INTRODUCTION:

Gamma (γ) - aminobutyric acid (GABA) is a non-protein amino acid that is widely distributed in microorganisms, plants and animals. GABA has potential as a bioactive component with variety of physiological functions and thus showed a great potential in applications of pharmaceuticals and functional foods¹. It is considered to be the major inhibitory neurotransmitter in the central nervous system (CNS). GABA is the product of decarboxylation of glutamate via l-glutamic acid decarboxylase (GAD), of glutamate and is thus an amino acid-derived neurotransmitter². Interestingly, GABA was derived from the same source as the brain’s most common excitatory neurotransmitter, glutamate, and sometimes acts in opposition to glutamate to achieve a balance between excitation and inhibition at the synaptic level.

Hence, consumption of food enriched GABA can improve memory and the learning abilities as well as control pain and anxiety. In addition, GABA could inhibit the growth of cancer cells and expressed anti-diabetic and antihypertensive effects in human³. However, the direction of supplementing of chemical GABA to food is considered unsafely. Thus, it is very necessary to come up with a natural method that can produce and increase GABA.

There have been many attempts to synthesize GABA chemically or biologically because of the beneficial functions of GABA and the increasing commercial demand¹. Lactic acid bacteria (LAB) are the safe microorganisms that were proved as GABA producing sources^4-7. Biosynthetic methods of GABA may be much more promising than chemical synthesis methods due to simple reaction procedure, high catalytic efficiency, mild reaction condition and environmental compatibility. The biosynthesis of GABA is one step reaction of decarboxylating glutamate to GABA, catalyzed by glutamate decarboxylase (GAD). Therefore, the study screened conditions for L. fermentum A01 in GABA production and then, we will be understood more about GABA production as alternative source of GABA for pharmaceutical and food industry.

MATERIALS AND METHODS:

Bacterial strain, media and culture conditions:

L. fermentum A01 was isolated from people inhabiting the central region of Vietnam. It was evaluated for their ability to produce GABA. Lactobacilli de Man, Rogosa, Sharpe (MRS) broth was autoclaved. The isolated bacteria were cultured in 5 mL MRS medium⁸, followed by incubation at 37°C for 24 hours with pH was maintained at 6.5.

Optimization of conditions for CLA production:

The morphology of the strain was observed under oil immersion (×1,000 in magnification) at light microscope level (Olympus CX21 BIM-SET6, Japan). A Gram staining was implemented to investigate the characteristics of the strains was modified⁹.

Cultivation of GABA-Producing Microorganism:

Glutamic acid was dissolved in distilled water aseptically and then added in sterilized MRS broth to obtain the final concentration at 25 mg/mL for 24, 48, 72 hours at 37°C to screen for the GABA-producing L. fermentum A01. After cultivation, the culture broth was centrifuged at 13000 rpm, 4°C for 10min, and the obtained supernatant was used for GABA analysis.

Extraction of GABA:

After cultivation, 5mL of culture broth of each condition was centrifuged at 13000 rpm, 4°C for 10min, and the supernatant was freeze-dried to evaporate all water and followed by extraction with chloroform, methanol and ethyl acetate. Sample (100 mg) was dissolved with 1 mL of chloroform and shaken thoroughly. After centrifugation at 13000 rpm in 10 min, chloroform layer was discarded, the liquid portion was removed totally by pipetting and evaporation. Dried extract was continually washed with chloroform, methanol and finally ethyl acetate to obtain partially pure GABA. GABA was diluted in distilled water for analysis.

Qualitative analysis by Thin Layer Chromatography (TLC):

Presence of GABA in supernatant was ﬁrstly analyzed by using TLC according to Sokovic Bajic et al.¹⁰ with modification. One drop of supernatant was spotted onto the TLC plates. Mobile phase was a mixture including methanol: chloroform: distilled water (5:2:2 in v/v). The Rf value was calculated as following:

Rf = migration distance by component/migration distance by solvent

Cultures of bacterial strains showed the same Rf value as the authentic standard GABA.

Qualitative analysis by High Performance Liquid Chromatography (HPLC):

According to GABA quantitation of Hayat et al.¹¹ with modification, GABA content was determined by a Shimadzu LC-8A preparative HPLC system (Shimadzu Corporation, Tokyo, Japan) equipped with an YMC C18 reverse-phase column with 5μm diameter, 250 mm length and 4.6mm internal diameter. Later, the sample was diluted and subjected to HPLC analysis. The injection volume was 20 μL with a flow rate of 0.6 mL/min. The HPLC mobile phase A was 1% trifluoroacetic acid (TFA). The pH of the mobile phase A was adjusted to 2.8 using triethylamine (TEA). HPLC mobile phase B was a mixture of isopropanol: acetonitrile. All mobile phases were passed through a 0.22 μm membrane filter. The column temperature was set up at 25°C. Sample injection volume was 20 µL and GABA was detected through a UV detector at 250 nm. The presence of GABA was confirmed by comparing the retention time of sample with the corresponding standard GABA. The amount of GABA was calculated by comparing the peak area with the peak area of corresponding standard GABA.

Statistical analysis:

Statistical Package for the Social Sciences (SPSS) software version 20.0 and Microsoft Excel was employed. Values (P < 0.05) refer to significant differences among experiments.

RESULTS AND DISCUSSION:

Screening of GABA-Producing ability in L. fermentum:

After checking GABA production on TLC, L. fermentum A01 exhibited strong spots for GABA production when MSG presenting in growth medium. The Rf value of L. fermentum A 01 was the same as the authentic GABA production (Fig.1).

Fig. 1: Thin Layer chromatography analysis of GABA production by L. fermentum A01 after 72-hour cultivation with MSG. (G): standard GABA; (LF): L. fermentum A01

Optimal condition for GABA production:

The time for cell growth and GABA production of bacterial strain was optimized by cultivation in 5 mL of MRS medium containing 1. mL MSG at 37°C for up to 72 h. Cell growth reached the stationary phase after 24 h of cultivation, whereas GABA production dramatically increased upon 72 h of cultivation by showing a clearer spot than that of 24 and 48 h cultivation according to TLC result. The dried supernatant collected after 72 h of culture from broth containing 25 mg/mL MSG was analyzed and further confirmed by HPLC analysis.

Quantitative analysis of GABA production:

GABA formation was quantitatively conﬁrmed using HPLC-UV. The data of retention time in case of L. fermentum A01 was approximately 21.3 min in samples, similarly to authentic GABA (21.326 min) (Table 1). The similar retention time indicated that GABA was produced in culture by this strain. Moreover, the peak area of interest GABA was calculated according to the concentration of standard GABA in ppm unit from chromatogram.

Table 1: Retention time of LAB in comparison with standard GABA

Lactic Acid Bacteria	Retention time
Lactobacillus fermentum	21.303
GABA standard	21.326

Fig. 2: HPLC spectra of standard GABA (A), Lactobacillus fermentum (B)

After qualitative analysis of GABA production, data demonstrated that L. fermentum A01 owned GABA-producing ability at high-efficiency. GABA yields in freeze-dried samples (170mg) of L. fermentum A 01 was 0.2278 mg after 72 h cultivation, more highly than 24 h and 48 h periods. Obviously, GABA yield was proportional to incubation time. By analysis, there was clear evidence that the extract of L. fermentum showed GABA produced extracellularly.

CONCLUSION:

L. fermentum A01 was evaluated for the ability to produce GABA at largest amount in 72 h cultured in MRS containing MSG (25 mg/mL). Detection of GABA-producing L. fermentum originated from human will be a benefit to pharmaceutical and food development with side effect limitation for human.

ACKNOWLEDGEMENT:

Thanks for the fund supported by Hochiminh City International University (HCM IU), Vietnam national university-Hochiminh city (VNU-HCM) for the research signed in the contract (17-BT-2017/HĐSV-QLKH) issued on February 1st, 2018.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 10.04.2020 Modified on 30.05.2020

Research J. Pharm. and Tech. 2021; 14(4):2188-2190.

DOI: 10.52711/0974-360X.2021.00387