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研究生:蕭嘉豪
研究生(外文):Hsiao, Chia-Hao
論文名稱:環境因子對大腸桿菌噬菌體生產之影響
論文名稱(外文):The effect of environmental factors on the bacteriophage ϕ103p productionof Escherichia coli O103
指導教授:楊芳鏘楊芳鏘引用關係
指導教授(外文):Yang, Fan-Chiang
口試委員:楊芳鏘林其昌洪志勳
口試委員(外文):Yang, Fan-ChiangLin, Chi-ChangHung, Chih-Hsun
口試日期:2013-01-20
學位類別:碩士
校院名稱:東海大學
系所名稱:化學工程與材料工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:96
中文關鍵詞:大腸桿菌噬菌體環境因子
外文關鍵詞:Escherichia coli,BacteriophageEnvironmental factors
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近年來對於迅速崛起多種的新病原菌種中,大多數新病原菌都擁有可抵抗抗生素的抗藥性因子,造成可使用的抗生素日益減少,甚至沒有效果可殺死或清除這些病原菌。目前有科學家以噬菌體治療的方式,發現能夠有效殺死這些新病原菌,而且對生物體不會有任何副作用。但是現階段要製備大量噬菌體的技術仍然不成熟。為了能夠充分了解製備噬菌體的方法,我們利用大腸桿菌作為宿主菌,並加入噬菌體感染大腸桿菌探討其生長環境的優缺點,希望建立日後噬菌體大規模量產的基本數據。
本實驗主要的目的是探討環境因子對感染大腸桿菌O103(Escherichia coli O103)之噬菌體(ϕ O103p)生產之影響,分別探討培養轉速、溫度、pH、添加金屬離子、培養基成分改變,並擴大為搖瓶實驗探討噬菌體生長之情形。實驗得知,搖瓶實驗中,原LB Broth培養基培養大腸桿菌的情況下,在200rpm、37℃下,加入噬菌體(109 PFU/ml),感染後120 min噬菌體斑數目PFU(plaque forming units)為1.70 x 109 PFU/ml;添加0.3g MgCl2 /per 100 ml於LB Broth培養基中,於200rpm、37℃下培養並加入噬菌體,感染後120 min,數目為4.08 x 109 PFU/ml. LB培養基中,培養大腸桿菌轉速為200 rpm,培養噬菌體時轉速改為150 rpm、37℃下加入噬菌體,感染後120 min,數目為5.00X 109 PFU/ml;。為了降低培養基成本,以酒糟(Thin Stillage)作為液態培養基,添加MgCl2於培養基中,於150 rpm、37℃下震盪培養大腸桿菌,並於5小時後加入噬菌體,感染後80分鐘噬菌體數目為1.92 x 1010 PFU/ml,是為LB Broth 培養基在200 rpm下培養的11.35倍。此外,本研究也進行小型發酵槽培養試驗,以酒糟作為液態培養基,在100rpm、37℃、無通氣量條件下培養大腸桿菌,5小時加入噬菌體感染後200分鐘,可達到1.00 x 109 PFU/ml。
關鍵字:大腸桿菌、噬菌體、環境因子

In recent years, some of so-called super bacteria have appeared, which normally had the characteristics of containing the genes resistant to antibiotics. One of novel ways for treatment of the disease caused by such bacteria is phage therapy. Phage therapy has been demonstrated to be effective for killing bacteria, and had no side effect to the organisms. However, the technology for large scale prodcution of phage has not been fully developed at present. The objective of this research was to investigate the effect of different cultural conditions on the production of phage. The Escherichia coli O103 was chosen as the host for the production of phage (ϕ O103p).
In this study, the influence of cultural conditions including temperature, pH, ions, medium compositions were investigated. Moreover, different types of cultural vessels were also compared to understand the feasibility of large scale production. In the sample-bottle culture of using, LB Broth at 200 rpm, 37℃ in shaker the level of phage PFU(plaque forming unit) reached to 1.70 X 109 PFU/ml, ) after infection 120 min. The addion of 0.3 g MgCl2 was proved to very effective to enhance the phage concentration to PFU 4.08 X 109 PFU/ml. In contrast, if the rotating speed decreased 150 rpm, the phage PFU rosed to 5.00 X 109 PFU/ml. In order to lower medium cost, thin stillage from a local distiller was used as media. Under the conditions of adding MgCl2, 150 rpm and 37℃ for 5 hours for E.coli growth, the phage PFU enhanced to the level of 1.92 x 1010 PFU/ml after infection of 80 minute, which was 11 times more than the control. A 5-liter stirred tank bioreactor was also used for the culture of E.coli and phage production. Using thin stillage culture to E.coli at 200 rpm, 37℃ 5 hours,add the phage, after infection 120 min, the concentration in fermenter reached to 1.00 x 109 PFU/ml.
Keywords: Escherichia coli, bacteriophage, environmental factors

目錄
誌謝..............................................................Ⅰ
摘要...............................................................Ⅱ
Abstract............................................................Ⅳ
目錄...............................................................Ⅵ
圖目錄.............................................................XI
表目錄...........................................................XIII第一章 緒論........................................................1
1-1 前言.........................................................1
1-2 研究動機與目的...............................................3
第二章 文獻回顧....................................................4
2.1 大腸桿菌簡介..................................................4
2.2 大腸桿菌的培養................................................5
2-2-1培養基成分................................................5
2-2-2以發酵方式培養大腸桿菌...................................5
2.3噬菌體的簡介.................................................6
2-4 噬菌體的應用................................................8
2-4-1 噬菌體療法之治療.........................................8
2-4-2食品方面中噬菌體的應用....................................9
2-4-3噬菌體對細菌的檢測.......................................10
2-4-4噬菌體對生物的呈現技術..................................10
2-4-5其他方面的噬菌體應用.....................................11
2-5 噬菌體的生產................................................12
2-5-1 噬菌體的培養及計算方式..................................12
2-5-2 噬菌體的成長範圍........................................13
2-5-3商業化生產噬菌體........................................14
2-5-4金屬離子的添加對噬菌體的影響............................17
第三章 實驗材料與方法.............................................19
3-1 實驗菌株與保存..............................................19
3-1-1大腸桿菌之菌株保存.......................................19
3-1-2噬菌體的保存.............................................21
3-1-3噬菌體的增量與濃縮.......................................21
3-2實驗儀器與藥品...............................................23
3-3實驗方法....................................................25
3-3-1 實驗架構................................................25
3-3-2 探討環境因子對大腸桿菌生長之影響........................26
3-3-2-1 大腸桿菌之隔夜菌製備.................................26
3-3-2-2 探討大腸桿菌生長曲線之測定...........................26
3-3-2-3 探討添加碳源對大腸桿菌生長之測定.....................26
3-3-2-4 搖瓶培養中大腸桿菌生長曲線之測定.....................27
3-3-2-5 搖瓶培養中以不同密閉方式培養大腸桿菌生長之測定.......27
3-3-2-6 搖瓶培養中不同震盪轉速對大腸桿菌生長之測定............27
3-3-2-7搖瓶實驗中不同Define medium對大腸桿菌生長之測定.......28
3-3-2-8 搖瓶實驗中豆漿成分對大腸桿菌生長之測定................29
3-3-2-9 搖瓶實驗中酒糟成分對大腸桿菌生長之測定................29
3-3-2-10小型發酵槽中通氣量對大腸桿菌生長之測定...............29
3-3-3 探討環境因子對噬菌體感染之影響...........................30
3-3-3-1 不同的MOI差異對噬菌體φO103p感染之測定..............30
3-3-3-2 探討不同pH值對噬菌體φO103p感染之測定................30
3-3-3-3 添加額外碳源對噬菌體φO103p感染之測定................31
3-3-3-4 探討震盪溫度對噬菌體φO103p感染之測定.................31
3-3-3-5 探討改變震盪轉速對噬菌體φO103p感染之測定..............32
3-3-3-6 探討添加金屬離子對噬菌體φO103p感染之測定............32
3-3-3-7 搖瓶實驗中探討最佳震盪轉速對噬菌體φO103p感染之測定..33
3-3-3-8 搖瓶實驗中探討高震盪溫度對噬菌體φO103p感染之測定....33
3-3-3-9搖瓶實驗中探討不同Define medium對噬菌體感染之測定.....34
3-3-3-10 搖瓶實驗中探討不同基質對噬菌體φO103p感染之測定.....34

3-3-3-11 搖瓶實驗中酒糟在最佳環境因素對噬菌體φO103p之影響...35
3-3-3-12 搖瓶實驗中酒糟在高震盪溫度下對噬菌體φO103p之影響...35
3-3-3-13小型發酵實驗中噬菌體φO103p生產之影響................35
第四章 結果與討論..................................................37
4-1 大腸桿菌液態培養實驗.........................................37
4-1-1大腸桿菌O103生長曲線之測定..............................37
4-1-2添加碳源對大腸桿菌菌株O103之影響........................40
4-1-3搖瓶培養中大腸桿菌菌株O103生成之測定與樣品瓶結果比較....42
4-1-4搖瓶培養不同密封方式對大腸桿菌之影響......................45
4-1-5搖瓶培養 Define medium對大腸桿菌生成之測定...............47
4-1-6搖瓶培養豆漿(Soy milk)對大腸桿菌生成之測定.................50
4-1-7搖瓶培養酒糟(Thin Stillage)對大腸桿菌生成之測定.............52
4-1-8 小型發酵槽在不同通氣量培養酒糟對大腸桿菌生成之測定.......54
4-2 噬菌體φO103p液態培養實驗...................................57
4-2-1 不同MOI值與噬菌體φO103p感染程度之影響.................57
4-2-2 不同pH值與噬菌體φO103p感染程度之影響..................59
4-2-3 添加碳源與噬菌體φO103p感染程度之影響....................61
4-2-4 震盪溫度與噬菌體φO103p感染程度之影響....................63
4-2-5 震盪轉速與噬菌體φO103p感染程度之影響...................66
4-2-6 添加金屬離子與噬菌體φO103p感染程度之影響...............71
4-2-7 搖瓶培養中震盪轉速與噬菌體φO103p感染程度之探討.........74
4-2-8 搖瓶培養實驗在42℃時與噬菌體φO103p感染程度之探討.......76
4-2-9 搖瓶培養實驗在不同Define medium時與噬菌體生產之影響.....78
4-2-10 搖瓶培養實驗在不同基質時與噬菌體φO103p感染程度之探討..80
4-2-11 搖瓶實驗以最佳環境因子對噬菌體φO103p感染程度之探討....82
4-2-12 搖瓶實驗以42℃改變轉速對噬菌體φO103p感染程度之探討...84
4-2-13 小型發酵槽對噬菌體φO103p感染程度之探討................86
第五章 結論與未來展望..............................................87
5-1結論........................................................87
5-2未來展望....................................................91
參考文獻...........................................................92

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