Isolation,Purification And Antibacterial Properties Of Bacteriocin From Bacillus Subtilis S1-2

Bacillus subtilis secretes one or more antibacterial active substances during the metabolic process.An ideal substitute for antibiotic bacteriostatic preparations,so Bacillus subtilis has become the main research object in the food and medical industries.In this study,Bacillus subtilis S1-2 with antibacterial activity,which was isolated and identified from traditional natural fermented soybean paste in Northeast China in the early stage of the laboratory,was studied.The research results are as follows:The whole genome sequencing results of Bacillus subtilis S1-2 showed that the size of the strain was 4076387 bp,and the GC content accounted for 43.82%.The number of predicted genes is 4000,and the gene sequence contains 86 t RNAs,30 r RNA,91 s RNA and 34 tandem repeat regions;the antibacterial genes of Bacillus subtilis S1-2 and six major databases(NR,Swiss-Prot,Pfam,Egg NOG,GO and KEGG)were compared and analyzed,and it was found that Bacillus subtilis S1-2could produce 6 kinds of antibacterial substances,namely subtilosin A,surfactin,fengycin,Mycosubtilosin,baclysin,bacillaene.In the study on the isolation and purification of bacteriocin secreted by Bacillus subtilis S1-2,the crude protein was concentrated by rotary evaporation at 42 °C by ethyl acetate extraction.The combined strategy of Sephadex-G50 and high performance liquid chromatography was used to purify the crude extract,and Staphylococcus aureus was used as the bacteriostatic indicator to verify the bacteriostatic activity of the extract.The purified substance was verified by Tricine-SDS-PAGE gel electrophoresis,the molecular weight was below 4.1 Kd,and it was identified as subtilosin A by LC-MS mass spectrometry.At the same time,the antibacterial spectrum study found that the bacteriocin only has inhibitory effect on Gram-positive pathogens.The research on the stability and antibacterial mechanism of bacteriocin showed that: subtilisin has good stability to temperature,pH,protease and other factors,and the minimum antibacterial diameter reaches 10 cm;the minimum inhibitory concentration of bacteriocin is 0.625 mg/ ml,the growth inhibition curve of Staphylococcus aureus showed that bacteriocin started to inhibit its growth at 4 h,and was completely suppressed at 12 h without regrowth,and the inhibitory effect was stable;bacteriocin would affect the formation of Staphylococcus aureus biofilm,it is more obvious when the concentration of bacteriocin is higher than 1×MIC;at the same time,the scanning electron microscope image can further verify that the bacteriocin produced by Bacillus subtilis S1-2 will destroy the cell membrane of Staphylococcus aureus,forming holes on the surface and damaging the cells.The content of antibacterial substances produced by strains is often affected by experimental factors.Single factor and response surface methodology were used to explore the optimal culture conditions for bacterial strains S1-2 to produce bacteriocin.The test results showed that the fermentation time,the rotation speed of the shaking table and the inoculum amount were the key factors to determine the activity of bacteriocin.The optimal culture conditions of strain S1-2 were obtained through the response surface analysis center combination design test: the fermentation time was23.164 h,the inoculum amount was 4.454%,and the rotation speed was 168.327r/min.The results of three sets of repeated tests under this condition showed that the content of antibacterial protein increased from 0.22 mg/ml to 0.36 mg/ml.The model had good predictive ability and the results were valid and reliable.