Study On Screening Of Bacteriocin-producting LacticAcid Bacteria,Purification And Characterization Of Bacteriocin

A bacteriocin producing strain WH12-2-1 was screened from several lactic acid bacteria which were isolated from traditional products in Inner Mongolia by scrip method and cylinder-dish method. The supernatant of WH12-2-1 had strong bacteriostatic activity against several gram-positive bacteria and gram-negative bacteria. The strain was identified as Lactobacillus pentosus by physiological-biochemical testing and molecular-biological testing. Factors that affect L. pentosus WH12-2-1 producing bacteriocin and the biological features of bacteriocin were analyzed. And purification of bacteriocin was studied. The results were as follows:⑴Factors that affected L. pentosus WH12-2-1 producing bacteriocin involved indicator and its density, category of medium, culture time, addition of organic solvent and other antibiotic substances. The supernatant of L. pentosus WH12-2-1 in MRS for 48h had good bacteriostasis effect on Bacillus cereus that its viable cell numbers were 2.0×106 cfu/mL. Adding Tween 80 or other antibiotic substances could preferably enhance bacteriostatic effect of the bacteriocin.⑵The biological features of bacteriocin were as follows , bacteriocin produced by L. pentosus WH12-2-1 was steady and still active in acid or weak acid , its optimum pH was 2.0 , and had good thermal stability between pH 2.0 and pH 6.0. The bacteriocin was sensitive to trypsin and chymosin but insensitive to pepsin and papain. Bacteriostatic spectrum of the bacteriocin was very wide, it could inhibit not only lactic acid bacteria but also gram-positive bacteria (B.cereus,Staphylococcus aureus,Listeria monocytogenes) and gram-negative bacteria (Escherichia coli,Salmonella typhimurium,Shigella flexneri). The mechanism of bacteriocin was bacteriostasis rather than sterilization, as shown by a obvious decrease about 2-3 orders of magnitude in the viable cell numbers of B.cereus over a period of 6-10h. And compared to a nisin producing strain L. lactis PCM2379 , antimicrobial effect of bacteriocin produced L.pentosus WH12-2-1 was basically equal to it.⑶Purification of bacteriocin was as follows.The bacteriocin was purified by ultrafiltration and Sephadex G-25 column chromatography. After the broth was ultrafiltrated, some substances which the molecular weight was 3KDa -5KDa had a powerful bacteriostatic effect to B.cereus ; then by Sephadex G-25 column chromatography,three kinds of composition were obtained, composition of the second summit had the strongest bacteriostatic activity. The results indicated, the bacteriostatic activity composition would be a kind of small molecular polypeptide, the molecular weight of which was between 3KDa and 5KDa. Composition of the second summit will be studied further.