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研究生:洪文凱
研究生(外文):HUNG WEN-KAI
論文名稱:黑殭菌素之分離、生物檢定及安全性評估
論文名稱(外文):Isolation, Bioassay and Safty Evaluation of Destruxins
指導教授:曾耀銘、王順成
指導教授(外文):TZENG, YEW-MIN
學位類別:碩士
校院名稱:大葉大學
系所名稱:食品工程研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:1995
畢業學年度:83
語文別:中文
論文頁數:78
中文關鍵詞:黑殭菌黑殭菌素分離生物檢定口服急毒性
外文關鍵詞:MetarhiziumDestruxinsIsolationBioassayToxicity﹒
相關次數:
  • 被引用被引用:6
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本研究目的為本土黑殭菌株所含黑殭菌素的分離純化、鑑定及其安全性及
藥效等相關問題之探討,依序分三部分進行,第一部分進行菌株鑑定、菌素
分離純化及鑑定。菌株鑑定主要以掃描式電子顯微鏡鑑定紅胸葉蟲分離之
本土黑殭菌株為黑殭菌小孢子變種。產率方面以固態培養之黑殭菌素產率
為4.7mg/g,液態培養0.33mg/mL。第二部分將固態及液態培養分離純化之
黑殭菌素經二倍系列稀釋2倍至64倍),以三齡初之甜菜夜蛾進行藥效測定,
結果顯示黑殭菌素具高的殺蟲毒效,固態萃取之黑殭菌素較液態萃取之黑
殭菌素尤具高的殺蟲毒效,顯示不同方式培養產生之菌素其生物藥效不同,
主要因素係固態培養分離純化之黑殭菌素含A+E 量為液態培養者七倍。第
三部分利用固態培養分離純化之黑殭菌孢子及黑殭菌素進行對大白鼠口服
急毒性之安全性評估,試驗以108黑殭菌孢子依胃管法以單一劑量處理大白
鼠,經21天之觀察,黑殭菌孢子在大白鼠體內並未產生增殖的現象,且於第
一天時發現85﹪以上的黑殭菌孢子皆由糞便中排出,試驗期間大白鼠外觀
臨床之觀察及體重均與對照組無顯著不同。解剖供試鼠隻之心、腎、腦、
肺、肝、脾及淋巴結亦無黑殭菌孢子感染,此菌對大白鼠不具毒性亦無感
染性,另以固態培養分離純化之黑殭菌素600mg/Kg 對大白鼠進行相同試
驗,初步結果顯示黑殭菌素於此劑量下對動物為一低毒性。
Destruxins(DTXs)can be produced by the green muscardine fungus
﹐Metarhizium anisopliae var﹒anisopliae in culture media. In
this study﹐Czapek-Dox medium enriched with 0.5%bacto-peptone
was used for shake flask production of destruxins.The
inoculated medium in 500 mL flask was held at 27℃﹐agitated
with a rotary shaker of 150rpm﹐incubated for 14-19 days(
Liquide state fermentation﹐LSF).Plastic bag containing
200-300g of autoclaved polished-rice was inoculated and
incubated at 28℃﹐24 hours l ight-period﹐for 14 days(Solid
state fermentation﹐SSF)﹒After incubation﹐both cultures were
subjected to extraction of destruxins by methylene chloride-
methanol(95:5).The crude extract was run on HPLC(Column:
waters 5C 18-AR﹐8×250mm)and separated with a linear gradient
of acetonitrile:water(30:70) to acetonitrile:water(70:30
)for 45 minutes﹒Six major peaks (6,15,19,23,25,33mins)were
collected by HPLC﹒Compounds from different retention times
were identified by FAB mass﹒Six destruxins(DA﹐DA2﹐DB﹐DMDB﹐
DD﹐DE)were isolated from both cultures﹐but the major
components were different﹒While﹐the total yields from LSF was
considerably ligher than SSF﹒ Comparsion of the biological
activity of destruxins from LSF and SSF showed that the
destruxins from SSF has a greater activity against Spodoptera
exigua larvae than destruxins from LSF﹒The mortalities of DE﹐
DA﹐DA2﹐DD﹐and DB against 3rd-instar larvae of Spodoptera
exigua were 43﹐18.33﹐11.86﹐10﹐and 2.27﹪ respectively﹒Rats
were orally dosed with Metarhizium anisopliae var﹒anisopliae
spore suspensions﹒These suspensions(108spore/ mL)produced no
adverse effects on the health of the test animals﹒There was no
evidence of infection in tissues﹒85﹪recovery of Metarhizium
anisopliae var﹒anisopliae spores was resulted from faces one
day after dosing.Results also demonstrated no toxicity of
destruxins(600mg/kg)to rats when administrated via oral route
of exposure﹒
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