| Chilled chicken has become the leading developing direction of Chinese raw poultry market due to the characteristics of nutritious,safe and healthful.Although the storage environment remains in a low-temperature through production to sale,it cannot inhibit the propagation of specific spoilage organism of chilled chicken completely.Pseudomonas is a psychrophilic Gram-negative bacterium,which played a key role in the spoilage of chilled chicken as an important bacteria that caused spoilage of proteinceous foods.The bacteria can regulate the behavior of the flora through quorum sensing,which in turn affects the spoilage process of food.Quorum sensing is a cell-to-cell communication mechanism used by bacteria to regulate their collective behaviors in a cell density-dependent manner.An increasing number of empirical evidences had demonstrated that there was a close relationship between bacterial quorum sensing and meat spoilage.Several bacterial characterists and spoilage characteristics are under control of signals-based quorum sensing system.Regulating meat spoilage by interfering with the quorum sensing system of spoilage bacteria has become a research hotspot in the field of meat preservationFirstly,isolated and identified spoilage bacteria in chilled chicken,and clarified the bacteriostatic effect and mechanism of chlorogenic acid;through chlorogenic acid treatment,the chlorogenic acid was studied on the quorum sensing of the dominant spoilage bacteria Pseudomonas fluorescens in chilled chicken regulation and inhibition of its spoilage properties;further study on the effect of chlorogenic acid on the expression of Pseudomonas fluorescens quorum sensing regulators and spoilage factors,and verify the function of GacA by constructing a gacA gene knockout strain,inferring chlorogenic acid's quorum sensing The inhibitory effect is achieved by inhibiting the global regulatory factor GacA in Pseudomonas fluorescens and then regulating the quorum sensing regulator luxl/R.GacA may be the target for chlorogenic acid to inhibit the decay of Pseudomonas fluorescens,while chlorogenic acid may become a novel quorum sensing inhibitor.This study enriches the theory of food storage and preservation that targets group sensing,and at the same time provides new ideas and ways for the preservation of chilled chicken.This study includes the following three parts:1.The spoilage bacteria of chilled chicken were isolated and identified.Study the antibacterial mechanism of chlorogenic acid on Pseudomonas fluorescens,Escherichia coli and Salmonella enteritidis.The dominant spoilage bacteria was identified as Pseudomonas fluorescens.The results showed that chlorogenic acid had bactericidal action on three spoilage bacterias.Furthermore,the morphology and membrane permeability of indicator bacteria exposed to chlorogenic acid were characterized by scanning electron microscope(SEM),confocal laser scanning micrographs(CLSM)and extracellular ATP analysis.The results showed that the antimicrobial effect of chlorogenic acid on spoilage bacteria was mainly completed by increasing membrane permeability,thereby leading to leakage of the intracellular components2.Chromobacterium violaceum CV026 strain was used as a detection model to confirm the inhibitory activity of quorum sensing.The signal molecule was detected by HPLC-MS/MS The effect of different concentration of chlorogenic acid on spoilage characteristics such as siderophore,extracellular protease and extracellular lipase production as well as bacterial motility of Pseudomonas fluorescens were measured,a class of signal molecule was added to the culture simultaneously.The results showed that chlorogenic acid could reduce the AHLs released by Pseudomonas fluorescens.Chlorogenic acid could effectively inhibit siderophore production,extracellular protease and lipase,swarming and swimming motility of Pseudomonas fluorescens.The concentration was positively correlated with its inhibitory effect.These results showed that chlorogenic acid had a great inhibitory activity,this study provides a basis for expanding the industrial application of chlorogenic acid as a novel inhibitor of spoilage bacteria.3.GacA as a global regulatory factor and Luxl/R as an important regulatory protein participate in the quorum sensing system mediated by AHLs.Using homologous recombination technology to construct knockout strain P.fluorescens ?gacA and chlorogenic acid to treat the knockout strain.Through the detection of extracellular enzymes,siderophilic bacteria,exercise characteristics and related gene expression,we had explored the changes of bacterial spoilage characteristics before and after the gacA gene was knocked out.So as to verify the role of the regulatory protein GacA and the effect of chlorogenic acid.The results showed that the gacA gene knockout had no significant effect on the normal growth state of the bacteria,and chlorogenic acid treatment had no significant change in the characterization of the bacterial spoilage characteristics and the expression of related genes.This indicated that chlorogenic acid regulates the quorum sensing system of Pseudomonas fluorescens by GacA and then inhibits its spoilage activity. |
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