生物学杂志

生物学杂志 ›› 2022, Vol. 39 ›› Issue (2): 110-.doi: 10.3969/j.issn.2095-1736.2022.02.110

• 技术方法 • 上一篇    下一篇

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

  

  1. 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
  • 出版日期:2022-04-18 发布日期:2022-04-18
  • 通讯作者: 李祝，博士，教授，研究方向为微生物与生化药物研究，E-mail：zhuliluck@163.com
  • 作者简介:汤鑫鑫，硕士研究生，研究方向为生物与医药，E-mail：ttq0101@126.com
  • 基金资助:
    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)

Establishment of determination method and optimization of fermentation conditions of erucamide in Bacillus megaterium L2

  1. 1.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;
    2.Institute of Products Detection & Determination of Guizhou Province, Guiyang 550003, China
  • Online:2022-04-18 Published:2022-04-18

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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(R2=0.997 4)，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。

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

Abstract: 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 (R2=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. megaterium. The fermentation medium and conditions of B. megaterium L2 significantly increased erucamide production, they provided reliable support for the subsequent research of erucamide.

Key words: erucamide, Bacillus megaterium, gas chromatography-mass spectrometry (GC-MS), response surface method, optimization fermentation

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