Effect of soaking and gaseous treatment on GABA content in germinated brown rice Noriko Komatsuzaki a,b,c, * , Kikuichi Tsukahara b , Hidechika Toyoshima c , Tadanao Suzuki c , Naoto Shimizu a , Toshinori Kimura a a Graduate School, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan b Domer, Inc., 3-3-19 Tokiwagi, Ueda, Nagano 386-0027, Japan c National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan Received 22 April 2005; accepted 28 October 2005 Available online 20 December 2005 Abstract To establish a new method of processing germinated brown rice (GBR), we processed grain of cultivars with a large germ by soaking and gaseous treatment. After soaking for 3 h and gaseous treatment for 21 h at 35 °C, the content of c-aminobutyric acid (GABA) in GBR (24.9 mg/100 g) was higher than that by the conventional soaking method (10.1 mg/100 g). Although the number of microorgan- isms on the surface of the GBR increased during soaking, steaming for 20 min and ethanol treatment for 3 min completely sterilized the GBR and did not reduce the amount of GABA. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: GABA; Germination; Brown rice with a large germ; Gaseous treatment 1. Introduction c-aminobutyric acid (GABA) is a free amino acid widely distributed in nature, and is a neurotransmitter in the brain and spinal cord of mammals (Manyam, Katz, Hare, Kan- ifefski, & Tremblay, 1981). GABA is produced primarily by the decarboxylation of L-glutamic acid, catalyzed by the enzyme, glutamate decarboxylase (GAD) [EC4.1.1.15] (Mayer, Cherry, & Rhodes, 1990). GABA has several physiological functions such as neurotransmission and induction of hypotensive effects, diuretic effects, and tran- quilizes effects (Jakobs, Jaeken, & Gibson, 1993; Okada et al., 2000; Omori et al., 1987). Germinated brown rice (GBR) extracts containing GABA inhibited cancer cell proliferation (Oh & Oh, 2004). Methods to increase GABA concentrations of food have been studied up to today. For example, GABA is higher in Gabaron tea, an anaerobically incubated tea (Sawai et al., 1999; Tsushida & Murai, 1987), in bean sprouts (soybean, black gram, green gram) treated with carbon dioxide (Katagiri & Shimizu, 1989), and in brown rice processed by high-pressure treatment (Kinefuchi, Sekiya, Yamazaki, & Yamamoto, 1999a). GBR is produced by soaking brown rice grains in water to promote germination, and GABA accumulates during this process. GBR contains vitamins, minerals, fiber, and effective components such as phytic acid (Graf & Empson, 1987; Hunt, Johnson, & Juliano, 2002) and ferulic acid (Tian, Nakamura, & Kayahara, 2004). Although normal brown rice has high nutritional value, its popularity is low because it cannot be cooked in a conventional rice coo- ker. However, GBR is easily cooked and the texture is softer than that of brown rice. Therefore, GBR could become a popular healthy food. In this study, we examined the accumulation of GABA in GBR and proposed a new method of processing. During the soaking of rice grains in water at 30–35 °C for more than 20 h such as in the commercial parboiling of rice, 0260-8774/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.10.036 * Corresponding author. Address: Graduate School, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan. E-mail address: norikoma@nfri.affrc.go.jp (N. Komatsuzaki). www.elsevier.com/locate/jfoodeng Journal of Food Engineering 78 (2007) 556–560 microorganisms multiply greatly (Bandara, Vithanege, & Bean, 1991). Methods of reducing microorganisms during the production of GBR include high-pressure treatment of rice (Kinefuchi, Sekiya, Yamazaki, & Yamamoto, 1999b), ultraviolet irradiation (Suzuki & Maekawa, 1999), and dis- infections by electrochemical treatment ( Feng et al., 2004). There are still problems in the effectiveness on the process- ing cost. Therefore, we investiga ted the effect of steaming and ethanol treatment after soaking stage on the disinfec- tions of GBR in order to solve those problems. 2. Materials and methods 2.1. Materials We used five cultivars of Oryza sativa L. ssp. Japon- ica:Haiminori and Oou 359, with a large germ, and Kos hi- hikari, Yumetsukushi,andNipponbare, with a normal size germ. All were harvested in 1999 or 2001. All samples were stored in a refrigerator at 4 °C for 6 months. 2.2. Measurement of hydration characteristics of GBR during soaking We investigated the hydration characteristics of Haimi- nori and Nipponbare brown rice. Grains were soaked in water at 35 °C. Samples were removed and weighed at specified times during soaking: 0.5, 1, 2, 3, 5, 10, 14, 18, 22, and 24 h. After soaking, the rice was dried at 135 °C for 3 h. The moisture content of each lot was calculated. 2.3. Preparation of GBR by soaking and gaseous treatment (GBR–SGT) Washed grains (100 g) of all five cultivars were soaked in water at 35 °C for 24 h. After germination, the wat er was drained off and grains were washed again. These grains were used as the control. Soaking and gaseous treatment (SGT) was carried out as shown in Fig. 1. Lots of washed rice grains (100 g) were soaked in water at 35 °C for 0.5, 1, 2, 3, 4, or 5 h, and then separately packed in six plastic boxes (12 cm · 8cm· 3 cm high) with a lid. These boxes were put in an incubator at 35 °C for 23.5, 23, 22, 21, 20, and 19 h, respectively. These grains were then taken out, frozen at À20 °C, and lyophilized. The lyophilized grains were pulverized to rice flour in a mill. The rice flour was used for amino acid analysis. 2.4. Analysis of free amino acids in brown rice and GBR Brown rice powder and GBR powder (2.5 g) were placed in a screw test tube containing 25 ml of 70% (v/v) ethanol solution. The mixture was vigorously mixed for 1 min at room temperature and then centrifuged at 8000g at 4 °C for 5 min. The supernatant was filtered (No. 2, filter paper, Whatman, Kent, England) and collected in a flask. The same volume of 70% ethanol solution was added to the precipitates as described above, and the extraction was repeated. The collected supernatant (50 ml) was dried with an evaporator (EYELA, Tokyo, Japan) at 40 °C. The res- idue was dissolved in 5 ml of lithium citrate elution buffer (pH 2.2) containing 1.5 g tri-lithium citrate, 19.8 g citric acid, 12.0 g LiCl and 20.0 g 2,2-thiodi ethanol (per) and then filtered through a 0.45 lm Millipore filter. Portions (20 ll) of the samples were injected into an amino acid ana- lyzer (LC-11 A, Yanako, Kyoto, Japan) to measure the amino acid contents. 2.5. Treatment of GBR by steaming and ethanol Washed rice grains (Nipponbare, 100 g) were soaked in water at 35 °C for 24 h. After germination, the water was drained off and the rice grains were washed again. The GBR was put in wire netting (15 cm diameter, 8 cm high), steamed for 10, 20, or 30 min in a steamer (Watanabe Co., Japan), and then soaked in 100 ml of 70% (v/v) ethanol solution for 3 min. The grains were frozen at À20 °Cand prepared for amino acid analysis as above. 2.6. Microbiological methods Steamed and ethanol-treated GBR (10 g) was put in a flask containing 100 ml of 0.85% sodium chloride solution. Total aerobic plate counts were determined on plate count agar (Nissui Pharmaceutical Co., Ltd., Japan) incubated for 48 h at 37 °C. Washing Drain Washing Packaging Gaseous treatment for germination (for 23.5, 23, 22, 21, 20, 19 h) Freezing for analysis Washing Incubator (35 ˚C) Soaking (at 35 º C for 0.5, 1, 2, 3, 4, 5 h) No exchange air Fig. 1. Production of germinated brown rice (GBR) by soaking and gaseous treatment (SGT). N. Komatsuzaki et al. / Journal of Food Engineering 78 (2007) 556–560 557 3. Results and discussions 3.1. Hydration characteristics of brown rice with large germ during soaking As Fig. 2 indicates, the moisture content of Nipponbare reached 35–36% after 24 h. In contrast, that of Haiminori was greater than 35% after 2 h, and reached 43.6% after 24 h. The GBR products contained normal 30–35% mois - ture. In raw wild rice, high moisture levels (35–50%) pro- moted mold and bacterial growth (Lindenfelser, Ciegler, & Hesseltine, 1978). The moisture content of Haiminori GBR should be kept under 35%, similar to that of normal brown rice. 3.2. Accumulation of free amino acids in GBR–SGT Fig. 3 shows GABA contents in Haiminori GBR treated by SGT. The highest GABA content (25.5 mg/100 g) occurred with soaking for 3 h and gaseous treatment for 21 h. The moisture content of this GBR was 36.9% (Fig. 2). Thus, 3 h was the optimal soaking time. Table 1 shows the amino acid contents in Haiminori brown rice (control), GBR (soaked at 35 °C for 24 h), and GBR–SGT (21 h gas at 35 °C). In GBR, contents of aspartic acid, serine, asparagine, and glutamic acid were decreased, and others were increased. Amino acids stored in brown rice as storage protein are decomposed by water absorption, changed into transportable amide, and supplied to the growing parts of the rice seedling (Lea, Robinson, & Stewart, 1990). Therefore, on water absorp- tion, glutamate decarboxylase (GAD) is activated, and glu- tamic acid is converted to GABA. In GBR–SGT, glutamine and glutamic acid were increased, although GABA was also increased. This result might indicate that the glutamic acid was synthesized by the glutamate synthase (GOGAT) glutamine synthetase (GS) cycle. The GS/GOGAT cycle plays an important role in anaerobic accumulation of GABA and alanine (Aurisano, Bertani, & Reggian i, 1995; Reggina, Nebuloni, & Brambilla, 2000). Moreover, GABA increases rapidl y in plant tissues in response to various forms of stress such as hypoxia (Roberts, Callis, Wemmer, Walbot, & Jardetzky, 1984), cold shock, and darkness (Servaites, Schrader, & Jung, 1979). We think that the SGT condition became hypoxic in this experiment. The GABA contents in the five cultivars of brown rice were a ll significantly higher in the SGT method than in the soaking treatment (Fig. 4). Oou 359 and Haiminori, both with a large germ, had a clearly higher GABA content than normal brown rice. 3.3. Evaluation of steaming and ethanol treatment of GBR To evaluate the effectiveness of disinfection by steaming and ethanol, we measured the aerobic plate counts (APC) of GBR. APC increased above 10 8 cfu g À1 after soaking 0 10 20 30 40 50 0 5 10 15 20 25 Soakin g time (h) Moisture content (%) Nipponbare Haiminori Fig. 2. Hydration characteristics of Nipponbare and Haiminori during soaking at 35 °C. 0 5 10 15 20 25 30 Soaked for 0.5 h Soaked for 1 h Soaked for 2h Soaked for 3 h Soaked for 4 h Soaked for 5 h GABA (mg/100 g FW) Fig. 3. GABA content in GBR (Haiminori) after different soaking times. Table 1 Free amino acid contents (mg/100 g FW) in Haiminori raw brown rice (control), germinated brown rice (GBR), and GBR treated by soaking and gaseous treatment Amino acid Control GBR GBR by SGT Asp 6.6 ± 1.04 1.2 ± 0.22 1.8 ± 0.61 Thr 1.0 ± 0.48 3.1 ± 0.76 6.0 ± 1.16 Ser 3.5 ± 0.29 2.0 ± 0.75 2.7 ± 0.42 Asn 7.1 ± 1.69 3.7 ± 0.55 7.0 ± 0.78 Glu 12.4 ± 3.06 4.5 ± 0.41 13.4 ± 3.57 Pro 1.9 ± 1.66 5.1 ± 0.67 8.4 ± 1.26 Gly 1.5 ± 0.89 4.3 ± 0.82 8.7 ± 1.50 Ala 12.2 ± 4.48 13.0 ± 2.00 25.6 ± 9.29 Val 0.8 ± 0.33 4.5 ± 0.76 12.3 ± 1.00 Cys 1.4 ± 0.51 1.9 ± 1.41 2.9 ± 0.70 Met 0.4 ± 0.40 2.2 ± 0.52 3.3 ± 1.01 I-Leu 0.7 ± 0.15 3.7 ± 0.67 5.8 ± 0.70 Leu 0.9 ± 0.17 6.4 ± 0.97 12.3 ± 1.31 Tyr 1.4 ± 0.39 4.1 ± 0.37 7.0 ± 0.33 Phe 1.0 ± 0.59 3.8 ± 0.37 5.5 ± 0.99 GABA 7.3 ± 2.05 10.1 ± 1.36 24.9 ± 4.00 Lys 3.9 ± 1.45 4.4 ± 0.84 9.6 ± 2.55 His 1.0 ± 0.30 2.4 ± 0.79 4.3 ± 0.99 Arg 4.9 ± 1.14 9.0 ± 3.06 10.6 ± 6.48 Total 67.0 ± 12.38 93.0 ± 13.34 178.7 ± 32.78 Data expressed as mean ± SD of three independent experiments. 558 N. Komatsuzaki et al. / Journal of Food Engineering 78 (2007) 556–560 at 35 °C for 24 h (Table 2). After steaming for 20 and 30 min, APC decreased from 10 8 to 10 2 cfu g À1 . But steam- ing and ethanol treatment for 20 and 30 min produced a zero APC. In general, Bacillus and molds such as Aspergillus are widely distributed in stored grain (Tsuruta, Ikekawa, & Saito, 1984). Although they die when treated at over 80 °C for 10 min (Rijal & Fujioka, 2003), it is possible that bacterial spores survive. High-pressure treatment reduced the viable bacterial count in brown rice, but num- ber of bacterial spores subsequently increased (Kinefuchi et al., 1999a, 1999b). Consequently, only the combination of steaming and ethanol treatment is completely effective. We measured the effect of steaming and ethanol treat- ment on the GABA content of GBR. Table 2 shows that the GABA content in GBR–SGT was 11.3 mg, and that GABA content was not affected by steaming and ethanol treatment. Watanabe, Maeda, Tsukahara, Kayahara, and Morita (2004) reported that GABA in bread containing brown rice and GBR was almost completely decomposed during the baking process. They suggested that heating decomposed the GABA; however, steaming at less than 100 °C had no effect in our experiment. 4. Conclusion Soaking and gaseous treatment (SGT) of Haiminori grains (with a large germ) gave the highest concentration of GABA in GBR. Microorganisms multiplied during soaking were elimi- nated by our recommended method, steaming for 20 min and ethanol treatment for 3 min, without reduction of GABA content. Acknowledgments This study was supported by the project, Development of Technology for Versatile Uses of Carbohydrates by Applying Glyco-Engineering, the Ministry of Agriculture, Forestry and Fisheries, Japan as a collaborative effort among industry, government, and academia. References Aurisano, N., Bertani, A., & Reggiani, R. (1995). 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