Screening And Identification Of Bacteriocin Producing Class? Bacteriocin And And Regulating Of PlnABC

Lactic acid bacteria is a kind of probiotics which is widely used in industry,agriculture and animal husbandry,food fermentation industry,medicine and so on.Therefore,the aim of this study is to screen out a broad spectrum bacteriocin and identify it,focuses on the factors affecting Class II can promote the bacteriocin,the best fermentation conditions of the strain were obtained.Specific tests are as follows:1.Screening and identification of Class II bacteriocins of lactic acid bacteria and Separation and purification of Class II bacteriocinThe main content of this study was to identify the strains of lactic acid bacteria by the Oxford cup method and MIC screening and 16 S RNA molecular identification,it has a significant inhibitory effect and a broad spectrum of antimicrobial effect of the strains,and select the best antibacterial effect of the strain L-4.The identification results of 16 S r DNA showed that L-4 molecules of lactic acid bacteria is Lactobacillus plantarum,the database number is KU572436.1.By PCR and agarose gel electrophoresis shows that the strains containing the quorum sensing system of Class II secretion of bacteriocins.At the beginning of bacteriocin extracts by molecular weight separation of bacteriocin by ultrafiltration system,then by ultra performance liquid chromatography separation and purification of protein,urea gel electrophoresis identification of the molecular weight of10 KDa.2.The influence of Tween80,MgSO₄ and EDTA on regulation of the expression of L-4The effects of different concentrations of Tween80 medium to culture MRS,EDTA and MgSO₄,its influence on the antibacterial activity of bacteriocin was detected by Oxford cup method and MIC experiment.To detect the expression regulation effect on Class II bacteriocin transporter plnABC by fluorescence quantitative PCR.0.3% Tween80?0.35g/L MgSO₄ and 0.8m M EDTA were added to the culture medium,they all have different degrees of promotion on the expression of bacteriocin transporter and the effect of EDTA on transporter expression was significant.3.Optimization of fermentation conditions of bacteriocinThe experimental factors: influence of fermentation time,fermentation pH,fermentation temperature,inoculation amount,rotation speed of bacteriocin production,by optimizing the fermentation conditions of the response surface method,thus the optimal fermentation parameters.The optimal fermentation conditions of Lactobacillus plantarum L-4: Incubation temperature: 37.08 C;culture time: 18.02 h;medium pH:4.5.The effects of Tween80,MgSO₄ and EDTA of lactic acid bacteria L-4 MIC and the diameter of inhibition zone,the results show that the maximum inhibitory circle of two strains of0.8m M EDTA indicator strains of Lactobacillus plantarum L-4 added to MRS culture medium after the diameter were 21.57 + 0.51 mm and 21.62 + 0.45 mm,MIC28mg/m L.Finally,by comparing the experimental results show that,under the optimal fermentation conditions and add 0.8m M EDTA in the culture environment,Lactobacillus plantarum L-4 antibacterial effect is best,the inhibition of Escherichia coli produced a diameter is 21.56 mm.