| The secondary metabolites of Bacillus subtilis are mostly small-molecule cyclic antibacterial lipopeptides.These cyclic antibacterial lipopeptides have the advantages of wide spectrum of inhibition,low resistance,non-toxicity,and degradability.Antimicrobial lipopeptides have long been tried in agriculture,animal husbandry,and medicine.They are considered green and safe to replace antibiotics.However,the research of antibacterial lipopeptides has the disadvantages of low yield and high cost,especially,the use of organic reagent.So antimicrobial lipopeptides cannot be produced industrially.In this study,we compared the antimicrobial lipopeptide production produced Bacillus subtilis HS-A38 and its mutagenized strains mutHS-245 and mutHS-301 that have been identified previously in the laboratory.Production of antibacterial lipopeptides produced by the three strains was performed.And the characteristics of antibacterial lipopeptides were analyzed.The main results are as follows:(1)The fermentation supernatants of Bacillus subtilis HS-A38 and mutagenized strains had antibacterial activities against Vibrio parahaemolyticus,Staphylococcus aureus,Pseudomonas aeruginosa,and Vibrio brilliant.Especially the supernatants,had more antibacterial activity against V.brilliant.The antibacterial lipopeptide products was purified by acid precipitation and methanol extraction from fermentation supernatants of the three strains.The results showed that the antibacterial lipopeptide production of mutHS-245 and mutHS-301 increased 26.0% and 31.3%,respectively,compared with the original strain.(2)After Tricine-SDS-PAGE,it was found that the molecular mass of the antibacterial lipopeptide was less than 3.3 kDa.And there were no other impurities.Through thin layer chromatography analysis(TLC),a total of three fluorescent spots were isolated.The ratio shifts(Rf)are 0.26,0.37,and 0.79,respectively.At the same time,by using Reversed-Phase High-Performance Liquid Chromatography(RP-HPLC),it was found that three purified groups were obtained,that is the peak A,peak B and peak C as the retention time was 23~26 min,27~31 min,and 33~39 min.According to the peak area normalization method,the peak A accounted for 48.69%(original strain),50.38%(mutHS-245),and 50.99%(mutHS-301)of the total area of each strain.(3)The antibacterial lipopeptide preparation was obtained by spray drying.It can be used as a feed additive in aquaculture feeds for aquatic animals and poultry.Mutagenic strain mutHS-245 was used as the starting strain to compare the effects of the production of antimicrobial lipopeptides by using the novel fermentation medium and the original fermentation medium.The results showed that the yield of antibacterial lipopeptides in the novel fermentation medium was increased by 17.9% compared to the original fermentation medium,reaching 54.72 g/L.Further study found when the optimized fermentation medium was used as the fermentation medium,the yield of the antibacterial lipopeptide preparation was 60.45 g/L.The antibacterial lipopeptide production of mutHS-245 and mutHS-301 increased 11.0% and 19.5%,respectively,compared with the original strain.After optimization,when 5% corn starch was used as a protective agent,the yield of antibacterial lipopeptide reached 44.90 g/L and the water content was only 0.11%.When the antibacterial lipopeptide product concentration was 20 mg/mL,there was a significant inhibitory effect on S.aureus and V.parahaemolyticus.(4)When the temperature is 25~80 °C,the antibacterial activity of antibacterial lipopeptide product is almost unchanged.The products also can tolerant strong acid and alkali environment.It also found that the antibacterial activity against S.aureus and V.brilliant had no obvious decrease after 1h of trypsin and pepsin treatment by the antibacterial lipopeptides.This means that the animal gastrointestinal proteases have no effect on the activity of the antimicrobial lipopeptides,and the products can be developed as animal feed additives in the future. |
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