Isolation And Identification Of Antibacterial Metabolites Co-cultured With Brevibacillus Laterosporus BL-21 And Bacillus Subtilis HNDF2

Members of the Bacillus genus are often considered as microbial factories for the production of a vast array of bioactive molecules which are able to inhibit the growth of phytopathogen.But the reports about co-cultures with two or more Bacillus sp.producing bioactive molecules and their applications of biological control are few.The co-culture of Brevibacillus laterosporus BL-21 and Bacillus subtilis HNDF2 was studied in this report.The final period of co-culture was determined by detecting the antimicrobial activity of the metabolites at different incubation time.The metabolites of pure culture and co-culture were comparatively analyzed by detecting the content of protein,ultraviolet absorption spectrum and thin layer chromatography(TLC).The antifungal compounds against Fusarium oxysporium were isolated from co-culture metabolites by CM-Sepharose Fast Flow ion exchange chromatography,Sephadex G-75,Sephadex G-25 gel filtration chromatography and high performance liquid chromatography(HPLC).The molecular weight was determined by MALDI-TOF-MS and the bioinformatics of the antifungal substances were analyzed.The stability of Sephadex G-25 antifungal compounds and the effect of F.oxysporium were studied.The results of antifungal activity against F.oxysporium of pure culture and co-culture metabolites showed that inhibition zone diameter of co-culture metabolites at 48 h was 28.50±0.5 mm,while that of pure culture of BL-21 was 20.33±2.89 mm and that of pure culture of HNDF2 was 9.33 ± 2.31 mm.Significant analysis indicated that the optimized co-culture time with the two strains should be for 48 h.The comparative analysis showed that the properties of metabolites of co-culture was similar to that of BL-21 pure culture,the protein content in co-culture liquid was not equal to the sum of those in BL-21 and HNDF2 pure culture.The results of examination on separation condition showed that CM-Sepharose Fast Flow was the most suitable packing substance used to separate the antagonistic molecules in co-culture liquid by ion-exchange chromatography,while the detection wavelength was 220 nm,the elution rate was 1.0 m L/min,the sample volume was 35 m L,eluent concentration was 1.0 mol/L,eluent volume was 200 mL.There were two peaks appeared after the co-culture metabolite was eluted by CMSepharose Fast Flow ion-exchange chromatography,but the antifungal compounds were only detected at the front part of the second peak.The bio-active part was collected and concentrated and then separated with Sephadex G-75 gel filtration chromatography.Only one peak was detected and the antifungal compound was still appeared at the front half part of the peak.After the antifungal compound was separated with Sephadex G-25 gel by filtration chromatography,we found that the antifungal compound was still at the first half part of the only peak.Finally,Twice HPLC method was used for further purification.During the first HPLC,the constituents were collected according to the antagonistic activity on F.oxysporium.After the second HPLC,two peaks were detected and collected,and then analysised by the MALDI-TOF-MS method.Result showed the molecular weight of two peaks were 1 304.604 and 1 292.730,respectively.The sequencing results of MALDI-TOF-MS showed that the two peaks were novel substances with low protein score among the known protein in database.The stability of Sephadex G-25 antifungal compounds and the effect of F.oxysporium showed that Sephadex G-25 antifungal compounds were thermolability,antifungal bioactivity decreased by 31.13 % at 60 ℃ and disappeared at 80 ℃.The pH stability of Sephadex G-25 antifungal compounds was good.The antifungal bioactivity decreased significantly with protease.The mycelium pigment of F.oxysporium were leaked and spore germination rate significantly reduced after treatment with Sephadex G-25 antifungal compounds.