Biotechnol Lett (2014) 36:9398

DOI 10.1007/s10529-013-1326-z

ORIGINAL RESEARCH PAPER

Optimization of c-amino butyric acid production in a newly

isolated Lactobacillus brevis
Tran Thi Thanh Binh Wan-Taek Ju
Woo-Jin Jung Ro-Dong Park

Received: 9 May 2013 / Accepted: 13 August 2013 / Published online: 28 September 2013
Springer Science+Business Media Dordrecht 2013

Abstract An isolate from kimchi, identified as

Lactobacillus brevis, accumulated c-aminobutyric
acid (GABA), a major inhibitory neurotransmitter, in
the culture medium. Optimal culture conditions for
growth of L. brevis and production of GABA were
6 % (w/v) L-glutamic acid, 4 % (w/v) maltose, 2 %
(w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl2,
2 g Tween 80/l, and 0.02 mM pyridoxal 50 -phosphate
at initial pH 5.25 and 37 C. GABA reached 44.4 g/l
after 72 h cultivation with a conversion rate 99.7 %,
based on the amount (6 %) of L-glutamic acid added.

Electronic supplementary material The online version of

this article (doi:10.1007/s10529-013-1326-z) contains supplementary material, which is available to authorized users.
T. T. T. Binh  W.-T. Ju  W.-J. Jung  R.-D. Park
Glucosamine Saccharide Materials-National Research
Laboratory (GSM-NRL), Division of Applied Bioscience
and Biotechnology, Institute of Agricultural Science and
Technology, Chonnam National University,
Kwangju 500-757, Korea
Present Address:
T. T. T. Binh
Faculty of Agriculture and Forestry, Tay Nguyen
University, Buon Ma Thuot, Dak Lak Province, Viet Nam
R.-D. Park (&)
Division of Applied Bioscience and Biotechnology,
College of Agriculture and Life Sciences, Chonnam
National University, Kwangju 500-757, Korea
e-mail: rdpark@jnu.ac.kr

GABA was purified using ion exchange column

chromatography with 70 % recovery and 97 % purity.
Keywords c-Aminobutyric acid  GABA 
Glutamic acid  Kimchi  Lactic acid bacterium 
Lactobacillus brevis

Introduction
c-Aminobutyric acid (GABA), a major inhibitory
neurotransmitter, is a non-protein amino acid that is
widely distributed in microorganisms, plants and
animals. It has several physiological functions, such
as induction of hypotension, a diuretic effect, and a
tranquilizer effect, particularly with regard to sleeplessness, depression, and autonomic disorders
observed during menopausal periods (Jakobs et al.
1993). GABA therefore has the potential as a bioactive
component in foods and pharmaceuticals. Some
GABA-containing foods, such as tea (Abe et al.
1995), soy products (Tsai et al. 2006) and rice germ
(Zhang et al. 2006), have been developed. The
consumption of GABA-enriched foods can depress
the systolic blood pressure in spontaneously hypertensive rats (SHRs) (Hayakawa et al. 2004) and mildly
hypertensive humans (Inoue et al. 2003).
Lactic acid bacteria (LAB) are generally regarded
as safe (GRAS) and have been extensively used in
dairy products, bread, fermented vegetables, meats
and fish (Lee et al. 2006; Leroy and De Vuyst 2004).

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Also, LAB have been used as probiotics due to their

properties such as immunomodulation, inhibition of
pathogenic bacteria, control of intestinal homeostasis,
resistance to gastric acidity, bile acid resistance, and
antiallergic activity (Hwanhlem et al. 2010; Tannock
2004). Thus, GABA produced by LAB in appropriate
foods can make full use of the health-promoting
properties of GABA and LAB themselves.
Some fermented products, such as dairy products
(Inoue et al. 2003), black raspberry juice (Kim et al.
2009), soymilk (Tsai et al. 2006) and kimchi (Seok
et al. 2008), have been enriched in GABA using
GABA-producing LAB as starter cultures. Some
GABA-producing LAB strains have shown potential
in large-scale fermentation for the production of
GABA (Komatsuzaki et al. 2005; Cho et al. 2007;
Yang et al. 2008).
In this study, a GABA-producing LAB, Lactobacillus brevis K203, was isolated and characterized
from kimchi. Culture conditions were then optimized
in order to enhance GABA productivity.

Materials and methods

Isolation and identification of GABA-producing
LAB
Lactobacillus brevis was isolated from kimchi and
identified by standard protocols (see Supplementary
data 1). It was given the strain designation of L. brevis
K203.

Biotechnol Lett (2014) 36:9398

beef extract and 0.5 % (w/v) yeast extract were

removed from the MRS medium and then 1 %
nitrogen sources of casein peptone, meat extract,
yeast extract, malt extract, skim milk, corn milk and
soybean powder were added (Kook et al. 2010). The
effects of NaCl, CaCl2, Tween 80 and pyridoxal 50 phosphate on GABA productivity were also tested.
Purification of GABA from the fermented broth
GABA was purified from the fermented culture broth
(200 ml) by centrifugation, decolorization using active
carbon, ethanol precipitation and Dowex 50WX4-200
ion exchange chromatography according to Li et al.
(2011) with modification.
Analysis of GABA
GABA contents were determined qualitatively by prestained TLC cellulose glass plates (Qiu et al. 2010)
and quantitatively according to Li et al. (2009) with
modifications. After development in n-butanol/acetic
acid/water (5:3:2, by vol.) containing 1.2 % (w/v)
ninhydrin, the plate was directly dried for color yield.
GABA spots were scratched out from the plate and
extracted with 4 ml 75 % (v/v) ethanol/0.6 % (w/v)
cupric sulfate (38:2, v/v) at 40 C. The absorbance of
the supernatant was read at 512 nm. Purity of the
product was also determined by HPLC (Li et al. 2009).

Results and discussion

Optimization of culture condition for GABA
production
MRS media containing 6 % (w/v) L-glutamic acid
(GA) at various initial pHs from 3.5 to 7.0 were used
for cultivation of L. brevis K203 at 37 C to test pH
effect on cell growth and GABA production (Cho et al.
2007). The effect of temperature was measured from
20 to 55 C at pH 5.25. For the effect of glutamate
concentration, MRS media containing 412 % (w/v)
GA and monosodium glutamate (MSG) were used (Li
et al. 2010). To compare GABA productivity for
various carbon sources, MRS media containing 6 %
(w/v) GA with glucose, maltose, sucrose, and soluble
starch at 1 and 2 % (w/v) were prepared. For the effect
of nitrogen, 1 % (w/v) protease peptone, 1 % (w/v)

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Isolation and identification of GABA producing

bacteria
Fourteen strains were isolated from kimchi as tentative
GABA producers (Supplementary Fig. 1a). Among
them, three strains K203, K304 and K305 produced
high concentration of GABA. Strain K203 completely
converted glutamate to GABA within 72 h cultivation
(Supplementary Fig. 1b) and was identified as L.
brevis. Currently, a few GABA-producing LAB
species have been isolated from kimchi, including L.
brevis OPY-1 (Park and Oh 2005), L. brevis GABA
100 (Kim et al. 2009), Lactococcus lactis B (Lu et al.
2009), and Lactobacillus buchneri MS (Cho et al.
2007).

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95

Fig. 1 The effect of the initial pH on cell growth (a) and GABA
production (b). Bacteria were grown in 100 ml flasks containing
MRS medium with 6 % GA at 37 C for 120 h. The 100 %
represents 33.8 mg/ml GABA

Optimization of culture conditions for GABA

production
Growth of L. brevis K203 reached the stationary phase
after 48 h at all pH values except those below pH 4.5
(Fig. 1a). The highest populations were at pH 5.0 and
5.25. The optimal initial pH for GABA production was
5.25 (Fig. 1b). GABA production rapidly decreased at
pH values below 4.5 or above 6.5, suggesting pH
dependence of cell growth and GABA production. At
the initial pH of 5.0 and 5.25, the entire GA was
converted to GABA within 96 h (data not shown).
This reaction would be carried out by a glutamic acid
decarboxylase that has been recognized in Lactobacillus spp. (Li and Cao 2010).
The optimum temperature for growth of L. brevis
K203 was 35 C. The strain grew well from 20 to
40 C but not above than 45 C (data not shown). The
highest production of GABA was obtained at 37 C
(34.8 mg/ml) and 40 C (33.1 mg/ml).
The GABA production by K203 was affected by the
concentration of GA or MSG (Fig. 2a). When GA

Fig. 2 Effect of glutamate (a) and carbon source (b) on GABA

production by L. brevis K203. Bacteria were grown in 100 ml
flasks containing MRS medium with GA or various carbon
source at initial pH 5.25 and 37 C for 120 h. The 100 %
represents 33.6 mg/ml (a) and 32.3 mg/ml GABA (b)

concentration increased from 4 to 6 % (w/v), GABA

production increased significantly. However, above
8 %, GABA production decreased. The extra high
concentration of GA was inhibitory for the bacterium
growth and for GAD activity.
Maltose (4 %) was the best carbon source for
GABA production with a 16 % increase of GABA
(from 27.8 to 32.3 mg/ml), compared to standard
MRS medium (2 % D-glucose) (Fig. 2b). The optimal carbon sources for GABA production varied
according to the LAB strains, for examples, 1 %
glucose for L. buchneri (Cho et al. 2007), 5.5 %
glucose for L. brevis NCL912 (Li et al. 2009) and
4 % sucrose for L. sakei B2-16 (Kook et al. 2010).

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Fig. 3 Effects of nitrogen sources (1 %) (a) and nitrogen

concentration (0.54 %) (b) on GABA production by L. brevis
K203. Bacteria were grown in 100 ml flasks containing MRS
medium with 6 % GA and nitrogen source at initial pH 5.25 and
37 C for 120 h. The 100 % represents 34.0 mg/ml (a) and
41.7 mg/ml GABA (b)

Addition (1 %) of casein peptone, meat extract and

yeast extract gave high GABA production (31.334 mg/
ml) (Fig. 3a). Exceptionally low production of GABA
was observed from skim milk, malt extract, soybean
powder and corn meal. Simple, soluble and digest-ready
nitrogen sources were better for the growth of L. brevis
K203 and GABA production. GABA production was
also affected by the concentration of nitrogen source
(Fig. 3b). GABA production increased according to the
concentration; 32.6, 34 and 41.7 mg/ml at 0.5, 1 and
2 % of yeast extract, respectively. However, at 4 %
yeast extract, it decreased to 38.4 mg/ml. The same
patterns of GABA production were observed for casein
peptone and meat extract (Fig. 3b). In a previous study,
3 % (w/v) soy peptone was the optimal nitrogen source
for GABA production with L. brevis NCL912 (Li et al.
2009).
Lactobacillus brevis K203 grew well up to 07 %
(w/v) NaCl which was expected as it was isolated from
kimchi, a salty fermented vegetable food. NaCl at 1 %

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Biotechnol Lett (2014) 36:9398

Fig. 4 Time course of cell growth (a), pH change (b), and

GABA production (c) during fermentation in a jar fermenter
(3 l) under the optimized condition. The pH was adjusted to 6.0
during cultivation (b)

(w/v) was the best for GABA production (42 mg/ml).

Addition of 1 % (w/v) CaCl2 also gave a 9 %
enhancement in GABA production. In case of L.
brevis NCL912, addition of CaCl2, MgSO4, vitamin C
and nicotinic acid had no significant effect on GABA
production (Li et al. 2009).
Addition of Tween 80 (2 ml/l), a growth-stimulating factor for LABs, enhanced GABA productivity
(36.3 mg/ml GABA). Addition of 0.01 and 0.02 mM
PLP, a coenzyme of glutamic acid decarboxylase,
gave 10 and 20 % enhancement of GABA production,
respectively, compared with no PLP addition.
The optimum conditions for GABA production by
L. brevis K203 are therefore as follows: 6 % (w/v)
glutamic acid, 4 % (w/v) maltose, 1 % (w/v) casein
peptone, 1 % (w/v) meat extract, 0.5 % (w/v) yeast
extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl2, 2 ml
Tween 80/l and 0.02 mM pyridoxal 50 -phosphate at
initial pH of 5.25 and 37 C.

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Table 1 Purification of GABA from the culture broth

Step

Amount

Recovery (%)

Purity (%)

Culture broth

190 ml

100

44.2

Decolorization

165 ml

88.8

52.6

Ethanol precipitation
IEC

50 ml
5.89 g

71
69.8

70
96.6

GABA production in a jar fermenter

When grown in a jar fermenter (3 l) under optimized
conditions, the bacterium reached the stationary phase
within 24 h (Fig. 4a). Accompanied with GABA formation, pH of the liquid medium increased rapidly and thus
pH adjustment was needed (Fig. 4b). Within 72 h
cultivation (24 h earlier than flask cultures), GABA
reached its highest concentration of 44.4 mg/ml
(Fig. 4c). The net conversion rate was calculated as
99.7 %, based on the theoretical GABA yield from 6 %
(w/v) GA. Until now, the highest conversion rate of GA to
GABA was 97.3 % by L. brevis NCL912 (Li et al. 2010).
GABA (5.89 g) was obtained as white powder from
200 ml culture broth, as summarized in Table 1. The
purification of GABA from the fermented broth was
completed with 69.9 % recovery and 96.7 % purity,
suggesting that protocol is simple but effective.

Conclusions
Lactobacillus brevis K203, a GABA-producing LAB,
was isolated and characterized from kimchi. Culture
conditions were optimized for GABA production with
L. brevis K203. Under the optimized condition, the
yield of GABA reached 44.4 g/l after 72 h fermentation with the bioconversion rate 99.7 %, the highest
record up to now. GABA was purified by ion exchange
chromatography with 69.9 % recovery and 96.7 %
purity from the culture broth.
Acknowledgments This work was supported by a grant from
the Next-Generation BioGreen 21 Program (No. PJ007983),
Rural Development Administration, Republic of Korea.

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