Production Of Quorum Sensing Signal Molecules And Effect On Food Spoilage In Pseudomonas Isolated From Food

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.