Purification And Partial Characterization Of A Bacteriocin Produced By Lactobacillus Pentosus C50-6

Ninety-one strains of lactic acid bacteria (LAB) isolated from traditional Sichuan fermented sausage and cured meat were tested for antagonistic activities by punch method. Cell-free supernatants from seven of these strains were shown to inhibit the growth of four indicators (Escherichia coli ATCC 25922,Staphylococcus aureus ATCC 25923,Micrococcus luteus 10209,Pseudomonas aeruginosa ATCC 27853,Bacillus subtilis), which contained both gram-positive bacteria and gram-negative bacteria, after eliminating the interferences of organic acid and H₂O₂. Strain C50-6 provided the most effective of inhibitory activity and was selected for further study. The inhibitory activity of cell-free supernatant from strain C50-6 decreased sharply after the treatment with trypsin and pepsin. It confirmed that this inhibitory material was a kind of protein and C50-6 was a broad-spectrum bacteriocin-producing strain. Its phenotype characteristics and 16S rDNA sequence analysis showed highly homology to Lactobacillus pentosus.Single factor level test was adopted to optimize the incubation condition of this bacteriocin. As a result, the highest production would be obtained at adding 2% of seeds into MRS broth (pH6.0), cultivating 36h at 30℃. The potency of cell-free supernatant from L. pentosus C50-6 under the optimal incubation condition was 1151IU/ml, resulted in 3.6-fold increase to the primal incubation condition.Contrasting the results of rough fractionation by ammonium sulfate precipitation, organic solvents precipitation, n-propanol extraction and pH adsorption, ammonium sulfate precipitation was finally chosen for rough fractionation of the bacteriocin. The bacteriocin was purified sequentially from culture supernatant, first precipitated with 70% saturation of solid ammonium sulfate, then purified by SP-Toyopeal-650M cation exchange chromatography, using gradient NaCl concentrtion stepwise elution with citrate buffer (pH4.4). The entire purifation protocol led to a 39-fold increase in the specific activity of the bacteriocin, and yield of 21.8%. The purified bacteriocin was identified as a ca. 2500Da peptide by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), and the protein band had strongly antibacterial activity.The culture supernatant and crude extract were characterized. The former showed activity at pH 2-5, and the latter showed activity at pH 2-6. The former maintained the most activity when heated 20min at 100℃, and the latter was stable when heated 20min at 121℃. Both of them were inactivated by papain and trypsin, and part-inactivated by proteinase K, but remained activity after treating by pepsin. The two had almost the same inhibition spectrum. Both of them could effectively inhibit gram-positive bacteria and gram-negative bacteria, could inhibit the growth of Lactobacillus pentosus G21-10 and Lactobacillus plantarum C21-6, and weakly inhibit the growth of Rhodotorula sp. and Penicillium sp.. The potency of crude extract increased remarkably, and it had more effectively inhibition to gram-positive bacteria than the culture supernatant. In a word, the bacteriocin produced by L. pentosus C50-6 is heat stable, acid and neutral stable, proteinaceous, and had a broad spectrum, seen to have the potential application value as a kind of food biological preservative.