2017 Volume 23 Issue 1 Pages 151-155
Rice endosperm enzyme activity affects the qualities of cooked rice. In this study, we aimed to investigate the differential localization of various enzymes in the endosperm of milled rice. Using hydrolytic enzyme analysis, we confirmed the activities of amylase, protease, and lipase. We milled brown rice to yields of 30 – 90%, at intervals of 10%, and then extracted enzymes from the milled rice. Total protein content was reduced by about 43% with 60% removal of the outer layer, resulting from the decrease in milling yield from 90 – 30%. However, the water-soluble protein content was only approximately 0.3% in both the inner and outer layers. Moreover, the activities of esterase (C4), acidic and alkaline phosphatase, leucine allyl amidase, naphthol-AS-BI-phosphodiester hydrolase, α- and β-galactosidases, N-acetyl-β-glucosaminidase, debranching enzyme, β-amylase, and α-glucosidase were similar between the inner and outer layers. In contrast, the activities of esterase lipase (C8), valine allyl amidase, cystine allyl amidase, trypsin, β-glucosidase, α-mannosidase, α-amylase, and polygalacturonase were greater in the outer layer. Thus, these results showed that measurement of rice endosperm enzyme activity could be used to assess enzyme localization.
There are various enzymes in the endosperm of rice grains. In previous studies, we showed that rice endosperm enzyme activity affects the taste and texture of cooked rice (Tsujii et al., 2007a, 2007b, 2009, 2010, 2012, 2013). Tran et al. have reported the relationship between cooked rice taste and α- and β-amylase activities (Tran et al., 2001, 2005). Besides, the relationship between α-glucosidase activity and cooked rice taste has been reported (Awazuhara et al., 2000; Kishio and Aoyagi, 2014). Chemometric analysis of the enzyme activity allowed for comparisons of the characteristics and activities of various carbohydrate-related enzymes in rice cultivars (Tsujii and Takano, 2015a). Interestingly, there is a positive correlation between the sensory evaluation score and rice endosperm enzyme activity, suggesting that the high palatability of cooked rice is correlated with increased enzyme activity (Tsujii and Takano, 2015b). However, negative correlations exist between ripening temperature and rice endosperm enzyme activities, suggesting that lower ripening temperatures were associated with greater enzyme activity.
In this study, we aimed to investigate the changes in enzyme activity and localization following rice milling. In particular, we investigated the activities of major enzymes related to the texture of cooked rice and of lipase and protease.
Rice samples Brown rice grains of Koshihikari (cultivar japonica) were produced in Chiba, Japan. Samples were obtained directly from a wholesaler in Tokyo, Japan (Kitoku Shinryo Co., Ltd.) and were stored at 4°C until use.
Methods of rice milling and properties measurement The yield (percentage by weight) of milled rice to original brown rice is used as a technical index in rice milling, and is termed the “milling yield”. Brown rice grain (200 g) was milled to different milling yields from 30 – 90%, at intervals of 10%, using a horizontal abrasive type rice-milling test machine at 915 rpm (TM-05; Satake Co. Ltd., Hiroshima, Japan). The surface roughness of the built-in roll was #46. Once the temperature of the roll reached 40°C, it was cooled down to room temperature by natural cooling. Table 1 shows the grain length (GL), grain width (GWh), grain thickness (GT), moisture, and total protein content. The 1000 kernel weight (GWt) was measured by conventional methods. The amount of nitrogen was determined using an Automatic High Sensitive NC Analyzer (SUMIGRAPH NC-220F; Sumika Chemical Analysis Service Ltd., Tokyo, Japan). Total protein content was obtained by multiplying the nitrogen value with a nitrogen-protein conversion factor of 5.95.
| sample | GL(mm) | GWh(mm) | GT(mm) | GWt(g) | RMR(%) | moisture(%) | TP(mg/g) |
|---|---|---|---|---|---|---|---|
| Brown rice | 4.86 | 2.93 | 2.03 | 22.21 | - | N.T. | N.T. |
| 90% | 4.80 | 2.86 | 1.97 | 19.51 | 87.8 | 17.7 | 51.9 |
| 80% | 4.62 | 2.80 | 1.92 | 18.65 | 84.0 | 16.1 | 48.9 |
| 70% | 4.27 | 2.69 | 1.86 | 15.87 | 71.5 | 14.9 | 40.9 |
| 60% | 3.71 | 2.53 | 1.83 | 13.43 | 60.5 | 12.8 | 35.9 |
| 50% | 3.05 | 2.36 | 1.82 | 10.32 | 46.5 | 11.5 | 36.8 |
| 40% | 2.68 | 2.23 | 1.80 | 8.45 | 38.0 | 11.2 | 32.2 |
| 30% | 2.45 | 2.08 | 1.71 | 7.13 | 32.1 | 10.8 | 30.0 |
GL: grain length GWh: grain width GT: grain thickness GWt: grain wight
RMR: rice millingrate TP: Total Protein NT.: NotTested
Extraction of crude enzyme Milled rice was added to water at a ratio of 1:1.8, and the mixture was left at room temperature for 1 h. This mixture was homogenized (15,000 rpm, 3 min; NS-51K, Microtec Co., Chiba, Japan) and placed in a reciprocal shaker for 1 h. The precipitate was then removed by centrifugation at 18,000 × g for 30 min. The supernatant was used as the crude enzyme for the determination of several enzyme properties. All processes were carried out at 4°C. Total protein content was measured by the Bradford assay (Bradford, 1976).
Measurement of enzyme activity for general hydrolytic enzymes The activities of a total of 19 types of enzymes were examined using a commercially available API-ZYM kit (BioMérieux Japan Ltd., Tokyo, Japan). The absorbance of the enzymatic reaction solution was measured us ing a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific K.K., Kanagawa, Japan).
Measurement of enzyme activity for enzymes involved in eating quality α-Amylase activity was assayed using the cereal α-amylase assay reagent method (Megazyme International Ireland Ltd., Wicklow, Ireland) (McCleary and Sheehan, 1987; Sheehan and McCleary, 1988). After 60 min of incubation at 37°C (pH 7.0), the reaction was stopped by the addition of 30% Tris. One unit (U) of α-amylase activity was defined as the amount of enzyme required to release 1 µmol p-nitrophenol in 1 min under the defined assay conditions.
β-Amylase activity was assayed using a Beta-Amylase Kit (Megazyme International Ireland Ltd. , Wicklow, Ireland) (McCleary and Codd, 1989). After 30 min of incubation at 37°C (pH 5.0), the reaction was stopped by the addition of 30% Tris. One unit of β-amylase activity was defined as the amount of enzyme required to release 1 µmol p-nitrophenol in 1 min under the defined assay conditions.
α-Glucosidase activity was assayed using a 60-µL reaction mixture containing 5 mM p-nitrophenyl-−-d-glucopyranoside (Sigma-Aldrich, St. Louis, MO, USA), 200 mM Mcllvaine buffer (pH 5.0), and an appropriate dilution of the enzyme preparation. After 30 min of incubation at 37°C, the reaction was stopped by the addition of 160 µL of 30% Tris, and the amount of released p-nitrophenol was then monitored. One unit of α-glucosidase activity was defined as the amount of enzyme required to release 1 µmol p-nitrophenol in 1 min under the defined assay conditions.
Debranching enzyme (DBE) activity was assayed using a 150-µL reaction mixture containing 1.0% pullulan, 200 mM Mcllvaine buffer (pH 6.0), and an appropriate dilution of the enzyme preparation. After 30 min of incubation at 40°C, the reaction was stopped by the addition of alkaline copper reagent, and the amount of residual sugars was measured using the Somogyi-Nelson method (Nelson, 1944; Somogyi, 1952). One unit of DBE activity was defined as the amount of enzyme required to release 1 µmol maltotriose in 1 min under the defined assay conditions.
Polygalacturonase (PG) activity was assayed using a 150-µL reaction mixture containing 0.5% pectic acid, 200 mM Mcllvaine buffer (pH 4.5), and an appropriate dilution of enzyme preparation. After 60 min of incubation at 50°C, the reaction was stopped by the addition of alkaline copper reagent, and the amount of residual sugars was measured by the Somogyi-Nelson method. One unit of PG activity was defined as the amount of enzyme required to release 1 µmol reducing sugars in 1 min under the defined assay conditions.
β-Galactosidase activity was assayed using a 60-µL reaction mixture containing 5 mM p-nitrophenyl-β-d-galactopyranoside (pNPβGal; Sigma), 200 mM Mcllvain buffer (pH 4.5), and an appropriate dilution of enzyme preparation. After 60 min of incubation at 37°C, the reaction was stopped by the addition of 160 µL of 30% Tris, and p-nitrophenol release was then monitored. One unit of β-galactosidase activity was defined as the amount of enzyme required to release 1 µmol p-nitrophenol in 1 min under the defined assay conditions.
Detection of amylase using zymography Amylase activity was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels containing 1.0% soluble starch (Nacalai Tesque, Inc. , Yokohama, Japan), following the procedure described by Laemmli (1970), with some modifications. Electrophoresis was carried out at 4°C, and SDS was removed using 2.5% Triton X-100 (Yamagata et al., 1988). The gels were incubated at 37°C overnight in 100 mM Mcllvain buffer (pH 5.0) containing 10 mM CaCl2, and were then stained with 0.01% (w/v) I2/0.1% (w/v) KI solution. Moreover, to remove the iodine bound to the starch, the stained gel was dipped in 2.5% Triton-X100 and washed with excess iodine stain.
Properties of rice samples prepared with different milling yields Proteins and lipids in rice grains are generally distributed within the external layer rather than the internal layer. First, we confirmed whether Koshihikari (cultivar japonica) rice exhibited this same tendency. As indicated in Table 1, the amount of total protein (TP) was decreased as the rice milling yield (RMR) decreased. TP content was reduced by about 43% with 60% removal of the outer layer, caused by the decrease in milling yield from 90 – 30%.
Localization of general hydrolytic enzymes To investigate the localization of general hydrolytic enzymes, enzymatic activities were detected in rice grains prepared with different milling yields. We measured the enzyme activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C15), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, chymotrypsin, acid phosphatase, phosphoamidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase by API-ZYM (Table 2). Most grains exhibited strong leucine arylamidase, phosphoamidase, and N-acetyl-β-glucosaminidase activities, whereas esterase (C4) activity was weak in grains with 80 – 90% milling yields. Valine arylamidase showed higher activities in grains prepared with milling yields of 80 – 90% than in those with milling yields of 30 – 70%. Esterase lipase (C8), cystine arylamidase, trypsin, chymotrypsin, β-glucuronidase, and β-glucosidase showed very weak activities in grains prepared with milling yields of 70 – 90%. No α-fucosidase or lipase (C15) activities were observed in any of the milled grains.
| sample | 1* | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 90% | 0.50** | 0.14 | 0.11 | n.d. | 1.13 | 0.76 | 0.18 | 0.18 | 0.07 | 0.49 | 1.24 | 0.33 | 0.64 | 0.11 | 0.41 | 0.17 | 0.68 | 0.37 | n.d. |
| 80% | 0.46 | 0.15 | n.d. | n.d. | 1.26 | 0.53 | 0.15 | 0.19 | 0.06 | 0.45 | 1.18 | 0.48 | 0.35 | 0.08 | 0.35 | n.d. | 0.87 | 0.15 | n.d. |
| 70% | 0.39 | 0.21 | n.d. | n.d. | 1.18 | 0.37 | 0.11 | 0.17 | 0.05 | 0.79 | 0.76 | 0.26 | 0.49 | n.d. | 0.39 | n.d. | 0.53 | 0.22 | n.d. |
| 60% | 0.53 | 0.19 | n.d. | n.d. | 1.16 | 0.23 | n.d. | n.d. | n.d. | 0.48 | 0.93 | 0.35 | 0.41 | n.d. | 0.31 | n.d. | 0.33 | 0.33 | n.d. |
| 50% | 0.52 | 0.34 | n.d. | n.d. | 1.36 | 0.35 | n.d. | n.d. | n.d. | 0.43 | 1.04 | 0.55 | 0.42 | n.d. | 0.44 | n.d. | 0.92 | 0.16 | n.d. |
| 40% | 0.56 | 0.15 | n.d. | n.d. | 1.34 | 0.32 | n.d. | n.d. | n.d. | 0.51 | 0.94 | 0.47 | 0.56 | n.d. | 0.45 | n.d. | 0.79 | 0.13 | n.d. |
| 30% | 0.17 | 0.25 | n.d. | n.d. | 1.24 | 0.17 | n.d. | n.d. | n.d. | 0.41 | 0.81 | 0.67 | 0.37 | n.d. | 0.55 | n.d. | 0.60 | 0.14 | n.d. |
Assay of enzymes involved in eating quality Recent studies have shown that some enzymes are involved in eating quality (Tsujii et al., 2013). The activities of the evaluated enzymes are shown in Table 3. The activities of α-amylase, polygalacturonase, and β-galactosidase were high in grains prepared at milling yields of 80% and 90%. Moreover, there was a 4-fold difference in the activities of α-amylase and β-galactosidase by the decrease of milling yield from 90 – 30%. In contrast, there were no significant differences in β-amylase, α-glucosidase, or DBE activities between the external and internal layers. The water-soluble protein content was only approximately 0.3% in both the inner and outer layers.
| sample | α-AMY | β-AMY | α-GLU | DBE | PG* | β-GAL | SP * * |
|---|---|---|---|---|---|---|---|
| 90% | 183 | 4099 | 923 | 8.7 | 1.02 | 1182 | 2.73 |
| 80% | 110 | 3998 | 917 | 10.9 | 1.09 | 769 | 2.78 |
| 70% | 67 | 4544 | 1236 | 12.7 | 0.34 | 448 | 2.73 |
| 60% | 48 | 4680 | 1102 | 11.3 | 0.19 | 430 | 2.75 |
| 50% | 50 | 4482 | 1104 | 13.2 | 0.07 | 390 | 2.75 |
| 40% | 43 | 4130 | 1116 | 12.9 | N.D. | 340 | 2.71 |
| 30% | 44 | 3621 | 1149 | 13.2 | N.D. | N.T. | 2.67 |
(U/g)
α-AMY: α-amylase β-AMY: β-amylase α-GLucosidase DBE: Debraching Enzyme PG: Polygalacturonase β-GAL: β-Glactosidase N.D.: Note detected N.T.: Not Tested SP: Soluble Protein
Electropherograms of amylase prepared using different milling yield grains The amylase isozyme patterns of grains prepared using different milling yields, as determined by SDS-PAGE, are presented in Figure 1. There were four isozymes (I, II, III, and IV), which had molecular weights of 270, 140, 57, and 44 kDa, respectively (Figure 1, left). Based on prior studies, isozymes I, II, and IV were likely α-amylase (Tsujii et al., 2013). Isozymes I and IV were detected in grains prepared with milling yields of 80% and 90%, whereas amylase isozyme II was detected in all samples as a low-intensity band. Isozyme III was likely not α-amylase but DBE because there is no α-amylase with a molecular weight of 57 kDa. In addition, the results of decolorization treatment by 2.5% Triton-X100 showed that a wide range of bands with molecular weights of approximately 57 – 75 kDa were all DBE (Figure 1, right). Notably, DBE isozymes were also detected in all samples.
Zymogram of amylases from different rice milling rate. Amylase activity was detected by SDS-PAGE containing 1.0% soluble starch. The gels were incubated at 37°C overnight in 100 mM Mcllvain buffer (pH 5.0) containing 10 mM CaCl2. They were stained with 0.01% (w/v) I2 / 0.1% (w/v) KI solution (Left). Moreover, in order to remove the iodine bound to the starch, dipped the stained gel was dipped in 2.5% Triton-X100, and washed with excess iodine staining (Light).
In this study, we found that there were various enzymes present in rice endosperm after milling and that these enzymes exhibited differences in localization. An analysis of the primary hydrolytic enzymes revealed the presence of amylase, protease, and lipase activity. Thus, these findings suggested that the presence of enzyme activity in the endosperm of dormant seeds persists even after milling in brown rice. Changes in enzyme activity or localization may affect the freshness and seed characteristics of the grain.
Tsuyukubo et al. demonstrated that the distribution of starch-degrading enzymes in dormant rice grains could be determined using specific antibodies against α-glucosidases, α-amylases, and β-amylases. The α-glucosidases were predominantly localized in the inner endosperm layers. In contrast, the α-amylases were mainly localized in the outer endosperm layers, and the β-amylases were distributed throughout the entire grain (Tsuyukubo et al., 2010).
Furthermore, we used zymography for measurement of enzyme activity to determine enzyme localization in the endosperm and showed that protease and lipase were localized in the endosperm.
The chain polysaccharide matrix structure of the cell walls and starches of the rice endosperm is decomposed during rice cooking, likely through the activities of endosperm amylases and cell wall degradation-related enzymes, thereby altering the hardness and adhesiveness of cooked rice (Tsujii et al., 2009; Tsujii et al., 2013). Tsuyukubo et al. demonstrated that rice grains contain starch-degrading enzymes, including α-glucosidases, α-amylases, β-amylases, pullulanase, and isoamylases. Moreover, studies have investigated the elution behaviors of these enzymes from rice grains into cooking water during rice cooking (Tsuyukubo et al., 2012).
In this study, we examined the localization of endosperm enzymes and inferred the functions of enzymes during rice cooking. For example, α-amylase, which was localized in the outer endosperm layer, was also released into the external solution during rice cooking, partially digested the outer layer starch, leached the malto-oligomer, and affected the texture and taste of the cooked rice. The DBE, which was localized to all endosperm layers, functioned to partially digest starches in all layers and affected the taste of the cooked rice. Thus, based on the localization of the enzyme in the rice grain, it is possible to estimate the changes in taste and quality characteristics of rice.
Acknowledgments We wish to thank Kitoku-Shinryo Co., Ltd. for supplying the rice samples. We also thank Tokyo University of Agriculture Applied Biological Sciences, Biological Department of Applied Chemistry Food Resources Physics and Chemistry Laboratory of the Graduates for assistance with experiments. Finally, we thank Kanako Oorui, Manami Tatsuta and Akira Miyake.
