Activities,Mode Of Action And Preliminary Structure Identification Of Bacillus Thuringiensis BRC-ZYR2 Bacteriocins

Bacteriocins are proteins,peptides or protein complexes with antibacterial or bactericidal activity which are synthesized and secreted by prokaryote.Bacteriocins have many advantages such as efficient,non-toxic,acid-resisting,thermostable,no residue,no drug resistance and free of affecting food flavor,therefore,they have a promising prospect in the area of food preservation and so on.This study used two food-borne pathogens Bacillus cereus(Bc)0938 and ATCC 10987 as indicator strains to evaluate the activities of Bacillus thuringiensis(Bt)by the modified agar-well diffusion method.The results showed that the pathogens were susceptible to 20 of 74 Bt and Bt BRC-ZYR2 had the best antibacterial activity of 137.34 U.Then 51 food-borne pathogens were used to evaluate the antibacterial spectrum of Bt BRC-ZYR2 and the results showed that the pathogens were insusceptible to Bt BRC-ZYR2.The cell-free fermentation supernatant of Bt BRC-ZYR2 showed no antibacterial effects against Bc 0938 in the first 8 h.When the strain was in the stable growth period of 34 h,the cell-free fermentation supernatant showed antibacterial effects against Bc 0938 and the antibacterial effects lasted to 72 h.The antibacterial effects were unstable and the highest antibacterial effect was 431.57 U in 64 h.The antibacterial effects declined after 64 h.The bacteriocin produced by Bt BRC-ZYR2 was initially-purified with ammonium sulfate precipitation.After 70%to 100%ammonium sulfate precipitation,the bacteriocin showed high antibacterial activities and tricine-SDS-PAGE indicated that the protein/polypeptide amounts of the 70%to 100%ammonium sulfate initially-purified bacteriocin were abundant.The bacteriocin produced by Bt BRC-ZYR2 was designated as bacteriocin ZYR2.In addition,the molecular weight of bactericion ZYR2 were estimated by SDS-PAGE and antibacterial activity was detected on gel.Its molecular weight was a little less than 6.5 kDa which accorded with the molecular weight of class II bacteriocins.The physical and chemical properties of initially-purified bacteriocin ZYR2 were studied.The antibacterial activity of initially-purified bacteriocin ZYR2 were not influenced by ?-amylase,lipase-?,lipase-?,RNase,lysozyme,catalase,trypsin,proteinase K,?-chymotrypsin-? and a-chymotrypsin-?.Since the bacteriocin could remain antibacterial activity after being cheated with proteinase K,suggesting the bacteriocin didn't contain C-terminal peptide bonds of aliphatic amino acid and aromatic amino acid and the bacteriocin might have peptide nature.The initially-purified bacteriocin ZYR2 remained strong antibacterial activity after exposure to 100 ? for 30 min and within pH 3 to 9,demonstrating its excellent thermostable property and pH resistance.The antibacterial patterns of initially-purified bacteriocin ZYR2 acting on cells and spores of Bc 0938 had been studied.After adding bacteriocin ZYR2,the OD600 of Bc 0938 medium declined from 1.76 to 0.52 while the OD600 of the control showed a J-type growth,suggesting that the cells of Bc 0938 ultimately lysed.The number of viable cells declined from 1010 CFU/mL to 106 CFU/mL while the number of viable cells of the control remained 1010 CFU/mL,indicating that bacteriocin ZYR2 had killed the Bc cells.Additionally,the OD600 of the spore culture was about 0.01 while the OD600 of the control showed a J-type growth,thus the initially-purified bacteriocin ZYR2 could inhibit the spore germination of Bc 0938.To sum up,the initially-purified bacteriocin ZYR2 was bacteriocidal and bacteriolytic.The initially-purified bacteriocin ZYR2 was further purified by Sephadex G-75 gel filtration chromatography,cellulose DEAE-52 anion-exchange chromatography and HPLC C18 chromatography.The total protein and activity of the pure antibacterial substance were 25.6 ?g/mL and 140.12 U,respectively.The recovery rate was 23.0%.The molecular weight of pure bacteriocin ZYR2 was 4.29 kDa by MALDI-TOF MS determination.The first-order diagram and second-order diagram of bacteriocin ZYR2 was gotten by LC-MS,and the matching protein could not be found through ProteinPilot software.