臺灣博碩士論文加值系統

腸炎弧菌為食品安全上重要的公共衛生問題，已有許多文獻證實酸性電解水 (acidic electrolyzed water, AEW) 可做為殺菌劑應用在食品，但其可能腐蝕食品加工器具和管路。相反地，微酸性電解水 (slightly acidic electrolyzed water, SlAEW) 較 AEW 在使用上安全。
本研究中所使用之試樣溶液為含有 80 mg/L 氯濃度的 SlAEW、AEW 和 NaOCl，磷酸緩衝鹽 (phosphate-buffered saline, PBS) 為控制組，在殺菌能力及萃取蛋白實驗中各試樣溶液之物理、化學特性可保持穩定。以 SlAEW 處理腸炎弧菌 180 秒，可使腸炎弧菌數量由 8.12 log CFU/mL 下降至平板計數法無法計數出，與 AEW 和 NaOCl 效果相當。同時使用 PBS 處理腸炎弧菌 180 秒，發現其不會對腸炎弧菌的數量造成太大的影響。
本研究中以SlAEW、AEW 和 NaOCl 處理腸炎弧菌後，比 PBS組之蛋白質濃度分別下降了 31.5、88.2 和 61.3%，由 SDS-PAGE 發現經試樣溶液處理後，圖譜確實有所變化，且發現蛋白質亮帶有消失的現象，顯示試樣溶液會破壞腸炎弧菌蛋白質，相較之下 SlAEW 破壞蛋白質的情形較輕微。
以二維電泳 (two-dimensional electrophoresis, 2DE) 分析腸炎弧菌整體蛋白質後，選取表現量明顯上調和下調的蛋白質點利用 MALDI-Q-TOF 及 LC/MS/MS 進行蛋白質定性，結果共比對到 7 個蛋白質，經過次氯酸鈉處理後表現量上調的 Outer membrane protein OmpK，為弧菌噬菌體 KVP40 的接受器；表現量下調的 Adenylate kinase、Phosphoglycerate kinase、Glyceraldehyde-3-phosphate dehydrogenase A 和 Enolase，和能量代謝相關；表現量下調的 Glyceraldehyde-3-phosphate dehydrogenase A 和 Elongation factor Tu，和蛋白質合成相關；表現量上調的 Outer membrane protein U，和滲透壓調節相關。
本論文證實，腸炎弧菌經過微酸性電解水、酸性電解水和次氯酸鈉處理後，可抑制 ATP 及蛋白質合成相關之蛋白，並對細菌造成無法負荷之滲透壓，使菌體死亡。

Vibrio parahaemolyticus remains a public health concern. Acidic electrolyzed water (AEW) has been reported to possess antimicrobial activity as a bactericide for foods but there are some side effects. As a result, slightly acidic electrolyzed water (SlAEW) has recently gained attention in food industry for strong microbial inactivation while it reduces the corrosion of processing equipment.
The testing solutions in this research were SlAEW, AEW and NaOCl with 80 mg/L available chlorine concentration (ACC), and we use phosphate-buffered saline (PBS) as a control. The results showed that the physical and chemical properties of testing solutions were stable in the procedures of disinfecting effectiveness and protein extracting experiments. Survivors of V. parahaemolyticus BCRC 10806 after 180-s treatments with SlAEW, AEW and NaOCl could be decreased to non-detectable level in a total plate count method. Otherwise, numbers of V. parahaemolyticus remains the same after PBS treatment. In our studies, we use SlAEW, AEW and NaOCl to treat the V. parahaemolyticus. After treantments with SlAEW, AEW and NaOCl, the protein concentrations decreased 31.5, 88.2 and 61.3% when comparing to the level of PBS group, and the patterns of SDS-PAGE were changed indeed. The protein band numbers of SDS-PAGE were decreased in the testing solution treated groups, and this indicated that testing solution may destroy the proteins of V. parahaemolyticus. However, SlAEW destroyed proteins of V. parahaemolyticus less than AEW and NaOCl.
The two-dimensional electrophoresis (2DE) revealed 1 and 4 proteins were up-regulated after treated with AEW and NaOCl, respectively. Otherwise, there were 9 , 7 and 3 proteins down-regulated after the cells were treated with SlAEW, AEW and NaOCl, respectively.
After the analysis of 2DE, these significantly regulated proteins were determined by MALDI-Q-TOF and LC/MS/MS mass spectrometry and their physiological functions were discussed. The up-regulated protein after treatment with NaOCl was Outer membrane protein OmpK which serves as a receptor for a vibriophage, KVP40. The down-regulated proteins related with energy metabolism were Adenylate kinase, Phosphoglycerate kinase, Glyceraldehyde-3-phosphate dehydrogenase A and Enolase. The down-regulated proteins related with protein synthesis were Glyceraldehyde-3-phosphate dehydrogenase A and Elongation factor Tu. The down-regulated protein related with osmotic pressure regulation was Outer membrane protein U.
These indicate that the pathways of ATP and protin synthesis were inhibited and the intolerant osmotic pressure were created when V. parahaemolyticus BCRC 10806 treated with SlAEW, AEW and NaOCl.

壹、前言 1
貳、文獻整理 5
一、 腸炎弧菌 5
1. 分佈情形 5
2. 一般特性 6
3. 致病情形 7
4. 預防與治療 10
二、 酸性電解水 11
1. 生成原理 11
2. 生成特性 11
3. 貯存特性 12
4. 殺菌特性 12
5. 缺點 13
三、 微酸性電解水 13
1. 生成原理 14
2. 殺菌機制 14
3. 安全性 15
四、 蛋白質體學 16
1. 二維電泳 16
2. 鑑定技術 17
參、實驗設計 20
肆、 材料與方法 21
一、材料 21
1. 腸炎弧菌 21
2. 酸性電解水 21
3. 微酸性電解水 21
二、藥品 21
1. 藥品來源 22
2. 培養基與試樣溶液配方 24
3. 抽取蛋白質所需藥劑 26
4. 電泳所需藥劑 26
5. 蛋白質染色配方 32
6. 膠內酵素水解 33
7. Zip-Tip 純化 34
三、方法 34
1. 菌株的保存 34
2. 菌種活化與培養 35
3. 生長曲線之測定 35
4. 試樣溶液物理、化學特性測定 35
5. 試樣溶液殺菌能力測定 36
6. 整體蛋白質萃取 36
7. 蛋白質去鹽與濃縮 37
8. 蛋白質濃度定量 38
9. SDS-PAGE 38
10. 二維電泳 39
11. 蛋白質固定與染色 (Fixation & Staining) 41
12. 二維電泳膠片掃描及影像比對 42
13. 膠內酵素水解 (In-gel digestion) 43
14. Zip-Tip microcolumns 之蛋白質純化 44
15. MALDI-Q-TOF 質譜儀分析 45
16. LC/MS/MS 質譜儀分析 45
17. 蛋白質序列比對 46
伍、結果 49
一、微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液之物理、化學特性變化 49
二、微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液對腸炎弧菌之殺菌能力比較 50
三、以 SDS-PAGE 分析微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液處理腸炎弧菌後之蛋白質圖譜 50
四、以二維電泳分析微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液處理腸炎弧菌後之蛋白質圖譜 52
五、以質譜儀鑑定相關蛋白質之身份 53
陸、討論 56
一、微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液之物理、化學特性變化 56
二、微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液對腸炎弧菌之殺菌能力比較 57
三、以 SDS-PAGE 分析微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液處理腸炎弧菌後之蛋白質圖譜 58
四、以二維電泳分析微酸性電解水、酸性電解水、次氯酸鈉和磷酸緩衝鹽溶液處理腸炎弧菌後之蛋白質圖譜 60
五、以質譜儀鑑定相關蛋白質之身份 61
柒、結論 66
捌、圖表 68
玖、參考文獻 91

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