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研究生:胡逸廷
研究生(外文):Yi-Ting Hu
論文名稱:從環境分離和分析鑑定在水產養殖應用上具抑菌能力之益生菌
論文名稱(外文):Isolation and characterization of pathogen-inhibiting probiotics from the environments for aquaculture application
指導教授:陳俊堯陳俊堯引用關係
指導教授(外文):Chun-Yao Chen
學位類別:碩士
校院名稱:慈濟大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:95
中文關鍵詞:益生菌水產養殖
外文關鍵詞:probioticaquaculture
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長久以來對抗水產動物疾病的方法大量依賴抗生素,但是長期過度的施用藥物,卻造成了具有抗藥性菌株的演化。我們嘗試由環境中分離能抑制魚類病原菌的細菌,以生態平衡的方法來控制病原菌。我們從海水與海鱺腸道中分離出 985 株細菌,其中 21 株在瓊脂擴散法 (agar-diffusion assay) 中證實對海鱺的病原菌雀鯛光桿菌弒魚亞種 (Photobacterium damselae piscicida) 菌株248、�鴽Q斯頓氏菌 (Listonella anguillarum) 菌株 P3 和吳郭魚病原菌創傷弧菌 (Vibrio vulnificus) 菌株93U204 具有抑制效果,進一步將這 21 株對斑馬魚以腹腔注射進行毒性測試後選出 11 株無毒性或低毒性的環境菌,再從中選取 2 株分泌型與 2 株接觸型進行測試,並從中挑出抑制病原菌能力最強的 L19 做進一步的研究。
利用 16S rDNA 序列分析,L19 鑑定為 Pantoea 種名。其培養之上清液能有效的抑制創傷弧菌及雀鯛光桿菌生長,共同培養實驗中也能有效抑制病原菌的生長。穿透式電子顯微鏡的觀察中也發現 L19 會對造成病原菌表面形態的改變。我們證實了上清液中的抑制物質為一對蛋白�� K 與胰蛋白�﹞ㄠ虓P,分子量小於 10 kD 耐酸之物質。L19 除了能直接抑制病原菌的生長外,我們發現加入此菌株能誘使已附著在黏液表面的雀鯛光桿菌弒魚亞種形成聚集並脫離表面的現象,這可能是另一種阻止病原菌的機制。益生菌 L19 可在養殖的低養條件下長時間存活,而 L19 的浸泡處理也可以提升斑馬魚在養殖環境下感染創傷弧菌後的存活率。益生菌 L19 具有廣效性抑菌能力與耐低養份環境的特性,根據以上結果未來將進一步評估並利用於養殖現場。
Combating microbial pathogens in aquaculture industry has been heavily relied on application of antibiotics, and as a result, this approach has fostered the rise of resistant bacteria. We attempted to use environmental and commensal bacteria as an ecological control agent against fish pathogens. Nine hundred and eighty five isolates were obtained from seawater and cobia intestine, 21 of them shown inhibitory activity against Photobacterium damselae piscicida, Listonella anguillarum or Vibrio vulnificus. Eleven isolates were proved to be non-pathogenic in zebrafish safety test. Two isolates demonstrating cell-associated and two demonstrating secreted pathogen inhibition activities were selected and compared, and isolate L19 was selected for further study due to its high potency.
L19 was identified as Pantoea ananatis by 16S rDNA analysis. Inhibition against V. vulnificus and P. damselae piscicida can be achieved by co-culture with L19 culture supernatant or live bacteria. L19 supernatant treatment damaged pathogen cell surface, as revealed by transmission electron microscopy. The inhibitory substance in culture supernatant is protease insensitive and acid resistant, and can pass the 10-kD molecular sieve. Addition of L19 can induce aggregation of P. damselae piscicida, which may provide additional protection. L19 reduced mortality in zebrafish receiving immersion challenge of V. vulnificus, and can persist in low nutrient environment for at least 15 days. These characteristics make L19 eligible for further field test in aquaculture as a pathogen-inhibiting probiotic.
中文摘要……………………………………………………………………………………………Ⅰ
英文摘要……………………………………………………………………………………………Ⅱ

第一章、研究背景
1. 水產養殖與細菌性疾病…………………………………………………………………1
2. 水產養殖與益生菌………………………………………………………………………6
3. 研究目的…………………………………………………………………………………10

第二章、由環境中篩選益生菌
1. 緒論………………………………………………………………………………………11
2. 材料與方法………………………………………………………………………………12
3. 結果………………………………………………………………………………………19
4. 討論………………………………………………………………………………………23

第三章、益生菌 L19 特性分析
1. 緒論………………………………………………………………………………………26
2. 材料與方法………………………………………………………………………………27
3. 結果………………………………………………………………………………………34
4. 討論………………………………………………………………………………………41

參考文獻……………………………………………………………………………………………68
附錄…………………………………………………………………………………………………87
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