Antioxidant Activity and Taste-active Component Distribution in the Bran Layer of Rice Grain

Abstract

Rice bran contains a higher level of bioactive and taste-active components than polished rice. The outer (inedible bran) layer of rice grains will be fractionated and analyzed to determine the distribution pattern of the various compounds in the bran layer. The phenol content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and lipophilic oxygen radical absorbance capacity (ORAC) were higher in fractions obtained from the outer layer, decreasing upon moving inwards. The hydrophilic ORAC was the highest in the second most outer layer. Although the concentrations of sucrose, umami amino acids, sweet amino acids, bitter amino acids, and gamma amino butyric acid were higher in the inner fractions of the bran layer close to the outside of the polished rice, their distribution patterns differed slightly. The obtained results indicated that the antioxidant and taste-active compounds were localized in different portions of the bran layer.

Introduction

Rice is a widely consumed staple food, particularly in Asia, where brown rice is commonly polished to ∼90% of the original weight (polished rice) before the preparation of cooked rice. Although rice bran, a by-product obtained from the milling process of brown rice, is used in rice oil, mushroom beds, and feedstuffs, part of the rice bran has yet to be utilized in Japan. Brown rice consists of an inedible bran layer of rice grain (embryo buds, pericarp, testa, aleurone layer, and sub-aleurone layer) and edible polished rice (starch endosperm) (Hoshikawa, 1973)ⁱ. This inedible bran layer is rice bran.

Rice bran is known to contain numerous compounds that exhibit health benefits, with examples including ferulic acid, gamma oryzanol, inositol, phytin, and tocoperol (Taniguchi et al., 2012; Sharif et al., 2014; Gul et al., 2014; Ravichanthiran et al., 2018). Butsat and Siramornpun (2010) also demonstrated that rice bran could be considered a valuable source of bioactive components exhibiting high antioxidant properties. Interestingly, the localization of several antioxidant components present in rice bran has been determined, and their distribution patterns in the outer layer of rice kennel were found to differ (Goufo and Trindade, 2015; Liu et al., 2015). Thus, it is important to clarify to the distribution profiles of the antioxidant activity in the bran layer of rice grain to ensure its effective utilization. In addition, the majority of these studies were carried out on pigmented or indicia rice. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was examined in two categories, i.e., the outer and inner bran fractions of purple rice, and the observed antioxidant activity was attributed to the presence of anthocyanins (Jang and Xu, 2009). However, these results may not apply to non-pigmented Japonica rice cultivars, such as koshihikari (a representative cultivar of Japonica rice in Japan). Furthermore, the above-mentioned studies on the distribution of antioxidant components and the DPPH radical scavenging activity were carried out on samples from only 2 or 3 bran fractions polished from the outer layer of rice kernel. The oxygen radical absorbance capacity (ORAC) can be used to evaluate the antioxidation capacity against lipid hydroperoxides, and that this resembles the in vivo mechanism (Apak et al., 2016; Huang et al., 2005). However, the distribution of this capacity in the outer layer of brown rice remains unclear. Therefore, it is necessary to precisely separate the outer layer of brown rice into fractions and to clarify their antioxidant activities (DPPH radical scavenging activity and ORAC). Indeed, to date, no ORACs, such the lipophilic (L)-ORAC, the hydrophilic (H)-ORAC, and the total (T)-ORAC (the sum of L-OLAC and H-ORAC), have been analyzed for these fractions.

In terms of the taste-active components present in rice, free amino acids and sugars are the main compounds, with their concentrations being higher in rice bran than in polished rice (Konishi et al., 1996). In this context, Saikusa et al. (1994) reported that with the exception of Arg, all free amino acids were most abundant in the outermost layer of the rice milled to 95% of its original weight. In addition, almost all amino acids slightly decreased as the degree of milling increased (Liu et al., 2017). On the other hand, Matsuzaki et al. (1992) speculated that Glu and Asp, which are the umami taste amino acids, were responsible for improving the flavor of cooked rice. Brown rice has also been found to contain higher concentrations of free sugars (i.e., sucrose and glucose) and free amino acids compared with polished rice (Tran et al., 2004), and it has been reported that polished rice still containing part of the aleurone layer has a stronger umami flavor than ordinary polished rice (Tajima et al., 1992). However, to the best of our knowledge, analysis of the distribution pattern of taste-active components, such as free amino acids and sugars, in the outer layer of brown rice has yet to be carried out in detail. In addition, no study exists that comprehensively evaluates the taste (umami, sweetness, and bitterness) of each fraction from the outer layer of brown rice based on the amino acid content.

Thus, we herein aim to evaluate the distribution pattern of the antioxidant activity and the taste-active components present in the bran layer of rice grain. The outer layer of brown rice will therefore be precisely fractionated into six fractions 1–6, and the total phenolic contents (TPCs), DPPH radical scavenging activity and ORAC will be determined for each fraction. The free amino acids and sugars will also be analyzed.

Materials and Methods

Sample preparation    Brown rice of the Japonica variety, Koshihikari, produced in 2015 was used for the purpose of this study. The rice (10.0 kg, 21.0 °C) was polished to 88% in a vertical rice mill (VP-32T, Yamamoto Co., Ltd, Yamagata, Japan) at 24.0 °C. During the milling process, each fraction was composed of 2% polishing from the outer layer of the brown rice weight (w/w) (i.e., fractions 1–6; flours with layers from 98–100, 96–98, 94–96, 92–94, 90–92, and 88–90% polishing, respectively). After milling, the temperature of the 88% polished rice was 41.2 °C. Each fraction mainly consisted of the following tissues: Fraction 1, embryo buds and pericarp; Fraction 2, pericarp; Fraction 3, pericarp and testa; Fraction 4, testa; Fraction 5, aleurone layer. The sub-aleurone layer in the aleurone layer consisted of the inner fractions of the bran layer close to the outside of the polished rice. Fraction 6 is the surface layer of polished rice (starch endosperm). Equal weights of fractions 1–6 were blended to give fraction 7, which represented reconstructed rice bran. The 88% polished rice sample was then floured using a lab mill (TUBE MILL control, IKA, Staufen, Germany) at 5 000 rpm for 30 s (fraction 8).

Moisture content and crude protein content    The moisture content was measured using an oven-drying method (3 h at 135 °C) to calculate the analyzed values on a dry basis. The crude protein content was calculated based on the nitrogen value measured using an elemental analyzer (Max CN, Elementar Analysensysteme GmbH, Langenselbold, Germany) based on the Pregl-Dumas technique (Patterson, 1973).

Measurement of the TPC and the antioxidant activities    The TPC was determined according to the method of Folin and Ciocalteu (1927) with slight modifications. Briefly, a sample (50 mg) extracted with 80% aqueous ethanol (1 mL). After the addition of the Folin-Ciocalteu reagent, the absorbance was measured at 750 nm, using a spectrophotometer (V-570, JASCO Corporation, Tokyo, Japan). The TPC was expressed in mg gallic acid equivalent (GE)/g DW sample using gallic acid as the standard.

The DPPH radical scavenging activity was measured according to the method of Katsube et al. (2004) with slight modifications. The sample was prepared as described above for TPC determination. After the addition of the DPPH solution to the diluted extract, the absorbance of the mixture was recorded at 520 nm, using a spectrophotometer (V-570, JASCO Corporation). The activity was expressed as µmol Trolox equivalent (TE)/g DW sample using Trolox as the standard.

The ORAC was determined according to the methods of Prior et al. (2003) and Watanabe et al. (2010) with slight modifications. For the lipophilic (L)-ORAC, a sample (100 mg) was extracted with a mixture of hexane and dichloromethane (4 mL, 1:1). After the addition of the ORAC reaction solutions to the diluted extract, the fluorescence intensity (excitation at 485 nm and emission at 528 nm) was recoreded using a microplate fluorometer (Fluoroskan Ascent FL, Thermo Fisher Scientific, Waltham, MA, USA). For the hydrophilic (H)-ORAC, the L-ORAC air-dried residue was extracted with mixture of acetone/water/acetic acid (10 mL, 700:295:5, v/v/v). Analysis was carried out as described above for L-ORAC, except for the solution volumes. The ORAC values were expressed in µmol TE/g DW sample using Trolox as the standard.

Free amino acids and sugars    Each fraction (1.0 g) was homogenized with 0.1 N HCl (10 mL) on ice. Following centrifugation, an aliquot of the supernatant was defatted and deproteinized using hexane and acetonitrile, respectively. The sample was dried prior to reconstitution in a mixture of 0.1 N HCl (95 µL) and 2.5 mM norleucine (5 µL, internal standard). After the derivatization, the sample solution (10 µL) was analyzed using an Acquity UPLC system (Waters, Milford, MA, USA). The derivatization and analytic conditions employed were as outlined in the manufacturer's instruction (Waters).

Each fraction (1.0 g) was stirred with an 80% aqueous ethanol solution (20 mL) at 200 rpm for 2 h at room temperature. After centrifugation, the supernatant was mixed with the same volume of acetonitrile. Analysis was performed by HPLC (isocratic mode) with an Asahipak NH2P-50 column (i.d. 4.6 mm × 250 mm, Showa Denko K.K., Tokyo, Japan) and a refractive index detector (L-3350, Hitachi High-Tech Science Corporation, Tokyo, Japan) using 75% acetonitrile as the mobile phase. The column temperature and flow rate were set at 40 °C and 1.0 mL/min, respectively. Sugars (fructose glucose, lactose, galactose, maltose, and sucrose, FUJIFILM Wako Pure Chemicals Corporation, Osaka, Japan) were quantitated using the peak areas of the individual sugar compounds and calibration curves computed from the peak areas of the standard sugars.

Statistical analyses    Tukey's multiple-range test was applied to determine the differences between the mean values of triplicate determinations using SPSS Statistics 23.0 (IBM Japan, Ltd., Tokyo, Japan). The significant levels were set at 5%.

Results and Discussion

TPC, DPPH radical scavenging activity, and ORAC    Figure 1 shows the TPCs in the rice flour samples with different polishing fractions. The TPCs in fractions 1, 2, and 3 (embryo buds, pericarp, and testa) obtained from the outer layer of brown rice were determined to be 35.8, 35.6, and 33.0 mg GE/g DW, respectively. These values are significantly higher than those of the other fractions, with the exception of fraction 7 (the combined fraction, p < 0.05). The TPC value was found to decrease upon moving towards the inner direction of the brown rice. The embryo buds and pericarp are generally contained in fraction 1. As the polishing ratio decreased, the contents of total free phenolics and nine individual phenolics in brown rice significantly decreased (Liu et al., 2015). In addition, as detailed below, amino acids and sugar that react with Folin-Ciocalteu reagent were highly distributed in other fractions. Our results therefore strongly suggest that larger amounts of polyphenolic compounds are present in the most outer layers of brown rice due to their greater abundance in the pericarp, and embryo buds compared to the other tissues. For reference, we note that the value for polished rice was 0.5 mg GE/g DW. This value was significantly less than that of fraction 6, which is a fraction of the most out layer of polished rice, suggesting that the levels of polyphenolic compounds in the most outer layers of polished rice was higher than those of the in the central part of polished rice.

Fig. 1.

Total phenolic contents (TPCs) in the rice flour samples with different polishing ratios: 1, 98–100; 2, 96–98; 3, 94–96; 4, 92–94; 5, 90–92; 6, 88–90; 7, 88–100; and 8, 0–88%. The values shown represent the mean ± the standard deviation (SD) for determinations carried out in triplicate. Different letters indicate significant differences at p < 0.05. ND, not detected.

Figure 2 shows the DPPH radical scavenging activity of the rice flour samples with different polishing fractions. In addition, the DPPH radical scavenging activities of fractions 1 (embryo buds and pericarp) and 2 (pericarp) were determined to be 17.8 and 18.0 µmol TE/g DW, respectively, which are significantly higher than those of the other fractions (with the exception of fraction 3) (p < 0.05). Overall, the DPPH radical scavenging activity was also found to decrease upon moving towards the inner direction of the brown rice. No DPPH radical scavenging activity was observed for fraction 6 (88–90% polishing ratio) or for the polished rice sample. In addition, as shown in Figure 1, the distribution pattern of the phenolic compounds in brown rice was similar to that of the DPPH radical scavenging activity. As both the TPC assay and measurement of the DPPH radical scavenging activity are based on electron transfer (Huang et al., 2005), our results suggest that the outer layer of brown rice contains higher concentrations of electron-donating compounds, and in particular, polyphenolic compounds.

Fig. 2.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities of the rice flour samples with different polishing fractions. The sample numbers are as outlined for Figure. 1. The values shown represent the mean ± the standard deviation (SD) for determinations carried out in triplicate. Different letters indicate significant differences at p < 0.05. ND, not detected.

Figure 3 shows the ORAC in the rice flour samples with different polishing fractions. More specifically, the highest and lowest L-ORAC values were obtained for fractions 1 (embryo buds and pericarp) and 8 (polished rice), respectively (83 and 2 µmol TE/g DW, respectively), with the L-ORAC value again decreasing upon moving toward the inner direction of the brown rice sample. Tocopherols, tocotorienols, and gamma oryzanol compounds, which are major lipophilic antioxidants, have been previously reported to be concentrated in the outer layer of the bran (Goufo and Trindade, 2017). In addition, tocopherols are known to be abundant in embryo buds (Yu, et al., 2007). As a result, the high L-ORAC value in fraction 1 (embryo buds and pericarp) was attributed to an abundance of these lipophilic compounds. In contrast, the H-ORAC values of fractions 2 (pericarp) and 3 (pericarp and testa) (304 and 279 µmol TE/g DW, respectively) were significantly higher than those of fractions 5–8 (p < 0.05), while the T-ORAC value of fraction 2 (pericarp) (356 µmol TE/g DW) was significantly higher than those of the other fractions, with the exceptions of fractions 1 (embryo buds and pericarp) and 3 (pericarp and testa) (p < 0.05). It was also found that the distribution pattern of H-ORAC in the outer layer of brown rice differed slightly from those of the polyphenolic compounds and the DPPH radical scavenging activity (Figures 1 and 2). In the previous study, as the increasing degree of milling of japonica increased from 0% to 2.6%, the contents of ferulic acid protocatechuic acid, caffeic acid, and chlorogenic acid did not change, while the contents of (+)-catechin and coumaric acid decreased (Liu et al., 2015). The differences in these distribution patterns for the polyphenolic compounds present in the outer layer of brown rice are therefore likely to account for the slight differences in the distribution profiles between both the H-ORAC and T-ORAC and both the polyphenolic content and the DPPH radical scavenging activity. In contrast, since the ORAC assay is based on hydrogen atom transfer (Huang et al., 2003; Huang et al., 2005), the obtained results suggest that compared to fraction 1 (embryo buds and pericarp), fractions 2 (pericarp) and 3 (pericarp and testa) contain a greater number of hydrophilic compounds that can donate hydrogen atoms. Therefore, although the outer layer of brown rice contains high levels of polyphenolic antioxidants, their compositions in the various fractions likely differ. As such, the antioxidants present in each fraction must be identified and quantified to clarify the antioxidant compositions. Furthermore, since the ORAC is imitative of an antioxidative mechanism in vivo (Apak et al., 2016; Huang et al., 2005), the components exhibiting antioxidation ability in vivo are likely present in abundance in fractions 2 (pericarp) and 3 (pericarp and testa).

Fig. 3.

Oxygen radical absorbance capacity (ORAC) values for the rice flour samples with different polishing fractions: (a) lipophilic (L)-ORAC, (b) hydrophilic (H)-ORAC, and (c) total (T)-ORAC. The sample numbers are as outlined for Figure. 1. The values shown represent the means ± SD for determinations carried out in triplicate. Different letters indicate significant differences at p < 0.05.

Sugar content    Figure 4 outlines the sugar contents of the rice flour samples with different polishing fractions. It was found that the sucrose content was highest in fraction 4 (testa) (7.64 g/100 g DW), and decreased toward both the inner and outer directions of brown rice (p < 0.05). Overall, the sucrose level in the polished rice was 0.1 mg/g, while glucose, lactose, fructose, and maltose were not detected in any of the fractions.

Fig. 4.

Sucrose contents in rice flour samples with different polishing fractions. The sample numbers are as outlined in Figure 1. The values shown represent the means ± SD for determinations carried out in triplicate. Different letters indicate significant differences at p < 0.05.

Crude protein content    Figure 5 presents the results for the crude protein content determination in the rice flour samples with different polishing fractions. As indicated, fraction 4 (testa) contained the highest content of crude protein (16.4 g/100 g DW) followed by fractions 5 (aleurone layer) (14.9 g/100 g DW) and 3 (pericarp and testa) (14.4 g/100 g DW). The crude protein content decreased toward both the inner and outer directions of brown rice (p < 0.05), following the same trend observed for the sugar content. Resurreccion et al. (1979) have reported the highest protein fraction in high-protein rice was the sub-aleurone layer. Thus, the present study comfirmed the protein content was higher in the inner fractions of the bran layer (sub-aleurone layer) close to the outside of the polished rice in the present study.

Fig. 5.

Crude protein contents in rice flour samples with different polishing fractions. The sample numbers are as outlined in Figure 1. The values shown represent the means ± SD for determinations carried out in triplicate. Different letters indicate significant differences at p < 0.05.

Free amino acids    Table 1 lists the free amino acid compositions of the rice flour samples with different polishing fractions. With the exception of ornithine, all amino acids were present in the greatest abundance in either fraction 3 (pericarp and testa) or 4 (testa). The total amounts of Glu and Asp, which give the umami taste, were significantly higher in fraction 3 (pericarp and testa) than in fractions 1, 6, and 8 (p < 0.05). In addition, the total amounts of sweet tasting amino acids (Ser, Gly, Thr, Ala, and Pro) and bitter tasting amino acids (Arg, Lys, His, Phe, Tyr, Leu, Ile, Met, and Val) were significantly higher in fractions 3 (pericarp and testa), 4 (testa), and 5 (aleurone layer) than in the other fractions (with the exception of fraction 7) (p < 0.05). Moreover, the total amounts of free amino acids in fractions 3 (pericarp and testa), 4 (testa), and 5 (aleurone layer) were also significantly higher than in the other fractions, again with the exception of fraction 7 (p < 0.05). During cereal seed germination, alfa-amylase in the aleurone layer plays an important role in sugar formation to promote root and shoot growth (Kaneko et al., 2002). Thus, as germination requires amino acids and sucrose, these compounds may be abundant in fractions 3–5, which include the aleurone layer. In terms of the gamma amino butyric acid content, a compound that has been shown to exhibit various health benefits (Diana et al., 2014), the highest levels were detected in fractions 3 (pericarp and testa), 4 (testa), and 5 (aleurone layer), while the lowest level was detected in fraction 8 (polished rice) (p < 0.05). The fraction with a polishing ratio of 78–90%, which corresponded to the surface layers of the polished rice, contained 50–60% reducing sugars, 60% sucrose, and 100% Glu or Asp in polished rice (Sugiyama et al., 1995), although these levels decreased significantly upon washing the rice prior to cooking (Kasai et al., 2000). In addition, a taste-active amino acid were relatively abundant in fractions 3–5 (Figure 4 and Table 1), and these results were supported by a previous study describing the effect of the remaining aleurone layer on the umami taste of polished rice (Tajima et al., 1992). Thus, a slight elevation in the rice polishing ratio may increase the quantities of these compounds in rice after washing and cooking. Furthermore, the use of wash-free rice is able to suppress the loss of water-soluble components such as sugars and free amino acids. As such, the use of such a rice in addition to a slightly higher polishing ratio may lead to improvements in the rice palatability, taste, and functionality due to the increased abundance of taste-active and bioactive components.

Table 1. Free amino acids in rice flour with different polishing fractions (mg/100 g DW)

Amino acid	1	2	3	4	5	6	7	8
His	0.43 ± 0.02 c	0.46 ± 0.06 bc	0.60 ± 0.04 a	0.57 ± 0.05 ab	0.56 ± 0.08 abc	0.49 ± 0.01 abc	0.55 ± 0.01 abc	0.08 ± 0.01 d
Ser	2.57 ± 0.23 cd	2.81 ± 0.41 bcd	3.88 ± 0.32 a	3.91 ± 0.43 a	3.62 ± 0.47 ab	2.04 ± 0.15 d	3.35 ± 0.19 abc	0.37 ± 0.01 e
Arg	3.69 ± 0.49 c	4.08 ± 0.89 bc	6.67 ± 0.59 a	7.83 ± 1.10 a	7.79 ± 1.58 a	4.10 ± 0.26 bc	6.22 ± 0.24 ab	0.29 ± 0.00 d
Gly	0.61 ± 0.05 c	0.67 ± 0.10 bc	1.00 ± 0.09 a	1.04 ± 0.11 a	0.95 ± 0.09 a	0.47 ± 0.01 c	0.85 ± 0.01 ab	0.10 ± 0.01 d
Asp	12.89 ± 0.95 cd	14.22 ± 1.39 bc	17.95 ± 1.69 a	16.61 ± 1.10 ab	14.96 ± 1.25 abc	9.95 ± 0.47 c	15.10 ± 1.12 abc	2.34 ± 0.13 d
Glu	16.15 ± 0.97 bc	16.91 ± 1.62 b	21.52 ± 1.61 a	21.28 ± 1.42 a	19.85 ± 2.00 ab	12.21 ± 1.62 c	19.73 ± 1.15 ab	1.90 ± 0.47 d
Thr	0.71 ± 0.06 d	0.81 ± 0.11 cd	1.23 ± 0.07 ab	1.29 ± 0.14 a	1.17 ± 0.13 ab	0.56 ± 0.03 d	1.02 ± 0.04 bc	0.06 ± 0.00 e
Ala	3.51 ± 0.29 c	3.83 ± 0.46 c	5.20 ± 0.48 ab	5.25 ± 0.47 a	4.73 ± 0.14 ab	2.59 ± 0.14 d	4.34 ± 0.12 bc	0.41 ± 0.02 e
GABA	2.75 ± 0.59 b	3.09 ± 0.76 b	4.88 ± 1.28 a	5.52 ± 0.79 a	5.05 ± 0.13 a	2.36 ± 0.20 b	3.29 ± 0.19 b	0.24 ± 0.04 c
Pro	1.10 ± 0.09 d	1.22 ± 0.16 cd	1.74 ± 0.11 ab	1.81 ± 0.16 a	1.60 ± 0.05 ab	0.77 ± 0.06 d	1.45 ± 0.03 bc	0.11 ± 0.01 e
Orn	0.08 ± 0.00 a	0.08 ± 0.02 a	0.08 ± 0.03 a	0.05 ± 0.01 a	0.05 ± 0.01 a	0.07 ± 0.02 a	0.07 ± 0.01 a	0.01 ± 0.01 b
Cys	ND	ND	ND	ND	ND	ND	ND	ND
Lys	0.65 ± 0.06 c	0.73 ± 0.16 bc	1.11 ± 0.17 a	1.10 ± 0.18 a	1.07 ± 0.14 ab	0.93 ± 0.09 ab	1.05 ± 0.11 ab	0.08 ± 0.02 d
Tyr	0.53 ± 0.05 cd	0.65 ± 0.09 bc	0.88 ± 0.09 a	0.86 ± 0.14 a	0.74 ± 0.01 ab	0.34 ± 0.01 d	0.70 ± 0.05 abc	0.07 ± 0.00 e
Met	0.15 ± 0.10 ab	0.16 ± 0.07 ab	0.36 ± 0.15 a	0.30 ± 0.18 ab	0.15 ± 0.07 ab	0.16 ± 0.04 ab	0.32 ± 0.05 ab	0.06 ± 0.01 b
Val	0.85 ± 0.07 c	0.99 ± 0.14 c	1.38 ± 0.16 a	1.29 ± 0.16 ab	1.10 ± 0.02 bc	0.54 ± 0.02 d	1.12 ± 0.04 abc	0.08 ± 0.02 e
Ile	0.33 ± 0.04 bc	0.36 ± 0.07 bc	0.54 ± 0.09 a	0.54 ± 0.06 a	0.50 ± 0.09 ab	0.22 ± 0.02 c	0.45 ± 0.03 ab	0.01 ± 0.01 d
Leu	0.34 ± 0.04 c	0.38 ± 0.06 c	0.62 ± 0.06 ab	0.70 ± 0.06 a	0.63 ± 0.04 a	0.27 ± 0.02 c	0.51 ± 0.02 b	0.04 ± 0.00 d
Phe	0.22 ± 0.10 a	0.16 ± 0.14 a	0.43 ± 0.19 a	0.21 ± 0.10 a	0.29 ± 0.11 a	0.23 ± 0.07 a	0.23 ± 0.16 a	0.21 ± 0.06 a
Umami	29.04 ± 1.88 cd	31.12 ± 2.91 bc	39.47 ± 3.28 a	37.89 ± 2.48 ab	34.81 ± 3.24 abc	22.16 ± 2.07 d	34.84 ± 2.16 abc	4.24 ± 0.61 e
Sweetness	8.51 ± 0.71 cd	9.34 ± 1.24 bc	13.06 ± 1.07 a	13.30 ± 1.31 a	12.07 ± 0.83 a	6.42 ± 0.39 d	11.02 ± 0.14 ab	1.06 ± 0.01 e
Bitterness	7.20 ± 0.75 cd	7.98 ± 1.55 bc	12.59 ± 1.43 a	13.39 ± 1.75 a	12.84 ± 1.80 a	7.28 ± 0.36 c	11.16 ± 0.10 ab	0.92 ± 0.06 d
Total	47.58 ± 3.88 cd	51.62 ± 6.46 bc	70.08 ± 6.90 a	70.15 ± 5.79 a	64.83 ± 2.99 a	38.30 ± 2.62 d	60.38 ± 1.78 ab	6.47 ± 0.57 e

Sample numbers are the same as in Fig. 1.

GABA: gamma Aminobutyric acid

Orn: Ornithine

Umami: Asp+Glu

Sweetness: Ala+Gly+Pro+Ser+Thr

Bitterness: Arg+Lys+His+Phe+Tyr+Leu+Ile+Met+Val

Total: Sum of free amino acids

ND, Not detected

Different letters indicate significant differences at p < 0.05

Conclusion

We herein reported the fractionation and analysis of the outer (inedible bran) layer of rice grains to determine the distribution pattern of the various bioactive and taste-active compounds present in the bran layer. The total phenolic content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and lipophilic oxygen radical absorbance capacity (ORAC) were found to be most abundant in the fractions of the outermost layer. However, the highest hydrophilic ORAC was found in the subsequent layer. Although the inner fractions of the bran layer that are close to the outside of the polished rice contain higher levels of sucrose, umami amino acids, sweet amino acids, bitter amino acids, and gamma amino butyric acid, there were slight differences in their distribution patterns. More specifically, the obtained results indicate that the antioxidant and taste-active compounds were localized in different portions of the bran layer. Thus, since compared to polished rice, rice bran contains greater quantities of taste-active compounds and bioactive components with high antioxidant properties, the distribution of these species determined herein are of importance to maximize the utilization of inedible rice bran.

Acknowledgements    This study was supported in part by a Grant-in-Aid (Grant Number CHUGOKU1507016) for projects to support the advancement of strategic core technologies from the Small and Medium Enterprise Agency of Japan.

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