Screening Of The Bacteriocin-producing Lactobacillus Plantarum Isolated From Pickle And Studying On Biological Characteristics Of The Bacteriocin

Bacteriocins are proteinaceous antibacterial compounds produced by bacterias. This paper is aimed to screen the bacteriocin-producing Lactobacillus plantarum which prossess the prospect as natural food preservative.There were 119 lactic acid bacteria strains including 53 lactococcus stains and 66 lactobacillus strains isolated from the pickle in fermentation anaphase. Lactobacillus C6 was determined the indicative strain and Lactobacillus C8,C10,C18 and C25 were determined the possible inhibitory strains by antibacterial test between any two strains. Some mutants were obtained by UV mutation treatment on four possible inhibitory strains. A mutant strain, C8-1, was determined the bacteriocin-producing strain after testing the inhibitory effect of protein salting-out product and eliminating orgnic acid and hydrogen peroxide. Inhibitory strain C8-1 and indicative strain C6 belong to Lactobacillus plantarum according to the indification of morphology, physiology and biochemistry and 16S rDNA sequence. The 16S rDNA homology of C8-1 shared 99% with Lactobacillus plantarum WCFS1.Fermentation conditions for Lactobacillus plantarum C8-1 were optimizated and the results showed that the optimal temperature was 30°C, the optimal broth initial pH was 6.5 and the optimal incubation time was 28h; Sucrose was the optimum carbon source and soya peptone was the optimum nitrogen source, each addition is 2% and 1.5%; The optimal additions of beef extrat and Tween80 were 1.5% and 3‰. There was no synthesis of bacteriocin using triammonium citrate as the unique nitrogen source. The titer of this bactericin was 278.24 IU/mL after optimizing fermentation conditions which was 10.7 times higher than the original.Using the method of 70% or 40%~70% saturated ammonium sulphate salting out separated the proteins in the supernatant of Lactobacillus plantarum C8-1. Then the protein concentrates were obtained respectively through treating the products of salting-out with dialysis and concentration. The result of Tricine-SDS-PAGE showed that there was a target protein band with Mr 16,500.The bacteriocin of Lactobacillus plantarum C8-1 had a broad antimicrobial spectrum which not only include gram-positive bacteria for example Staphylococcus aureus and most of LABS used in this experiment but also include gram-negative bacteria such as E.coli, Pseudomonas, Sphingobacterium,Flavobacterium,etc. This bacteriocin had a good temperature stability in ?20°C~121°C. The antimicrobial activity loss was less than 6% when temperature treatment at 4°C for 5d, ?20°C for 5d and 60°C, 80°C, 100°C, 121°C for 15 minutes. With the increase of pH, the antimicrobial activity was gradually decreased and could not be showed at pH≥6. This bacteriocin was partly inactivated when treated with pepsin, papain and trypsin. Based on the above conclusions, the bacteriocin of Lactobacillus plantarum C8-1 had the conditions and prospect as the natural food preservative.