摘要
[目的]优化枯草芽孢杆菌NTGB-178发酵工艺,为提高发酵液中的生物量与芽孢产量,降低该菌产品制剂生产成本提供技术支撑。[方法]采用响应面法(RSM)对NTGB-178发酵工艺进行优化。在单因素试验基础上,利用Plackett-Burman设计试验,筛选出影响NTGB-178芽孢产量的主要影响因子。利用最陡爬坡试验,确定逼近存在最大响应值的中心点。最后利用Design-Expert软件设计Box-Behnken试验,对其结果进行多元二次回归拟合,建立响应面方程,绘制响应面图,并对影响发酵产孢的主要因素及其交互作用进行响应面分析和评价,确定最优的液体发酵培养条件。[结果]麸皮、NaCl、初始pH为影响NTGB-178芽孢产量的主要因素,方程预测最优水平为:麸皮10.35 g·L^(-1),NaCl 4.41 g·L^(-1),初始pH6.07,其他条件分别为:玉米粉12.5 g·L^(-1),豆粕25.0 g·L^(-1),CaCO_31.0 g·L^(-1),MgSO_4·7H_2O 2.0 g·L^(-1),接种量4%,装液量65 m L/250 m L,培养温度36℃,摇床转速220 r·min^(-1),培养时间36 h。经验证,优化后发酵液中生物量为6.55×109CFU·m L^(-1),芽孢产量为6.16×10~9CFU·m L^(-1),芽孢产量较优化前提高了8.48倍。最后在中试培养条件下,发酵36 h后芽孢产量最高为7.33×109CFU·m L^(-1),验证了摇瓶优化条件的稳定性。[结论]采用单因素试验与响应面法相结合的方法优化发酵工艺,可显著提高NTGB-178的生物量与芽孢产量,此发酵工艺为芽孢杆菌NTGB-178工业化扩大生产奠定了基础。
[Objectives] The objective of this study is to optimize the fermentation conditions of Bacillus subtilis NTGB-178 for improving the liquid fermentation of biomass and the spore yield, then provide a cost-effective fermentation technique. [ Methods ] Response surface methodology （RSM）was used to optimize the fermentation conditions. The main factors which affected the liquid fermentation were screened by Plackett-Burman design, based on the single factor experiment. The steepest ascent path was used to identify the central point of response surface and approach the optimal region. The Box-Behnken design was adopted to identify the optimal fermentation conditions by establishing a multiple quadratic regression equation, analyzing and evaluating the factors which affect the spore yield of B. subtilis NTGB-178. [ Results] Bran,NaC1 and initial pH were identified as the main factors. The optimum conditions,which were predicted by the response surface equation, were bran 10.35 g·L^-1 , NaC1 4.41 g· L^-l, and initial pH6.07. The other factors were as follows：corn flour 12.5 g·L^-1 ,bean pulp 25.0 g·L^-1 ,CaCO31.0 g·L^-l ,MgSO4 .7H20 2.0 g·L^-1 ,inoculation amount 4%,loaded liquid volume 65 mL/250 mL, rotation speed 220 r·min^-1 and cultivating at 36 % for 36 h. After verification,the biomass reached 6.55×109 CFU·mL^-1 ,and the spore yield reached 6.16×10^9 CFU.mL^-1,which was improved 8.48 times compared to the result before optimization. In the 100 L fermenter enlarge cultivations,the spore yield was 7.33×10^9 CFU. mL^-1. The stability of the optimal fermentation conditions wase indicated. [ Conclusions ] Single factor experiment and RSM were simultaneously used in the optimization of fermentation of B. subtilis NTGB-178 for the biomass and spore production,which was proved to be effective. This study laid the foundation for industrialized production of B. subtilis NTGB-178.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2017年第6期1031-1040,共10页
Journal of Nanjing Agricultural University
基金
国家重点研发计划项目(2016YFD0300904-2)
湖南省重点研发计划项目(2016NK2016)
关键词
枯草芽孢杆菌
优化
芽孢
响应面法
发酵工艺
Bacillus subtilis
optimization
spore
response surface methodology
fermentation technology
作者简介
郑双凤，硕士研究生，助理工程师。;谭石勇，博士，研究方向为植物营养与生物肥料，E-mail：Shiy-tan@hotmail．com。
