Screening Of Lactic Acid Bacteria Producing Antibacterial Peptides And Heterologous Expression Of Antibacterial Peptides

Antimicrobial peptides gradually become a promising drug for the future because of its wide antimicrobial spectrum, obvious antimicrobial activity and drug resistance.Lactic acid bacteria secrete antimicrobial peptides, also known as acteriocins, which are non-toxic, no residue, good stability and other advantages belonging to the natural food preservatives, have become the hotspots of current research. However, due to relatively low yield and high cost of antimicrobial peptide which secreted in lactic acid bacteria, it is not widely used in the food industry. This study isolated lactic acid bacteria from traditional fermented dairy products(mare’s milk yogurt powder and cheese), aiming to screen fine strains which producing antimicrobial peptides and then using genetic engineering methods, heterologous expression in Escherichia coli,provide approaches for mass production of antimicrobial peptides.(1) 79 strains were isolated from the traditional fermented milk(cheese, mare’s milk yogurt powder). 8 strains of those strains showed antimicrobial activity to Listeria monocytogenes. Through physiological and biochemical identification and16 S r RNA strain identification, 6 strains were identified as Lactobacillus plantarum and the other two strains were identified as Enterococcus faecium.(2) Using the double Oxford cup method to determine the titer of 8 strains above, the best strains 1-4 and 2-2 were identified, and they were used as experimental materials optimization conditions of antimicrobial peptides producing by lactic acid bacteria. The results showed that the optimum conditions for antimicrobial peptides synthesis were that culture time of 12 h-16 h, incubation temperature of 37 ℃, and the original p H value of 6-6.5, bacteria age of 14 h,inoculation amount of 108 CFU/m L. Under such conditions, the maximum bacterial concentration and the highest yield of antimicrobial peptides were achieved.(3) Primers were designed according to the nucleotide sequence of plantaricin E and plantaricin F, then amplified fragment was connected with plasmid p Cold-SUMO,and the two recombinant plasmids were constructed and induced to expresse. The optimized induction expression condition was determined: optimal expressing host cell was E. coli Arctic Express, the time of induction was 8 h, the temperature was15℃, and the concentration of IPTG was 0.2 m M. The two target protein expression verified in the supernatant by ultrasonic fragmentation, and the fusion proteins were produced and purified by natural conditions. When combined with the Ni-NTA resin,the target proteins were completely separated from the impurity protein, and theconcentration was large. The recombinant plasmid p Cold-SUMO-pln E fusion protein was successfully cut his-SUMO-tag, however the recombinant plasmid p Cold-SUMO-pln F fusion protein failed. Experimental study on inhibitory activity showed that two recombinant plasmids fusion protein lysate supernatant did not obviously inhibit Listeria monocytogenes, the fusion protein after purified have inhibition obviously. Before and after restriction enzyme digestion his-SUMO-tag,antimicrobial activity of fusion protein had no obvious change.In summary, the research results showed that plantaricin E and plantaricin F genes was successfully expressed in Escherichia coli and the purification of fusion protein both have inhibitory effect on Listeria monocytogenes. The yield antibacterial peptide was obviously improved by means of heterologous expression in E. coli. It provided a theoretical basis for the application of antimicrobial peptides in various fields.