Bioresource Technology 98 (2007) 1675–1679

Short Communication

Production of yogurt with enhanced levels of gamma-aminobutyric

acid and valuable nutrients using lactic acid bacteria
and germinated soybean extract
Ki-Bum Park b, Suk-Heung Oh a,¤

a
Department of Medicinal Biotechnology, Woosuk University, Jeonju 565-701, Republic of Korea
b
Department of Life Science and Technology, Graduate School, Woosuk University, Jeonju 565-701, Republic of Korea

Received 19 September 2005; received in revised form 6 June 2006; accepted 12 June 2006
Available online 18 October 2006

Abstract

Yogurt with high levels of gamma-aminobutyric acid (GABA), free amino acids and isoXavones was developed using lactic acid bacte-
ria (LAB) and germinated soybean extract. Fermented soya milk (GABA soya yogurt) produced with starter and substrate had the
GABA concentration of 424.67 g/g DW, whereas fermented milk produced by a conventional method had GABA less than 1.5 g/g DW.
The GABA soya yogurt also contained signiWcantly high levels of free amino acids and isoXavones compared with other conventional
yogurts. The results suggested that the Lactobacillus brevis OPY-1 and germinated soybean possessed a prospect to be applied in dairy
and other health products with high nutritive values and functional properties.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Gamma-aminobutyric acid (GABA); Germinated soybean; IsoXavones; Lactobacillus brevis; Yogurt

1. Introduction -decarboxylation of Glu catalyzed by the enzyme gluta-

Yogurt is a nutrient-rich fermented food made of milk,
containing various organic acids, peptones, peptides, other
trace activators and lactic acid bacteria. Yogurt has an
intestine-cleaning function to promote the proliferation of
intestinal lactic acid bacteria (Savaiano et al., 1984; Park
et al., 2003). Ingredients such as non-fat dry milk, soya pro-
tein, vegetables, sweet potato, pumpkin, plum, etc. are
sometimes added into Korean yogurts (Park et al., 2003;
Ko, 1989; Joo et al., 2001). Soybean is a very good source of
plant protein (Brunsgaard et al., 1994). Glutamic acid (Glu)
is one of the most abundant amino acids found in legumes
such as soybean, red bean and mung bean (Koh et al., 1997).
Gamma-aminobutyric acid (GABA) is a ubiquitous
non-protein amino acid which is produced primarily by the
*
Corresponding author. Tel.: +82 63 290 1433; fax: +82 63 290 1429.
E-mail addresses: shoh1573@yahoo.co.kr, shoh@woosuk.co.kr (S.-H.
Oh).
mate decarboxylase (GAD) (Satya Narayan and Nair,
1990). It is well known that GABA functions in animals as
a major inhibitory neurotransmitter (Krogsgaard-Larsen,
1989; Mody et al., 1994). GABA is involved in the regula-
tion of cardiovascular functions, such as blood pressure
and heart rate, and plays a role in the sensations of pain
and anxiety (Mody et al., 1994). The consumption of
GABA-enriched foods such as milk (Hayakawa et al.,
2004), soybean (Shizuka et al., 2004), tempeh (Aoki et al.,
2003), gabaron tea (Abe et al., 1995), red mold rice (Tsuji
et al., 1992), and Chlorella (Nakamura et al., 2000) has been
reported to depress the elevation of systolic blood pressure
in spontaneously hypertensive rats (SHRs).
It has been recently reported that: when chitosan is used
in the medium for the germination of brown rice, GAD
activity increases. As a consequence, germinated brown
rice with increased GABA concentration was produced
(Oh and Choi, 2000). GABA and some free amino acids
such as alanine in germinated brown rice were further

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.06.006
1676 K.-B. Park, S.-H. Oh / Bioresource Technology 98 (2007) 1675–1679
increased by joint treatment with chitosan and glutamic
acid (Oh and Oh, 2003). By applying such methods, germi-
nated soybean with enhanced levels of GABA and free
amino acids was produced, and its extract was used for pro-
ducing yogurt. The GABA content was also enhanced by
applying microorganisms with high GABA producing abil-
ity for yogurt. Here we report the methods and materials to
produce the yogurt with high levels of GABA, free amino
acids and isoXavones.
2. Methods
2.1. Microogranisms and media
Strains used in this study were Lactobacillus acidophilus
(KCCM 40265), Lactobacillus plantarum (KCTC 3105) and
Lactobacillus brevis OPY-1 (KFCC 11337). The L. brevis
OPY-1 strain was isolated from Kimchi and deposited to
Korea Culture Center of Microorganisms. Stock cultures
were maintained on agar plates containing 55 g/L of MRS
broth and 20 g/L of agar. Seed culture was conducted in
MRS broth medium. The initial pH of a medium was
adjusted to 6.2 and was not regulated during Xask culture.
The medium was sterilized in an autoclave at 121 °C and
1.5 psi for 20 min.
2.2. Cultivation of L. brevis OPY-1
L. brevis OPY-1 seed culture was prepared in a 100 mL
Xask with 10 mL MRS broth incubated at 30 °C and
150 rpm for 24 h. A 4% volume of seed culture was used as
its inoculum for the Xask culture. In order to investigate the
GABA production by L. brevis OPY-1, the Xask cultures
were carried out in a 250 mL Xask with 50 mL of MRS
broth with 1% (w/v) of monosodium glutamate (MSG) at
30 °C and 150 rpm for 24 h.
2.3. Producing germinated soybean and fermentation
substrate
Germinated soybean was produced as described else-
where (Oh and Oh, 2003). In brief, 50 g of commercial soy-
bean was germinated in an incubator with 100 mL solution
to which a chitosan/glutamic acid germination solution
(50 ppm chitosan dissolved in 5 mM glutamic acid) was
added at 25 °C. The germination solution was exchanged
for fresh solution at 12 h intervals until germination was
complete at 72 h. After removing the germinated soybean
from the solution, it was dried on a Wlter paper. The germi-
nated soybean was frozen in liquid nitrogen and ground
with a mortar and pestle as described (Oh and Oh, 2003).
Four volumes of double distilled water were added to the
soybean powder, and the mixture was sterilized in an auto-
clave at 121 °C and 1.5 psi for 20 min. The sterilized sample
was Wltered and treated with -amylase (Park and Oh,
2005) to use as a fermentation substrate.
2.4. Producing starter and yogurt
The procedures to produce starter and fermentation
were as described by Park and Oh (2005) with minor modi-
Wcations. The L. acidophilus, L. plantarum and L. brevis
OPY-1 strains were inoculated into Lactobacillus MRS
broth (4% v/v), and the inoculum was activated at three
times at 37 °C for 24 h to use as the starter for production of
yogurt. Powdered whole milk (18%) and skim milk (2%)
were added to the prepared fermentation substrate solution
and homogenized in a Warning blender for 5 min. After-
wards, it was sterilized in an autoclave for 20 min at 121 °C.
After the sterilized substrate was warmed to 30 °C, the
substrate was inoculated with the mixed strain starter
(L. acidophilus + L. plantarum + L. brevis OPY-1 strain,
1:1:3 v/v), and was fermented at 30 °C for 24 h.
2.5. GABA assay
Contents of GABA in the cell suspension of L. Brevis
OPY-1, germinated soybean extract and fermented GABA
soya milk were determined by HPLC (Waters, Milford,
MA) as described earlier (Oh and Oh, 2003; Park and Oh,
2005). GABA was extracted essentially as described by
Baum et al. (1996) with minor modiWcations (Oh and Oh,
2003). GABA contents were calculated using the Autochro
WIN program (Young-Lin, Seoul, Korea).
2.6. Measuring viable count
Sample (1 mL) was collected 4 h after inoculation and
diluted 10 fold with sterilized physiological saline. After
that 0.1 mL of aliquot was smeared on MRS plate count
agar using a micropipette and incubated for 24 h at 37 °C.
Visible colonies were then counted and the unit expressed
as CFU (colony forming unit)/mL.
2.7. Content of isoXavones in yogurt
The contents of isoXavones in GABA soya milk were
determined by HPLC (Waters) as described by Wang et al.
(2003) with minor modiWcations (Kim et al., 2004). IsoXav-
ones were extracted as described by Kim et al. (2004). Iso-
Xavone contents were calculated from standard calibration
curves generated by using standard genistein, daidzein and
glycitein.
2.8. Sensory evaluation of yogurt
To evaluate the sensory properties of the product, the
curd of yogurt, which was incubated at 20 °C for 20 h, was
broken and kept in a refrigerator at 4 °C for 5 h. After-
wards, 20 panelists evaluated its overall acceptability, taste,
odor, texture, etc., and each item was scored between 1 and
5 points, in which 1 is equal to worst and 5 is equal to best.
DiVerences in preferences between the conventional yogurt
and GABA soya yogurt were analyzed with Student’s T-test
 K.-B. Park, S.-H. Oh / Bioresource Technology 98 (2007) 1675–1679 1677

to evaluate signiWcance of the diVerences. Statistical analy- Table 1

ses were performed using SAS software version 8 (SAS Changes in the levels of GABA and free amino acids by the germination
of soybean
Institute, Cary, NC, USA).
Namea Soybean (mg/100 g) Germinated soybean (mg/100 g)
3. Results GABA 5.87 12.51
Asp 5.42 6.68
Glu 13.09 15.55
3.1. Growth and GABA production of L. brevis OPY-1
Lys 0.00 0.00
His 9.42 11.34
GABA produced by L. brevis OPY-1 was over 2.5 g/L Arg 3.91 2.40
and the rate of GABA production was 104.2 mg/L/h. The Ser 16.15 29.13
cell growth of L. brevis OPY-1 increased up to 6.2 of the Thr 3.89 14.32
Cys 0.00 0.00
optical density unit at 24 h of fermentation (Fig. 1).
Phe 2.12 8.77
Tyr 1.09 6.00
3.2. Production and characteristics of fermented GABA soya Gly 1.98 2.64
milk Ala 5.24 10.90
Pro 2.09 7.09
Val 0.78 2.10
In order to produce fermented GABA soya milk (GABA
Leu 2.08 3.15
soya yogurt), germinated soybean with enhanced levels of Ile 0.89 6.02
GABA and some free amino acids was produced (Table 1). Met 1.18 1.82
The germinated soybean extract was used to prepare a fer- a
GABA and free amino acids were analyzed by HPLC as described in
mentation substrate solution. Starter strains including the Methods.
L. brevis OPY-1 were inoculated into substrate solution,
which was again incubated somewhere between 20 h and 600
24 h to produce the Wnal product. The initial viable bacte-
Amino acid (nmole/mL)

rial number of the yogurt could not be recorded between
0 h and 4 h due to a short incubation term, but remarkably
increased by a time-dependent manner between 16 h and
20 h of fermentation. Maximum bacterial numbers were
recorded as 4.4 £ 108 colony forming units per ml of sample
at 20 h of fermentation. The initial levels of GABA in the
yogurt were also low, but increased in a time-dependent
manner, reaching almost maximal levels within 20 h
(415.34 g/g D.W.). Yogurt with the GABA of 424.67 g/
g D.W. was produced, whereas fermented milk made by
conventional method had the GABA of less than 1.5 g/
g D.W. The yogurt also contained isoXavones (genistein,
55 g/g F.W.; daidzein, 11 g/g F.W.; glycitein, 21 g/g F.W.)
and several free amino acids (Fig. 2), which have rarely
been detected in other conventional yogurts. Sensory scores
3000
2500
GABA (mg/L)
500
400
300
200
100
0
Asp Ser Glu Gly His Arg Ala Pro Tyr Cys Met Val Leu Ile Phe
Amino acid
Fig. 2. Comparison of free amino acid levels between GABA soya yogurt
and yogurt prepared by conventional laboratory methods. The closed bar
(䊏) and the open bar (䊐) show the amino acid levels of GABA soya
yogurt and conventional yogurt, respectively.
on a 5-point scale were higher for high-GABA soya yogurt
than conventional yogurt. The scores of GABA soya
7 yogurt in color, Xavor and acceptability were over 3.5,
whereas the scores of conventional yogurts were around
6 2.5. The p values of it were less than 0.001 in color, Xavor
5 and acceptability and less than 0.05 in taste, respectively,

2000 which was helpful to weigh the signiWcance of diVerence.

Abs (600nm)

4
1500 4. Discussions
3
1000
2 In the present study, we utilized lactic acid bacteria and
500 1
germinated soybean with high GABA-generating capacity
and developed yogurt with enhanced levels of GABA, iso-
0 0 Xavones and some amino acids. It has been reported that
6hr 12hr 18hr 24hr GABA synthesis in plants is controlled by various external
Cultivation time factors, e.g., physical stimulus, temperature, hypoxia, mois-
Fig. 1. Growth proWle and GABA production of L. brevis OPY-1. The
ture, stress, etc. (Brown and Shelp, 1997; Crawford et al.,
open bar (䊐) and the closed square (䊏) show the GABA levels and 1994). Thus plants, it may be suggested, utilize the GABA-
growth proWle, respectively. generating system to cope with environmental stress, and
1678 K.-B. Park, S.-H. Oh / Bioresource Technology 98 (2007) 1675–1679
their GABA-generating system is, as it has been reported,
closely connected to various other factors, such as glutamic
acid, GAD, calcium, calmodulin, etc. (Crawford et al., 1994;
Snedden et al., 1996; Oh and Cha, 2000). Germinated soy-
bean with an enhanced level of GABA was produced by
exploiting such systems, and its extract was used to produce
GABA soya yogurt with a view to making a functional,
nutritive product with high consumer satisfaction.
Functional and nutritive values were also enhanced by
applying microorganisms with a high GABA producing
ability to the production of the yogurt. We have estimated
the cost of the production of GABA soya yogurt as $1.3/L
which is a little higher than that of the traditional yogurt of
$1.0/L. Although the price is a little high, we expect that
consumer will choose that kinds of yogurt, respecting qual-
ity and functionality rather than price. It has been reported
that GABA production confers resistance to an acidic pH
in Lactococcus lactis and Escherichia coli (Sanders et al.,
1998; Castanie-Cornet et al., 1999). Moreover, it has been
believed that it can facilitate cell survival by maintaining
cellular pH, even under acidic environments, because GAD
must consume an H+ ion for GABA production. Similarly,
dairy products with high GABA and GAD activity are
capable of sustaining through the digestive system, by
which they made it possible to have probiotic eVects, and
also they may possess the same acid-stability properties
that are required to survive in the intestines. Additional ani-
mal and clinical studies are needed on the GABA soya
yogurt and lactic acid bacteria with a high GABA produc-
ing power with a particular emphasis on acid-stability and
bile resistance.
Recently, the research team found out that germinated
brown rice extracts with high content GABA markedly
stimulate immune response, and that the extracts can
repress or block the proliferation of cancer cells (Oh and
Oh, 2003, 2004). Hayakawa et al. (2004) showed that
GABA-enriched milk (1 nmol/mL) has lowered blood pres-
sure in spontaneously hypertensive and normotensive
Wistar-Kyoto rats. Therefore, the amount of GABA incor-
porated into the yogurt, it seems, is high enough to have
some functional value. The Lactobacillus strain and other
GABA producing lactic acid bacteria seem to show a pros-
pect to be applied in dairy and other health products that
can exploit the functional properties of GABA. It has pre-
viously been reported that free amino acids and oligopep-
tides in foods have such nutritional advantages due to their
rapid absorption (Kamiya, 2002; Aoyama et al., 1996),
muscle protein maintenance (Aoyama et al., 1996), and
antioxidative activity (Hoppe et al., 1997). Other various
physiological eVects in addition to antihypertensive eVects
can also be expected in GABA soya yogurt, but they still
need to be conWrmed by animal and clinical studies.
Acknowledgements
This work was supported by the Jeonbuk Bioindustry
Development Institute Grant funded by the Ministry of
Commerce, Industry and Energy (MOCIE), Jeollabuk-do
Provincial Government and Imsil–Gun.
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