Bioresource Technology 98 (2007) 1675–1679

Short Communication

Production of yogurt with enhanced levels of gamma-aminobutyricacid 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 Koreab 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 2006Available 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 theGABA 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 conventionalyogurts. The results suggested that the Lactobacillus brevis OPY-1 and germinated soybean possessed a prospect to be applied in dairyand 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

Yogurt is a nutrient-rich fermented food made of milk,containing various organic acids, peptones, peptides, othertrace activators and lactic acid bacteria. Yogurt has anintestine-cleaning function to promote the proliferation ofintestinal lactic acid bacteria (Savaiano et al., 1984; Parket al., 2003). Ingredients such as non-fat dry milk, soya pro-tein, vegetables, sweet potato, pumpkin, plum, etc. aresometimes added into Korean yogurts (Park et al., 2003;Ko, 1989; Joo et al., 2001). Soybean is a very good source ofplant protein (Brunsgaard et al., 1994). Glutamic acid (Glu)is one of the most abundant amino acids found in legumessuch as soybean, red bean and mung bean (Koh et al., 1997).

Gamma-aminobutyric acid (GABA) is a ubiquitousnon-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).

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2006.06.006

�-decarboxylation of Glu catalyzed by the enzyme gluta-mate decarboxylase (GAD) (Satya Narayan and Nair,1990). It is well known that GABA functions in animals asa major inhibitory neurotransmitter (Krogsgaard-Larsen,1989; Mody et al., 1994). GABA is involved in the regula-tion of cardiovascular functions, such as blood pressureand heart rate, and plays a role in the sensations of painand anxiety (Mody et al., 1994). The consumption ofGABA-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 (Tsujiet al., 1992), and Chlorella (Nakamura et al., 2000) has beenreported to depress the elevation of systolic blood pressurein spontaneously hypertensive rats (SHRs).

It has been recently reported that: when chitosan is usedin the medium for the germination of brown rice, GADactivity increases. As a consequence, germinated brownrice with increased GABA concentration was produced(Oh and Choi, 2000). GABA and some free amino acidssuch as alanine in germinated brown rice were further

1676 K.-B. Park, S.-H. Oh / Bioresource Technology 98 (2007) 1675–1679

increased by joint treatment with chitosan and glutamicacid (Oh and Oh, 2003). By applying such methods, germi-nated soybean with enhanced levels of GABA and freeamino acids was produced, and its extract was used for pro-ducing yogurt. The GABA content was also enhanced byapplying microorganisms with high GABA producing abil-ity for yogurt. Here we report the methods and materials toproduce the yogurt with high levels of GABA, free aminoacids and isoXavones.

2. Methods

2.1. Microogranisms and media

Strains used in this study were Lactobacillus acidophilus(KCCM 40265), Lactobacillus plantarum (KCTC 3105) andLactobacillus brevis OPY-1 (KFCC 11337). The L. brevisOPY-1 strain was isolated from Kimchi and deposited toKorea Culture Center of Microorganisms. Stock cultureswere maintained on agar plates containing 55 g/L of MRSbroth and 20 g/L of agar. Seed culture was conducted inMRS broth medium. The initial pH of a medium wasadjusted to 6.2 and was not regulated during Xask culture.The medium was sterilized in an autoclave at 121 °C and1.5 psi for 20 min.

2.2. Cultivation of L. brevis OPY-1

L. brevis OPY-1 seed culture was prepared in a 100 mLXask with 10 mL MRS broth incubated at 30 °C and150 rpm for 24 h. A 4% volume of seed culture was used asits inoculum for the Xask culture. In order to investigate theGABA production by L. brevis OPY-1, the Xask cultureswere carried out in a 250 mL Xask with 50 mL of MRSbroth with 1% (w/v) of monosodium glutamate (MSG) at30 °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 solutionto which a chitosan/glutamic acid germination solution(50 ppm chitosan dissolved in 5 mM glutamic acid) wasadded at 25 °C. The germination solution was exchangedfor fresh solution at 12 h intervals until germination wascomplete at 72 h. After removing the germinated soybeanfrom the solution, it was dried on a Wlter paper. The germi-nated soybean was frozen in liquid nitrogen and groundwith a mortar and pestle as described (Oh and Oh, 2003).Four volumes of double distilled water were added to thesoybean powder, and the mixture was sterilized in an auto-clave at 121 °C and 1.5 psi for 20 min. The sterilized samplewas 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 fermentationwere as described by Park and Oh (2005) with minor modi-Wcations. The L. acidophilus, L. plantarum and L. brevisOPY-1 strains were inoculated into Lactobacillus MRSbroth (4% v/v), and the inoculum was activated at threetimes at 37 °C for 24 h to use as the starter for production ofyogurt. Powdered whole milk (18%) and skim milk (2%)were added to the prepared fermentation substrate solutionand 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, thesubstrate 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. BrevisOPY-1, germinated soybean extract and fermented GABAsoya 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 byBaum et al. (1996) with minor modiWcations (Oh and Oh,2003). GABA contents were calculated using the AutochroWIN program (Young-Lin, Seoul, Korea).

2.6. Measuring viable count

Sample (1 mL) was collected 4 h after inoculation anddiluted 10 fold with sterilized physiological saline. Afterthat 0.1 mL of aliquot was smeared on MRS plate countagar using a micropipette and incubated for 24 h at 37 °C.Visible colonies were then counted and the unit expressedas CFU (colony forming unit)/mL.

2.7. Content of isoXavones in yogurt

The contents of isoXavones in GABA soya milk weredetermined 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 calibrationcurves generated by using standard genistein, daidzein andglycitein.

2.8. Sensory evaluation of yogurt

To evaluate the sensory properties of the product, thecurd of yogurt, which was incubated at 20 °C for 20 h, wasbroken 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 and5 points, in which 1 is equal to worst and 5 is equal to best.DiVerences in preferences between the conventional yogurtand 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-ses were performed using SAS software version 8 (SASInstitute, Cary, NC, USA).

3. Results

3.1. Growth and GABA production of L. brevis OPY-1

GABA produced by L. brevis OPY-1 was over 2.5 g/Land the rate of GABA production was 104.2 mg/L/h. Thecell growth of L. brevis OPY-1 increased up to 6.2 of theoptical density unit at 24 h of fermentation (Fig. 1).

3.2. Production and characteristics of fermented GABA soya milk

In order to produce fermented GABA soya milk (GABAsoya yogurt), germinated soybean with enhanced levels ofGABA and some free amino acids was produced (Table 1).The germinated soybean extract was used to prepare a fer-mentation substrate solution. Starter strains including theL. brevis OPY-1 were inoculated into substrate solution,which was again incubated somewhere between 20 h and24 h to produce the Wnal product. The initial viable bacte-rial number of the yogurt could not be recorded between0 h and 4 h due to a short incubation term, but remarkablyincreased by a time-dependent manner between 16 h and20 h of fermentation. Maximum bacterial numbers wererecorded as 4.4£ 108 colony forming units per ml of sampleat 20 h of fermentation. The initial levels of GABA in theyogurt were also low, but increased in a time-dependentmanner, reaching almost maximal levels within 20 h(415.34 �g/gD.W.). Yogurt with the GABA of 424.67 �g/g D.W. was produced, whereas fermented milk made byconventional 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 rarelybeen detected in other conventional yogurts. Sensory scores

Fig. 1. Growth proWle and GABA production of L. brevis OPY-1. Theopen bar (�) and the closed square (�) show the GABA levels andgrowth proWle, respectively.

0

500

1000

1500

2000

2500

3000

6hr 12hr 18hr 24hr

Cultivation time

GA

BA

(m

g/L)

0

1

2

3

4

5

6

7

Abs

(60

0nm

)

on a 5-point scale were higher for high-GABA soya yogurtthan conventional yogurt. The scores of GABA soyayogurt in color, Xavor and acceptability were over 3.5,whereas the scores of conventional yogurts were around2.5. The p values of it were less than 0.001 in color, Xavorand acceptability and less than 0.05 in taste, respectively,which was helpful to weigh the signiWcance of diVerence.

4. Discussions

In the present study, we utilized lactic acid bacteria andgerminated soybean with high GABA-generating capacityand developed yogurt with enhanced levels of GABA, iso-Xavones and some amino acids. It has been reported thatGABA synthesis in plants is controlled by various externalfactors, e.g., physical stimulus, temperature, hypoxia, mois-ture, stress, etc. (Brown and Shelp, 1997; Crawford et al.,1994). Thus plants, it may be suggested, utilize the GABA-generating system to cope with environmental stress, and

Table 1Changes in the levels of GABA and free amino acids by the germinationof soybean

a GABA and free amino acids were analyzed by HPLC as described inMethods.

Namea Soybean (mg/100 g) Germinated soybean (mg/100 g)

GABA 5.87 12.51Asp 5.42 6.68Glu 13.09 15.55Lys 0.00 0.00His 9.42 11.34Arg 3.91 2.40Ser 16.15 29.13Thr 3.89 14.32Cys 0.00 0.00Phe 2.12 8.77Tyr 1.09 6.00Gly 1.98 2.64Ala 5.24 10.90Pro 2.09 7.09Val 0.78 2.10Leu 2.08 3.15Ile 0.89 6.02Met 1.18 1.82

Fig. 2. Comparison of free amino acid levels between GABA soya yogurtand yogurt prepared by conventional laboratory methods. The closed bar(�) and the open bar (�) show the amino acid levels of GABA soyayogurt and conventional yogurt, respectively.

0

100

200

300

400

500

600

Asp Ser Glu Gly His Arg Ala Pro Tyr Cys Met Val Leu Ile Phe

Amino acid

Am

ino

acid

(nm

ole/

mL)

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 glutamicacid, 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 byexploiting such systems, and its extract was used to produceGABA soya yogurt with a view to making a functional,nutritive product with high consumer satisfaction.

Functional and nutritive values were also enhanced byapplying microorganisms with a high GABA producingability to the production of the yogurt. We have estimatedthe cost of the production of GABA soya yogurt as $1.3/Lwhich is a little higher than that of the traditional yogurt of$1.0/L. Although the price is a little high, we expect thatconsumer will choose that kinds of yogurt, respecting qual-ity and functionality rather than price. It has been reportedthat GABA production confers resistance to an acidic pHin Lactococcus lactis and Escherichia coli (Sanders et al.,1998; Castanie-Cornet et al., 1999). Moreover, it has beenbelieved that it can facilitate cell survival by maintainingcellular pH, even under acidic environments, because GADmust consume an H+ ion for GABA production. Similarly,dairy products with high GABA and GAD activity arecapable of sustaining through the digestive system, bywhich they made it possible to have probiotic eVects, andalso they may possess the same acid-stability propertiesthat are required to survive in the intestines. Additional ani-mal and clinical studies are needed on the GABA soyayogurt and lactic acid bacteria with a high GABA produc-ing power with a particular emphasis on acid-stability andbile resistance.

Recently, the research team found out that germinatedbrown rice extracts with high content GABA markedlystimulate immune response, and that the extracts canrepress or block the proliferation of cancer cells (Oh andOh, 2003, 2004). Hayakawa et al. (2004) showed thatGABA-enriched milk (1 nmol/mL) has lowered blood pres-sure in spontaneously hypertensive and normotensiveWistar-Kyoto rats. Therefore, the amount of GABA incor-porated into the yogurt, it seems, is high enough to havesome functional value. The Lactobacillus strain and otherGABA producing lactic acid bacteria seem to show a pros-pect to be applied in dairy and other health products thatcan 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 theirrapid absorption (Kamiya, 2002; Aoyama et al., 1996),muscle protein maintenance (Aoyama et al., 1996), andantioxidative activity (Hoppe et al., 1997). Other variousphysiological eVects in addition to antihypertensive eVectscan also be expected in GABA soya yogurt, but they stillneed to be conWrmed by animal and clinical studies.

Acknowledgements

This work was supported by the Jeonbuk BioindustryDevelopment Institute Grant funded by the Ministry of

Commerce, Industry and Energy (MOCIE), Jeollabuk-doProvincial Government and Imsil–Gun.

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