Screening Of Bacteriocin-producing Lactic Acid Bacteria And Studies On Its Bacteriocin, Leucocin B-K7

Bacteriocins of lactic acid bacteria(LAB) are ribosomally synthesized antimicrobial peptides or proteins produced by LAB. As a class of new biological preservatives, they are safe and healthy, and do not cause side-effect and drug resistance. In order to discover novel bacteriocin(s) produced by LAB, a bacteriocin-producing LAB was isolated from a chinese traditional fermented vagetable, pickle. The biological properties, related gene cluster, purification, mode of action and preliminary application in refrigerated milk of the bacteriocin, i.e. leucocin B-K7 which it produced were researched in this paper. The main contents and results are as follows.1. A bacteriocin-producing strain was isolated from pickle. It was named as Leuconostoc mesenteroides K7 based on 16 S rDNA analysis. The antimicrobial activity of the cell-free-supernatant of strain K7 was 80 AU·mL-1. The bacteriocin, i.e. leucocin B-K7 produced by the strain, exhibited antagonistic activity against the pathogen Listeria monocytogenes. It was sensitive to protease and displayed good thermal stability and broad active pH range.2. lcnB which encodes the precursor of leucocin B-K7 was amplified and sequenced. The sequence indicates that the mature peptide of leucocin B-K7 is identical to those of reported bacteriocins including leucocin B-Ta11 a, leucocin B-KM432 Bz and leucocin A-QU15, while one animo acid residue, i.e. D8 E in prepeptide is different from the three reported leucocins. The gene cluster of leucocin B-K7 was found to locate at a plasmid of 11049 bp of L. mesenteroides K7 by plasmid curing. The plasmid contains the genes functioning as putative replication initiation proteins(rep) and the genes encoding the bacteriocin precursor(lcnB), immunity protein(lcnI), ABC-transporters acting as a translocator(lcn T) and a secretion protein(lcnS), accessory protein for ABC-transporter(lcnX) and two hypothetic proteins of unknown function.3. Leucocin B-K7 was purified partially by cation-exchange chromatography using SP Sepharose Fast Flow and gel filtration chromatography using Sephadex G50, with the recovery of 67.8% and 28.2%, respectively. Tricine-SDS-PAGE revealed that the molecular weight of leucocin B-K7 was 3.5~6.5 kDa. Leucocin B-K7 inhibited the growth of L. monocytogenes CMCC 54005 at the minimum inhibitory concentration(MIC) of 0.028 mg·m L-1.4. The mode of action of leucocin B-K7 was analyzed. Leucocin B-K7 had no effect on the efflux of ATP from L. monocytogenes CMCC 54005 but triggered the efflux of K+ and the intracellular hydrolysis of ATP. It also dissipated the transmembrane electrical potential(ΔΨ) completely and the transmembrane pH gradient(ΔpH) partially.5. Leucocin B-K7 was added into L. monocytogenes CMCC 54005-contaminated milk at 80 AU·mL-1 at 4 ℃. The concentration of L. monocytogenes CMCC 54005 in this sample decreased by 2.5~3.9 log unit compared to that in non-bacteriocin control. The combination of leucocin B-K7 at 80 AU·m L-1 with glycine at 5 mg·m L-1 completely inhibited the growth of L. monocytogenes CMCC 54005 for 7 days at 4 ℃ in milk.In this paper, a bacteriocin-producing strain was isolated and the bacteriocin, leucocin B-K7 was researched preliminarily. The research indicates that it may be useful for improving the product safety of food.