丙酸发酵过程中四种有机酸快速、简便的分析方法

Fast and convenient determination method of four organic acids during propionic acid fermentation

产酸丙酸杆菌在发酵生产丙酸的同时也会伴随有机杂酸的产生，通过对4种有机酸的监测以期调控发酵条件，从而使丙酸高产。应用HPLC的方法分别研究了在不同流动相种类、pH、洗脱条件、流速、柱温条件下四种有机酸的分离状况，建立了快速、简便测定丙酸、乳酸、乙酸和丁二酸四种有机酸的方法。结果表明，色谱柱条件为COSMOSIL 5C18-PAQ（4．6mm×250mm），检测器为紫外检测器，紫外检测波长为210nm，流动相为K2HPO4缓冲液（0．02mol／L）和乙腈（0～1％梯度洗脱），流速1．0mL／min，柱温25℃，进样量10μL，采用外标法定量。在该条件下各物质线性回归方程相关系数均〉0．999，加样回收率均在95％～105％，精密度实验结果相对标准偏差均〈5．0％，表明该方法准确度高、精密度好，可为丙酸发酵过程中四种有机酸的监控及发酵调控提供数据参考。

Propionibacterium not only produces propionic acid during fermentation, but also produces organic acids. In order to obtain propionic acid with high production, four organic acids were monitored to regulate fermentation conditions. The separation conditions ofpropionic acid, lactic acid, acetic acid and succinic acid under different mobile phases, pH, elution conditions, flow rates, column temperatures were explored by HPLC. Then a fast and convenient method was set up to determine the contents of four organic acids. Results showed that the optimal conditions were column COSMOSIL 5C18-PAQ （4.6 mm×250 mm）, ultraviolet detector with wavelenth of 210 nm, 0.02 mol/L KH2PO4 and 0-1% acetonitrile as mobile phase, flow rate 1.0 ml/min, column temperature 25 ℃ and loading volume 10 μl, and the acids contents were quantified with external standard method. Under these conditions, the correlation coefficients R2 were all more than 0.999, the average adding recoveries were 95%-105%, RSD of precision tests results were all less than 5.0%. The method was with high accuracy and good precision, and it was suitable for four organic acids contents determination in propianic acid fermentation, which provided reference for monitoring and controlling four orangic acids in fermentation.