LWT - Food Science and Technology 184 (2023) 115014

Contents lists available at ScienceDirect

LWT
journal homepage: www.elsevier.com/locate/lwt

Gamma-aminobutyric acid (GABA) promotes characteristics of

Levilactobacillus sp. LB-2
Haifeng Li a, b, *, Bingbing Li a, Lishan Gao a, Ruping Ge a, Xinyu Cui a, Jia Zhou a, Zhijian Li b, c
a
School of Biological Engineering, Henan University of Technology, Zhengzhou, 450001, China
b
National Engineering Laboratory/Key Laboratory of Henan Province, Henan University of Technology, China
c
College of Food Science and Engineering, Henan University of Technology, Zhengzhou, 450001, China

A R T I C L E I N F O A B S T R A C T

Keywords: γ-Aminobutyric acid (GABA) is a pharmaceutical, bioactive amino acid that can be produced by lactic acid
GABA production bacteria (LAB), but the yield of GABA is still limited by several reasons. In this study, a GABA-producing LAB
Levilactobacillus sp. strain Levilactobacillus sp. LB-2 (OP890629) was isolated from a Chinese traditional dough fermentation starter of
Optimization
Jiaozi. When subjected to GABA production optimization applying one-factor-at-a-time (OFAT) and response
Response surface methodology
Steamed bread
surface method (RSM), the temperature, pH, and L-monosodium glutamate (L-MSG) concentration for GABA
production were set as 37 ◦ C, pH 4.5, and 2.5 g/L, respectively. The maximum amount of GABA produced by
strain Levilactobacillus sp. LB-2 was 49.42 g/L, which was 3.5-fold higher than that before optimization of 14.19
g/L. The GABA content of steamed bread made by Levilactobacillus sp. LB-2 significantly increased. Thus, strain
Levilactobacillus sp. LB-2 could be used for the production of GABA or applied for the development and
improvement of GABA-enriched foods.

Credit author Statement
Bingbing Li: conducted experiments, Lishan Gao conducted experi­
ments; Haifeng Li conceived and designed research; Bingbing Li:
contributed new reagents or analytical tools, Ruping Ge: contributed
new reagents or analytical tools, Xinyu Cui contributed new reagents or
analytical tools; Jia Zhou: analyzed data, Zhijian Li analyzed data;
Bingbing Li: wrote and edited the manuscript, Haifeng Li: wrote and
edited the manuscript. All authors read and approved the manuscript.
1. Introduction
Gamma-aminobutyric acid (GABA) is a non-protein amino acid
widely distributed in vertebrate animals, plants, and bacteria (Sharma
et al., 2021), and it is the end product of the decarboxylation of glutamic
acid (GA) in lactic acid bacteria (LAB) by the enzyme of glutamic acid
decarboxylase (GAD, EC 4.1.1.15) (Falah et al., 2021). Studies have
proven that GABA has various beneficial physiological functions such as
lowering blood pressure, anti-aging, anti-anxiety, tranquilizing,
improving sleep quality, strengthening the liver and kidney, alleviating
diabetes, enhancing immunity, preventing obesity, and promoting
alcohol metabolism in mammals (Cui et al., 2020; Dhakal et al., 2012; Li
et al., 2020; Ngo & Vo, 2019; Park et al., 2021; Sarasa et al., 2020;
Venturi et al., 2019; Wan-Mohtar et al., 2020). It also plays important
roles in the development of neural diseases such as schizophrenia, Alz­
heimer’s disease, Parkinson’s disease, and Huntington’s disease (Die­
z-Gutiérrez et al., 2020). Therefore, as a bioactive compound, GABA has
attracted the attention of researchers in the pharmaceutical and food
industries. The formulation of neurotransmitter GABA-enriched foods or
functional foods that can deliver GABA is an opening research and
development target for the food industry. These foods include dairy
products such as yogurt, cheese, fermented milk products, soybean, and
juice products. Therefore, the due acknowledgement should be given to
the mass production of GABA and its subsequent use in the modern food
industry as a bioactive food ingredient (Mancini et al., 2019).
GABA can be produced by chemical synthesis, plant enrichment, and
microbial fermentation (Wu et al., 2022). Given the serious environ­
mental pollution, unnatural and unsafe by-products associated with
chemical synthesis (Diana et al., 2014), and the low yields and unclear
mechanism of plant enrichment (Sahab et al., 2020), microbial
fermentation has been studied extensively. At present, it is considered as
the main GABA-producing method, which is simple, efficient, and
environmentally friendly (Cataldo et al., 2020).
GABA is primarily produced through the fermentation of bacteria,

* Corresponding author. School of Biological Engineering, Henan University of Technology, Zhengzhou, 450001, China.
E-mail address: hfli@haut.edu.cn (H. Li).

https://doi.org/10.1016/j.lwt.2023.115014
Received 28 March 2023; Received in revised form 21 June 2023; Accepted 23 June 2023
Available online 8 July 2023
0023-6438/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
H. Li et al.
fungi, and yeasts. Good GABA producers were composed of Levilacto­
bacillus brevis, Lactiplantibacillus plantarum, Lactobacillus paracasei,
Lactobacillus fermentum (Kim et al., 2022; Tanamool et al., 2019),
Enterococcus faecium BS5 (Bs et al., 2021), Saccharomyces cerevisiae
(Zhang et al., 2022), Aspergillus oryzae strain NSK (Wan-Mohtar et al.,
2019), and Kluyveromyces marxianus (Zhang et al., 2022). Among them,
LAB is generally regarded as a safe microorganism, and it has been re­
ported to synthesize larger amounts of GABA compared with other mi­
croorganisms (Falah et al., 2022). For example, GABA concentration
could reach 53.61 g/L in the medium produced by a strain of Levi­
lactobacillus brevis screened from kimchi (Liu et al., 2022).
The production of GABA usually begins in the logarithmic phase of
bacteria and increases with GAD activity during stabilization (Falah
et al., 2021; Sharafi and Nateghi, 2020). Bacterial GABA fermentation is
influenced by a number of factors, including substrate, pH, temperature,
and culture time. The carbon source and L-monosodium glutamate
(L–MSG) are the substrates for cell growth and GABA conversion, and
they are the main factors in GABA production (Phuengjayaem et al.,
2021). Recently, the GABA production properties of many probiotic LAB
strains have been investigated by Sharafi and Nateghi, 2020, but the
researches about increasing the GABA content in steamed bread made
by the LAB strains was little.
Steamed bread is a kind of traditional staple foods in China. Besides
flour and water, microbial fermentation starter is usually required to
make steamed bread (Kim et al., 2009; Laohasongkram et al., 2011). The
steamed bread prepared by yeast lacks the flavor and taste produced by
traditional mixed strain (TMS) starter cultures, which are preferred by
consumers (Li et al., 2014). The ratio of LAB to yeast in sourdough is
generally 100:1, demonstrating the dominant role played by LAB in
sourdough (Gobbetti, 1998). LAB can also produce volatile aroma
compounds to provide products with desirable flavour and improve the
safety of products (Suo et al., 2021). Therefore, LAB have been widely
applied in steamed bread production. The amount of GABA in the ma­
jority of fermented foods (especially for steamed bread) and beverages is
small. Thus, fermentation should be optimized to produce higher
amounts of GABA (Sahab et al., 2020) in food to meet the needs of
human health.
In the current study, a LAB strain Levilactobacillus sp. LB-2 was
selected for GABA production. In addition, the medium composition and
fermentation conditions of this strain were optimized by using one-
factor-at-a-time (OFAT) and response surface method (RSM) to
improve GABA concentration. Then, sourdough fermented by yeast and
Levilactobacillus sp. LB-2 was used to make steamed bread, thereby
laying the foundation for the development of GABA functional food.
2. Materials and methods
2.1. Strains and medium
The strains used in this study included LAB Levilactobacillus sp. LB-2,
Lactobacillus sp. LF-5, Leuconostoc sp. M1, Lactobacillus sp. K7, acetic acid
bacteria (AAB) Acetobacter sp. A3, and yeast S. cerevisiae Y5, and they
were all isolated from dough starters of Laomian and Jiaozi and stored in
a − 80 ◦ C refrigerator.
The MRS medium for LAB and AAB culture contained 10 g/L of
peptone, 10 g/L of beef extract, 5 g/L of yeast extract, 2 g/L of
K2HPO4⋅3H2O, 2 g/L of C6H17N3O7, 5 g/L of CH3COONa, 20 g/L of
glucose, 1 mL/L of tween 80, 0.58 g/L of MgSO4‧7H2O, and 0.25 g/L of
MnSO4‧H2O. The pH value of the medium was adjusted to 6.2–6.4 using
a pH meter (PHS–2F, Shanghai, China). The MRS medium was sterilized
using a high pressure steam sterilization pot (LDZX-50L, Shanghai,
China) at 115 ◦ C for 30 min. And the MRS solid medium was added with
20 g/L of agar and sterilized as previously described.
The YPD medium for yeast culture contained 20 g/L of peptone, 20
g/L of glucose, and 10 g/L of yeast extract. It was sterilized at 115 ◦ C for
30 min. The solid YPD medium was added with 20 g/L of agar and
2
LWT 184 (2023) 115014
sterilized as previously described.
Peptone, yeast extract powder and beef extract were all purchased
from Beijing Aobo Star Co., Ltd. (Beijing, China). Chemicals with
analytical grade like glucose, agar, CH3COONa, C6H17N3O7,
K2HPO4⋅3H2O, tween 80, MnSO4‧H2O, and MgSO4‧7H2O were all pur­
chased from Tianjin Kemi Ou Co., Ltd. (Tianjin, China).
2.2. Strain activation
The test strains were thawed and spread on several sterilized MRS
plates (Kantachote et al., 2017) and then incubated in a
constant-temperature incubator (DH-600, Beijing, China) at 37 ◦ C for
24 h. After being activated for two generations under the same condi­
tions, some of the plates were placed in a refrigerator (BCD-202M/TC,
Guangdong, China) at 4 ◦ C, and some of the single colony on the other
plates was selected for follow-up experiment.
2.3. Quantitative estimation of GABA in fermented broth
Each tested LAB strain was transferred to MRS liquid medium after
activation and incubated at 37 ◦ C for 24 h to logarithmic period. Then,
the strains were inoculated at 4% inoculum amount into a fermentation
medium and incubated at 37 ◦ C stewing for 24 h. In addition, AAB
Acetobacter sp. A3 was incubated in a shaker (BSD-TX270, Shanghai,
China) under a shaking condition of 160 r/min. The amount of GABA
produced by the LAB and AAB strains was determined quantitatively by
high-performance liquid chromatography (LC-2030C 3D Plus, Kyoto,
Japan) as the methodology described by Sharma et al., 2021 with slight
modification. Then, the strain with the highest GABA production ca­
pacity was selected.
2.4. One-factor-at-a-time-based culture optimization for GABA
production
First, the OFAT strategy was applied to determine the optimal carbon
and nitrogen source. The carbon source present in the defined MRS
medium (glucose) was replaced with sucrose, fructose, sodium acetate,
and soluble starch. Similarly, nitrogen sources (peptone and beef
extract) present in the defined MRS medium were replaced with
peptone, yeast powder, beef extract, urea, and potassium nitrate.
Second, the effect of substrate concentrations and ammonium citrate
addition on the GABA production of strain Levilactobacillus sp. LB-2 was
investigated by adjusting the concentration of L–MSG (food grade,
Shanghai, China) to 0.0, 2.5, 5.0, 7.5, and 10.0 g/L based on optimal
carbon and nitrogen sources, as well as the medium with and without
ammonium citrate. Other parameters were individually optimized by
altering within reasonable ranges, such as 10–55 g/L of sucrose con­
centration, 10–44 g/L of peptone concentration, 1–9 days of fermenta­
tion time, 1%–5% inoculation amount, and 3.8–8.0 initial pH.
2.5. Response surface method-based optimization for GABA production
Based on the results of OFAT experiments, three of the cultural fac­
tors that had a significant effect on GABA yield, namely, initial pH, su­
crose (g/L), and peptone (g/L), were selected as independent variables,
and other factors were fixed. Based on the Box–Behnken design idea, a
three-factor, three-level response surface analysis test was designed
using Design Expert 12 with GABA yield (g/L) as the response value to
determine the conditions for optimal GABA production from strain
Levilactobacillus sp. LB-2. The optimal GABA-producing conditions were
optimized by RSM for fermentation trials, and then the test results were
processed and analyzed using Design Expert 12 to determine the optimal
culture conditions. Moreover, the validity of the model was verified by
comparing the model predicted values with the experimental values.
H. Li et al.
2.6. Sourdough preparation and GABA-enriched steamed bread making
Four different treatments were set in accordance with distinct com­
binations of microbial starters (Li et al., 2021): CK (control check: with
no addition of starter), Y (only added with yeast: 108 CFU/g), YL (added
with 108 CFU/g of yeast and 107 CFU/g of Levilactobacillus sp. LB-2), and
YLA (added with 108 CFU/g of yeast, 107 CFU/g of Levilactobacillus sp.
LB-2, and 107 CFU/g of Acetobacter sp. A3).
Sourdoughs were prepared from flour (100 g), microbial starters, and
55 mL of sterile water. The sourdoughs were fermented at 32 ◦ C and
85% relative humidity for 1 h in a controlled fermenting box. Then, the
sourdoughs were rounded and molded manually and fermented again in
the fermenting box to verify the abovementioned parameters. After­
ward, the rounded dough samples were steamed for 25 min and stuffed
for 5 min. The quality of steamed breads was evaluated after cooling at
room temperature for 1 h.
2.7. Measurement of quality properties of steamed bread
2.7.1. Appearance quality
The appearance qualities of steamed bread such as specific volume
and height-to-diameter ratio were in accordance with the reported
methods (Li et al., 2014). The specific volume was determined using the
rape seed displacement method.
2.7.2. GABA content of the steamed bread
The method modified by Coda et al., 2010 was used. 5.0 g of steamed
bread was accurately weighed, added with 25 mL of distilled water,
stirred well, and then extracted in a water bath at 80 ◦ C for 2 h. The
extract was transferred into a centrifuge tube, and centrifuged at 8000
r/min for 10 min. Then, the supernatant was collected to analyze GABA
content.
2.8. Statistical analysis
All experiments were conducted in triplicate, and the results were
expressed as mean ± standard deviation. Statistical analysis was per­
formed using oneway ANOVA and then applied Tukey s-b’ s post hoc
test. The values were considered signiffcantly different when p < 0.05.
All statistical data were conducted using SPSS software (SPSS Inc., CH,
USA).
3. Results and discussions
3.1. Screening of the best GABA-producing microbial strain
As shown in Table 1, the six strains selected for this experiment could
produce GABA, but the yields were different, among which the highest
yield (14.19 g/L) was obtained from Levilactobacillus sp. LB-2. And the
strains belonged to Lactobacillus brevis or Levilactobacillus brevis always
had the highest potential of GABA production (Liu et al., 2022; Sharafi
Table 1
Comparison of GABA-production abilities of different microbial strains.
Strain GABAJ (g/L) GABAY (g/L)
a
Levilactobacillus sp. LB-2 14.19 ± 0.18 13.19 ± 0.22a
Lactobacillus sp. LF-5 13.94 ± 0.34a 11.80 ± 0.34b
Leuconostoc sp. M1 12.33 ± 0.31b 9.12 ± 0.30d
Lactobacillus sp. K7 9.37 ± 0.43d 9.39 ± 0.39d
Acetobacter sp. A3 – 10.74 ± 0.38c
S. cerevisiae Y5 10.45 ± 0.39c 11.08 ± 0.32b
Note: Values are presented as mean ± standard deviation of three individual
experiments. Different letters in the same column (a–d) represent significant
differences (P < 0.05).
GABAJ: GABA content produced by strains under static culture conditions;
GABAY: GABA content produced by strains under shaker culture conditions.
3
LWT 184 (2023) 115014
and Nateghi, 2020). Followed by Lactobacillus sp. LF-5 (13.94 g/L), and
the lowest yield (9.37 g/L) was obtained from Lactobacillus sp. K7. Under
shaking cultural conditions, the GABA yield of AAB Acetobacter sp. A3
reached 10.74 g/L.
Levilactobacillus sp. LB-2 had the best GABA production capacity
among the six tested strains, and strains of LAB with good GABA pro­
duction capacity were well studied previously (Lim et al. 2017).
Therefore, Levilactobacillus sp. LB-2 was selected as the functional strain
for subsequent experiments (see Fig. 1).
3.2. Effect of medium components on GABA production of
Levilactobacillus sp. LB-2
The composition of the medium significantly affects microbial GABA
production (Lim et al., 2017). In particular, medium components such as
carbon and nitrogen sources exert a strong influence on GABA produc­
tion (Edalatian Dovom et al., 2023; Lim et al., 2017). As shown in
Fig. 2A, sucrose was determined to be a good carbon source for GABA
production (14.48 g/L), and GABA concentration was the lowest when
sodium acetate was used as the carbon source. Previous studies have
reported that some strains of Levilactobacillus preferred sucrose over
glucose as the carbon source (Kook et al., 2010; Seo et al., 2013a).
The effect of nitrogen sources on GABA production by Levilactoba­
cillus sp. LB-2 was also investigated using MRS medium containing su­
crose as the carbon source. As shown in Fig. 2B, the GABA content
(14.92 g/L) produced by the strain with peptone as the nitrogen source
was the highest, whereas the addition of inorganic nitrogen sources had
no beneficial effect on GABA synthesis. Therefore, the optimum carbon
and nitrogen sources for GABA production by Levilactobacillus sp. LB-2
were determined as sucrose and peptone, respectively.
L–MSG is an ideal substrate for GABA production, and its concen­
tration directly affects the production of GABA (Lim et al., 2017; Park
et al., 2021). The effect of initial L–MSG concentration on GABA pro­
duction by Levilactobacillus sp. LB-2 was determined using the
as-optimized culture medium (modified MRS medium containing su­
crose and peptone) under a fixed culture condition (37 ◦ C). The
maximum GABA production (15.37 g/L) was obtained upon the addition
of 2.5 g/L of L–MSG (Fig. 2C), and as the L–MSG concentration increased
from to 5.0–10.0 g/L, the growth of the bacterium was inhibited and the
GABA yield decreased. It also have been reported that the relatively low
L–MSG concentration was optimal for the GABA production by bacteria
(Li et al., 2010; Lim et al., 2016; Yang et al., 2008). Therefore, low
concentrations of L–MSG can increase the efficiency of GABA
Fig. 1. Growth curve of Levilactobacillus sp. LB-2 in the MRS medium.
H. Li et al.
Fig. 2. Effects of carbon sources (A), nitrogen sources (B), MSG concentrations (C), ammonium citrate (D), sucrose concentrations (E), and peptone concentrations
(F) on the GABA production by Levilactobacillus sp. LB-2.
production. By contrast, at high concentrations of L–MSG, large amounts
of L–MSG increase the osmotic pressure of cells and disrupt bacterial
metabolism, inhibiting the bacterial growth and glutamate decarbox­
ylase activity (Edalatian Dovom et al., 2023; Komatsuzaki et al., 2005).
Although L–MSG is regarded as a requisite substrate for GABA genera­
tion, the optimum concentration of L–MSG is different for various mi­
croorganisms in GABA production (Edalatian Dovom et al., 2023;
Komatsuzaki et al., 2005; Li et al., 2010).
With regard to the effect of sucrose and peptone concentrations,
GABA production of the strain in the fermentation medium increased
with the increase of the amount of sucrose added, with a maximum
GABA content of 18.48 g/L as 35 g/L of sucrose was added. When the
sucrose concentration was more than 35 g/L, the GABA content may
decrease (Fig. 2E). The GABA content in the fermentation medium also
increased with the addition of peptone in the range of 10–32 g/L, and it
4
LWT 184 (2023) 115014
reached the maximum value of 22.18 g/L when 32 g/L of peptone was
added. At high peptone concentration, the GABA content decreased
(Fig. 2F). Although the abundance of carbon and nitrogen sources is
favorable to the growth of the bacteria, excessive nutrients will lead to
strong division of the bacteria themselves, thereby inhibiting the syn­
thesis or activation of related enzymes and affecting the conversion of
the substrates.
With regard to the effect of ammonium citrate, GABA content
increased with the addition of ammonium citrate from the 1st day until
the 9th day. By contrast, in the group without ammonium citrate, GABA
production reached the maximum of 24.73 g/L at the 5th day and then
decreased evidently at the 9th day (Fig. 2D).
H. Li et al.
3.3. Effect of culture conditions on GABA production of Levilactobacillus
sp. LB-2
With the optimum medium substrate, the GABA production of Levi­
lactobacillus sp. LB-2 reached 22.29 g/L at the 1st day and reached the
maximum value of 22.36 g/L at the 5th day, after which the GABA
content started to decrease with fermentation (Fig. 3A). In addition, no
significant difference in GABA content was observed at the 1st day and
5th day. Considering the long fermentation time, which will inevitably
increase the production cost, the GABA produced by the strain Levi­
lactobacillus sp. In the current study could be harvested after 24 h of
fermentation (Wan-Mohtar et al., 2020). When the inoculation amount
was 3%, the GABA content reached a peak of 28.22 g/L (Fig. 3B). The
competition between the bacteria would intensify when the increase of
inoculation; therefore, GABA production decreased with the decrease of
bacterial growth (Liew et al., 2005).
Microbial biosynthesis of GABA is strictly regulated by pH, and it
plays a significant role in the fermentation of bacteria (Lu et al., 2009).
The investigation of the initial pH effect on GABA production by Levi­
lactobacillus sp. LB-2 revealed that GABA was produced well within a pH
range between 3.8 and 8.0, and the maximum GABA production of
34.06 g/L was exited when the initial pH was 5.5. GABA production and
cell growth of Levilactobacillus sp. LB-2 were strongly inhibited at pH 8.0.
Consistent with these results, the pH conditions for most LAB strains
Fig. 3. Effects of culture time (A), inoculation amount (B), and initial pH (C) on the GABA production by Levilactobacillus sp. LB-2.
5
LWT 184 (2023) 115014
producing GABA have been optimized within the pH range of 4.5–6.0
(Seo et al., 2013b), such as pH 5.0 for L. buchneri, pH 5.25 for L. brevis,
and pH 5.31 for L. plantarum (Binh et al., 2014; Cho et al., 2007; Taja­
badi et al., 2015).
The selected S. cerevisiae grew at an initial pH of 4.0 and 35 ◦ C for 60
h, and the GABA production was 4.31 g/L (Zhang et al., 2022). In a
strain of L. plantarum from fermented fish products and incubated in
MRS medium at pH 5–6 for 48 h, the maximum GABA production
reached 15.74 g/L (Tanamool et al., 2019). The key parameters,
including 30 ◦ C, initial pH 5, 100 g/L of sucrose, combination of yeast
extract and GA, and C8: N3, generated the highest GABA (3278.31
mg/L) of Aspergillus oryzae strain NSK in a koji fermentation (Wan-­
Mohtar et al., 2019). The highest production of GABA (4.31 g/L) from
Kluyveromyces marxianus was obtained under the following optimized
conditions: culture temperature of 35 ◦ C, fermentation time of 60 h, and
initial pH of 4.0 (Zhang et al., 2022).
After optimizing the medium substrate and fermentation conditions
via the OFAT strategy, the GABA production of strain Levilactobacillus sp.
LB-2 increased by 2.5 times, from 14.19 to 34.06 g/L.
3.4. Further optimization for GABA production by RSM
The RSM is a three-dimensional spatial surface composed of the re­
sults obtained from the interaction of the test factors. The OFAT-based
H. Li et al. LWT 184 (2023) 115014

optimization results revealed that sucrose and peptone were the best Table 3
carbon and nitrogen sources, respectively. Based on Box–Behnken’s Designs and responses of Box-Behnken.
central combination design principle, a three-factor, three-level Test number X1 X2 X3 GABA production (g/L)
response surface analysis test was designed with 17 test points. Seven­
1 − 1 − 1 0 42.56
teen sets of tests can be divided into two categories (1–12 are analysis 2 1 − 1 0 18.76
factor tests and 13–17 are central tests) to estimate the experimental 3 − 1 1 0 36.70
error, with initial pH, nitrogen source, and carbon source as independent 4 1 1 0 20.19
variables and GABA content as the response value. The selection of the 5 − 1 0 − 1 39.38
6 1 0 − 1 23.94
test factors and levels is shown in Table 2, and the arrangement of the 7 − 1 0 1 37.70
response surface analysis tests and GABA content (g/L) obtained from 8 1 0 1 22.98
each test is shown in Table 3. 9 0 − 1 − 1 32.52
The interaction effect of these factors on GABA production concen­ 10 0 1 − 1 35.63
11 0 − 1 1 32.30
tration by Levilactobacillus sp. LB-2 was analyzed by response surface
12 0 1 1 28.77
analysis. Using GABA yield (Y) as the response value, the regression 13 0 0 0 42.92
equation was expressed as follows: 14 0 0 0 42.81
15 0 0 0 43.42
Y = 43.81 − 8.81Х1 − 0.6074Х2 − 1.21Х3 + 1.82 Х1Х2 + 0.1788Х1 Х3 − 16 0 0 0 44.92
1.66Х2Х3 − 7.78Х21 − 6.48Х22 − 5.03Х23 17 0 0 0 44.99

The negative coefficients of pH (− 8.81), sucrose (− 0.6074) con­

centrations, and peptone (− 1.21) indicated that the low level of these
Table 4
factors contributed to GABA production.
Analysis of variance (ANOVA) for GABA production.
The significance of the regression coefficient was proven using
Source Sum of DF Mean F- p-value
ANOVA (P < 0.05; Table 4). The F-value of 44.69 with Model Prob > F
Squares Square value
less than 0.0001, and the regression coefficient of determination R2 =
0.9829. In addition, the correction coefficient R2Adj = 0.9609, indicating Model 1258.11 9 139.79 44.69 <0.0001 significant
X1 620.45 1 620.45 198.36
that the results of the model fitting test are a good fit and can be applied
<0.0001
X2 2.95 1 2.95 0.9435 0.36
to the analysis and prediction of the optimization of GABA-producing X3 11.80 1 11.80 3.77 0.09
conditions. In this case, X1 as the most significant factor with less than X1X2 13.28 1 13.28 4.25 0.08
0.0001 as Model Prob > F. The “Lack of Fit F-value” of 5.06 and P-value X1X3 0.1278 1 0.13 0.04 0.85
X2X3 11.04 1 11.04 3.53 0.10
of 0.0757 indicated that the Lack of Fit was not significantly related to
X21 254.82 1 254.82 81.47 <0.0001
the pure error. There was a 7.57% chance that such a large “Lack of Fit F- X22 176.55 1 176.55 56.44 0.0001
value” could occur because of noise. The misfit term of the model is not X23 106.48 1 106.48 34.04 0.0006
significant, and the non-significant lack of fit also indicated that the Residual 21.90 7 3.13
model fitted. Therefore, this quadratic model suitably predicted GABA Lack of 17.33 3 5.78 5.06 0.08 not
Fit significant
production by Levilactobacillus sp. LB-2. Optimal levels of each factor
Pure 4.47 4 1.14
and the effects of their interactions on GABA production are illustrated Error
by three-dimensional contour plots (Fig. 4). Cor Total 1280.00 16
In this study, GABA production begins in the exponential growth
phase and increases near to the stationary phase, which is consistent
with GAD being an intracellular enzyme produced in response to acidic completely depends on species (Sharafi and Nateghi, 2020).
conditions (Lu et al., 2009). In addition, GABA production occurs under GABA production also increased with the increase of sucrose con­
acidic conditions (Edalatian Dovom et al., 2023). As shown in Fig. 4, centration and peptone concentration (Fig. 4). The maximum GABA
within the pH range of 3.8–7.2, the content of GABA initially increased production was reached when sucrose was 32.7 g/L and peptone was
and then decreased, and the content of GABA was the highest in pH 4.5 30.7 g/L (P < 0.05). Carbon resource has a direct effect on the properties
(P < 0.05). The optimum level of pH to keep GAD active is 4–5 (Li et al., and yield of fermentation, and it is the most important compound in the
2010). In a study on the efficiency of GABA production by L. brevis AK03, culture medium used to produce microbial metabolites. The effect of the
the highest amount of GABA production was obtained at pH 4.5, which carbon source on GABA production is the high demand of the strain
was 983 and 25,359 mmol in the two culture media of MRS and GA (Falah et al., 2021).
containing MRS, respectively (Li et al., 2010; Wu et al., 2018). There are Three-dimensional surface plots were generated to estimate the ef­
researchers identified and characterized the GABA-producing LAB and fect of the combination of independent variables on GABA production
claimed that most LAB strains can produce the highest amount of GABA using Design Expert. The effects of pH and sucrose (Fig. 4A), pH and
in the pH range of 4–5 at 30 ◦ C–50 ◦ C and in the presence of GA (Li et al., peptone (Fig. 4B), and sucrose and peptone (Fig. 4C) on GABA pro­
2010). The highest amount of GABA production by L. plantarum was duction were determined when the other factors were fixed at the center
obtained at pH 5, or GABA production with L. plantarum was achieved at point. When the concentration of peptone was kept constant, the GABA
pH 5–5.5 (Komatsuzaki et al., 2008; Park et al., 2021). In addition, the level increased with the increase of sucrose (P < 0.05), but increasing pH
amount of effective pH for the maximum production of GABA led to low amounts of GABA (Fig. 4A). Fig. 4B presents the 3D plot
showing the dependency of GABA concentration on peptone and pH,
keeping the third factor (sucrose) at its central level of 35 g/L. The graph
predicted that GABA concentration was 40.57 g/L (P > 0.05) when pH
Table 2
Factors and level takes for response surface analysis.
was 5.5 and peptone was 32 g/L. However, GABA concentration at lower
pH remained approximately constant by increasing peptone. Fig. 4C
Variables Range and Levels
shows that the amount of GABA formed increased with the increase of
− 1 0 1 peptone and sucrose concentrations (P > 0.05).
X1 pH 3.8 5.5 7.2 Denser and more elliptical contour lines indicate a significant
X2 Sucrose (g/L) 15 35 55 interaction between the two factors, whereas the opposite indicates a
X3 Peptone (g/L) 20 32 44 non-significant interaction. Data analysis showed a maximum value in

6
H. Li et al. LWT 184 (2023) 115014

Fig. 4. Response surface plots using different variables on the GABA production by Levilactobacillus sp. LB-2. (A) The combined effects of pH and sucrose con­
centrations. (B) The combined effects of pH and peptone concentration. (C) The combined effects of sucrose and peptone concentrations.

7
H. Li et al.
the regression model, and the optimal conditions for GABA production
were as follows: initial pH of 4.51, sucrose 32.71 g/L, and peptone
30.65 g/L. The maximum value of GABA production predicted from this
model was determined to be 46.47 g/L. And the optimal culture con­
ditions for maximum GABA production of 21.44 mM by strain Lacto­
bacillus brevis HYE1 were determined to be 2.14% (w/v) maltose, 4.01%
(w/v) tryptone, and an initial pH of 4.74 (Lim et al., 2017).
In verifying the reliability of the test, the above mentioned optimal
conditions were used for the GABA production validation test. Consid­
ering the convenience of practical operation, the optimal conditions
were modified as follows: initial pH value of 4.5, sucrose 32.7 g/L, and
peptone 30.7 g/L. The GABA content in the two parallel tests was 49.81
and 49.03 g/L, respectively, and the average GABA concentration was
the actual mean value, which was not significantly different from the
predicted value, indicating that the parameters of this response surface
optimization test were accurate and reliable. Thus, this test had great
application potential.
3.5. Quality properties of steamed bread
3.5.1. Quality of the steamed bread
The steamed breads from the four treatment groups had the basic
form of a steamed bread with a smooth, fluffy, and elastic surface, except
for those from the CK treatment group. The steamed breads made using
the sourdough containing the strain of Levilactobacillus sp. LB-2 (YL &
YLA) had a smooth surface as well as dense and uniform longitudinal
aperture, without wrinkles.
The specific volume represents the fluffiness, and the height-to-
diameter ratio is related to the verticality and fullness of the steamed
bread. The results shown in Table 5 indicated that the steamed bread in
YL treatment had the highest height-to-diameter ratio. The highest
specific volume determined in the steamed bread from YLA treatment (P
< 0.05) may be related to the beneficial interactions of the metabolism
of LAB and AAB during fermentation. Studies have shown that the
increased specific volume and softened texture of the steamed bread
could be attributed to the gas-holding capacity of gluten in the dough
(Wu et al., 2012). Based on previous reports, the higher the specific
volume of bread, the lower the staling time (Luo et al., 2018). Therefore,
LAB and AAB added in the starter from the YLA treatment could play an
active role on the quality of the steamed bread.
3.5.2. GABA contents of the steamed bread
The GABA content of the steamed bread from the YLA treatment
group was approximately 5.18 mg/g, which was the highest among all
the treatment groups. On the contrary, the GABA content of the steamed
bread from the YL treatment group was 4.95 g/L, which was signifi­
cantly higher than that from the Y and CK treatments. No significant
difference in GABA content of the steamed bread was observed between
the YL and YLA treatment groups, and they were both more than 2.1
times than that of the CK treatment group (2.37 mg/g). The results
indicated that the steamed bread made from the doughs supplemented
with Levilactobacillus sp. LB-2 had a higher GABA content and better
quality. In addition, TMS fermentation can improve the quality of
steamed bread (Li et al., 2014; Sha et al., 2023), and similar results were
found by Xing et al. (2019).
4. Conclusion
In this study, a strain of Levilactobacillus sp. LB-2 with good GABA
production ability was screened, and the fermentation conditions were
optimized by OFAT and RSM. The final GABA yield was 49.42 g/L,
which was about 3.5 times higher than that before optimization (14.19
g/L). The GABA content in the steamed bread made by strain Levi­
lactobacillus sp. LB-2 and yeast was significantly higher than that of the
CK (without addition of microbial fermentation starter) and Y (only
fermented by yeast) treatment groups. Therefore, the strain
8
LWT 184 (2023) 115014
Table 5
The quality characteristics of the steamed bread from different treatments.
Treatment Specific volume Height-to-diameter ratio GABA content (mg/g)
d b
CK 0.69 ± 0.03 0.58 ± 0.03 2.37 ± 0.15c
Y 2.01 ± 0.02c 0.64 ± 0.02a 3.57 ± 0.25b
YL 2.15 ± 0.01b 0.66 ± 0.01a 4.95 ± 0.18a
YLA 2.36 ± 0.03a 0.62 ± 0.02ab 5.18 ± 0.25a
Note: Values are presented as mean ± standard deviation of three individual
experiments. Different letters in the same column (a–d) represent significant
differences (P < 0.05).
CK: blank without addition of bacteria; Y: addition with S. cerevisiae Y5 only; YL:
addition with S. cerevisiae Y5 mixed with Levilactobacillus sp. LB-2; YLA: addition
with mixture of S. cerevisiae Y5, Levilactobacillus sp. LB-2 and Acetobacter sp. A3.
Levilactobacillus sp. LB-2 has a good potential to enrich fermented
products using GABA. The results of this study would encourage further
research on the health properties of Levilactobacillus sp. LB-2 GABA food
in vivo.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This research was supported by the fund of National Engineering
Laboratory/Provincal Key Laboratory of Food Science Discipline, Henan
University of Technology (NL2021010 and NL2022009), National Nat­
ural Science Foundation of China (No. 42107139) and Young Key
Teachers Training Program of Henan University of Technology and
Young Key Teachers Cultivation Plan in colleges and universities of
Henan province (2019GGJS-088).
References
Binh, T. T. T., Ju, W. T., Jung, W. J., & Park, R. D. (2014). Optimization of
γ-aminobutyric acid production in a newly isolated Lactobacillus brevis. Biotechnology
Letters, 36(1), 93–98. https://doi.org/10.1007/s10529-013-1326-z
Bs, S., Thankappan, B., Mahendran, R., Muthusamy, G., Femil Selta, D. R., &
Angayarkanni, J. (2021). Evaluation of GABA production and probiotic activities of
Enterococcus faecium BS5. Probiotics and Antimicrobial Proteins, 13, 993–1004.
https://doi.org/10.1007/S12602-021-09759-7
Cataldo, P. G., Villegas, J. M., de Giori, G. S., Saavedra, L., & Hebert, E. M. (2020).
Enhancement of γ-aminobutyric acid (GABA) production by Lactobacillus brevis CRL
2013 based on carbohydrate fermentation. International Journal of Food Microbiology,
333, Article 108792. https://doi.org/10.1016/j.ijfoodmicro.2020.108792
Cho, Y. R., Chang, J. Y., & Chang, H. C. (2007). Production of gamma-aminobutyric acid
(GABA) by Lactobacillus buchneri isolated from Kimchi and its neuroprotective effect
on neuronal cells. Journal of Microbiology and Biotechnology, 17(1), 104–109.
Coda, R., Rizzello, C. G., & Gobbetti, M. (2010). Use of sourdough fermentation and
pseudo-cereals and leguminous flours for the making of a functional bread enriched
of γ-aminobutyric acid (GABA). International Journal of Food Microbiology, 137(2–3),
236–245. https://doi.org/10.1016/j.ijfoodmicro.2009.12.010
Cui, Y., Miao, K., Niyaphorn, S., & Qu, X. (2020). Production of gamma-aminobutyric
acid from lactic acid bacteria: a systematic review. International Journal of Molecular
Sciences, 21(3), 995. https://doi.org/10.3390/ijms21030995
Dhakal, R., Bajpai, V. K., & Baek, K. H. (2012). Production of GABA (γ-aminobutyric
acid) by microorganisms: a review. Brazilian Journal of Microbiology, 43, 1230–1241.
https://doi.org/10.1590/S1517-83822012000400001
Diana, M., Quílez, J., & Rafecas, M. (2014). Gamma-aminobutyric acid as a bioactive
compound in foods: a review. Journal of Functional Foods, 10, 407–420. https://doi.
org/10.1016/j.jff.2014.07.004
Diez-Gutierrez, L., San Vicente, L., Barron, L. J. R., del Carmen Villaran, M., &
Chavarri, M. (2020). Gamma-aminobutyric acid and probiotics: multiple health
benefits and their future in the global functional food and nutraceuticals market.
Journal of Functional Foods, 64, Article 103669. https://doi.org/10.1016/j.
jff.2019.103669
H. Li et al.
Edalatian Dovom, M. R., Habibi Najafi, M. B., Rahnama Vosough, P., Norouzi, N., Ebadi
Nezhad, S. J., & Mayo, B. (2023). Screening of lactic acid bacteria strains isolated
from Iranian traditional dairy products for GABA production and optimization by
response surface methodology. Scientific Reports, 13(1), 440. https://doi.org/
10.1038/S41598-023-27658-5
Falah, F., Vasiee, A., Alizadeh Behbahani, B., Tabatabaee Yazdi, F., & Mortazavi, S. A.
(2021). Optimization of gamma-aminobutyric acid production by Lactobacillus brevis
PML1 in dairy sludge-based culture medium through response surface methodology.
Food Science and Nutrition, 9(6), 3317–3326. https://doi.org/10.1002/FSN3.2304
Falah, F., Vasiee, A., Tabatabaei-Yazdi, F., Moradi, S., & Sabahi, S. (2022). Optimization of
γ-aminobutyric acid (GABA) production by Lactobacillus spp. from agro-food waste.
Biomass Conversion and Biorefinery. https://doi.org/10.1007/S13399-022-02361-Z
Gobbetti, M. (1998). The sourdough microflora: interactions of lactic acid bacteria and
yeasts. Trends in Food Science & Technology, 9(7), 267–274. https://doi.org/10.1016/
S0924-2244(98)00053-3
Kantachote, D., Ratanaburee, A., Hayisama-ae, W., Sukhoom, A., & Nunkaew, T. (2017).
The use of potential probiotic Lactobacillus plantarum DW12 for producing a novel
functional beverage from mature coconut water. Journal of Functional Foods, 32,
401–408. https://doi.org/10.1016/j.jff.2017.03.018
Kim, Y., Huang, W., Zhu, H., & Rayas-Duarte, P. (2009). Spontaneous sourdough
processing of Chinese Northern-style steamed breads and their volatile compounds.
Food Chemistry, 114(2), 685–692. https://doi.org/10.1016/j.foodchem.2008.10.008
Kim, J., Yoon, Y. W., Kim, M. S., Lee, M. H., Kim, G. A., Bae, K., et al. (2022). Gamma-
aminobutyric acid fermentation in MRS-based medium by the fructophilic
Lactiplantibacillus plantarum Y7. Food Science and Biotechnology, 31(3), 333–341.
https://doi.org/10.1007/S10068-022-01035-W
Komatsuzaki, N., Nakamura, T., Kimura, T., & Shima, J. (2008). Characterization of
glutamate decarboxylase from a high γ-aminobutyric acid (GABA)-producer,
Lactobacillus paracasei. Bioscience, Biotechnology, and Biochemistry, 72(2), 278–285.
https://doi.org/10.1271/bbb.70163
Komatsuzaki, N., Shima, J., Kawamoto, S., Momose, H., & Kimura, T. (2005). Production
of γ-aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional
fermented foods. Food Microbiology, 22(6), 497–504. https://doi.org/10.1016/j.fm.
2005.01.002
Kook, M. C., Seo, M. J., Cheigh, C. I., Pyun, Y. R., Cho, S. C., & Park, H. (2010). Enhanced
production of gamma-aminobutyric acid using rice bran extracts by Lactobacillus
sakei B2-16. Journal of Microbiology and Biotechnology, 20(4), 763–766.
Laohasongkram, K., Poonnakasem, N., & Chaiwanichsiri, S. (2011). Process development
of shelf-stable Chinese steamed bun. Journal of Food Process Engineering, 34(4),
1114–1124. https://doi.org/10.1111/j.1745-4530.2009.00531.x
Li, H., Fu, J., Hu, S., Li, Z., Qu, J., Wu, Z., et al. (2021). Comparison of the effects of acetic
acid bacteria and lactic acid bacteria on the microbial diversity of and the functional
pathways in dough as revealed by high-throughput metagenomics sequencing.
International Journal of Food Microbiology, 346, Article 109168. https://doi.org/
10.1016/J.IJFOODMICRO.2021.109168
Li, H., Qiu, T., Huang, G., & Cao, Y. (2010). Production of gamma-aminobutyric acid by
Lactobacillus brevis NCL912 using fed-batch fermentation. Microbial Cell Factories, 9
(1), 1–7. https://doi.org/10.1186/1475-2859-9-85
Li, Y., Chen, X., Shu, G., & Ma, W. (2020). Screening of gamma-aminobutyric acid-
producing lactic acid bacteria and its application in Monascus-fermented rice
production. Acta Scientiarum Polonorum Technologia Alimentaria, 19(4), 387–394.
https://doi.org/10.17306/J.AFS.2020.0868
Liew, S. L., Ariff, A. B., Raha, A. R., & Ho, Y. W. (2005). Optimization of medium
composition for the production of a probiotic microorganism, Lactobacillus
rhamnosus, using response surface methodology. International Journal of Food
Microbiology, 102(2), 137–142. https://doi.org/10.1016/j.ijfoodmicro.2004.12.009
Li, Z., Li, H., Deng, C., & Liu, C. (2014). Effect of mixed strain starter culture on
rheological properties of wheat dough and quality of steamed bread. Journal of
Texture Studies, 45(3), 180–186. https://doi.org/10.1111/jtxs.12064
Lim, H. S., Cha, I. T., Lee, H., & Seo, M. J. (2016). Optimization of γ-aminobutyric acid
production by Enterococcus faecium JK29 isolated from a traditional fermented foods.
Microbiology and Biotechnology Letters, 44(1), 26–33. https://doi.org/10.4014/
mbl.1512.12004
Lim, H. S., Cha, I. T., Roh, S. W., Shin, H. H., & Seo, M. J. (2017). Enhanced production of
gamma-aminobutyric acid by optimizing culture conditions of Lactobacillus brevis
HYE1 isolated from kimchi, a Korean fermented food. Journal of Microbiology and
Biotechnology, 27(3), 450–459. https://doi.org/10.4014/jmb.1610. 10008
Luo, D., Wu, R., Zhang, J., Zhang, K., Xu, B., Li, P., … Li, X. (2018). Effects of ultrasound
assisted dough fermentation on the quality of steamed bread. Journal of Cereal
Science, 83, 147–152. https://doi.org/10.1016/j.jcs.2018.07.016
Liu, W., Li, H., Liu, L., Ko, K., & Kim, I. (2022). Screening of gamma-aminobutyric acid-
producing lactic acid bacteria and the characteristic of glutamate decarboxylase
from Levilactobacillus brevis F109-MD3 isolated from kimchi. Journal of Applied
Microbiology, 132(3), 1967–1977. https://doi.org/10.1111/jam.15306
Lu, X., Xie, C. H., & Gu, Z. (2009). Optimisation of fermentative parameters for GABA
enrichment by Lactococcus lactis. Czech Journal of Food Sciences, 27(6), 433–442.
https://doi.org/10.17221/45/2009-cjfs
Mancini, A., Carafa, I., Franciosi, E., Nardin, T., Bottari, B., Larcher, R., et al. (2019). In
vitro probiotic characterization of high GABA producing strain Lactobacilluas brevis
DSM 32386 isolated from traditional “wild” Alpine cheese. Annals of Microbiology,
69, 1435–1443. https://doi.org/10.1007/s13213-019-01527-x
Ngo, D. H., & Vo, T. S. (2019). An updated review on pharmaceutical properties of
gamma-aminobutyric acid. Molecules, 24(15), 2678. https://doi.org/10.3390/
molecules 24152678
LWT 184 (2023) 115014
Park, S. J., Kim, D. H., Kang, H. J., Shin, M., Yang, S. Y., Yang, J., et al. (2021). Enhanced
production of γ-aminobutyric acid (GABA) using Lactobacillus plantarum EJ2014
with simple medium composition. LWT-Food Science and Technology, 137, Article
110443. https://doi.org/10.1016/j.lwt.2020.110443
Phuengjayaem, S., Kuncharoen, N., Booncharoen, A., Ongpipattanakul, B., &
Tanasupawat, S. (2021). Genome analysis and optimization of γ-aminobutyric acid
(GABA) production by lactic acid bacteria from plant materials. Journal of General
and Applied Microbiology, 67(4), 150–161. https://doi.org/10.2323/
JGAM.2020.10.002
Sahab, N. R., Subroto, E., Balia, R. L., & Utama, G. L. (2020). γ-Aminobutyric acid found
in fermented foods and beverages: Current trends. Heliyon, 6(11), Article e05526.
https://doi.org/10.1016/j.heliyon.2020.e05526
Sarasa, S. B., Mahendran, R., Muthusamy, G., Thankappan, B., Selta, D. R. F., &
Angayarkanni, J. (2020). A brief review on the non-protein amino acid, gamma-
amino butyric acid (GABA): Its production and role in microbes. Current
Microbiology, 77(4), 534–544. https://doi.org/10.1007/s00284-019-01839-w
Sharafi, S., & Nateghi, L. (2020). Optimization of gamma-aminobutyric acid production
by probiotic bacteria through response surface methodology. Iranian Journal of
Microbiology, 12(6), 584. https://doi.org/10.18502/IJM.V12I6.5033
Sharma, P., Sharma, A., Singh, J., Singh, N., Singh, S., Tomar, G. S., … Nain, L. (2021).
Co-production of gamma amino butyric acid (GABA) and lactic acid using
Lactobacillus plantarum LP-9 from agro-residues. Environmental Technology &
Innovation, 23, Article 101650. https://doi.org/10.1016/J.ETI.2021.101650
Seo, M. J., Lee, J. Y., Nam, Y. D., Lee, S. Y., Park, S. L., Yi, S. H., … Lim, S. L. (2013a).
Production of γ-aminobutyric acid by Lactobacillus brevis 340G isolated from Kimchi
and its application to skim milk. Food Engineering Progress, 17(4), 418–423. https://
doi.org/10.13050/foodengprog.2013.17.4.418
Seo, M. J., Nam, Y. D., Lee, S. Y., Park, S. L., Yi, S. H., & Lim, S. I. (2013b). Expression and
characterization of a glutamate decarboxylase from Lactobacillus brevis 877G
producing γ-aminobutyric acid. Bioscience, Biotechnology, and Biochemistry, 77(4),
853–856. https://doi.org/10.1271/bbb.120785
Sha, H. Y., Wang, Q. Q., & Li, Z. J. (2023). Comparison of the effect of exopolysaccharide-
producing lactic acid bacteria from sourdough on dough characteristics and steamed
bread quality. International Journal of Food Science and Technology, 58(1), 378–386.
https://doi.org/10.1111/ijfs.15881
Suo, B., Chen, X., & Wang, Y. (2021). Recent research advances of lactic acid bacteria in
sourdough: Origin, diversity, and function. Current Opinion in Food Science, 37,
66–75. https://doi.org/10.1016/j.cofs.2020.09.007
Tajabadi, N., Ebrahimpour, A., Baradaran, A., Rahim, R. A., Mahyudin, N. A.,
Manap, M. Y. A., … Saari, N. (2015). Optimization of γ-aminobutyric acid
production by Lactobacillus plantarum Taj-Apis362from honeybees. Molecules, 20(4),
6654–6669. https://doi.org/10.3390/molecules20046654
Tanamool, V., Hongsachart, P., & Soemphol, W. (2019). Screening and characterisation
of gamma-aminobutyric acid (GABA) producing lactic acid bacteria isolated from
Thai fermented fish (Plaa-som) in Nong Khai and its application in Thai fermented
vegetables (Som-pak). Food Science and Technology, 40, 483–490. https://doi.org/
10.1590/fst.05419
Venturi, M., Galli, V., Pini, N., Guerrini, S., & Granchi, L. (2019). Use of selected
lactobacilli to increase γ-Aminobutyric acid (GABA) content in sourdough bread
enriched with amaranth flour. Foods, 8(6), 218. https://doi.org/10.3390/
foods8060218
Wan-Mohtar, W. A. A. Q. I., Ab Kadir, S., Halim-Lim, S. A., Ilham, Z., Hajar-Azhari, S., &
Saari, N. (2019). Vital parameters for high gamma-aminobutyric acid (GABA)
production by an industrial soy sauce koji Aspergillus oryzae NSK in submerged-liquid
fermentation. Food Science and Biotechnology, 28, 1747–1757. https://doi.org/
10.1007/s10068-019-00602-y
Wan-Mohtar, W. A. A. Q. I., Sohedein, M. N. A., Ibrahim, M. F., Ab Kadir, S., Suan, O. P.,
Weng Loen, A. W., … Ilham, Z. (2020). Isolation, identification, and optimization of
γ-aminobutyric acid (GABA)-producing Bacillus cereus strain KBC from a
commercial soy sauce moromi in submerged-liquid fermentation. Processes, 8(6),
652. https://doi.org/10.3390/pr8060652
Wu, M., Ding, J., Zhang, Z., You, S., Qi, W., Su, R., et al. (2022). Kinetic modeling of
gamma-aminobutyric acid production by Lactobacillus brevis based on pH-dependent
model and rolling correction. Chinese Journal of Chemical Engineering, 50, 352–360.
https://doi.org/10.1016/J.CJCHE.2022.05.021
Wu, C. H., Hsueh, Y. H., Kuo, J. M., & Liu, S. J. (2018). Characterization of a potential
probiotic Lactobacillus brevis RK03 and efficient production of γ-aminobutyric acid in
batch fermentation. International Journal of Molecular Sciences, 19(1), 143. https://
doi.org/10.3390/ijms19010143
Wu, C., Liu, R., Huang, W., Rayas-Duarte, P., Wang, F., & Yao, Y. (2012). Effect of
sourdough fermentation on the quality of Chinese Northern-style steamed breads.
Journal of Cereal Science, 56(2), 127–133. https://doi.org/10.1016/j.jcs.2012.03.007
Xing, X., Suo, B., Yang, Y., Li, Z., Nie, W., & Ai, Z. (2019). Application of Lactobacillus as
adjunct cultures in wheat dough fermentation. Journal of Food Science, 84(4),
842–847. https://doi.org/10.1111/1750-3841.14496
Yang, S. Y., Lü, F. X., Lu, Z. X., Bie, X. M., Jiao, Y., Sun, L. J., et al. (2008). Production of
γ-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under
submerged fermentation. Amino Acids, 34, 473–478. https://doi.org/10.1007/
s00726-007-0544-x
Zhang, L., Yue, Y., Wang, X., Dai, W., Piao, C., & Yu, H. (2022). Optimization of
fermentation for γ-aminobutyric acid (GABA) production by yeast Kluyveromyces
marxianus C21 in okara (soybean residue). Heliyon, 45(7), 1111-1123. https://doi.
org/10.1007/s00449-022-02702-2.