Study On The Fermentation Process Of Producing Active Substance Against Citrus Blue And Green Mold By Gymnopus Sp.

Jiangxi is one of the main producing area of citrus fruits. Green and blue mold are the cause of major diseases of post-harvest decay of citrus fruits. Chemical fungicides have generated pathogen resistance and environmental residual. Study on the biological source germicide is getting more and more attention. A macrofungal strain Gymnopus sp. with activity against citrus green and blue molds were found in our laboratory, lacking the basic data of nutrition growth requirements of the wild strains and the metabolism of antimicrobial substances restricted the development and utilization of this wild strain.This paper determined the anti-fungal activity by the method of mycelium growth rate and microscopic observation. The medium composition and culture conditions were optimized by the single factor test, Plackett-Burman, steepest ascent test and Box-Behnken design. Organic solvent extraction, silica gel column chromatography and semi preparative HPLC were used to separate and purificate the antimicrobial active substance. The results showed the Gymnopus sp. fermentation broth has inhibitory effect on 13 kinds of plant pathogens and the EC50 value against Penicillium italicum and Penicillium digitatum are 69.25?g/mL and 56.70?g/mL, respectively. The minimum inhibition concentration?MIC? and the minimum fungicidal concentration?MFC? of this two pathogens were 200 ?g/mL and 250 ?g/mL, respectively, which was lower than the positive control of imazalil of 500?g/mL and it has some toxic effects on the mycelium of Penicillium italicum. Optimization of the liquid fermentation process are potato 280 g/L, glucose 30g/L, bran 8.25g/L, MgSO₄·7H₂O 0.41g/L, KH₂PO₄ 2g/L; VB1 3?g/mL,NAA 0.5?g/mL and GA 2?g/mL. Fermentation period was 11 days, inoculation volume 5mL/100 mL, shaking speed 180r/min, culture temperature 28?, liquid medium volume 100mL/250 mL with the anti-pathogen activity of the fermentation broth increased from 42.26% to 55.77%. 15 L fermentor enlarge culture can shorten the fermentation period from 11 d to 9d. However, the addition of vitamins and plant growth regulator in the fermentation period was shortened to 6d, which was more benefit to the growth and metabolism of the strain. Preliminary proved silica gel chromatography separation elution phase for producing antifungal substance is dichloromethane:butyl alcohol=16:1.High performance liquid chromatography elution procedure is as follows: sample volume 20?L, mobile phase 40%⁵5% acetonitrile, flow rate 1.2mL/min, detection wavelength 207 nm.