利用响应面分析法优化贝莱斯芽孢杆菌TCS001的发酵条件

Fermentation condition optimization of Bacillus velezensis TCS001 using response surface methodology

贝莱斯芽孢杆菌Bacillus velezensis TCS001是一种新型生防细菌,为了提高其生防作用,本文开展了培养基发酵条件优化研究。以TCS001发酵滤液对黄瓜灰霉病菌Botrytis cinerea的抑制率为因变量,先从NB、BPY、YT、BP、LB和SOB 6种基础发酵培养基中筛选最佳基础培养基,再利用Plackett-Burman (PB)试验筛选出影响发酵滤液抑菌率的关键因素;通过最陡爬坡路径法确定主要影响因素的响应中心点,利用Box-Behnken设计和响应面分析法获得最优发酵条件;通过活体植株喷雾法测定发酵液对黄瓜灰霉病菌的抑制活性,进一步明确其优化效果。最终获得最佳基础培养基为NB培养基,发酵条件为:蛋白胨10.0 g,葡萄糖12.5 g,牛肉浸膏4.0 g,酵母粉1.0 g,蒸馏水1 000 mL,装液量(体积分数) 40%,种子培养时间16 h,接种量(体积分数) 3%,转速164 r/min,温度25℃,pH 7.0,发酵培养时间36 h。离体抑菌活性试验验证发现,优化培养的发酵滤液抑菌活性最强,抑制率达90.9%;活体盆栽抑菌试验表明,优化培养的发酵液对黄瓜灰霉病的病斑抑制率为74.7%,比优化前提高了36.9%。该结果为贝莱斯芽孢杆菌有效的活性产物分离提取及结构鉴定等后续研究提供了理论基础。

Bacillus velezensis(TCS001)is a new biocontrol bacterium.The fermentation condition optimization of TCS001 was conducted to improve its biocontrol effect.To obtain the best inhibitory rate of TCS001 fermentation filtrate against pathogenic fungus Botrytis cinerea,six media,including NB,BPY,YT,BP,LB,and SOB medium,were screened.The key factors of fermentation filtrate inhibition rate were investigated using Plackett-Burman(PB)design.Moreover,the central axises of these factors were determined using steepest-ascent method.Optimal fermentation conditions were determined by Box-Behnken design(BBD)and Response Surface Methodology(RSM).Besides,the inhibitory activity of the fermentation broth against B.cinerea was evaluated via artificial spray.The NB medium was found as the best initial fermentation medium for TCS001.And its optimal fermentation parameters were as follows:10.0 g of peptone,12.5 g of glucose,4.0 g of beef extract,1.0 g of yeast extract,1 000 mL of distilled water,loading volume of liquid 40%(V/V),inoculum amount3%(V/V),164 r/min of shaking speed at 25°C,initial pH 7.0,16 h of strain age,and 36 h of fermentation incubation.The in vitro antibacterial activity test showed that the optimized fermentation broth had the strongest inhibition rate of 90.9%.The disease control experiment showed that the inhibition rate on the disease lesions of B.cinerea was 74.7%,which was 36.9%higher than that before the optimization.The results have provided a theoretical basis for the subsequent research on the separation and extraction of active products and structural identification of TCS001.