電解水 (Electrolyzed water, EW) 已在食品工業上被認為是良好的抑菌劑，但因為只能清潔食品表面以及接觸有機物後有效氯降解而限制了消毒效果，因此透過結合另一非熱加工技術—高壓加工 (High pressure processing, HPP) 減少或消除微生物，來彌補電解水在應用上的不足。然而，前人少有針對電解水結合高壓加工之欄柵處理方式的研究主題進行探討，因此本研究藉此深入了解電解水結合高壓加工處理對李斯特菌的失活效率與相關殺菌機制之探討。
本研究主要是使用微酸性電解水結合高壓加工的欄柵方法 (hurdle method) 來處理食品常見病原菌 Listeria monocytogenes，比較蛋白質外漏、DNA 外漏、脫氫酶相對活性和超氧化物歧化酶活性來探討不同濃度電解水和 (或) 壓力處理對 L. monocytogenes 菌體之損害程度，結果所示使用有效氯濃度 30 ppm、壓力 300 MPa 以及欄柵方法有效氯濃度 20 ppm 結合 200 MPa 可從起始菌量 7 log CFU/mL 降至 3-5 log CFU/mL，高有效氯濃度 50 ppm、壓力 400 MPa 以及欄柵方法有效氯濃度 20 ppm 結合 300 MPa 之下細菌無法存活，在欄柵方法之處理下對細菌細胞壁和細胞膜破壞劇烈而造成最嚴重的蛋白質外漏，在 DNA 測定的部分則是高有效氯濃度促使細菌細胞膜通透性增加而明顯加劇 DNA 外漏現象，另外在提高壓力後則是可顯著降低脫氫酶相對活性，在超氧化物歧化酶活性測定時高有效氯濃度形成過多自由基以及高壓誘發活性氧的產生，皆能造成其活性降低，具濃度效應。從 SDS-PAGE 發現微酸性電解水組及欄柵方法組別之菌體蛋白質亮帶有較多明顯的消失及變模糊。透過無標記定量串聯式質譜分析方法得到在微酸性電解水與高壓環境下的蛋白質體學資訊，總共鑑定到 380 種蛋白質受到不同殺菌方式所影響，篩選出的差異表現蛋白 (differentially expressed proteins) 中，表現量上調的蛋白以核醣體蛋白為主，與穩定核醣體轉譯運作以維持細胞正常功能有關，表現量下調的蛋白多為接合酶，對細菌蛋白質的生合成和菌體細胞壁的形成等之生理活性造成影響。
本研究發現微酸性電解水結合高壓之欄柵方法會破壞菌體之細胞膜、蛋白質、DNA、酵素活性，且經蛋白體學分析，欄柵方法對胞內的核醣體與細胞壁合成等功能有更明顯的影響，引發菌體死亡，因此考量微生物性的食品安全下，酸性電解水結合高壓處理可以提高對食品病原菌的失活效率，呈協同效應 (synergistic effect)，將此欄柵技術應用於食品工業上為具有潛力發展的趨勢。

The use of electrolyzed water (EW) has been one of common bactericidal methods in food processing. Take account of the sanitization only on food surface and the lost of chlorine after exposure to organic matters, the effect of EW disinfection on food is limited. EW in combination with sanitizing techniques such as high pressure processing (HPP) is applied to overcome difficulties on a variety of food products. There is still lacke of hurdle treatment (EW+HPP) to improve the inactivation efficiency of microorganisms except for Bacillus cereus spores. Therefore, the objective of this study was to investigate the effect of HPP + SAEW (slightly acidic electrolyzed water) on the inactivation of Listeria monocytogenes. The purpose of this study is to use SAEW, HPP and hurdle method to treat foodborne microorganism L. monocytogenes. Results show that 30 ppm SAEW, 300 MPa HPP and SAEW 20 ppm + HPP 200 MPa cannot fully kill L. monocytogenes but can significantly reduced 3-5 log CFU/mL of bacterial cell number. Besides, 50 ppm SAEW, 400 MPa HPP and SAEW 20 ppm + HPP 300 MPa could completely inhibit L. monocytogenes growth, and significantly cause protein and DNA leakage. The relative activity of dehydrogenase and superoxide dismutase could be reduced after SAEW, HPP and SAEW+HPP treatment. From SDS-PAGE, the protein bands were getting faint even disappeared after treatments, especially for SAEW and SAEW+HPP. The label-free LC-MS/MS profiling identified 380 proteins which are collected from cell pellets of L. monocytogenes after different treatments. Ribosomemal protein was the main expression in up-regulated differentially expressed proteins, and cytoplasmic ligase was significantly influenced in down-regulated differentially expressed proteins.
This study demonstrated that slightly acidic electrolyzed water and high pressure processing would destroy the cell membrane, intracellular protein, DNA and enzyme activity of L. monocytogenes. According to proteomic analysis, the hurdle method would greatly affect cell functions such as translation and cell wall synthesis leading to death. It indicates synergistic effects of combined SAEW and HPP on inactivation of pathogen and have the potential on food processing industry.

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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