Critical Optimized Conditions for Gamma-Aminobutyric Acid (GABA)-Producing Tetragenococcus Halophilus Strain KBC from a Commercial Soy Sauce Moromi in Batch Fermentation

Abstract

Gamma-aminobutyric acid (GABA) has several health-promoting qualities, leading to a growing demand for natural GABA production via microbial fermentation. The GABA-producing abilities of the new Tetragenococcus halophilus (THSK) isolated from a commercial soy sauce moromi were proven in this investigation. Under aerobic conditions, the isolate produced 293.43 mg/L of GABA after 5 days of cultivation, compared to 217.13 mg/L under anaerobic conditions. Critical parameters such as pH, monosodium glutamate (MSG), and sodium chloride (NaCl) concentrations were examined to improve GABA yield. MSG had the most significant impact on GABA and GABA synthesis was not suppressed even at high NaCl concentrations. Data showed that a pH of 8, MSG content of 5 g/L, and 20% NaCl were the best culture conditions. The ultimate yield was improved to 653.101 mg/L, a 2.22-fold increase (293.43 mg/L). This design shows that the bacteria THSK has industrial GABA production capability and can be incorporated into functional food.

1. Introduction

Gamma-aminobutyric acid (GABA) is an attractive compound due to its multiple reported bioactivities against hypertension [1], diabetes [2], cancer, and inflammation [3]. It was not only proven to be an antioxidant, anti-microbial, and anti-allergy, but can also help with regulating blood pressure, immune system stimulation, anxiety reduction, improved sleep, and muscular growth hormone enrichment [3,4,5]. GABA is a non-protein amino acid produced mainly from glutamate by glutamate decarboxylase (GAD) [3,5,6,7,8]. Bacteria, plants, and animals all contain GABA. In mammals’ central nervous systems, it is a key inhibitory neurotransmitter. Even though GABA is present in a wide range of fruits and vegetables, its concentration is modest (0.03–2.00 mmol/g fresh weight) [9] compared to the daily dose of 10 to 26 mg of GABA which has been shown to provide beneficial effects such as lowering blood pressure and depression and improving sleep [10,11]. In addition, GABA can also be synthesized chemically; however, it is more costly and considered unnatural and more hazardous due to the corrosive nature of the reagent used [12]. Therefore, natural food enrichment with GABA, with the possible aim of preventing the diseases and disorders mentioned earlier through diet, is being increasingly explored. Several studies investigated the production of GABA-rich foods by microbial fermentation. This includes honey syrup [13], sourdough bread [14], fermented whey-based formulate [15], water dropwort [16], fermented sea tangle [17], fermented buffalo milk [18], and idli, a fermented food [19].

The production of GABA by microorganisms is at the center of these studies. Although some mold species such as Rhizopus and Aspergillus [20,21] can produce GABA, lactic acid bacteria (LAB) are still the most often exploited microorganisms for GABA production due to their widespread use in fermented foods [22]. For these reasons, the extensive screening of LAB from new sources and with potential GABA capabilities is desirable. Fermented foods high in L-glutamate are major sources of GABA-producing LAB isolates such as kimchi [23], Sufu [24], fermented fish [25], fermented mulberry fruits [26], fermented yogurt [27], and, recently, soy sauce [20,28,29,30].

Soy sauce is a condiment made of two well-known steps, koji, a short-term fermentation stage, and moromi, a long-term fermentation stage that consists of 18%–20% salt concentrations [31]. Because of the non-sterile environment of soy sauce production and the extended fermentation duration that can reach up to 4 years, the microbial community consisting of bacteria, fungi, and yeast is very diverse, which makes it an ideal isolation source [5]. Previous efforts led to the successful isolation of two GABA-producing strains from soy sauce and their optimization, namely, a koji-originated Aspergillus oryzae strain NSK [20,28] and a moromi-based Bacillus cereus strain KBC [30]. The goal of this investigation was to see if the newly discovered LAB Tetragenococcus halophilus strain KBC (THSK), which was isolated from soy sauce moromi, has any GABA-like properties [29]. Additionally, several factors that regulate the biochemical properties of GAD can influence GABA synthesis, for example, fermentation temperature, fermentation time, initial pH, and media additives as well as the strain used, which was found to vary substantially [32].

Therefore, the optimization of these factors is crucial for increasing GABA yield. In this regard, the effect of several culture conditions on THKC GABA potential was exploited, namely, the initial pH, MSG, and NaCl concentrations using Response Surface Methodology (RSM), which is proven to be statistically accepted, as well as information on the effect of independent and combined variables (pH, MSG, and NaCl) on the response (GABA yield). This is the first research to the best of our knowledge that harnesses the GABA potential of the newly found THSK and optimizes it using RSM. The findings of this study will contribute to expanding the range of applications for LAB as starter cultures and the potential for GABA enrichment of soy sauce or other health-conscious fermented foods.

2. Materials and Methods

2.1. Chemicals

GABA standard, De Man, Rogosa, Sharpe (MRS) agar and broth, monosodium glutamate (MSG), sodium chloride (NaCl), acetic acid, triethylamine (Sigma-Aldrich, St. Louis, MO, USA), sodium acetate (Chemiz, Selangor, Malaysia), and acetonitrile (Fisher Scientific, Hampton, NH, USA) were all used in this experiment.

2.2. Strains

The novel bacterial strain THSK was procured from a commercial Malaysian soy sauce company where it was previously isolated from the moromi stage and identified as reported [29] (Figure 1). THSK was subcultured and maintained in MRS slants and agar plates supplemented with 5% NaCl to maintain a working culture. These THSK plates were inspected and sub-cultured regularly to avoid contamination. When not in use, the THSK master strain was kept overnight in MRS broth supplemented with 5% NaCl; then, 500 μL was placed into a 2mL glass vial containing 500 μL of 50% v/v glycerol and stored at −80 °C.

2.3. Inoculum Preparation

A loopful of THSK stock culture was thawed, streaked onto the agar plate of MRS + 5% NaCl and then incubated at 30 °C for 48 to 72 h in both aerobic and anaerobic conditions. The agar plates were incubated in anaerobic jars supplied with Anaerocult A (Merck) to maintain anaerobic conditions. A single colony of THSK was inoculated using 100-mL Erlenmeyer flasks with sterilized MRS broth supplemented with 5% NaCl and incubated overnight at 30 °C in aerobic and anaerobic conditions. For the following experiments, a 10% of THSK inoculum with an approximate 106 CFU/mL cell count was employed.

2.4. Growth Curve

The growth curve of the selected strain was monitored by preparing 150 mL of MRS broth + 5% NaCl using Erlenmeyer flasks of 250 mL inoculated with 10% of THSK and then incubated at 30 °C for 100 rpm. A volume of 1 mL of the suspension culture was aliquoted from day 0 to day 7 to determine the optical density at 600nm (OD) using a spectrophotometer (Jenway spectrophotometer 7305). Alternatively, 1 mL of the culture was harvested for the determination of GABA content. This experiment was replicated in an anaerobic atmosphere with Anaerocult A in anaerobic jars.

2.5. GABA Capabilities of the Selected Strain

As previously stated, the GABA potential of THSK was determined by collecting 1 mL of the suspension culture from day 0 to day 7. The supernatant was collected and filtered using a 0.22 μm pore-size nylon filter before being stored in 1.5 mL HPLC vials to test the GABA concentration after centrifuging the sample for 15 min at 1000× g (4000 rpm in an F-45-12-11 rotor, MiniSpin, Eppendorf AG, Hamburg, Germany) at room temperature (22 to 25 °C). Under anaerobic conditions, the GABA capacities of THSK were also studied.

2.6. GABA Optimization

To optimize the GABA production of the selected bacterial strain and analyze the effect of different culture conditions, Design Expert Version 12 (Stat-Ease, Inc., Minneapolis, MN, USA), a well-known statistical and analytical method, was applied. Three levels of three factors (−1, 0, and 1) Box−Behnken design was used to investigate the effect of the following factors: initial pH medium (3–8), NaCl concentrations (5–20%), and MSG concentrations (1–5 g/L) against the produced GABA yield (

Table 1. The selected factors and levels used for RSM optimization of GABA.

Variables	Range and Levels
	−1	0	1
pH	3.0	5.0	8.0
MSG (g/L)	1.0	3.0	5.0
NaCl (%)	5.0	12.5	20.0

). A total of twenty runs were created (

Table 2. Box–Behnken design of the three selected factors and the generation of predicted and actual GABA yield (mg/L) of T. halophilus strain KBC.

Run	Variables			Actual *	Predicted
	pH	MSG (g/L)	NaCl (%)	GABA (mg/L)	GABA (mg/L)
1	8.00	5.00	20.00	653.10 ± 50.05	591.70
2	8.00	3.00	12.50	645.81 ± 32.38	546.44
3	3.00	3.00	12.50	534.77 ± 4.66	526.22
4	3.00	1.00	5.00	573.77 ± 6.94	480.97
5	5.50	3.00	20.00	525.58 ± 1.93	549.70
6	8.00	1.00	20.00	532.02 ± 6.34	527.91
7	5.50	3.00	5.00	474.23 ± 18.08	522.97
8	5.50	5.00	12.50	564.00 ± 11.68	568.22
9	5.50	3.00	12.50	559.36 ± 5.69	536.33
10	5.50	3.00	12.50	559.36 ± 5.69	536.33
11	5.50	3.00	12.50	559.36 ± 5.69	536.33
12	5.50	3.00	12.50	559.36 ± 5.69	536.33
13	8.00	1.00	5.00	429.35 ± 11.23	501.19
14	3.00	5.00	20.00	490.00 ± 6.34	571.48
15	3.00	5.00	5.00	575.18 ± 4.88	544.75
16	3.00	1.00	20.00	489.78 ± 45.53	507.69
17	5.50	3.00	12.50	559.36 ± 5.69	536.33
18	5.50	1.00	12.50	442.75 ± 142.34	504.44
19	5.50	3.00	12.50	495.17 ± 151.52	536.33
20	8.00	5.00	5.00	504.32 ± 40.4	564.97

* Results are the average of three replications with ± SD.

), and the experiment was carried out using 100-mL Erlenmeyer flasks with 50-mL MRS broth under 100 rpm agitation.

2.7. Analytical Methods

• GABA content

Centrifugation of GABA samples was carried out at 4000 rpm for 15 min. The supernatant was then filtered through a 0.22 μm pore-size nylon filter prior to injection into high-performance liquid chromatography (HPLC) equipped with a Hypersil Gold C-18 column (250 × 4.6 mm I.D., particle size 5 μm; Thermo Scientific, Meadow, UK). GABA separation was achieved as previously described in [30].

2.8. Statistical Analysis

All experiments were carried out in triplicate and all results were expressed as means ± SD. Analysis of variance (ANOVA) was used to assess significant differences between samples using Design Expert 12.0 software with a p-value of less than 0.05.

3. Results and Discussion

3.1. Growth of Tetragenococcus Halophilus Strain KBC

The THSK was successfully identified from the 80-days moromi sample at 50% depth of the soy sauce solid-state fiberglass fermentation tank [29]. Thereafter, it was tested for growth under aerobic and anaerobic conditions. THSK isolates exhibited good growth under both conditions and were considered facultative anaerobic bacteria, as reported in the literature [33,34]. The isolates showed slightly better growth in the presence of oxygen, reaching the maximum cell concentration indicated by the highest OD600 values (ca. 1.5) before attaining a stationary phase. Under anaerobic conditions, the highest OD600 values were achieved at (ca. 1.1). The absence of oxygen seems to affect the growth of THSK moderately.

3.2. GABA Capabilities of THSK

GABA production has been reported in various microbes, including Aspergillus, Bacillus, Monascus, Haematococcus, and yeast [35]. However, due to their ubiquitous use in fermented products and their high activity of endogenous GAD, LAB remains the main microorganism studied for the biosynthesis of GABA, using, in particular, Lactobacillus, Lactococcus, and Enterococcus species. For this study, the novel THSK previously isolated from soy sauce moromi was monitored for its potential to produce GABA. These novel isolates have never been previously recognized as GABA-producing microorganisms to the best of our knowledge. Figure 2 shows the GABA yield produced by the bacteria in MRS broth supplemented with 5% NaCl under aerobic and anaerobic conditions for 7 days. It can be observed that THSK exhibited the potential to produce a considerable amount of GABA under both conditions. In the absence of oxygen, the maximum GABA concentrations were observed on day 4 with a value of 217.13 mg/L, while under aerobic conditions, the GABA amount was moderately higher, reaching 293.43 mg/L on day 4 of the fermentation period. Although it is stated in the literature that anaerobiosis may help enhance GAD gene expression [36], the reduced GABA amount could be explained by the diminishing growth of the isolates in the absence of oxygen, as observed in Figure 2. The highest yield of GABA for both conditions was reached at the stationary phase of growth, which was also observed for the B. cereus strain KBC [30], Lactobacillus plantarum DSM19463 [37], and Lactobacillus fermentum strains [38]. During the exponential and stationary phases, however, GABA synthesis by the A. oryzae strain NSK, isolated from soy sauce koji, was evident [20]. It is assumed that the GAD enzyme generated in the exponential phase requires some modification prior to becoming active in the stationary phase [37]. After 4 days of fermentation, GABA concentrations gradually decrease. This could be due to the limitations of nutrients in MRS broth which are essential in producing GABA. Furthermore, GABA could be utilized for survivability by the microorganisms. This was observed with Saccharomyces cerevisiae which uses GABA as a nitrogen source. This was also the case for Pseudomonas and Bacillus cereus [21,30].

3.3. Optimization of GABA Yield

Culture conditions have been proved to have a substantial impact on increasing GABA production yield. In this study, fermentation conditions, namely NaCl (A), pH (B), and MSG concentration (C) (input variables) on GABA yield (measured response), were optimized using the Box−Behnken design. The following equation demonstrates the relationship between the determined GABA yield and the three fermentation factors, where A (NaCl), B (pH), and C (MSG) are labeled as A, B, and C in Equation (1).

$$\begin{array}{ll}\mathrm{GABA}\mathrm{yield}=& +543.61+13.36\times A+10.11\times B+31.89\times C\\ & +52.58\times A\times B+5.61\times A\times B+5.61\times A\times C\\ & +24.30\times B\times C-36.14\times A^2+54.25\times B^2-32.67\times C^2\end{array}$$

(1)

Table 3. Analysis of variance (ANOVA) from obtained results of GABA production by T. halophilus strain KBC.

Source	Sum of Squares	Df	Mean Square	F Value	p-Value Prob > F
Model	51340.71	9	5704.52	4.82	0.0109	significant
A-NaCl	1785.70	1	1785.70	1.51	0.2475
B-pH	1022.27	1	1022.27	0.86	0.3746
C-MSG	10171.51	1	10171.51	8.59	0.0150
AB	22116.60	1	22116.60	18.69	0.0015
AC	252.18	1	252.18	0.21	0.6543
BC	4724.24	1	4724.24	3.99	0.0737
A2	3591.31	1	3591.31	3.03	0.1121
B2	8092.15	1	8092.15	6.84	0.0258
C2	2935.42	1	2935.42	2.48	0.1464
Residual	11836.08	10	1183.61
Lack of Fit	8402.14	5	1680.43	2.45	0.1742	not significant
Pure Error	3433.95	5	686.79
Cor Total	63,176.79	19
Standard Deviation = 34.40		Mean = 536.33			Adequate Precision = 0.6440
R2 = 0.8127		Adjusted R2 = 0.6440

shows the statistical analysis of variance ANOVA used to determine the model’s significance, and Table 2 shows the experimental results of the 20 RSM runs. The p-value (p = 0.0109) indicates that the experimental data obtained fit well with the model (p < 0.05). The coefficient of determination (R²) was used to evaluate the quality of the model. The values of R² and the adjusted R² were 0.8127 and 0.6440, respectively, which indicates that the model could explain 81.27% of GABA variation while a minor remainder (18.73%) could not be predicted by the model. The Lack of Fit was insignificant, and its p-value was 0.1742 (p < 0.05). All these values indicate that the reliability and general quality of the model were highly acceptable in predicting the GABA yield.

The results given in Table 2 show that the variation among the three different variables affects the final yield of GABA. The factors A (NaCl) and B (pH) had no significant influence on the GABA yield, with p-values of 0.2475 and 0.3746, respectively. The most significant factor (MSG) had a p-value of less than 0.05 (p = 0.0150). The effect of each factor and its interactions on the generated GABA yield is illustrated in Figure 3 and Figure 4. The highest GABA produced after RSM optimization of 653.101 mg/L was obtained from Run number 1, consisting of 20% NaCl, 5 g/L MSG, and an initial pH of 8.

3.4. Effect of Culture Media

3.4.1. Effect of NaCl Concentrations

Due to their potential to produce GABA, several microbes were isolated from diverse fermented food sources. As previously stated, these isolation sources, such as kimchi, cheese, and soy sauce, are typically high in L-glutamate, have an acidic environment, and can often have a high NaCl concentration [39]. For the present study, THSK was isolated from soy sauce moromi where NaCl concentrations ranged from 18–20% [5,31]. Therefore, the potential effect of NaCl on GABA yield was explored using different concentrations of 5–20% of NaCl. As shown in Figure 3 and Table 3, this factor exhibited less influence on GABA (p = 0.2475) than MSG; it appears that an increase in salt concentrations led to an increase in GABA yield, which was sustained even at 20% of NaCl concentrations. This correlated with findings stating that GAD activity was detected in Synechocystis under osmotic stress [40]; similar to Lb. plantarum, L10-11 was isolated from fermented fish (Plaa-som), where its GABA generation was not affected by the presence of NaCl 7% [25]. Moreover, GABA synthesis considerably increased under NaCl stress using Kocuria kristinae isolated from Kedong sufu, however, the strain’s growth seemed to be affected by high concentrations of NaCl [24]. THSK can be therefore prioritized to produce GABA while withstanding high salt concentrations reaching 20%.

3.4.2. Effect of Initial pH

Another essential component for GABA production is pH, which influences microbial growth and GAD activity [39]. Disruption of the GAD enzyme is noticed at a lower or higher initial pH, leading to a reduced GABA yield [38]. It is stated that GAD activity helps bacterial cells withstand low pH stress by exchanging hydrogen ions and glutamate from the medium, increasing the extracellular pH [37,39,41,42]. However, various studies demonstrated different pH ranges for different strains; for example, Lactobacillus plantarum DSM19463 produced a maximum GABA of 4.83 mM at pH 6.0 [37]. Similarly, once the condition hit pH 6, Weissella hellenica SB105 produced a substantial amount of GABA compared to when the pH was adjusted to 3, 4, or 5, where <100 mg/100 mL of GABA was produced [9]. In addition, Wan-Mohtar et al. [30] observed that a B. cereus strain KBC successfully produced 3.4 g/L of GABA under pH 7.0. In an earlier study, Lactobacillus brevis GABA 100 yielded maximum GABA under a low pH value of 3.5 [43]. Conversely, other research showed that the pH of culture media had no significant effect on GABA formation. This was observed for Streptococcus thermophilus fmb5 [44] and Lactobacillus brevis ANP7-6, ANP7-6, and SB109 [9]. The effect of pH on optimal GABA yield using THSK was investigated by altering the initial pH medium values from 3.0 to 8.0. With increasing pH values, GABA concentrations rose to a maximum of 653.101 mg/L at pH 8.0. It can be inferred that the pH factor has a strain-specific influence, and the ideal pH for optimal GABA synthesis is fully dependent on species and varies widely [32].

3.4.3. Effect of MSG Concentration

MSG generates L-glutamate by hydrolysis and is a well-known factor for GABA synthesis as a substrate for cell proliferation and GABA production; L-glutamate is a necessary precursor for GABA production. However, it is unable to be produced in significant amounts by GABA-producing microorganisms. A supply of exogenous L-glutamate through MSG is thus needed in the fermentation process [39].

MSG is broken down into two components: sodium and glutamate. The enzyme glutamate decarboxylase (GAD) and the cofactor pyridoxyl pyrophosphate (PLP) turn the latter into GABA and release CO₂ as a byproduct, resulting in the GABA shunt. The components of the GABA shunt usually consist of a set of enzymes that convert GABA to succinate for use in the tricarboxylic acid cycle (TCA) and energy production. After GABA is produced from glutamate by a GAD enzyme, it is converted to succinic semialdehyde (SSA) by 4-aminobutyrate transaminase (GABA-T), and then to succinate by a succinic semialdehyde dehydrogenase to produce α-ketoglutarate following the TCA cycle [28,30,38,45,46].

Literature verified that significant concentrations of MSG (6 to 15%) are generally required to boost GABA production by inducing the regulation of gadB [47]. High MSG concentrations, on the other hand, have been shown to decrease and degrade GABA synthesis by activating GABA-T. Similarly to pH, the optimal MSG concentrations for producing GABA vary depending on the microorganisms [30,39,45]. Rayavarapu et al. [38] were able to generate 5.34 g/L of GABA with 1.5% MSG using Lactobacillus fermentum. Moreover, Kwon et al. [16] achieved 10 mg/mL of GABA with 3% MSG by Lactobacillus plantarum K154. In further research by [47], GABA yield was markedly enhanced in Lactobacillus plantarum CGMCC 1.2437T where it reached 721.35 mM using 100 mM of MSG. Interestingly, in our previous study [30], MSG exhibited less influence on optimizing GABA in B. cereus strain KBC (p-value = 0.2101) and was also noticed in the GABA production of Streptococcus salivarius subsp. thermophilus Y2 that was unaffected by MSG concentrations ranging from 10 to 20 g/L [48]. The THSK was used to study the improvement of generated GABA from MSG utilizing varied concentrations ranging from 1 to 5 g/L. In our current investigation, MSG had the greatest impact on GABA yield. GABA yield increased dramatically as MSG concentration was increased (Figure 4), reaching a maximum of 653.101 mg/L of GABA at 5 g/L of MSG. It is then concluded that the artificial administration of MSG into the fermentation media considerably elevates GABA accumulation for THSK.

3.5. Highlights of Relevant Literature against the Current Work

Table 4. GABA-producing abilities of critical microorganisms from fermented food sources.

Microorganism	Isolation Source	Culture Media	GABA Production mg/L	Optimization Method	Reference
Tetragenococcus halophilus strain KBC	Soy sauce moromi	MRS broth and 5% NaCl	293.4	Unoptimized	This study
Tetragenococcus halophilus strain KBC	Soy sauce moromi	MRS broth, MSG 5 g/L, and 20% NaCl	653.1	Response surface methodology	This study
Tetragenococcus halophilus strain KBC	Soy sauce moromi	Soybeans and 20% molasses	159.0	Unoptimized	[29]
Bacillus cereus strain KBC	Soy sauce moromi	MRS broth	532.7	Unoptimized	[30]
Bacillus cereus strain KBC	Soy sauce moromi	MRS broth and MSG (5 g/L)	3393.0	Response surface methodology	[30]
Bacillus cereus strain KBC	Soy sauce moromi	Soybeans and 5% molasses	161.0	Unoptimized	[29]
Aspergillus oryzae NSK	Soy sauce koji	Glutamic acid	194.0	Unoptimized	[28]
Aspergillus oryzae NSK	Soy sauce koji	Cane molasses	354.1	One-factor-at-a-time optimization	[20]
Aspergillus oryzae NSK	Soy sauce koji	Sucrose, yeast extract, glutamic acid, and C8:N3	3278.3	One-factor-at-a-time optimization	[4]
Aspergillus oryzae NSK	Soy sauce koji	Soybeans and organic wheat flour	1290.0	Unoptimized	[29]
Lactobacillus fermentum	Palm wine	Soymilk	5340.0	Response surface methodology	[38]
Streptococcus thermophilus 84C	Nostrano cheese	Milk	84.0	Unoptimized	[10]
Lactobacillus plantarum L10-11	Fermented fish (Plaa-som)	MRS broth	15740.0	One-factor-at-a-time optimization	[25]
Lactobacillus delbrueckii ssp. bulgaricus	NP	MRS broth	36.1	Response surface methodology	[32]

shows the GABA-producing abilities of a variety of microorganisms from recent studies isolated from various sources, as determined by the researchers. As previously discussed, the maximum GABA yield varies significantly from one strain to another. It depends on a few parameters of the culture conditions and the strain itself, among other things, as well as the different biochemical properties and genetic organization of GAD [39]. The GABA synthesis of several LAB was higher than that produced by THSK, including Lactobacillus plantarum L10-11 isolated from fermented fish, which made 157,40 mg/L of GABA in one batch. Lactobacillus fermentum, which was isolated from palm wine, similarly produced 5340 mg/L.

Furthermore, the GABA production of the B. cereus strain KBC, which was recovered from the same soy sauce moromi as the current strain, was higher than the above (3393.02 mg/L). THSK, on the other hand, produced significantly more GABA than the strains Streptococcus thermophilus 84C isolated from nostrano cheese (84 mg/L) and Lactobacillus delbrueckii ssp. bulgaricus (84 mg/L) used as controls (36.07 mg/L). Furthermore, compared to our nearest study where THSK was able to produce 159 mg/L, the current effort was efficient in increasing the GABA abilities of the strain to 653.101 mg/L by investigating and optimizing different culture conditions (pH, MSG, and NaCl concentrations).

Previous studies have also confirmed that a daily intake of 10 mg of GABA has been shown to be beneficial for lowering blood pressure in hypertensive patients [10] while 26 mg of GABA helped depression and improved sleep [11]. This indicates that the use of the selected isolate may be sufficient for obtaining the benefits of the reported health properties of GABA. According to the Food and Drug Administration (FDA), the chosen LAB strain, which is considered GRAS, was able to show considerable results under high salt concentrations (20%), which can be advantageous for producing GABA-rich soy sauce or potential functional foods.

Aside from the mentioned culture condition optimization, further research is needed to enhance GABA bioconversion and the production yield of different strains through genetic engineering by allowing recombination with the high L-glutamate producing abilities of certain strains such as Corynebacterium glutamicum [49], over-expression of the GAD gene [50] as well as the combination of different, high GABA-producing strains through co-culturing fermentation [39].

4. Conclusions

The novel THSK bacteria was shown to have significant GABA-producing abilities, under both aerobic and anaerobic conditions. Under aerobic conditions, the selected strain produced a moderately higher yield than the control strain. The findings revealed that MSG had the most significant impact on GABA levels of the tested substances. Under the following conditions: pH 8, MSG 5 g/L, and NaCl 20%, we increased the GABA production of THSK in MRS broth from 293.43 to 653.101 mg/L, which is a 2.22-fold increase. In addition to having varying GABA potentials, different LAB have variable fermentation profiles, which is vital for their use as starter cultures because they have other fermentation profiles. As a result, the identification of novel GABA-producing strains is critical. THSK can be a helpful starter culture to use in promoting GABA-rich soy sauce or fermented foods in conjunction with other cultures.