2014 年 20 巻 1 号 p. 161-165
We observed clear differences in glucose accumulation in milled grains between 8 japonica and 8 indica rice (Oryza sativa L.) cultivars subjected to hot-water (60°C) treatment for 30 min. The mean glucose content increased rapidly, from 8.9 to 123 mg/100 g DW in japonica grains and 4.6 to 54 mg/100 g DW in indica grains. The difference in glucose content between the two ecotypes corresponded to the observed significant difference in hydrolytic activities on gelatinized starch, which were much higher in japonica grains. Whereas, the mean α-glucosidase activity of the two ecotypes did not significantly differ. There was a significant correlation between the glucose content under hot-water treatment and glucose liberating enzyme activity.
Rice is the major cereal food in Asian countries. In Japan, production consists mainly of japonica cultivars, which have a starchy grain quality. The levels of chemical components such as reducing sugars and amino acids are closely associated with palatability (Maruyama et al., 1983; Tajima et al., 1994). Glucose is the main reducing sugar and accumulates during cooking through the action of endogenous hydrolytic enzymes. Glucose accumulation is optimally stimulated by preheating at 40 to 60°C (Kasai et al., 2000; Awazuhara et al., 2000). Endogenous hydrolytic enzymes, mainly α-glucosidases, are affected by milling properties and temperature before cooking (Mabashi et al., 2010a, 2010b). Furthermore, these reports revealed that α-glucosidase was the most important endogenous enzyme for starch degradation in milled rice grains, and that its activity was highest at 60°C and at a milling yield of 85% to 90%. Iwata et al. (2001) also reported that α-glucosidase activity affected the physicochemical properties of cooked rice and differed among cultivars. Recently, Nakamura et al. (2012) reported that a physical property (adhesion) was correlated with enzyme activities (cellulase and xylanase) and they found discriminative DNA bands by PCR amplification of these enzymes. Tsujii et al. (2007) also reported the amylase activity of glutinous rice grains was correlated with the paste viscosity. Most interestingly, Tsujii et al. (2013) revealed that the activity of amylase isozymes affects the palatability of cooked rice and suggested that the enzymes in rice endosperm are potential useful markers of palatability.
In this study, we aimed to clarify varietal and ecotypical differences in glucose accumulation caused by the activity of endogenous hydrolytic enzymes.
Rice materials We used 8 japonica cultivars (Asahi, Koshihikari, Hatsunishiki, Nipponbare, Norin 22, Sasanishiki, Snow Pearl, and Yamadanishiki) and 8 indica cultivars (Amber, Bluebelle, British Honduras Creole [BHC], Hadsaduri, Kinandang, Modan, Panbira, and Taichung Native 1 [TN-1]) The 16 cultivars were grown in the paddy field of Yamagata University in 2010 with inorganic fertilizer applied at a standard rate of 80 kg N, 35 kg P, and 66 kg K per ha. Panicle samples were harvested 40 days after flowering, dried in a greenhouse to a grain water content of about 14%, and stored at 4°C for about one month. Before analysis, about 2 g of brown rice grain was milled in a small milling machine (Pearlest; Kett Electric, Tokyo, Japan) for 50 s to 90%. Table 1 shows the 1000-grain weight and actual milling yield of each cultivar
| Cultivar | 1000-grain weight (g) | Milling yield (%)NS | |
|---|---|---|---|
| Brown ricNS | Milled riceNS | ||
| japonica | |||
| Koshihikari | 21.43 | 19.28 | 89.97 |
| Hatsunishiki | 21.02 | 18.57 | 88.36 |
| Asahi | 26.52 | 24.01 | 90.56 |
| Sasahishiki | 22.22 | 20.17 | 90.76 |
| Nipponbare | 24.20 | 22.70 | 93.79 |
| Norin 22 | 20.09 | 18.09 | 90.08 |
| Snow Pearl | 24.42 | 22.19 | 90.88 |
| Yamadanishiki | 26.15 | 24.27 | 92.80 |
| japonica average | 23.26 | 21.16 | 90.90 |
| indica | |||
| Hadsaduri | 17.67 | 16.23 | 91.87 |
| TN-1 | 21.14 | 18.74 | 88.62 |
| Modan | 26.04 | 23.04 | 88.47 |
| Panbira | 20.74 | 18.43 | 88.84 |
| Kinandang | 26.43 | 24.28 | 91.88 |
| Bluebelle | 18.92 | 16.93 | 89.49 |
| Amber | 17.22 | 15.24 | 88.46 |
| BHC | 23.60 | 21.23 | 89.98 |
| indica average | 21.47 | 19.26 | 89.70 |
| Mean | 22.36 | 20.21 | 90.30 |
NS: Not significantly different between ecotypes at p<0.05 by Student's t-test.
Hot-water treatment For hot-water treatment, 100-mg samples were placed in 1.7-mL tubes. The grains were rinsed twice with 0.2 mL distilled water, and 0.15 mL Milli-Q water was added to each tube. The grains were soaked at room temperature for 20 min and then heat-treated at 60°C for 30 min in a tube incubator (GTU-1615; TAITEC, Nagoya, Japan).
Extraction of soluble sugars Samples (100 mg on a dry-weight [DW] basis) of untreated and hot-water-treated milled grains were homogenized in 1.5 mL of 70% ethanol using a mortar and pestle for 3 min, and then collected into 1.7-mL tubes. The tubes were heat-treated at 98°C with refluxing for 30 min to release soluble sugar molecules, and then centrifuged at 12 000 ×g for 20 min. Each supernatant was transferred to a fresh tube and vacuum-dried with centrifugation (6000 ×g) at 40°C.
Analysis of soluble sugars The dried sugar pellets were dissolved in 0.6 mL Milli-Q water and analyzed by high-performance liquid chromatography (HPLC; Hitachi LaChrom, Tokyo, Japan). A 20 µL sample solution was injected in the sugar analysis column (SP 0810; Shodex, Tokyo, Japan) with an elution solution of distilled Milli-Q water. The flow rate was 0.6 mL/min. Sugars were detected with a refractive index detector (L-7490; Hitachi). We used 0.2 and 2.0 % glucose as standard solutions. The analysis was conducted in triplicate.
Analysis of endogenous enzyme activities To induce endogenous enzymes and prepare enzyme extracts, we treated 300 mg of grains with 0.45 mL water at 60°C for 30 min to swell the grains. Next, 0.8 mL of 50 mM Na2HPO4 (pH 5.0) containing 5 mM dithiothreitol and 100 mM NaCl was added to each tube and the grains were ground using a narrow glass bar until the starch was suspended uniformly in the tube. The samples were centrifuged at 12 000 ×g for 20 min at 4°C, and the supernatants were removed and used as the crude enzyme solution.
To measure α-glucosidase activity, we used maltose as the substrate, as described by Mabashi et al. (2010a). In brief, 0.4 mL of 0.5% maltose solution containing 5 mM CaCl2 was added to 0.2 mL of the enzyme solution, and the mixture was incubated at 60°C for 20 min. The mixture was then cooled on ice, and 1 mL of 99.5% ethanol was added to stop the reaction. The product (glucose) was measured by HPLC (Hitachi) with a KS 801 column (Shodex). We defined one unit of enzyme activity as the amount necessary to liberate 1 µg glucose per min per 1 g of milled grains. Maltose solution incubated without enzyme solution and supplemented with the same volume of buffer solution was used as a reference.
To measure glucose-liberating activity in gelatinized starch (by α-amylase, β-amylase, and α-glucosidase combined), we used rice amylopectin as the substrate, obtained from the grains of a glutinous cultivar (Dewasansan) lacking amylose. The grains were milled to 90% and ground with a mortar and pestle, and the powder was sieved through a 200-µm mesh. The powder (1 g) was dissolved in 100 mL of 50 mM Na2HPO4 (pH 7.0) containing 5 mM CaCl2, and autoclaved at 120°C for 20 min to gelatinize the starch and inactivate endogenous hydrolytic enzymes. We then measured hydrolytic enzyme activities as above. Gelatinized starch solution incubated without enzyme solution and supplemented with the same volume of buffer solution was used as a reference.
Changes in glucose contents of hot-water-treated grains Hot-water treatment caused a significant increase in average grain glucose levels: from 6.8 mg/100 g DW in the untreated grains to 88.4 mg/100 g DW in the treated grains (Table 2). The increase in the glucose content of treated grains significantly differed between the japonica and indica cultivar groups. The mean glucose content in japonica cultivars increased from 8.9 to 123 mg/100 g DW, and that in the indica cultivars increased from 4.6 to 54 mg/100 g DW. The glucose levels in hot-water-treated grains also differed among cultivars within each group. The glucose contents of treated grains of japonica cultivars Asahi, Koshihikari, Snow Pearl, and Yamadanishiki were each > 100 mg/100 g DW; Koshihikari had the highest glucose content. On the other hand, the indica cultivar contents were < 100 mg/100 g DW, except for Hadsaduri, which had a value of approximately 100 mg/100 g DW.
| Cultivar | Glucose content (mg/100 g DW) | |
|---|---|---|
| Untreated grains | Hot-water-treated grains* | |
| japonica | ||
| Koshihikari | 8.3±8.1 | 214.7±17.3 |
| Hatsunishiki | 15.3±2.5 | 77.6±16.1 |
| Asahi | 9.5±2.3 | 167.0±5.8 |
| Sasahishiki | 7.3±2.1 | 91.3±9.2 |
| Nipponbare | 6.3±0.8 | 80.9±8.3 |
| Norin 22 | 7.7±1.4 | 95.3±27.0 |
| Snow Pearl | 10.5±2.1 | 110.3±13.0 |
| Yamadanishiki | 6.5±2.1 | 146.0±22.2 |
| japonica average | 8.9±2.7 | 122.9±14.8 |
| indica | ||
| Hadsaduri | 5.1±1.3 | 99.6±36.7 |
| TN-1 | 3.6±0.4 | 91.3±11.5 |
| Modan | 4.9±0.6 | 39.4±14.5 |
| Panbira | 3.3±1.1 | 43.7±5.5 |
| Kinandang | 4.4±1.4 | 31.2±5.0 |
| Bluebelle | 3.5±0.6 | 44.0±12.8 |
| Amber | 5.1±2.4 | 30.1±17.0 |
| BHC | 6.8±1.7 | 52.1±2.3 |
| indica average | 4.6±1.2 | 53.9±13.2 |
| Mean | 6.8±2.0 | 88.4±14. |
Values are means ± SD. *: Significantly difference between ecotypes at p<0.05 by Student's t-test.
Variation in endogenous hydrolytic enzyme activities We investigated the activities of starch-hydrolyzing enzymes that might result in glucose accumulation. There was no significant difference between the mean α-glucosidase activities of the japonica and indica cultivar groups, though there were some differences observed among cultivars (Fig. 1). However, there was a significant difference in liberated glucose from gelatinized glutinous starch between the groups (Fig. 2): the mean activity of the japonica cultivars was higher than that of the indica cultivars, though variation was observed in members of both groups. These hydrolytic activities are considered to be the combined activities of α-amylase, β-amylase, and α-glucosidase.
α-Glucosidase activity of hot-water-treated rice grains.
One unit of enzyme activity was defined as the amount of enzyme necessary to liberate 1 µg glucose per min per g of milled grains from maltose solution substrate. Values are presented as means ± SD. There was no significant difference between ecotypes at p < 0.05 by Student's t-test. The dark column indicates the values of japonica cultivars and the light column indicates the values of indica cultivars. TN-1, Taichung Native 1; BHC, British Honduras Creole.
Hydrolytic enzyme activity of hot-water-treated rice grains.
One unit of enzyme activity was defined as the amount of enzymes (α-amylase + β-amylase + α-glucosidase) necessary to liberate 1 µg glucose per min per g of milled grains from amylopectin solution substrate. Values are presented as means ± SD. There was a significant difference between ecotypes at p<0.05 by Student's t-test. The dark column indicates the values of the japonica cultivars and the light column indicates the values of indica cultivars. TN-1, Taichung Native 1; BHC, British Honduras Creole.
The levels of glucose accumulation in grains treated with hot water at 60°C for 30 min were much greater in japonica cultivars than in indica cultivars. Kasai et al. (2000) also reported the accumulation of reducing sugars (mainly glucose) during cooking; the largest increase was at 40 to 60°C. Mabashi et al. (2007) found the greatest accumulation of glucose at 60°C. Tsuyukubo et al. (2010) suggested that three major endogenous enzymes—α-glucosidase, α-amylase, and β-amylase—have a role in starch hydrolysis to produce reducing sugars, and that the three enzymes have distinct distributions in rice grains: α-glucosidase occurs in the inner endosperm, α-amylase in the outer layer of endosperm, and β-amylase throughout the grain. From our data, we assume that during hot-water treatment, the starch (amylose and amylopectin) in milled grain is hydrolyzed by endogenous α-amylase and β-amylase to maltose, which is further catalyzed by α-glucosidase to glucose.
Although, there was no significant difference between the mean α-glucosidase activities of the two ecotypes, the japonica grains showed significantly higher hydrolytic enzyme activities on gelatinized glutinous starch than the indica grains. These results are consistent with the difference in mean glucose accumulation between groups. The correlation coefficient between the glucose contents of the hot-water-treated grains and the endogenous hydrolytic activities was significant (r = 0.763, p < 0.05). Because there was no significant difference in the activity of α-glucosidase between groups, the difference in glucose accumulation is thought to be due to hydrolytic enzymes other than α-glucosidase, namely α-amylase, β-amylase and debranching enzyme.
Tsujii et al. (2013) reported a relationship between endosperm amylase and the amount of sugars (reducing sugars and total sugars) leached during rice cooking. They found that α-amylase activity and debranching enzyme activity was positively correlated with the amount of sugar leached during rice cooking. Their data suggested that sugars were leached during rice cooking by the action of starch hydrolytic enzymes such as α-amylase and debranching enzyme. Our data also suggest that in the hot-water-treated rice at 60°C for 30 min, a similar condition to the early stage of rice cooking, the amount of liberated glucose increases in proportion to the activities of endogenous hydrolytic enzymes other than α-glucosidase.
Acknowledgements The authors thank Mr. S. Uchida and Mr. T. Hyogo for their help with the measurement of sugars by HPLC.
