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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
Department of Medicinal Biotechnology, Woosuk University, Jeonju 565-701, Republic of Korea 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 bacteria (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 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 protein, 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: firstname.lastname@example.org, email@example.com (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 glutamate 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 regulation 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
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, germinated soybean with enhanced levels of GABA and free amino acids was produced, and its extract was used for producing yogurt. The GABA content was also enhanced by applying microorganisms with high GABA producing ability 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 elsewhere (Oh and Oh, 2003). In brief, 50 g of commercial soybean 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 germinated 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 autoclave 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 modiWcations. 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. Afterwards, 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). IsoXavones were extracted as described by Kim et al. (2004). IsoXavone 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. Afterwards, 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
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to evaluate signiWcance of the diVerences. Statistical analyses were performed using SAS software version 8 (SAS Institute, 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/L and the rate of GABA production was 104.2 mg/L/h. The cell growth of L. brevis OPY-1 increased up to 6.2 of the optical 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 (GABA soya yogurt), germinated soybean with enhanced levels of GABA and some free amino acids was produced (Table 1). The germinated soybean extract was used to prepare a fermentation substrate solution. Starter strains including the L. brevis OPY-1 were inoculated into substrate solution, which was again incubated somewhere between 20 h and 24 h to produce the Wnal product. The initial viable bacterial 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 7 6
Table 1 Changes in the levels of GABA and free amino acids by the germination of soybean Namea GABA Asp Glu Lys His Arg Ser Thr Cys Phe Tyr Gly Ala Pro Val Leu Ile Met Soybean (mg/100 g) 5.87 5.42 13.09 0.00 9.42 3.91 16.15 3.89 0.00 2.12 1.09 1.98 5.24 2.09 0.78 2.08 0.89 1.18 Germinated soybean (mg/100 g) 12.51 6.68 15.55 0.00 11.34 2.40 29.13 14.32 0.00 8.77 6.00 2.64 10.90 7.09 2.10 3.15 6.02 1.82
a GABA and free amino acids were analyzed by HPLC as described in Methods.
600 Amino acid (nmole/mL) 500 400 300 200 100 0
Asp Ser Glu Gly His Arg Ala Pro Tyr Cys Met Val Leu Ile Phe
Fig. 2. Comparison of free amino acid levels between GABA soya yogurt and yogurt prepared by conventional laboratory methods. The closed bar ( ) and the open bar ( ) show the amino acid levels of GABA soya yogurt and conventional yogurt, respectively.
4 1500 3 1000 500 0 6hr 12hr 18hr 24hr Cultivation time 2 1 0
on a 5-point scale were higher for high-GABA soya yogurt than conventional yogurt. The scores of GABA soya yogurt in color, Xavor and acceptability were over 3.5, whereas the scores of conventional yogurts were around 2.5. The p values of it were less than 0.001 in color, Xavor and 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 and germinated soybean with high GABA-generating capacity and developed yogurt with enhanced levels of GABA, isoXavones and some amino acids. It has been reported that GABA synthesis in plants is controlled by various external factors, e.g., physical stimulus, temperature, hypoxia, moisture, stress, etc. (Brown and Shelp, 1997; Crawford et al., 1994). Thus plants, it may be suggested, utilize the GABAgenerating system to cope with environmental stress, and
Fig. 1. Growth proWle and GABA production of L. brevis OPY-1. The open bar ( ) and the closed square ( ) show the GABA levels and growth proWle, respectively.
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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 soybean 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 quality 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 animal and clinical studies are needed on the GABA soya yogurt and lactic acid bacteria with a high GABA producing 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 pressure in spontaneously hypertensive and normotensive Wistar-Kyoto rats. Therefore, the amount of GABA incorporated 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 prospect to be applied in dairy and other health products that can exploit the functional properties of GABA. It has previously been reported that free amino acids and oligopeptides 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. References
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