高效杀蚊苏云金芽孢杆菌BRC-LLP29的发酵优化

苏云金芽孢杆菌BRC-LLP29为新型高效杀蚊菌株,应用快速有效的数学统计方法对其杀蚊毒力的发酵培养进行优化.通过单因素筛选确定最佳碳源为葡萄糖、麦芽糖、可溶性淀粉,氮源为typetone、大豆蛋白胨、干酪素;最佳金属离子为Mg²⁺、Al³⁺.采用二水平Placlkett-Burman设计对影响毒力的8因素进行显著性筛选,获得培养基成分中3个重要影响因子:葡萄糖、干酪素和Al₂（SO₄）₃;运用爬坡路径法对这3种因子进行试验,获得3种重要因子的最适浓度范围;通过响应面分析法得到3个重要因子的交互作用和最佳条件,确定BRC-LLP29菌株最佳毒力水平的发酵培养基为:葡萄糖19.8g/L、干酪素28.4g/L、Al₂（SO₄）₃1.2g/L、MgSO₄ 2g/L、K₂HPO₄ 3g/L、CaCO₃ 0.5g/L,优化后毒力水平达到致死率61.11%,与响应面数学模型的预测值只有5.91%的误差.发酵条件优化结果表明:发酵温度为31°,发酵初始pH为7.0,摇瓶装量为40mL/250mL三角瓶,每瓶的接种量为3.5%,发酵72h,对致倦库蚊最终致死率达到最高为83.33%.

Fermentation Optimization of Novel Mosquitocidal Bacillus Thuringiensis Strain BRC-LLP29

BRC-LLP29 is a novel and mosquitocidal Bacillus thuringiensis strain.Statistical methods were used to optimize the fermentation conditions.According to single factorial experiments,glucose,maltose and soluble starch were the most suitable carbon sources,while typetone,casein and soy peptone were the best nitrogen sources.Meanwhile,Mg²⁺ and Al³⁺ were both the best metal ions.It also showed in Plackett-Burman design that glucose,soy peptone and Al₂（SO₄）₃were the key three factors on toxicity in culture medium.The path of steepest ascent experiment was employed to approach the optimal region of the medium concentration.The optimal combined concentration and mutual effect of the three factors were optimized by response surface methodology（RSM）.It showed that the best media composition were glucose 19.8 g/L, casein 28.4g/L,Al₂（SO₄）₃1.2g/L,MgSO₄2g/L,K₂HPO₄3g/L and CaCO₃ 0.5g/L,respectively. Based on this condition,the corrected mortality to Culex quinquefasciatus was 61.11%,which was only 5.91%error from the forecast by RSM.In additon,analysis for culture conditions indicated that 31℃was the most suitable fermentation temperature.When BRC-LLP29 was cultured for 72h with 250mL shake flasks,7.0 of the initial pH and 3.5%of the density,the mortality of C. quinquefasciatus could reach to 83.33%