醋酸菌辅酶Q10的RP-HPLC测定方法及提取工艺优化

RP-HPLC determination and extraction optimization of CoQ10 in acetic acid bacteria

建立了一种测定辅酶Q10(CoQ10)含量的反相高效液相色谱方法(RP-HPLC)并优化了其提取工艺。色谱测定使用Diamonsil反相C18柱(4.6 mm×250 mm,5μm),流动相甲醇与乙醇的体积比为20∶80,检测波长275 nm,柱温35℃,进样体积20μL,流速1.0 mL/min。在此条件下,CoQ10在1~100μg/mL质量浓度范围内的线性关系良好(R^2=0.9992),平均回收率为97.07%,相对标准偏差(RSD)为2.93%,可很好地应用于醋酸菌CoQ 10含量的测定。在单因素实验的基础上,采用响应面分析法,建立了巴氏醋杆菌中CoQ 10酸热法提取的二次多项式回归方程,确定了CoQ 10的最优提取工艺:热处理温度为91℃,HCl溶液物质的量浓度为0.08 mol/L,热处理时间为38 min。采用该工艺提取的巴氏醋杆菌样品中CoQ 10的质量分数为114.81μg/g,与理论值115.66μg/g相近,相同样品的CoQ 10提取值为优化前的368.83%,说明优化后的提取工艺可以有效提取醋酸菌中CoQ 10。

Coenzyme Q 10(CoQ 10)is one of the most important evaluation index for the metabolism ability of acetic acid in acetic acid bacteria.In this investigation,a reversed-phase high performance liquid chromatography(RP-HPLC)method was established to determine the content of CoQ 10 in acetic acid bacteria.It was performed on a Diamonsil C18 column(4.6 mm×250 mm,5μm)with column temperature setting at 35℃.Mobile phase was composed with methanol and ethanol in a volume ratio of 20∶80.The flow rate was 1.0 mL/min and the detective wavelength was 275 nm.Under these conditions,the calibration curve of CoQ 10 demonstrated good linear relationship in the range of 1~100μg/mL(R 2=0.9992)with the relative standard deviation(RSD)of 2.93%and the average recovery rate of 97.07%,proving the effectiveness of this method in determining CoQ 10 content in acetic acid bacteria.On the basis of single factor experiments,a quadratic polynomial rgression equation of CoQ 10 extraction in acetic acid bacteria was constructed using the response surface methodology.The optimal extraction conditions were established as follow:the temperature was 91℃,the concentration of hydrochloric acid was 0.08 mol/L,the extraction time was 38 min.In these optimized conditions,the content of CoQ 10 extracted from acetic acid bacteria(114.81μg/g)was close to the theoretical value(115.66μg/g),and was 368.83%higher than the extracted value before optimization.The results can be used to further utilize acetic acid bacteria and develop functional CoQ 10 products.