| The thesis proved the synergistic inhibition behavior of plantaricin E/F(PlnEF)and L-lactic acid on gram negative pathogen.We focused on the inhibition behavior and mechanism of PlnEF and lactic acid on Aeromonas hydrophila LPL-1.The results of the research provided a theoretical foundation for application of bacteriocin producing bacteria in seafood and aquaculture against gram-negative pathogens.The results and conclusions including:(1)Lactobacillus paraplantarum L-ZS9 produced two kinds of antimicrobial substances-lactic acid and class Ⅱb bacteriocin PlnEF.9 gram-negative pathogen were used as indicator bacteria,synergistic inhibition and bactericidal behavior of PlnEF and lactic acid on these bacteria were illuminated by plate diffusion method,the optical density method and the flow cytometry analysis.In addition,the results showed that PlnEF and lactic acid synergistically reduced the motility,biofilm formation,adhesion to colon cancer cell HT-29,proteolytic activity,and cell cytotoxicity of A.hydrophila LPL-1.(2)PlnEF act on A.hydrophila whose outer membrane disrupted by lactic acid.We analyzed the LPS release,changes of membrane potential and the distribution of PlnEF.The results demonstrated that lactic acid injured the outer membrane of A.hydrophila,releasing LPS,which enabled distribution of PlnEF to cell membrane.PlnEF act on A.hydrophila with injured outer membrane,dissipating membrane potential,enhancing the membrane permeability and ATP leakage.Scanning electron microscopy and transmission electron microscopy analysis revealed the morphology changes of A.hydrophila cells.The results showed that PlnEF lengthened the cells and induced out membrane vehicles.Lactic acid alone caused cell deformation,outer membrane damage,plasmolysis,and intracellular DNA aggregation.PlnEF combined with lactic acid caused more severe cell deformation,plasmolysis.In addition,the inner membrane were damaged,and a part of DNA aggregated as high electron density particles.(3)PlnEF and lactic acid interfere the energy metabolism,inhibited DNA repair and reduced the protein folding accuracy of A.hydrophila.Differential proteomics of cells treated by PlnEF and lactic acid individually and in combination were analyzed.The results showed that cells treated with lactic acid responded adaptively to the sublethal acid condition.Intracellular metabolites flew to the TCA cycle,which could increase energy synthesis;In order to ensure the supply of protein,expression of polynucleotide phosphorylase was downregulated to avoid mRNA degradation.In cells treated with PlnEF and lactic acid:The TCA metabolism reduced,and the metabolites flow to EMP pathway with lower energy consumption;DNA ligase were down regulated,suggesting the DNA repair inhibition;The expression of EF-Tu were upregulated to insure protein translation;The expression of ThrRS and GraB increased significantly,suggesting that they are potential intracellular targets for PlnEF. |
