通过诱变所获得的乳酸菌突变株较多,经96孔板发酵后用传统的分光光度计法测定γ-氨基丁酸(γ-aminobutyric acid,GABA)的含量效率低、成本高。为快速、高效地测定GABA的含量,基于Berthelot反应,该实验采用酶标仪法测定GABA含量并对影响该方法的主要因素进行优化。确定了在测定GABA的适宜反应体系中0.2 mol/L pH10.0硼酸盐缓冲液为0.75 mL,6%的重蒸苯酚为2.5 mL,有效氯5.2%的NaClO溶液为2 mL时,酶标仪测定可获得较高的GABA含量,同时进行了精密度试验。最后,通过对诱变后的75株突变株经96孔板发酵后,分别使用分光光度计与酶标仪测定发酵液中GABA的含量并进行拟合,以验证该方法的可行性。得出2种方法的相关系数R=0.949 3,显示2种测定方法具有很好的一致性。该实验结果表明酶标仪法能简便、快速、准确地测定发酵液中GABA的含量,为后续高通量选育GABA乳酸菌奠定了基础。

In view of the mutants of lactic acid bacteria obtained by mutagenesis were massive and the traditional determination of GABA content in 96-well plates through spectrophotometer was low efficiency and high cost,microplate reader was used to determine the content of GABA quickly and efficiently herein and the main influencing factors of the method was optimized based on the Berthelot reaction. The optimum condition for the GABA determination was as follows: 0. 75mL 0. 2mol /L pH10. 0 borate buffer,2. 5 mL 6% redistilled phenol,and 2mL NaClO solution with5. 2% available chlorine. Under this condition,the higher concentration of GABA could be detected by microplate reader. The precision test was implemented simultaneously. Finally,in order to verify the feasibility of this method,75 mutant strains were fermented in 96-well plates and then respectively measured by spectrophotometer and microplate reader. The data regression showed a significant relationship between these two methods,and the correlation was0. 9493. The experimental results showed that microplate reader can simply,rapidly and accurately measure the content of GABA. It also laid the foundation for high-throughput selecting and breeding of high-yield GABA from lactic acid bacteria in future.