臺灣博碩士論文加值系統

電解水被認為是對病原菌非常有效的殺菌劑，已廣泛的使用於食品工業上。本研究主要使用微酸性電解水 (slightly acidic electrolyzed water) 和微鹼性電解水 (slightly basic electrolyzed water)，以高有效氯濃度 (60 ppm) 和低濃度 (20 ppm)，處理食品常見病原菌 Escherichia coli，並萃取其蛋白質，和使用 SDS-PAGE 電泳技術找出不同分子量之蛋白質亮帶，再以串聯式質譜儀 (LC-MS/MS) 來分析蛋白質，再利用生物資訊資料庫來進行比對，找出菌體的蛋白質變化，來推測微酸性、微鹼性電解水對 E. coli 之殺菌機制。另一部分以蛋白質外漏，鉀離子外漏，DNA 外漏以及脫氫酶活性來探討不同濃度與性質之電解水處理對 E.coli 菌體損害程度。結果闡明微酸性與微鹼性電解水之最小抑菌濃度均為 30 ppm 有效氯濃度。微酸性電解水主要氯系化合物為次氯酸水 (HOCl)
而微鹼性電解水為次氯酸根離子 (OCl-)。二種濃度有效氯之微酸性、微鹼性電解水及次氯酸鈉溶液皆能將起始菌量 9 log CFU/mL 殺滅至 2 log CFU/mL 以下。20 ppm 微酸性和微鹼性電解水處理後之菌體再重新培養 12 小時，受損的菌體可以繼續生長。因此低濃度有效氯之電解水無法完全殺滅 E. coli 但卻能降低初始菌數，延長遲滯期約 10 小時並減緩生長作用。高濃度有效氯的微酸性電解水對菌體鈉離子、DNA 與蛋白質造成最嚴重的外漏現象以及最低的脫氫酶活性。從SDS-PAGE 發現電解水及次氯酸鈉處理組別，菌體蛋白質亮帶有明顯的消失及變得模糊，且隨著有效氯濃度提高更趨明顯。經過質譜分析鑑定出來的蛋白質，發現多為能量代謝、蛋白質合成相關酵素，以及外膜蛋白，和熱休克蛋白和一些胺基酸的合成蛋白，表示微酸性電解水會對這些蛋白質和酵素造成影響，進而使菌體死亡。
本研究發現微酸性和微鹼性電解水會破壞大腸桿菌之細胞膜，並推測微酸性電解水可能亦會破壞大腸桿菌的能量代謝、蛋白質生成系統，而菌體也會生成熱休克蛋白、穀胱甘肽和一些胺基酸來保護自身，抵抗外界的氧化壓力。

Slightly acidic electrolyzed water (SAEW), considered as a broad-spectrum and high-performance bactericide are increasingly applied in the food industry. In this
study, high levels of available chlorine concentration (ACC) at 60 ppm and low levels ACC at 20 ppm of slightly acidic electrolyzed water (SAEW) and slightly basic electrolyzed water (SBEW) were treated with major food-borne pathogen Escherichia coli. After treatment, the bacterial protein were extracted and applied in the SDS-PAGE analysis. The differential protein bands were cut, trypsin digested and identified by tandem mass spectrometry coupled with bioinformatics. Meanwhile, protein leakage, intracellular potassium leakage, DNA leakage and dehydrogenase activity were analyzed to evaluate bacterial damage status. The results demonstrated that the minimum inhibitory concentration were 30 ppm ACC for both SAEW and SBEW. The predominate chlorine species were hypochlorous acid and hypochrolite ion for SAEW and SBEW, respectively. The 60 and 20 ppm ACC of SAEW, SBEW
and NaOCl solution can destroy E. coli to less than 2 log CFU/mL from initial 9 log CFU/mL. However, 20 ppm cannot fully kill E. coli but can significantly reduced initial bacterial cell number, extend lag phase to 10 hr and reduce growth rate. The most severe leakage of DNA, protein and potassium as well as the lowest dehydrogenase activity for E. coli was the 60 ppm SAEW treatment. The protein bands were getting faint even disappeared after treatments, especially for 60 ppm SAEW.The protein dentification by tandem mass spectrometry was found to be
mostly energy metabolism, protein synthesis related enzymes, outer membrane proteins, and chaperone proteins and some amino acid synthesis protein, indicating that slightly acidic electrolyzed water will affect these proteins and enzymes.This study demonstrated that slightly acidic and slightly basic electrolyzed water will destroy the cell membrane of E. coli, and we speculated that slightly acidic electrolyzed water may also damage the energy metabolism, protein production system of E. coli, and E. coli also produce chaperone protein, glutathione and some amino acids to protect themselves against slightly acidic electrolyzed water.

III

目錄
壹、 前言...............................................1
貳、 文獻回顧...........................................2
一、 食品病原菌...................................2
(一) 簡介....................................2
(二) 大腸桿菌 (Escherichia coli)............3
二、 酸性、微酸性和鹼性電解........................5
(一) 生成原理.................................5
(二) 儲存性質................................ 6
(三) 殺菌機制及影響因子....................... 6
(四) 應用範圍................................ 7
(五) 其他可以直接用於食品之殺菌劑...............8
三、 蛋白質分析技術...............................9
(一) 簡介....................................9
(二) 蛋白質分析方法和應用......................9
(三) 質譜法和應用............................10
參、 實驗設計..........................................12
1. SAEW 和 SBEW 殺菌效果探討 ............ 12
2. SAEW 和 SBEW 殺菌機制探討探討 .........13
肆、 材料與方法........................................14
一、 實驗材料....................................14
(一) 試驗菌株................................14
(二) 試樣溶液之製備..........................14
(三) 菌株培養藥品............................14
(四) 菌體分析藥品............................14
(五) 蛋白質萃取藥品..........................15
(六) 蛋白質定量..............................15
(七) SDS-PAGE ..............................15
(八) LC-MS/MS ..............................15
二、 實驗方法....................................15
(一) 菌株保存................................15
(二) 菌種活化與培養..........................16
(三) 電解水物化性質之測定.....................16
(四) 電解水氯化合物型態測定...................16
(五) 生長曲線測定............................16
(六) 電解水殺菌能力之測定.....................16
(七) 式樣溶液最小抑制濃度之測定...............16
(八) 電解水對病原菌鉀離子外漏之影響............17
(九) 電解水對病原菌 DNA 外漏之影響 .........17
(十) 測量電解水對病原菌 TTC-dehydrogenase 相對
活性...................................17
(十一) 電解水對病原菌蛋白質表現之影響..........17
(十二) 以質譜儀分析蛋白質電泳圖譜..............19
伍、 結果與討論........................................22
一、 微酸性電解水、微鹼性電解水對大腸桿菌之殺菌能力.22
(一) 最小抑制濃度............................22
(二) 吸光光譜掃描............................22
(三) 有效氯濃度 60、20 ppm 微酸性電解水和微鹼性
電解水對大腸桿菌之殺菌效力...............23
二、 微酸性電解水、微鹼性電解水對大腸桿菌之影響.....25
(一) 大腸桿菌之生長曲線圖.....................25
(二) 大腸桿菌經過式樣溶液處理後鉀離子流失比率...25
(三) 大腸桿菌經過式樣溶液處理後 DNA 流失比 ..26
(四) 大腸桿菌經過有效氯濃度 20 ppm 微酸性和微
鹼性電解水處理後 TTC-dehydrogenase 相對活
性.....................................27
三、 以 SDS-PAGE 分析微酸性電解水、微鹼性電解水、次
氯酸鈉和磷酸緩衝溶液處理大腸桿菌後之蛋白質圖譜.29
(一) 蛋白質含量變化..........................29
(二) SDS-PAGE 蛋白質圖譜 ...................29
四、 以質譜儀鑑定相關蛋白質之身分..................31
(一) 外膜蛋白................................31
(二) 熱休克蛋白..............................32
(三) tRNA 合成酶 ...........................32
(四) 甘露糖和麩胺酸的生合成、代謝..............33
(五) 胺基酸的生合成..........................34
(六) 能量代謝系統............................34
陸、 結論..............................................36
柒、 參考文獻..........................................37
捌、 圖表..............................................46

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