Determination of coronatine in fermentation broth of Pseudomonas syingae and its application

In order to improve the efficiency of engineering strain improvement, a simple, rapid and efficient dispersive liquid-liquid microextraction combined with high performance liquid chromatography(HPLC) method was developed for the content determination of coronatinein fermentation broth of Pseudomonas syingae. Factors influencing the extraction efficiency were optimized, including the protein precipitation method, type and volume of the extraction solvent, type and volume of the dispersive solvent, extraction time and centrifugation intensity. The optimized extraction conditions were as follows: 2.0 mL acetone (dispersive solvent), 400 μL chlorobenzene (extraction solvent), extraction time 5 min, centrifugation at 5 000 r/min for 5 min. The detection conditions for HPLC were as follows: V(methanol ) : V(0.5% acetic acid aq.)=70 : 30 at a flow rate of 1 mL/min was used as a mobile phase in isocratic elution mode, the injection volume was 5 μL for all the solutions and the detection was performed at the wavelength of 220 nm at 30 ℃. Within the range of 3.2-100 mg/L, the peak area of coronatine had a good linear relationship with its mass concentration, and the correlation coefficient (r) was 0.999 8. The detection limit of this method was 2.1 mg/L and the quantitative limit was 6.5 mg/L. At the spiked levels of 6.5, 25 and 100 mg/L, the recoveries of coronatine in the fermentation broth of P. syingae ranged from 95% to 98%, and the relative standard deviations (n = 5) ranged from 2.7% to 5.1%. The method can be used for the determination of coronatine in the fermentation broth of P. syingae.