Purification And Characterization Of Bacteriocin Produced By Lactobacillus Pentosus LS1

Bacteriocin is a small molecule biologically active antibacterial peptide,which is synthesized in the ribosome of some bacteria and released outside the cell.Bacteriocin can kill or inhibit prokaryotes,and still has a strong inhibitory effect on some antibiotic-resistant bacterial strains.Bacteriocin still has a rapid and potent effect even at low doses while it does not significantly change the microbiota of the human intestine,which is different from synthetic drugs and chemical preservatives,,bacteriocin.Its new mode of action and ability to inhibit drug-resistant strains have attracted much attention in food preservation.There are many kinds of bacteriocins.Pentocin is characterized by high heat resistance,small molecular weight,and stability under acidic or weak alkaline conditions.It has a broad spectrum of bacteriostasis and is strong against some food pathogens.It has great application value in the food and pharmaceutical industries.The strain LS1 deposited in the laboratory was identified as Lactobacillus pentosus through16S r DNA identification.The medium components and culture conditions for bacteriocin production were optimized,the response surface optimization results were as follows:culture temperature 36.3?,culture time 43 h,initial p H 6.5 and inoculum 2.9%.On this basis,the effect of medium components on bacteriocin production was determined by orthogonal experiment.The optimal combination was 3.0%glucose,2.0%compound nitrogen source and 0.25%Tween 80.Besides,the bacteriocin of Lactobacillus pentosus LS1 fermentation liquor was isolated and purified.Its fermentation liquor was precipitated by 60%saturated ammonium sulfate for supernatant,was desalinated and purified by Sephadex G-10 column chromatography,purified with SP Sepharose Fast Flow,and purified by Superdex 30 Increase finally.After purification,the specific activity was 1238.1 AU/mg,which was 110.5 times that of the original bacteriocin.The bacteriocin has a molecular mass of 1123.8 Da and the N-terminal sequence was ACDFCFCGMK by LC-MS/MS.The antimicrobial spectrum and biological characteristics of Lactobacillus pentosus LS1 were determined.The results showed that Pentocin LS1 had antimicrobial activity against some gram-negative and positive food putrefying bacteria.In addition,Pentocin LS1 still retained 80.2%of antibacterial activity heating at 100°C for 15 minand had strong activity in the range of p H2.0?8.0.It was sensitive to proteolytic enzymes,and insensitive to amylase,lipase ca talase,and some chemistry reagent.The results showed that Pentocin LS1 has potential application value in food preservation.Fluorescent probe labeling was used to study the bacteriostatic mechanism of Pentocin LS1against Listeria monocytogenes.It was found that Pentocin LS1 disrupted cell transmembrane potential and p H gradient,leading to the leakage of intracellular K~+,ATP and phosphate salt ions of Listeria monocytogenes caused the death of the bacteria eventually.In this paper,we studied the identification of the strain,optimization of culture medium and culture conditions,isolation and purification of bacteriocin,physical and chemical properties,and the inhibitory mechanism against Listeria monocytogenes.It is of great significance for the development of high-efficiency,non-toxic,stable and pollution-free biological preservatives.