Study On The Antibacterial Activity And Mechanism Of Phenyllactic Acid Combined With Common Food Preservative And Organic Acid

Phenyllactic acid(PLA)is a novel natural substance found in recent years,which shows broad-spectrum antibacterial properties against gram positive and gram negative bacteria.However,the antibacterial activity of PLA is lower than other biological preservatives such as nisin and ε-PL,so that the high cost of PLA used as a biological preservative limits its commercial application.Therefore,the present study was to screen a preservative that could produce a synergistic effect with PLA,thereby reducing the cost and the dosage of PLA.At the same time,the synergistic antibacterial mechanism of PLA and synergistic agent was studied to provide theoretical basis for the combined use of PLA and preservatives in food preservation.Two common foodborne pathogens Listeria monocytogenes 10403 s and Escherich coli ATCC 44752 were used as indicators.The minimal inhibitory concentrations were determined by broth micro-dilution method and the interaction of PLA with nine kinds of food preservatives was determined using the checkerboard test,furthermore,the combination under the condition of optimal ratio of synergistic inhibitory effect was investigated by the time-kill curve.The results showed that five preservatives which could synergy with PLA against E.coli were selected,including sodium benzoate,potassium citrate,lactic acid,acetic acid and citric acid.Time-kill curve showed that PLA combinated with lactic acid,acetic acid,potassium sorbate,sodium benzoate and citric acid against E.coli for 24 h,viable counts were reduced by 4.24,3.41,2.85,2.79 and 2.43 on logarithm,respectively.Two kinds of organic acids,lactic acid and acetic acid,which could produce synergistic effect with PLA against L.monocytogenes was screened.Time-kill curve showed that PLA combinated with lactic acid and acetic acid against L.monocytogenes for 24 h,viable counts were reduced by 5.29 and 4.88 on logarithm,respectively.The dosage was decreased 75% of PLA and 50% of sodium benzoate,potassium acetate,lactic acid,acetic acid and citric acid when PLA was in combination with these food preservatives,which greatly reduced the dosage and the cost of preservatives.It was found that PLA,lactic acid and acetic acid were able to increase the permeability of the outer membrane of E.coli,which was labeled by NPN probe,and the damage increased after combined use.The change of PI fluorescence signal was detected by flow cytometry.It was showed that when 1/4MIC concentration of PLA was combined with 1/2MIC concentration of lactic acid or acetic acid,the damage rate of L.monocytogenes membrane integrity was 96.4% and 87.1%,which were higher than MIC concentration of PLA,lactic acid or acetic acid.Therefore,it showed a significant synergistic effect when a lower concentration of PLA combined with lactic acid or acetic acid,whose effect was equivalent or even more than the effect of MIC concentration of PLA,lactic acid or acetic acid.However,the damage rates were less than 10% whether PLA,lactic acid and acetic acid alone or combined use on the membrane integrity of E.coli,indicating that there was no significant effect.The effects of PLA,lactic acid and acetic acid alone or combined use on the ultrastructure of the cells were observed by scanning electron microscope.A large number of depression and shrinkage appeared on cell surface indicated that the effect of lower concentration of PLA combined with lactic acid or acetic acid was more than those were used alone.It was further proved that there was a synergistic effect of PLA combined with lactic acid or acetic acid on the cell membrane.In addition,it was found that PLA,lactic acid and acetic acid could be embedded with DNA by fluorescence spectroscopy.And a synergistic effect on DNA was produced,which increased the DNA fluorescence quenching,when PLA was combined with lactic acid or acetic acid.Therefore,it was speculated that the synergistic mechanism of PLA combined with lactic acid and acetic acid was to enhance the destruction effect of each other on the cell membrane permeability and integrity.Then,because of the damage of cell membrane it was more easily for PLA,lactic acid and acetic acid accessible to the cell interior,and binding with DNA,affecting the stability of DNA,and thus playing a synergistic antibacterial effect.