伊枯草菌素A发酵过程中游离氨基酸的HPLC分析

doi:10.13560/j.cnki.biotech.bull.1985.2018-0012

生物技术通报 ›› 2018, Vol. 34 ›› Issue (8): 151-158.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0012

伊枯草菌素A发酵过程中游离氨基酸的HPLC分析

赵杰^1,2, 岳华^1,2, 苟学磊^1,2, 周金燕¹, 谭红¹

1. 中国科学院成都生物研究所中国科学院环境与微生物重点实验室,成都 610041;
2. 中国科学院大学,北京 100039

收稿日期:2018-01-03 出版日期:2018-08-26 发布日期:2018-09-04
作者简介:赵杰,女,硕士研究生,研究方向：微生物发酵及代谢产物分离提取;E-mail：zjyhm0920@126.com
基金资助:
国家重点研发计划（2017YFD0200900）,四川省科技支撑计划项目（2016NZ0032）

Analysis of Free Amino Acids During Fermentation of Iturin A by High Performance Liquid Chromatography

ZHAO Jie^1,2, YUE Hua^1,2, GOU Xue-lei^1,2, ZHOU Jin-yan¹, TAN Hong¹

1. Key Laboratory of Environmental and Applied Microbiology,Chengdu Institute of Biology,Chinese Academy of Sciences,Chengdu 610041;
2. University of Chinese Academy of Sciences,Beijing 100039

Received:2018-01-03 Published:2018-08-26 Online:2018-09-04

摘要/Abstract

摘要： 建立一种快速、高效测定游离氨基酸含量的异硫氰酸苯酯（PITC）柱前衍生高效液相色谱法,并利用此方法分析检测iturin A发酵过程中游离氨基酸的动态变化。以异硫氰酸苯酯（PITC）为衍生化试剂,采用Venusil-AA（4.6 mm × 250 mm,5 μm）氨基酸分析专用柱,并优化HPLC检测色谱条件。结果表明：梯度洗脱程序、流动相pH值、色谱柱温对分析时间、色谱峰分离及峰型具有重要影响。当最优色谱条件为：流动相A为0.1 mol/L无水乙酸钠缓冲溶液（pH6.4±0.1）-乙腈（66∶5）,流动相B为乙腈-水（4∶1）,流速1.0 mL/min,检测波长254 nm,色谱柱温40℃,梯度程序洗脱,35 min内可完全分离16种氨基酸,且各氨基酸在一定浓度范围内线性关系良好（R²均大于0.9986）,加标回收率在83.84%-108.02%之间,RSD值均小于2.77%。该方法耗时短、操作简便、准确可靠,具有良好的精密度和稳定性。通过此方法研究分析伊枯草菌素A发酵过程中各游离氨基酸含量变化规律,发现其氨基酸浓度变化规律大致分为三类。

关键词: 异硫氰酸苯酯, 高效液相色谱法, 游离氨基酸, 伊枯草菌素A

Abstract: A fast and efficient HPLC with phenylisothiocyanate（PITC）as the pre-column derivatization to detect the concentrations of free amino acids was established and utilized to analyze the dynamic change patterns of free amino acids in the fermentation process of iturin A. The method using PITC as pre-column derivatizing reagent for HPLC analysis,liquid chromatography conditions were optimized. Amino acids were separated on a Vensuil-AA amino acid analysis column（4.6 mm × 250 mm,5 μm）,with optimal conditions：0.1 mol/L sodium acetate buffer（pH 6.4±0.1）-acetonitrile（66∶5）as mobile phase A and acetonitrile -water（4∶1）as mobile phase B by gradient elution,the flow rate was 1.0 mL/min,the UV detection wavelength was set at 254 nm,and chromatographic column temperature was 40℃. The results showed that gradient elution program,pH of the mobile phases and chromatographic column temperature had important effects on the analysis time of amino acids,peak separation and peak shapes. Sixteen amino acids were separated drastically in 35 min under the optimal chromatographic conditions. In addition,each amino acid was separated with fine linearity（R²> 0.9986）in a range of the test concentration,and the average recoveries were 83.84% to 108.02% with RSD less than 2.77%. The method was accurate and reliable with favorable precision and stability. Based on this method,the dynamic changes of free amino acids during the fermentation of iturin A was studied,by which three different changing trends of various amino acids were revealed.

Key words: phenylisothiocyanate, high performance liquid chromatography, free Amino acids, iturin A

赵杰, 岳华, 苟学磊, 周金燕, 谭红. 伊枯草菌素A发酵过程中游离氨基酸的HPLC分析[J]. 生物技术通报, 2018, 34(8): 151-158.

ZHAO Jie, YUE Hua, GOU Xue-lei, ZHOU Jin-yan, TAN Hong. Analysis of Free Amino Acids During Fermentation of Iturin A by High Performance Liquid Chromatography[J]. Biotechnology Bulletin, 2018, 34(8): 151-158.

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伊枯草菌素A发酵过程中游离氨基酸的HPLC分析

Analysis of Free Amino Acids During Fermentation of Iturin A by High Performance Liquid Chromatography

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