Purification,Characteristics And Amino Acid Sequence Of Bacteriocin Produced By Lactobacillus Salivarius SJH-8

The use of antibiotics for prevention and treatment of pathogens and other diseases has made an important contribution to human,animal and plant health.However,for its difficulty in degradation and has induced a variety of resistance genes,antibiotics have caused a serious threat to the environment and human health.Bacteriocin,a kind of polypeptide or protein,has the potential to replace the use of antibiotics with the characterisation to be enzymatic hydrolysis.Thus,it is a kind of environmental friendly antibacterial substances.The resource of lactic acid bacteria in China is very rich,which has made the research become a hot spot for stable,efficient,broad-spectrum and non-resistant bacteriocin in recent years.The research is mainly concentrated on the separation and purification,biological characteristics and mechanism of antimicrobial action.While the information for amino acid sequence and gene sequence is less.So it is difficult to quantify it,which brings some difficulties to its application.Therefore,this research was mainly foused on develop a new type of bacteriocin,study its amino acid sequence and gene sequence to provide a strong prerequisite for the future heterologous expression of recombinant engineering strains to produce bacteriocinIn this study,lactic acid bacteria were isolated from the intestinal tract of aquatic animals in the Pearl River estuary.Vibro parahaemolyticus and Staphylococcus aureus were used to screen strains with broad antimicrobial activity.Then the biological characteristics of the bacteriocin produced by the lactic acid bacteria were studied and the bacteriocin was isolated and purified.The amino acid sequence of the bacteriocin was deduced by SDS-PAGE and MALDI-TOF/TOF mass spectrometry.Based on the information,target genes for the bactriocin expression was amplified with specific primers.The acquirement of the gene for expressing the bacteriocin provides the basis for the application for instead of antibiotics and prerequisites for its production.The results were as follows:1.97 strains of lactic acid bacteria were screened from the intestinal tract of healthy aquatic animals of tilapia,white spot fish and white shrimp from the Pearl River estuary.With V.parahaemolyticus ATCC 17802 and S.aureus ATCC 35844 as the indicator bacteria,after antibacterial activity preliminary and re-screening,four strains were obtained which have antibacterial activity to the two indicator bacteria.For the strain SJH-8 had the best antimicrobial activity,so it's confirmed to be the test bacteria for research bacteriocin.Based on the 16 S rDNA sequence,the lactic acid bacteria SJH-8 was identified as a strain of Lactobacillus salivarius.2.After the ammonium sulfate precipitation,the antimicrobial active part for Gram-negative bacteria was still in the supernatant,while the precipitation had a strong antibacterial activity to a variety of Gram-positive pathogens,with no antimicrobial activity to the Gram-negative strains.The active ingredient in the precipitated sample could be completely hydrolyzed by trypsin,and the activity of by papain,pepsin and protease K was weakened,but still existed.It had strong thermal stability and pH stability,in other words,it still retained good activity after treated at 121? for 15 min or at pH2.0 and pH10.0 for 2 h.3.The partial amino acid sequence of bacteriocin SJH-8 was deduced by SDS-PAGE and MALDI-TOF/TOF mass spectrometry.It was confirmed that the bacteriocin was a Class IIb peptide composed of SJH-8? and SJH-8?.According to the gene of abp118,the specific primers were designed,by which the target gene was amplified with SJH-8 DNA as the template.Finally the gene sequence of the bacteriocin was obtained.