Screening Of Lactic Acid Bacteria With Inhibitory Activity Against Quorum Sensing Of Aeromonas Hydrophila And Its Inhibitory Effects

Aeromonas hydrophila is gram-negative pathogenic bacteria which can cause the disease of aquatic animals.Its pathogenicity is closely related to the quorum sensing system.Lactic acid bacteria(LAB)can produce reuterin,nisin and acids metabolites to control pathogenic bacteria and spoilage organisms.However,the study about LAB as quorum sensing inhibitor(QSI)has rarely been reported at home and abroad.In this paper,lactic acid bacteria with good antagonistic activity against A hydrophila QS were screened from traditional fermented vegetables,and the mechanism of inhibiting the QS and the inhibition of the active substance of QS were explored,which laid the foundation for the development of a kind of lactic acid bacteria biologics to inhibit the group induction of A.hydrophila.The main results were as follows:1.A total of 135 LAB strains were isolated from traditional fermented vegetables.The six strains LAB with strong antagonistic activity toward quorum sensing of A.hydrophila were screened using the filter paper method,and strain SCT-2 has the strongest effect,and it was identified as Lactobacillus plantarum by physiological and biochemical test and 16S rRNA sequence analysis.The QS antibacterial components of cell-free supernatants(CFS)from strain SCT-2 were analyzed using protease sensitivity test,acid test and heat-treated test.The QS inhibitory substance produced by LAB was preliminarily determined as bacteriocins.2.The antagonism mechanism of strain SCT-2 CFS to quorum sensing of A.hydrophila is preliminarily studied by double layer agar diffusion method.The results showed that the secretory volume of proteolytic and siderophore of A.hydrophila treated with 8 mg/mL crude extract were decreased by 27.18%and 22.11%,respectively,and the degradation rate of AHLs of A.hydrophila was 32.27%.It also obviously inhibited the swarming and swimming of A.hydrophila.3.The paper studied the inhibitory effect of Lb.plantarum SCT-2 on the formation of A.hydrophila biofilm.The results showed that the surface of Lb plantarum SCT-2 and A.hydrophila had both the ability of hydrophobicity and auto-aggregation,which could form biofilm.After co-cultivation of the two strains,the strain SCT-2 was found to effectively inhibit the growth and reproduction of A hydrophila on the surface of polytetrafluoroethylene(PTFE).When the crude extract of SCT-2 metabolites reached 8 mg/mL,the inhibition rate of A.hydrophila biofilm was 45.16%.4.The QS antimicrobial components from CFS of the strain SCT-2 were separated,purified and identified.These results showed that inhibitory substance was extracted from CFS by ethyl acetate and the inhibition zone diameter reached 21.96 mm.The inhibitory substance was further purified by ultrafiltration system,moderate purification and high performance liquid chromatography(HPLC).The fration at 4.8 min from HPLC had inhibitory activity of quorum sensing of A hydrophila.The SDS-PAGE showed that the molecular mass of the bacteriocin was between 1.0?3,3 ku.The mass spectrometry(MS)result showed that the molecular mass of the bacteriocin was 0.86 ku.