Screening Of Lactic Acid Bacteria Producing Broad Spectrum Bacteriocin And Regulation Of High Expression Of Bacteriocin

Food preservative plays an important role in food shell-life and food quality. Chemical preservatives give priority in food industry presently, it may cause hidden trouble. Nisin is used widely as one of a natural preservative, but it can inhibit G+ bacteria only and can not inhibit G- bacteria, yeast and fungus. Pediocin as the typical representation of Classâ…¡a bacteriocin has been a focus duo to its characteristics of broad antimicrobial spectrum, high stability and good dispersibility. However, one of the bottleneck to industrialized Classâ…¡a bacteriocin is low output. Therefore, screening of lactic acid bacteria producing Classâ…¡a bacteriocin and study on purification, biological information, physic-chemical characteristic, production condition optimization, synthesize regulation, modification and effects on application of bacteriocin were developed.According to the conserved sequence -YGNGV- of Classâ…¡a bacteriocin, establish high throughout protocol to screening of LAB producing Classâ…¡a bacteriocin based on Colony-PCR. Based on this platform, Lactobacillus paracasei-J23 was isolated as candidate with excellent activity and broad antibacterial spectrum of 275 lactic acid bacteria from Chinese traditional fermented food, and identified by Biolog system and 16SrDNA technic. The antibacterial peptide was confirmed and named as Bac- J23. Bac-J23 was purified by three steps including absorb by host, cation exchange and hydrophobic chromatogram. Purification folds and recovery rate were 45.1% and 16.4% respectively. Tricine-SDS-PAGE and HPLC test appear to single band with molecular weight 6.56KD. The results of amino acid sequence assay shows that N-sequence of Bac-J23 is (K, N, D)-Y-G-N-V-G(V)-V- (A, F)-(V, F), which is the typical character of Classâ…¡a bacteriocin.Results of physic-chemical and biological characteristic showed Bac-J23 contains abundant hydrophobic amino acid and an unidentified amino acid. Theoretical and real isoelectric point and were 9.145 and 8.9 respectively. The extinction coefficient of Bac-J23 was 27850L/mol/cm. Thermodynamics properties of Bac-J23 indicated the denaturalization temperature and enthalpy were 86.35℃and 16.49J/g respectively. Bac-J23 is stable under acidic condition and sensitivity to Proteinase K, Trypsin and Papain, but resistant to pepsin, lipase and amylase. The contents of–SH and–S-S- were 10.18 mol/106g and 21.15 mol/106g, respectively. Prediction of subcellular localization and transmembrane domains shows that Bac-J23 is a secretion peptide and no transmembrane domains. Homogeneity predication suggests Bac-J23 may be one of an unidentified peptide though it is similar to Enterocin E-760 produced by Lactobacillales, Enterococcaceae and Enterococcus.pH, temperature and inoculation size dependent property were observed when Lactobacillus paracasei-J23 produced Bac-J23. The optimum condition to Bac-J23 synthesize were 36.8℃, pH5.2 and 2.13×10⁴CFU/ml inoculum size。The optimum culture medium were sucrose 20g/L,yeast extraction 0.5%,beef extraction1%,KH₂PO₄ 20g/L,citric acidammonium 0.2%,MgSO₄ 0.2g/L,MnSO₄ 0.25g/L。Under the optimum condition, the production of Bac-J23 was improved 1.68 folds and reached 5400 U/ml. QS system to regulation Bac-J23 biosynthesis was identified, and it was induced by Bac-J23 with threshold from 2.5×10^-10M to 6.4×10^-8M, the saturation yield was 3200U/ml. Temporal effectiveness was observed that Bac-J23 must be added before stationary phase when it was used as inducer. Established Lactobacillus paracasei-J23 resting cell system to select extrinsic inducers to synthesize Bac-J23. Glycerin, Cys, Glycin and pyruvic acid were regard as inducers, but no function with Glu, Tyr and Ala. E.coli and Bacillus subtilis can improve Bac-J23 yield when co-cultured with Lactobacillus paracasei-J23. The inducers exist in the fermentation broth and the cell of Lactobacillus paracasei-J23 respectively. The inoculation size with induce function were 1.8×10⁴CFU/ml for E.coli and 5.2×10⁴CFU/ml for Bacillus subtilis, the yield of Bac-J23 can be improved 2-6 folds.Proteolyses of Bac-J23 by both Proteinase K and Trypsin yield fragments of various molecular weights.The Proteinase K cleavage yields peptides of molecular weight about 4.5KD and 1.9KD, while the comparable pieces in a Trypsin digest have apparent weights of about 3.8KD and 2.5KD. The digests lose almost all of their binding ability and in vivo activity but the in vitro activity is augmented. The effects of food components and food-related environment on Bac-J23 activity showed that Casein and lecithin appear to be negative effect on Bac-J23 activity. EDTA and NaCl can enhance Bac-J23 activity, while Lactobacillus paracasei-J23 can not produce Bac-J23 above 6%NaCl. About 50% activity was kept when stored at normal temperature for one year, which suggests the excellent reserve stability of Bac-J23.