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ABSTRACT A total of 50 lactic acid bacteria (LAB) isolates from Iranian traditional dairy products (Motal and Lighvan cheeses, and artisanal yogurt) were screened for gamma-aminobutyric acid

(GABA) production. Firstly, a rapid colorimetric test was performed to evaluate the glutamate decarboxylase (GAD) activity among the LAB isolates examined. Thin layer chromatography (TLC)

was then performed on selected strains to identify isolates with high/moderate GABA producing capacity, and a GABase micro-titer plate assay was employed to quantify GABA. Finally, two

_Lactococcus_ (_Lac._) _lactis_ strains were selected for GABA production optimization via Response Surface Methodology (RSM) following Central Composite Design (CCD). Forty-one out of the

50 isolates showed GAD activity according to the colorimetric assay. Eight isolates displayed strong GAD activity, while nine showed no activity; low to moderate GAD activity was scored for

all other isolates. GABA production was confirmed by TLC in all isolates with high GAD activity and in four selected among isoaltes with moderate activity. Among the _Lactococcus_ strains

tested, _Lac. lactis_ 311 and _Lac. lactis_ 491 were the strongest GABA producers with amounts of 3.3 and 1.26 mM, respectively. These two strains were subjected to GABA production

optimization applying RSM and CCD on three key variables: Monosodium glutamate concentration (MSG) (between 25 and 150 mM), incubation temperature (between 25 and 37 °C), and pH (between 4.0

and 5.0). Optimal conditions for GABA production by _Lac. lactis_ 311 and _Lac. lactis_ 491 of temperature, pH and MSG concentration were, respectively, 35.4 and 30 °C, pH 4.5 and 4.6, and

MSG concentration of 89 and 147.4 mM, respectively. Under the above conditions, the amount of GABA produced by _Lac. lactis_ 311 and _Lac. lactis_ 491 was 0.395 and 0.179 mg/mL,

respectively. These strains and the optimal culture conditions determined in this study could be used for the biotechnological production of GABA or applied in food fermentations for the

development of naturally GABA-enriched foods. SIMILAR CONTENT BEING VIEWED BY OTHERS OPTIMIZING ALPHA-AMYLASE FROM _BACILLUS AMYLOLIQUEFACIENS_ ON BREAD WASTE FOR EFFECTIVE INDUSTRIAL

WASTEWATER TREATMENT AND TEXTILE DESIZING THROUGH RESPONSE SURFACE METHODOLOGY Article Open access 06 November 2023 A REFINED MEDIUM TO ENHANCE THE ANTIMICROBIAL ACTIVITY OF POSTBIOTIC

PRODUCED BY _LACTIPLANTIBACILLUS PLANTARUM_ RS5 Article Open access 07 April 2021 INDUSTRIAL PRODUCTION AND FUNCTIONAL PROFILING OF PROBIOTIC _PEDIOCOCCUS ACIDILACTICI_ 72 N FOR POTENTIAL

USE AS A SWINE FEED ADDITIVE Article Open access 29 April 2025 INTRODUCTION Lactic acid bacteria (LAB) are among the most essential and common Gram-positive bacteria which are not only

widely distributed in nature and naturally exist in traditional fermented foods, but also extensively involved in industrial food fermentations due to having a reputation of being “Generally

Recognized As Safe” (GRAS). The use of pure cultures of lactic acid bacteria for fermentation of cucumbers, cabbage, olives, and other products has been explored for several decades with

varying degrees of success. However, at present pure cultures are exploited only on a limited commercial scale for these commodities1,2,3,4. LAB also offer special functions that are

considered beneficial, such as antioxidant and antimicrobial activities, as well as the formation of bioactive compounds such as, among others, peptides, organic acids, and short-chain fatty

acids5,6,7,8,9. Because of their significant beneficial effects, there are currently noteworthy researches on LAB and attract many industrial interest10. Gamma-aminobutyric acid (GABA) is

considered as one of the well-known bioactive compounds; it is a four-carbon, non-protein amino acid formed by different organisms including microorganisms, plants, and animals3,11,12. This

amino acid-derived compound serves as the main inhibitory neurotransmitter in the mammalian central nervous system and, as such, it leds to hypotension, contributes to gut-to-brain signaling

and has tranquilizer effects7,13,14,15,16. Several studies show that GABA contributes to other physiological functions, such as regulating of sleeplessness and depression, enhancing the

plasma level of growth hormone, controlling the quorum-sensing systems by acting as a signal molecule between eukaryote cells and pathogens, reducing the inflammation in rheumatoid

arthritis10,17,18,19. As a bioactive compound, the application of GABA varies from pharmaceuticals to functional fermented foods20. Therefore, it is an ever-growing demand for highly

effective GABA biosynthesis, and hence, substantial efforts have been made in this field. Biological methods applying microorganisms are more promising; thus many GABA-enriched products are

obtained by fermentation21,22,23,24,25. Biosynthesis of GABA from glutamate occurs by the action of the glutamate decarboxylase (GAD), a pyridoxal 5-phosphate-dependant enzyme (EC 4.1.1.15)

that is responsible for the conversion of L-glutamate into GABA3,24. The wide distribution of GAD among eukaryotes (plants, animals, and fungal strains) and prokaryotes has been reported by

several studies26,27. However, LAB, besides all beneficial and technological properties, have been introduced as the most promising group of microorganisms in which they are capable of

producing a high level of GABA due to high GAD activity28. The effort to screen for new GABA-producing LAB still attracts attention, though numerous strains have already been isolated and

characterized. Thus, it seems that further research on the isolation and characterization of GABA-producing LAB is demanded to provide novel starters for the nutraceutical and functional

fermented foods industry10. The diversity of new GABA-enriched foods ranges from cereal-based to dairy-fermented products, including sourdough, bread, cheese, fermented tea and vegetables,

traditional Asian fermented foods, and dairy and soy products1,29. As mentioned above, by further screening, the isolation sources should be diversified as much as possible, including

traditional fermented foods to achieve new GABA-producing LAB strains. This leads to a more comprehensive application area and higher flexibility of starter cultures11. According to the

literature, raw milk cheeses have been identified as a valuable source of microbial biodiversity and new LAB strains with health-promoting properties14. Though currently, there are several

different types of researches and industrial interest in the biological examination of the potential of traditional dairy LAB30,31,32. So far, no GABA-producing LAB have been reported from

Iranian traditional fermented foods. Recently Azizi et al. (2017) have claimed that most of the isolates from Motal cheese could be used as starters or adjunct-starters in novel fermented

functional foods. Besides having antimicrobial properties33, the production of bioactive compounds, such as GABA, could potentially substantiate the functionality claimed for such cultures.

Hence, the present study aims to screen for GABA-producing LAB from various Motal and Lighvan cheeses and yogurt samples which are among the preeminent and most appreciated traditional

Iranian dairy products. MATERIALS AND METHODS CHEMICAL AND MEDIA The commercial GABase kit and all chemicals such as NADP + (Nicotinamide adenine dinucleotide phosphate), α-ketoglutarate,

monosodium glutamate (MSG), Triton X-100®, and GABA were purchased from Sigma-Aldrich (St. Louis, MO, USA). M17 and de Man, Rogosa, and Sharpe (MRS) broth media were obtained from Quelab

(Quelab Laboratories Inc., Canada). LAB STRAINS AND GROWTH CONDITIONS A total of 50 LAB isolates previously isolated from Iranian traditional dairy products (Motal cheese33, Lighvan

cheeses31,34, and Iranian yogurt35), identified using molecular techniques and 16S rRNA gene sequencing, were screened for GABA production (Table 1). All examined isolates, including

lactobacilli (_Lactobacillus, Lactiplantibacillus_, and _Levilactobacillus_) (27 isolates), _Lactococcus_ (20 isolates), and _Streptococcus_ (3 isolates), were kept frozen in MRS

(lactobacilli) or M17 (lactococci and streptococci) broth with 15% glycerol at -80 °C. Pre-cultures were made by adding 20 µL of stock culture to 10 mL fresh medium. Lactobacilli strains

were grown in MRS medium, while M17 medium was used for lactococci and _Streptococcus_ (_St._) _thermophilus_ strains. Based on the types, incubations were done at 30 or 37ºC for 24–48 h

under aerobic or anaerobic conditions. QUALIFICATION SCREENING OF LAB ISOLATES DETERMINATION OF GAD ACTIVITY Glutamic acid decarboxylase (GAD) assay is a rapid colorimetric test used to

assess the GABA-producing potential among LAB isolates. Sample preparation for GAD activity determination was done as described by Lacroix et al.36. An overnight grown culture of each

isolate (5 mL) was washed with 0.9% (w/v) NaCl solution and centrifuged at 5000 × g (20 min, 25 ºC). Cells were suspended in 500 µL of GAD reagent solution (pH = 4), which consists of

L-glutamic acid (1 g), Triton X-100 (300 µL), NaCl (90 g), and bromocresol green (0.05 g) in 1 L deionized water. After anaerobic incubation for 4 h at 37ºC, GAD activity was visually

examined by color change. The development of a greenish or blueish color was considered as low or high GAD activity, respectively. However, no color change (yellow) was considered with no

activity36. THIN LAYER CHROMATOGRAPHY (TLC) To identify isolates with high and moderate GABA producing ability, a TLC assay was performed as described by Lee et al.37. Briefly, strains were

inoculated in their respective broth medium supplemented with 0.5% monosodium glutamate (MSG) and incubated at their optimal temperature for 24 h. To test the presence of GABA, supernatants

were collected by centrifugation (8000 × g, 4ºC, 5 min) and then 2 µL of each filtered supernatant was spotted on a TLC plate (Silica gel 60 F254). TLC was conducted using n-butanol:acetic

acid:water ratio of 5:2:2 (v/v/v). The silica gel plate was subsequently developed by spraying with a 2% ninhydrin solution and heating at 105ºC for 5 min. DNA EXTRACTION AND STRAIN TYPING

BY REP-PCR Total genomic DNA was extracted from GABA-producing isolates using a commercial kit (High Pure PCR Template Preparation Kit; Roche, Basel, Switzerland), following the

manufacturer’s instructions. To assess the interspecies diversity between isolates, repetitive extragenic profiling PCR (rep-PCR) was done using BoxA2R (5´-ACGTGGTTTGAAGAGATTTTCG-3´) as

reported by Koeuth et al.38. In brief, reactions were performed in 25 µL volume containing 2 µL purified DNA, 12.5 µL Red master mix (Ampliqon, Odense, Denmark), 9.3 µL Nuclease-free water,

and 1.2 µL of the primer. Thermal conditions were as follows: an initial step of denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation and annealing at 94 °C for 29 s, 40 °C

for 1 min, and at 68 °C for 8 min; with a final extension at 68 °C for 10 min. PCR products were then electrophoresed using 1.5% agarose (Merck, Darmstadt, Germany) gel at 75 V for 90 min.

DNA safe stain (0.75 µL/100 mL) (Sina Clon Bio Science, Tehran, Iran) was used for visualization of the gel and the bands photographed under UV light by Gel Doc System. The similarity of the

patterns was expressed by the Simple Matching (SM) coefficient, and the clustering was performed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method, with the

Multivariate Statistical Package (MVSP) software version 3.13d31. GABA DETERMINATION Quantitative measurment of GABA in broth medium was done by a GABase microtiter plate assay as described

by Tsukatani et al.39. Bacterial isolates were grown in MRS broth medium containing 0.5% MSG for 2 days at their optimal growth temperature. The reactive mixture contained 750 mM sodium

sulfate, 10 mM dithiothreitol, 1.4 mM NADP + , 2.0 mM α-ketoglutarate in Tris–HCl buffer (80 mM, pH 9.0) and GABA-T (Gamma-aminobutyrate glutamate aminotransferase) (Sigma-Aldrich). The

mixture was added to each well (in a 96-well microtiter plate) in a final volume of 90 µL. The initial absorbance was read at 340 nm with a microplate reader (BioTek, ELX 808) before adding

a 10 µL sample of a standard GABA solution. The substrate of NADP + turns to NADPH in the presence of GABA and α-ketoglutarate after incubation at 30-37ºC for 1 h. To calculate the GABA

content formed by each isolate, the difference between initial and final absorbances at 340 nm was compared to standard curves of calibrated GABA solutions. OPTIMIZATION OF GABA PRODUCTION

USING RESPONSE SURFACE METHODOLOGY (RSM) Optimization of GABA production in MRS medium and studying the interaction of key parameters were done using response surface methodology (RSM). A

Central Composite Design (CCD) of RSM was applied to optimize the 3 effective variables: MSG concentration (between 25 and 150 mM), incubation temperature (from 25 to 37 °C), and pH

(4.0–5.0). Varying these three independent parameters, a total of 20 experiments for each isolate were designed and performed. The statistical software Design Expert 10.0.7.0 was used for

data analysis12,40. Laboratory-scale fermentations were set up to investigate optimal parameters for GABA production by selected isolates. Bacterial isolates were inoculated into falcon

tubes containing 50 mL MRS broth medium with different concentrations of MSG with initial pH adjustment and incubated at different temperatures for 48 h to reach optimum GABA production.

After 48 h of incubation, samples were withdrawn from the each falcon tube to determine GABA content. STATISTICAL ANALYSIS To analyze the results related to the potential of GABA production

by examined bacteria, Duncan’s one-way analysis of variance (ANOVA) was applied using the SPSS software (ver. 16), while to optimize the conditions of GABA production, RSM with Design Expert

10.0.7.0 software was used. RESULTS SCREENING OF GAD ACTIVITY Total number of LAB isolates and the results of their previous identification31,33,34,35 are presented in Table 1. These

isolates include members of lactobacilli (of the genera _Lactobacillus, Lactiplantibacillus_, and _Levilactobacillus_) (27 isolates), _Lactococcus_ (20 isolates), and _Streptococcus_ (3

isolates). The GAD activity of the 50 isolates from Iranian traditional dairy products are also summarized in Table 1. The cultures of the isolates _Levilactobacillus_ (_Lb._) _brevis_ M2,

M4 and M12; _Streptococcus_ (_St._) _thermophilus_ 43, and _Lac. lactis_ 261, 311, 454, and 491 developed a strong blue color, consistent with a high GAD activity. No color change was

observed for the isolates M3, M6, M7, M8, M9, M13, M15, M17, and M19; thus these are considered not to have GAD activity. Other isolates with greenish-blue, green and greenish-yellow color

were considered as having medium, low and very low GAD activity, respectively. Fourteen isolates, including all high and some with moderate GAD activity were selected for subsequent

experiments. GAD assay results revealed that most of the GAD-positive isolates were isolated from cheese (Lighvan and Motal); whereas, only one isolate from a traditional yogurt exhibited

high GAD activity. GENETIC ANALYSIS OF ISOLATES Lactobacilli, _Streptococcus_ and _Lactococcus_ species were targeted by rep-PCR to evaluate the intramolecular diversity among the isolates,

as well as to assess whether isolates with GAD activity belonging to the same species were replicates or independent strains. Further, typing of the strains could serve for selecting

different strains for GABA production optimization. The different patterns of similarity were clustered using the SM coefficient by the UPGMA method. Given the reproducibility of the assay

around 94 (Fig. 1), isolates sharing a percentage of similarity > 94% were considered to belong to the same strain. Figure 2 a, b, c and d are the profiles obtained from 3 isolates of

_Lb. brevis_, 4 isolates of _Lac. lactis_, 3 isolates of _St. thermophilus_ and 4 isolates of _Lb. delbrueckii. Lb. brevis_ M2 and _Lb. brevis_ M12 strains showed 100% similarity. _Lac.

lactis_ 311 and _Lac. lactis_ 454 strains had 100% similarity. _St. thermophilus_ 74 and _St. thermophilus_ 43 also had 100% similarity. These strains can be multiple isolates.

_Lactobacillus_ (_Lb._) _delbrueckii_ 69 and _Lb. delbrueckii_ 67 also showed very high homology. The relation between the intra-species diversity with GABA production in these species

showed that GABA production was significantly different between isolates with high similarity and comparable between isolates with low similarity. Unlike other examined strains, there was no

significant difference in GABA production in _St. thermophilus_ isolates with high and low similarity. CONFIRMATION OF GABA BY THIN LAYER CHROMATOGRAPHY Isolates that showed no GAD activity

by the colorimetric method were excluded from further analyses. Based on the results of GAD activity, we selected 14 isolates corresponding to the species _St. thermophilus_ (three), _Lb.

delbrueckii_ (four), _Lac. lactis_ (four), _Lb. brevis_ (three) for further studies. The GABA production was confirmed by observing red spots on the TLC plates (Fig. 3). The spot mobility

from the culture supernatant of the 14 isolates was consistent with GABA, as a retention factor (Rf) value (0.77 cm) of the sample matched with the GABA standard. Thus, all 14 selected

isolates were considered as GABA producers. GABA’s of strains show the same retention factor (Rf) value as the GABA standard (= 0.77 cm). The retention factor was defined as the ratio of the

distance traveled by the center of a spot to the distance traveled by the solvent front. QUANTITATIVE GABA PRODUCTION MEASUREMENT GABA production of selected strains was quantified using a

GABase microtiter plate enzymatic determination. As shown in Table 1, GABA production varied widely ranging from 0.01 to 0.36 mg/L (equivalent to 0.12 and 3.52 mM). All _Lb. brevis_ isolates

from Motal cheese exhibited a high ability to produce GABA (> 1 mM), while the highest GABA concentration was produced by _Lb. brevis_ M4. Our findings further revealed that all isolates

belonging to _Lac. lactis_ have a medium to high GABA producing ability, while all _Lb. delbrueckii_ cultures presented a low level of this ability. RSM DESIGN Although _Lac. lactis_

isolates produced less GABA than the _Lb. brevis_ isolates, the former species is the most economically-important starter component in cheese43, which prompted us to select two different

strain of this species, _Lac. lactis_ 311 and _Lac. lactis_ 491, for GABA production optimization by RSM methodology. The selected two _Lac. lactis_ strains are different strains according

to the rep-typing results.As stated in the corresponding section, a CCD (central composite design) was performed based on a single variable factor test at a time and considering three

independent variables: MSG concentration (mM), Temperature (°C), and pH. The 20 different combination set ups based on CCD and the corresponding GABA concentration for both predicted and

experimental values are listed in Tables 2 and 3, respectively. The interaction effect of these factors on GABA production concentration by _Lac. lactis_ 311 is described by the following

equation: $$\begin{aligned} {\text{R}} = & 0.{18} + 0.0{23} \times {\text{A}} + 0.0{19} \times {\text{B}} + {5}.{\text{429E}} - 00{3} \times {\text{C}} + {2}.{\text{363E}} - 00{3} \times

{\text{AB}} - {1}.{\text{937E}} \\ - 00{3} \times {\text{AC}} + {3}.{\text{863E}} - 00{3} \times {\text{BC}} - 0.0{16} \times {\text{A}}^{{2}} - 0.0{2}0 \times {\text{B}}^{{2}} - 0.0{13}

\times {\text{C}}^{{2}} \\ \end{aligned}$$ (1) where R is the concentration of GABA (mg/mL) and A, B and C are coded values of the independent variables, viz., MSG, temperature and pH,

respectively. Three-dimensional surface plots were generated to estimate the effect of the combinations of the independent variables on the GABA production using Design Expert software. The

3D plot in Fig. 4A shows the interaction effect of MSG and temperature, keeping pH at its central level 4.5. According to the 3D plot, predictably, the GABA concentration increased with

increasing MSG concentration and the increase of temperature. Nevertheless, in the low MSG concentration, increasing GABA concentration was more sharply compared to a higher concentration.

The maximum concentration (3.83 mM) was observed with MSG in concentration of 89 mg/mL and temperature of 35.4 °C. Figure 4B presents the 3D plot showing the dependency of GABA concentration

on MSG concentration and pH, keeping the third factor (temperature) at its central level of 31 °C. The graph predicted that the highest GABA concentration by relevant strain at 35.4 °C was

observed with the mid pH (4.5) and MSG concentration (89 mg/ml). However, GABA concentration at lower pH remained approximately constant by increasing MSG concentration. The 3D curve showing

the interaction effect of temperature and pH has shown in Fig. 4C. The low GABA concentration was observed with the lowest temperature and lowest pH. The maximum GABA concentration (3.83 

mM) was observed with pH (4.5) and temperature (35.4 °C). Equation 2 explains the interaction effect of the independent variables on GABA concentration by _Lac. lactis_ with strain code 491:

$$\begin{aligned} {\text{GABA}} = & 0.{39} + 0.0{32} \times {\text{A}} + 0.0{48} \times {\text{B}} + {6}.{\text{314E}} - 00{3} \times {\text{C}} - 0.0{11} \times {\text{AB}} -

{7}.{\text{459E}} \\ - 00{4} \times {\text{AC}} - 0.0{14} \times {\text{BC}} - 0.0{19} \times {\text{A}}^{{2}} - 0.0{43} \times {\text{B}}^{{2}} - 0.0{28} \times {\text{C}}^{{2}} \\

\end{aligned}$$ (2) Figure 5A showed that with increasing the concentration of monosodium glutamate and increasing the temperature, the amount of GABA formed increased. This trend is in

agreement with the results reported elsewhere by other authors41,42. An increase in the amount of GABA was observed with increasing MSG and pH to 89 mg/ml and 4.59, respectively, at a

constant temperature (Fig. 5B). When the concentration of monosodium glutamate was kept constant, the GABA level increased sharply with increasing temperature, but increased pH led to the

production of low amounts of GABA (Fig. 5C). The incubation temperature is another important factor that may affect GABA production. In addition to the stability and biological activity of

the enzyme, temperature also affects the thermodynamic equilibrium of the reaction. SELECTION OF OPTIMAL TREATMENT AND VALIDATION OF MODEL The results of GABA production by two examined

strains showed the optimal conditions for GABA production by _Lac. lactis_ 311 and _Lac. lactis_ 491 had a temperature of 35.4 and 30 °C, a pH of 4.5, 4.6 and a concentration of 89 and 147.4

 mM of MSG, respectively. In the above conditions, the amount of GABA production by these two strains was 3.83 and 1.73 mM, respectively. Examination of the results in Fig. 6 shows that the

points with a good approximation are on the straight line and the values obtained experimentally are in a good agreement with the values predicted by the model and showed the accuracy of the

equation model. DISCUSSION Regarding the Motal cheese isolates in this study, the results are consistent with previously reported levels by Ebadi Nezhad et al. (2020)44. GABA production

begins in the exponential growth phase and increases near to the stationary phase due to an increased in GAD activity, which agrees with GAD being an intracellular enzyme produced in

response to acidic conditions45. The nutrient sources required for the GABA production by bacteria include the primary sources of carbon, nitrogen (including GABA substrate, glutamic acid)

and minerals46. To avoid the use of replicates from the optimization studies, a strain-level typing scheme by rep-PCR was employed. Knowing intra-species diversity also helps to know how

diverse the extend of GABA production is in closely-related and unrelated strains of the same species10. As for many other phenotypic traits, large phenotypic variations have occasionally

been reported for genetically closely-related LAB strains47. Typing of LAB isolates from Koozeh and Lighvan cheese by rep-PCR with BOXA2R primer and typing of GABA-producing LAB isolates

with rep-PCR by (GTG)5 primer has been previously reported by others10,31. As in this work, high intraspecies diversity was reported in the referred studies. Presumptive detection of GABA

production by LAB species by TLC, followed by its confirmative identification and quantification by High-performance liquid chromatography (HPLC) has been previously reported to be a

successful strategy by Zhang et al.48. These authors reported that the mobility spot of the culture supernatant of BC114 strain was basically consistent GABA standard and could be

preliminarily identified as GABA within a negligible margin of error. The measured sample from the fermentation broth (a complex matrix), may also result in incomplete agreement with pure

GABA standards48. The strain under investigation, _Lb. plantarum_ BC114, showed the highest GABA production as measured by HPLC, which reached a value of 1.52 ± 0.07 g/L48. Tanamool et al.49

also used the TLC method to screen GABA-producing lactic acid bacteria strains. These authors found that among 44 LAB, the isolates L10-11 clearly produced the highest GABA based on the TLC

results49. In our results, 14 isolates among 50 LAB were considered as GABA producers. Valenzuela et al.25 showed the ability of _Lb. brevis_ to produce GABA is consistent with other

reported levels for _Lb. brevis_ LMG6906 (0.29 g/L). _Lb. brevis_ CECT8183 isolated from Spanish cheese by Diana et al.17 produced 0.1 g/L of GABA. _Lb. brevis_ BJ20 produced 0.002 g/L in a

fermented sea tangle solution, which is a popular traditional marine food in Korea based on brown seaweed50. _Lb. brevis_ PM17 was among the most potent studied isolates by Franciosi et

al.9. By contrast, no significant GABA production was detected for any _St. thermophilus_ strain isolated from yogurt products, compared to lactobacilli and _Lactococcus_ species in study.

According to the literature, large numbers of strains from these genera from various fermented foods are indicated as GABA-producing LAB13. There are many publications reporting variable

GABA production levels by different LAB species. Among other factors, production could be affected by the GABA detection methods. For instance, _Lb. delbrueckii_ PR1 (63 mg/kg) and _Lac.

lactis_ PU1 showed the highest GABA concentrations9. The results of Ly et al.10 showed _Lb. futsaii_, _Lb. namurensis_ and _Lb. plantarum_ to produce high amounts of GABA in the range of

1.7–2 g/L. Hwang et al.51 have examined GABA production by two _Lac. lactis_ strains in MRS containing 5% MSG at 30 °C. Maximum GABA production was observed after 40 h (1.37 g/L). Redruello

et al.52 studied the effect of 6 _Lac. lactis_ GABA-producing strains in cheese making. GABA accumulated at concentrations up to 0.457 g/Kg in cheese. Galli et al.53 revealed GABA content of

their fermented milks by two mix starters (_Lac. lactis_ and _Lb. rhamnonus_ or _Lb. paracasei_) 0_._185 and 0.319 g/L, respectively by adding 249 mg/L MSG. Santos-Espinosa et al.54

demonstrated the highest GABA concentration (0.086 g/L) in fermented milk with _Lac. lactis_ L-571 at 37 °C with 3 g/L of glutamate substrate. In a study conducted by Tajabadi et al.55 the

optimum conditions for maximum GABA production by _Lb. plantarum_ Taj-Apis362 were an initial glutamic acid concentration of 497.97 mM, culture temperature of 36 °C, initial pH of 5.31 and

incubation time of 60 h, which produced 0.74 g/L of GABA. GABA-producing ability is linked to the activity of GAD enzyme in LAB56. The source of LAB might affect GAD activity and GABA

production capacity, as it was speculated that acidified foods could probably be the natural niche of GABA producers10. Li et al.57 identified and characterized the GABA-producing lactic

acid bacteria and claimed that most LAB strains are able to produce the highest amount of GABA in the range of pH 4 to 5 at 30 to 50 °C and in the presence of glutamic acid. For this reason,

this temperature and pH range was used in their study57. GABA production occurs under acidic conditions. LAB metabolism mainly leads to the production of organic acids such as lactic and

acetic acid. This means that they have to frequently face acidic stress and therefore, they have developed different mechanisms to cope with acidic conditions such as the GAD pathway.

Laroute et al.58 claimed the activation of this pathway occurred after changing the pH to 4.6 and during the stationary phase. Their results are consistent with our results which optimum pH

for GABA production in two examined isolates is 4.5 and 4.6. As reported by Laroute et al.58 a dose of 152 mM glutamate, increased the production of GABA by _Lac. lactis_ NCDO 2118_._ This

dosage of glutamate is similarly optimum dose for GABA production in our examined isolate (_Lac. lactis_ 491). The findings of our work showed _Lac. lactis_ 311 produced more GABA in lower

concentration than _Lac. lactis_ 491. The probable reason that lower concentrations of MSG can increase the efficiency of GABA production in comparison to higher concentrations of it, is

that large amounts of MSG increase the osmotic pressure of cells and disrupt bacterial metabolism, leading to a decrease in GABA efficiency by bacteria 59. Although the optimal

concentrations of MSG are different for various microorganisms in GABA production, some researchers have proven that excessive MSG could inhibit cell growth and minimize GABA

production60,61. Lu et al.62 showed that the highest concentration of GABA production by _Lac. lactis_ isolated from kimchi was at 34° C (3.68 g/L). These results are similar to our results

about _Lac. lactis_ 311. However, they stated that the optimal pH of GABA production was in the range of 7–862, which was not in compliance with the results obtained in our study.

CONCLUSIONS In the present study, firstly, LAB isolates were screened for GABA production according to a qualitative method based on a colorimetric assay. Significant variability in GABA

production was encountered among the different species and strains tested. GABA-producing strains were then subjected to a TLC analysis to detect strains with high GABA producing ability.

Response surface methodology was finally used to optimize conditions for maximum GABA production in two _Lac. lactis_ strains, taking into account the variables affecting the response. The

selected strains and the culture conditions producing the highest amounts of this bioactive compound could be used to develop GABA-enriched functional foods or in other biotechnological

applications. DATA AVAILABILITY The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. ABBREVIATIONS * LAB: Lactic acid

bacteria * GRAS: Generally recognized as safe * GABA: Gamma-aminobutyric acid * GAD: Glutamate decarboxylase * NADP: Nicotinamide adenine dinucleotide phosphate * MSG: Monosodium glutamate

* TLC: Thin layer chromatography * rep-PCR: Repetitive extra genic profiling PCR * RSM: Response surface methodology * CCD: Central composite design * Rf: Retention factor REFERENCES * FDA.

GRAS Exemption Claim for gamma-Aminobutyric acid (GABA) for Use as a Food Ingredient in the United States (U.S.). . (FDA), Food and Drug Administration Co, Pharma Food International; 2015. *

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J._ 41(1), 48–52 (2008). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS The research of the corresponding author is supported by a grant from Ferdowsi University of

Mashhad (N.2/45302). FUNDING This work was supported by [Ferdowsi University of Mashhad] (Grant Numbers [2/45302]). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Food Science

and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad (FUM), Mashhad, Iran Mohammad Reza Edalatian Dovom, Mohammad Bagher Habibi Najafi, Paria Rahnama Vosough, Neda Norouzi 

& Seyyed Javad Ebadi Nezhad * Instituto de Productos Lácteos de Asturias (IPLA-CSIC), 33300-Villaviciosa, Asturias, Spain Baltasar Mayo Authors * Mohammad Reza Edalatian Dovom View

author publications You can also search for this author inPubMed Google Scholar * Mohammad Bagher Habibi Najafi View author publications You can also search for this author inPubMed Google

Scholar * Paria Rahnama Vosough View author publications You can also search for this author inPubMed Google Scholar * Neda Norouzi View author publications You can also search for this

author inPubMed Google Scholar * Seyyed Javad Ebadi Nezhad View author publications You can also search for this author inPubMed Google Scholar * Baltasar Mayo View author publications You

can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.R.E.D.: Supervision, Project administration, Writing – review & editing. M.B.H.N.: Supervision, Writing—review

& editing. N.N.: Investigation, Writing—original draft. P.R.V.: Investigation, Writing—review & editing. S.J.E.N.: Investigation, Writing—review & editing, B.M.: review &

editing. CORRESPONDING AUTHOR Correspondence to Mohammad Reza Edalatian Dovom. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

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CITE THIS ARTICLE Edalatian Dovom, M.R., Habibi Najafi, M.B., Rahnama Vosough, P. _et al._ Screening of lactic acid bacteria strains isolated from Iranian traditional dairy products for GABA

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