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研究生:吳志聰
研究生(外文):Chih-tsung, Wu.
論文名稱:蠟蚧輪枝菌固態發酵培養生產孢子之探討
論文名稱(外文):Study on the Production of Verticillium Lecanii spores by Solid State Fermentation
指導教授:吳宗正吳宗正引用關係曾耀銘曾耀銘引用關係
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:119
中文關鍵詞:蠟蚧輪枝菌回應曲面法固態發酵幾丁質分解酵素
外文關鍵詞:Verticillium LecaniiResponse surface methodologySolid state fermentationchitinase
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蠟蚧輪枝菌(Verticillium lecanii)是一種分布極廣,寄生眾多農業害蟲的病原真菌,當與寄主細胞或是類似幾丁質、幾丁聚醣的物質接觸時,會誘導促成幾丁質相關酵素的合成。也是在生物性農藥中很早被開發出來的微生物殺蟲劑,在過去研究中被人發現其孢子產量與殺蟲效果有著密不可分的關係,惟尚未見有系統的研究。因此本研究即針對此一關係,以下述三個部分予此深入探討:1.蠟蚧輪枝菌基本培養相關條件對產孢量影響之探討,2.找出影響蠟蚧輪枝菌產孢量之最適培養基,3.利用高壓間歇除熱固態發酵槽擴大培養生產孢子。
在基本相關培養條件方面,以白米40g、米糠55g製成太空包時,每包添加50 mL的無菌水,控制溫度在24℃,相對溼度90%,進行發酵培養14天,可得產孢量為2.1´109 #/g。
有關最適培養基部分是利用回應曲面法( response surface methodology, RSM ),回歸出一階和二階模式,從模式中觀察培養基各成分對產孢量的影響,找到最適培養基之各成分的組成分別為白米58.8 g、米糠34g。其孢子產量之理論計算值為8.33´109 #/g;實際以太空包培養所得之產孢量為8.72´109 #/g,結果顯示回應曲面法具有良好的可靠性。
傳統的固態發酵培養方法因受限於通氣及散熱效率問題,總是無法提高培養基厚度跟產孢量,本研究提出一經改良設計的新型高壓間歇除熱固態發酵系統。結果顯示以白米及米糠為培養基,控制在溫度24~26℃、溼度70%以上,壓力幅度0.5 atm,發酵培養14天,其產孢量及培養基厚度均可提升兩倍以上。
另外,加入稻殼當作膨鬆劑以利於通氣及散熱,以進行白米、米糠、稻殼三種培養基組成的太空包交叉實驗,發現培養基組成成分為白米30g、米糠30g、稻殼30g時,溫度24℃、相對溼度70%以上,可獲得最大產孢量為4.75 ×1010 #/g,同時發現米糠對產孢量的影響遠大於白米。
關鍵字:蠟蚧輪枝菌、回應曲面法、固態發酵、幾丁質分解酵素
Verticillium Lecanii (V.L.) is generally recognized as the pathogenic fungi that most wildly spread and parasite in many agricultural insects. The important metabolic characteristic is that they will produce inducible chitinolytic related enzymes when they contact with host insect cells or chitinolytic and chitosanic analogous. This genus is the most earier item used as microbial pesticide in the past time. The previous studies showed that their pesticide effectiveness highly related with the amount and quality of spore, but no further systematic study to establish this relationship was found.
In order to clarify the relationship between the spore quantity and pesticide effectiveness, this study was devoted with three major parts :1. The optimize culture conditions for the production of V.L. spores; 2. The most suitable medium composition for spores production; 3. Scale-up the process by using innovated high pressure periodic breath function of solid state fermentation technique.
Several important significant results were established in this study. First, the medium composited with 40g rice, 55g rice bran, and 50 ml aseptic water, culture conditions with 24℃, R.H. 90% would produce the largest amount of spores during 14 days fermentation. The final spore production was 2.1 ×109#/g. Second, Response surface methodology (R.S.M.) was applied to search the relationship between the medium composition and the amount of spore production, the theoretical result was that the medium contained with rice 58.8g and rice bran 34g, the spore amount will be 8.72 ×109#/g. Comparing with the experiment result of 8.33 ×109#/g spores, it showed that the R.S.M. technique had high reliability and usefulness for this kind of study. Third, High-pressuse periodic breath SSF shows excellent capability to improve the spore production and enlarge the thickness of medium mash.It was found that on the conditions of 24~26℃, 70%R.H., 0.5 atm pressure for 14 days fermentation, the amount of spore production could be increased two folds.
Finally, this study also found that the unhusked rice was the major contributor to the spore production during SSF process. At 24℃, 70%R.H., the unhusked rice which served as leavening agent, will produce the largest amount of spores with 4.75 ×109#/g in the medium composed of 30g rice, 30g rice bran and 30g unhusked rice.
Keyword: Verticillium Lecanii, Response surface methodology, Solid state fermentation, chitinase.
授權書……………………………………………………………………i
學位審定書………………………………………………………………ii
中文摘要………………………………………………………………...iii
英文摘要…………………………………………………………………v
謝誌……………………………………………………………………...vi
目錄……………………………………………………………………..vii
表目錄…………………………………………………………………...ix
圖目錄…………………………………………………………………...xi
第一章 緒論
一、前言…………………………………………………………………1
二、文獻回顧……………………………………………………………4
第二章 蠟蚧輪枝菌以太空包固態發酵培養生產孢子之培養條件探討
一、前言………………………………………………………………..28
二、實驗材料及方法…………………………………………………..29
三、結果與討論………………………………………………………..33
四、結論………………………………………………………………..38
第三章 蠟蚧輪枝菌以固態發酵生產孢子之最適培養基探討
一、前言………………………………………………………………..50
二、材料與方法………………………………………………………..51
三、結果與討論………………………………………………………..57
四、結論………………………………………………………………..61
第四章 利用高壓間歇除熱固態發酵槽發酵擴大生產蠟蚧輪枝菌孢子
一、前言………………………………………………………………..76
二、高壓間歇除熱固態發酵槽的背景及原理………………………..77
三、材料與方法………………………………………………………..82
四、結果與討論………………………………………………………..86
五、結論………………………………………………………………..90
第五章 總結論………………………………………………………..107
第六章 未來與展望…………………………………………………..109
參考文獻………………………………………………………………111
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