Screening Of Antibacterial Peptide Producing Lactic Acid Bacteria And Expression Of Antibacterial Peptide PlnF Gene

In this study,Lactic acid bacteria isolated from fermented foods,such as glutinous rice dough,corn noodle,chili sauce,potherb mustard pickles,and stinky tofu in Northeast China.The 7 strains with antimicrobial activity were screened through the Oxford cup diffusion method and identified shown that the seven strains were apart of Lactobacillus plantarum,Lactobacillus pentosus,and Lactobacillus paracasei through 16s rRNA gene analysis.After the supernatant of lactic acid bacteria treated with proteinase K,pepsin,and papain,the antibacterial effect almost disappeared.And the ammonium sulfate precipitation confirmed that strains produced antibacterial substances belonging to the protein family.And the inhibition zone of concentrate of T8 against pathogens significantly increased?>13 mm?.Most strains of antibacterial were highly resistant to heat?65-121°C?,acid?pH 2-6?,and dissolved in alcohol.The antimicrobial effect of the most strains treated with the Tween-80 surfactant,however,was significantly reduced and the antibacterial property of T4 even lost.In bacterial growth inhibition experiments,the colony count of Staphylococcus aureus was up to 10¹⁵ CFU/mL in the 3×de Man,Rogosa,and Sharpe?MRS?group,and this value was more than that in the 3×S6supernatant group(10¹² CFU/mL)and the control group(10¹⁰ CFU/mL)at 12 h.This finding showed that lactic acid bacteria elicited antibacterial effects and possibly promoted bacterial growth in MRS.The study provided a basis for the selection of antimicrobial peptides and the development and utilization of lactic acid bacteria.The lactic and bacteria with antibacterial effect of Staphylococcus aureus isolated from the fermented food as samples and verification by PCR amplification has antimicrobial peptide genes of PlnF,PlnE,PlnN,PlnJ,and PlnK.In order to verify what kinds of antimicrobial peptides have a very antibacterial effect on Staphylococcus aureus Then designed a primer to introduce Nco I and Xho I sites into both ends of the antibacterial peptide genes with the treated of pET28a plasmid to obtain recombinant plasmid.The experiment results show that the inhibition zone of the pET28a-peptide was but the PlnF after 0.5 mM IPTG inducted 6 h and cell after crushing with 8 M urea denaturation and renaturation with Ni purified remarkably increased to 13.43±0.21 mm compare with the control group.The expression of the antimicrobial peptide PlnF from the Lactobacillus plantarum was cloned into the Lactococcus lactis cloning expression vector,and its application can be expected and directly used in food preservation.In Lactococcus lactis pNZ8149/NZ3900expression vector,the PlnF and extracellular signal peptide gene were cloned to pNZ8149vector to construct the recombinant plasmid.Further in the electroporation condition of 200?and 25?F,the linearized plasmid was transformed into NZ3900.After the serious of verified and expression,the PlnF was successfully expressed and the arose diffusion assay showed that the diameter of inhibition zone of Staphylococcus aureus was reached up 14.03±0.23 mm with the incubation time.The recombinant protein displayed a molecular weight of approximately5.8-7.8 kDa,which is consistent with expectations,as determined by Tricine-SDS-PAGE.Further,verified by nanoLC-ESI-MS/MS showed prokaryotic expression,and the application of anti-bacterial peptide has a certaind that PlnF protein expression in Lactococcus lactis pNZ8149/NZ3900 vector correctly of extracellular expression.Moreover,the purity and extraction of antimicrobial peptides can be effectively improved by the expression of promotion effect.