Optimizing A PCR Screening Method For Class Ⅱa Antimicrobial Peptides

Consumers are increasingly concerned about the relation of between foodadditives and healthy. The natural and traditional foods which are withoutpreservatives and chemicals, are favored. Biopreservatives are trend in the future.Antimicrobial peptide is promising and attractive agents that is used asbiopreservatives in the food industry because of its nature and safety. Traditionalscreening strategies are time-consuming and labor-intensive, so researchers need toexplore and develop more rapid and higher-throughput approaches for identifyingpotential bacteriocins.In our previous study, we built an initial PCR screening method for class Ⅱabacteriocins. The lactic acid bacteria isolated from traditional fermented food werescreened and obtained a strain and proved that have antibacterial effect. Show that thismethod is feasible. However, this method is still the obvious, lack of high-throughscreening qualities.This study optimized an initial PCR screening method for class Ⅱa bacteriocinsin order to improve the efficiency of screening in further. In this study, the speciesprimers screening method and degenerate primers screening method were compared.The aim of this study is to build the foundation for high-throughput screening model.In this study, the method of extracting metagenomic DNA from the fermentedmilk was established and optimized. Twelve fermented mare milk samples wereextracted metagenomic DNA and used for screening of class Ⅱa bacteriocins based onthe PCR method. This method eliminated the need for pre-isolation of bacteria, andimproved the efficiency of screening bacteriocins. Three positive fermented maremilk samples were determined. Then118lactic acid bacteria were isolated andpurified from positive samples. Three strains were identified by means of PCRmethod. The lactic acid bacteria of positive PCR were detected antimicrobial activityby means of traditional well well-diffusion method. These strains showedantimicrobial activity to Listeria sp. The genomic DNA of strain C71were used as atemplate for amplifying antimicrobial peptide genes. These genes were linkedrespectively to the expression vector pCold-SUMO. The four recombinant plasmidswere successfully constructed. The expression conditions were optimized. Theoptimal induction time was8h, and the concentration of IPTG was0.2mmol/L.Antimicrobial peptide genes were expressed successfully in psychrophilic Escherichia coli. The result indicated that leader peptide of antimicrobial peptide had nosignificant effect on its antibacterial activity. This study evaluated the effect of celldisruption through the boiling method, lysate dissolution method, and sonicationmethod. Sonication breaks thoroughly the cell and maximize protect the integrity andactivity of antimicrobial peptides. The results showed that supernatant hadantimicrobial activity to Listeria sp. Compared with the original lactic acid bacteria,antimicrobial peptides expressed significantly increased in prokaryotic. It has acertain potential applications in food industry.