| Enterobacter cloacae and Enterococcus faecalis are widely present in the environment and intestinal tract of animals.Therefore,the surfaces of carcasse are easily contaminated by these bacteria when livestock and poultry were slaughtered.These bacteria had a strong ability of biofilm formation.Biofilms were complex multicellular aggregates formed by bacterial cells.When biofilms were formed on the surface of processing equipment or food products,the bacterial cells in the biofilm can better tolerate various unfavorable factors including antibacterial agents for which the bacterial cells were difficult to be completely inactivated and contaminated the contact foods.As a result of food spoilage or the outbreak of foodborne diseases,the control of biofilm contamination in recent years had been got extensive attention in food industry.The phenyllactic acid(PLA)was a new biological antibacterial agent with broad-spectrum antibacterial effect which was discovered in recent years.However,there were few studies on antibacterial and antibiofilm activities of PLA on the above two spoilage bacteria.In this paper,the antibacterial and bactericidal effects and mechanism of PLA on these two strains and their biofilms were studied using E.cloacae C4 and E.faecalis R612-Z1 isolated from poultry meat products as the target strains.It provided more scientific basis of PLA application in the control of food-borne bacteria and biofilm contamination,and provided a theoretical basis for the future development and utilization of novel biological antibacterial agents.The specific research contents and results of this paper were listed as follows:(1)Both of the minimal inhibitory concentrations of PLA against E.cloacae C4 and E.faecalis R612-Z1 were 5 mg/mL.Bacterial activity experiments showed that all the tested PLA solutions(1.25,2.5,5,10 mg/mL)had bactericidal effects on E.cloacae C4 and E.faecalis R612-Z1,and the bactericidal effects were better as the increasing of the PLA concentrations.Scanning electron microscopy and transmission electron microscopy revealed that the cell membranes of PLA-treated groups were damaged.With the increase of PLA concentrations,the cell potential,extracellular ATP and UV-absorbing substance concentrations increased significantly(p<0.05).Confocal laser scanning microscopy and flow cytometry results showed that the cell membrane structure had been completely damaged after treated with 10 mg/mL PLA,and most cells were death.These results showed that the antibacterial effects of PLA on these two strains were mainly due to the cell membrane damages and leakage of the intracellular content.(2)The total bacterial RNA was extracted from E.cloacae C4 and E.faecalis R612-Z1cells in the logarithmic growth phase after treated by sub-MIC concentrations of PLA(2.5mg/mL)for 1 h,respectively.The results of transcriptome sequencing showed that many metabolic pathways were changed in the PLA-treaed bacterial cells,indicating that PLA treatment affects not only cell membrane integrity,but some biochemical metabolic processes in the cells.The expression levels of genes which were related to fatty acid synthesis and metabolism were changed.Since the phospholipids structures in cell membrane were mainly composed by fatty acids,it indicated that the PLA-treated cells changed the fatty acids synthesis and metabolism to adapt cell membrane damages.(3)E.cloacae C4 and E.faecalis R612-Z1 had the highest biofilm formation at 48 h.The treatment of sub-MIC of PLA(2.5 mg/mL)had no significant effect on the bacterial counts in E.cloacae C4 biofilm(p>0.05),no significant effect on the bacterial counts in E.faecalis R612-Z1biofilm at the concentration of 1.25 mg/mL,while had significant inhibition of bacterial counts(p<0.05)at 2.5 mg/mL.Treating of PLA(1.25,2.5 mg/mL)to the E.cloacae C4 and E.faecalis R612-Z1 cells biofilm formed on the surface of stainless steel had significant inhibition of bacterial counts(p<0.05).Determination of biofilm formation by the crystal violet method and the contents of extracellular polysaccharides,proteins and nucleic acids showed that the experimental concentration of PLA could inhibit the biofilm formation and reduce the production of extracellular substances.The biofilm morphology was observed by scanning electron microscopy and biofilm thickness was confirmed by confocal microscopy.The biofilm thickness was reduced and its dense structure was destroyed under PLA treatment.The results of real-time PCR showed that the gene expression levels related to bacterial movement ability and extracellular polysaccharide synthesis were significantly decreased.This showed that after treating with PLA can reduce the bacterial counts in biofilm,cell adhesion and production of extracellular polysaccharides,proteins and nucleic acids.(4)Different concentrations of PLA(0,1.25,2.5,5,10 mg/mL)had different bactericidal effect on E.cloacae C4 and E.faecalis R612-Z1 biofilms formed for 3 days and 7 days.After treated with 10 mg/mL PLA for 30 min,the bacterial counts in E.cloacae C4 biofilm that formed for 3 d and 7 d were decreased 2.69 and 3.53 log CFU/cm~2,and those in E.faecalis R612-Z1 biofilm were decreased 4.39 and 4.47 log CFU/cm~2.After PLA treatment,the internal bacteria cells in biofilm structure were found to be damaged shown by CLSM images.PLA had a significant bactericidal effect on E.cloacae C4 and E.faecalis R612-Z1 biofilm formed on the stainless steel surfaces.After treated with 10 mg/mL PLA for 30 minutes,all the bacteria cells on the stainless steel surfaces were killed.These results showed that PLA can penetrate the biofilm and act on the bacteria cells in the biofilm through to achieve bactericidal action.According to the results above,the mechanism of PLA act on bacteria is to destroy its membrane structure,cause leakage of intracellular substance,change the intracellular and extracellular potential so as to achieve a bactericidal effect.In addition,PLA at the sub-inhibitory concentrations inhibit the biofilm formation of E.cloacae C4 and E.faecalis R612-Z1,and have a good bactericidal effect on the formed biofilm. |
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