Differences and similarities of albumins from japonica rice bran, japonica, indica, and javanica rice endosperm in the suppressive effect of postprandial blood glucose elevation

Abstract

Suppression of rapid increase in blood glucose level using food components can prevent diabetes. To use functional food components from natural resources, information regarding the plant varieties and plant parts where the component exists is important for the effective extraction and the effective use of biological resources. We have previously found that proteins from the albumin fraction of rice endosperm are indigestible and adsorb glucose to suppress postprandial blood glucose elevation in vivo. However, the variety and location of this functional protein remain unknown. We investigated whether water‐soluble fraction from the endosperm of indica and javanica rice and red and middle bran of japonica rice contains functional albumin observed in the endosperm of japonica. Middle bran was found to possess an indigestible albumin with the same molecular weight as that in the endosperm of japonica and adsorb a large amount of glucose. Middle‐bran albumin suppressed postprandial blood glucose elevation after oral glucose loading in animal experiments, as well as the rice‐endosperm albumin. As middle bran is the outer layer of the endosperm, many functional components are distributed. Our study can aid the effective use of middle bran, which is predominantly discarded in the process of making rice wine, as a functional food material for the prevention of diabetes mellitus.

INTRODUCTION

The number of diabetic patients has risen to over 400 million and is expected to reach 600 million by 2040 (Yang et al., 2021). Type 2 diabetes, which accounts for ~90% of diabetes cases, causes serious complications including retinopathy, nephropathy, and neuropathy. To avoid the complications of diabetes, early treatment or prevention by management of postprandial blood glucose level is important (Ceriello, 2005; Gong et al., 2020). Although there are currently many drugs for the treatment of diabetes, some have been reported to cause side effects, such as hypoglycemia (American Diabetes Association, 2021). Thus, safe food-derived components that suppress the increase in postprandial blood glucose levels are promising to prevent Type 2 diabetes.

Some food components have been reported to suppress the rapid increase in postprandial blood glucose levels, such as catechins (Takahashi et al., 2020), other polyphenols (Cao et al., 2019; Takemori et al., 2022), honey (Ahmad et al., 2008), almond (Choudhary et al., 2009), and cinnamon (Solomon & Blannin, 2009). However, these components have characteristic flavors like bitterness, sweetness, and woody smell limiting their use in food. For cereals, proteins from wheat (Buonocore et al., 1977), buckwheat (Ninomiya et al., 2018; Ninomiya, Yamaguchi, et al., 2022), barley (Weselake et al., 1983), and rye (Granum, 1978) are reported to inhibit α-amylase to suppress postprandial blood glucose elevation after ingestion of starch-based food. To use functional components in food, information regarding the plant varieties and plant parts where the components exist is important for their effective extraction and effective use of biological resources.

We have found that albumin from rice endosperm (REA) effectively suppresses the postprandial blood glucose elevation after both glucose and starch administration in rats (Ina et al., 2016). REA barely inhibits mammalian α-amylase although some other cereal proteins inhibit mammalian α-amylase to suppress glucose production. REA contains an indigestible 16-kDa protein that adsorbs glucose like dietary fiber, which is considered to inhibit glucose absorption in the gastrointestinal tract to aid the excretion of glucose (Ina et al., 2016). In fact, REA remains a 14-kDa high-molecular peptide (HMP) even after digestion by pepsin and trypsin. HMP adsorbs glucose probably by noncovalent bonds like hydrogen bonds as well as intact REA in vitro and suppresses postprandial blood glucose elevation in vivo (Ina et al., 2020). Thus, the suppressive effect of oral administration of REA on blood glucose elevation is partially due to glucose adsorption by HMP. In addition, REA affords less than 2-kDa peptides (LMP) during digestion, which also suppress postprandial blood glucose elevation by inhibiting the expression of the glucose transporter SGLT1 (Ina et al., 2020). Moreover, REA possesses desirable features of a food material, including high water solubility and heat resistance (Ina et al., 2019); and is expected to be a functional food material for the management of postprandial blood glucose level. These functional proteins from REA have been extracted only from the endosperm of japonica rice (Oryza sativa subsp. japonica); little is known about rice varieties and the parts from which such proteins can be extracted.

Rice (Oryza sativa) has more than thousand varieties worldwide. The major rice varieties include japonica (Oryza sativa subsp. japonica), indica (Oryza sativa subsp. indica), and javanica (Oryza sativa subsp. javanica). Japonica rice has round grains and is mainly grown in relatively cool temperate regions such as Japan and Korea. Indica rice shapes long grains and is produced mostly in tropical regions, such as Pakistan, Brazil, and Sri Lanka, where it is more widely produced than japonica rice (Koizumi et al., 2021). Javanica rice shows intermediate characteristics between those of japonica and indica (Taira et al., 1988).

Rice grains are the seeds of rice and consist of chaff and caryopses. The chaff of rice protects it from the invasion of insects and fungi and is removed for eating as it is inedible. The caryopsis of rice is called brown rice, an edible part of rice, and is usually polished for consumption because of its low digestibility due to its rich dietary fiber. Brown rice is polished to 90% of its weight and produces both white rice at a polishing rate of 90% and bran, which is referred to as red bran (Figure 1). Red bran consists of a seed coat, pericarp, and aleurone layers. White rice mainly consists of an endosperm and is a popular ingredient of staple diets, desserts, and tea. Other bran is also produced during the polishing process to produce rice wine (sake). When white rice is further polished to 80% of the weight of brown rice, middle bran is produced (Figure 1). The middle bran refers to the outer layer of white rice. White bran is produced when rice with a polishing rate of 80% is polished to 70% of the brown rice weight. Bran is rich in proteins, vitamins, minerals, dietary fiber, high-quality lipids, and unique phytochemicals, including γ-oryzanol (Ochiai et al., 2016; Sharif et al., 2014), and exhibits functional properties, including antioxidative activity (Kobayashi et al., 2019; Wattanasiritham et al., 2016), inhibitory activities against platelet aggregation (Wong et al., 2016), and suppressive effects on blood pressure increase (Shobako & Ohinata, 2020). Despite the functionality of rice bran, it is predominantly discarded and rarely used effectively. Most rice proteins are distributed around the outer layer of white rice, including the red and middle bran (Ogawa et al., 2001). Thus, the functional proteins similar to REA may exist in the red and middle bran.

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In this study, we explored proteins with the suppressive effect of blood glucose elevation in indica and javanica rice, as well as in red and middle bran. Intact REA has a major 16-kDa protein, which is highly indigestible and remains a 14-kDa peptide even after treatment with digestive enzymes. In addition, REA adsorbs glucose like dietary fiber. We evaluated the molecular weight, digestibility, and glucose-adsorbability of albumin in the endosperm of indica and javanica rice, as well as in the red and middle bran of japonica rice. Albumin with high indigestibility and glucose-adsorbability was subjected to animal experiments to investigate its suppressive effect on postprandial blood glucose elevation in rats. The albumin was further analyzed to identify the protein structure using LC–MS/MS and a protein database.

EXPERIMENTAL PROCEDURES

Chemical reagents

Pancreatin from porcine pancreas and carboxymethylcellulose sodium salt (CMC) were purchased from Sigma-Aldrich (Merck). 2-Mercaptethanol was purchased from NACALAI TESQUE Inc. All other chemical reagents were purchased from FUJIFILM Wako Pure Chemical Corporation.

Materials

Japonica rice (Oryza sativa subsp. japonica, polishing ratio: 90%) was purchased from Nakatani Farm Co. Ltd. Indica rice (Oryza sativa subsp. indica) was purchased from uchinobeikoku Co., Ltd. Javanica rice (Oryza sativa subsp. javanica) was purchased from El Colmado GK. The rice was roughly powdered using a home grinder for 30 s and then finely powdered using a mill (MPW-G008, MAKINO mfg. Co., Ltd.) for 10 s to afford powder with ~10 μm diameter. The obtained powder was used for further experiments without sieving.

Red-bran and middle-bran (Oryza sativa subsp. japonica) powders were provided by Meiji Rice Delica Corporation.

Extraction of rice albumins

Rice albumins were prepared as previously described (Ina et al., 2016). Powdered rice or rice-bran powder (600 g each) was immersed in 3 L of 100 mM citric acid buffer at pH 6.0 and 4°C for 16 h. The suspension was centrifuged at 15000g and 4°C for 5 min, and the collected supernatant was heated at 60°C to denature and precipitate undesired and heat-sensitive proteins. The suspension after heating was centrifuged at 15000g and 4°C for 5 min, and proteins in the supernatant were precipitated by adding 40% saturated ammonium sulfate. The precipitate was collected after centrifugation at 15000g and 4°C for 1 h and then suspended in distilled water. The suspension was dialyzed against distilled water at 4°C for 2 days and then centrifuged at 15000g and 4°C for 15 min. The supernatant was lyophilized and the obtained powder was designated as albumin, which was stored at −20°C until use. Albumin obtained from japonica rice, indica rice, javainca rice, red-bran powder, and middle-bran powder were designated japonica rice-endosperm albumin (Jp-REA), indica rice-endosperm albumin (In-REA), javanica rice-endosperm albumin (Jv-REA), japonica rice-red-bran albumin (Jp-RRBA), and japonica rice-middle-bran albumin (Jp-RMBA), respectively. The protein concentrations of Jp-REA, In-REA, Jv-REA, Jp-RRBA, and Jp-RMBA were determined using the BCA method were 80.7%, 73.1%, 63.3%, 89.1%, and 81.5%, respectively.

SDS-PAGE

Rice albumin was dissolved in distilled water to obtain a protein concentration of 2 mg/mL. The solution was mixed with 2 × sample buffer (pH 6.8, 65 mM Tris–HCl, 2% SDS, 10% glycerol, 0.0025% bromophenol blue, 5% 2-mercaptethanol, 200 mM DTT) in equal amounts and boiled for 5 min. Then, the solution (10 μL) and molecular weight marker (5 μL, TEFCO Co.) were loaded onto a 16% acrylamide gel (ATTO CORPORATION). Electrophoresis was performed under the following conditions: 50 V (100 mA) for 30 min and 150 V (100 mA) for 65 min. The separated proteins were stained with Coomassie Brilliant Blue.

Digestibility of rice albumins

Digestibility of rice albumins was evaluated according to our previous study (Ina et al., 2016) with some modifications. In brief, albumins (50 mg protein each) were dissolved in 5 mL of 0.1 mg/mL pepsin solution at pH 2 adjusted by HCl. The solution was constantly shaken at 100 cycles/min at 37°C for 2 h and 25 μL aliquot of the solution was collected. The reaction was stopped by the addition of NaHCO₃. Furthermore, the solution was constantly shaken at 100 cycles/min at 37°C for 6 h in the presence of 5 mg pancreatin. A 25 μL aliquot of the solution was collected every 2 h and mixed with 100 μL of distilled water and 125 μL of 2 × sample buffer (pH 6.8), which was promptly boiled for 5 min to stop the reaction. These boiled samples were subjected to SDS-PAGE.

Glucose-adsorbability of albumins

The glucose adsorbability of albumins was evaluated as described in our previous study (Ina et al., 2016). Jp-REA, In-REA, Jv-REA, Jp-RRBA, Jp-RMBA, guar gum, and CMC were used as the test samples. Briefly, 8 mg/mL sample and 100 mM glucose in distilled water were loaded into the upper chamber of the dialysis unit (Slide-A-Lyzer MINI Dialysis Unit with a cutoff molecular weight of 3500; Thermo Fisher Scientific K.K.), and distilled water was loaded into the lower chamber. The unit was constantly shaken at 37°C, and the glucose concentration in the lower chamber unit was determined using Glucose CII Test Wako (Wako Pure Chemical Industries). The amount of glucose adsorbed on the sample was calculated according to our previous study and expressed as grams per gram of protein (Ninomiya, Ina, et al., 2022).

Animal experiments

A total of 27 seven-week-old male Wistar rats were purchased from Japan SLC, Inc. After acclimatization, they were randomly divided into three groups: Control, Jp-REA, and Jp-RMBA, of nine rats each. All animal experiments were performed in accordance with the Guidelines for Animal Experiments of the College of Bioresource Sciences of Nihon University (approval number: AP20BRS026-1). The rats were acclimated for 1 week before the experiment. The rats were allowed free access to standard chow (CE-2; CLEA Japan, Inc.) and tap water during the acclimation period. The room temperature and humidity were maintained at 23 ± 1°C and 50 ± 5%, respectively, with a 12 h light/dark cycle (8:00–20:00).

Oral glucose tolerance test

Oral glucose tolerance test (OGTT) was conducted according to our previous paper (Ina et al., 2016). In brief, blood was collected from the tail vein after rats were fasted overnight, which was designated as the blood sample at 0 min. Phosphate-buffered saline (PBS) solution containing 1 g/kg body weight (BW) of glucose with Jp-REA or Jp-RMBA (200 mg-protein/kg BW) was immediately administered by gavage (5 mL/kg BW). A PBS solution containing only 1 g/kg BW glucose was administered to the control group. Blood was collected from the tail vein 15, 30, 45, and 90 min after the oral administration of glucose. Blood samples were collected in heparinized capillary tubes containing aprotinin and kept on ice. The blood was centrifuged at 1300g and 4°C for 10 min to collect the plasma, which was stored at −80°C until use. Blood glucose levels were measured using a blood glucose meter (4239R1006; ForaCare Inc.). Plasma insulin levels were measured using the LBIS Rat Insulin ELISA Kit (U-E type, FUJIFILM Wako Shibayagi Corporation). The area under the curve (AUC) was calculated for blood glucose and plasma insulin levels according to Wolever and Jenkins (Wolever & Jenkins, 1986).

Two-dimensional electrophoresis and tryptic digestion

Two-dimensional electrophoresis was performed as previously described (Ina et al., 2016). First, 5 μg of Jp-REA or Jp-RMBA were dissolved in 155 μL of 60 mM Tris–HCl buffer (pH 8.8 containing 0.5% (vol/vol) ZOOM Carrier Ampholytes pH 3–10 (Thermo Fisher Scientific) and 0.02% (wt/vol) bromophenol blue, and then the solution was added to a ZOOM IPG Runner Cassette (Thermo Fisher Scientific K.K.). ZOOM strips (pH 3–10, Thermo Fisher Scientific K.K.) were used for first-dimensional electrophoresis. The strip was allowed to swell in the sample solution for at least 1 h in a sample loading device (ZOOM IPG Runner Cassette). First-dimensional electrophoresis was performed in ZOOM IPG Runner Mini-Cell (Thermo Fisher Scientific K.K.) at 175 V for 20 min, 175–2000 V for 45 min, and 2000 V for 60 min. SDS-PAGE was performed in a 4%–12% 2D-NuPAGE Bis-Tris ZOOM gel (Thermo Fisher Scientific K.K.). Protein spots were visualized using silver staining. Tryptic digestion was performed according to the method described elsewhere (Mori et al., 2012).

LC–MS/MS

LC–MS/MS was performed using an Advance LC (Bruker) equipped with Q Exactive (Thermo Fisher Scientific K.K.). The analytical column was a Zaplous alpha Pep C18 (0.2 × 50 mm, 3 μm, 120 Å, AMR incorporated). The mobile phase of the LC system consisted of Solvent A (0.1% formic acid–water) and Solvent B (acetonitrile). Digested protein solution (1 μL) was injected and eluted with gradient buffer B (5%–65% for 20 min, 65%–95% for 1 min, 95% for 3 min, 95%–5% for 1 min, and 5% for 5 min). The flow rate was 1.5 mL/min, the column temperature was 35°C, and the analysis time was 30 min. The scan mass range was set to m/z 350–2000.

Identification

Protein identification was performed using Proteome Discoverer 2.28 (Thermo Fisher Scientific K.K.) by matching the amino acid sequence information of the protein corresponding to “Oryza sativa japonica Taxi ID = 39947 2021-09-01” on NCBI (). When multiple proteins corresponded to each spot, the protein with the highest coverage (%) was identified as the protein of each spot.

Statistics

Numerical data are expressed as the mean ± SE of in vivo experiments, or the mean ± SD in the in vitro experiment. The significance of the differences between groups was evaluated by one-way ANOVA followed by Dunnett's test for in vivo experiments or Tukey's test for in vitro experiments.

RESULTS AND DISCUSSION

Molecular weight and digestibility of rice albumins

To identify functional proteins that suppress blood glucose elevation in In-REA, Jv-REA, Jp-RRBA, and Jp-RMBA, protein composition (Figure 2) and digestibility (Figure 3) were investigated by SDS-PAGE. Major bands of Jp-REA appeared at ~16 kDa was hydrolyzed to be mainly 14-kDa band after digestion with pepsin for 2 h and pancreatin for 6 h. These results are in accordance with those of our previous study (Ina et al., 2016). Other rice albumins, In-REA, Jv-REA, Jp-RRBA, and Jp-RMBA possessed a 16-kDa band as well. The 14-kDa band was mainly observed after digestion in In-REA, Jv-REA, and Jp-RMBA. While 14-kDa band of Jp-RRBA faded after only pepsin digestion for 2 h, that of In-REA, Jv-REA, and Jp-RMBA remained. Although Jp-RRBA included 16-kDa proteins, as well as Jp-REA and other albumins, this protein seemed to have a different structure from other albumins.

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Glucose-adsorbability of rice albumins

In our previous study, REA and its high-molecular-weight fraction, digested REA (HMP), adsorbed glucose. To describe the functional features of In-REA, Jv-REA, Jp-RRBA, and Jp-RMBA, the glucose adsorption ability of these albumins was evaluated. Figure 4 shows the time course of glucose concentration in the lower chamber of the unit. Glucose concentration gradually increased over the experimental period. The glucose concentrations of In-REA and Jv-REA were higher than that of Jp-REA and CMC at the final time point of 150 min. The glucose concentrations of Jp-RRBA and Jp-RMBA were lower than that of Jp-REA and similar to that of guar gum at the final time point 150 min. This indicated that Jp-RRBA and Jp-RMBA adsorbed glucose like guar gum. Figure 5 shows the calculated amount of glucose adsorbed onto rice albumins. The amounts of adsorbed glucose onto Jp-REA, In-REA, Jv-REA, Jp-RRBA, and Jp-RMBA were 521.1 ± 88.0 mg/g-protein, 124.4 ± 5.2 mg/g-protein, 160.6 ± 30.1 mg/g-protein, 893.8 ± 46.5 mg/g-protein, and 977.4 ± 45.4 mg/g-protein, respectively. The amount of glucose adsorbed onto Jp-RRBA and Jp-RMBA was significantly higher than that adsorbed onto Jp-REA (p < 0.05). Protein compositions were different among the albumins (Figure 2), which may affect the difference in glucose adsorbability. Jp-RRBA and Jp-RMBA exhibited the highest glucose adsorbability, but Jp-RRBA may not be effective in vivo because it was mostly hydrolyzed upon digestion (Figure 3). On the other hand, a 14-kDa peptide remained after the digestion of Jp-RMBA by pepsin and pancreatin. Jp-RMBA is more likely to suppress blood glucose elevation in vivo than Jp-RRBA; therefore, we used Jp-RMBA for the OGTT.

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The effects of Jp-REA and Jp-RMBA on the blood glucose and the plasma insulin levels in OGTT

Figure 6 presents the time course of the blood glucose levels after oral administration of glucose with or without Jp-REA or Jp-RMBA and the AUC. The blood glucose level peaked 15 min after oral administration of glucose in the control group, while the peak shifted after 15 min in the Jp-REA and Jp-RMBA groups (Figure 6a). The blood glucose levels 15 min after glucose administration in the control, Jp-REA, and Jp-RMBA groups were 178 ± 5, 153 ± 4, and 150 ± 4 mg/dL, respectively. Blood glucose levels 15 min after glucose administration in the Jp-REA and Jp-RMBA groups were significantly lower than those in the control group (p < 0.05). The AUC of the blood glucose levels in the control, Jp-REA, and Jp-RMBA groups were 3245 ± 268 mg·min/dL, 3253 ± 390 mg·min/dL, and 3384 ± 233 mg·min/dL, respectively (Figure 6b). The AUC was not significantly different between the groups. Figure 7 presents the time course of plasma insulin levels and AUC. The plasma insulin levels 15 min after glucose administration in control, Jp-REA, and Jp-RMBA groups were 0.82 ± 0.11 ng/mL, 0.57 ± 0.05 ng/mL, and 0.49 ± 0.11 ng/mL, respectively (Figure 7a). Plasma insulin level 15 min after glucose administration in the Jp-RMBA group was significantly lower than that in the control group (p < 0.05). The AUC of the plasma insulin levels in control, Jp-REA, and Jp-RMBA groups were 24.11 ± 4.46 ng·min/mL, 20.00 ± 2.04 ng·min/mL, and 20.22 ± 3.3 ng·min/mL, respectively (Figure 7b). The AUC of Jp-REA and Jp-RMBA was lower than that of the control group; however, the differences were not significant (p < 0.05).

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Identification of proteins in Jp-REA and Jp-RMBA using LC–MS/MS

Figure 8 presents two-dimensional SDS-PAGE electrophoreses of Jp-REA and Jp-RMBA. Eight spots in Jp-REA and 13 spots in Jp-RMBA were subjected to protein identification by LC–MS/MS. Tables 1 and 2 show the proteins identified from each spot. Proteins from major spots No. 6 and 7 of Jp-REA were identified as Os07g0214300 by ordered locus name, which was the same as major spot No. 9 in Jp-RMBA. Although some spots were identified as proteins that differed between Jp-REA and Jp-RMBA, the major protein was Os07g0214300, which is a seed storage protein located in protein body II (PB-II) in the endosperm cells (Kurokawa et al., 2014; Lang et al., 2010; Zhou et al., 2017). Os07g0214300 is also present in various species, including indica rice (Teshima et al., 2010), which is consistent with the result that In-REA contains an indigestible 16-kDa protein. The protein content in the endosperm is lower inside and decreases with the polishing ratio (Anzawa et al., 2013). Since the middle bran is produced from the outer layer of white rice, Os07g0214300 may be localized in the outer layer of the rice endosperm.

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TABLE 1 Proteins identified in Jp-REA.

Spot No.	Accession No.	Protein name	Matched peptides	Coverage (%)	MW	Calc. pI
1	Q8H4L8	Os07g0214600	8	41	17	8.03
2	Q8H4L8	Os07g0214600	2	15	17	8.03
3	Q01882	Os07g0214300	1	4	17.9	7.84
4	Q8H4L8	Os07g0214600	2	18	17	8.03
5	Q01882	Os07g0214300	7	52	17.9	7.84
6	Q01882	Os07g0214300	8	58	17.9	7.84
7	Q01882	Os07g0214300	8	52	17.9	7.84
8	Q8H4M4	Os07g021380	2	17	17.3	8.34

TABLE 2 Proteins identified in Jp-RMBA.

Spot No.	Accession No.	Protein name	Matched peptides	Coverage (%)	MW	Calc. pI
1	Q01882	Os07g0214300	5	39	17.9	7.84
2	Q0DT04	Os03g0277500	6	56	15	5.86
3	A3BHT2	Uncharacterized protein	5	39	15.3	6.95
4	Q01882	Os07g0214300	3	23	17.9	7.84
5	Q01882	Os07g0214300	7	46	17.9	7.84
6	Q01882	Os07g0214300	5	39	17.9	7.84
7	Q01882	Os07g0214300	10	58	17.9	7.84
8	Q01882	Os07g0214300	5	36	17.9	7.84
9	Q01882	Os07g0214300	8	55	17.9	7.84
10	P29421	Os04g0526600	6	24	21.4	8.37
11	P29421	Os04g0526600	14	55	21.4	8.37
12	Q01882	Os07g0214300	5	36	17.9	7.84
13	B9FYC8	Uncharacterized protein	5	20	27.4	7.58

The suppressive effects of both Jp-REA and Jp-RMBA on blood glucose elevation in rats were possibly due to Os07g0214300 and the mechanisms can be common. Previous studies have shown that Jp-REA suppresses postprandial blood glucose elevation by promoting glucose excretion in the gastrointestinal tract and inhibiting the expression of SGLT1 (Ina et al., 2020). These effects were probably due to hydrolysates of Os07g0214300, HMP, and LMP (Ina et al., 2020). The digestibility of Os07g0214300 was not evaluated in vivo and oral digestion was unconsidered in this study. Thus, it is unproven that HMP remains in the intestinal cavity. However, Ina et al. found that there are many disulfide bonds in the structure of Os07g0214300 and 14 kDa HMP still remained even after 6-h digestion by pancreatin in vitro in this study (Figure 3). Although the digestibility of Os07g0214300 should be evaluated in vivo as well, we assume that Os07g0214300 is indigestible and the mechanism of action of Jp-REA and Jp-RMBA to inhibit postprandial blood glucose elevation may also be the same.

Much research has been conducted on the effective utilization of biomass from food-derived waste products, such as rice bran (Dessie et al., 2020). This study can contribute significantly to the effective utilization of unutilized resources and enhance the value of rice bran as a functional food material with a suppressive effect on blood glucose elevation.

CONCLUSIONS

The functional 16-kDa protein similar to Jp-REA with the suppressive effect on blood glucose elevation was found in indica and javanica rice (In-REA and Jv-REA), and in japonica rice-middle bran (Jp-RMBA), but not in japonica rice-red bran (Jp-RRBA). Jp-RMBA adsorbed more glucose than In-REA and Jv-REA and suppressed postprandial rapid blood glucose elevation in rats as well as Jp-REA. A major factor responsible for the effect of Jp-RMBA was identified as Os07g0214300, which is the same major protein in Jp-REA. Since Jp-RMBA is an albumin fraction of middle bran, which is the outer layer of white rice, Os07g0214300 seems to be localized in the outer layer of the endosperm. Our study can aid the effective use of middle bran, which is predominantly discarded in the process of making rice wine, as a functional food material for the prevention of diabetes mellitus.

AUTHOR CONTRIBUTIONS

Rio Ogawa carried out the research, analyzed the data, and wrote the first draft of the manuscript, Chiaki Sugimoto and Aya Hamada carried out the research and analyzed the data, Kazumi Ninomiya and Shigenobu Ina developed a method and supervised, Yusuke Yamaguchi conceived and designed the study, contributed to the manuscript final drafting, Hitoshi Kumagai developed a method and supervised, Hitomi Kumagai conceived and designed the study, funding acquisition, and supervised the conduct of this study. All authors reviewed the manuscript draft and revised it critically on intellectual content. All authors approved the final version of the manuscript to be published.

ACKNOWLEDGMENTS

This work was supported by JSPS KAKENHI Grant Number 20K05883, and Ryoshoku Kenkyu-kai. Red rice bran and middle bran were kindly provided from Meiji Rice Delica Corporation.

FUNDING INFORMATION

This work was supported by JSPS KAKENHI Grant Number 20K05883, and a research grant from Ryoshoku Kenkyu-kai.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest.

ETHICS STATEMENT

All animal experiments were performed in accordance with the Guidelines for Animal Experiments of the College of Bioresource Sciences of Nihon University (approval number: AP20BRS026-1).

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