Study On Inhibition Of Lactobacillus Plantarum Y42 Culture Supernatant Aginst Listeria Monocytogenes Aiofilm Formation

Listeria monocytogenes is an important food-borne pathogen,ubiquitously distributed in nature and can form biofilms in the food processing environment.Organic acids,hydrogen peroxide,bacteriocins and other metabolites produced by Lactobacillus are safe,highly effective and non-resistance and are good choices for controlling L.monocytogenes infection.In this paper,the inhibitory effect of Lactobacillus culture supernatant against planktonic and biofilm L.monocytogenes was studied.Firstly,the diameter of inhibition zone of Lactobacillus plantarum Y42 determined by Oxford cup double-layer plate diffusion method was 21.64 mm,significantly higher than those of other strains.The culture supernatant of L.plantarum Y42 was lyophilized and then re-dissolved in sterile distilled water to obtain a concentration gradient of 240 to 30 mg / mL and the minimal inhibitory concentration(MIC)of the re-dissolved solution was determined as 60 mg/mL by micro-well plate resazurin color method.The 1/2×MIC of L.plantarum Y42 culture supernatant did not inhibit the growth of L.monocytogenes.Therefore,the inhibition effect of L.plantarum Y42 culture supernatants with concentrations of 1/2×MIC,1/4×MIC,1/8×MIC,1/16×MIC and 1/32×MIC against biofilm formation of L.monocytogenes can eliminate the interference caused by the antibacterial effect of L.plantarum Y42 culture supernatants.The quantitative result of microtiter plate crystal violet staining indicated that L.monocytogenes was a medium biofilm-forming strain.Ciprofloxacin and levofloxacin were used to determine the antibiotic susceptibility difference between planktonic and biofilm L.monocytogenes.It was found that the L.monocytogenes biofilm tolerance to ciprofloxacin and levofloxacin both were 128 times higher than that of the planktonic L.monocytogenes.When culture supernatant of L.plantarum Y42 at a sub-inhibitory concentration interacted with ciprofloxacin and levofloxacin respectively against the L.monocytogenes biofilm,the minimum biofilm removal concentration(MBEC)of ciprofloxacin and levofloxacin both were reduced to 1/2-1/4 of that antibiotics used alone.Microtiter plate crystal violet staining was used to quantify the biofilm formation inhibitory and biofilm scavenging effects of L.plantarum Y42 culture supernatants at sub-inhibitory concentrations against L.monocytogenes.The results showed that L.plantarum Y42 culture supernatants at concentrations of 1/2×MIC,1/4×MIC,1/8×MIC,1/16×MIC and 1/32×MIC all significantly inhibited the biofilm formation of L.monocytogenes,and effectively cleared the pre-prepared mature L.monocytogenes biofilm.When L.plantarum Y42 culture supernatants had a concentration of 1/8×MIC,the inhibition rate of the biofilm formation of L.monocytogenes reached a maximum of 40.75% and the clearance rate to mature biofilm reached a maximum of 32.24%.The effect of L.plantarum Y42 culture supernatant at sub-inhibitory concentrations on the morphology and biomass of L.monocytogenes biofilm formation on the coverslip was qualitatively observed by optical microscopy and scanning electron microscopy.It was found that L.plantarum Y42 culture supernatant at sub-inhibitory concentrations effectively reduced the adherence and aggregation of L.monocytogenes on coverslips and the secretion of extracellular polymeric substances,thereby inhibited the elongation and thickening of biofilm even caused the collapse of the three-dimensional structure of the biofilm,so the endophyte was detached from the original biofilm and became a planktonic organism again.The Pearson correlation coefficient was used to analyze the correlation between the inhibition of L.plantarum Y42 culture supernatants on cell surface properties of L.monocytogenes and the inhibition of L.plantarum Y42 culture supernatant against L.monocytogenes biofilm formation.The simple correlation analysis results showed that the culture supernatant of L.plantarum Y42 inhibited the biofilm formation of L.monocytogenes and had no significant correlation with the inhibition of autoaggregation and cell surface hydrophobicity of L.monocytogenes.Instead,it interferes with L.monocytogenes biofilm formation by reducing the motility of L.monocytogenes and the secretion of extracellular proteins and extracellular polysaccharides by L.monocytogenes.In addition,L.plantarum Y42 culture supernatants at sub-inhibitory concentrations also inhibited L.monocytogenes adherence to HT-29 cells.The crude protein component 3 and crude protein component 4 obtained by salting out 60% and 80% ammonium sulfate in the culture supernatant of L.plantarum Y42 have a good inhibitory effect on the biofilm formation of L.monocytogenes.In conclusion,the culture supernatant of L.plantarum Y42 had a good inhibitory effect on planktonic and biofilm L.monocytogenes.However,the fundamental mechanism of L.plantarum Y42 culture supernatant inhibiting L.monocytogenes biofilm formation remains to be further studied.