鹅膏菌产抑菌物质培养条件优化及安全性分析

Optimization of Cultivation Conditions and Security Analysis of Antifungal Substance from Amanita sp.

经前期试验验证鹅膏菌菌株AV对杨树烂皮病病原菌金黄壳囊孢菌有抑制作用,通过比色法和生物量测定结果得出其抑菌物质产生的最佳营养条件。在供试营养物质中,乳糖为最佳碳源,蛋白胨为最佳氮源,方差分析结果表明,碳源是鹅膏菌AV生物量及其抑菌物质产生的最主要影响因素,与其他营养元素差异显著(α=0.05)。培养基最佳组合为:①乳糖30.0 g/L,蛋白胨0.5 g/L,MgSO41.5 g/L,KH2PO44.5 g/L;②乳糖10.00 g/L,蛋白胨0.50g/L,MgSO40.75 g/L,KH2PO44.50 g/L;③乳糖30.0 g/L,蛋白胨2.0 g/L,MgSO41.50 g/L,KH2PO43.0 g/L。液体发酵培养时,菌株AV产生抑菌活性物质的最适培养条件为:pH值5～6,温度25℃,接种量1.25%,装液量50%,摇床转数180 r/min,培养时间20 d。当温度超过75℃,pH大于9或小于4时,或者紫外照射时间超过4 h都会使抑菌物质活性降低。小鼠毒性试验结果表明,小鼠饲喂菌株AV菌丝体,LD50为418.70 mg/kg,95%可信限为453.11～386.90 mg/kg,抑菌物质控制在2 g/L以内对小鼠无影响,因此,在进行病原菌防治时,为减少对环境中其他生物的影响,菌株AV抑菌物质质量浓度应低于2 g/L。

Previous experiment proved the inhibiting effects of strain AV of Amanita on Cytospora chrysosperma.The optimal substrates for antifungal substance were obtained by the colorimetric method and biomass determination.In the tested nutrients,lactose was the optimal carbon source,and peptone was the optimal nitrogen source.Carbon source was the main influence factor on the biomass of strain AV of Amanita and the activity of antifungal substance from strain AV of Amanita sp.,and there was significant differences（α=0.05） between the carbon source and other nutrients.The optimal nutrient combinations were obtained as lactose 30.0 g/L,peptone 0.5 g/L,MgSO4 1.5 g/L and KH2PO4 4.5 g/L;lactose 10.00 g/L,peptone 0.50 g/L,MgSO4 0.75 g/L and KH2PO4 4.50 g/L;lactose 30.0 g/L,peptone 2.0 g/L,MgSO4 1.5 g/L and KH2PO4 3.0 g/L.The optimal fermentation conditions for antifungal activity were obtained as pH 5-6,fermentation temperature 25 degrees C,inoculum concentration 1.25%,liquid medium volume 50%,rotation speed 180 r/min,and fermentation time 20 d.The antifungal activity would be reduced at 75 degrees C when pH was more than 9 or less than 4.The antifungal activity also decreased when the antifungal substance was irradiated with ultraviolet radiation for more than 4 h.The toxicity test on mice indicated that the median lethal dose（LD50） of the mycelium of the strain AV of Amanita was 418.70 mg/kg（95% credible limit 453.11 to 386.90）.No influence on mice was observed when the concentration of antifungal substance was below 2 g/L.Therefore,the concentration of antifungal substance from strain AV of Amanita should be less than 2 g/L in order to ensure ecological security during the control of pathogens.