【作者】 綦国红；

【导师】 董明盛；

【作者基本信息】 南京农业大学 ， 食品科学， 2006， 博士

【摘要】 细菌细胞产生信号分子并释放到环境中去，当环境中信号分子的浓度达到某一阈值后，细菌细胞开启细胞密度依赖的特定基因的表达机制，这一现象被称为“群体感应（quorum sensing，简称QS）”。N-酰基-高丝氨酸内酯类（AHLs）化合物是革兰氏阴性菌群体感应系统中最典型的一类信号分子。细菌细胞通过产生AHL进行交流信息，协调群体行为，调控某些特定的生理性状的表达，这一研究领域日益受到人们的关注。假单胞菌（Pseudomonas）是导致高蛋白食品腐败的重要细菌。研究表明，嗜铁素的产生和蛋白酶活性是假单胞菌最重要的致腐因素，导致腐败食品特有的金属光泽和腐臭味。近年来对人类条件致病菌铜绿假单胞菌（Pseudomonas aeruginosa）以及植物致病菌的研究表明，嗜铁素产生及蛋白酶活性是由AHL调控的。但目前国内外对食源假单胞菌群体感应信号分子的产生及其与嗜铁素产生、蛋白酶活性之间的关系未见报道。本文对食源假单胞菌N-酰基-高丝氨酸内酯类群体感应信号分子的产生及其对食品腐败的影响进行了研究。为进一步确定群体感应机制在食品腐败中的作用，以期为构建基于干扰腐败细菌群体感应系统作为新靶点的食品防腐保鲜策略提供理论依据。主要结果如下：1利用细菌生物感应器Chromobacterium violaceum CVO26和Agrobacteriumtumefaciens A136（pCF218／pCF372）建立了琼脂扩散法（平行画线法、报告平板法）、细菌生物感应器与薄层层析相结合（TLC-Biosensor）以及β-半乳糖苷酶活法检测AHL的生物学检测方法，该法能够简单、快速检测AHL。2根据AHL信号分子的典型特征，建立HPLC／MS法检测、鉴定AHL信号分子的方法。结果表明，在没有标准品的条件下，HPLC／MS是一种有效鉴定AHL信号分子的方法，检测的灵敏度明显高于经典的细菌生物感应器检测AHLs的方法：3通过生理生化鉴定与16S rDNA序列分析相结合的方法，对源于食品的3株革兰氏阴性菌FML05-1、FML05-2、FML05-3进行鉴定，菌株FML05-1、FML05-2为荧光假单胞菌（Pseudomonas fluorescens），FML05-3为恶臭假单胞菌（Pseudomonas putida）；4利用细菌生物感应器对3株菌AHL产生情况进行检测，结果表明3株菌均产生AHLs信号分子。为进一步确定3株菌所产生的AHL的种类，对3株菌的培养上清进行TLC-biosensor分析，发现FML05-1和FML05-2至少产生两种AHLs分子，主要信号分子是N-3-氧代-辛酰基-高丝氨酸内酯（N-3-oxo-C₈-HSL），而FML05-3没有检测到任何斑点；5利用HPLC／MS方法对3株菌产生的AHLs分子进一步鉴定，结果表明，FML05-1菌株产生N-3-oxo-C8-HSL和N-3-oxo-C10-HSL，FML05-2菌株产生N-3-oxo-C10-HSL、N-3-oxo-C8-HSL、C6-HSL和C4-HSL，而FML05-3菌株产生C6-HSL和C4-HSL。由此可见，利用TLC-biosensor方法没有得到鉴定的AHL分子，HPLC／MS方法得到鉴定；6对菌株在生长过程中所产生的AHLs的活性变化进行研究，发现随着菌体密度的增加，AHLs浓度也随之增加，在菌体生长至对数中末期或稳定期时达到最大，随之AHLs的浓度开始下降。同时其培养液中的pH值呈现上升的趋势。同时研究了pH值条件对AHL稳定性的影响，结果发现，在碱性条件下AHL的稳定性下降。当pH值为8.5，反应时间为8h时，AHLs活性降至约为原来的50％。因此，菌体生长后期AHL活性下降与环境中pH值的升高有关；7研究了不同温度及碳源对食源假单胞菌AHLs产生的影响。结果表明，在25℃条件下，菌株FML05-1和FML05-2，产生长链和短链两种AHL信号分子，而在4℃条件下，主要产生长链AHL分子。而且在不同碳源（葡萄糖，果糖，木糖，麦芽糖等）的AB培养基中生长，所产生的AHLs分子种类也不相同。表明在不同的环境条件（碳源及温度）下假单胞菌产生不同种类的AHLs；8对3株腐败菌的致腐因素进行检测，发现FML05-2具有很强的蛋白水解活性，并且在脱脂牛奶中生长还产生大量的嗜铁素，但在LB中生长时不产生嗜铁素；菌株FML05-1蛋白水解活性相对FML05-2较弱，在LB及脱脂牛奶中生长均不产生嗜铁素，而菌株FML05-3不表达上述2种致腐因素；9利用产aiiA蛋白酶的菌株对假单胞菌FML05-2所产生的AHL分子进行降解，发现FML05-2对脱脂牛奶的致腐作用明显减弱，嗜铁素的产量与蛋白酶活性明显下降。说明FML05-2嗜铁素的产生和蛋白酶活性与AHL的产生及浓度有关。为建立以干扰腐败细菌群体感应系统为靶点的食品防腐保鲜新策略提供理论依据。更多还原

【Abstract】 Bacterial cells trigger particular gene expression in a cell-density-dependent manner by producing ,secreting molecules that accumulate in the enviroment in proportion to cell densiy when the density reaches a threshold level, a phenomenon termed ’quorum sensing（QS）’.In general, this communication system is mediated by the production of N-acyl-homoserine lactones signal molecules in Gram-negative bacteria. It become an interest topic that the bacterial cell communicated with each other to coordinate the population behavior and to regulate the expression of some physiological traits by producing the AHL.Pseudomonas was an important bacteria that caused spoilage of proteinaceous foods. It was demonstrated production siderophore, protase activity were most important factors that spoiled the food, and caused the food the characteristic metal brightness and offensive off odors. In resent years, it was reported that the production siderophore, protase activity in the human opportunitic pathogen Pseudomonas aeruginosa were regulated by AHL .But there was no study on the production of quorum sensing signal molecules and relationship between production siderophore, protase activity and signal molecules in Pseudomonas isolated from food.In this paper, it was studied on the production of quorum sensing signal molecules and the effect on the food spoilage in pseudomonas isolated from food.This study laid the foundation for the role of quorum sensing in the food spoilage and new strategies for food preservation based on interfering in quorum sensing of spoilage bacteria. The main results were as follows:1. It was established the biological methods of agar diffusion assay（streak assays in parallel, reporter agar assays）, thin-layer chromatography combination with biosensor analysis andβ-galactosidase activity assay that detected AHL based on the biosensor Chromobacterium violaceum CVO26, Agrobacterium tumefaciens A136 （pCF218/pCF372） .It was simpler and faster manner to detect the AHL using the biosensor.2. The method of detection and identification AHL based on HPLC/MS was developed according to the typical structure characteristic of AHL molecules.The results showed that it was powerful method to determinate the AHL under no standards. The sensitivity of HPLC/MS detection was higher compared to the classical AHL analysis of biosensor.3. The three gram-negative bacteria FML05-1、FML05-2、FML05-3 were isolated from the food. The strains FML05-1、FML05-2 were identified as Pseudomonas fluorescens and FML05-3 identified as Pseudomonas putida by analysis of 16S ribosomal DNA gene sequence, physiological and biochemical characteristics.4. Three strains produced communication signals involved in quorum sensing by assays based on the biosensor A. tumefaciens A136 （pCF218/pCF372） ,C. violaceum CVO26. The strains FML05-1 and FML05-2 were found to produce two type of AHLs in the supernatant and the main signal molecules were N-3-oxo-C₈-AHL by the analysis of TLC-biosensor assays, but no spots were detected in FML05-3.5. The AHL signal molecules producued by three strains were further identified by the HPLC/MS.The results showed that the strain FML05-1 produced N-3-oxo-C8-HSL and N-3-oxo-C10-HSL; the strain FML05-2 produced the N-3-oxo-C10-HSL ,N-3-oxo-C8-HSL, C6-HSL; the FML05-3 produced the C6-HSL and C4-HSL. So the AHL molecules unidentified by the method of TLC-biosensor were identified by the method based on the HPLC/MS.6. The change of AHL activity in strains along the cell growth was detected. The result showed that the AHLs activity rose with the population cell density and reached maximal value when the cells growth entered the exponential end phase or stationary phase, then decreased with growth. At the same time, the pH in the culture of three strains increased throughout the growth. So the stability of AHLs was studied in different pH conditions. It was presented that the AHLs signal molecules became unstable in alkaline conditions. Nearly 50% degradation occured at pH=8.5 when reaction time was 8h. The increase in pH might be responsible for reduction of AHL levels in the cultures.7. The influence of carbon source, temperature on the AHLs profile produced by food-derived Pseudomonas were tested. The results showed that the strains FML05-1 and FML05-2 produced the long chain AHLs and short chain AHLs at 25℃. However, the long chain AHLs were produced mainly when the strains were grown at 4℃. The strains produced the different types of AHLs when grown in the AB medium of different carbon source（such as, glucose, fructose, xylose, maltose）. Overall, it was demonstrated the effect of environmental parameters （temperature and carbon source） on AHL profile production by food-derived Pseudomonas.8. The factors （Protase ,siderophore（s）） involved in food spoilage were assayed. The results showed that the FML05-2 was capable of the proteolytic activity and production siderophore（s） in skim milk, FML05-1 showed the vague proteolytic activity, was unable to produce the siderophore in skim milk and LB. But FML05-3 did not express the traits above.9. The AHLs produced by FML05-2 were degraded by the strain of the expression aiiA enzyme .The results demonstrated that the spoilage of the skim milk that caused by the FML05-2 was suppressed and decrease in the production of siderophore and proteinase by FML05-2 was observed. This indicated that there were relationship between production of siderophore, proteinase activity and production, concentration of AHL.This study lays the foundation for new strategies of food preservation based on interfering in quorum sensing of spoilage bacteria.更多还原