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研究生:林雪麗
研究生(外文):Lin, Hsueh-Li
論文名稱:分離和鑑定來自枯草芽孢桿菌E20醱酵豆粉之抗菌胜肽,並應用於改善白蝦養殖之弧菌症
論文名稱(外文):Isolation and characterization of antimicrobial peptide derived from Bacillus subtilis E20-fermented soybean meal and use it for the prevention of Vibrio infection in shrimp aquaculture
指導教授:劉俊宏劉俊宏引用關係
指導教授(外文):Liu, Chun-Hung
口試委員:黃承輝陳淑美胡紹揚
口試委員(外文):Huang, Chen-HueiChen, Shu-MeiHu, Shao-Yang
口試日期:2017-07-07
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:水產養殖系所
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:76
中文關鍵詞:Bacillus subtilis E20醱酵豆粉白蝦抗菌胜肽弧菌
外文關鍵詞:Bacillus subtilis E20fermented soybean mealwhite shrimpantimicrobial peptidesVibriosis
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本實驗證實枯草芽孢桿菌E20醱酵豆粉 (Bacillus subtilis E20-fermented soybean meal, FSBM)中存在抗菌胜肽 (Antimicrobial peptides, AMPs),其對Vibrio alginolyticus (VA)和V. parahaemolyticus (VP)具有良好的抑菌活性。透過10 kDa分子篩、5 kDa分子篩、Sephadex-G15膠體層析及高效液相層析儀 (Reversed phase high-performance liquid chromatography,RP-HPLC)方法,成功由FSBM分離出AMPs,並將純化後的醱酵豆粉抗菌胜肽 (FSB-AMP)以逆相奈米超效液相色譜 (RP-UPLC)電噴霧離子化串聯質譜 (ESI-MS/MS)鑑定其序列為HTSKALLDMLKRLGK,並證實其對VA和VP之殺菌活性較其他AMPs要來的高。抑菌活性評估顯示,FSB-AMP對VA和VP之最小抑菌濃度 (Minimal inhibitory concentrations, MIC)為72.5及72.5 μM。而透過掃描式電子顯微鏡觀察VA和VP之外觀型態改變,及螢光顯微鏡搭配碘化丙啶染色 (Propidium iodide, PI),證實FSB-AMP會造成VA和VP細胞膜破裂而導致細胞死亡。隨後,將FSB-AMP加入白蝦 (Litopenaeus vannamei)飼料中,並餵食白蝦,發現可提升白蝦抵抗VP之感染,其效果如同白蝦攝食含15%魚粉被枯草芽孢桿菌E20-FSBM取代之飼料一樣。而枯草芽孢桿菌E20-FSBM,可提高白蝦感染弧菌後之存活率,可能是由於內含FSB-AMP或FSBM中可提高白蝦腸道免疫相關基因,包括anti-lipopolysaccharide factor isoform 1 (alf1)、crustin (crus)、lysozyme (lys)、penaedin 3 (pen3)及peritrophin (ptn)表現量的物質有關。總而言之,枯草芽孢桿菌E20醱酵豆粉除是良好的飼料原料外,亦具有健康改善的生物功能,可用於養殖蝦類弧菌症的防治。
Bacillus subtilis E20-fermented soybean meal (FSBM) was found to produce antimicrobial peptides (AMPs) with great antimicrobial activity against Vibrio alginolyticus (VA) and V. parahaemolyticus (VP). Three AMPs were purified with 10-kDa and 5-kDa ultrafiltrations, Sephadex G-15 column and reverse-phase high-performance liquid chromatography (RP-HPLC). The FSB-AMP, HTSKALLDMLKRLGK, identified by an RP-nano-ultrapure liquid chromatography (UPLC) electrospray ionization (ESI)-tandem mass spectroscopic (MS/MS) analysis exhibited the highest bactericidal activity against VA and VP compared to the others. The antimicrobial activity assessment indicated that FSB-AMP inhibited the growth of VA and VP with minimal inhibitory concentrations of 72.5 and 72.5 μM. Alterations in the morphology of VA were observed by scanning electronic microscopy, and membrane disruption of VA and VP was confirmed by fluorescent microscopy with propidium iodide staining. The FSB-AMP was then incorporated into the diet of white shrimp, Litopenaeus vannamei, and a protective effect in shrimp against VP infection was recorded as well as for shrimp fed a diet containing 15% fish meal replaced by B. subtilis E20-FSBM. The survival of shrimp improved by B. subtilis E20-FSBM was due to the enhanced immune gene expressions of alf1, crus, pen3 and ptn in intestine of shrimp and AMPs in FSBM. Results demonstrated that B. subtilis E20-FSBM could be a biofunctional ingredient to prevent Vibriosis in shrimp aquaculture.
目錄
摘要…… I
Abstract III
目錄 ……………………………………………………………………VI
第一章 前言 1
第二章 文獻回顧 3
2. 1白蝦之簡介 3
2. 1. 1 白蝦的分類及習性 3
2. 1. 2 白蝦之養殖現況 4
2. 1. 3 白蝦養殖面臨之困境 4
2. 2 抗菌胜肽 (Antimicrobial peptides, AMPs) 5
2. 2. 1 AMPs之結構 5
(1) 線性的陽離子α-螺旋 (α-helical) AMPs 5
(2) 陽離子β-摺疊 (β-sheet) AMPs 6
(3) 陽離子線性延伸 (Extended) AMPs 6
2. 2. 2 AMPs之作用機制 6
(1) 圓筒穿鑿模式 (Barrel-stave model) 6
(2) 地毯模式 (Carpet model) 7
(3) 環孔模式 (Toroidal pore model) 7
2. 2. 3 醱酵食品之生物功能 9
第三章 材料與方法 12
3. 1 實驗設計 12
3. 2 菌株之來源及培養條件 13
3. 3 FSBM之製備 14
3. 4 FSBM及SBM粗胜肽之製備 14
3. 5 FSBM及SBM之抑菌活性分析 14
3. 5. 1 抑菌活性分析用病原菌株 14
3. 5. 2 抑菌活性分析 17
3. 6 AMPs之純化 17
3. 6. 1 膠體過濾層析儀 (Gel filtration chromatography) 17
3. 6. 2 逆向高壓液相層析儀 (Reversed-phase high performance liquid chromatography, RP-HPLC) 18
3. 7 胜肽鑑定及分析 19
3. 8 胜肽合成 19
3. 9 抑菌濃度分析 19
4. 1 掃描式電子顯微鏡觀察細菌外部形態的改變 20
4. 1. 1 藥品配製 20
4. 1. 2 樣品前處理 20
4. 2 螢光顯微鏡之分析細菌細胞膜之通透性 23
4. 3 AMPs對白蝦感染V. parahaemolyticus的保護評估 23
4. 3. 1 實驗飼料之製作 23
4. 3. 2 實驗飼料成份分析 26
4. 3. 2. 1 水分含量分析 26
4. 3. 2. 2 粗蛋白分析 26
4. 3. 2. 3 粗脂質分析 27
4. 3. 2. 4 灰份分析 27
4. 3. 2. 5 實驗用蝦蓄養與感染試驗 28
4. 4 枯草芽孢桿菌E20醱酵豆粉及AMPs對白蝦腸道免疫基因表現 28
4. 4. 1 白蝦腸道之總量核醣核酸萃取 29
4. 4. 2 反轉錄合成 (Reverse transcription, RT) 29
4. 4. 3 相對定量聚合酶鏈鎖反應 (Relative real-time quantitation PCR) 30
4. 5 統計分析 32
第四章 結果 33
4. 1 SBM和FSBM之抑菌活性分析 33
4. 2 枯草芽孢桿菌 E20醱酵豆粉之AMPs純化及AMPs身份鑑定 35
4. 3 FSB-AMP之抑菌活性分析 39
4. 4 細菌細胞膜之通透性 41
4. 5 分析FSB-AMP及枯草芽孢桿菌E20醱酵豆粉對白蝦感染 V. parahaemolyticus 之影響 45
4. 6 枯草芽孢桿菌E20醱酵豆粉及AMPs對白蝦腸道免疫基因表現之分析 47
第五章 討論 49
第六章 結論 54
參考文獻 55
作者簡介 64


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