Gas release-based prescreening combined with reversed-phase HPLC quantitation for efficient selection of high-γ-aminobutyric acid (GABA)-producing lactic acid bacteria

Abstract

High γ-aminobutyric acid (GABA)-producing lactobacilli are promising for the manufacture of GABA-rich foods and to synthesize GRAS (generally recognized as safe)-grade GABA. However, common chromatography-based screening is time-consuming and inefficient. In the present study, Korean kimchi was used as a model of lactic acid-based fermented foods, and a gas release-based prescreening of potential GABA producers was developed. The ability to produce GABA by potential GABA producers in de Man, Rogosa, and Sharpe medium supplemented with or without monosodium glutamate was further determined by HPLC. Based on the results, 9 isolates were regarded as high GABA producers, and were further genetically identified as Lactobacillus brevis based on the sequences of 16S rRNA gene. Gas release-based prescreening combined with reversed-phase HPLC confirmation was an efficient and cost-effective method to identify high-GABA-producing LAB, which could be good candidates for probiotics. The GABA that is naturally produced by these high-GABA-producing LAB could be used as a food additive.

Dairy Streptococcus thermophilus improves cell viability of Lactobacillus brevis NPS-QW-145 and its γ-aminobutyric acid biosynthesis ability in milk

“…The predicated amino acids sequence of amplified gadA (316 aa) only have 164 aa in common with that of amplified gadB (307 aa) after ClustalW alignment. Besides the above genetic analysis, we have confirmed high GABA production from this organism 15 . These sequences were further used to design qPCR primers for quantifying the expression of GAD genes in L. brevis 145 as shown in Table 1 .…”

High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter

“…Currently, chromatography-based techniques such as amino acid analyzer (AAA), liquid chromatography (LC), gas chromatography (GC) and thin layer chromatography (TLC) are still the first choice for determining GABA content in various food samples (Li et al, 2010a). AAA and TLC techniques do not require the derivatization process for GABA, but TLC method does not provide enough accuracy for quantifying GABA; LC method normally requires pre-column derivatization with dansyl chloride by an addition of chromophore to GABA that could be detected by fluorescence spectroscopy (Wu et al, 2015b); For GC method, pre-column derivatization of GABA is usually achieved with norvaline to form a volatile compound, which could be separated and detected by GC equipped with a flame ionization detector (FID) or mass spectrometry (MS) (Kagan et al, 2008). Above methods are time-consuming due to tedious sample preparation processes according to analytical protocols; this requires high work-load for screening GABA-producing microorganisms, and is not economical nor rapid.…”

“…Currently, prescreening methods are normally developed based on the decarboxylation of glutamate into GABA by glutamate decarboxylase; this reaction eliminates a proton and also produces carbon dioxide. Thus, glutamate decarboxylase-based microtiter plate assay (Tsukatani et al, 2005), pH indicator method (Yang et al, 2006) and gas release-based assays (Wu et al, 2015b) are suitable for high-throughput pre-screening of GABA producers with remarkable improvements in testing time and economic practice.…”

Gamma-aminobutyric acid (GABA) production in milk fermented by specific wild lactic acid bacteria strains isolated from artisanal Mexican cheeses

“…The GABA production in aqueous extracts from FM was quantified by reversed-phase high-performance liquid chromatography in a Series 1260 Infinity HPLC system (Agilent Technology, Waldbronn, Germany). Aliquots of 2 mL were centrifuged (Eppendorf, Hauppauge, NY, USA) at 12000×g for 10 min at room temperature to remove solids and bacterial cells and were then ultrafiltered through membranes with a pore size of < 3 kDa (Pall Life Sciences, Port Washington, NY, USA) (Wu and Shah 2016). Afterwards, a derivatization of the extracts was made in a precolumn following the methodology of the 6-aminoquinolyl-n-hydroxy succinimidyl carbamate commercial kit AccQ-Tag (Waters Corporation, Ciudad de México, México).…”