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研究生:林偉誠
研究生(外文):Wei - Chen Lin
論文名稱:應用StreptomycespadanusPMS-702生產Fungichromin最適化培養條件之探討
論文名稱(外文):Optimization of the cultivation conditions on the fungichromin production from streptomyces padanus PMS-702
指導教授:劉永銓
指導教授(外文):Yung-Chuan Liau
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系
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:2004
畢業學年度:92
語文別:中文
論文頁數:101
中文關鍵詞:生物防治鏈黴菌治黴色基素多烯類大環內酯生物檢定分析回應曲面法
外文關鍵詞:Streptomycesfungichromin
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本研究中,使用具有抗真菌活性的鏈黴菌菌株Streptomyces padanus PMS-702,進行大環多烯類抗生素fungichromin之醱酵生產研究。此抗生素對於立枯絲核菌、蕃茄晚疫病有良好的抑制效果,也可應用於如腫瘤等之臨床醫療上。初步利用搖瓶實驗作培養之結果,由醱酵液中所得之fungichromin的產量約為0.005 %(W/V)。利用溶劑置換及減壓濃縮的方式,可製得fungichromin的純度為約91%。不同的氮、碳源組合之液體培養基中,以黃豆粉加葡萄糖的試驗組,所得fungichromin的產量為最高;利用回應曲面實驗設計法探討產生fungichromin最適之醱酵培養基組成為,1.1 %脫脂黃豆粉、1.1 % 葡萄糖、0.044%碳酸鈣;培養基最適初始pH和醱酵溫度,分別為5.6和31.7℃。在以上的條件作醱酵培養,可得到較佳的fungichromin濃度,其統計預估濃度值為 11mg/100ml,與實際實驗觀測值,(fungichromin平均濃度)可達11.2 ± 0.3 mg/ml相接近。本研究結果提供Streptomyces padanus PMS-702產生fungichromin之最適條件(包括培養基組成、醱酵培養條件及純化製備等),有助於fungichromin實際生產製程之開發。
In this study, a novel discovered strain named Streptomyces padanus PMS-702, harbouring antifungal activity, was used to produce polyene macrolide antibiotic, fungichromin in various cultivation conditions. The antibiotic fungichromin was found to be an active ingredient for the control of Rhizoctonia damping-off of cabbage and tomato late blight. It was also found useful in the clinical cancer treatment. In the preliminary fermentation experiment, the fungichromin concentration in the broth was about 0.005%(w/v). For obtaining pure fungichromin, solvent replacement and crystallization through vacuum evaporation were applied to obtain fungichromin purity of 91%. For determining the medium components, various carbon and nitrogen sources were employed in the fermentation. The result displayed that soybean and glucose were the best components for the fungichromin production. Next, the response surface methodology(RSM) was used to optimize medium composition. Hopefully, the improvement of fermentation conditions could bring about an efficient and economical fungichromin fermentation process.
目錄
中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
表目錄 Ⅵ
圖目錄 Ⅶ
第一章、緒論 1
1- 1、前言 1
1-2、文獻回顧 4
1-2-1、鏈黴菌的分類與特性簡介 4
1-2-2、鏈黴菌的生物特性 6
1-3、鏈黴菌與病害生物防治 7
1-3-1、鏈黴菌屬產生抗生物質之情形 10
1-3-2、鏈黴菌所產生的抗生物質之簡介及特性 13
1-4鏈黴菌、PMS-702菌株簡介 17
1-4-1、鏈黴菌PMS-702之應用 18
1-5、fungichromin的簡介 19
1-5-1、fungichromin的相關研究 20
1-5-2、fungichromin萃取純化方式 22
1-5-3、回應曲面法之應用 24
第二章、fungichromin之標準品製備 34
2-1、前言 34
2-2、材料與方法 35
2-3、fungichromin純化與分析方法 36
2-4、結果與討論 41
第三章、S. padanus PMS-702生產fungichromin的培養基探討 48
3-1、前言 48
3-2、材料與方法 49
3-2-1、基礎培養基培養情形 49
3-2-2、氮源置換 53
3-2-3、碳源置換 56
3-2-4、S. padanus PMS-702生產fungichromin之時間曲線 58
3-3結果與討論 60
第四章、回應曲面法設計治黴色基素最適培養基之探討 63
4-1、前言 63
4-2、材料與方法 64
4-2-1、回應曲面實驗設計法 64
4-2-2、回應曲面法之原理 64
4-2-3、二水準因子設計原理 65
4-2-4、陡升路徑法原理 68
4-2-5、中心混成設計原理 69
4-2-6、回應曲面模式適切性之統計檢驗 69
4-3、實驗設計 70
4-3-1、分析方法 70
4-3-2、二水準因子設計 70
4-3-3、陡升路徑法 71
4-3-4、中心混成設計 73
4-4、結果與討論 74
第五章、結論與展望 88
5-1、結論 88
5-2、未來展望 90
參考文獻 91
表目錄
表1-1、1990年後應用放線菌防治植物病害 16
表1-2、Streptomyces sp. PMS-702菌株、S. galbus CCRC-12166菌株及S. padanus CCRC-12168菌株之生理及生化特性 29
表1-3、大環內酯類化合物之共軛雙鍵數目及最大紫外光吸收光譜值 31
表1-4、fungichromin之理化性質 32
表1-5、fungichromin與lagosin及cogomycin的分子式及物理性 33
表3-1、DNS試劑之組成 50
表3-2、fungichromin於置換碳氮源之培養基的時間曲線 59
表4-1、二水準因子設計反應參數實驗值之範圍 82
表4-2、二水準因子設計之結果 82
表4-3、陡升路徑之實驗結果 83
表4-4、中心混成設計反應參數實驗值之範圍 83
表4-5、中心混成實驗設計之結果 84
表4-6、中心混成實驗設計之回歸分析表 85
表4-7、二階RSM模式之變異係數分析(ANOVA) 86
表4-8、二階RSM模式之最大值 87
圖目錄
圖1-1、Streptomyces padanus PMS-702菌株在Inorganic salts starch agar (ISP4)培養基培養21天的形態 25
圖1-2、Streptomyces padanus PMS-702菌株在燕麥粉培養基平板生長14天後,於掃描式電子顯微鏡下之形態幾丁質 26
圖1-3、fungichromin之結構式 27
圖1-4、利用Streptomyces padanus strain PMS-702濾液純化所得daidzein, fungichromin及sterol glycoside對於Rhizoctonia solani之效果 28
圖2-1、本實驗所使用的醱酵槽系統 38
圖2-2、醱酵培養中的S.padanus PMS-702 (200倍率放大) 39
圖2-3、fungichromin的純化流程圖 40
圖2-4、經萃取處理後,物質最後形態 42
圖2-5、萃取物質之UV圖譜 (稀釋100倍) 42
圖2-6、文獻中的fungichromin圖譜 43
圖2-7、乙酸乙酯萃取物質之UV圖譜 43
圖2-8、文獻之fungichromin HPLC圖譜 45
圖2-9、萃取物質: fungichromin之HPLC圖譜fungichromin 46
圖2-10、fungichromin之濃度檢量線 47
圖3-1、還原糖吸光值之校正曲線 51
圖3-2(a)、S. padanus PMS-702於基礎培養基的時間曲線 52
圖3-2(b)、S. padanus PMS-702於基礎培養基的時間曲線 53
圖3-3、fungichromin於各置換氮源之培養基之產量影響 55
圖3-4、fungichromin於各置換源之培養基之產量影響 57
圖3-5、生產fungichromin之反應時間曲線 60
圖3-6、S. padanus PMS-702的殘糖量及乾重之時間曲線 62
圖4-1、回應曲面法實驗設計流程圖 66
圖4-2、RSM實驗設計之概念圖 67
圖4-3、RSM陡升路徑之概念圖 69
圖4-4、葡萄糖與碳酸鈣對fungichromin之回應曲面圖 77
圖4-5、脫脂黃豆粉與碳酸鈣對fungichromin之回應曲面圖 77
圖4-6、葡萄糖與脫脂黃豆粉對fungichromin之回應曲面圖 78
圖4-7、pH與碳酸鈣對fungichromin之回應曲面圖 78
圖4-8、葡萄糖與pH對fungichromin之回應曲面圖 79
圖4-9、pH與脫脂黃豆粉對fungichromin之回應曲面圖 79
圖4-10、pH與Temperature對fungichromin之回應曲面圖 80
圖4-11、Temperature與碳酸鈣對fungichromin之回應曲面圖 80
圖4-12、葡萄糖與Temperature對fungichromin之回應曲面圖 81
圖4-13、Temperature與脫脂黃豆粉對fungichromin之回應曲面圖 81
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