纸层析-酶标仪法测定发酵液中γ-氨基丁酸  

Determination of γ-aminobutyric acid in fermentation broth by paper chromatography-microplate reader

作　　者：张敏 薛正莲 余飞 刘艳 王洲 ZHANG Min;XUE Zhenglian;YU Fei;LIU Yan;WANG Zhou(Microorganism Fermentation Engineering and Technology Research Center of Anhui Province, Institute of Biologic & Chemical Engineering, Anhui Polytechnic University, Wuhu241000, China)

机构地区：[1]安徽工程大学生物与化学工程学院微生物发酵安徽省工程技术研究中心

出　　处：《食品与发酵工业》2019年第15期262-267,共6页Food and Fermentation Industries

摘　　要：建立纸层析-酶标仪法对微孔板发酵液中γ-氨基丁酸产量进行测定。对影响纸层析-酶标仪法测定结果的重要因素进行优化,并建立相应的分光光度法。优化后层析液中显色剂-茚三酮的浓度为8g/L,展开剂-正丁醇、冰醋酸与水3者体积比为2∶1∶1;洗脱液中乙醇的体积分数为75%,乙醇与质量浓度为6g/LCuSO4·5H2O的体积比为39∶1;γ-氨基丁酸质量浓度在1~7g/L之间,吸光度与样品浓度线性关系良好(R2=0.9990),平均回收率为98.2485%,重复性RSD为2.6255%。纸层析-酶标仪法能快速、准确地测定微孔板发酵液中γ-氨基丁酸产量,为后续高通量选育γ-氨基丁酸高产菌株奠定了基础。A paper chromatography-microplate reader method was established to determine the yield of γ-aminobutyric acid in microplate fermentation broth. Important factors that affected the performance of paper chromatography-microplate reader method were optimized, and a corresponding spectrophotometric method was established. The optimized chromatography solution contained 8 g/L ninhydrin, and n-butanol, glacial acetic acid and water were in a volume ratio of2∶1∶1. Moreover, the optimized eluent had 75%( v/v ) ethanol, and the volume ratio of ethanol to 6 g/L pentahydrate copper sulfate was39∶1. It was found that the absorbance and γ- aminobutyric concentrations showed a good linearity ( R2=0.999 0) within1~7 g/L . The average recovery rate of the method was 98.249%, and the RSD was2.626%. Therefore, the paper chromatography-microplate reader method could rapidly and accurately determine the production of γ-aminobutyric acid in microplate fermentation broth, which lays a foundation for high-throughput selecting and breeding of strains with high γ-aminobutyric acid yield.

关 键 词：Γ-氨基丁酸 测定 纸层析 酶标仪 

分 类 号：TQ929[轻工技术与工程—发酵工程] TS218