Study On The A Ntibacterial Activity And The Mechanism On Food-borne Pathogens Of The Combination Of Phenyllactic Acid And Organic Acids

Phenyllactic acid(PLA)is a novel natural antibacterial agent discovered in recent years.It has broad-spectrum antibacterial activity and can be produced by lactic acid bacteria.However,the antibacterial acitivity of PLA was relatively weak and large dose was needed in use,which limits its commercial application.Previous studies in our laboratory have showed that the antibacterial activity of PLA could be enhanced when it was co-existed with organic acid.However,the synergistic antibacterial activity and mechanism of PLA combined with organic acids remained unknown.Therefore,the combined bacteriostasis effect of PLA and organic acids was studied,in order to find out the organic acids which showed synergistic antibacterial effect with PLA.Then the synergistic antibacterial mechanism of PLA and organic acids was investigated,which provided a theoretical basis for the application of PLA in food industry.This study selected the main organic acids that Lactic acid bacteria had metabolized such as lactic acid,acetic acid,citric acid,butyric acid,succinic acid,malic acid,propionic acid,tartaric acid,and oxalic acid as the research subjects.The minimal inhibitory concentration of PLA and organic acids on Listeria monocytogenes,Staphylococcus aureus,Escherich coli and Salmonella typhimurium were investigated.The MIC of PLA was range from 1.25 to 2.5 mg/mL,while the MIC of the organic acids to each indicator was 0.5～2.5 mg/mL,and the minimum inhibitory concentration of acetic acid to E.coli could be as low as 0.5mg/mL.Then,a combination of PLA and each organic acid was used to perform combined bacteriostatic studies on each indicator bacteria.The combination of PLA and acetic acid,succinic acid and malic acid exerted a synergistic bacteriostatic effect on the four indicator bacteria.The combined use of PLA with citric acid,butyric acid,and tartaric acid will produce synergistic bacteriostasis effects on the three indicator bacteria.Choosing L.monocytogenes as the indicator of gram-positive bacteria to investigate the synergistic antibacterial mechanism of PLA in combination with lactic acid and acetic acid from the aspects of cell membrane,DNA and protein expression,respectively.The effect of PLA combined with lactic acid and acetic acid on the apoptosis of L.monocytogenes cells showed that the combined use of organic acids caused more than 80%of cell apoptosis and showed a synergistic effect;The Zeta potential of L.monocytogenes was significantly changed by PLA and lactic acid and acetic acid,and the surface charge of the bacteria was negatively changed to positive;Using DiSC3(5)as fluorescence probes for transmembrane potential investigated the effect of PLA combined with lactic acid and acetic acid on L.monocytogenes.The results showed that the combination of PLA with lactic acid and acetic acid will dissipate the cell membrane potential,thus consume the transmembrane potential.At the same time,PLA and lactic acid,acetic acid and acetic acid membrane permeability were studied with SYTO-9 and PI fluorescent probes.The effect of fluorescence microscopy showed that the membrane permeability was significantly impaired by each organic acid treatment.Flow cytometry results showed that PLA combined with lactic acid and acetic acid exerted a synergistic destructive effect on cell membrane permeability;TEM results of PLA and lactic acid,acetic acid in combination showed that the cell density of L.monocytogenes decreased,but the cell membrane was not severely damaged.In DNA,the dispersion of gel-blocking electrophoresis bands indicates that the degradation of DNA occurred,and the DAPI probe labeling results shows that the synthesis of DNA in vivo can be affected when PLA was combined with lactic acid or acetic acid.In the expression of protein,SDS-PAGE electrophoresis results showed the increase,disappearance,darken or lighten of protein bands,it is speculated that the combination of PLA and lactic acid,acetic acid will affect the protein expression of L.monocytogenes.E.coli was used as a gram negative indicator bacteria,and the bacteriostasis mechanism of PLA incombination with lactic acid and acetic acid was investigated from cell membrane,DNA and protein respectively.The apoptosis rate when PLA combined with lactic acid and acetic acid was lower than 30%.PLA,lactic acid,acetic acid could significantly change the surface charge of E.coli and the combination have a synergistic effect on the Zeta potential.The results indicate that PLA,lactic acid,and acetic acid dissipate the transmembrane potential and show a synergistic effect;The effects of the combination of organic acids on the membrane permeability of E.coli cells were observed with PI and SYTO-9 fluorescent probe labeling flow cytometry and fluorescence microscopy.The results showed that the change of cell membrane permeability of each organic acid was little.The results of TEM showed that the combination of PLA with lactic acid and acetic acid destroyed the cytoplasm of E.coli cytoplasm more seriously,but did not significantly destroy the integrity of the cell membrane.In terms of DNA,the results of gel block electrophoresis showed that PLA and lactic acid could reduce the brightness of the DNA band and disperse the strip.The results of DNA synthesis by DAPI probe showed that the combined use of organic acid could reduce the synthesis of double chain DNA,and the synergistic effect of PLA and acetic acid and acetic acid was shown.After treatment with organic acids for 4 h,there was no significant change in the species and quantity of E.coli protein.Therefore,it was speculated that the combination of lactic acid and lactic acid and acetic acid had no effect on the species and quantity of protein.