Study On Quorum-Sensing Signal Of Spoilage Bacteria In Raw Chicken Meat

Quorum sensing has been found in a diverse group of bacteria which communicate with each other by producing signaling molecules known as auto-inducers. Many gram negative bacteria produce acylated homoserine lactones (AHLs) as signal molecules. As the cell density increases, the concentration of AHLs increases. When a significant concentration of signal has accumulated, AHLs interact with the receptor proteins and initiate expression of particular genes. Species, which cannot or fail to produce AHLs, show higher sensitivity to AHLs indentical or similar to the AHLsthey produce themselves. Interestingly, several bacterial which are typically associated with food spoilage are able to produce AHLs, and AHLs have been detected in a variety of different food products, such as fish, meat, milk and vegetable. Furthermore, several bacterial characterists, such as bioluminescence, spore, bifofilm formation and extracellular enzyme activity, are under control of AHL-based quorum sensing system. However, the quorum-sensing signals and regulation mechanism involved in meat born spoilage bacteria is unclear. Therefore, in the present research we identified the AHL-producing spoilage bacteria, studied the change of AHLs in raw chicken meat, and evaluated the influence of AHLs concentration in environment on bacterial characteristic. We would like to find the role of AHL-based quorum sensing system in food contamination, and provide a theory basis for development of bacterial reduction technology. The whole work includes five parts, the specific contents and results are as follows:1. Effect of temperature fluctuation on the storage characteristic of raw chicken during chiller storageOn the basis of temperatures found in some domestic and retail store refrigerators, the experiment was carried out at 4℃,0-4℃ and 4-10℃. In order to determine the spoilage bacteria in raw chicken under chiller conditions, we investigate the effect of temperature fluctuation on the storage characteristics by bacterial culture and polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) methods. Fluctuating chiller temperatures have no significant effect on pH and total volatile basic nitrogen (TVB-N), while have been shown to alter the diversity of micro-flora. The bacterial growth rate was reduced when the temperature decreased. When temperature was reduced to 4 from 0℃, both the growth rates of lactic acid bacteria (LAB) and Enterobacteriaceae decreased, however, LAB was able to rapidly adapted, unlike Enterobacteriaceae. According to the denaturing gradient gel electrophoresis (DGGE) profiles, Staphylococcus spp., Pseudomonas spp., Acinetobacter spp., Carnobacterium spp., Aeromonas spp. and Weissella spp. were the dominate bacteria during storage. The profiles also showed that Shewanella spp. and Psychrobacter spp. were appeared in samples stored at 0 and 4℃. At the end of strage, the main bacteria were Acinetobacter spp., Pseudomonas spp., Carnobacterium spp., Aeromonas spp. and Weissella spp.2. Isolation and identifying AHL-producing spoilage bacteria from raw chicken meatThe purpose of the present work was to obtain the AHL-produceing bacteria, which present as the special spoilage bacteria of raw chicken. Bacterial culture and VITEK 2 Auto Microbic System was used for bacterial isolation and identification, whereas bioassay was used for AHLs detection. The results show that there were two Citrobacter braakii strains, one Escherichia coil strains and three Pseudomonas aeruginosa strains, and all the probabilities are more than 90%. Two P. aeruginosa strains were able to produce AHLs. Moreover, the strain with the highest probability can produce AHLs in LB, minced chicken and chicken broth.3. Development of relative quantification of AHLsIn order to develop an AHLs quantification method based on bioassays of AHLs biosensors, nonline regression was used to describe the relationship between the β-galactosidase and AHLs concentration. AHLs were extracted with acidified ethyl acetate, the solvent was evaporated and the residue was dissolved in water and heated at 100℃ for 15 min. Thin-layer chromatography combined with Agrobacterium tumefaciens KYC55-based bioassays indicated the presence of C4-, C6-, C8- and C12-homoserine lactones in the extract. The C8-AHL and C6-AHL, as the main AHLs production, were used to develop a standard curve. The standard curve was described by the following equation: OD42o=exp[-0.26-0.88/(AHLs concentration+0.37)]. The standard curve was able to calculate the concentration of AHLs extract at the 0-6 ng/ml level. In order to evaluate the performance of model, three different different types of AHLs (C8-AHL, C6-3-oxo-AHL and C6-AHL) were used. The result showed that the predicted values were reliable.4. Kinetics of AHLs producted by Pseudomonas aeruginosaThe objective of this part is to evaluate the AHL-production kinetics of P. aeruginosa. According to the P. aeruginosa growth curves in chicken broth and minced chicken, the lag phase, exponential phase and stationary phase can be caculated. Based on the AHLs quantification method which was developed in chapter three, AHLs were detected during bacterial growth. According to the TLC profies, AHLs type produced by P. aeruginosa varied with storage time. Although storage temperature had no influence on AHLs type, it affected the AHLs concentration. During late exponential and early stationary phase, the concentration of AHLs reached a peak, and P. aeruginosa produced most kinds of AHLs. AHLs accumulated in the exponential phase, but decreased in the stationary phase. The Weibull model can be user to caculate the AHLs concentration in chicken broth or minced chicken by P. aeruginosa growth.5. Effect of AHLs on Pseudomonas aeruginosa activity and attachmentIn present study the influence of AHLs concentration on bacterial activity and attachment was evaluated. In LB, addition of 0.68 ng/mL AHLs has no effect on the growth and biofilm formation of P. aeruginosa. When the addition of AHLs was equale or higher than 2.03 ng/mL, bacterial growth rate increased but biofilm formation decreased. The effect of AHLs extract on bacterial growth, heat flow, enzyme activity and attachmen was evaluate at 2.03 and 3.38 ng/mL AHLs in chicken broth. AHLs extract increased bacterial growth rate, but it also accelerated decay speed and shortened bacterial growth time. The heat flow profile also indicated that AHLs extracts enhanced the activity of P. aeruginosas. Moreover, AHLs addition increased the protease activities, but has no effect on lipase activities. The actin, troponin-T and tropomyosin were hydrolyzed faster with higher concentration of AHLs. Fruthmore, the attachment of P. aeruginosa was decreased, and no biofilm was detected in samples with AHLs extracts.