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短短芽胞杆菌(Brevibacillus brevis)FJAT-0809-GLX是广谱生防菌。羟苯乙酯是该菌株发酵液中主要功能成分之一,为了建立一种快速、简单、有效的纯化分离方法对其进行定量检测,本研究建立了测定短短芽胞杆菌FJAT-0809-GLX发酵液羟苯乙酯的高效液相色谱(high performance liquid chromatography,HPLC)法。以甲醇∶水(65∶35)作流动相洗脱,在波长254 nm进行检测,进样量为10μL。结果表明,羟苯乙酯在1.0~20μg/m L的范围内呈良好的线性关系,相关系数达0.998 7,最低检测浓度为1.0×10-15μg/m L。将营养琼脂(NA)培养基发酵液提取的羟苯乙酯样品,在不同时间进行检测,结果表明,48 h内出峰时间和峰面积稳定,说明该方法在48 h内检测检测发酵液均比较稳定。添加不同水平羟苯乙酯的回收率在96.2%~105.3%之间,平均相对标准偏差(relative standard deviation,RSD)为0.8%,回收率较稳定。利用优化发酵培养基培养短短芽胞杆菌FJAT-0809-GLX,经该方法检测显示,其羟苯乙酯出峰时间为3.12 min,峰面积为136。根据标准曲线计算得出该发酵液中羟苯乙酯含量约为2.111μg/m L。该方法简便、快速、灵敏度高,可用于短短芽胞杆菌FJAT-0809-GLX发酵液羟苯乙酯的检测分析。本研究结果为短短芽胞杆菌功能成分的追踪检测奠定基础。
Brevibacillus brevis FJAT-0809-GLX is a broad spectrum biocontrol agent. Hydroxyphenylacetate is one of the main functional components in the fermentation broth of the strain. In order to establish a rapid, simple and effective purification method for the quantitative determination of the fermentation broth, this study established a method for the determination of the fermentation of B. brevis FJAT-0809-GLX Liquid high-performance liquid chromatography (HPLC) method. The mobile phase was eluted with methanol: water (65:35) and detected at a wavelength of 254 nm with an injection volume of 10 μL. The results showed that there was a good linear relationship between the concentration of HPP and 1.0 ~ 20μg / m L with the correlation coefficient of 0.998 7 and the minimum detection concentration of 1.0 × 10-15μg / m L. The results showed that the peak time and peak area were stable within 48 h, indicating that the method of detection and detection of fermentation broth within 48 h relatively stable. The recovery of different levels of ethylparaben was between 96.2% and 105.3%, the relative standard deviation (RSD) was 0.8%, and the recovery was stable. Bacillus subtilis FJAT-0809-GLX was cultured by optimized fermentation medium, and its peak time was 3.12 min and the peak area was 136 by this method. Calculated according to the standard curve in the fermentation liquid hydroxyethyl ethyl ester content of about 2.111μg / m L. The method is simple, rapid and sensitive, and can be used for the detection and analysis of the ethylparaben in fermentation broth of B.subtilis FJAT-0809-GLX. The results of this study laid the foundation for the follow-up detection of functional components of B.subtilis.