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研究生:蔡泰和
研究生(外文):Tai-Her Tasi
論文名稱:探討真菌Aspergillusterreus在深層醱酵生產lovastatin過程中菌株增長形態之變化
論文名稱(外文):Investigation on morphological changes of lovastatin production by submerged cultures
指導教授:賴龍山賴龍山引用關係
指導教授(外文):Long-Shan T. 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
論文頁數:89
中文關鍵詞:真菌醱酵溶氧介質pH/DO控制菌絲增長形態
外文關鍵詞:fungal fermentationoxygen vectorpH/DO controlmorphology
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摘 要
本研究在搖瓶實驗中確定起始孢子濃度107spores/ml與接種量15﹪等條件,之後,再藉由添加2.5%之溶氧介質(正十二烷,簡稱C12)並配合醱酵過程中溶氧控制,探討菌株增長型態之改變與對lovastatin生產的影響。
在搖瓶實驗中添加C12之lovastatin產量(504.8mg/l),高於沒有添加者約1.7倍,推究其原因為C12提高溶氧,有助於菌株吸收氧氣並進而誘導其菌絲顆粒形成一緊密球狀菌絲體其粒徑分佈為1.3mm-0.65mm,且球狀菌絲之顆粒均一性較高(平均直徑約0.8mm),而菌絲顆粒密度為 880 pellet/ml。但是在醱酵槽中,添加等量的C12會產生高溶氧狀態(DO>60%或以上)並產生形態不規則(星形)之菌絲體,其菌絲顆粒密度雖高達 1050 pellets/ml,但lovastatin之產量僅達未添加C12之10%,推究其原因主要是高溶氧之限制。
另由pH之控制進行實驗,而醱酵槽中,由反應第48小時後開始控制pH,其結果以未有pH控制者較佳,推論主導其菌絲體形成以及誘導二次代謝物生產之關鍵酵素之pH最適化控制策略仍待探討。此外,在真菌A. terreus 深層醱酵過程中,實驗證實pH與DO之交互作用影響真菌細胞之增長形態變化和lovastatin之產量。

Abstract
In the studies, a 107 spores/ml of initial spore density and a 15% of inoculum from seed to production were applied throughout the experiments. We then applied an adding of 2.5% n-dodecane, coupled with DO (dissolved oxygen) controls, to examine its effects on the changes of morphology of Aspergillus terreus and the associated secondary metabolite production in submerged cultures.
In shaking-flask experiments, adding a 2.5﹪C12 enhanced lovastatin production by a factor of 1.7-fold (504.8mg/l), compared with no adding. Base on experimental observations, this oxygen vector increased dissolved oxygen tension, enhanced DO utilization and hence induced the formation of uniform and compact pellets, with a pellet diameter distribution from 0.65-1.3mm. The average pellet diameter was 0.8mm; meanwhile, pellet density was calculated as 880pellets/ml. On the contrary, adding a 2.5% C12 resulted in formation of a number of star-like pellets in a 5L fermentor operation. Although the pellet density was as high as 1050 pellets/ml, it resulted in a 90% decrease of lovastatin production. This phenomenon was mainly attributed an inhibition to lovastatin synthesis resulting from high DO levels in broth.
Except DO controls, no benefits were obtained in term of lovastatin production while coupled with additional pH controls starting 48 hr later of fermentations. Results of such experiments showed a higher lovastatin production in a pH self-stabilized case, which implied that the pH strategies controlling activities of key enzymes, which probably dominated the pellet formation of A. terreus and induced its associated secondary metabolite production, needed to be sought. Besides, the pH-DO interactions influencing cellular morphology and lovastatin production by A. terreus were experimentally proved to be true.
【Key Words:Aspergillus terreus, lovastatin, morphology, pellet formation】

目 錄
中文摘要………………………………………………….....Ⅰ
英文摘要……………………………………………………...Ⅱ
表目錄………………………………………………………...Ⅲ
圖目錄………………………………………………………...Ⅳ
第一章緒論
1.1 前言……………………………………………….……..1
1.2 研究目的………………………………………….……..4
第二章文獻回顧
2.1 降血脂劑……………………………………………....7
2.2 真菌醱酵生理………………………………………...12
2.3真菌二次代謝物生產……………………………..17
2.3.1 一次代謝物……………………..……………....17
2.3.2 二次代謝物…………………….…………….....17
2.3.3 真菌之型態變化………………………….….....19
2.4 溶氧介質…………………..…………….…………..23
2.5 醱酵放大技術………………………………………….24
第三章材料與方法
3.1 菌株………………………………………………….…30
3.2 實驗藥品…………………………………………….…31
3.2.1 培養基……………………………………......…31
3.3 實驗儀器設備……………………………………..….32
3.4 實驗方法…………………………………………..….33
3.4.1 搖瓶實驗……………..……………………....…33
3.5 醱酵槽試驗……………………………………….....34
3.5.1 醱酵槽溶氧介質添加試驗…….…………….....34
3.6 分析方法…………………………………………..….36
3.6.1 細胞乾重……………………………………......36
3.6.2 萃取……………………………………………....36
3.7 定量分析………………………………..…………..37
3.7.1 HPLC分析條件……………..………………......37
3.7.2 殘糖總濃度分析(RSC)…………………….....37
第四章結果與討論
4.1 搖瓶實驗……………………………………………..40
4.1.1 起始孢子濃度效應……………………………....42
4.1.2 接種量之效應. …………………...…. ……...42
4.1.3 溶氧介質之效應……………………...…...…..43
4.2 5升醱酵槽實驗………………………………..…….46
4.2.1 添加溶氧介質(C12H26)之實驗……………....47
4.2.2 pH控制實驗………………………….……..…..48
4.2.3 真菌型態變化探討 …………………...…....…50
第五章結論與建議
5.1 結論…………..……………………………………..53
5.2 建議………………………………………………....54
附錄
一.不同降血脂劑之化學結構圖……………………………..72
二.lovastatin分析校正曲線圖…………………………....73
三.殘糖濃度分析校正曲線圖…………………………..…..73
四.5升醱酵槽操作步驟………………………………….....74
符號說明……………………………………………………...80
參考文獻……………………………………………………….81

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