Study On Co-cultivation Of Aeromonas Salmonicida And Pseudomonas Fluorescens Based On Quorum Sensing System

The spoilage of aquatic products is a relatively complicated process,and the growth and metabolism of microorganisms are considered to be an important factor leading to the spoilage potential of aquatic products.Studies have shown that most aquatic products cause rot-causing bacteria to participate in the expression of rot-causing factors by secreting N-acyl-homoserine lactones（AHLs）signal molecules.This process is regulated by the quorum sensing system.Therefore,investigating the spoilage mechanism and targeted regulation of spoilage bacteria in aquatic products based on quorum sensing system provide a new strategy for aquatic products preservation.At present,the research in related relatively more attention is paid to the isolation of strains,the regulation of spoilage factors and the development of inhibitors,and there is less research on the interaction between bacteria groups and interspecific quorum sensing.In this study,Aeromonas salmonicida and Pseudomonas fluorescens was the main test strains,and analyzed the interaction between the two strains under co-culture conditions based on the quorum sensing system and volatile organic compounds of the strains.The main conclusions are as follows:1.The quorum signals and co-culture with specific spoilage organism of aquatic products by the biosensor method and Gas Chromatography-Mass Spectrometer method A.salmonicida CS12,isolated from spoiled sea bass,were studied and identified.The results indicated that CS12 mainly produced C₆-HSL of signaling molecules with density-dependent.When co-cultured with P.fluorescens PF03,the bacterial density and the activity of AHLs was significantly inhibited.These results showed that P.fluorescens PF03 had a great inhibitory activity in co-culture.2.Study on interaction between two bacterias by comparing Changes of growth of strains,production of AHLs and motility during co-culture.The results showed that there was competition between bacteria in mixed cultured,and the growth inhibition effect was best in 1:1 inoculation culture.The strain CS12 has no motility activity,its AHLs during co-culture can promote the migration of PF03.The AHLs of strain regulated by the quorum sensing system with density-dependent,the AHLs ability is the strongest at 48 h,and the AHLs of PF03and co-cultured is inhibited after the stationary phase.The volatile odors of alone and co-cultured are similar after the logarithmic growth phase,its main components are nitrogen oxides,aromatics,ethanol and carbonyl compounds.3.The inhibitory activity of cell-free supernatant and AHLs extract of P.fluorescens on A.salmonicida CS12 was investigated by biosensor method and Oxford cup method.The results showed that the cell-free supernatant of PF03 can effectively inhibit growth of A.salmonicidum CS12.In the early stage of bacterial culture,the external addition of PF03-AHLs can promote the growth of A.salmonicidum CS12.PF03-AHLs extract has broad-spectrum bacteriostasis,and it can produce significant inhibitory effects on Chromobacterium violaceum CV026,Escherichia coli,Staphylococcus aureus and CS12.The activity was regulated by the quorum sensing system,and was the strongest at 48 h.The AHLs extract of co-cultured also had inhibitory activity against CS12.4.Analysis of volatile organic compounds of strains CS12 and PF03co-cultured by electronic nose and HS-SPME-GC-MS.The results showed that the composition and proportion of volatile organic compounds produced during co-culture are compared with PF03.Indole accounts for the highest proportion,followed by phenol,dimethyl disulfide,dimethyl trisulfide.The main volatile organic compounds of strain CS12 are indole and dimethyl disulfide,the external addition of PF03-AHLs can promote the production of indole and different types of volatile organic compounds,and most of them are used in pharmaceutical intermediates or in the production of pesticides,fungicides and edible flavors and additives of food,such as 3-Aminophenylacetylene,1-Acetylindole,2,4-Di-t-butylphenol,Methyl tetradecanoate,its have certain inhibitory activity.