铬天青S法结合高效液相色谱法测定甲烷氧化菌素含量

Determination of Methanomycin Content by Chrome Azurol S Method Combined with High Performance Liquid Chromatography

目的:建立由M.trichosporium OB3b发酵获得的甲烷氧化菌素(Mb)在发酵过程与发酵成熟阶段的含量测定方法。方法:分别考察了CuSO4、HDTMA、磷酸缓冲液添加量以及络合时间等因素对铬天青法检测Mb含量的影响;在铬天青S法对发酵后冻干粉Mb含量确定的基础上,为调控Mb的发酵过程,通过高效液相色谱法对发酵前4d每24h的Mb含量进行测定。结果:在CuSO4、HDTMA、磷酸缓冲液添加量分别为1.2、4.0、2.0mL及络合时间为15min的条件下,铬天青S法测得纯化的1L发酵液中Mb的含量为26.91μmoL,最低检测限为0.042μmoL/L。通过与高效液相色谱法的结合,确定在发酵24、48、72、96h时发酵液中Mb含量分别为1.008、1.336、3.125、7.500μmoL/250mL。结论:本文所建立的方法稳定、重现性好,可用于Mb在发酵不同阶段含量的测定。

Objective:To establish a determination method of the content of methanobactin( Mb) obtained by fermentation of M.trichosporium OB3b during fermentation process and fermentation ripening stage.Methods:The effects of addition of CuSO4 , HDTMA,phosphate buffer and complexation time on the content of Mb were investigated.Based on the determination of Mb content in freeze-dried powder after fermentation by chrome azurol S method,in order to control the Mb fermentation process, the Mb content of every 24 h in the first 4 days during fermentation was determined by high performance liquid chromatography.Results:The content of CuSO4 ,HDTMA and phosphate buffer was 1.2,4.0,2.0 mL and the complexation time was 15 min,the content of Mb in the purified 1 L fermentation broth was 26.91 μmolL,and the minimum detection limit was 0.042 μmol /L.Combining with high performance liquid chromatography,the Mb content in the fermentation broth was determined to be 1.008,1.336,3.125,7.500 μmol /250 mL at 24,48,72 and 96 h,respectively.Conclusion:The method established was stable and had good reproducibility,which could be used for the determination of Mb content in different stages of fermentation.