摘要
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CuSO_4、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4 d每24 h的Mb含量进行测定。结果:在CuSO_4、HDTMA、磷酸缓冲液添加量分别为1.2、4.0、2.0 mL及络合时间为15 min的条件下,铬天青S法测得纯化的1 L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、96 h时发酵液中Mb含量分别为1.008、1.336、3.125、7.500μmoL/250 mL。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 CuSO_4,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 CuSO_4,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.
引文
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