巨大芽孢杆菌中芥酸酰胺测定方法的建立及发酵条件优化  

Establishment of determination method and optimization of fermentationconditions of erucamide in Bacillus megaterium L2

作　　者：汤鑫鑫 谢毓丹 肖洋 孙冉 李祝 邓丽 姚蒋庞 TANG Xinxin;XIE Yudan;XIAO Yang;SUN Ran;LI Zhu;DENG Li;YAO Jiangpang(Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Collaborative Innovation Center for Mountain Ecology&Agro-Bioengineering(CICMEAB),College of Life Sciences/Institute of Agro-bioengineering,Guizhou University,Guiyang 550025,China;Institute of Products Detection&Determination of Guizhou Province,Guiyang 550003,China)

机构地区：[1]贵州大学,生命科学学院/农业生物工程研究院,山地植物资源保护与保护种质创新教育部重点实验室,山地生态与农业生物工程协同创新中心,贵阳550025 [2]贵州省产品质量检验检测院,贵阳550003

出　　处：《生物学杂志》2022年第2期110-115,130,共7页Journal of Biology

基　　金：贵州省科技计划项目(黔科合支撑[2021]193号);国家自然科学基金项目(31660533)。

摘　　要：利用气相色谱质谱联用技术(GC-MS)建立巨大芽孢杆菌L2中芥酸酰胺的测定方法,采用单因素试验和响应面法对其培养基成分和发酵条件进行研究。通过Design-Expert 8.06对蛋白胨添加量、温度、pH值进行三因素响应面试验设计,以芥酸酰胺产量为响应值,最后进行响应面分析。在GC-MS色谱条件下,芥酸酰胺含量与峰面积呈现良好线性关系(R^(2)=0.9974),精密度、重复性和稳定性的相对标准偏差(RSD)均低于10%,回收率82.78%~98.14%,测得巨大芽孢杆菌发酵液样品中芥酸酰胺含量为0.940 mg/L。在葡萄糖浓度为5 g/L、蛋白胨添加量为16.40 g/L、NaCl添加量为3 g/L、接种量为2.0%、培养温度为34.8℃、摇床转速为150 r/min、初始pH值为5.80的最优培养条件下,芥酸酰胺产量为1.325 mg/L,比优化前提高了39.03%。结果可对巨大芽孢杆菌发酵液中芥酸酰胺的含量测定提供参考依据,获得芥酸酰胺产量明显提高的巨大芽孢杆菌L2发酵培养基和发酵条件,为芥酸酰胺的后续研究提供理论基础。A method for the determination of erucamide in B.megaterium L2 was established by gas chromatography-mass spectrometry(GC-MS),and then single factor experiments and response surface methods were used to study the fermentation medium and fermentation conditions.Design-expert 8.06 was used to design a three-factor response surface experiment for peptone addition,temperature,and pH.The response value was erucamide yield,and finally response surface analysis was performed.Under GC-MS chromatographic conditions,the erucamide content and peak area showed a good linear relationship(R^(2)=0.9974),the relative standard deviations(RSD)of precision,repeatability and stability were all lower than 10%,and the recovery rate was 82.78%-98.14%,and the content of erucamide in the fermentation broth of B.megaterium was determined to be 0.940 mg/L.The optimal fermentation conditions were as follows:glucose concentration of 5 g/L,peptone concentration of 16.40 g/L,NaCl concentration of 3 g/L,inoculation 2.0%,culture temperature 34.8℃,shaking speed 150 r/min,initial pH 5.80.Under these conditions,the yield of erucamide was 1.325 mg/L,which was 39.03%higher than that before optimization.It can provide a reference for the determination of erucamide in B.megateriu m.The fermentation medium and conditions of B.megaterium L2 significantly increased erucamide production,they provided reliable support for the subsequent research of erucamide.

关 键 词：芥酸酰胺 巨大芽孢杆菌 气相色谱-质谱法(GC-MS) 响应面法 发酵优化 

分 类 号：Q939.9[生物学—微生物学]