于春欣, 刘少金, 谭伟明, 李召虎, 段留生. 丁香假单胞菌发酵液中的冠菌素含量测定方法及其应用[J]. 农药学学报, 2020, 22(3): 561-566. DOI: 10.16801/j.issn.1008-7303.2020.0022
    引用本文: 于春欣, 刘少金, 谭伟明, 李召虎, 段留生. 丁香假单胞菌发酵液中的冠菌素含量测定方法及其应用[J]. 农药学学报, 2020, 22(3): 561-566. DOI: 10.16801/j.issn.1008-7303.2020.0022
    YU Chunxin, LIU Shaojin, TAN Weiming, LI Zhaohu, DUAN Liusheng. Determination of coronatine in fermentation broth of Pseudomonas syingae and its application[J]. Chinese Journal of Pesticide Science, 2020, 22(3): 561-566. DOI: 10.16801/j.issn.1008-7303.2020.0022
    Citation: YU Chunxin, LIU Shaojin, TAN Weiming, LI Zhaohu, DUAN Liusheng. Determination of coronatine in fermentation broth of Pseudomonas syingae and its application[J]. Chinese Journal of Pesticide Science, 2020, 22(3): 561-566. DOI: 10.16801/j.issn.1008-7303.2020.0022

    丁香假单胞菌发酵液中的冠菌素含量测定方法及其应用

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

    • 摘要: 为了辅助提高工程菌种改良的效率,建立了一种简单、快速、高效的分散液-液微萃取-高效液相色谱检测丁香假单胞菌发酵液中冠菌素的分析方法。优化了蛋白沉淀法、萃取剂的种类和体积、分散剂的种类和体积、萃取时间和离心强度等对萃取率的影响。确定最优萃取条件为:以2.0 mL丙酮作为分散剂,以400 μL氯苯为萃取剂,萃取5 min,在5 000 r/min下离心5 min。高效液相色谱的检测条件为:流动相为V (甲醇) : V (0.5%乙酸水溶液) = 70 : 30,等梯度洗脱,流速1.0 mL/min,柱温30 ℃,检测波长220 nm。在3.2~100 mg/L范围内,冠菌素的峰面积与其质量浓度间呈良好的线性关系,相关系数 (r) 为0.999 8。方法的检出限为2.1 mg/L,定量限为6.5 mg/L。在6.5、25和100 mg/L添加水平下,冠菌素在丁香假单胞菌发酵液中的回收率在95%~98%之间,相对标准偏差 (n = 5) 在2.7%~5.1%之间。该方法可用于丁香假单胞菌发酵液中冠菌素含量的测定。

       

      Abstract: 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.

       

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