Analysis Of The Antibacterial Activity And Physicochemical Properties Of The Metabolites Of Three Actinomycetes

Five actinobacteria strains,HD-006,HD-010,HD-087,HD-103 and HD-109,were isolated by Key Laboratory of Microbiology,College of Life Sciences,Heilongjiang University.Strains HD-087?HD-103 and HD-109 were screened out according to their significant anti-fungus activity.to a variety of plant pathogenic fungus.The aim of this study was to determine their antibiotic production period,anti-fungus spectrum and the physicochemical properties of their fermentation broth.The inhibitory mechanisms of their fermentation broth to Fusarium oxysporum f.sp.Glycines were also preliminary studied,and the antifungal substances in the fermentation liquid of 3 strains were preliminary separated and analysised.The main results of this study are as follows:DBY medium is determined to be the optimal antibiotic fermentation medium of 3strains of Streptomyces.In 250mL flask,the optimum volume of fermentation broth of strain HD-087 and HD-103 are 60mL,and its optimum fermentation time is 96h.While the optimum volume of fermentation broth of HD-109 is 30m L,and its optimum fermentation time is 144h.All of the fermentation supernatant of three Streptomyces strains have broad anti-fungus spectrums for their strong inhibitory effect against F.oxysporum and other plant pathogenic fungus.But all of these fermentation supernatant have no inhibitory effect against Pythium aphanidermarum?Edson?Fitzpatrick and Phytophthora capsici.The fermentation supernatant of strain HD-087 and HD-103 have good heat stability and pH stability,yet the stability of fermentation supernatant of strain HD-109is worse than the other two strains under acidic conditions.The fermentation supernatant of HD-087 can be relatively resistant to UV radiation,but that of HD-103and HD-109 are sensitive because their bioactivities are total lost under UV radiation for 3h.Bioactivity of these fermentation supernatant declined slowly when they were stored at 4?and-20?,this result indicate that these temperature are suitable for a long time preservation of antifugal activity.After organic solvent treatment,antibacterial activities of the fermentation supernatant of 3 strains of Streptomyces are significantly declined.Fermentation supernatant of 3 strains are sensitive to protease K.Bioactive substances in the fermentation supernatant of HD-087,HD-103 and HD-109 are separated by ammonium sulfate precipitating,the results show that antifungal substances mainly concentrate on the 60%?80%saturation of?NH₄?₂SO₄.Then the crude extract is separated by DEAE-Sepharose Fast Flow anion exchange resin,the column was washed with Tris?0.05M,pH8.5?to remove any unbound proteins,and the bound proteins were eluted with a linear gradient of increasing Na Cl?0–2 M?.Crude extract of HD-087 is carried out using exchange resin and two peaks are obtained,and crude extract of HD-103 is carried out using exchange resin and three peaks are obtained,crude extract of HD-109 is carried out using exchange resin and one peak is obtained.Each peak was assayed for antimicrobial activity against F.oxysporum f.sp.Glycines,with all peaks demonstrating activity.The discovery after ultrafiltration,Bio-active protein with large molecular weight.Non-denaturing electrophoresis indicates that molecular weight of 669k Da bands of 3 strains of Streptomyces have obvious antibacterial activity.Tricine-SDS-PAGE electrophoresis indicates that activity bandsof 669k Da are denatured and degraded,proving they are proteins.Treated with the fermentation supernatant of 3 strains of Streptomyces,the growth of F.oxysporum f.sp.Glycines mycelium is suppressed and the morphology of mycelium is abnormal.Dry weight tests of mycelium show that EC₅₀0 of fermentation supernatant of HD-087,HD-103 and HD-109 against F.oxysporum f.sp.Glycines is2287?g/mL,1152?g/mL and 1362.5?g/mL,respectively.All of the fermentation supernatant of 3 strains can restrain the germination of F.oxysporum microconidium to 100%when the fermentation supernatant concentration is 624?L/mL.Fermentation supernatant of three Streptomyces strains against F.oxysporum's mycelium,reaults show that conductivities of extracellular fluid of F.oxysporum mycelium were not significant change.By observing below microscope,mycelium treated by fermentation supernatant with control have no significant difference.which indicated fermentation broth of three Streptomyces can't change the permeability of mycelium cell.