Comparison of chemical compositions and bioactive compounds of germinated rough rice and brown rice

Food Chemistry 122 (2010) 782–788

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Comparison of chemical compositions and bioactive compounds of germinated rough rice and brown rice Anuchita Moongngarm *, Nattawat Saetung Department of Food Technology and Nutrition, Faculty of Technology, Mahasarakham University, Muang, Mahasarakham, Thailand

a r t i c l e

i n f o

Article history: Received 16 December 2009 Received in revised form 25 January 2010 Accepted 10 March 2010

Keywords: Germinated brown rice Germinated rough rice Brown rice Bioactive compounds c-Aminobutyric acid

a b s t r a c t The aim of the study was to compare changes in the chemical compositions and bioactive compounds of germinated rough rice and germinated brown rice. Ungerminated rice (brown rice) and germinated rice extract powder were also prepared, for comparison purposes. In general, the concentration of crude protein, total free amino acids, a-tocopherol, c-oryzanol, thiamine, niacin and pyridoxine, in the germinated rough rice and the germinated rice extracted powder, were significantly higher, than those of the germinated brown rice and the ungerminated rice, whilst there was no significant difference in the levels of crude fat, carbohydrate and ash. The amino acid contents of the germinated rice products were also investigated and differences were found amongst these samples. The most significant changes, in c-aminobutyric acid, glycine, lysine and leucine, were observed in the germinated rough rice and the germinated rice extracted powder. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Germination of cereal and legume seed is an economical processing technology. A number of studies have documented its advantages and health benefits. Recently, germinated brown rice (GBR), or pre-germinated brown rice (pre-GBR), has been seen as one of the most interesting germinated cereal products and it has gained a great deal of attention, especially in Asian countries. The product is achieved by soaking the whole kernel of brown rice in water until its embryo begins to bud. During the process of germination, the chemical compositions of the rice change drastically, because the biochemical activity produces essential compounds and energy, for the formation of the seedling. Hydrolytic enzymes are activated and these decompose large molecular substances, such as starch, non-starch polysaccharides and proteins, to small molecular compounds. These processes result in an increase of simple sugars, peptides and the amino acids of germinated seeds, such as wheat (Yang, Basu, & Ooraikul, 2001), barley (Rimsten, Stenberg, Andersson, Andersson, & Aman, 2003) and rice (Saman, Vázquez, & Pandiella, 2008). Apart from changing the level of nutrients, the biochemical activities, which occur during germination, can also generate bioactive components and some of these possess antioxidants, such as ascorbic acid, tocopherols, tocotrienols and phenolic compounds, thus resulting in an increase of antioxidant

* Corresponding author. Address: Department of Food Technology and Nutrition, Faculty of Technology, Muang, Mahasarakham University, Mahasarakham 44000, Thailand. Tel./fax: +66 43 743 135. E-mail address: [email protected] (A. Moongngarm). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.03.053

activity (Fernandez-Orozco et al., 2008; Frias, Miranda, Doblado, & Vidal-Valverde, 2005). Important bioactive compounds in GBR showed a significant improvement after germination, for example, c-aminobutyric acid (GABA), dietary fibre, ferulic acid, tocotrienols, magnesium, potassium, zinc, c-oryzanol and prolylendopeptidase inhibitor (Kayahara, Tsukahara, & Tatai, 2000). When these GBR compounds were compared, with those of milled rice, they were 10 times greater for GABA, nearly four times greater for dietary fibre, vitamin E, niacin and lysine and three times greater for thiamine, pyridoxine and magnesium (Kayahara et al., 2000). The nutritional value of numerous seeds is also improved, through an increase in essential amino acids, protein digestibility, amino acid bioavailability (Nakamura, Tian, & Kayahara, 2004; Sangronis & Machado, 2007), vitamins (thiamin, riboflavin, niacin, ascorbic acid) (Frias et al., 2005) and a decrease in some antinutrients, such as phytic acid (Ghavidel & Prakash, 2007; Idris, AbdelRahaman, ElMaki, Babiker, & El Tinay, 2006). However, from several viewpoints in our preliminary study, germination of whole grain rough rice would be more effective, than that of brown rice, in order to enhance the concentration of nutrients and bioactive compounds, in addition to the ease of performing the germination process. The germination of brown rice often encountered difficulties, since there was a low germination rate and spoilage of non-germ kernels and broken kernels. After the husk is removed, some kernels may be broken and this can lead to easy access by enzymes and microorganisms and as a result they spoil, when they are soaked in water. In addition, the embryo of brown rice is no longer protected by the husk and thus it is exposed to air and light, which causes oxidation to take place and

A. Moongngarm, N. Saetung / Food Chemistry 122 (2010) 782–788

various enzymatic and non-enzymatic reactions are initiated (Warwick, Farrington, & Shearer, 1979). For instant, within the intact grain, lipids are stable. However, during and after de-husking, lipids undergo several reactions and generate free fatty acids leading to an odour defect (Galliard, 1986). Physical and biochemical damage of the embryo during de-husking of rough rice and also the deterioration of biological compounds, in the brown rice kernel, impacts on its germination capability. Furthermore, in order to obtain a product, which had a finer texture and also contained a higher level of chemical components, the germinated rough rice extract powder was prepared, by further treating the germinated rough rice, using enzymes activated during germination. Although similar studies have proposed for the benefits of germinated rice, more studies are necessary to provide more information to consumer. Therefore, changes in chemical compositions and bioactive compounds, amongst the ungerminated brown rice, the germinated brown rice, the germinated rough rice and the germinated rough rice extract powder, were compared, in the present study. 2. Materials and methods 2.1. Rice samples Rough rice of Oryza sativa L., cultivar RD-6 (a popular waxy rice cultivar for consumption in the Northeast of Thailand) was selected for the study and it was purchased from a local rice-milling factory in Mahasarakham province, Thailand. The preparation of germinated rice samples followed the methods studied and reported by Saetung (2006). Ungerminated rice (UGR), or brown rice, was prepared by removing the husk of the ungerminated rough rice, using a laboratory de-husker. 2.2. Germinated brown rice (GBR) preparation Rough rice was dehusked to brown rice and then 5 kg of brown rice was steeped in distilled water, at room temperature (28–30 °C) for 12 h. The steeping water was changed every 4 h and drained at the end of soaking. The steeped rice kernels were distributed on double layers of cotton cloth and placed in plastic basket. This basket was then covered by double layers of cotton cloth. The germination took place in a germinating chamber for 24 h, at 28–30 °C, with 90–95% relative humidity, using an automatic sprinkler. The germinated seeds were dried at 50 °C, to approximately 10% of moisture content. 2.3. Germinated rough rice (GRR) preparation Rough rice (5 kg) was soaked in tap water at room temperature for 48 h, to 40 ± 2% of moisture content and the water was changed every 8 h. The rice seeds were distributed in plastic baskets, in the same manner as performed in the GBR preparation, except that they were germinated for 48 h. The germinated seeds were dried at 50 °C, to approximately 10% of moisture content. The hull, root and shoot were separated, using laboratory de-husker, in order to obtain a GRR. All samples were finely grounded (40 mesh), prior to analysis and preparation of the germinated rough rice extract powder. The samples were stored at 20 °C, until used. The germination rate was investigated, by following the method of Jiamyangyuen and Ooraikul (2008), but with some modifications. The rice grains were considered as a germinated seed, when the young radicle or primary root (white root emerging from the lower end of the rice seed) was visible. Approximate 200–300 grains, from those in the germinated basket, were sampled and these germinated seeds

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were counted. The results of three replicates were calculated, as the percentage of germinated kernels to total kernels used. 2.4. Germinated rough rice extract powder (GRP) preparation The preparation of GRP followed the method reported by Sangsopha (2008). Briefly, ground GRR was added to distilled water (1:5 w/v). It was adequately mixed and incubated at 40 °C, for 30 min. Subsequently, the temperature was raised to 50 °C and held for another 30 min, before being heated up to 60 °C and held for 1 h. The final temperature was raised to 80 °C, for 10 min, in order to inactivate the enzyme activity. The slurry was cooled down to approximately 50 °C and then homogenised and filtered twice through cheese cloth. The extracted slurry was subjected to freeze drying, in order to obtain GRP. The sample was stored at 20 °C and used for analysis. 2.5. Determination of chemical compositions 2.5.1. Determination of proximate compositions, total free amino acids (TFAA), total sugars and reducing sugar The moisture content of samples was determined, by oven-drying at 105 °C, to a constant weight. Crude protein, crude fat, crude fibre, ash and total free amino acids were determined, using the standard methods of Association of Official Analytical Chemists (AOAC) (1990). The crude protein content was calculated from nitrogen content, using the Kjeldahl method (Gerhardt, Germany) and multiplied by a factor of 5.95. Crude fat was measured, by extracting the ground rice samples with petroleum ether, using the Soxhlet apparatus (Buchi, Switzerland). The crude fibre was determined by extraction with hot acid and base, using a crude fibre extractor (Velp Scientifica, Italy). The ash content was investigated by ashing the sample at 550 °C, in a muffle furnace. Available carbohydrate (nitrogen-free extract) was obtained, by difference. Total sugars and reducing sugars were determined, by following the method of Dubois, Gilles, Hamilton, Rebers, and Smith (1956) and Somogyi (1952), respectively. All the determinations were undertaken in triplicate and expressed as a percent of dried matter (DM) basis. 2.5.2. Determination of thiamin, niacin, and pyridoxine The method of Erbas, Certel, and Uslu (2005) was applied, with minor modifications. The 6 g of ground samples were mixed with 10 ml n-hexane and 16 ml HPLC grade water. The mixture was homogenised (Ultra-Turrax T50 homogenizer, USA) at 12,000 rpm, for 10 min and then centrifuged at 3250g for 30 min. The aqueous phase was filtered through a Whatman No. 42 filter paper and a 0.45 lm membrane filter, sequentially. Then, 20 ll of supernatant were injected onto HPLC system (Spectra physic 100, USA) equipped with a UV–Vis detector (Spectra physics, 8000, USA), in which the wavelength was set to 254 nm, in absorbance mode. An analytical column (Water symmetry, 5 lm, C18, 150  4.6 mm) was used, with an isocratic solvent system consisting of 97% of 50 mM KH2PO4 and 3% acetonitrile. The flow rate was 1 ml/min. The vitamin standards were prepared in a mobile phase. Peaks were verified by adding the standard vitamins to samples and each peak area was calculated, in relation to a standard peak area. The results were calculated on a dried weight basis. 2.6. Determination of bioactive compounds 2.6.1. Determination of total phenolic compounds (TPC) The samples (100 g) were extracted, by stirring with methanol 250 ml for 3 h. The extracted samples were then filtered through Whatman No.1 filter paper, the residue was washed with 100 ml methanol and the extracts were pooled. The extracts were evapo-

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rated to dryness under vacuum, using a rotary evaporator. The residues were dissolved with 10 ml of methanol and used for total phenolic compounds. The determination of total phenolic content was performed as gallic acid equivalents (mg/100 g), by using the Folin–Ciocalteau phenol reagent and this followed the method described by Iqbal, Bhanger, and Anwar (2005). The diluted methanol extracts (0.2 ml) were added, with 0.8 ml of Folin–Ciocalteau phenol reagent and 2.0 ml of sodium carbonate (7.5%), in the given order. The mixtures were vigorously vortex-mixed and diluted to 7 ml of deionised water. The reaction was allowed to complete for 2 h in the dark, at room temperature, prior to being centrifuged for 5 min at 1259 g. The supernatant was measured at 765 nm, on a Shimazu spectrophotometer. Methanol was applied, as a control, by replacing the sample. Gallic acid was used as a standard and the results were calculated as gallic acid equivalents (mg/100 g) of the sample. The reaction was conducted in triplicate and the results were averaged. 2.6.2. Determination of phytic acid Phytic acid was determined, by following the method of GarciaEstepa, Guerra-Hernandez, and Garcia-Villanova (1999) and Febles, Arias, Hardisson, Rodriquez-Alvarez, and Sierra (2002). 2.6.3. Determination of a-tocopherol and c-oryzanol analysis The extraction and determination of a-tocopherol and c-oryzanol were performed, according to the method of Chen and Bergman (2005), with some modifications. The ground rice sample (100 g) was mixed with 5 ml methanol for 10 min, using a magnetic stirrer and it was further sonicated for 10 min, using a sonicator (Vibra Cell, USA). The mixture was centrifuged for 10 min at 825 g and filtered (0.45 lm) and analysed by reversed phase (RP)-HPLC, as described below. Three extraction replications were used for each sample. The extracts of rice were analysed by HPLC, using the modified method of Rogers et al. (1993). The system consisted of an HPLC (Waters 2690 Alliance, USA) connected to both fluorescence and UV detectors, thus allowing simultaneous measurement of tocopherols (fluorescence, Ex = 298 nm: EM = 328 nm), and coryzanols (UV 325 nm). A water symmetry analytical column (C18, 150  3.9 mm, 5 lm) and a gradient mobile phase (1.0 ml/ min) were used, in order to separate the compounds of interest. The initial mobile phase conditions were acetonitrile (60%), methanol (35%) and water (5%), which ran isocratic for 5 min and then it changed, within 3 min, to 0% water, 40% methanol and 60% acetonitrile and then it was isocratic for an additional 2 min, prior to changing linearly to acetonitrile (22%) and methanol (78%), over the next 10 min. This held for 15 min, before running to initial conditions. The total HPLC run time was 45 min. 2.7. Amino acid analysis All samples were analysed, using the EZ: faast physiological kits for gas chromatography–flame ionisation detection (GC–FID) (Phenomenex, California, USA). The rice samples were hydrolysed with concentrated HCl and analysed directly. All steps, including the SPE sample cleanup, derivatisation, and analysis, were performed, as described in the kit’s manual. All analyses were performed on the Agilent 6890 GC (Palo Alto, California, USA) equipped with FID detector and using Chemstation software (Agilent). The GC column used was the ZB-AAA GC column, which was provided in the EZ: faast kits and standard analysis conditions were used, as described in the kit’s manual. The concentrations of GABA in the rice were prepared and measured, using RP-HPLC (Bosch, Alegria, & Farre, 2005; Liu, Chang, Yan, Yu, & Liu, 1995). The derivertised samples (5 ll) were injected onto the HPLC system (Waters Alliance 2695 with a heater (Waters, USA) and equipped with a Water

2475 fluorescence detector (fluorescence, Ex = 250 nm: EM = 395 nm). The column was AccQ-Tag (3.9  150 mm, 4 lm), used with AccQ-Tag eluent A, acetonitrile and deionized water. The flow rate was 1 ml/min. 2.8. Amylase activity

a-Amylase activity was determined, by using the Megazyme method. Amylase activity is the amount of enzyme activity which, acting under the conditions of the method, is expressed as U/g dry ground sample. 2.9. Statistical analysis All experiments were conducted in triplicate and the results are expressed as mean ± SD. The statistical examination of the data was performed, using the SPSS version 11.5 programme. Mean values of chemical compositions and bioactive compounds, within different germinated rice, were compared, using an analysis of the variance (ANOVA) test. These means were compared, using the Duncan Multiple Range Test and p < 0.05 was applied, in order to establish significant differences. 3. Results and discussion 3.1. Germination rate The germination conditions of the rough rice and the brown rice followed the method optimised and reported by Saetung (2006). The germination rate of the rough rice (94.7%) was significantly higher than that of the brown rice (84.3%), by approximately 10%. This may result from the fact that, in the rough rice, all parts of the dormant seed were intact, when the moisture and temperature were allowed to reach the optimum points and then germination occurred. In general, the germination performance of the rough rice was easier and it needed less intensive care. It is very probable that it could be produced in a larger amount, at any time, compared to germination of the brown rice, despite the fact that it took a longer time to germinate. 3.2. GRP characteristics GRP was processed, using partial enzymatic hydrolysis and water extraction, in order to treat the ground GRR. Enzymes, for example, a-amylase and protease, developed during germination and these were involved as hydrolytic agents. After the GRR was treated and passed through several steps, as mentioned in the method section, a white (and slightly brown) fine powder was obtained and designated as ‘germinated rough rice extract powder’ (GRP). As a result, the main part of the slurry was retained, except for large particles of hull and bran, which had been separated. In addition, only parts of the rice starch were hydrolysed during germination and extraction, because the activity of a-amylase in the germinated rice was lower than that in the germinated barley and sorghum. The a-amylase activity of GRR, in this study, was 24.9 U/g, whereas a-amylase in germinated red sorghum and millet, reported by Traore, Mouquet, Icard-Verniere, Traore, and Treche (2004), was 56 and 42 U/g. The GRP acquired also indicated a high solubility in warm water (data not shown), compared to that of ground GRR and GBR. 3.3. Content of proximate compositions The chemical content of UGR, GBR, GRR and GRP are presented in Table 1 and the percent change of some chemical compositions

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A. Moongngarm, N. Saetung / Food Chemistry 122 (2010) 782–788 Table 1 Comparison of chemical components of ungerminated rice (UGR), germinated brown rice (GBR), germinated rough rice (GRR), germinated rough rice powder (GRP) (dry weight basis). GBR

GRR

GRP

Proximate compositions (%) Moisture content 9.44 ± 0.76a Crude protein 6.98 ± 0.07b Crude fatns 1.20 ± 0.68 79.2 ± 2.08 Carbohydratens 1.96 ± 0.11 Ashns Crude fiberns 1.13 ± 0.16 Total sugar 0.91 ± 0.03c Reducing sugar 0.19 ± 0.04c TFAAA 2.11 ± 0.56b

Parameter

UGR

8.86 ± 0.95a 8.98 ± 0.27a 1.23 ± 0.68 77.7 ± 2.49 2.06 ± 0.11 1.22 ± 0.26 1.88 ± 0.13c 0.81 ± 0.19c 3.12 ± 0.55b

9.21 ± 0.34a 9.31 ± 0.19a 1.19 ± 0.44 76.8 ± 3.19 2.19 ± 0.18 1.17 ± 0.04 2.54 ± 0.17b 1.21 ± 0.13b 5.89 ± 0.91a

7.18 ± 0.56b 9.51 ± 0.32a 1.61 ± 0.47 78.5 ± 3.88 2.16 ± 0.24 1.03 ± 0.13 14.6 ± 0.11a 10.9 ± 0.28a 6.24 ± 0.87a

B vitamins (mg/100 g) Thiamine 0.23 ± 0.02a Niacin 7.66 ± 0.14b Pyridoxine 0.76 ± 0.08b

0.12 ± 0.02b 4.47 ± 0.18c 0.66 ± 0.04c

0.27 ± 0.06a 11.2 ± 0.42a 1.35 ± 0.27a

0.21 ± 0.04a 10.5 ± 0.62a 1.39 ± 0.18a

1.15 ± 0.08b

0.92 ± 0.08a

0.98 ± 0.04a

84.3 ± 6.35c 0.86 ± 0.08b

98.6 ± 7.43b 1.36 ± 0.04a

110 ± 5.20a 1.50 ± 0.38a

84.0 ± 5.93b

104 ± 7.88a

106 ± 7.08a

Bioactive compounds Phytic acid 1.32 ± 0.07c (g/100 g) TPCA (mg/100 g) 70.3 ± 8.31c a-Tocopherol 0.93 ± 0.18b (mg/100 g) c-Oryzanol 66.0 ± 5.93b (mg/100 g)

Means within rows followed by the same letter are not significant different at p < 0.05. A TFAA = total free amino acids; TPC = total phenolic compounds. ns no significant difference.

sitions in this study were close to those reported by Heinemann, Fagundes, Pinto, Penteado, and Lanfer-Marquez (2005), who found values of protein, crude fat and ash of brown rice (O. sativa) ranging from 6.34% to 7.42%, 2.37% to 3.02% and 1.15% to 1.29%, respectively. The variation of chemical contents relies on a number of factors, such as varieties, water supply handling, fertiliser application, harvesting and storage management. At germination, there was a significant increase of crude protein, total sugars, reducing sugar and total free amino acid contents, whilst no significant difference was observed, amongst the rice samples, in the level of available carbohydrate, crude fat, crude fibre and ash. The highest level of total sugars and reducing sugar was found in GRP, followed by that of GRR (Table 1). The level of crude protein in GRR and GRP was comparable, but higher than that of UGR and GBR, by 33.4% and 3.67%, respectively, in GRR and by 36.3% and 5.9%, respectively, in GRP. This could be due to the fact that, during the germination process, several enzymes are activated and some non-protein nitrogen substances, such as nucleic acids, are produced: therefore, these can cause protein levels to be increased. The increase of total free amino acids, after germination, was a result of the degradation of protein by protease and a synthesis of new enzymes, which helped to liberate the free amino acid. A similar observation was reported by Traore et al. (2004). There was no significant decrease of crude fat and ash level, even though lipids could be hydrolysed during germination and used to produce the necessary energy for the biochemical and physicochemical modifications, which occurred in the seed.

B vitanins

100 GBR

80 GRR

60

Retention (%)

is showed in Fig. 1. The major chemical compositions, crude protein, crude fat, crude fibre, ash and total free amino acid contents of UGR, were 6.98%, 1.20%, 1.13%, 1.96%, and 2.11%, respectively. The levels of total sugars and reducing sugar were 0.91% and 0.188%, respectively. The protein content of this study was similar to that studied by Kennedy and Burlingame (2003), who reported the protein content of unpolished rice (brown rice) in O. sativa varieties varying from 4.5% to 15.9%. Moreover, some chemical compo-

GRP

40 20 0 -20

300

-40 GBR

250

-60

GRR

Thiamine

Niacin

Pyridoxine

GRP

Bioactive compounds

80 150

60 GBR

Retention (%)

Retention (%)

200

100

50

GRR

40

GRP

20 0

0

Crude protein

Total sugar

Reducing sugar

TFAA

-20 -40

Fig. 1. Changes in chemical compositions of germinated brown rice (GBR), germinated rough rice (GRR) and germinated rough rice extract powder (GRP) indicated as retention contents (%), compared with that of ungerminated rice (UGR). The change of total sugars in GRP was 1502% and changes in the reducing sugar in GBR, GRR and GRP, was 437%, 863% and 5690%, respectively. TFAA refers to total free amino acids.

TPC

α-tocopherol

γ-oryzanol

Phytic acid

Fig. 2. Changes in B vitamins and bioactive compounds of germinated brown rice (GBR), germinated rough rice (GRR) and germinated rough rice extract powder (GRP), indicated as retention contents (%), were related to that of ungerminated rice. TPC refers to total phenolic compounds.

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The level of reducing sugar in GRR was significantly higher, than that in UGR and GBR, by approximately 17.25 times (863%) and 1.58 times (79.2%), respectively, whilst that of GBR was 8.74 times (437%) higher, than that of UGR. The same trends were found in the level of total sugars (Fig. 1). The increase of reducing sugar and total sugars is due to the starch degradation presumably being involved in the initial action of a-amylase on the starch granules, since a-amylase showed a greater increase in activity (discussed in next section). The other hydrolyses probably assisted in the complete hydrolysis to simple sugar and reducing sugar, such as the action of invertase, which hydrolysis sucrose, to glucose and fructose. According to Traore et al. (2004), glucose and fructose were the major reducing sugars in germinated sorghum, millet and corn, where the level of glucose and fructose of sorghum was increased by 89.6 folds (4478%) and 30.34-folds (1517%), respectively. 3.4. Contents of thiamine, niacin, and pyridoxine The content of thiamine, niacin and pyridoxine, in the rice samples, are given in Table 1. The content of thiamine and niacin, in the ungerminated rice, was near to those reported by Deepa, Singh, and Naidu (2008), who found that the content of thiamine and niacin of non- medicinal rice (IR64) was 0.40 and 4.68 mg/100 g, respectively. The germination of the brown rice brought about a significant reduction of thiamine content (47.8%) (Fig. 2), compared to that of UGR, which may be due to the effect of soaking the de-hulled rice seed and changing the water, which led to a leaching out of water soluble vitamins, whereas there was no significant effect of soaking and water changing on the B vitamins in the rough rice, because it was protected by the hull. On the contrary, in the case of germination of the rough rice, the level of thiamin was slightly increased, but not significantly. Similar trends were observed in the niacin and pyridoxine content in GBR, whilst the content of these two vitamins were significantly increased in GRR and GRP. 3.5. Contents of total phenolic, phytic acid, a-tocopherol and coryzanol The total phenolic contents (TPC) was determined, by following a modified Folin–Ciocalteu reagent method and the results were expressed as gallic acid equivalents (Table 1). The percent change is shown in Fig. 2. Significant differences were observed for TPC, amongst the rice samples. GRP contained the highest amount of TPC, followed by GRR, GBR, and UGR (110, 98.6, 84.3, and 70.3 mg/100 g, respectively. TPC, reported by Iqbal et al. (2005), was in the range of 251–359 mg/100 g of the rice bran extracts. The level of TPC, in the present study, was lower, because the concentration of TPC was high in the bran layer. However, in this study, the phenolic content, determined, came from the whole kernel. Moreover, the change of TPC was also dependent on the type of phenolic compounds, which dominated in the different rice cultivars. Tian, Nakamura, and Kayahara (2004) found that 6-O-feruloylsucrose and 6-O-sinapoylsucrose were the major soluble phenolic compounds, in brown rice and there were significant decreases during germination for 24 h, whilst the levels of free ferulic acid and sinapinic acid increased significantly. The effect of germination, on the phytic acid content, is shown in Table 1 and Fig. 2. The phytic acid content, for all the germinated rice samples, was reduced significantly by 12.9%, 30.3%, and 25.8%, in GBR, GRR, and GRP, respectively. GRR and GRP were more effective in reducing phytic acid, compared with that of GBR. A reduction in phytic acid contents of cereals seeds, by germination, has been frequently reported (Centeno et al., 2001; Liang, Han, Nout, & Hamer, 2008). This reduction has been attributed to an increase

of phytase activity and therefore, it generates a lower molecular weight of inositols (Centeno et al., 2001). The concentration of a-tocopherol and c-oryzanol was also affected by the germination and treatment processes (Table 1). The levels of a-tocopherol and c-oryzanol, in UGR, were 0.93 and 66 mg/100 g, respectively, which slightly differed from those studied by Ha et al. (2006) (1.46 and 35 mg/100 g for a-tocopherol and c-oryzanol, respectively), depending on the rice cultivars. After germination and extraction, the amount of a-tocopherol of GRR and GRP was increased significantly, by 46.2% and 61.3%. Similarly, the content of c-oryzanol was increased by 57.6% and 60.7%, respectively, whilst that of GBR was slightly reduced, but not significantly, in relation to that of UGR (Fig. 2). 3.6. Amino acid analysis The amino acid content of UGR, GBR, GRR and GRP, measured by using the GC–FID method, is indicated in Table 2 and the percent changes, in amino acid content, is shown in Fig. 3. The predominant amino acids, of all the rice samples, were non essential amino acids (NEAA). In UGR, the glutamic acid content showed the highest amount, followed by alanine, and aspartic acid, with the amount of 961, 748 and 731 mg/100 g, respectively. This result was similar to that of Roohinejad et al. (2009). The germination significantly increased the content of almost all the amino acids, except histidine, methionine and threonine, glutamic acid, aspartic acid and serine in GBR (Table 2 and Fig. 3). The same trend was reported by Shu, Frank, Shu, and Engel (2008). In the case of EAA, it was sufficiently interesting to consider the increase of some essential amino acids, since some of these acids limited the amino acid in the ungerminated rice, but after passing through the germination process and water extraction, the acids improved significantly, for example, lysine increased by 71.6% in GBR, 138% and 139% in GRR and GRP, respectively, which is in agreement with a previous study, on soaked brown rice, by Saikusa, Horino, and Mori (1994). When the level of EAA, in GBR, was compared with that of GRR, the content of leucine, lysine, phenylalanine and valine, was significantly higher by 32.2, 38.8, 16.7 and 51.1, respectively. GABA is one of the most interesting compounds in germinated rice, since it can prevent and/or avert diseases; it plays a vital role

Table 2 Amino acid contents (essential amino acids (EAA) and non essential amino acids (NEAA)) in ungerminated rice (UGR), germinated brown rice (GBR), germinated rough rice (GRR), germinated rough rice powder (GRP) (mg/100 g dry weight). Amino acids

UGR

GBR

GRR

GRP

EAA Histidine Isoleucine Leucine Lysine Methioninens Phenyalanine Threoninens Valine

221 ± 17.5ab 136 ± 23.7b 695 ± 31.0c 331 ± 21.3c 103 ± 13.9 330 ± 14.7b 255 ± 26.4 539 ± 45.4c

204 ± 16.8b 146 ± 54.3ab 1210 ± 52.6b 568 ± 52.2b 93.4 ± 17.2 360 ± 19.8b 235 ± 13.4 723 ± 35.6b

215 ± 33.8b 186 ± 22.6a 1599 ± 92.5a 788 ± 66.3a 119 ± 10.5 420 ± 22.8a 245 ± 32.8 1092 ± 79.9a

266 ± 48.3a 195 ± 39.6a 1523 ± 30.3a 792 ± 44.3a 110 ± 9.53 441 ± 42.7a 232 ± 21.1 1017 ± 46.2a

NEAA Alanine Aspartic acid GABA Glycine Glutamic acid Hydroxyprolinnens Proline Serinens Tyrosine

748 ± 82.2b 731 ± 31.5b 23.8 ± 1.74c 561 ± 74.5c 961 ± 30.2b 158 ± 16.5 329 ± 12.8b 399 ± 19.6 298 ± 9.46b

1537 ± 133a 602 ± 34.9c 68.4 ± 4.43b 1362 ± 100b 853 ± 43.2c 154 ± 27.1 355 ± 31.3b 384 ± 25.0 324 ± 21.7b

1734 ± 103a 844 ± 26.8a 115 ± 9.12a 1769 ± 257a 1530 ± 128a 169 ± 15.3 646 ± 44.6a 372 ± 18.7 398 ± 18.6a

1712 ± 118a 907 ± 51.6a 118 ± 14.21a 1792 ± 162.5a 1489 ± 159a 178 ± 24.6 699 ± 23.2a 395 ± 26.0 422 ± 27.8a

Means within rows followed by the same letter are not significant different at p < 0.05. ns no significant difference.

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45

Essential amino acids

Alpha amylase activity (U/g DM)

140

Retention (%)

120 GBR

100

GRR

80

GRP

60 40 20 0 -20 His

Ile

Leu

Lys

Met

Phe

Thr

Val

40 35 GBR

30 GRR

25 20 15 10 5 0

Non-essential amino acids

400

0

48

72

96

Germination time (h)

350 GBR

Fig. 4. Comparison of a-amylase activity, between germinated brown rice (GBR) and germinated rough rice (GRR).

300 GRR

Retention (%)

24

250

GRP

200

as cultivar and growing location. The activity of GRR was found to be higher, than that of GBR, in all germination times.

150 100

4. Conclusions

50 0 -50 Ala

Asp

GABA

Gly

Glu

Pro

Ser

Tyr

Fig. 3. Changes in the amino acid contents of germinated brown rice (GBR), germinated rough rice (GRR) and germinated rough rice extract powder (GRP), indicated as retention contents (%), were related to that of ungerminated rice.

in the central nervous system, as an inhibitory neurotransmitter; and it has a hypotensive effect on blood pressure (Xu, Hua, & Godber, 2001). The concentration of GABA also remarkably increased, by 188%, 381%, and 395%, in GBR, GRR and GRP, respectively, compared with that of UGR. When considering the amount of GABA only, in GBR and GRR, it was revealed that GRR contained a significantly higher GABA content, by 67.5%. These results were similar to those reported by Ohtsubo, Suzuki, Yasui, and Kasumi (2005) and Shu et al. (2008). A GABA level is related to the content of glutamic acid, as GABA is synthesised by decarboxylation of glutamic acid (Bak, Schousboe, & Waagepetersen, 2006). However, a small of variation, in the GABA obtained, can be varied by several factors, such as cultivar, germination temperature, light intensity and germination time.

3.7. a-Amylase activity Changes in a-amylase activity, during germination of the rice grain, are presented in Fig. 4. The activity of a-amylase, of both GBR and GRR, showed a steady increase, with advancement of the germination time, for up to 72 h germination time and subsequently it reached the maximum value of 21.0 and 42.5 U/g DM, in GBR and GRR, respectively, after 96 h germination time. On the contrary, the activity of a-amylase, in the ungerminated rice, was very low (0.17 U/g DM), because the rice grain was dormant and therefore the enzyme was inactive. Similar trends of a-amylase activity were observed, in a study by Veluppillai, Nithyanantharajah, Vasantharuba, Balakumar, and Arasaratnam (2009). The large variation, in amylase activity, depends on a number of factors, such

Germination caused significant changes in several chemical compositions, bioactive compounds and amino acids in GRR when related to those of UGR and GBR. The treatment of GRR, with enzymes activated during germination, could improve some chemical components and remarkably increase the reducing sugar and total sugars. GRR and GRP could be a potent source of nutrients and bioactive compounds, since it contains a mixture of phenolic compounds, E vitamins, oryzanols and perhaps other bioactive compounds, which were not determined in this study. The results suggest that GRR and GRP could be used and this would open up a useful opportunity for the functional food industry and in addition consumption of these products would afford health benefits to consumers. Through comparison of the germination of brown rice and rough rice, in terms of economy, the production of germinated rice, when using rough rice, would probably lower production costs, due to the lower price of rough rice and its higher germination rate and consequent higher production yield. In terms of germination operation, the germination of rough rice would be more suitable for development and application, at an industrial level. Acknowledgements The authors gratefully acknowledge the Toray Foundation of Thailand and Mahasarakham University for providing financial support. References Association of Official Analytical Chemists (AOAC) (1990). Official methods of analysis of the association of official analytical chemists. Washington, DC, USA: AOAC. Bak, L. K., Schousboe, A., & Waagepetersen, H. S. (2006). The glutamate/GABAglutamine cycle: Aspects of transport, neurotransmitter homeostasis and ammonia transfer. Journal of Neurochemistry, 98(3), 641–653. Bosch, L., Alegria, A., & Farre, R. (2005). RP-HPLC determination of tiger nut and orgeat amino acid contents. Food Science and Technology International, 11(1), 33–40. Centeno, C., Viveros, A., Brenes, A., Canales, R., Lozano, A., & de la Cuadra, C. (2001). Effect of several germination conditions on total P, phytate P, phytase, and acid phosphatase activities and inositol phosphate esters in rye and barley. Journal of Agricultural and Food Chemistry, 49(7), 3208–3215.

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