Optimization of culture conditions for and assessment of kimchi-originated lactic acid bacterial isolates toward their extracellular GABA-producing ability

Abstract

The objectives of this study were to optimize the medium and culture conditions using a strong γ-aminobutyric acid (GABA) producer as a reference lactic acid bacterial strain, to screen and identify GABA-producing lactic acid bacterial isolates from kimchi, and to determine their extracellular GABA-producing abilities. Thin-layer chromatography was used to screen GABA-producing bacterial isolates and high-performance liquid chromatography was used to evaluate the bacterial GABA production abilities. Species-specific polymerase chain reaction analyses were used to identify GABA-producing bacterial isolates. The optimal medium and culture conditions were found to be the modified Man-Rogosa-Sharpe (MRS) broth (with an initial pH of 6.5) containing 4% sucrose, 5% glutamate, and 1% yeast extract at 37 °C for 5 days. After incubation under the optimized culture conditions, 217 kimchi bacterial isolates were screened to evaluate their respective GABA-producing abilities. Screening the 217 kimchi bacterial isolates identified 24 GABA-producing lactic acid bacterial isolates (11%): Lactobacillus plantarum (17), Lactobacillus brevis (six), and Leuconostoc mesenteroides (one), indicating that only a small proportion of the strains produce GABA in the culture broth. The extracellular GABA-producing abilities of the bacterial strains identified in this study varied even within the same species, ranging from 5.8 to 101.7 mM among the 17 GABA-producing L. plantarum isolates and from 8.5 to 88.6 mM among the six GABA-producing L. brevis isolates. In summary, three species of the 24 kimchi GABA-producing bacterial isolates were identified, including one rare species (L. mesenteroides) and the two most dominant species (L. brevis and L. plantarum).

Headnote

ABSTRACT

The objectives of this study were to optimize the medium and culture conditions using a strong γ-aminobutyric acid (GABA) producer as a reference lactic acid bacterial strain, to screen and identify GABA-producing lactic acid bacterial isolates from kimchi, and to determine their extracellular GABA-producing abilities. Thin-layer chromatography was used to screen GABA-producing bacterial isolates and high-performance liquid chromatography was used to evaluate the bacterial GABA production abilities. Species-specific polymerase chain reaction analyses were used to identify GABA-producing bacterial isolates. The optimal medium and culture conditions were found to be the modified Man-Rogosa-Sharpe (MRS) broth (with an initial pH of 6.5) containing 4% sucrose, 5% glutamate, and 1% yeast extract at 37 °C for 5 days. After incubation under the optimized culture conditions, 217 kimchi bacterial isolates were screened to evaluate their respective GABA-producing abilities. Screening the 217 kimchi bacterial isolates identified 24 GABA-producing lactic acid bacterial isolates (11%): Lactobacillus plantarum (17), Lactobacillus brevis (six), and Leuconostoc mesenteroides (one), indicating that only a small proportion of the strains produce GABA in the culture broth. The extracellular GABA-producing abilities of the bacterial strains identified in this study varied even within the same species, ranging from 5.8 to 101.7 mM among the 17 GABA-producing L. plantarum isolates and from 8.5 to 88.6 mM among the six GABA-producing L. brevis isolates. In summary, three species of the 24 kimchi GABA-producing bacterial isolates were identified, including one rare species (L. mesenteroides) and the two most dominant species (L. brevis and L. plantarum).

Keywords: Lactobacillus brevis; Lactobacillus plantarum; Leuconostoc mesenteroides; Optimization; γ-aminobutyric acid (GABA)

INTRODUCTION

Lactic acid bacteria (LAB) catabolize carbohydrates into lactic acid as a major end metabolite during fermentation and possess distinct acid-resistance mechanisms. Among them, the glutamic acid decarboxylase (GAD) system serves as a fitness determinant in LAB for acid resistance (Wu and Shah, 2017). This bacterial group can decarboxylate L-glutamic acid into γ-aminobutyric acid (GABA), which is catalyzed by GAD. GABA is a free nonproteinaceous amino acid that is found in animals, plants, and microorganisms, including LAB (Somkuti et al., 2012).

In the vertebrate central nervous system, GABA is known to function as a major inhibitory neurotransmitter. In addition, clinical implications of GABA ingestion has led to positive correlations with various important physiological functions, including tranquilization and anti-anxiety functions, although whether the orally administered GABA can enter the blood-brain barrier is still a contradictory issue to be clarified (Somkuti et al., 2012).

Consequently, various functional food supplements containing GABA are commercially mass-produced. To produce such GABA-rich foods, food-grade bacteria, such as LAB, require utilization in various fermentations because chemically-synthesized GABA is a prohibited food additive in developed countries (Wu and Shah, 2017).

Therefore, most studies on GABA-producing LAB have been accomplished to isolate individual strains and determine their GABA-producing abilities under the specific fermentation conditions of the respective LAB (Li and Cao, 2010; Dhakal et al., 2012; Wu and Shah, 2017). In addition, recent reviews report various GABA-producing LAB strains isolated from different food and environmental sources, while focusing on the high GABA producers (Dhakal et al., 2012; Wu and Shah, 2017; Luo et al., 2021). Moreover, most of the LAB isolates ranked as high GABA producers have been identified as Lactobacillus plantarum and Lactobacillus brevis (Wu and Shah, 2017).

Kimchi is a traditional Korean fermented vegetable-based dish made from Korean cabbage (baechu) and other minor ingredients. (Patra et al., 2016). High numbers of LAB are produced in kimchi during fermentation. Metagenomic analysis of kimchi showed that its fermentation was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella (Jung et al., 2014). More recent metagenomic studies have suggested that five Lactobacillus species, including L. plantarum, L. brevis, and L. sakei, three Leuconostoc species, including L. mesenteroides, two Weissella species, and one Pediococcus species dominantly enriched kimchi fermentation (Jung et al., 2014; Swain et al., 2014).

Kimchi or other pickled vegetables are acid-based fermented foods and often the habitats of various GABA producers. LAB communities in kimchi are highly diverse, with Leuconostoc, Lactobacillus, and Weissella as the three most abundant genera (Jung et al., 2014). GABA-producing LAB strains have been screened in various fermented foods, and numerous LAB strains of the genus Lactobacillus have been isolated compared with other genera, such as Leuconostoc (Wu and Shah, 2017). Although many LAB isolates have been evaluated for their respective GABA-producing abilities, rare GABA-producing LAB strains from kimchi, an important LAB source, still require screening.

This study first aimed to find the optimal medium and culture conditions to augment GABA production in the culture broth by the GABA-producing LAB isolates of kimchi. Second, a total of 217 bacterial isolates from kimchi were screened to isolate both rare and dominant (such as the Lactobacillus species) GABA-producing LAB strains. Finally, GABA-producing LAB strains were identified, and their respective GABA-producing potentials were determined.

MATERIALS AND METHODS

Bacterial strains and their identification

Lactobacillus sakei subsp. sakei ATCC 15521T was used as a reference strain to determine the optimal medium and culture conditions. A collection of 217 kimchi bacterial isolates were obtained from the Food Microbiology Laboratory (Chung-Ang University, Ansung, South Korea). Species-specific polymerase chain reaction (PCR) methods were used to identify GABA-producing LAB isolates, as described previously (Cho et al., 2009; Kim and Kim, 2014).

Medium and culture conditions for GABA production

The reference strain L. sakei subsp. sakei ATCC 15521T was used to optimize the culture conditions. This strain was cultured in modified de Man-Rogosa-Sharpe (MRS) broth (Difco, Becton Dickinson Co., Sparks, MD, USA) containing 4% of various extra carbon sources (sucrose, maltose, galactose, glucose, fructose, or lactose), 0-5% monosodium glutamate (MSG), and 1% yeast extract, with initial pH values between 3.5 and 8.5 for 7 days at 37 °C with shaking. After the optimization step, 217 kimchi bacterial isolates were evaluated for their respective GABA-producing abilities using thin-layer chromatography (TLC), and the produced GABA was quantified by highperformance liquid chromatography (HPLC).

Qualitative determination of GABA produced in the culture broth

After incubation under the optimized culture conditions (in the modified MRS broth with an initial pH of 6.5 containing 4% sucrose, 5% glutamate, and 1% yeast extract at 37 °C for 5 days), the broth culture was clarified by centrifugation coupled with filtration through a 0.45-µm syringe filter. GABA production (in the filtered supernatant) was qualitatively evaluated on silica gel 60 F254 TLC plates (Merck, Darmstadt, Germany), as described previously (Thwe et al., 2011). GABA and glutamate were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All chemical reagents used in this study were of analytical or HPLC grade.

Determination of GABA-producing capabilities

The glutamate and GABA in the culture supernatant were quantified using a reversed-phase HPLC system equipped with a Nova-Pak C18 analytical column (3.9 mm × 150 mm, 4 µm particle size; Waters, Milford, MA, USA), a Waters 474 scanning fluorescence detector (Waters), a Waters 600S controller (Waters), and Clarity Lite software (DataApex, Prague, Czech Republic), as described previously (Jin et al., 2013).

Data analysis

Glutamate and GABA quantification data were obtained from three independent measurements and presented as mean ± standard deviation. The quantitative data were statistically analyzed, as described previously (Jin et al., 2013).

RESULTS

Addition of 4% sucrose to increase GABA production

Various extra carbon sources (sucrose, maltose, galactose, glucose, fructose, and lactose) were added (4%) to the culture broth containing 5% glutamate to evaluate their respective effects on GABA production by L. sakei subsp. sakei ATCC 15521T (Fig. 1). GABA production increased maximally to 275.2 mM after 5 days of cultivation when 4% sucrose was added to the culture broth (p < 0.05). However, the reference strain yielded less GABA production (below 200.0 mM) when each of the other tested carbon sources was added.

Five-day cultivation to increase GABA production

The effect of incubation time on GABA production by L. sakei subsp. sakei ATCC 15521T was monitored from the initial cultivation point to 7 days when grown in the modified MRS broth (Fig. 2). GABA rapidly increased from days 2 to 5 (p < 0.05) in the modified MRS broth, while glutamate gradually disappeared from initial cultivation until day 5 (p < 0.05). High GABA production (above 275.0 mM) was maintained between days 5 and 7; therefore, the optimal cultivation period for GABA production was found to be 5 days.

Addition of 5% glutamate to increase GABA production

Glutamate was used as a substrate to produce GABA. To maximize GABA production by L. sakei subsp. sakei ATCC 15521T, the effect of the initial substrate levels of 0 to 6% was monitored in the culture broth after 5 days of cultivation (Fig. 3). GABA production rapidly increased from 8.5 to 274.9 mM (p < 0.05) when the initial substrate content gradually increased from 3 to 5% (p < 0.05). However, high GABA production (above 270.0 mM) slightly decreased when the substrate level was increased to 6%. Therefore, GABA was maximally produced by the reference strain when 5% glutamate was added as the substrate for the GAD enzyme.

Initial pH of 6.5 to increase GABA production

To maximize GABA production by L. sakei subsp. sakei ATCC 15521T, the effect of an initial pH of 3.5-8.5 on the GABA level was monitored in the culture broth (Fig. 4). GABA gradually increased as the initial pH was increased over the range of 3.5-6.5 (p < 0.05), while the unconverted substrate gradually decreased to 22.2 mM (p < 0.05). However, high GABA production (270.0 mM) rapidly decreased when the initial pH was increased from 6.5 to 8.5 (p < 0.05), while the unconverted substrate rapidly increased (p < 0.05). Therefore, GABA was maximally produced by the reference strain when the initial pH of the culture broth was 6.5.

Screening and identifying extracellular GABA-producing bacterial isolates

Qualitative screening of 217 bacterial strains was performed to isolate the strains capable of producing extracellular GABA. The extracellular GABA-producing ability of L. sakei subsp. sakei ATCC 15521T is shown in Fig. 5. Twenty-four (11%) isolates were determined to be putatively positive in the screening experiment.

For this study, species-specific PCR reactions using previously published primer sets for L. plantarum, L. brevis, and L. mesenteroides (Cho et al., 2009; Kim and Kim, 2014) could identify the 24 GABA-producing isolates. Representative species-specific PCR fragments amplified from each bacterial species are shown in Fig. 6. Each target PCR fragment could be species-specifically generated from the 24 GABA-producing isolates by the three speciesspecific PCR primer sets, and only one species-specific PCR fragment was amplified from each isolate. Therefore, each isolate was identified by PCR reactions using the three species-specific PCR primer sets. Among the 24 GABA producers, 17 were identified as L. plantarum, six as L. brevis, and one as L. mesenteroides.

Determining extracellular GABA-producing abilities

To determine their GABA-producing abilities, the 24 putatively-positive strains were subjected to a quantitative analysis of GABA production in the culture broth (Table 1). Lactobacillus plantarum KC-Q1 exhibited the highest extracellular GABA production capacity (102.0 mM) among the positive strains. Isolates KC-D13 (89.0 mM) and KC-I60 (71.0 mM) produced GABA in higher concentrations than the other isolates in this study and were identified as L. brevis. The extracellular GABAproducing abilities of the GABA-producers identified in this study varied even within the same species when they were grown in the culture broth containing 5% MSG, ranging from 5.8 to 101.7 mM among the 17 GABAproducing L. plantarum isolates and from 8.5 to 88.6 mM among the six GABA-producing L. brevis isolates.

DISCUSSION

Groups of LAB possessing specific physiological activities have long been extensively utilized in food industries (Wu and Shah, 2017). LAB produce organic acids, including lactic acid and acetic acid, ethanol, aroma compounds, bacteriocins, exopolysaccharides, and several enzymes. These constituents may positively impact the sensory characteristics of the final products, improving texture, enhancing microbial safety, and extending shelf life.

LAB have been considered as generally-regarded-as-safe (GRAS) organisms in the food fermentation industry since the status of GRAS was introduced by the USA Food and Drug Administration (Wu and Shah, 2017). Besides generating a tremendous self-survival challenge, organic acids (mostly lactic acid) produced by these microorganisms lead to rapid acidification of the raw materials during fermentation (Leroy and De Vuyst, 2004; Wu and Shah, 2017). Therefore, self-protection of the LAB from the acidic environment is pivotal, and various kinds of acidresistance mechanisms are used by these microorganisms to neutralize the acid stress (Liu et al., 2015). Among these mechanisms, the GAD system is one of the most crucial (Gong et al., 2019).

Various LAB strains with GABA-producing potential have been studied in numerous foods or environments over the past three decades (Wu and Shah, 2017; Gao et al., 2019; Luo et al., 2021). In this study, we focused on the LAB species that have been reported as GABA producers based on GABA yield confirmation, with only one isolate representative of each GABA-producing LAB species listed in Table 2. Table 2 shows GABA-producing LAB strains that have been identified in a variety of fermented foods and belong to 36 different LAB species, namely three Bifidobacterium species, five Enterococcus species, 18 Lactobacillus species, two Lactococcus species, three Leuconostoc species, one Pediococcus species, two Streptococcus species, and two Weissella species. It is evident that only a fraction of the strains in a reported LAB species can produce GABA from its substrate. In other words, only a fraction of the strains belonging to each of the 36 LAB species may possess the GAD system. This scenario implies that there may be interstrain variation rather than interspecies variation in their GABA-producing potential (Lyu et al., 2018; Zhuang et al., 2018; Gao et al., 2019).

Owing to the human and animal health benefits conferred by the genus Lactobacillus, over 200 species of Lactobacillus are extensively used in the food industry (Salvetti et al., 2018). Consistent with the recognition of L. plantarum and L. brevis as the main GABA-producing LAB (Wu and Shah, 2017), in this study, L. brevis KC-D13 and KC-I60, and L. plantarum KC-L10 and KC-Q1 (which secreted 88.6, 71.4, 69.5, and 101.7 mM of GABA into the culture broth, respectively) represented the highest GABA producers among the 217 kimchi bacterial isolates. The GABA-producing abilities of 36 GABA-producing LAB strains, each representing 36 different species, including the two main GABA-producing LAB species, are listed and compared in Table 2. In addition, 18 (50%) out of the 36 different species are members of Lactobacillus (Table 2). Therefore, Lactobacilli represents the most dominant group of LAB that possess GABA-producing ability, even in kimchi. In contrast, as mentioned above, the Leuconostoc species strains possessing GABA-producing potential have been rarely reported. In this study, numerous kimchioriginated bacterial strains, including LAB, were screened, and a rare GABA-producing LAB species, L. mesenteroides, was isolated. Here, we also revealed that the reference strain L. sakei subsp. sakei ATCC 15521T can produce 275.2 mM of GABA in the culture broth under the optimized conditions. Although 36 LAB species possessing GABAproducing ability have been identified and characterized, further screening studies are necessary.

CONCLUSIONS

In this study, the culture conditions were optimized for the reference strain L. sakei subsp. sakei ATCC 15521T to produce a high GABA concentration. Consequently, the optimal medium and culture conditions for the reference strain were found to be the modified MRS broth with an initial pH of 6.5 containing 4% sucrose, 5% glutamate, and 1% yeast extract at 37 °C for 5 days. Under these conditions, the reference strain produced up to 275.2 mM of GABA in the culture broth and was evaluated to be a strong GABAproducer. Under the optimal conditions, 24 LAB strains (11%) with GABA-producing ability were isolated after 217 kimchi-originated bacterial strains were screened, indicating that only a small proportion of the strains produce GABA in the culture broth. Among the 24 GABA-producers, 17 isolates belonged to L. plantarum, six to L. brevis, and one to L. mesenteroides, respectively. The extracellular GABAproducing abilities of the GABA-producers identified in this study varied even within the same species, ranging from 5.8 to 101.7 mM among the 17 GABA-producing L. plantarum isolates and from 8.5 to 88.6 mM among the six GABAproducing L. brevis isolates. Besides, the four best GABA producers belonged to L. plantarum (two) and L. brevis (two), the two most dominant GABA-producing LAB species. In addition, one GABA producer belonged to L. mesenteroides, a rare GABA-producing LAB species. Nonetheless, further screening studies are needed to identify and characterize more LAB species possessing GABA-producing ability.

ACKNOWLEDGMENTS

This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) [grant number 2018R1D1A1B07051318], and the Chung-Ang University research grant in 2022. Outstanding technical assistance was provided by Yan Jin during various stages of manuscript preparation.

Conflict of interest

The authors declare no conflicts of interest.

Authors' contributions

Keun-Sung Kim contributed to the conception/design, coordination, and supervision of the research and to the drafting and revision of the manuscript. Jin-Sung Lee performed the analysis and interpretation of the results and revised the manuscript. Both authors critically reviewed and agreed on the final version of the manuscript before submission.

Sidebar

Received: 21 March 2023; Accepted: 21 October 2023

Footnote

*Corresponding author:

Keun-Sung Kim, Department of Food Science and Technology, Chung-Ang University, Ansung, Kyungki-do 456-756, South Korea E-mail: [email protected]

References

REFERENCES

Barla, F., T. Koyanagi, N. Tokuda, H. Matsui, T. Katayama, H. Kumagai, T. Michihata, T. Sasaki, A. Tsuji and T. Enomoto. 2016. The γ-aminobutyric acid-producing ability under low pH conditions of lactic acid bacteria isolated from traditional fermented foods of Ishikawa Prefecture, Japan, with a strong ability to produce ACE-inhibitory peptides. Biotechnol. Rep. (Amst). 10: 105-110.

Barrett, E., R. P. Ross, P. W. O'Toole, G. F. Fitzgerald and C. Stanton. 2014. This article corrects: γ-Aminobutyric acid production by culturable bacteria from the human intestine. J. Appl. Microbiol. 116: 1384-1386.

Cho, K. M., R. K. Math, S. M. A. Islam, W. J. Lim, S. Y. Hong, J. M. Kim, M. G. Yun, J. J. Cho and H. D. Yun. 2009. Novel multiplex PCR for the detection of lactic acid bacteria during kimchi fermentation. Mol. Cell. Probes. 23: 90-94.

Dhakal, R., V. K. Bajpai and K. H. Baek. 2012. Production of Gaba (γ-aminobutyric acid) by microorganisms: A review. Braz. J. Microbiol. 43: 1230-1241.

Gangaraju, D., V. R. Murty and S. G. Prapulla. 2014. Probioticmediated biotransformation of monosodium glutamate to γ-aminobutyric acid: Differential production in complex and minimal media and kinetic modelling. Ann. Microbiol. 64: 229-237.

Gao, D., K. Chang, G. Ding, H. Wu, Y. Chen, M. Jia, X. Liu, S. Wang, Y. Jin, H. Pan and H. Li. 2019. Genomic insights into a robust gamma-aminobutyric acid-producer Lactobacillus brevis CD0817. AMB Express. 9: 72.

Gao, Q., Q. Duan, D. Wang, Y. Zhang and C. Zheng. 2013. Separation and purification of γ-aminobutyric acid from fermentation broth by flocculation and chromatographic methodologies. J. Agric. Food Chem. 61: 1914-1919.

Gong, L., C. Ren and Y. Xu. 2019. Deciphering the crucial roles of transcriptional regulator GadR on gamma-aminobutyric acid production and acid resistance in Lactobacillus brevis. Microb. Cell Fact. 18: 108.

Hiraga, K., Y. Ueno, S. Sukontasing, S. Tanasupawat and K. Oda. 2008. Lactobacillus senmaizukei sp. Nov., Isolated from Japanese pickle. Int. J. Syst. Evol. Microbiol. 58: 1625-1629.

Jin, W. J., M. J. Kim and K. S. Kim. 2013. Utilization of barley or wheat bran to bioconvert glutamate to γ-aminobutyric acid (GABA). J. Food Sci. 78: C1376-C1382.

Jung, J. Y., S. H. Lee and C. O. Jeon. 2014. Kimchi microflora: History, current status, and perspectives for industrial kimchi production. Appl. Microbiol. Biotechnol. 98: 2385-2393.

Kim, M. J. and K. S. Kim. 2014. Tyramine production among lactic acid bacteria and other species isolated from kimchi. LWT-Food Sci. Technol. 56: 406-413.

Komatsuzaki, N., J. Shima, S. Kawamoto, H. Momose and T. Kimura. 2005. Production of γ-aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods. Food Microbiol. 22: 497-504.

Lee, K. W., J. M. Shim, Z. Yao, J. A. Kim and J. H. Kim. 2018. Properties of kimchi fermented with GABA-producing lactic acid bacteria as a starter. J. Microbiol. Biotechnol. 28: 534-541.

Leroy, F. and L. De Vuyst. 2004. Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci. Technol. 15: 67-78.

Li, H. and Y. Cao. 2010. Lactic acid bacterial cell factories for gammaaminobutyric acid. Amino Acids. 39: 1107-1116.

Liu, Y., H. Tang, Z. Lin and P. Xu. 2015. Mechanisms of acid tolerance in bacteria and prospects in biotechnology and bioremediation. Biotechnol. Adv. 33: 1484-1492.

Luo, H., Z. Liu, F. Xie, M. Bilal, L. Liu, R. Yang and Z. Wang. 2021. Microbial production of gamma-aminobutyric acid: Applications, state-of-the-art achievements, and future perspectives. Crit. Rev. Biotechnol. 41: 491-512.

Ly, D., S. Mayrhofer, I. B. Agung Yogeswara, T. H. Nguyen and K. J. Domig. 2019. Identification, classification and screening for γ-amino-butyric acid production in lactic acid bacteria from Cambodian fermented foods. Biomolecules. 9: 768.

Lyu, C., W. Zhao, C. Peng, S. Hu, H. Fang, Y. Hua, S. Yao, J. Huang and L. Mei. 2018. Exploring the contributions of two glutamate decarboxylase isozymes in Lactobacillus brevis to acid resistance and γ-aminobutyric acid production. Microb. Cell Fact. 17: 180.

Nejati, F., C. G. Rizzello, R. Di Cagno, M. Sheikh-Zeinoddin, A. Diviccaro, F. Minervini and M. Gobbetti. 2013. Manufacture of a functional fermented milk enriched of angiotensin-I converting enzyme (ACE)-inhibitory peptides and γ-amino butyric acid (GABA). LWT-Food Sci. Technol. 51: 183-189.

Patra, J. K., G. Das, S. Paramithiotis and H. S. Shin. 2016. Kimchi and other widely consumed traditional fermented foods of Korea: A review. Front. Microbiol. 7: 1493.

Phuengjayaem, S., N. Kuncharoen, A. Booncharoen, B. Ongpipattanakul and S. Tanasupawat. 2021. Genome analysis and optimization of γ-aminobutyric acid (GABA) production by lactic acid bacteria from plant materials. J. Gen. Appl. Microbiol. 67: 150-161.

Ratanaburee, A., D. Kantachote, W. Charernjiratrakul and A. Sukhoom. 2013. Selection of γ-aminobutyric acid-producing lactic acid bacteria and their potential as probiotics for use as starter cultures in Thai fermented sausages (Nham). Int. J. Food Sci. Technol. 48: 1371-1382.

Ribeiro, S. C., M. F. P. Domingos-Lopes, C. Stanton, R. P. Ross and C. C. G. Silva. 2018. Production of γ-aminobutyric acid (GABA) by Lactobacillus otakiensis and other Lactobacillus sp. isolated from traditional Pico cheese. Int. J. Dairy Technol. 71: 1012-1017.

Salvetti, E., H. M. B. Harris, G. E. Felis and P. W. O'Toole. 2018. Comparative genomics of the genus Lactobacillus reveals robust phylogroups that provide the basis for reclassification. Appl. Environ. Microbiol. 84: e00993-18.

Santos-Espinosa, A., L. M. Beltrán-Barrientos, R. Reyes-Díaz, M. Á. Mazorra-Manzano, A. Hernández-Mendoza, G. A. González-Aguilar, S. G. Sáyago-Ayerdi, B. Vallejo-Cordoba and A. F. González-Córdova. 2020. Gamma-aminobutyric acid (GABA) production in milk fermented by specific wild lactic acid bacteria strains isolated from artisanal Mexican cheeses. Ann. Microbiol. 70: 12.

Siragusa, S., M. De Angelis, R. Di Cagno, C. G. Rizzello, R. Coda and M. Gobbetti. 2007. Synthesis of gamma-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses. Appl. Environ. Microbiol. 73: 7283-7290.

Somkuti, G. A., J. A. Jr. Renye and D. H. Steinberg. 2012. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110. J. Ind. Microbiol. Biotechnol. 39: 957-963.

Song, H. Y. and R. C. Yu. 2018. Optimization of culture conditions for gamma-aminobutyric acid production in fermented adzuki bean milk. J. Food Drug Anal. 26: 74-81.

Sun, T., S. Zhao, H. Wang, C. Cai, Y. Chen and H. Zhang. 2009. ACE-inhibitory activity and gamma-aminobutyric acid content of fermented skim milk by Lactobacillus helveticus isolated from Xinjiang koumiss in China. Eur. Food Res. Technol. 228: 607-612.

Swain, M. R., M. Anandharaj, R. C. Ray and R. Parveen Rani. 2014. Fermented fruits and vegetables of Asia: A potential source of probiotics. Biotechnol. Res. Int. 2014: 250424.

Thuy, D. T. B., A. Nguyen, K. S. Khoo, K. W. Chew, M. Cnockaert, P. Vandamme, Y. C. Ho, N. D. Huy, H. H. Cocoletzi and P. L. Show. 2021. Optimization of culture conditions for gammaaminobutyric acid production by newly identified Pediococcus pentosaceus MN12 isolated from "mam nem", a fermented fish sauce. Bioengineered. 12: 54-62.

Thwe, S. M., T. Kobayashi, T. Luan, T. Shirai, M. Onodera, N. Hamada-Sato and C. Imada. 2011. Isolation, characterization, and utilization of γ-aminobutyric acid (GABA)-producing lactic acid bacteria from Myanmar fishery products fermented with boiled rice. Fish. Sci. 77: 279-288.

Woraharn, S., N. Lailerd, B. S. Sivamaruthi, W. Wangcharoen, S. Sirisattha, S. Peerajan and C. Chaiyasut. 2016. Evaluation of factors that influence the L-glutamic and γ-aminobutyric acid production during Hericium erinaceus fermentation by lactic acid bacteria. CyTA-J. Food. 14: 47-54.

Wu, Q. and N. P. Shah. 2017. High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter. Crit. Rev. Food Sci. Nutr. 57: 3661-3672.

Yang, H., R. Xing, L. Hu, S. Liu and P. Li. 2016. Accumulation of γ-aminobutyric acid by Enterococcus avium 9184 in scallop solution in a two-stage fermentation strategy. Microb. Biotechnol. 9: 478-485.

Yang, S. Y., F. X. Lu, Z. X. Lu, X. M. Bie, Y. Jiao, L. J. Sun and B. Yu. 2008. Production of gamma-aminobutyric acid by Streptococcus salivarius subsp. Thermophilus Y2 under submerged fermentation. Amino Acids. 34: 473-478.

Zhao, A., X. Hu, L. Pan and X. Wang. 2015. Isolation and characterization of a gamma-aminobutyric acid producing strain Lactobacillus buchneri WPZ001 that could efficiently utilize xylose and corncob hydrolysate. Appl. Microbiol. Biotechnol. 99: 3191-3200.

Zhuang, K., Y. Jiang, X. Feng, L. Li, F. Dang, W. Zhang and C. Man. 2018. Transcriptomic response to GABA-producing Lactobacillus plantarum CGMCC 1.2437T induced by L-MSG. PLoS One. 13: e0199021.