凝结芽孢杆菌发酵液中有机酸成分反相高效液相色谱法建立及其测定

Construction and determination of organic Acids in fermentation broth of bacillus coagulans by reversed phase high performance liquid chromatography

【目的】建立分离5种有机酸标准品的反相高效液相色谱法,通过此方法分析凝结芽孢杆菌发酵液中有机酸代谢产物。【方法】选择优化好的液相色谱条件:色谱柱为Venusil ASB C18(5μm,250 mm×4.6 mm),流动相为乙腈0.02mol/L磷酸二氢钾缓冲溶液(磷酸水溶液调pH 2.5),流速1.0mL/min,柱温28℃,检测波长为210nm。【结果】此方法能够快速、准确分离5种混合酸标准液;检测出4种凝结芽孢杆菌发酵液中的有机酸,分别是L-乳酸、乙酸、丙酸、甲酸;相对标准偏差为1.2%~2.9%,回收率87.5%~107.5%,各种有机酸的线性相关系数r范围在0.999 1~0.999 6之间。【结论】建立一种快速分离5种有机酸的方法,并检测出凝结芽孢杆菌发酵液有机酸的组成。

[Objective]To establish a reversed phase high performance liquid chromatographic （RP-HPLC） method for the simultaneous separation of five organic acid standards, The organic acid metabolites of the fermentation broth of Bacillus coagulans were analyzed by this method. [Methods]The optimized HPLC conditions is as follows： Venusil ASB C18 column （5μm, 250ram lows）was used and mobile phase contained acetonitrile-0.02 mol/L potassium dihydrogen phosphate buffer solution （pH 2.5 adjusted with phosphoric acid solution） . The flow rate was 1.0 mL/min and column temperature was 28℃. Detection wavelength was at 210 nm. [Results]This method could rapidly and accurately separate in five kinds of mixed standards. Meanwhile, four kinds of organic acids is include L-lactic acid, acetic acid, propionic acid and formic acid in the fermentation broth of Bacillus. The spiked recoveries were between 87.5% and 107.5% with the relative standard deviations （RSDs） of 1.2% -2.9%. The linear correlation coefficients were from 0.9991 to 0.9996 for all organic acids. [Conclusion] A method for rapid separation of five organic acids was established, and the types of organic acids in the fermentation broth of Bacillus coagulans could be determined.