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 of 2∶1∶1. Moreover, the optimized eluent had 75% (v/v) ethanol, and the volume ratio of ethanol to 6 g/L pentahydrate copper sulfate was 39∶1. It was found that the absorbance and γ-aminobutyric concentrations showed a good linearity (R2=0.999 0) within 1~7 g/L . The average recovery rate of the method was 98.249%, and the RSD was 2.626%. 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.
ZHANG Min
,
XUE Zhenglian
,
YU Fei
,
LIU Yan
,
WANG Zhou
. Determination of γ-aminobutyric acid in fermentation broth by paper chromatography-microplate reader[J]. Food and Fermentation Industries, 2019
, 45(15)
: 262
-267
.
DOI: 10.13995/j.cnki.11-1802/ts.020844
[1] LIANG F, HATANAKA Y, SAITO H, et al. Differential expression of gamma-aminobutyric acid type B receptor-1a and -1b mRNA variants in GABA and non-GABAergic neurons of the rat brain [J]. Journal of Comparative Neurology, 2015, 416(4):475-495.
[2] MEKONNEN D W, FLGGE U I, LUDEWIG F. Gamma-aminobutyric acid depletion affects stomata closure and drought tolerance of Arabidopsis thaliana[J]. Plant Science, 2016, 245:25-34.
[3] CONTI F, MELONE M, DE B S, et al. Neuronal and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in human cerebral cortex: with a note on its distribution in monkey cortex [J]. Journal of Comparative Neurology, 2015, 396(1):51-63.
[4] BOONBURAPONG B, LALOKNAM S, INCHAROENSAKDI A. Accumulation of gamma-aminobutyric acid in the halotolerant cyanobacterium Aphanothece halophytica under salt and acid stress [J]. Journal of Applied Phycology, 2016, 28(1):141-148.
[5] KEHR J, UNGERSTEDT U. Fast HPLC estimation of gamma-aminobutyric acid in microdialysis perfusates: effect of nipecotic and 3-mercaptopropionic acids [J]. Journal of Neurochemistry, 1988, 51(4):1 308-1 310.
[6] WU Q, SHAH N. Gas release-based pre-screening combined with reversed-phase HPLC quantitation for efficient selection of high-γ-aminobutyric acid (GABA)-producing lactic acid bacteria [J]. Journal of Dairy Science, 2015, 98(2):790-797.
[7] MEEPLOY M, DEEWATTHANAWONG R. Determination of γ-aminobutyric acid (GABA) in Rambutan fruit cv. Rongrian by HPLC-ELSD and separation of GABA from rambutan fruit using dowex 50W-X8 column [J]. Journal of Chromatographic Science, 2016, 54(3):445-452.
[8] ROY U, STUTE L, C HFLING, et al. Sex- and age-specific modulation of brain GABA levels in a mouse model of Alzheimer's disease [J]. Neurobiology of Aging, 2017, 62:168-179.
[9] ALBERT B, MENNY K, NICOLE G, et al. The transporter GAT1 plays an important role in GABA-mediated carbon-nitrogen interactions in Arabidopsis[J]. Frontiers in Plant Science, 2015, 6.
[10] TAKEDA, SAHORI, YAMANO, et al. Rapid determination of 4-aminobutyric acid and L-glutamic acid in biological decarboxylation process by capillary electrophoresis-mass spectrometry [J]. Journal of Separation Science, 2015, 35(2):286-291.
[11] HAGI T, KOBAYASHI M, NOMURA M. Metabolome analysis of milk fermented by γ-aminobutyric acid-producing Lactococcus lactis[J]. Journal of Dairy Science, 2016, 99(2):994-1 001.
[12] CALDERON G D, BRATOEFF E, RAM REZ L E, et al. Effect of flutamide and two novel synthetic steroids on GABA, glutamine and some oxidative stress markers in rat brain and prostate [J]. Andrologia, 2011, 43(4):225-232.
[13] 杨晶晶,曲媛,崔秀明.γ-氨基丁酸的制备方法与含量测定研究进展[J].食品工业科技,2014,35(3):351-356.
[14] WEIWEI D, XIAONING N, MENGLOU L. Efectos de tutin y sus derivados en el GAD y GABA-T en Pseudelatia separata (Lepidoptera: Noctuidae)[J]. Revista Colombiana De Entomologia, 2012, 38:81-86.
[15] 苗雨田,杨悠悠,李颂,等.全自动氨基酸分析仪法快速测定乌龙茶中γ-氨基丁酸[J].分析测试技术与仪器,2015,21(4):231-235.
[16] KUMAR S, PUNEKAR N S, SATYANARAYAN V, et al. Metabolic fate of glutamate and evaluation of flux through the 4-aminobutyrate (GABA) shunt in Aspergillus niger[J]. Biotechnology & Bioengineering, 2015, 67(5):575-584.
[17] LIGNY C L D, REMIJNSE A G. Efficiency of chromatographic procedures. Part IV: The efficiency of paper chromatography on various kinds of Whatman paper and of thin-layer chromatography on various kinds of cellulose powder for the separation of amino-acids [J]. Recueil des Travaux Chimiques des Pays-Bas, 1967, 86(4):421-435.
[18] PAULETTA G, DEFRANCESCHI A. Studies on the amino-acid metabolism of Mycobacterium tuberculosis. I. Preliminary investigation by paper chromatography [J]. Biochimica et Biophysica Acta, 1952, 9(3):271-282.
[19] LI H, QIU T, CAO Y, et al. Pre-staining paper chromatography method for quantification of γ-aminobutyric acid [J]. Journal of Chromatography A, 2009, 1 216(25):5 057-5 060.
[20] CHONG Z, JING L, LIN C, et al. Biosynthesis of γ-aminobutyric acid by a recombinant Bacillus subtilis strain expressing the glutamate decarboxylase gene derived from Streptococcus salivarius ssp. thermophilus Y2 [J]. Process Biochemistry, 2014, 49(11):1 851-1 857.
[21] LIGNY C L D, NIEBOER H, VIJLDER J J M D, et al. Separation of some DL-amino acids into their optical isomers by paper adsorption chromatography [J]. Recueil des Travaux Chimiques des Pays-Bas, 2015, 82(2):213-224.
[22] FRANCISCO F L, SAVIANO A M, PINTO T D J A, et al. Development, optimization and validation of a rapid colorimetric microplate bioassay for neomycin sulfate in pharmaceutical drug products [J]. Journal of Microbiological Methods, 2014, 103:104-111.
[23] 余飞,孙俊峰,刘鹏飞,等.弗氏链霉菌产硫酸新霉素高通量选育模型的建立及优化[J].食品与发酵工业,2019,45(8):162-167.
[24] 叶子弘, 陈春. 生物统计学[M]. 北京:化学工业出版社, 2012: 100-109.
[25] RICHTER F, SCHEIB U S, MEHLHORN J, et al. Upgrading a microplate reader for photobiology and all-optical experiments [J]. Photochem Photobiol Sci, 2015, 14(2): 270-279.
[26] DUELLMAN T, BURNETT J, YANG J. Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader [J]. Analytical Biochemistry, 2015, 473:34-40.
