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研究生:蔡幸宜
研究生(外文):Shing-Yi Tasi
論文名稱:真菌Aspergillusterreus二次代謝生產lovastatin之培養條件最適化研究
論文名稱(外文):Investigation on the Optimun Culture Conditions for the Production of Lovastatin by Aspergillus terreus
指導教授:賴龍山賴龍山引用關係
指導教授(外文):Long-Shan Lai
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:應用化學系碩士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
中文關鍵詞:真菌醱酵移瓶時機
外文關鍵詞:Aspergillus terreusfermentationcross-over timing
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摘 要
本研究探討真菌Aspergillus terreus ATCC 20542 醱酵生產其二次代謝產物lovastatin之培養條件最適化。實驗上以醱酵液pH值與菌體增長二者的變化趨勢來探討均衡的菌株生長,結果顯示由一階生長轉移至二階生產之移瓶時機以16小時、第二階段培養基起始pH值6.5、以及連續醱酵7天可得到最佳產率。探討生產培養基組成份時發現碳源以乳糖(7-10%)最佳,甘油次之,葡萄糖較差。複合式碳源(1:1)以乳糖:甘油產量較乳糖:葡萄糖和甘油:葡萄糖為高。再以26-3部分因子實驗,結果顯示:乳糖、酵母萃取物、KH2PO4、 KCl、PEG2000對產量lovastatin有顯著影響。而中心混成實驗中,可看出乳糖/ KH2PO4、乳糖/ KCl、酵母萃取物/ KCl、KH2PO4/ KCl二因子之間的交互作用。所求得生產槽培養基的最適化濃度為(g/l)乳糖109.29、酵母萃取物10.92、脫脂黃豆粉1.5、KH2PO4 1.52、KCl 1.114 、PEG2000 0.452 ,此時模式預測產量為1.16g/l,而6重複之實驗結果為1.14g/l,標準差為0.067 g/l。

Abstract
In this study, we investigated the optimum culture conditions for the production of lovastatin by Aspergillus terreus ATCC 20542. A two-stage fermentation technique was applied. The trends of the pH value and the packed cell volume in broth were used to study a balanced growth for a determination of the cross-over timing. Experimental results at 28℃ and 200rpm showed that the cross-over time was 16 hours, meanwhile one could transfer the most actively growing mycelium in seed to the production stage. An initial pH value 6.5 was found suitable for the lovastatin production carried out in 7-day fermentations. Preliminary studies on the production medium showed that 7%-10% lactose was the carbon source of choice, followed by glycerol and then glucose. Besides, experiments using multiple sources of carbon revealed that 1:1 of lactose/glycerol were better than the combinations of lactose/glucose and glucose/glycerol.
A 26-3 fractional factorial design showed that lactose, yeast extract, KH2PO4, KCl, and PEG 2000 significantly affected the titre of lovastatin. Experiments designed by the central composite model explained that there were interactions between lactose/ KH2PO4. Lactose/KCl, yeast extract/KCl, and KH2PO4/KCl. The optimum production medium was found to be lactose (109.29g/l), yeast extract (10.92g/l), defatted soybean meal (1.5g/l), KH2PO4 (1.52g/l), KCl (1.114g/l), and PEG 2000 (0.452g/l). The model predicted 1.16g/l meanwhile 6-replication results was 1.14g/l. Standard deviation was 0.067 g/l.

目 錄
中文摘要 I
英文摘要 II
誌謝 III
表目錄 IX
圖目錄 I
第一章 前言
1.1、研究動機 1
1.2、本研究目的 3
第二章 文獻回顧
2.1、降低膽固醇的藥物 4
2.2、降血脂劑(HMG-CoA RIS) 4
2.3、培養基實驗設計 6
2.3.1、二水準因子設計 8
2.3.2、陡升路徑法 9
2.3.3、中心混成設計CCD 10
2.3.4、回應曲面模式適切性之統計檢定 10
第三章 材料與方法
3.1、菌株 17
3.2、實驗藥品 17
3.3、實驗儀器與設備 18
3.4、實驗設計方法 18
3.5、搖瓶實驗 18
3.5.1、全醱酵液萃取 19
3.5.2、lovastatin之定性分析 19
3.5.3、lovastatin之校正曲線 20
第四章 結果與討論
4.1、種槽階段 23
4.2、生產槽階段 25
4.2.1、最適醱酵天數的探討 25
4.2.2、生產槽培養基初始酸鹼值之
探討 25
4.2.3、生產槽培養基碳源之探討 26
4.2.4、生產槽培養基脫脂黃豆粉含量
之探討 27
4.3、實驗設計 26
4.3.1、生產槽培養基的實驗設計 28
4.3.2、部分因子實驗(T1實驗) 28
4.3.3、陡升路徑實驗(T2實驗) 29
4.3.4、中心混成實驗(T3實驗) 30
第五章 結論與建議
5.1、結論 32
5.2、建議 33
參考文獻 61
附錄STATISTICA的操作步驟 68
目錄
表4.2 T1中各變數的階層與相對應之濃度 34
表4.3 26-3部分因子設計(T1)及其實驗結果 35
表4.4 26-3部分因子設計(T1)之迴歸分析表. 36
表4.5 RSM陡升路徑(T2)之實驗設計圖 37
表4.6 RSM陡升路徑(T2)之實驗結果 38
表4.7 T3中各變數的階層與相對應濃度 39
表4.8 中心混成設計(T3)及實驗結果 40
表4.9 中心混成設計(T3)之迴歸分析表 41
圖目錄
圖2.1 RSM實驗設計之概念圖…………………………16
圖3.1 lovastatin標準品之校正曲線…………………..21
圖4.1 種槽階段pH和PCV之變化……………………43
圖4.2 A.terreus ATCC 20542 在搖瓶實驗中,醱酵參數之
變化速率(a)lovastatin產量(b)單位時間產物
產出之變化 ………………………………………………44
圖4.3 A.terreus ATCC 20542 在搖瓶實驗中,不同的起始pH值對
lovastatin的影響(a)pH(b)ppm(c)PCV%………..45
圖4.4 A.terreus ATCC 20542 在搖瓶實驗中,不同的碳源對
lovastatin(a)ppm(b)PCV%的影響…………………...46
圖4.5 A.terreus ATCC 20542 在搖瓶實驗中,不同的乳糖含量對
lovastatin(a)pH (b)ppm的影響…………………..…47
圖4.6 A.terreus ATCC 20542在搖瓶實驗中,添加甘油對lovastatin(a)pH(b)ppm(c)PCV%的影響……………………48
圖4.7 A.terreus ATCC 20542在搖瓶實驗中,複合式的碳源對
lovastatin(a)ppm(b)PCV%的影響………………….49
圖4.8 A.terreus ATCC 20542在搖瓶實驗中,不同的脫脂黃豆粉含
量對lovastatin的影響(a)ppm(b)PCV% ……………50
圖4.9 乳糖和酵母萃取物之回應曲面及等高線圖……………51
圖4.10 乳糖和磷酸二氫鉀之回應曲面及等高線圖 …………….52
圖4.11 乳糖和氯化鉀之回應曲面及等高線圖………………….53
圖4.12 乳糖和PEG2000之回應曲面及等高線圖 ……………54
圖4.13 酵母萃取物和磷酸二氫鉀之回應曲面及等高線圖……..55
圖4.14 酵母萃取物和氯化鉀之回應曲面及等高線圖…………..56
圖4.15 酵母萃取物和PEG2000之回應曲面及等高線圖……..57
圖4.16 磷酸二氫鉀和氯化鉀之回應曲面及等高線圖…………58
圖4.17 磷酸二氫鉀和PEG2000之回應曲面及等高線圖………59
圖4.18 氯化鉀和PEG2000之回應曲面及等高線圖……………60

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