Isolation Of Brevibacillus Brevis YNP-1 And Preliminary Identification Of Its Antibacterial Substance

Brevibacillus brevis is a kind of spore-producing and strictly aerobic gram-positive bacteria,which is widely distributed in plant rhizosphere soil,seawater,wetlands,silt and other ecological environment.And its antimicrobial substance produced by antagonistic strain has many utilities in the pharmaceuticals.For example,it can be used as a food preservative,feed additive,and agricultural biocontrol agent.Kiwifruit canker disease is a devastating bacterial disease that causes a decli-ne in the yield and quality of kiwifruit.The main purpose of this study was to isolate Bacillus sp.strains with strong antibacterial activity against Pseudomon as.syringae pv.actinidiae S01,a pathogen of kiwifruit canker,from the natural environment.We established a laboratory-scale fermentation process for antibac-terialsubstances produced by strains and explored some physicochemical propert-ies of crude extracts of antimicrobial substances,separated and purified antimicrobial substances,and identified the type and primary structure of the antimicrobial substances.The main results were as following:?1?Three strains were isolated from 12 samples of soil,exhibited antibacterial activities against Pseudomonas.syringae pv.actinidiae S01.YNP-1was identified as Brevibacillus brevis according to its physiological and biochemical characteristics which measured by 16 SrDNA sequencing analysis and it has the strongest antibacterial activities of three under natural conditions.?2?The fermentation process and medium formula of B.brevis YNP-1 was optimized by the single factor experiment,Plackett-Burman design,steepest ascent test and response surface design.We established the best laboratory-scale fermentation condition:dextrin concentration 15 g/L,peptone concentration 23.0g/L,CaCl₂ concentration 1.5 g/L,Tween-20 addition 0.99%,68.0 h,pH 7.5,30.0°C,190.0 rpm,strain inoculation amount 8%,medium liquid volume 76.0mL.The inhibition zone diameter of the antibacterial substance?bacteriostatic activity?produced by B.brevis was reached 25.8 mm.under the best condition,which was 33%higher than the activity of the antibacterial substance before optimization.?3?To investigate the physicochemical properties of the antibacterial substance,the antibacterial substance produced by the fermentation of B.brevis YNP-1 was extracted with ethanol at low temperature,which was freeze-dried for 24 h and 0.1 g of the dried sample was dissolved in 1 mL of phosphate buffer?PBS,30 mmol/L?.The result reveal that the antibacterial substance of B.brevis YNP-1 was insensitive to temperature,proteasek and papain,and it can keep activity though boiled in water at 100°C for more than 10 minutes.Not only changeable pH from 3 to11,but storage at low-temperature for long-term,the activity of the antibacterial substance is almost unchanged.Antibacterial substance also can endure ultraviolet radiation in a certain period of time.Its maximum absorption wave length is around 280 nm,and has broad spectrum antibacterial properties,exhibiting strong antagonism against Gram positive bacteria and Gram-negative bacteria.?4?The antibacterial substance was isolated from antibacterial crude extracts by dialyzing for 48 hours using a dialysis bag with a molecular weight of 8¹4 kDa,Sephadex G-150 gel filtration,DEAE-52 anion chromatography,and reversed-phase high-performance liquid chromatography?RP-HPLC?.The results showed that the main antibacterial components produced by the Brevibacillus brevis YNP-1 was mostly metal peptidase M4 or N-acetyl transfer-ase of contained a GNAT family,or a mixture of both.