Characterization Of Bacteriocin Produced By Enterococcus Faecium Isolated From Soypaste

Bacteriocins produced by lactic acid bacteria is a kind of natural food preservative, which can inhibit the growth and reproduction of pathogenic bacteria and spoilage bacteria in food.There are various of microorganisms in traditional fermented soybean paste,which contains abundant resources of lactic acid bacteria.Enterococcus faecium is a kind of lactic acid bacteria, which can exist in fermented soybean products. Therefore, Oxford agar diffusion method was used firstly to screened spherical lactic acid bacteria, and then 16S rDNA sequence analysis was used to determine strain which can product bacteriocin.And then, analysed the characteristics of Enterococcus faecium and bacteriocin.This research provided an important theoretical basis for the development and application of new natural food preservatives and feed additives.This experiment collected 13 samples of traditional fermented soybean paste from six regions in Liaoning Province.First of all, separating and purificating of lactic acid bacteria from fermented soybean paste. And then screened lactic acid bacteria which can product bacteriocin by the Oxford agar diffusion method.Finally identified species of lactic acid bacteria which can product bacteriocin through 16S rDNA sequence analysis.The results showed that there were 164 strains isolated from all the samples which contained 84 strains of staphyl lactic acid bacteria. Preliminary screening results showed that there are 24 strains had good antibacterial activity to Staphylococcus aureus, and finally obtained 1 strain of lactic acid bacteria which can product bacteriocin. And then 16S rDNA sequence analysis showed that this strain was Enterococcus faecium, and named it Enterococcus faecium R1.Further test, analysed the characteristics of Enterococcus faecium R1 and bacteriocin. First determined the antimicrobial spectrum of Enterococcus faecium R1 by the Oxford agar diffusion method, and measured the stability of the bacteriocin in different pH value, temperature and protease. Secondly, the tolerance of Enterococcus faecium R1 on artificial gastric, intestinal juice and bile salt were studied to judge the probiotic of that strain. Thirdly, the detection of drug resistance and virulence genes of R1 were carried out to evaluate its safety. Finally, the kind of the bacteriocin was determined by PCR and gene cloning. Test results showed that the bacteriocin has inhibitory effect on most of indicator bacteria, especially on Listeria monocytogenes, antimicrobial spectrum widely. The bacteriocin producted by Enterococcus faecium R1 was EntP. This bacteriocin was stable in acidic conditions, and has good thermal stability, protease sensitive. Enterococcus faecium R1’s survival rates were 44.10% and 86.35% in simulated gastric juice for pH2.5 and pH3.0. Respectively, it is survival rates were 32.21% and 87.72% in simulated intestinal fluid and containing 0.3% bile salt culture medium for 6h, the strain has good tolerance to simulated gastric juice and bile salt. This strain was not resistant to common antibiotics, and it did not contain the common virulence genes.lt was potential safety.The results showed that the strain has good probiotic properties and safety. Bacteriocin generated of that strain has widely antibacterial range and good stability. This research provided an important theoretical basis for the development and application of new natural food preservatives and feed additives.