Isolation, Purification And Antibacterial Properties Of Bacteriocin Lac-B23Producing By Lactobacillus Plantarum

Preservative and fresh keeping are very important ways to ensure food securityduring the process of food preservation. Lactic acid antimicrobial peptide can replacechemical preservatives due to its properties such as non-toxic, no residue,high-efficiently and inhibit or killing spoilage and pathogenic bacterium. Nowadays,Nisin is the only antimicrobial peptide approved used in food industry, however, itsnarrow antibacterial spectrum and poor dispersity limited wide range of application, soit's a hot spot to research and development new bacteriocins. In this work, alactobacillus strain which can steadily produced antimicrobial substance namedLactobacillus plantarum B23was screened from those preserved strains of LAB. Thereal inhibiting substance was peptide named Lac-B23after research and analysis.Properties of Lac-B23were studied such as antimicrobial activity, pH stability, thermalstability and resistant to protease after purification of it with different methods. Theresults are as followsSix strains with antimicrobial property screened from300strains of LAB by theagar diffusion bioassay, one of the six has better antimicrobial activity against Listeriamonocytogenes and Staphylococcus aureus. The strain was Lactobacillus plantarumwith99%similarity as Lactobacillus plantarum, identified by morphology,16Sribosomal DNA sequencing and API50CH strips, then submitted to Genebank and theregister number is KC292491.1. Proving the antibacterial substance was peptide byexcluding hydrogen peroxide experiments and substances hydrolysis by protease.Three steps were used to purify Lac-B23. Fractional precipitation resultsdemonstrated that the best concentration of ammonium sulphate was80%, whichcontains more Lac-B23than that by adsorption-desorption. The purity of lac-B23was796.54fold and with2.76%yield than untreated through adsorption-desorption methodfollowed by cation exchange chromatograph, compared to827.01fold and11.36%yield by80%ammonium sulphate precipitation followed by cation exchangechromatograph. Using HPLC to purify Lac-B23during the high degree purificationprocess, two samples which got by different methods both exit a single peak at about25min, therefore Lac-B23can be got at25min. Lac-B23, molecular weight6.73kDa analyzed by Tricine SDS-PAGE, has significant antimicrobial activity. LC-MS analyzedshowed that Lac-B23might be a new peptide.The antimicrobial activity and characteristic experiments of Lac-B23showed thatLactobacillus plantarum B23has the highest value of OD600and best antimicrobialactivity in the16h of fermentation. Lac-B23has high tolerance stability within the pHrange from4.0to12.0, has the antimicrobial activity between25to121?, however,antimicrobial activity decrease significantly when temperature higher than90?;Lac-B23resistant to papain and pepsin, However, proteases like protein k, protein E andtrypsin were found to degrade it. The antimicrobial activity decrease when added K+,Ca2+, Zn2+, Mg2+and Cu2+, whereas Fe2+and Mn2+increase that activity. Addedskimmed milk has negative effect on antimicrobial activity, but ethanol has the positiveeffect. Raise the concentration of Nacl decreased of antimicrobial activity, but inhibitoryeffect receded when the concentration of Nacl higher than10%.