論文名稱(外文):The preliminary study of antibacterial mechanism of low molecular weight chitosan by using differentially susceptible strains of Staphylococcus aureus and Escherichia coli
指導教授:林世斌林世斌引用關係
指導教授(外文):Shih-Bin Lin
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:生物技術研究所碩士班
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:91
中文關鍵詞:幾丁聚醣低分子量幾丁聚醣幾丁質酶溶菌酶纖維酵素抗菌活性
外文關鍵詞:chitosanlow molecular weight chitosanchitinaselysozymecellulaseantibacterial activity
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幾丁質(chitin)、幾丁聚醣(chitosan)及幾丁聚醣水解物具組織相容、生物活性等特性,近年來在生物技術、生物醫學、藥理、農業等領域上受到廣泛的重視。本實驗室使用自木黴菌(Trichoderma viride, BCRC 32054)誘發之幾丁質酶(chitinase)與市售溶菌酶(lysozyme)及纖維酵素(cellulase)三種酵素,透過其不同的水解機制,對不同去乙醯度(DD80、DD92)的幾丁聚醣進行水解,製備出分子量、抗菌性、溶解度、電荷密度不同的低分子量幾丁聚醣(LMWC)。幾丁聚醣的去乙醯度明顯影響酵素的水解行為,經過水解24小時後,各組合產生之LMWC之內生性黏度分子量(MV)大小排列依序為:DD92_lys(24.8 kDa)、DD80_lys(14.8 kDa)、DD92_chit(13.5 kDa)、DD80_chit(9.6 kDa)、DD80_cel(8.1 kDa)及DD92_cel(6.9 kDa)。LMWC之溶解度與其分子量大小相關而與其製備所使用之酵素無關。本實驗以96微孔盤抗菌試驗對各LMWC進行抗菌試驗,結果顯示LMWC_lys具較佳的抗菌效果,但溶解度不佳,而LMWC_cel雖具較佳的溶解度,但不具抗菌效果。利用DD92及chitinase所製備的LMWC則有高溶解度及抗菌效果,故選擇其作為後續抗菌機制探討之用。微生物培養增殖分析儀的抗菌試驗乃針對3株S. aureus (BCRC 10451、10780、10781) 及2株E. coli (BCRC 10314、10675) 進行探討,結果顯示不同品系的菌種對LMWC具不同程度的敏感性。在機制探討的結果顯示,界達電位(zeta potential)關係著細菌與LMWC的吸附力,當菌體的負電荷與正電荷的LMWC電位差距越大,其彼此間的吸附力也跟著提升,導致抗菌性也有增強。透過SEM觀察,對LMWC較敏感的品系,S. aureus(BCRC 10451)和E. coli (BCRC 10314)其菌體表面會被LMWC所包覆。若將細菌表面所吸附的LMWC洗去,可發現菌體遭受破壞及孔洞形成。而菌體DNA萃取的分析結果,同樣指出BCRC 10780的DNA最難萃取,意味著其細胞壁可能較原本堅固,不易破壞。綜合上述結果推測細菌與LMWC作用後,會有電中和現象,且彼此會因為相互吸附使得細菌被包覆,初期以抑制細菌分裂達到抑菌的效果,也可入侵造成細菌細胞壁使結構改變,造成細菌死亡。
Chitin, chitosan and chitosan hydrolysates possess histocompatibility and biological activity, in addition, they have been widely applied in the biotechnology, biomedical, pharmacological and agricultural fields recently. In this study, the low molecular weight chitosans (LMWCs) were prepared by using crude chitinase (induced from Trichoderma viride), commercial lysozyme and cellulase. Due to these enzymes have different hydrolysis mechanism on chitosan with different DD, a diverse characteristics of LMWC including Mw, antibacterial activity, solubility, zeta potential could prepared from DD of 80 and 92%. The intrinsic viscosity molecular weight (MV) of LMWC form each of the combination was decreased steeply in 24 hours (from 370 kDa to 13-37 kDa) and, the order of MV were DD92_lys (24.8 kDa), DD80_lys (14.8 kDa), DD92_chit (13.5 kDa), DD80_chit (9.6 kDa), DD80_cel (8.1 kDa) and DD92_cel (6.9 kDa). The solubility of LMWC was affected strongly by Mw but not the enzyme by which LMWC was prepared. The minimum inhibition concentration (MIC) of LMWC was done by 96 microplate method. The results show that LMWC_lys has better antibacterial activity and poor solubility; in opposition, LMWC_cel had better solubility but ineffective antibacterial activity. The LMWC made from DD92 and chitinase had high solubility and good antibacterial activity. Therefore, the LMWC_DD92_chit was used for the following antibacterial activity study. The antibacterial activity test performed by using biophotorecorder method against three strains of S. aureus (BCRC 10451, 10780, 10781) and two strains of E. coli (BCRC 10314, 10675) revealed that various strains of antibacterial action of bacteria had different susceptibility to LMWC. The antimicrobial action of LMWC was strongly related to zeta potential. The more charge disparity between positive LMWC and negative bacteria, the more adsorptive power between each other, which led to higher antibacterial activity. SEM was used to observe the interaction between LMWC and the most sensitive strains S. aureus (BCRC 10451) and E. coli (BCRC 10314), and results revealed bacteria were heavily coated with LMWC. While the LMWC coat was washed away with acetic buffer, the surface integrity of bacteria was destroyed which resulted in the membrane poration on the surface. The bacterial DNA extraction analysis showed that DNA of BCRC 10780 was the most difficult to be extracted. It suggested that cell wall of BCRC 10780 was thicker, more rigid and not easy to be destroyed. In the early stage, the surface covering of LMWC can possibly cease cell division by charge neutralization. It could also invade into and cause deterioration of cell wall which leads bacteria to death.
目錄
中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅸ
表目錄 XII
第一章、前言 1
第二章、文獻回顧 3
2.1. 幾丁質 3
2.2. 幾丁聚醣 5
2.3. 幾丁聚醣水解物 6
2.3.1. 製備方法 6
2.3.2. 化學法 7
2.3.3. 酵素法 7
2.3.4. 常見的水解酵素 7
2.3.4.1. 幾丁質酶 8
2.3.4.2. 溶菌酶 9
2.3.4.3. 纖維酵素 10
2.4. 幾丁聚醣及其水解物的功能與應用 12
2.4.1. 降低脂肪與膽固醇 12
2.4.2. 提高免疫力 13
2.4.3. 抗腫瘤活性 13
2.4.4. 抗氧化性 13
2.4.5. 促進傷口癒合、凝血 14
2.4.6. 幾丁聚醣及其水解物的抗菌活性 14
2.5. 影響抗菌活性之因子 15
2.5.1. 去乙醯度 15
2.5.2. 分子量 16
2.5.3. 溶解度 16
2.5.4. 電荷密度 17
2.6. 目前研究的抗菌機制 17
2.7. 細菌細胞壁的介紹 19
2.7.1. 革蘭氏陽性菌 20
2.7.2. 革蘭氏陰性菌 21
2.7.3. 革蘭氏陽性菌與陰性菌比較 23
第三章、實驗材料與方法 24
3.1. 研究架構 24
3.2. 實驗器材 25
3.2.1. 儀器及設備 25
3.2.2. 樣品 26
3.2.3. 實驗菌株 26
3.2.4. 藥品清單 27
3.2.5. 商購酵素 27
3.3. 實驗方法 27
3.3.1. 幾丁質酶粗酵素之誘發 27
3.3.2. LMWC製備 29
3.3.3. LMWC分析 29
3.3.3.1. 分子量測定 29
3.3.3.2. 溶解度 30
3.3.3.3. 粒徑與界達電位分析 30
3.4. 抗菌活性試驗與機制探討 31
3.3.4.1. 菌株培養 31
3.3.4.2. 菌種鑑定 31
3.3.4.3. 不同pH值培養條件下的影響 32
3.3.4.4. 抑菌試驗 32
3.3.4.5. 菌體吸附試驗 33
3.3.4.6. 掃瞄式電子顯微鏡 33
3.3.4.7. Genomic DNA之抽取(使用ZR Fungal/Bacterial DNA Kit進行萃取) 34
第四章、結果與討論 36
4.1. LMWC分析 36
4.1.1. 分子量與粒徑變化 37
4.1.2. 溶解度測定 38
4.1.3. 界達電位測定 39
4.2. 水解產物抗菌活性分析 40
4.2.1. 96微量孔盤 (微量試驗) 40
4.2.2. 微生物培養增值分析儀 (增量試驗) 43
4.3. 菌體吸附試驗 44
4.4. 掃瞄式電子顯微鏡 46
4.5. 菌體DNA萃取分析 49
第五章、總結 50
第六章、圖表 52
第七章、附錄 71
第八章、參考文獻 76



圖目錄
圖一、幾丁質結構 4
圖二、α- chitin的排列方式 4
圖三、β- chitin的排列方式 4
圖四、幾丁聚醣結構 6
圖五、在酸性下帶正電荷的幾丁聚醣 6
圖六、幾丁質酶作用不同去乙醯度幾丁聚醣之機制 9
圖七、溶菌酶水解作用於兩種型態的幾丁聚糖 10
圖八、幾丁寡糖-單體混合物對B.cereus和E. coli的殺菌反應模擬
機制 19
圖九、革蘭氏陽性菌結構 21
圖十、革蘭氏陰性菌結構 22
圖十一、分子量變化對溶解度與表面電位之影響 53
圖十二、三種品系金黃色葡萄球菌在濃度在400 ppm LMWC_DD92_ chit下之生長曲線圖 55
圖十三、三種品系金黃色葡萄球菌在濃度在800 ppm LMWC_DD92_ chit下之生長曲線圖 56
圖十四、二種品系大腸桿菌與濃度在100 ppm LMWC_DD92_chit下之生長曲線圖 57
圖十五、二種品系大腸桿菌與濃度在200 ppm LMWC_DD92_chit下之生長曲線圖 58
圖十六、在不同pH值下金黃色葡萄球菌BCRC 10451的生長情形 59
圖十七、在不同pH值下金黃色葡萄球菌BCRC 10780的生長情形 60
圖十八、在不同pH值下金黃色葡萄球菌BCRC 10781的生長情形 61
圖十九、根據懸浮液殘菌變化量評估不同濃度LMWC對3株金黃色葡萄球菌吸附作用變化情形 62
圖二十、根據懸浮液殘菌變化量評估不同濃度LMWC對2株大腸桿菌吸附作用變化情形 63
圖二十一、正常狀態下金黃色葡萄球菌的電子顯微 64
圖二十二、LMWC與金黃色葡萄球菌 (BCRC 10451) 作用1小時(A)並比較20 mM醋酸緩衝液沖洗後(B)的電子顯微圖 65
圖二十三、LMWC與金黃色葡萄球菌 (BCRC 10781) 作用1小時(A)並比較20 mM醋酸緩衝液沖洗後(B)的電子顯微圖 66
圖二十四、LMWC與金黃色葡萄球菌 (BCRC 10780) 作用1小時(A)並比較20 mM醋酸緩衝液沖洗後(B)的電子顯微圖 67
圖二十五、LMWC與大腸桿菌 (BCRC 10314) 作用1小時(A)並比較20 mM醋酸緩衝液沖洗後(B)的電子顯微圖 68
圖二十六、LMWC與大腸桿菌 (BCRC 10675) 作用1小時(A)並比較20 mM醋酸緩衝液沖洗後(B)的電子顯微圖 69
圖二十七、萃取3株金黃色葡萄球菌DNA並比較其量 70


表目錄
表一、利用幾丁質酶、纖維素、溶菌酶水解不同去以醯度幾丁聚醣所製備之水解產物及其物理特性探討 52
表二、E. coli及S. aureus之界達電位利用幾丁質酶、纖維酵素、溶菌酶水解所製備的LMWC之最低抑制濃度的關係 54
表三、界達電位分散體系穩定的標誌 71
表四、3株金黃色葡萄球菌Biolog之GP2 MicroPlate微生物鑑定套組的96種碳水化合物生化特性試驗結果 72
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