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研究生:陳怡安
研究生(外文):Chen, Yi-An
論文名稱:微酸性電解水結合高壓技術對李斯特菌的殺菌效果之探討
論文名稱(外文):Studies on Inactivation of Listeria monocytogenes by Slightly Acidic Electrolyzed Water Coupled with High Pressure Processing
指導教授:陳泰源陳泰源引用關係
指導教授(外文):Chen, Tai-Yaun
口試委員:黃書政張心儀黃登福龔瑞林陳冠文
口試委員(外文):Huang, Shu-ChenChang, Hsin-YiHwang, Deng-FwuKong, Zwe-LingChen, Guan-Wen
口試日期:2020-01-13
學位類別:碩士
校院名稱:國立臺灣海洋大學
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:2019
畢業學年度:108
語文別:中文
論文頁數:107
中文關鍵詞:李斯特菌微酸性電解水高壓技術蛋白質體學差異表現蛋白
外文關鍵詞:Listeria monocytogeneselectrolyzed waterhigh pressure processingproteomicsdifferentially expressed proteins
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摘要....................................................I
Abstract...............................................II
壹、前言................................................1
貳、文獻回顧.............................................3
一.食品病原菌............................................3
(一). 食品中毒之簡介...................................3
(二). 李斯特菌 (Listeria monocytogenes)...............3
1. 生理特性........................................3
2. 分布範圍、汙染與傳染途徑..........................4
3. 潛伏期與常見症狀.................................4
4. 致病因子........................................4
5. 預防措施........................................5
二.電解水...............................................5
(一). 生成原理........................................5
(二). 貯存特性........................................6
(三). 殺菌機制及影響因子...............................6
(四). 應用範圍........................................7
1. 蔬果與其他農產品.................................7
2. 禽畜產品........................................8
3. 水產品..........................................8
4. 動物飼養場清潔...................................9
5. 食品加工設備.....................................9
6. 醫療............................................9
(五). 其他可直接用於食品和食品接觸面之殺菌劑.............10
1. 直接用於食品.....................................10
2. 用於食品接觸面...................................10
三.高壓加工技術..........................................11
(一). 高壓加工原理....................................11
(二). 高壓加工之特色..................................11
(三). 高壓殺菌及影響因子...............................12
(四). 應用範圍........................................13
1. 蔬果產品.........................................13
2. 乳製品...........................................13
3. 禽畜產品.........................................14
4. 水產品...........................................14
四.欄柵技術..............................................15
(一). 簡介與目的......................................15
(二). 電解水結合高壓技術之應用.........................15
五.蛋白質體學分析技術.....................................16
(一). 簡介............................................16
(二). 硫酸十二酯鈉-聚丙醯胺凝膠電泳 (sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS-PAGE)...........17
(三). 質譜法 (Mass spectrometry, MS)..................17
(四). 蛋白質無標記定量 (label-free quantification).....18
參、實驗設計.............................................20
一.微酸性電解水 (SAEW) 結合高壓技術 (HPP) 之殺菌能力探討....20
二.微酸性電解水 (SAEW) 結合高壓技術 (HPP) 之殺菌機制探討....21
肆、材料與方法...........................................22
一.實驗材料..............................................22
(一). 試驗菌株........................................22
(二). 電解水之製備....................................22
(三). 高壓處理之材料與設備.............................22
(四). 菌株培養藥品....................................22
(五). 菌體分析藥品....................................22
(六). 蛋白質萃取藥品..................................22
(七). 蛋白質定量......................................22
(八). 蛋白質去鹽......................................23
(九). SDS-PAGE.......................................23
(十). LC-MS/MS.......................................23
二.實驗方法..............................................23
(一). 菌株保存........................................23
(二). 菌種活化與保養..................................23
(三). 電解水物化性質之測定.............................24
(四). 生長曲線測定....................................24
(五). 殺菌處理之方法..................................24
(六). 培養基對殺菌影響之測定...........................24
(七). 殺菌能力之測定..................................24
(八). 對病原菌 DNA 外漏之影響..........................25
(九). 對病原菌蛋白質外漏之影響.........................25
(十). 對病原菌 TTC-dehydrogenase 相對活性之測定........25
(十一). 對病原菌超氧化物歧化酶 (superoxide dismutase, SOD) 之抑制效果..........................................25
(十二). 對病原菌蛋白質表現之影響........................25
1. 各式殺菌方法處理試驗菌株..........................26
2. 菌體蛋白質之萃取.................................26
3. 蛋白質去鹽.......................................26
4. 蛋白質濃度定量...................................26
5. SDS-PAGE........................................26
(十三). 以質譜儀分析蛋白質損傷程度......................27
1. 菌體蛋白質之萃取.................................27
2. 蛋白質濃度定量...................................27
3. 溶液相蛋白質水解 (In-solution digestion).........27
4. Zip-Tip microcolumns 之蛋白質純化................28
5. LC-MS/MS 分析...................................28
6. 蛋白質定性與定量之軟體分析........................28
伍、結果與討論...........................................31
一.微酸性電解水、高壓技術以及欄柵方法對李斯特菌之殺菌能力....31
(一). 培養基對殺菌效果之影響...........................31
(二). 比較不同有效氯濃度與壓力大小對李斯特菌生長之影響....32
二.微酸性電解水結合高壓技術對李斯特菌之影響.................34
(一). 李斯特菌之生長曲線圖.............................34
(二). 李斯特菌經殺菌處理後之蛋白質流失率................34
(三). 李斯特菌經殺菌處理後之 DNA 流失率.................35
(四). 李斯特菌經殺菌處理後之 TTC-dehydrogenase 相對活性.36
(五). 李斯特菌經殺菌處理後之 SOD 比活性比較.............37
三.以 SDS-PAGE 分析微酸性電解水結合高壓技術處理李斯特菌之蛋白質圖譜....................................................39
四.以質譜儀分析與無標記定量鑑定相關蛋白質...................41
(一). 以無標記定量後不同殺菌方式處理組間之相關性.........41
(二). 不同殺菌方式處理組間之差異表達蛋白質和生物資訊學分析42
1. 以無標記定量後不同殺菌方式處理組間之差異表達蛋白質...42
2. 差異表現蛋白之功能性分析..........................44
陸、結論.................................................47
柒、參考文獻.............................................48
捌、圖表.................................................65
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