Chemical and functional components in different parts of rough rice (oryza sativa l[1] ) beforeandaftergermination

Chemical and functional components in different parts of rough rice (Oryza sativa L.) before and after germination Hyun Young Kim a , In Guk Hwang b , Tae Myoung Kim c , Koan Sik Woo d , Dong Sik Park b , Jae Hyun Kim b , Dae Joong Kim c , Junsoo Lee a , Youn Ri Lee e , Heon Sang Jeong a, ⇑ a Department of Food Science and Technology. Chungbuk National University, Cheongju 361-763, Republic of Korea b Department of Agrofood Resources, National Academy of Agricultural Science, Suwon 441-857, Republic of Korea c College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea d Department of Functional Crop, National Institute of Crop Science, Miryang 627-803, Republic of Korea e Department of Food and Nutrition, Daejeon Health Sciences College, Daejeon 300-711, Republic of Korea article info Article history: Received 2 October 2011 Received in revised form 21 December 2011 Accepted 21 February 2012 Available online 1 March 2012 Keywords: Rough rice Germination Seed parts Chemical components Functional component abstract This study investigated the changes in chemical and functional components in different parts of rough rice seed (Oryza sativa L.) before and after germination. Rough rice was separated into hull, brown rice, and sprout, and then analysed for crude protein, crude lipid, free sugars, fatty acids, phytic acid, vitamin E, c -oryzanol and c -aminobutyric acid (GABA). Before germination, the crude protein content of rough rice was 97.28 mg/g, whereas after germination, it increased to 105.14 mg/g. The phytic acid content was decreased after germination, but glucose, which was absent before germination, increased to 11.45 mg/g in brown rice and 8.82 mg/g in rough rice. After germination, linoleic acid increased whereas oleic and palmitic acid decreased in brown rice. The GABA content showed the highest increase from 15.34 to 31.79 mg/100 g in the rough rice part after germination. The c -oryzanol content in rough rice and brown rice increased 1.13 and 1.20-fold after germination, respectively. The vitamin E content increased from 3.21 to 3.93 mg/100 g in rough rice. The sprout had high vitamin E (5.45 mg/g) and c -oryzanol (9.91 mg/g) content. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Rice (Oryza sativa, L.) is the common name for more than 20 an- nual species in the grass family and is the main food of almost half of the world’s population. The rice seed, or caryopsis, consists mainly of the seed coat, embryo, and endosperm. Rice bran (the seed coat) contains protein, B complex vitamins, and vitamin E and K, while polished rice (without the seed coat) contains about 25% carbohydrate, with trace amounts of iodine, iron, magnesium, and phosphorus, and only small amounts of protein and fat (Madamba & Lopez, 2002; Ponciano & Richard, 2005). Rice bran contains many valuable substances, such as vitamin E ( a -tocopherol and tocotrienol) and c -oryzanol. The major component of vitamin E in rice bran is a -tocopherol, which is an antioxidant that can lower the risk of cancer and coronary heart disease (Zhimin, Na, & Samuel, 2001), and is also reported to prevent Alzheimer’s disease and many allergies (Nakamura, Tian, & Kayahara, 2004). Germination is an effective and common process used to im- prove the nutritional quality of cereals consumed around the world (Lee et al., 2007a). The germination process is affected by external factors such as germination time and absence or presence of light, both of which can aid or inhibit germination in relation to the re- serve (nutrition content) within the seed (Ridge, 1991). During germination, some seed reserves are degraded and used for respi- ration and synthesis of new cell constituents for the developing embryo, thereby causing significant changes in the biochemical, nutritional, and sensory characteristics of the cereal (López- Amorós, Hernandez, & Estrella, 2006). New compounds, such as c -aminobutyric acid (GABA), c -oryzanol, and useful amino acids, are synthesised during germination (Ang, 1991; Woo & Jeong, 2006). Lee et al. (2007a) reported changes in reducing sugars, total sugars, free amino acids, and crude protein content of rough rice before and after germination. Changes in the phenolic content and radical scavenging activity have also been reported (Lee, Woo, Kim, Son, & Jeong, 2007b). However, few studies have reported changes in the chemical and functional components of different parts of rough rice before and after germination. Therefore, the objective of the present study 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.02.138 ⇑ Corresponding author. Address: Department of Food Science and Technology, Chungbuk National University, 52 Naesurodong, Heungduk-gu, Cheongju, Chung- buk 361–763, Republic of Korea. Tel.: +82 43 261 2570; fax: +82 43 271 4412. E-mail address: hsjeong@chungbuk.ac.kr (H.S. Jeong). Food Chemistry 134 (2012) 288–293 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem was to analyse the chemical and functional components of the seed parts (i.e., hull, brown rice, and sprout) before and after germinated rough rice. We examined the crude protein, crude lipid, free sugars, fatty acids, phytic acid, vitamin E, c -oryzanol, and c -aminobutyric acid content. 2. Material and methods 2.1. Rough rice and sample preparation The rough rice (cv. Ilpumbyeo, O. sativa, L.) was grown at the National Institute of Crop Science, Rural Development Administra- tion, Suwon, Korea, during the 2010 growing season. The seed was soaked in water at 15 °C, and the water was changed every 24 h. Three days after germination, the seed was separated into three parts (hull, brown rice, and sprout including the embryo), dried at 60 °C for 24 h, and then ground in a food processor (J. World Tech., Korea). Samples (rough rice seed, hull, brown rice, and sprout) were kept at À20 °C and protected from light prior to fur- ther use. The powdered samples were then passed through a 100- mesh sieve and the chemical and functional components were analysed. 2.2. Analysis of crude protein and lipids The standard method of AOAC (1990) was used for determina- tion of crude protein and lipid content. The crude protein content was measured with the Kjeldahl method (AOAC, 950.09) and the crude lipid content was obtained after incineration using the Soxhelt method (AOAC, 963.15). 2.3. Analysis of free sugars Free sugars were analysed by extracting 5 g of homogenised sample in 20 ml water for 10 min, filtering this through a 0.45- l m membrane, and injecting the filtrate into an HPLC (Waters 2695, New Castle, DE, USA). The analytical conditions followed the method of Park, Gil, and Kim (2002). A carbohydrate analysis column (4.6 Â 150 mm; Waters), RI detector (Waters 2414; Waters), and acetonitrile: water 75:25 (v/v) mobile phase at a flow rate of 1 ml/min were used. 2.4. Analysis of fatty acids Fatty acids in the sample extract were trans-esterified to methyl esters (FAMEs) using a base-catalysed transesterification followed by a Borontrifluoride-catalysed esterification according to AOCS (1998, Official methods). The FAMEs (1.5 l l) were injected into a gas chromatograph (Agilent 6850 GC, Agilent Palo Alto, CA, USA) equipped with a 30 m capillary column coated with HP-INNOWAX (0.25 mm film thickness, Agilent). The injector temperature was set at 250 °C and the flame ionisation detector temperature was 300 °C. The initial oven temperature was 120 °C and was pro- grammed to rise to 230 °Cat5°C/min. Nitrogen gas (99.999%) was used as carrier gas at a velocity of 1.3 cm/s. Fatty acid methyl esters were identified based on retention times in relation to authentic lipid standards and fatty acid compositions were ex- pressed as area percentage of total fatty acids. 2.5. Analysis of phytic acid The amount of phytic acid in the different parts of rough rice be- fore and after germination was measured using a UV spectropho- tometer (DU-650; Beckman Coulter, Fullerton, CA) at a wavelength of 500 nm, according to the modified method of Haung and Lantzsch (1983). The phytic acid level was calculated based on a standard curve. 2.6. Analysis of vitamin E The vitamin E content of methanolic extracts from different parts of rough rice seeds was determined according to the proce- dure described by Lee, Suknark, Kluvitse, Phillips, and Eitenmiller (1998), with some modifications. In brief, an aliquot of each meth- anolic extract was evaporated under N 2 gas. The residues were re- dissolved in n-hexane, filtered, and analysed using normal phase HPLC (Younglin Inc., Seoul, Korea). Tocopherols and tocotrienols were analysed using an LiChrosphere-Diol 100 column (4.0 Â 250 mm, i.d. 5 l m) with a hexane:isopropanol (98.7:1.3, v/ v) mobile phase at a flow rate of 1 ml/min. Peaks were detected by fluorescence using an excitation wavelength of 290 nm and an emission wavelength of 330 nm. 2.7. Analysis of c -oryzanol c -Oryzanol was analysed using HPLC (Thermo Separation Prod- ucts, San Jose, CA, USA) with a UV detector at 325 nm. The metha- nolic extracts were evaporated under N 2 gas. The residue was dissolved in n-hexane and then analysed. The sample extracts were separated on a Nova-Pak C18 column (3.9 Â 150 mm; Waters) using a modified version of Rogers et al. (1993) method. The extractions were performed using initial mobile phase conditions of 50% MeOH, 40% acetonitrile, 5% water, and 5% dichloromethane, at a flow rate of 1.0 ml/min for 5 min. The mobile phase was chan- ged linearly to methanol, acetonitrile, water, and dichloromethane at a ratio of 45:45:5:5 (v/v/v) over the next 10 min. After 30 min, the mobile phase was changed linearly to a ratio of 40:45:5:10 (v/v/v) and held for 60 min before returning to the initial conditions. 2.8. Analysis of c -aminobutyric acid (GABA) c -Aminobutyric acid (GABA) content was extracted according to the method of Oh and Oh (2003) with a slight modification. Briefly, the mixture of organic solution (CH 3 OH 5 ml: CHCl 3 10 ml: H 2 O 5 ml) was added to the pulverised grains (1.0 g). The aqueous solution layer containing GABA was obtained through centrifugation (2800g,4°C, 10 min), and then the supernatant was freeze dried. GABA was measured by a spectrophotometric as- say at 340 nm (Zhang & Brown, 1997). 2.9. Statistical analysis Statistical analysis was carried out using SPSS version 11.5 (SPSS Inc., Chicago, IL, USA). The results are expressed as means ± standard deviations. Student’s t-tests for unpaired data were used for all measured parameters to determine the signifi- cance of the changes before and after germination. 3. Results and discussion 3.1. Crude protein and lipids The changes in crude protein of the different parts of rough rice seed before and after germination ranged from 38 ± 1.21 mg/g in the hull to 105 ± 2.62 mg/g in the brown rice (Fig. 1). During germi- nation, the crude protein content of rough rice slightly increased from 97 ± 2.73 mg/g before to 105 ± 2.62 mg/g after germination, whereas the brown rice protein content slightly decreased (p > 0.05), but the hull content increased significantly from H.Y. Kim et al. /Food Chemistry 134 (2012) 288–293 289 38 ± 1.21 mg/g to 50 ± 2.16 mg/g (p < 0.01). Most storage proteins in rice grain are found in the endosperm, and brown rice contains about 83 mg/g protein (Matz, 1996); these results are similar to those reported by Jones and Lookhart (2005). The increase of protein content may confer nutritional advantage on the germinated rough rice. The increase of protein content by germination could be attrib- uted to net synthesis of enzyme protein which might have resulted in the production of some amino acids during protein synthesis (Marero et al., 1989; Uwaegbute, Iroegbu, & Eke, 2000). The crude lipid was highest in the sprout (6 ± 0.18%) after germination (Fig. 2), while in the hull it increased from 0.6 ± 0.12% to 1.1 ± 0.06% after germination (p < 0.05), but decreased slightly in the brown rice. Both the crude protein and crude lipid content increased after germination, probably because of the biosynthesis of new com- pounds during germination. These results agree with research re- ported for sesame (Hahma, Park, & Lo, 2009), soybean (Park et al., 2002), and germinated brown rice (Anuchita & Nattawat, 2010). 3.2. Free sugars The changes in free sugar content of different parts of rough rice seed before and after germination are shown in Table 1. Fructose and sucrose were found before germination, and fructose and glu- cose found after germination. Total free sugar content increased after germination. Glucose which was absent in rough rice seed and brown rice before germination increased to 8.82 and 11.45 mg/g after germination, respectively. Sucrose content was 0.55 mg/g in rough rice seed and 0.65 mg/g in brown rice before germination but disappeared after germination. In the sprout, glu- cose and sucrose were absent but the fructose content was 0.61 mg/g. In this study, the increase in free sugar content after germination agrees with other reports on rough rice germination (Nakamura et al., 2004). Ayernor and Ocloo (2007) reported that the reducing sugar content increased significantly (p < 0.05) during rice germination up to nine days. It has been reported that free sugars increase after germination because of starch hydrolysis (Kazanas & Fields, 1981). 3.3. Fatty acids Fatty acids are very efficient sources of energy and several fatty acids are known to have potent physiological effects (Kim, Kho, Lee, Kim, & Lee, 2001). The fatty acid compositions of different seed parts before and after germination are shown in Table 2. Palmitic, oleic, and linoleic acids were the major fatty acids (80%), and stea- ric and linolenic acids were minor fatty acids. The total fatty acid content did not differ before and after germination. The sprout contained palmitic acid (23.41%), oleic acid (38.21%), and linoleic acid (18.13%). The linoleic acid content of brown rice after germi- nation increased from 17.40% to 21.99% (p < 0.05). After germina- tion, the oleic acid content of the rough rice and hull increased from 42.99% to 44.00% and from 42.92% to 44.22%, respectively. 3.4. Phytic acid The phytic acid contents of different parts of rough rice before and after germination are shown in Fig. 3. The phytic acid de- creased significantly after germination (p < 0.05). The phytic acid content of rough rice decreased from 3.57 to 2.17 mg/g, and that of brown rice decreased from 4.34 to 3.42 mg/g (p < 0.05). The sprout part that was absent before germination was 0.26 mg/g. The decrease in the phytic acid content after germination may be attributed to leaching out into soaking water (Abdullah, Baldwin, & Minor, 1984). Other researchers have reported that the decrease in phytic acid content due to an increase in phytase activity of ger- minated grains ( Borade, Kadam, & Salunkhe, 1984; Rao & Deosthale, 1982). Phytase activity was found during the germination of grains, which hydrolyse phytate to phosphate and myoinositol phos- phates. A lot of researches on the damaging effects of phytic acid have been published (Spencer & Karmer, 1988) but other results showed that phytates possess possible ability to reduce the risks of heart disease and cancer (Cornforth, 2002). 3.5. c -Oryzanol The beneficial effects of c -oryzanol on human health have gen- erated global interest in developing simple methods for its separa- tion from natural sources, such as crude rice bran oil, rice bran oil soap stock, rice bran acid oil, or biodiesel residue from rice bran (Zullaikah, Melwita, & Ju, 2009). c -Oryzanol is a mixture of 10 esters of triterpene alcohols (Zhimin et al., 2001) and can be used to reduce blood cholesterol, to treat nerve imbalances as an antioxidant or preservative (Murase & Iishima, 1963; Sasaki et al., 1990). In all parts of rough rice, the amount of c -oryzanol ranged from 0.19 to 9.91 mg/g (Fig. 4). After germination, the c -oryzanol content of rough rice and brown rice increased 1.13-fold and 1.2-fold, respec- tively (p < 0.05). The c -oryzanol content of the sprout was 9.91 mg/ g after germination. It is thought that an increase in c -oryzanol occurs in the embryo following rough rice germination. Previous studies have reported that the c -oryzanol content of brown rice grown is influenced by site and season (Miller & Engel, 2006). ** 0 20 40 60 80 100 120 Rough rice Hull Brown rice Sprout Crude protein (mg/g) BG AG Fig. 1. Changes in crude protein content of different parts of rough rice (Oryza sativa L.) before (BG) and after germination (AG). Results are expressed as the average of triplicate samples with mean ± SD. ⁄ p < 0.01; Significantly different by paired t-test, significantly different by Students t-test between before and after germination. * 0.0 2.0 4.0 6.0 8.0 Rough rice Hull Brown rice Sprout Crude lipid (%) BG AG Fig. 2. Changes in crude lipid content of different parts of rough rice (Oryza sativa L.) before (BG) and after germination (AG). Results are expressed as the average of triplicate samples with mean ± SD; ⁄ p < 0.05; Significantly different by paired t-test, significantly different by Students t-test between before and after germination. 290 H.Y. Kim et al. /Food Chemistry 134 (2012) 288–293 3.6. Vitamin E The changes in the vitamin E (tocopherols) content of different parts of rough rice b efore and after germination are show n in Table 3. The a -, b, and c -tocopherol content differed in different parts of the rough rice seed during germination. The tocopherols identified in rough rice agree with those found by Choi, Jeong, and Lee (2007) for black rice, who reported 2.64 mg/100 g vitamin E. The total vitamin E content in the hull and brown rice increased from 0.17 mg/100 g and 3.02 mg/100 g before germination to 1.30 mg/ 100 g and 3.06 mg/100 g after germination, respectively (p < 0.01). Only a -tocopherol (0.09 mg/100 g) and c -tocotrienol (0.08 mg/100 g) were found in the hull before germination, whereas after germination b- and c -tocopherol and a -tocotrienol were found. The a - and b-tocotrienol content in brown rice in- creased from 0.78 and 0.02 mg/100 g before germination to 1.19 and 1.43 mg/100 g after germination (p < 0.01), respectively. An in- crease of a -tocopherol content after germination should increase the vitamin E bioactivity in the sprout. However, further investiga- tions are needed to confirm the activity and bio-availability of sprout tocopherols, and the optimum germination conditions needed to maintain the quality of tocopherols in the germinated sprout. 3.7. c -Aminobutyric acid (GABA) c -Aminobutyric acid (GABA), a non-protein amino acid, is widely distributed along with eukaryotes and prokaryotes. It is known as one of the main inhibitory neurotransmitters in the sym- pathetic nervous system and plays an important role in cardiovas- cular function (Wang, Tsai, Lin, & Ou, 2006). Therefore, searching GABA-rich foods becomes one of the focuses in the field of func- tional food research. Change in the GABA content is enhanced in the germination state, so allowing time for germination during processing can help improve rice quality. As shown in Fig. 5, the GABA contents of different part of rough rice were increased after germination. The GABA content increased from 15.34 before to 31.79 mg/100 g after germination in rough rice, and the content Table 1 Changes in free sugar content of different parts of rough rice (Oryza sativa L.) before and after germination (unit:mg/g). Parts Fructose Glucose Sucrose Total free sugar Before germination Rough rice 0.25 ± 0.011 ND 0.55 ± 0.013 0.79 ± 0.024 Hull ND a ND ND ND Brown rice 0.25 ± 0.009 ND 0.65 ± 0.001 0.90 ± 0.011 After germination Rough rice 0.37 ± 0.006 8.82 ± 0.098 *** ND 9.20 ± 0.105 *** Hull ND ND ND ND Brown rice 0.30 ± 0.008 11.45 ± 0.103 *** ND 11.75 ± 0.132 *** Sprout 0.61 ± 0.005 ND ND 0.61 ± 0.005 Results are expressed as the average of triplicate samples with mean ± SD. a ND: Not detected. *** p < 0.001; Significantly different by paired t-test, significantly different by Students t-test between before and after germination. Table 2 Changes in fatty acid content of different parts of rough rice (Oryza sativa L.) before and after germination (unit:%). Parts Palmitic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) Linolenic acid (C18:3) Before germination Rough rice 17.53 ± 0.535 1.33 ± 0.566 42.99 ± 0.159 18.98 ± 0.190 1.32 ± 0.062 Hull 17.84 ± 0.015 1.18 ± 0.303 42.92 ± 0.479 18.94 ± 0.223 1.26 ± 0.031 Brown rice 25.83 ± 0.627 3.42 ± 0.627 37.88 ± 0.819 17.40 ± 0.958 3.46 ± 0.363 After germination Rough rice 19.38 ± 0.117 * 1.15 ± 0.468 44.00 ± 0.548 * 17.74 ± 0.024 1.19 ± 0.014 Hull 18.09 ± 0.361 0.99 ± 0.093 44.22 ± 0.477 * 18.36 ± 0.005 1.22 ± 0.018 Brown rice 23.70 ± 0.017 2.26 ± 0.057 30.08 ± 0.020 21.99 ± 0.010 * 3.38 ± 0.030 Sprout 23.41 ± 0.040 2.17 ± 0.195 38.21 ± 0.138 18.13 ± 0.041 1.25 ± 0.024 Results are expressed as the average of triplicate samples with mean ± SD. * p < 0.05; Significantly different by paired t-test, significantly different by Students t-test between before and after germination. * * 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Rough rice Hull Brown rice Sprout Phytic acid (mg/g) BG AG Fig. 3. Changes in phytic acid contents on different parts of rough rice (Oryza sativa L.) before and after germination. Results are expressed as the average of triplicate samples with mean ± SD; ⁄ p < 0.05; Significantly different by paired t-test, signif- icantly different by Students t-test between before and after germination. * * 0 2 4 6 8 10 12 Rough rice Hull Brown rice Sprout γ-Oryzanol (mg./g) BG AG Fig. 4. Changes in c -oryzanol contents of different parts of rough rice (Oryza sativa L.) before (BG) and after germination (AG). Results are expressed as the average of triplicate samples with mean ± SD. ⁄ p < 0.05; Significantly different by paired t-test, significantly different by Students t-test between before and after germination. H.Y. Kim et al. /Food Chemistry 134 (2012) 288–293 291 of hull, brown rice and sprout after germination increased to 3.34, 26.84, and 6.04 mg/100 g, respectively, compared with that of be- fore germination. These results were similar to those reported by Anuchita and Nattawat (2010). The GABA content is related to the amount of glutamic acid, as GABA is synthesised by the decar- boxylation of glutamic acid (Lee et al., 2007a, 2007b). GABA is one of the most interesting compounds in germinated rice. 4. Conclusions It is well established that enzymatic activity and functional components increase in cereal through the process of germination (Woo & Jeong, 2006). Thus, the cereal’s functional quality can be improved using germination as part of the processing method (Yang, Basu, & Ooraikul, 2001). Germination caused significant changes in several chemical and functional compositions of differ- ent parts of germinated rough rice. The chemical and functional components were determined for rough rice, hull, brown rice, and sprout parts before and after germination. Functional compo- nents, such as vitamin E, c -oryzanol, and GABA contents of rough rice, hull, brown rice, and sprout part increased significantly after germination. After germination, the total vitamin E contents of rough rice, hull, and brown rice parts increased 1.28, 7.65, and 1.01 times, those of GABA increased 2.35, 1.69, and 2.23 times, and those part of c -oryzanol increased 1.13, 1.67, and 1.2 times, respectively. The vitamin E, GABA, and c -oryzanol content in sprout part were 5.45, 6.037 and 9.91 mg/g, respectively. Oxidative stress is related to diabetes and diabetic complications, and fat- soluble vitamins, such as vitamin A, vitamin E diminish the lipid content of blood plasma in patients with non-insulin-dependent diabetes mellitus (Lee et al., 2007a). The evaluation of GABA in germinated brown rice is important when looking to enhance the dietary supplements effect on human health, because GABA is responsible for various biological activities. 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Changes in the chemical and functional components of Korean rough rice before and after germination. Food Science and Biotechnology, 16, 1006–1010. Table 3 Changes in vitamin E contents of different parts of rough rice (Oryza sativa L.) before (BG) and after germination (AG) (unit: mg/100 g). Parts a -T a -T3 b-T b-T3 c -T c -T3 Total BG Rough rice 0.63 ± 0.007 0.31 ± 0.006 – – – 1.88 ± 0.042 2.82 ± 0.049 Hull 0.09 ± 0.061 – – – – 0.08 ± 0.002 0.17 ± 0.003 Brown rice 1.08 ± 0.041 0.78 ± 0.003 0.03 ± 0.003 0.02 ± 0.001 0.09 ± 0.002 1.02 ± 0.024 3.02 ± 0.097 AG Rough rice 0.93 ± 0.017 0.58 ± 0.016 0.08 ± 0.001 – – 2.03 ± 0.031 3.62 ± 0.169 Hull 0.64 ± 0.027 ** 0.15 ± 0.005 *** 0.07 ± 0.002 * – 0.10 ± 0.001 ** 0.34 ± 0.028 ** 1.30 ± 0.059 *** Brown rice 0.39 ± 0.035 1.19 ± 0.090 ** 0.02 ± 0.001 1.43 ± 0.010 *** 0.03 ± 0.003 – 3.06 ± 0.046 ** Sprout 4.72 ± 0.015 0.24 ± 0.005 0.21 ± 0.007 0.06 ± 0.001 0.14 ± 0.002 0.08 ± 0.003 5.45 ± 0.023 Results are expressed as the average of triplicate samples with mean ± SD. Significantly different by paired t-test, significantly different by Students t-test between before and after germination. * p < 0.05. ** p < 0.01. *** p < 0.001. ** ** 0.0 10.0 20.0 30.0 40.0 Rough rice Hull Brown rice Sprout GABA (mg/100g) BG AG Fig. 5. 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