Biochemical Engineering Journal 41 (2008) 48–52

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Isolation of ␥-aminobutyric acid-producing bacteria and optimization of

fermentative medium
Xiaoxue Lu, Zhigang Chen, Zhenxin Gu ∗ , Yongbin Han
College of Food Science & Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Ten ␥-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) strains were isolated from kimchi
Received 20 October 2007 and yoghurt. The strain B, isolated from kimchi showed the highest GABA-producing ability (3.68 g/L)
Received in revised form 23 February 2008 in MRS broth with 1% monosodium glutamate (MSG). Strain B was identified as Lactococcus lactis subsp.
Accepted 13 March 2008
lactis. The GABA-producing ability of L. lactis B was investigated using brown rice juice, germinated soybean
juice and enzymolyzed skim milk as medium compositions. The D-optimal mixture design was applied to
Keywords:
optimize the ratio of the three kinds of components in the media. The results showed that when the mixing
Amino acid
ratio of brown rice juice, germinated soybean juice and enzymolyzed skim milk was 33:58:9 (v:v:v), the
Lactic acid bacteria
Fermentation
maximum GABA yield of L. lactis B was 6.41 g/L.
D-optimal mixture design optimization © 2008 Elsevier B.V. All rights reserved.
Biotransformation
Food engineering

1. Introduction grade GABA production by using synthetic or semi-synthetic media,

␥-Aminobutyric acid (GABA), a non-protein amino acid, is
widely distributed among eukaryotes and prokaryotes. It is known
as one of the major inhibitory neurotransmitters in the sympathetic
nervous system and plays an important role in cardiovascular func-
tion [1–5]. Therefore, searching GABA-rich foods becomes one of
the focuses in the field of functional food research.
GABA synthesis by plants through adding glutamic acid or gluta-
mate has been reported recently [6,7]. However, the yields of GABA
accumulated by fruit and vegetables were low. GABA production
through fermentation, believed to be convenient and efficient has
been gradually applied in food industry. E. coli was used for produc-
ing GABA firstly [8,9], but many hidden troubles and dangers existed
by E. coli fermentation. Some shiga toxin-producing E. coli are pri-
marily food-borne pathogens that can cause severe and potentially
fatal human disease [10]. So several microorganisms [11–17] gener-
ally regarded as safe including lactic acid bacteria (LAB) have been
widely researched and applied in GABA production over the last few
years. Lactobacillus brevis [11,13,18], Lactobacillus paracasei [12] and
Lactoccocus lactis [19] were the most studied LAB species for GABA-
rich foods and pharmaceuticals synthesis in many researches. The
GABA-producing LAB was mostly cultivated in synthetic or semi-
synthetic media [12,20,21]. Because of safety problems for food
∗ Corresponding author. Tel.: +86 25 8439 6293; fax: +86 25 8439 6293.
E-mail address: guzx@njau.edu.cn (Z. Gu).
separation and purification of GABA were needed after fermenta-
tion, which would increase the cost of production. Natural media
are composed of complex unknown organic substances. Many foods
including dairy products, fruit and vegetables can be used as nat-
ural medium, but their application in GABA production by LAB is
little explored [18,22,23].
In this study, LAB with high GABA-producing ability were
isolated and screened from kimchi and yoghurt; brown rice, ger-
minated soybeans and skim milk were applied as natural medium
components and their optimum ratio for LAB to produce GABA was
investigated by D-optimal mixture design. To our knowledge, this
was the first time that a D-optimal mixture design was used in the
area of natural medium optimization.
2. Materials and methods
2.1. Materials
Brown rice was kindly supplied by Mayang Oil Co. Ltd. in Mayang
County, Hunan Province, China. Yoghurt and germinated soy-
beans were bought from the local supermarket (Weigang, Nanjing
city, Jiangsu Province, China). The kimchi was made from Chi-
nese cabbage and radish bought from the supermarket. ␣-Amylase
(4000 U/g) was purchased from Beijing Double Spin Microbial
Medium Plant, China. Glucoamylase (100,000 U/g) was from Shang-
hai Chemical Company, China. Papain (2000 U/mg) was purchased
from Nanjing Scigene Technology Co. Ltd., China. Skim milk powder

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.03.005
 X. Lu et al. / Biochemical Engineering Journal 41 (2008) 48–52 49

was bought from Bright Dairy Co. Ltd., China. All other chemicals Table 1
Independent variables and their coded and actual values used for optimization in
used were also from commercial sources.
D-optimal mixture design experiment

Factors Symbol Code levels

2.2. Isolation and identification of GABA-producing LAB
1 2/3 1/2 1/3 1/6 0
Kimchi and yoghurt were used as isolation sources of LAB. About Brown rice juice X1 0.500 0.350 0.275 0.200 0.125 0.050
0.5 mL samples were inoculated into 100 mL flasks with 50 mL Germinated soybean juice X2 0.900 0.750 0.675 0.600 0.525 0.450
MRS broth containing 1% MSG. The flasks were incubated at 30 ◦ C Enzymolyzed skim milk X3 0.500 0.350 0.275 0.200 0.125 0.050

and 37 ◦ C for samples from kimchi and yoghurt respectively, for

24 h without shaking. Two hundred microliters aliquots of 107 -fold Table 2
diluted enrichment culture were taken and spread onto MRS plates. Observed and predicted values of GABA yield obtained in D-optimal mixture design
After incubation at 30 ◦ C and 37 ◦ C for 48 h, single colonies were experiment

picked up and streaked on MSR plates. 12% (w/v) skim milk was Mixture Level GABA yield (g/L)
used for maintenance of LAB strains. X1 X2 X3 Actual valuea Predicted value
Identification of the GABA-producing strain was done by
1 0 1 0 3.91 ± 0.30 4.02
its physiological and biochemical characteristics and 16S rDNA
2 1 0 0 5.45 ± 0.66 5.60
sequence analysis [24]. The 16S rDNA was sequenced by Shenggong 3 0 1/2 1/2 4.00 ± 0.51 4.26
Co. Ltd., China. 4 0 0 1 3.81 ± 0.32 3.97
5 1/2 0 1/2 6.03 ± 0.47 6.21
6 1/2 1/2 0 6.11 ± 0.50 6.29
2.3. Optimization of fermentative medium for LAB to produce 7 2/3 1/6 1/6 6.14 ± 0.43 6.39
GABA 8 1/6 1/6 2/3 5.61 ± 0.38 5.17
9 1/3 1/3 1/3 6.04 ± 0.26 5.94
2.3.1. Fermentation substrate preparation 10 1/6 2/3 1/6 5.67 ± 0.24 5.21
11 1 0 0 5.93 ± 0.40 5.60
Brown rice was milled into fine powder and passed through a 12 0 0 1 4.07 ± 0.36 3.97
60-mesh sieve. After adding five volumes of double distilled water, 13 0 1 0 4.06 ± 0.51 4.02
the rice powder was gelatinized at 95 ◦ C for 40 min. When cooling 14 0 1/2 1/2 4.12 ± 0.32 4.26
to 60 ◦ C, the rice paste was added with 0.5% (w/w) ␣-amylase and a
Results are mean values of triplicate determinations ± standard deviation.
incubated at 60 ◦ C for 60 min. The liquefied rice slurry was homog-
enized through the colloid mill twice to get an intermixture. After design. If the mixture region is not a simplex, or if mixture vari-
the mixture was adjusted to pH 4.5 with citric acid solution, 0.3% ables with process variables are combined, the design will be built
(w/w) glucoamylase was added and incubated at 60 ◦ C for 2 h. Then according to the D-optimal principle, which consists in a sub-set
the mixture was adjusted to pH 7.0 by NaOH solution, papain 0.1% of candidate points which define a maximal volume region in the
(w/w) was added and incubated at 60 ◦ C for 3 h. Finally, the above multidimensional space [25].
enzymolysis liquid was centrifugated at 4000 rpm for 10 min. The The D-optimal mixture design was applied to define the opti-
supernatant was taken and adjusted to 4 ◦ Brix. mum ratio of the three culture medium materials: brown rice juice,
Germinated soybeans with the same weight of distilled water germinated soybean juice and enzymolyzed skim milk, for LAB
were boiled for 10 min to inactivate lipoxyganase and wet-milled by to produce GABA. The experimental design and statistical analy-
a triturator for 30 s. The crushed germinated soybeans were treated sis were performed using Stat-Ease software (Design-Expert 6.0.10
with 0.1% (w/w) papain at 60 ◦ C for 3 h. Germinated soybean juice Trial, Delaware, USA Echip, 1993). A three-factor mixture design
was obtained by centrifugation (4000 rpm for 10 min) and adjusted was chosen to evaluate the optimum combination of the three
to 4 ◦ Brix. kinds of natural medium components. Table 1 illustrated the fac-
Twelve percent (w/v) skim milk was added with 0.1% papain tors and the levels of each factor under investigation. Brown rice
and incubated at 60 ◦ C for 3 h. Then the enzymolyzed milk was cen- juice, germinated soybean juice and enzymolyzed skim milk were
trifugated at 4000 rpm for 10 min. The supernatant was adjusted to represented as X1 , X2 and X3 , respectively. The proportional lev-
4 ◦ Brix as a fermentation substrate. els of fermentation substrate for brown rice juice were set at
0.05–0.5 (5–50), germinated soybean juice 0.45–0.9 (45–90) and
enzymolyzed skim milk 0.05–0.5 (5–50). X1 + X2 + X3 = 100% (100).
2.3.2. Fermentation conditions
The three kinds of juice used as fermentation substrate for
The medium for LAB seed culture was MRS broth. Two hundred
GABA-producing LAB were mixed according to the experiment
and fifty milliliters flask containing 100 mL MRS broth was inocu-
design (Table 2), and GABA yield in the culture broth was the
lated and incubated at 30 ◦ C for 20 h. The seed culture contained
response value. The complete design consisted of 14 combinations
approximately 108 CFU/mL.
including six minimum model points (treatments 1–6), four lack of
MSG was added to the sterile fermentation media (pH 7.0) at
fit estimation points (treatments 7–10) and four replicates of the
1% concentration. After inoculated with 2% (v/v) seed culture, the
mixtures 1–4 (treatments 11–14).
250 mL conical flasks with 100 mL fermentative media were kept
The polynomial used in the present work is shown in Eq. (1).
at 30 ◦ C for 6 d without shaking. All the media were sterilized at
The responses function (Y) of the special cubic model is partitioned
121 ◦ C for 20 min before use.
into linear, interactive and cubic term components

2.3.3. Experimental design


p
 
Ŷ = bj xj + bhj xh xj + bhjk xh xj xk ,
Mixture design has been successfully applied in various fields
j=1 h<j h<j<k
such as fermentation industry, pharmaceuticals and others. In a
⎧
mixture design where the composition is the factor of interest, ⎪
⎨ pxj ≥ 0
the levels cannot be chosen arbitrarily. All fractions of the compo-  (j = 1, 2, 3, . . . , p) (1)
nents must sum to unity. There are three types of classical mixture ⎪ xj = 1
designs: simplex-lattice design, simplex-centroid design and axial
⎩
j=1
50 X. Lu et al. / Biochemical Engineering Journal 41 (2008) 48–52

Table 3
Physiological and genetical characteristics of strain B

Parameters or tests Characteristics

Gram staining Gram-positive

Catalase activity −a
Aerobic growth +b
Anaerobic growth +
Growth at pH 9.6 −
Growth in 4% NaCl +
Growth in 6.5% NaCl −
Spore None
Litmus milk reactions Acid curd

Fermentation
Glucose +
Fig. 1. GABA yield of different LAB strains cultivated in MRS broth with 1% MSG for Lactose +
6 d. Raffinose −
Sorbitol −
Glucose +
where bj is defined as the linear coefficient; bhi the interactive coef- Lactose +
ficient and bhjk the cubic coefficient. Xh , Xj and Xk represent levels of Mannitol +
the independent variables while P equal to the number of the tested Salicin +
factors (P = 3). The analysis of variance (ANOVA) tables are gener- Growth temperature (◦ C)
ated and the effect and regression coefficients of individual linear, 10 +
interactive and cubic terms are determined. The significances of all 45 −
terms in the polynomial are judged statistically by computing the 65 −

F-value at a probability (P) of 0.001, 0.01 or 0.05. The regression Hydrolyzation

coefficients are used to make statistical calculations to generate Esculin +
Starch −
contour map from the regression model.
Arginine +
Hippuric acid −
2.4. Analytical methods
Homology of 16S rDNA sequences 99% with Lactococcus lactis subsp. lactis
K337 (AB118035)
The GABA contents in the culture medium were measured using
a
The strain cannot grow or cannot utilize the sugar into acid or cannot hydrolyze
an amino acids analyzer (Hitachi L-8900, Japan. 4.6 mm × 60 mm (#
the chemical substances.
2622). Column temperature: 57 ◦ C. Reactor temperature: 136 ◦ C). b
The strain can grow or can utilize the sugar into acid or can hydrolyze the
Cells were removed from the culture by centrifugation (10,000 rpm chemical substances.
for 10 min at 4 ◦ C). The 0.5-mL supernatant was diluted with 0.5 mL
10% trichloroacetic acid and centrifugated at 10,000 rpm for 10 min.
The supernatant was diluted with 200-fold distilled water. Ten 16S rDNA sequence and physiological characters (Table 3). Then L.
milliliters of samples were injected into the amino acids analyzer. lactis B was further explored with D-optimal mixture experiment
Free amino acids were assayed using the ninhydrine col- to find the optimum medium with brown rice juice, germinated
orimetric method. Reducing sugars were analyzed applying soybean juice and enzymolyzed skim milk.
3,5-dinitrosalicylic acid (DNS) method. Total sugars were assayed
with sulphuric acid–phenol method. And the soluble proteins were 3.2. Several main nutritional characteristics of the three different
measured by colorimetric method with coomassie brilliant blue culture components
G-250 as colorimetric liquid [26].
The main nutritional components of the three different cul-
2.5. Statistics ture substrates were analyzed (Table 4). There were the most
reducing sugars in brown rice juice and free amino acids in ger-
The GABA yield was taken as responsive value. Data reported minated soybean juice. It could be indicated that the brown rice
were mean ± standard deviation (S.D.). The statistics significance juice and enzymolyzed skim milk could offer a mass of carbon
was evaluated using Student’s t-test and P < 0.05 was taken source for GABA production while the germinated soybean juice
as significant. A second-order polynomial regressed equation and enzymolyzed skim milk could offer nitrogen source for GABA
was established on the basis of analysis of mixture experimen- production.
tal data, and the optimum ratio of the medium components
was found using the Design-Expert 6.0.10 software optimization 3.3. Optimization of fermentative medium for Lactoccocus lactis
toolbox. B producing GABA

3. Results and discussion
3.1. Screening and identification of GABA-producing LAB
10 LAB strains, produing ␥-aminobutyric acid in MRS culture
medium were isolated from kimchi and yoghurt. Two of the strains
(Y3 and Y4 ) were bacilli while the other eight were cocci. Screening
results were shown in Fig. 1. Strain B (isolated from kimchi) showed
the highest GABA-producing ability (3.68 g/L) in MRS broth. The
strain B was identified as Lactococcus lactis subsp. lactis based on
The GABA yields produced in the mixtures from the three dif-
ferent fermentation substrates were shown in Table 2. Significant
differences of GABA yield were found among the different mixtures.
The lower GABA yields were found in low content of brown rice juice
trials and the highest GABA yield was found in appropriate ratio
of brown rice juice, germinated soybean juice and enzymolyzed
skim milk. These results indicated that the mixing of these three
kinds of juice could improve the GABA yield from L. lactis B and
reflected the importance of medium optimization to attain higher
GABA concentration.
 X. Lu et al. / Biochemical Engineering Journal 41 (2008) 48–52 51

Table 4
Main nutritional characteristics of different culture components

Indexesa Culture components

Brown rice juice Germinated soybean juice Enzymolyzed skim milk

Total sugars (g/L) 79.50 ± 0.41 12.96 ± 0.52 42.71 ± 0.21

Reducing sugars (g/L) 77.79 ± 1.20 7.95 ± 0.19 34.85 ± 0.17
Soluble proteins (␮g/mL) 68.47 ± 0.00 36.62 ± 0.02 34.41 ± 0.03
Free amino acids (␮g/mL) 123.55 ± 0.05 2084.97 ± 3.89 894.51 ± 0.83
a
Results are mean values of triplicate determinations ± standard deviation.

Table 5
Analysis of variance (ANOVA) for the regression (Eq. (2)).

Source Sum of squares Degree of freedom Mean squares F-value P-value

Model 11.81 5 2.36 23.12 <0.001

Linear Mixture 7.35 2 3.68 35.97 <0.001
X1 X2 2.26 1 2.26 22.16 0.002
X1 X3 2.09 1 2.09 20.41 0.002
X2 X3 0.11 1 0.11 1.05 0.335
Residual 0.82 8 0.10
Pure Error 0.17 4 0.04
Lack of fit 0.65 4 0.16 3.95 0.106

Corrected total 12.63 13

Polynomial model describing the correlation between GABA and
the three variables was presented as follows:
Y = −6.35X1 + 2.97X2 + 0.79X3 + 29.25X1 X2
+ 28.07X1 X3 + 5.32X2 X3
The value of R2
and adjusted R2 ,
being measures of fitness of the
regressed model, were 0.935and 0.895 respectively, which sug-
gested that the experimental data were in good agreement with
predicted values. It could be seen in Eq. (2) that X1 had remarkable
negative effect on the GABA yields while X2 and X3 had strong pos-
itive effects. The absolute value for the coefficient of X1 was larger
than those of X2 and X3 , indicating that the linear term influence of
brown rice juice (X1 ) was more than those of germinated soybean
juice (X2 ) and enzymolyzed skim milk (X3 ). The function was not
linear, indicating the importance of interactions among X1 , X2 and
X3 . The coefficients of X1 X2 and X1 X3 were larger than that of X2 X3
showing that the combination effects of brown rice juice and germi-
nated soybean juice (X1 X2 ) and brown rice juice and enzymolyzed
skim milk (X1 X3 ) were main factors influencing GABA production.
Table 5 showed the analysis of variance (ANOVA) for the regres-
sion model (Eq. (2)). In this experiment, the obtained model was
very significant (P < 0.001) according to ANOVA given in Table 5 and
could be used to navigate the design space. On the basis of the t-test
(2) and P values, the interaction between brown rice juice and germi-
nated soybeans juice was significant (X1 X2 , P = 0.002) as well as the
interaction between brown rice juice and enzymolyzed skim milk
(X1 X3 , P = 0.002), which was in accordance with the polynomial
model described before.
To obtain more detailed information, the contour plot and tri-
angular surface for the effect of the independent variables on GABA
yield were shown in Fig. 2. The diagram described the variation
on the response of GABA yield as a function of the mixture com-
position. The zone of optimization, as shown in the superimposed
contour plot, depicted the brown rice juice between 0.25 and 0.40,
germinated soybean juice between 0.50 and 0.65 and enzymolyzed
skim milk between 0.05 and 0.15. When the optimum ratio of the
three components was as follows: brown rice juice 0.33, germi-
nated soybeans juice 0.58 and enzymolyzed skim milk 0.09, the
largest predicted GABA yield was 6.41 g/L on the basis of Eq. (2).

Fig. 2. Contour plot (A) and triangular surface (B) for the effects of brown rice juice, germinated soybean juice and enzymolyzed skim milk on GABA yield. X1 denotes the
brown rice juice, X2 the germinated soybean juice and X3 the enzymolyzed skim milk.
52 X. Lu et al. / Biochemical Engineering Journal 41 (2008) 48–52
The experiment result showed the GABA concentration in the opti-
mum medium was 5.99 g/L higher than that in the medium without
inoculation after autoclaving (0.42 ± 0.08 g/L). Also, the GABA yield
was much higher than that in MRS medium (3.68 g/L) shown in
Fig. 1 and in medium made of glucose, soybean flour and corn steep
liquor (3.63 g/L) [27].
The mixture of three kinds of natural juice could provide more
nutrition for LAB growth and GABA production than single one and
synthetic media. First, the complex medium supplied ample car-
bon and nitrogen sources (Table 4). Secondly, there were plenty of
vitamins in the optimum medium which were necessary for the
growth of L. lactis B. Brown rice contain more nutritional com-
ponents, such as phytic acids, vitamin E and B, than the ordinary
milled rice [28]. Germinated soybeans are rich in vitamin C, vitamin
B12 , carotene, riboflavin, and niacin. Moreover, germinated soybean
juice and enzymolyzed skim milk are rich in many kinds of amino
acids and peptides [29]. Thirdly, the optimum medium in this study
could supply various mineral elements, such as Fe, Cu, Mn, Zn, etc. It
showed that the components of optimum medium were beneficial
to the synthesis of GABA by L. lactis B and had hygienical functions
after fermentation.
Nowadays, people are paying more and more attention to food
and pharmaceutical securities. Natural media must be in high
demand for replacing synthetic media in food industry due to leg-
islative action and consumer concern in the near future. Because
GABA-producing LAB was cultivated in the medium consisting of
edible natural materials in this study, the fermentation liquid could
be directly used as functional food without further extraction and
purification.
4. Conclusion
Ten strains of LAB which accumulated GABA in MRS broth were
isolated from kimchi and yoghurt. One strain, identified as Lactococ-
cus lactis subsp. lactis B showed the highest GABA-producing ability
(3.68 g/L) in MRS broth with 1% MSG. The D-optimal mixture design
was applied to optimize the ratio of the natural materials of brown
rice, germinated soybean juice and enzymolyzed skim milk for L.
lactis B to produce GABA. The observed GABA results were close to
the predicted values. It was showed that when the mixing ratio of
brown rice juice, germinated soybean juice and enzymolyzed skim
milk was 33:58:9 (v:v:v), the GABA yield of L. lactis B was the high-
est (6.41 g/L). The optimum medium obtained in this paper would
be expected to prove useful on further development for functional
food.
Acknowledgement
We wish to acknowledge the Foundation of Nanjing Agricultural
University (Grant no. 080-804071) for financial support.
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