Studies On Purification And Properties Of Antifungal Compound Produced By Streptomyces Sp.182-2

The fermented broth of Strain 182-2, for its superior capacity of inhibiting pathogens (Alternaria alternata (Fries) Keissler, Alternaria solani, Alternaria alternata f. sp. mali, etc.),controlling disease and enduring bad conditions, is of great practical value. A was made of the purification of antifungal substance produced by Streptomyces sp.182-2, and of its characteristics of inhibiting pathogens. The results are as follows:(1) The pretreatment of fermentation broth was conducted adopting such techniques as oxalic acid acidification and acetone sediment. The mycelium, the remaining medium components and the other metabolites in the broth were removed.(2) The pretreated fermentation broth was purified and decolorized by activated carbon adsorption. The optimum adsorption conditions for the antifungal substance in shake-flask were defined as follows:1.5mm columnar activated carbon used as adsorption agent to active substance; the adsorption temperature,28℃; adsorption time under 60r/min,4h; proportion of active carbon and broth,0.5g/mL. The optimum elution conditions for the antifungal substance in shake-flask were defined as follows:eluting agent, 50% acetone; elution temperature,28℃; elution time under 60r/min,4h. The decolorization rate of the antifungal substance, after the treatment, reached 77%. Meanwhile, the diameter of the inhibition zone was up to 42.0 mm at the concentration of 2000μg/mL.(3) The crude extracts of the fermetation broth was purified by macroporous resin adsorption. The separation conditions of macroporous resin column chromatography were as follows:macroporous resin H103; sample volume,20μL (2mg/mL); adsorption flow rate,0.5mL/min; eluting agent,50% acetone; elution flow rate, 1.0mL/min. After being isolated by the macroporous resin chromatography, two eluting peaks were obtained, both having obvious inhibition effect on target pathogen. The results indicated that the antifungal substance may contain at least two active components. Because the antifungal activity of peakⅠwas weaker and the collected volume was less than those of peakⅡ, we collected peakⅡfor the further study.(4) Studies were carried out of the inhibiting pathogen characteristics of the active componentⅡ. In vitro, it had obvious inhibition effect on Alternaria alternata at the concentration of 2000μg/mL and the diameter of inhibition zone was up to 58.0 mm. The experiment of antibiotic action showed that the minimum inhibitory concentration (MIC) was 80μg/mL and the minimum bactericidal concentration (MBC) was 160μg/mL. The active componentⅡhad the highest inhibition rate to mycelial growth and spore germination 48h after treatment, the EC50 being 31.9 and 42.2μg/mL respectively. When the pathogen spore or hypha was treated by the componentⅡ, tube and hypha grew abnormally.