利用响应面法优化贝莱斯芽孢杆菌ZJ20发酵参数

Optimization of Bacillus velezensis ZJ20 fermentation parameters by response surface methodology

【目的】优化贝莱斯芽孢杆菌的培养条件,以提高其菌落数和抗菌活性,为该菌株工业化生产提供理论依据。【方法】以贝莱斯芽孢杆菌ZJ20为供试菌株,采用单因素试验,研究培养基、pH、温度、转速对ZJ20菌落数和抗菌活性的影响;在此基础上,以菌落数为考察指标,采用响应面法对pH、温度、转速3个因素进行进一步的优化,得到了贝莱斯芽孢杆菌ZJ20的最佳培养条件;最后在温室采用盆栽试验,研究最佳培养条件得到的贝莱斯芽孢杆菌ZJ20对杂交竹梢枯病的防治效果。【结果】单因素试验结果显示,供试培养基中,NB培养基(牛肉膏3g,蛋白胨10g,氯化钠5g,琼脂20g,蒸馏水1L,pH 7.0～7.2)上贝莱斯芽孢杆菌ZJ20的菌落数和抑菌圈直径均最高,分别为4.11×108 CFU/mL和6.10mm,且显著高于其余培养基,说明NB培养基更有利于贝莱斯芽孢杆菌的生长。当pH为4～10时,随着pH的增加,贝莱斯芽孢杆菌ZJ20的菌落数和抑菌圈直径均先增后降,其中当pH为7～8时ZJ20的菌落数和抑菌圈直径均显著高于其他处理;在此基础上的复筛结果显示,7.0～7.4为贝莱斯芽孢杆菌的最优pH。当转速为140～200r/min时,随着转速的增加,贝莱斯芽孢杆菌ZJ20的菌落数和抑菌圈直径均先增后降,其中当转速为160～180r/min时,菌落数和抑菌圈直径均较高。温度为22～30℃时,随着温度的升高,菌落数和抑菌圈直径均先增加后下降,其中当温度为22～28℃时菌落数和抑菌圈直径均较高。响应面试验结果显示,3个因素中,转速对菌落生长和繁殖的影响最大,其次是温度,影响最小的是pH,贝莱斯芽孢杆菌ZJ20最优培养条件为:pH 7.27,转速160r/min,温度28℃,在此条件下测定的菌落数为769×107 CFU/mL。盆栽试验结果显示,优化培养条件下得到的ZJ20悬液防治效果明显提高,均超过50%。【结论】得到了贝莱斯芽孢杆菌的最佳培养条件,这不仅使其菌落数增加,而且极大地增强了该菌的抗病活性。

【Objective】This study optimized the culture conditions of Bacillus velezensis ZJ20 for enhancing the quantity and antibiotics activity to provide basis for the industrial production of this strain.【Method】The effects of culture medium,pH,temperature and speed on the quantity and activity of B.velezensis ZJ20 were studied by single factor method.On this basis,the quantity was used as the index,the three factors of pH,temperature and speed were further optimized by response surface method,and the optimum culture conditions of B.velezensis ZJ20 were obtained.Finally,the control effects of B.velezensis ZJ20 under the optimal conditions were studied by pot culture in greenhouse.【Result】The results of single factor method showed that the colony number and inhibition zone diameter of B.velezensis ZJ20 in NB medium(beef extract 3 g,peptone 10 g,NaCl 5 g,agar 20 g,distilled water 1 L,and pH 7.0-7.2)were the highest with the values of 4.11×10^8 CFU/mL and 6.10 mm,which were significantly higher than the other culture media.Thus,NB medium was to the best for the growth of B.velezensis ZJ20.With the increase of pH from 4 to 10,the colony number and inhibition zone diameter of B.velezensis ZJ20 increased first and then decreased with the peak values at pH 7-8.The rescreening showed that 7.0-7.4 was the best pH to B.velezensis ZJ20.When the speed was 140-200 r/min,the colony number and inhibition zone diameter of B.velezensis ZJ20 increased first and then decreased with the increase of speed.The colony number and inhibition zone diameter with the speed of 160-180 r/min were higher than other treatments.With the increase of temperature during 22-30℃,the colony number and inhibition zone diameter increased first and then decreased with the peak values at 22-28℃.The response surface method showed that speed had most influence on the growth and breeding of this strain,followed by temperature,while pH had the minimum influence.The optimum culture conditions of B.velezensis ZJ20 were pH 7.27,speed 160 r/min,and temperature 28℃.Under these conditions,the colony numbers were 769×10^7 CFU/mL.In the pot experiment,the control effect of the bacterial suspension of optimized parameters was significantly heightened and exceeded 50%.【Conclusion】Optimization culture conditions of B.velezensis were obtained,which not only increased the quantity,but also significantly enhanced the disease resistance.