Comparative Analysis Of Antimicrobial Proteins In Co-culture Medium Of Brevibacillus Laterosporus BL-21 And Bacillus Subtilis HNDF2 And Pure Culture Medium

Biopesticide has been widely applied in agricultural production in recent years because it has overcome the disadvantage of traditional chemical pesticide that can pollute the environment,be harmful to human and animal,and cause the pathogenic bacterium resistance to drug.But the development and application of active microbial agents are limited because of the affection by their environmental adaptability and shorter shelf life.The metabolites of microorganism resource has more obvious advantages in preparation of different dosage forms and efficacy stability of controlling plant disease.With the development of co-culture technology,discovering the novel antimicrobial substance which are produced by cooperative metabolism or induction effect occurred between two or more microbes have already attracted more attentionsThe co-culture of Brevibacillus laterosporus BL-21 and Bacillus subtilis HNDF2 was studied in this report.The stability of co-culture liquid,the antifungal spectrum and antibacterial mechanism were analyzed.The antifungal proteins of pure-culture and coculture were isolated,purified and contrastive analyzed by isolation technique and ESIMS,which have laid the foundation for the further study of structural characteristics of antimicrobial substance and practical application.Accoording to the study of the effect of generationsto co-culture activity,the antifungal activity of metabolites produced by Bacillus subtilis HNDF2 was stable from the first generation to the fifth generation;but the antimicrobial activity of the culture substance produced by Bre.laterosporus BL-21 was unstable and the second generation had better activity.It is a remarkable fact that the co-culture liquid with the second generation of the two strains showed the strongest antimicrobial activity.Through the study of co-culture liquid stability,it showed that the antimicrobial activity of co-culture metabolites was stable when it was under the condition of UV and sunlight.It hadbetter thermal stability aftertreated at 40? and 60? for a long time,but the antifungal activity had decreased by 22.6% aftertreated at 80? in 30 minutes.Moreover,the antimicrobial activity of co-culture liquid changed very little when treated with methanol and ethanol,so the metabolites was well tolerated with organic solvent.The result of determination of antifungal spectrum showed that the co-culture liquid could inhibit the growth of ten kinds of plant pathogenic fungus,especially it had strong inhibitory effect against Alternaria alternata and Fusarium graminearum.It could restrain the spore germination of F.graminearum,and the inhibition ratio was 43.5% and had the best inhibitory effect on the expansion of the pathogenic fungus colony with inhibition ratio of 64% when its titre level was 49.2AU/m L,and it showed the best inhibitory effect on mycelium dry weight for the inhibition ratio of 77.8%when the titre was 21.4AU/m L.It had significant effect on mycelium cell permeability,and conductivity value of mycelium extracellular fluid increased obviously after treated with the co-culture liquid at the different titre level.Through the preliminary comparison and analysis of antimicrobial proteins in pureculture and co-culture liquid under the same fermentation condition,the protein content in the pure broth of Bre.laterosporus BL-21 was the highest and that of the pure broth of B.subtilis HNDF2 was less,and that of co-culture liquid was 2495?g/m L..Through the contrastive analysis of SDS-page,the protein bands in B.subtilis HNDF2 was less,and the protein bands of Bre.laterosporus BL-21 were similar to that of co-culture liquid although some remarkble differences also exist.Through the contrastive analysis of antimicrobial proteins in pure-culture and coculture liquid by ammonium sulfate precipitation,two particular bands were observed at170 KDa and 10 KDa in co-culture liquid lane when ammonium sulfate precipitation was40% to 50%.When ammonium sulfate precipitation was 50% to 60%,there was an obvious particular band at 55 KDa in co-culture liquid lane.When ammonium sulfate precipitation was 60% to 70%,co-culture and pure-culture liquid all had bands at 60 KDa and 15 KDa,but the protein abundance of pure-culture was higher than that of co-culture liquid,and pure-culture liquid had an obvious particular band at 37 KDa.The antimicrobial proteins which were under 3KDa in pure-culture and co-culture liquid were isolated and purified by CM-Sepharose Fast Flow ion exchange chromatography,Sephadex G-25 gel filtration chromatography and HPLC,and peptidase M23 was identified by ESI-MS in pure-culture liquid,while polyketide synthase and locillomycin synthase A were isolated and identified in co-culture liquid.It showed that low-molecular-weight antimicrobial substances of pure-culture and co-culture liquid were significantly different.