周正俊臺灣大學：食品科技研究所江明倫Chiang, Ming-LunMing-LunChiang2007-11-272018-06-292007-11-272018-06-292007http://ntur.lib.ntu.edu.tw//handle/246246/56320在本研究中，以臨床分離之菌株腸炎弧菌690，於42℃下或5%乙醇中進行次致死熱震及乙醇處理。探討次致死熱震及乙醇處理之腸炎弧菌菌體脂肪酸組成、菌體型態及其於不同培養基上之復甦。測試次致死熱震及乙醇處理對腸炎弧菌蛋白質表現、耐熱性溶血素 (TDH) 產量及其暴露於後續不同環境壓力期間存活之影響。此外，並檢測次致死熱震及乙醇處理對腸炎弧菌超氧岐化酶 (SOD)、觸酶 (CAT) 及其於不同氮源和碳源下生長之影響。 結果發現，熱震處理之腸炎弧菌菌體中飽和脂肪酸與不飽和脂肪酸之比值 (SFA/USFA) 顯著地高於控制組菌體。在熱震處理之腸炎弧菌菌體上呈現細胞壁凹陷和細胞破裂之傷害。熱震處理之腸炎弧菌菌體於TCBS和APS agar上之復甦顯著地少於在TSA-3% NaCl復甦者。此外，熱震處理減少腸炎弧菌對有機酸 (25 mM) 之忍受性，然而增加腸炎弧菌於0.1% NaCl中之存活，並使得所試菌體對20% NaCl較敏感。 次致死乙醇處理之腸炎弧菌菌體於TCBS上之復甦顯著地少於在TSA-3% NaCl復甦者。腸炎弧菌經次致死乙醇處理後發現菌體懸浮上清液中之核酸和蛋白質物質有顯著地增加。在次致死乙醇處理之腸炎弧菌菌體上亦出現細胞破裂、皺摺和細胞壁凹陷之損傷情形。次致死乙醇處理之腸炎弧菌菌體相較於控制組菌體在4℃下之存活率並無差異，但對-18℃則具較高之敏感性。此外，次致死乙醇處理提高腸炎弧菌菌體對47℃之耐熱性和在8%乙醇中之忍受性。 次致死乙醇處理減少腸炎弧菌菌體飽和脂肪酸與不飽和脂肪酸之比值 (SFA/USFA) 及在添加有6或7% NaCl TSA上之復甦程度。此外，次致死乙醇處理增加腸炎弧菌在過氧化氫 (20 ppm) 中之存活，然而卻使所試菌體對結晶紫 (3 ppm)、高NaCl (20%) 和有機酸 (25 mM) 較不具抵抗性。 一維電泳分析顯示，腸炎弧菌菌體有三種蛋白質，分子量為93、77和58 kDa，相較於控制組菌體，發現受次致死熱震及乙醇處理所誘導。二維電泳分析顯示，次致死熱震及乙醇處理影響腸炎弧菌共28種蛋白質之表現，其中有4種蛋白質，分子量為94、32.1、26.7和25.7 kDa，其表現受次致死熱震及乙醇處理而增加。此外，免疫轉印分析顯示，在所試控制組、次致死熱震及乙醇處理之菌體中有GroEL類蛋白質之存在，分子量為61 kDa。次致死熱震及乙醇處理之菌體所產生之GroEL類蛋白質量較高於控制組菌體，然而試驗中並未在腸炎弧菌菌體中偵測出DnaK類蛋白質。 次致死熱震及乙醇處理降低腸炎弧菌超氧岐化酶和觸酶之活性程度。膠體活性染色分析中並未偵測出觸酶之表現，然而在控制組、次致死熱震及乙醇處理之腸炎弧菌菌體中偵測出一種超氧岐化酶。試驗中亦發現熱震處理30-60分鐘及次致死乙醇處理45-60分鐘提高所試菌體耐熱性溶血素之合成。次致死乙醇處理和控制組之腸炎弧菌菌體在TSB-3% NaCl中之生長及耐熱性溶血素產量均相同，然而直到培養200分鐘前，熱震處理之菌體其生長及耐熱性溶血素產量則較低。此外，次致死乙醇處理之菌體在所試含不同氮源和碳源培養基中之生長與控制組菌體相似，然而熱震處理之菌體其遲滯期則顯現延長之情形。In the present study, Vibrio parahaemolyticus 690, a clinical strain, was subjected to either heat shock at 42℃ or ethanol shock in the presence of 5% ethanol. The fatty acid profile, morphology and recovery of the heat-shocked and ethanol-shocked cells of V. parahaemolyticus on different media were investigated. The effects of heat shock and ethanol shock on the protein expression, thermostable direct hemolysin (TDH) production and survival of V. parahaemolyticus during subsequent exposure to various environmental stresses were examined. Furthermore, the superoxide dimutase (SOD), catalase (CAT) activities and growth behavior of V. parahaemolyticus in the presence of various nitrogen and carbon sources as affected by heat shock and ethanol shock were also determined. It was found that the ratio of saturated fatty acid to unsaturated fatty acid (SFA/USFA) observed in the heat-shocked cells of V. parahaemolyticus was significantly higher than that in the control cells. Extensive cell-wall pitting and cell disruption, representing cell-surface damage, were also observed on the heat-shocked cells. The recovery of the heat-shocked cells of V. parahaemolyticus was significantly less on TCBS (thiosulfate-citrate-bile salts-sucrose agar) and APS agar (alkaline peptone salt broth supplemented with 1.5% agar) than on TSA-3% NaCl (tryptic soy agar supplemented with 3% NaCl). Heat shock decreased the tolerance of V. parahaemolyticus to organic acids (25 mM). While heat shock increased the survival of V. parahaemolyticus in the presence of 0.1% NaCl and made the test organism more susceptible to 20% NaCl. The recovery of the ethanol-shocked cells of V. parahaemolyticus was significantly less on TCBS than on TSA-3% NaCl. A significantly marked increase of protein and nucleic acid materials in the supernatant of the cell suspension was found after cells of V. parahaemolyticus were subjected to ethanol shock. Extensive cell disruption, wrinkling and cell-wall pitting, indicative of cell-surface damage were also noted on the ethanol-shocked cells. The ethanol-shocked cells of V. parahaemolyticus exhibited a similar yet higher susceptibility at 4℃ and -18℃ compared with the control cells. Moreover, there was a marked increase in the thermal tolerance (47℃) and resistance to 8% ethanol with cells of V. parahaemolyticus after ethanol shock. Ethanol shock decreased the SFA/USFA in cells of V. parahaemolyticus. The recovery of the ethanol-shocked cells on TSA plus 6% or 7.5% NaCl was significantly less than the control cells. Furthermore, ethanol shock increased the survival of V. parahaemolyticus in the presence of H2O2 (20 ppm), while made the test organism less resistant to crystal violet (3 ppm), high NaCl (20%) and organic acids (25 mM). Analysis with one-dimensional SDS-PAGE indicated that three proteins with a molecular mass of 93, 77, and 58 kDa were found to be induced by heat shock and ethanol shock for V. parahaemolyticus compared with the control cells. Analysis with two-dimensional electrophoresis revealed that heat shock and ethanol shock affected the expression of a total of 28 proteins in V. parahaemolyticus. Among them four proteins with a molecular mass of 94, 32.1, 26.7 and 25.7 kDa were enhanced by heat shock and ethanol shock. Furthermore, immunoblotting analysis showed the presence of a GroEL-like protein with a molecular mass of 61 kDa in the heat-shocked, ethanol-shocked and control cells of the test organism. The heat-shocked and ethanol-shocked cells produced GroEL-like protein in a larger quantity than the control cells. However, DnaK-like protein was not detectable in cells of V. parahaemolyticus. Heat shock and ethanol shock reduced the level of SOD and CAT activities in V. parahaemolyticus. Gel activity staining assay failed to detect the expression of CAT, while one SOD enzyme was detected in the control, heat-shocked and ethanol-shocked cells of V. parahaemolyticus. Heat shock for 15-60 min and ethanol shock for 45-60 min were found to enhance the synthesis of TDH by the test organism. The growth and TDH produced by the ethanol-shocked and control cells of V. parahaemolyticus in TSB-3% NaCl were identical, while a lower level of growth and TDH production was detected with the heat-shocked cells until after 200 min of cultivation. Furthermore, the growth of the ethanol-shocked cells was similar to that of the control cells, while the heat-shocked cells exhibited an extended lag period in the media with various nitrogen and carbon sources examined.摘要------------------------------------------------------i Abstract------------------------------------------------iii 目錄-----------------------------------------------------vi 圖目錄---------------------------------------------------xi 表目錄-------------------------------------------------xiii 第一章 總論-----------------------------------------------1 前言------------------------------------------------------2 文獻整理--------------------------------------------------4 1. 腸炎弧菌 (Vibrio parahaemolyticus)---------------------4 1.1. 腸炎弧菌之生長與生化特性-----------------------------4 1.2. 腸炎弧菌之環境分佈與傳播媒介-------------------------5 1.3. 腸炎弧菌之食品中毒-----------------------------------6 1.3-1. 腸炎弧菌食品中毒之發生-----------------------------7 1.3-2. 腸炎弧菌食品中毒之症狀與致病因子-------------------7 1.3-3. 腸炎弧菌食品中毒之預防與治療----------------------10 1.4. 影響腸炎弧菌存活之因素------------------------------10 1.4-1. 溫度----------------------------------------------11 1.4-2. pH值及有機酸--------------------------------------12 1.4-3. 氯化鈉--------------------------------------------13 1.4-4. 抗生素--------------------------------------------14 1.4-5. 其他----------------------------------------------14 2. 壓力反應 (Stress response)----------------------------15 2.1. 壓力反應之發生--------------------------------------15 2.2. 壓力蛋白質------------------------------------------16 2.3. 交叉保護作用----------------------------------------17 3. 熱震反應 (Heat shock response)------------------------18 3.1. 熱震基因之調控--------------------------------------19 3.2. 熱震蛋白質之功能------------------------------------20 3.3. 熱震蛋白質之誘導表現--------------------------------22 3.4. 熱震處理引發菌體之耐熱性----------------------------24 3.5. 熱震處理引發菌體之交叉保護作用----------------------25 4. 乙醇壓力反應 (Ethanol stress response)----------------26 4.1. 乙醇之性質與抑菌機制--------------------------------26 4.2. 菌體對乙醇忍受性及其脂肪酸組成之相關性--------------28 4.3. 乙醇壓力蛋白質之誘導表現----------------------------29 4.4. 次致死乙醇處理引發菌體之交叉保護作用----------------30 參考文獻-------------------------------------------------36 Chapter 2 Fatty acid composition, cell morphology and responses to challenge by organic acid and sodium chloride of heat-shocked Vibrio parahaemolyticus------------------55 1. Abstract----------------------------------------------56 2. Introduction------------------------------------------58 3. Materials and methods---------------------------------61 3.1. Microorganism and the preparation of inoculum-------61 3.2. Heat shock treatment--------------------------------61 3.3. Organic acid and NaCl challenge studies-------------62 3.4. Analysis of fatty acids-----------------------------62 3.5. Electron microscopy---------------------------------63 3.6. Enumeration of V. parahaemolyticus------------------64 3.7. Statistical analysis--------------------------------64 4. Results and discussion--------------------------------66 4.1. Effect on fatty acid profile------------------------66 4.2. Morphology of heat-shocked cells of V. parahaemolyticus-----------------------------------------68 4.3. Survival in the presence of various organic acids---68 4.4. Survival in the presence of low and high NaCl contents-------------------------------------------------70 4.5. Recovery of heat-shocked V. parahaemolyticus on different media------------------------------------------72 5. Conclusion--------------------------------------------74 6. References--------------------------------------------82 Chapter 3 Response of Vibrio parahaemolyticus to ethanol shock----------------------------------------------------86 1. Abstract----------------------------------------------87 2. Introduction------------------------------------------89 3. Materials and methods---------------------------------92 3.1. Microorganism and the preparation of inoculum-------92 3.2. Growth inhibition studies---------------------------92 3.3. Ethanol shock treatment-----------------------------93 3.4. Cellular material leakage studies-------------------93 3.5. Electron microscopy---------------------------------94 3.6. Challenge studies-----------------------------------94 3.7. Enumeration of V. parahaemolyticus------------------95 3.8. Statistical analysis--------------------------------96 4. Results and discussion--------------------------------97 4.1. Effect of ethanol concentration---------------------97 4.2. Recovery of ethanol-shocked cells with different media----------------------------------------------------97 4.3. Cell leakage and SEM micrograms of V. parahaemolyticus-----------------------------------------98 4.4. Susceptibility to 8% ethanol------------------------99 4.5. Survival at low temperature------------------------101 4.6. Thermal tolerance----------------------------------102 5. Conclusion-------------------------------------------104 6. References-------------------------------------------113 Chapter 4 Ethanol shock changes the fatty acid profile and survival behavior of Vibrio parahaemolyticus in various stress conditions---------------------------------------116 1. Abstract---------------------------------------------117 2. Introduction-----------------------------------------118 3. Materials and methods--------------------------------121 3.1. Microorganism and the preparation of inoculum------121 3.2. Ethanol shock treatment----------------------------121 3.3. Analysis of fatty acids----------------------------122 3.4. Challenge studies----------------------------------123 3.5. Enumeration of V. parahaemolyticus-----------------124 3.6. Statistical analysis-------------------------------124 4. Results and discussion-------------------------------125 4.1. Effect of ethanol shock on fatty acid profile of V. parahaemolyticus----------------------------------------125 4.2. Effect of ethanol-shock on the recovery of V. parahaemolyticus on TSA plus various amounts of NaCl----126 4.3. Effect of ethanol shock on H2O2 tolerance of V. parahaemolyticus----------------------------------------127 4.4. Effect of ethanol shock on acid tolerance of V. parahaemolyticus----------------------------------------128 4.5. Effect of ethanol shock on the susceptibility of V. parahaemolyticus to crystal violet----------------------130 4.6. Effect of ethanol shock on the viability of V. parahaemolyticus in the presence of high NaCl content---131 5. Conclusion-------------------------------------------133 6. References-------------------------------------------142 Chapter 5 Comparison on the protein expression in Vibrio parahaemolyticus 690 subjected to sublethal heat and ethanol shock treatments--------------------------------147 1. Abstract---------------------------------------------148 2. Introduction-----------------------------------------149 3. Materials and methods--------------------------------152 3.1. Microorganism and the preparation of inoculum------152 3.2. Heat shock and ethanol shock treatments------------152 3.3. Preparation of cell extract------------------------153 3.4. One-dimensional SDS-PAGE---------------------------153 3.5. Two-dimensional electrophoresis--------------------154 3.6. Gel staining and image analysis--------------------155 3.7. Immunoblotting-------------------------------------155 4. Results and discussion-------------------------------157 4.1. Heat-shocked and ethanol-shocked proteins analyzed with one-dimensional SDS-PAGE---------------------------157 4.2. Heat-shocked and ethanol-shocked proteins analyzed with two-dimensional electrophoresis--------------------159 4.3. Characterization of DnaK- and GroEL-like proteins--162 5. Conclusion-------------------------------------------165 6. References-------------------------------------------172 Chapter 6 Expression of superoxide dismutase, catalase and thermostable direct hemolysin by heat-shocked and ethanol-shocked Vibrio parahaemolyticus as well as their growth in the presence of various nitrogen and carbon sources-----180 1. Abstract---------------------------------------------181 2. Introduction-----------------------------------------183 3. Materials and methods--------------------------------186 3.1. Microorganism and the preparation of inoculum------186 3.2. Preparation of heat-shocked and ethanol-shocked V. parahaemolyticus----------------------------------------186 3.3. Preparation of cell lysate-------------------------187 3.4. Superoxide dismutase assay-------------------------187 3.5. Catalase assay-------------------------------------188 3.6. Native-PAGE and gel activity staining--------------189 3.7. Determination of TDH production--------------------189 3.8. Growth study with various nitrogen and carbon sources---------------------------------------------------------190 3.9. Statistical analysis-------------------------------191 4. Results and discussion-------------------------------192 4.1. Heat shock and ethanol shock affect the level of SOD and CAT activities--------------------------------------192 4.2. Gel activity staining of SOD and CAT---------------193 4.3. Heat shock and ethanol shock affect TDH production-195 4.4. Growth and TDH production by heat-shocked and ethanol-shocked V. parahaemolyticus in TSB-3% NaCl--------------197 4.5. Growth of heat-shocked and ethanol-shocked V. parahaemolyticus in the presence of various nitrogen and carbon sources------------------------------------------198 5. Conclusion------------------------------------------------------------------------------------------------------201 6. References-------------------------------------------208 附錄----------------------------------------------------215 M.-L. Chiang et al. / International Journal of Food Microbiology 104 (2005) 179-187-------------------------215 M.-L. Chiang et al. / Food Microbiology 23 (2006) 461-467----------------------------------------------------------2243320869 bytesapplication/pdfen-US腸炎弧菌熱震處理次致死乙醇處理復甦脂肪酸蛋白質耐熱性溶血素Vibrio parahaemolyticusHeat shockEthanol shockRecoveryFatty acidProteinThermostable direct hemolysinthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/56320/1/ntu-96-F90641018-1.pdf