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研究生:王妙鐶
研究生(外文):Miao-Huan Wang
論文名稱:由TrichodermaparceramosumBissettNo.017所產真菌抑制劑之分離純化及其特性探討
論文名稱(外文):Purification and Characterization of an Antifungal Compound Produced by Trichoderma parceramosum Bissett No. 017
指導教授:蔡國珍
指導教授(外文):Guo-Jane Tsai
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:93
中文關鍵詞:真菌抑制劑致病性酵母菌TrichodermaCandida albicans
外文關鍵詞:Antifungal agentPathogenic yeastTrichodermaCandida albicans
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Candida albicans 為人體之機緣性致病酵母菌,本研究以C. albicans為作用目標菌,從附著在海洋固體表面及土壤中,篩選得最具潛力之真菌抑制劑生產菌株,經鑑定為 Trichoderma parceramosum Bissett No. 017,分別探討碳源、氮源、鹽類及培養溫度與時間對其生產真菌抑制劑之影響,並以此最適條件來生產真菌抑制劑。當培養液以 glucose 為碳源、以 beef extract 為氮源時最有利於此菌生產真菌抑制劑。添加 0.2﹪NaCl 可增加真菌抑制劑之產生。在 1﹪glucose,1.1﹪beef extract 及 0.2﹪NaCl 之培養液中,此菌之最適生長溫度為25-30℃,125 rpm 培養7天後,培養液中真菌抑制劑活性達最高。此粗真菌抑制劑對高溫之耐受性高,在 100℃ 加熱10分鐘仍保有67﹪活性。此外,此真菌抑制劑不受 trypsin、pronase、protease K、carboxypeptidase A 等蛋白分解酵素及 α-amylase、lysozyme、pectinase 等醣類分解酵素之影響,但對 α-glucosidase、dextranase 及 papain 敏感,推測此真菌抑制劑可能為一帶有醣基及胜肽鍵結的化合物。於 pH 2∼10 時,此粗真菌抑制劑的抗菌效果不受外在 pH 值改變所影響。此真菌抑制劑之分子量介於 3000 ~ 10000 Da;冷藏或冷凍六週活性不變。除了 C. albicans 外,此真菌抑制劑對 Candida albicans CCRC 21538、Filobasidiella neoformans var. neoformans CCRC 20533及 Trichosporon beigelii CCRC 21675 等三株病原酵母菌及 Aspergillus niger CCRC 30201、Aspergillus parasiticus CCRC 30117、Penicillium minioluteum CCRC 32639及 Penicillium minioluteum CCRC 32645 等四株病原黴菌均具極強的抗菌活性。經超過濾濃縮及 G-25 膠體過濾管柱層析處理後,可得到部分純化之真菌抑制劑,其耐熱性略低於未純化狀態,對環境中酸鹼變化的耐性更高,對 Candida albicans CCRC 20513、Candida albicans CCRC 21538、Candida glabrata CCRC 20586、Candida guilliermondii CCRC 20862、Candida krusei CCRC 21543、Candida lusitaniae CCRC 21740、Cryptococcus neoformans var. neoformans CCRC 22241 等七株酵母菌及Penicillium minioluteum CCRC 31698 及 Penicillium minioluteum CCRC 32639 等兩株黴菌亦有極強之抗菌活性。動物感染試驗顯示口服此真菌抑制劑可有效提高小白鼠及大白鼠對抗 C. albicans 感染之能力。

Candida albicans is an opportunistic pathogenic yeast of humans. Among thousands of fungal isolates from various substrate exposed to seawater and from soil, an isolate identified as Trichoderma parceramosum Bissett No. 017 was screened out for the production of anti- C. albicans agent. The effects of carbon and nitrigen source, salts and cultivation temperature on the production of this antifungal agent were investigated. Glucose and beef extract were the best carbon source and nitrogen source in the medium for production of antifungal agent. The addition of 0.2﹪NaCl stimulated the production of this antifungal agent. The optimal production temperature was 25℃ in the medium composed by 1﹪glucose,1.1﹪beef extract and 0.2﹪NaCl. The crude antifungal agent was thermal stable, with residual activity being 67% after heating at 100℃ for 10 min. It was stable over a pH range of 2 to 10. It was resistant to trypsin, pronase, protease K, carboxypeptidase A, α-amylase, lysozyme, and pectinase, but susceptible toα-amylase、lysozyme and pectinase. Its molecular weight was about 3000 ~ 10000 Da.This antifungal agent showed strong activity against various pathogenic fungi including Candida albicans CCRC 21538, Filobasidiella neoformans var. neoformans CCRC 20533, Trichosporon beigelii CCRC 21675, Aspergillus niger CCRC 30201, Aspergillus parasiticus CCRC 30117, Penicillium minioluteum CCRC 32639, and Penicillium minioluteum CCRC 32645. After ultrafiltration and Sephadex G-25 gel filtration chromatography, the partial purified antifungal agent was obtained. It was less thermal stable but more pH stable than the crude agent. It also showed strong activity against Candida albicans CCRC 20513, Candida albicans CCRC 21538, Candida glabrata CCRC 20586, Candida guilliermondii CCRC 20862, Candida krusei CCRC 21543, Candida lusitaniae CCRC 21740, Cryptococcus neoformans var. neoformans CCRC 22241, Penicillium minioluteum CCRC 31698, Penicillium minioluteum CCRC 32639. The oral ingestion of this antifungal agent can increase the resistance of mice and rats against the infection of C. albicans.

壹、前言
貳、文獻整理
一、真菌
1.真菌的重要性
2.真菌感染疾病
二、抗生素
1.抗生素之定義與分類
2.真菌抑制劑
三、菌株抗藥性
1.抗藥性
2.機制
四、新抗真菌藥物之純化與特性
1. 人工合成真菌抑制劑
2. 天然動植物之萃取、分泌物
3. 利用微生物生長分泌
參、材料設備與實驗方法
一、材料設備
二、實驗方法
(一)真菌抑制劑生產菌株之培養與鑑定
(二)培養液組成及培養條件對抑制劑產量之影響
(三)利用 No. 017 菌株生產真菌抑制劑及活性鑑定
(四)粗真菌抑制劑之基本特性測定
1.熱安定性之測試
2. pH安定性之測試
3.酵素對粗真菌抑制劑活性的影響
4.粗真菌抑制劑之分子量預估
5.真菌抑制劑儲藏溫度與時間之影響
6.真菌抑制劑之拮抗菌譜 (antimicrobial spectrum)
(五)真菌抑制劑之純化與分離
1.粗真菌抑制劑之濃縮
2. Sephadex G-25膠體過濾 (gel filtration) 管柱層析
3.冷凍乾燥及其殘存活性評估
4.蛋白質定量分析
5.總醣量分析
(六)純化後真菌抑制劑之基本特性
1.溫度安定性之測試
2. pH安定性之測試
3.純化後真菌抑制劑之拮抗菌譜 (antimicrobial spectrum)
(七)動物實驗
肆、結果與討論
(一)真菌抑制劑生產菌株之培養與鑑定
(二)粗真菌抑制劑之生產條件
(三)粗真菌抑制劑之基本特性測定
(四)、粗真菌抑制劑對數種致病性真菌之抗菌效果
(五)真菌抑制劑之純化與分離
(六)純化後真菌抑制劑之基本特性
(七)動物實驗
伍、結論
陸、參考文獻

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