Study On Overproduction Of Subtilosin A By Bacillus Subtilis LF-11

The increasing drug resistance of bacteria has made the search for new antibiotic alternatives an urgent problem in the livestock and biomedical fields.Antimicrobial peptides can directly damage bacterial cell membranes or cell walls,and are not easy to develop tolerance,so they have attracted much attention,but there is currently a problem of low yield.Subtilosin A produced by Bacillus subtilis possesses a strong inhibitory effect on Listeria monocytogenes,Porphyromonas gingivalis,Shigella sonnei and other pathogenic bacteria,and has antiviral and anti-biofilm forming activities,which shows great application potential in food,medicine,animal husbandry and other aspects,but its maximum yield was only 42 mg/L,thus its application was greatly limited.Taking Bacillus subtilis LF-11 as the research object,this paper first identified the bacteriostatic substance produced by the strain and optimized the fermentation conditions to improve the yield,further investigated the preparation process of the bacteriostatic substance,and finally explored its physiological effect on the producing bacteria and anti-inflammatory effect on intestinal epithelial cells,which laid the foundation for the industrial production and application of this strain and its bacteriostatic substance.The landmark results achieved are as follows:1.Antimicrobial substance produced by B.subtilis LF-11 was identified.By proteinase K sensitivity experiment combined with TRINCINE-SDS-PAGE analysis,the bacteriostatic substance was determined to be a polypeptide.The pure product was then prepared by acid precipitation combined with reversed-phase high performance liquid chromatography,and by mass spectrometry combined with genome mining,finally it was identified as subtilosin A and named subtilosin A-11.2.The fermentation production conditions of subtilosin A-11 were optimized.First,the carbon source was optimized and the fermentation kinetic curves of subtilosin A-11 produced by B.subtilis LF-11 showed that glycerol significantly increased the yield of subtilosin A-11 by raising biomass and prolonging the exponential growth period.Then,the amount of yeast powder and peptone was optimized,and the optimal medium composition was determined:20 g/L glycerol,10 g/L yeast powder,5 g/L peptone and 5 g/L sodium chloride.The influence of initial pH and shaker speed was subsequently determined,and it was showed that neutral pH and high dissolved oxygen conditions were favorable for the production of subtilosin A-11.Finally,the amplification experiment was carried out in a 5L fermenter,and the yield of subtilosin A-11 reached 515.90±6.79 mg/L when incubated at 37℃ for 36h when the aeration was set to 1 vvm,which was the highest level reported.3.A method for efficient preparation of subtilosin A-11 from fermentation broth was established.By measuring the precipitation yield at various pH and the elution conditions of subtilosin A-11 in reversed-phase column chromatography,the separation strategy of acid precipitation combined with reverse-phase column chromatography gradient elution was determined.that is,the pH of the fermentation broth was adjusted to 3.0 to collect the precipitate,and the recovery of subtilosin A-11 could reach 94.11%and the purity was 50.00%.Then reverse-phase column chromatography was performed to elute with 30%acetonitrile for the best effect;After collecting the eluate and lyophilizing,the pure subtilosin A-11 product was obtained,and the yield could reach 58.39%and the purity was 95.55%.4.The cell activity of subtilosin A-11 was explored.In the view of limitations of genetic manipulation of B.subtilis LF-11,B.Subtilis 168 was chosen as the subject and found that the knockout of the sbo-alb gene cluster significantly reduced spore production and addition of subtilosin A-11 could significantly improve spore formation,revealing the promoting effect of sbo-alb gene cluster and its product subtilosin A-11 on spore formation,which proved that the production of bacteriocin was closely related to spore formation.At the same time,it was found that low concentrations of subtilosin A-11 could significantly downregulate the transcription level of inflammatory factors caused by Salmonella,thus it was speculated that subtilosin A-11 had a protective effect on intestinal cells.