Studies On Bacterial Quorum-Sensing In Spoilage Of Chilled Pork

China is a big country of pig breeding and pork consumption in which the main consumption pattern is fresh meat. Due to the characteristics of nutritious, safe and healthful, chilled pork has become the leading developing direction of fresh meat.Though the temperature environment remains 0-4℃through production to marketing, it cannot inhibit the propagation of psychrophilic microorganisms which leads to a short shelf life of chilled pork. Based on that, China has been severely restricted in the development of production and domestic sales market of chilled pork.The corruption of chilled pork results from the interaction of specific spoilage microorganisms.Pseudomonas aeruginosa is a kind of Gram-negative bacterium,a psychrophilic microorganism which causes the corruption of high-protein food,and it plays an important role in spoilage of chilled pork. It is the Gram-negative bacterium that give rise to food spoilage based on the research of recent years. And N-3-acyl-homoserine lactones (AHLs) have been detected in corruption food. Therefore this assay is aimed to select P.aeruginosa which produce signal molecules of AHLs in putrid chilled pork, and extract the AHLs using an Agrobacterium tumefaciens A136(pCF218/pCF372) plate screening model. Then the researcher simulated the microenvironment of putrid chilled pork, investigated the intervention of Pseudomonas and its quorum sensing(QS)in degradation of pork protein extracts in chilled pork. It provides research basis of the role that QS plays in meat corruption and antisepsis and preservation tactics which base on the interference with corruption bacterial quorum sensing as a target of chilled meat. Main conclusions of the tests are as follows:1,Selective medium were used to study the initial microflora of pallet packaging chilled pork, then the interaction and changes of growth and decline of microorganisms in pallet packaging chilled pork were investigated under the storage temperature of 4℃. The results showed:the initial microflora of main microorganisms in chilled pork was Pseudomonas and lactobacillus under aerobic condition. Total bacterial count increased under the storage temperature of 4℃; as the storage time extended, the growing speed of Pseudomonas was the highest, and the bacterial count was the most. The number of Escherichia Coli and Staphylococcus grew at different degrees, reached a higher logarithm value at the end of the experiment. During the experiment, growing speed of lactobacillus was the slowest; its proportion kept falling during the period and it lost the status of dominant bacteria at the end of the experiment. Pseudomonas had a negative correlation with lactobacillus while Escherichia Coli had a positive correlation with Staphylococcus.2,66 strains Pseudomonas were gained from chilled pork under low temperature storage and corrupted chilled pork sample using Pseudomonas isolation medium 15 strains Pseudomonas that produce AHLs were gained using A.tumefaciens A136 (pCF218/pCF372) reported plates for secondary screening. Then one Pseudomonas strain that gained the highest yield of AHLs was identified by combining 16S rRNA molecular biology identification with conventional bacterial morphological and biochemical methods. Finally the one was identified as Pseudomonas aeruginosa.The representative 16S rRNA sequence of Pseudomonas was selected from GeeBank. DNAMAN software was used to structure Phylogenetic tree. Properties that related to food spoliage, such as proteolytic activity and biofilm formation ability of the P. aeruginosa, were analyzed at the same time.And study shows that it can generate biofilm and has a strong capacity of protein forming.3,AHLs that were synthesis by P.aeruginosa C272, together with microorganisms that caused spoilage of chilled pork,were added to the extracts from pork protein.Using SDS-polyacrylamide gel electrophoresis (SDS-PAGE),AHLs were found to mediate the biodegradation process of chilled pork protein extracts that were caused spoilage by microorganisms. The results showed that AHLs extracted from P.aeruginosa C272 has a promote effect on sarcoplasmic protein degradation of itself,Staphylococcus aureus, Escherichia coli,Lactobacillus plantarum.Especially AHLs had a most promote effect on S. aureus and a secondary effect on P.aeruginosa C272 itself and L.plantarum while the effect on E.coli was not so significant. According to the atlas, AHLs were found to help degradation of protein whose molecular weight is about30-40kDa. However, AHLs from P.aeruginosa. C272 had different regulations of myofibrillar protein degradation.They stimulated myofibrillar protein degradation of P.aeruginosa C272, and had a certain promotion of E.coli on degradation of myofibrillar protein whose molecular weight is about 105kDa and 34kDa, while they helped S.aureus and L.plantarum restrict degradation of myofibrillar protein. 4,A research about the effects of different microbiota on the degradation of pork protein was carried out by mimicking various pork spoilage microbiota with SDS-PAGE. The results indicated that both sarcoplasmic protein and myofibrillar protein would be degraded under various microbiota containing P.aeruginosa C272 and other main spoilage organism. The degradation extent of swine sarcoplasmic protein under different microbiota and single species of microbiota were almost without differences, and the situation was the same between different microbiota.The degradation conditions were complex under various microbiota,but the atlas indicated that myofibrillar protein degradation was suppressed to different extent under the microbiota with Pseudomonas and with four strains the degradation was minimum.Antagonism of the four strains to suppress each other's degradation activity may contribute to the situation.