杯伞(Clitocybe sp.)培养条件Box-Behnken法优化研究

Optimization of Cultivation Conditions for Exopolysaccharide and Mycelial Biomass by Clitocybe sp. Using Box-Behnken Design

在Plackett-Burman试验设计结果基础上,采用Box-Behnken响应曲面法(responsesurfacemethodology,RSM)对影响杯伞(Clitocybesp.)AS5.112发酵胞外多糖与生长的关键培养条件(培养温度、时间和装液量)进行了探讨。结果表明,在培养温度为24.3～25.8℃、时间为9.7～10.2d和装液量为76.0～90.0ml条件下,每毫升发酵醪可获得>1253.00μg胞外多糖;而在培养温度为23.8～24.8℃、时间为9.6～10.3d、装液量为71.0～98.0ml范围内,每毫升发酵醪可获得8.32mg菌丝生长量。通过对胞外多糖曲面方程和菌丝干重二次多项回归方程解逆矩阵得知,在培养温度、时间和装液量分别为25.0℃、9.9d和83.4ml时,杯伞AS5.112胞外多糖的最大预测值为1265.45μg·ml-1发酵醪,在上述自变量分别为24.4℃、9.9d和87.1ml时,菌丝浓度可达8.50mg·ml-1发酵醪;在24.5℃、9.9d和84.7ml的条件下,每毫升发酵醪可同时获得1261.60μg胞外多糖和8.47mg菌丝量。验证试验证实了该方程的预测值与实际值之间具有较好的拟合度。

Response surface methodology (RSM) based on a three-level three-factor Box-Behnken design of experiment was used to optimize the exopolysaccharide content (EPC) and the mycelium biomass in submerged cultivation by Clitocybe sp. AS 5.112. The critical factors selected for the investigation were temperature, time of cultivation and volume of medium, based on the results of previous Plackett-Burman design. By analyzing the response surface plots, the optimum ranges of cultivation temperature, time and medium volume for obtaining over 1 253.00 μg·ml-1 of EPC were in 24.3-25.8℃, 9.7-10.2 d and 76.0-90.0 ml, respectively. While for obtaining over 8.32 mg·ml-1 of dry cell weight (DCW), the above variables would be in the range of 23.8- 24.8℃, 9.6-10.3 d and 71.0-98.0 ml, respectively. By solving the inverse matrix from the quadratic regression equations, the optimal conditions for obtaining 1 265.45 μg·ml-1 of EPC were 25.0℃, 9.9 d and 83.4 ml, to gain 8.50 mg·ml-1 of DCW were 24.4℃, 9.9 d and 87.1 ml. In order to obtain the maximum yield of EPC and DCW at the same time, the above conditions would be 24.5℃, 9.9 d and 84.7 ml, respectively, in this situation, the maximum predicted EPC and DCW were 1 261.60 μg·ml-1 and 8.47 mg·ml-1, respectively. The experimental data under various conditions have validated the theoretical values.