Professional Documents
Culture Documents
Short Communication
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
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 2.4. Producing starter and yogurt
acid (Oh and Oh, 2003). By applying such methods, germi-
nated soybean with enhanced levels of GABA and free The procedures to produce starter and fermentation
amino acids was produced, and its extract was used for pro- were as described by Park and Oh (2005) with minor modi-
ducing yogurt. The GABA content was also enhanced by Wcations. The L. acidophilus, L. plantarum and L. brevis
applying microorganisms with high GABA producing abil- OPY-1 strains were inoculated into Lactobacillus MRS
ity for yogurt. Here we report the methods and materials to broth (4% v/v), and the inoculum was activated at three
produce the yogurt with high levels of GABA, free amino times at 37 °C for 24 h to use as the starter for production of
acids and isoXavones. 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-
2. Methods
wards, it was sterilized in an autoclave for 20 min at 121 °C.
After the sterilized substrate was warmed to 30 °C, the
2.1. Microogranisms and media
substrate was inoculated with the mixed strain starter
(L. acidophilus + L. plantarum + L. brevis OPY-1 strain,
Strains used in this study were Lactobacillus acidophilus
1:1:3 v/v), and was fermented at 30 °C for 24 h.
(KCCM 40265), Lactobacillus plantarum (KCTC 3105) and
Lactobacillus brevis OPY-1 (KFCC 11337). The L. brevis
2.5. GABA assay
OPY-1 strain was isolated from Kimchi and deposited to
Korea Culture Center of Microorganisms. Stock cultures
Contents of GABA in the cell suspension of L. Brevis
were maintained on agar plates containing 55 g/L of MRS
OPY-1, germinated soybean extract and fermented GABA
broth and 20 g/L of agar. Seed culture was conducted in
soya milk were determined by HPLC (Waters, Milford,
MRS broth medium. The initial pH of a medium was
MA) as described earlier (Oh and Oh, 2003; Park and Oh,
adjusted to 6.2 and was not regulated during Xask culture.
2005). GABA was extracted essentially as described by
The medium was sterilized in an autoclave at 121 °C and
Baum et al. (1996) with minor modiWcations (Oh and Oh,
1.5 psi for 20 min.
2003). GABA contents were calculated using the Autochro
WIN program (Young-Lin, Seoul, Korea).
2.2. Cultivation of L. brevis OPY-1
2.6. Measuring viable count
L. brevis OPY-1 seed culture was prepared in a 100 mL
Xask with 10 mL MRS broth incubated at 30 °C and Sample (1 mL) was collected 4 h after inoculation and
150 rpm for 24 h. A 4% volume of seed culture was used as diluted 10 fold with sterilized physiological saline. After
its inoculum for the Xask culture. In order to investigate the that 0.1 mL of aliquot was smeared on MRS plate count
GABA production by L. brevis OPY-1, the Xask cultures agar using a micropipette and incubated for 24 h at 37 °C.
were carried out in a 250 mL Xask with 50 mL of MRS Visible colonies were then counted and the unit expressed
broth with 1% (w/v) of monosodium glutamate (MSG) at as CFU (colony forming unit)/mL.
30 °C and 150 rpm for 24 h.
2.7. Content of isoXavones in yogurt
2.3. Producing germinated soybean and fermentation
substrate The contents of isoXavones in GABA soya milk were
determined by HPLC (Waters) as described by Wang et al.
Germinated soybean was produced as described else- (2003) with minor modiWcations (Kim et al., 2004). IsoXav-
where (Oh and Oh, 2003). In brief, 50 g of commercial soy- ones were extracted as described by Kim et al. (2004). Iso-
bean was germinated in an incubator with 100 mL solution Xavone contents were calculated from standard calibration
to which a chitosan/glutamic acid germination solution curves generated by using standard genistein, daidzein and
(50 ppm chitosan dissolved in 5 mM glutamic acid) was glycitein.
added at 25 °C. The germination solution was exchanged
for fresh solution at 12 h intervals until germination was 2.8. Sensory evaluation of yogurt
complete at 72 h. After removing the germinated soybean
from the solution, it was dried on a Wlter paper. The germi- To evaluate the sensory properties of the product, the
nated soybean was frozen in liquid nitrogen and ground curd of yogurt, which was incubated at 20 °C for 20 h, was
with a mortar and pestle as described (Oh and Oh, 2003). broken and kept in a refrigerator at 4 °C for 5 h. After-
Four volumes of double distilled water were added to the wards, 20 panelists evaluated its overall acceptability, taste,
soybean powder, and the mixture was sterilized in an auto- odor, texture, etc., and each item was scored between 1 and
clave at 121 °C and 1.5 psi for 20 min. The sterilized sample 5 points, in which 1 is equal to worst and 5 is equal to best.
was Wltered and treated with -amylase (Park and Oh, DiVerences in preferences between the conventional yogurt
2005) to use as a fermentation substrate. and GABA soya yogurt were analyzed with Student’s T-test
K.-B. Park, S.-H. Oh / Bioresource Technology 98 (2007) 1675–1679 1677
500
rial number of the yogurt could not be recorded between
0 h and 4 h due to a short incubation term, but remarkably 400
increased by a time-dependent manner between 16 h and 300
20 h of fermentation. Maximum bacterial numbers were
recorded as 4.4 £ 108 colony forming units per ml of sample 200
at 20 h of fermentation. The initial levels of GABA in the 100
yogurt were also low, but increased in a time-dependent
0
manner, reaching almost maximal levels within 20 h Asp Ser Glu Gly His Arg Ala Pro Tyr Cys Met Val Leu Ile Phe
(415.34 g/g D.W.). Yogurt with the GABA of 424.67 g/ Amino acid
g D.W. was produced, whereas fermented milk made by
Fig. 2. Comparison of free amino acid levels between GABA soya yogurt
conventional method had the GABA of less than 1.5 g/ and yogurt prepared by conventional laboratory methods. The closed bar
g D.W. The yogurt also contained isoXavones (genistein, (䊏) and the open bar (䊐) show the amino acid levels of GABA soya
55 g/g F.W.; daidzein, 11 g/g F.W.; glycitein, 21 g/g F.W.) yogurt and conventional yogurt, respectively.
and several free amino acids (Fig. 2), which have rarely
been detected in other conventional yogurts. Sensory scores 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,
3000
whereas the scores of conventional yogurts were around
2500 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,
GABA (mg/L)
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, Commerce, Industry and Energy (MOCIE), Jeollabuk-do
closely connected to various other factors, such as glutamic Provincial Government and Imsil–Gun.
acid, GAD, calcium, calmodulin, etc. (Crawford et al., 1994;
Snedden et al., 1996; Oh and Cha, 2000). Germinated soy- References
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