Fermentation, Isolation, Purification And Properties Of Antifungal Active Substances Produced By Paenibacillus Polymyxa Cp-S316

Paenibacillus polymyxa Cp-S316, an antimicrobial bacterium, was isolated from soil of Mountaintai and further identified. Its sequences of 16S rDNA was registed at Genbank with accession number AY292989. In the research of the antimicrobial bacterium, it showed broad inhibitory spectrum and great inhibition against animal and plant pathogenic bacteria and fungi. The shake-flask fermentation properties of Cp-S316 were also excellent, and antibacterial and antifungal active substances were produced in the same medium, but the production of both active substances fluctuated greatly with different medium. The antibacterial active substances were isolated, purified, and their structure was identified. The fermentation conditions and medium for antifungal active substances were optimized, and the route of isolation and purification for antiftmgal active substances was established. Meanwhile, some physical-chemical and biological properties of active substances were characterized with the sample of crude antifungal active substances. The main results were as follows.1 Through the cultivation in shake-flask fermentation, the effects of environmental conditions and nutrition on production of antifungal active substances produced by Cp-S316 were studied.The optimum carbon, nitrogen, C/N, inorganic salt, and fermentation conditions were confirmed by one-factor-at-a-time. In the optimization of medium, the influences of lactose, peptone, NaNO₃ and MgSO₄ on active substances production were first evaluated using a fractional factorial design. The components tested had significant effect on active substances production, peptone and MgSO₄ influenced active substances production positively, while lactose and NaNO₃ affected active substance production negatively. The path of steepest ascent was used to approach the optimal region of the medium composition. In the last step, the optimal concentrations of components were determined by central composite design and response surface analysis.The components of optimum medium for production of antifungal active substances were: lactose 12.29g, peptone 17.53g, NaNO₃ 0.39g, MgSO₄ 4.52g, potato 100g, H₂O 1L.The optimized fermentation conditions for production of antifungal active substances were: pre-inoculation time 20h; inoculum's volume 2% (v/v); solution amount 50mL/250mL; fermentation temperature 30℃; initial pH 6.3-6.5; incubation time 48h.The titer of optimized medium and fermentation conditions got to 4687.709μg/mL, 25.86% higher than that of one-factor-at-a-time experiment, and 305.04% higher than that of basal medium.2 Isolation and purification of antifungal active substances were operated. The absorption and desorption capacities of three kinds of macroporous adsorptive resins for active substances were researched. The macroporous adsorptive resin that had good absorption and desorption capacities was further investigated on desorption solution, and the optimum desorption solution was chosen. The results showed that adsorption and desorption capacities of macroporous adsorptive resin X-5 for active substances were the best. The saturated absorption quantity of X-5 for active substances was 4.815×10⁴μg/g, with the optimum desorption solution chosen—0.01 mol/L HC1-70% ethanol desorbing in static state[desorption solution:resin=2:1(v/m)], and the desorption rate could get to 94.28%.The desorption liquid was concentrated by vaporization and vacuum freezy-desiccation. Antifungal active substances were further isolated gradually by acetone fractionation, DEAE-Sepharose Fast Flow ion exchange column chromatography, CM-Sepharose Fast Flow ion exchange column chromatography with 0～0.5mol/LNaC1 as grads desorption solution. The active substances contained less impurity were got by Sephadex G50 column chromatography.Finally antifungal active substances were refined by reversed-phase high performance liquid chromatography (RP-HPLC). The method of RP-HPLC was set up for preparation of the antifungal active substances. The conditions of RP-HPLC were suggested as SinoChrom ODS-BP C₁₈ column(300×10.0mm, 10μm), detection wavelength 210nm, the mobile phase gradient elution of acetonitrile-buffer(23:77,v/v) at 30℃, with the buffer containing 0.1% TFA, flow rate 1.0mL/min, the volume of injection 200μL. One antifungal active substance component was separated under the condition.3 Physical-chemical and biological properties of antifungal active substances were explored. The antifungal active substances treated 100min at 100℃or 20min at 121℃, indicated some good properties for application, they were stable to heat, keeping integrated activity. And they were also stable to ultraviolet radiation, not sensitive to trypsin and proteinase K, and partial sensitive to chloroform. It also showed higher activity in the solution with pH 4-7, and didn't indicate activity in alkali solution. Great prospect of application and exploitation was expected for their simple extraction and stable physical-chemical characteristics. Inhibition or sterilization mechanism was researched by the investigation of Alternaria alternate mycelium treated by antifungal active substances. Observed by microscope, the growth of mycelium was strongly inhibited, and the mycelium became bitty and easy to ruptured, some mycelium indicated transparent and even the protoplast treated appeared agglomerate or overflowed. Observed by feeding experiment of antifungal active substances, they had no bad influence on the growth of silkworm, the antifungal active substances were safe to silkworm, so they had good biology safety.