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目的:鉴于厚垣孢子为主要活性成分的木霉菌剂具有抗逆性强、存活期长、易贮藏等特点,更容易满足实际生产需求,本文利用系统的液体发酵策略,优化木霉菌高产厚垣孢子液体发酵条件,同时降低发酵成本,指导未来木霉菌厚垣孢子生防菌剂研发。创新点:采用从开放到闭合的递进式液体发酵筛选优化模式,通过Plackett-Burman设计和Box-Behnken优化设计确定影响木霉菌产厚垣孢子的关键因子及最佳发酵条件,确定玉米粉作为基础培养基,添加甘油和调节p H相结合的液体发酵策略,在降低发酵成本的同时提高厚垣孢子产量。方法:以玉米粉为基础培养基,通过Plackett-Burman筛选实验,从8个候选的发酵因子中初步确定玉米粉、甘油和发酵液起始pH值三个显著因子。进而利用响应面中的Box-Behnken设计(表5)三个因素为自变量,厚垣孢子产量为响应值,通过Design Expert 7.0软件对实验数据进行多项式回归分析,建立多元二次回归方程。通过方差分析,最终确定木霉菌SH2303高产厚垣孢子液体发酵工艺的最佳参数。结论:确定了影响厚垣孢子形成的三个关键的发酵因子:玉米粉、甘油和起始pH值。获得厚垣孢子形成最佳的液体发酵条件:玉米粉62.86 g/L,甘油7.54 ml/L,起始p H 4.17,发酵6天。实际最大厚垣孢子产量4.5×10~8孢子/ml,与模型预测值3.6×10~8孢子/ml相当。厚垣孢子产量与优化前相比提高8倍,可用于指导低成本的木霉菌厚垣孢子新型生防菌剂开发。
OBJECTIVE: In view of the fact that Trichoderma sphalerothecium is the main active ingredient, Trichoderma has the characteristics of strong resistance, long life, easy storage and so on. It is easier to meet the actual production needs. In this paper, the liquid fermentation strategy of the system is used to optimize Trichoderma longum Spore liquid fermentation conditions, while reducing the cost of fermentation to guide the future development of Trichoderma chlamydospores biocontrol agents. Innovative point: The optimization mode of progressive liquid fermentation screening from open to closed was used to determine the key factors influencing Trichoderma chlamydosporium production and the optimal fermentation conditions by Plackett-Burman design and Box-Behnken optimization design. The basal medium, the liquid fermentation strategy of adding glycerin and adjusting the pH of pH, increases the chlamydospore yield while reducing the cost of fermentation. Methods: Based on the maize meal-based medium, Plackett-Burman screening experiments were conducted to determine three significant factors of initial pH of corn meal, glycerol and fermentation broth from eight candidate fermentation factors. Furthermore, using the Box-Behnken design in the response surface (Table 5) as the independent variable and chlamydospore yield as the response value, polynomial regression analysis was performed on the experimental data using Design Expert 7.0 software to establish the multiple quadratic regression equation. By ANOVA, the best parameters of the fermentation process of high-yield chlamydosporium liquid of Trichoderma SH2303 were finally determined. Conclusion: Three key fermentation factors affecting chlamydospore formation were identified: corn flour, glycerol and initial pH. Chlamydospore obtained the best liquid fermentation conditions: corn flour 62.86 g / L, glycerol 7.54 ml / L, the initial p H 4.17, fermentation 6 days. The actual maximum chlamydospore yield was 4.5 × 10 ~ 8 spores / ml, which was equivalent to the predicted value of 3.6 × 10 ~ 8 spores / ml. Chlamydospore production increased by 8 times compared with that before optimization, which can be used to guide the development of low cost Trichoderma chlamydospores new biocontrol agent.