Potential for Preventing Diabetes and Dementia by Consuming Unpolished Rice Blended with Black Unpolished Rice and Super-Hard Rice

Type-2 diabetes, which may prompt Alzheimer’s disease, can be regulated by diet. We investigated the palatability and efficacy of consuming two diets rich in β-glucan or resistant starch by blending ordinary polished rice and polished waxy barley (5:5) or by blending ordinary unpolished rice, unpolished pigmented rice and polished super-hard rice (2:4:4), then subjecting the blends to high-pressure treatment before cooking. After subjects consumed these diets for 12 weeks, consumers of the former rice blend had significantly less body fat, lower blood glucose levels. The latter blend showed higher antioxidant capacity and inhibition against β-secretase than control, and the subjects, consuming the latter blend, showed not only lower blood glucose levels, but also a higher ratio of amyloid β 42/40 peptide than the subjects consuming the traditional rice diet. Long-term consumption of the latter blended rice diets might inhibit rises in postprandial blood glucose levels and impede amyloid β production. Abbreviations: Ae Amyloid High-Pressure Rice with Unpolished Rice And Super-Hard Rice (Ratio KWB Rice: Rice with Waxy Barley (Ratio 5:5), BGL: Blood Glucose AUC: Under Rising Limb of a Glucose Curve for Postprandial Blood Glucose, AUCi: Area Under The Rising Limb of an Insulin Curve, FBG: Fasting Blood Glucose Level, RS: Resistant Starch, BFP: Fat LDL: Lipoprotein

Introduction pharmacological interventions to prevent or delay its progression to complications [2]. The WHO and Food and Agriculture Organization of the United Nations recommend foods with low glycemic index (GI) to prevent diabetes [3]. The concept of GI was introduced by Jenkins et al. [4] as a ranking system for carbohydrates based on their immediate impact on blood glucose levels, and low GI and glycemic load diets have more recently been widely recommended for the prevention of chronic diseases including diabetes, obesity, cancer and heart disease, [5] because the resulting glycemic index classification of foods provided a numeric physiologic classification of relevant carbohydrate foods in the prevention and treatment of diseases such as diabetes [6]. The glycemic effect of food depends on numerous factors, including the structure of amylose and amylopectin [7]. The amylopectin found in amylose extender (ae) rice mutants (e.g., "Chikushikona85"cultivar) contains more longchain glucans than normal (non-mutant) rice, thereby making the texture of the mutant rice grains very hard and non-sticky after boiling, which renders it less palatable than traditional rice [8]. Wada et al. showed that the rise in blood sugar after consuming cookies made from "Chikushi-kona85" rice flour was significantly less than when consuming cookies made from the traditional rice cultivar [9] Several studies have reported the development of highly resistant starch (RS) rice [10,11] (via physical or chemical mutation), as well as high-amylose and high-dietary fiber rice [12].
Barley, used as food, malt, and as feedstock in Japan, is rich in β-glucan. Beta-glucan is a linear polysaccharide of glucose monomers and water-soluble dietary fiber (SDF). Several recent studies, in both hypercholesterolemic and healthy subjects, found that the daily consumption of 5 g of β-glucan significantly lowers total and LDL cholesterol in serum [13,14]. Waxy barley cultivars are a typical component of specialized foods. It imparts a sticky texture to foods and contains a total β-glucan content that is higher than normal isotypes of barley [15].
The production of short-chain fatty acids in β-glucan may also be a probable mechanism behind its observed metabolic effects.
Moreover, fermentation of oat β-glucan has been shown to yield larger amounts of propionate, which has been shown to significantly inhibit cholesterol synthesis in humans [16][17][18].
Several studies report that long-term consumption of RS may lower fasting cholesterol and triglyceride levels [19,20]. Robertson et al. showed that 30g RS/d added to the habitual diet of healthy subjects resulted in a significant decline in subcutaneous abdominal adipose tissue, non-esterified fatty acid (NEFA), and glycerol release [21].
High-pressure treatment (HPT), a technological process that limits the negative effects of food preparation on hydro-soluble vitamins, is recognized as being very useful in preserving nutritional quality in foods [22,23]. Yamakura et al. [24] showed that subjecting rice to HPT before cooking results in more free amino acids and stickier cooked rice.
In previous studies, we developed a novel method for inhibiting abrupt postprandial blood glucose levels (BGL) in Sprague-Dawley rats by feeding them ae-mutant rice (super-hard rice) soaked with functional food ingredients that had been subjected to HPT before preparation [25]. Furthermore, when we pre-prepared (with HPT) "Koshihikari" rice blended with black rice (HPT KO) at a ratio of 6:4 (using a randomized, single-blind, crossover-designed study) , we found that BGL in subject at 90 and 120 min after ingesting the cooked HPT KO was significantly lower than BGL following ingestion of the cooked "Koshihikari" (polished) rice alone. In fact, the increase in amyloid β40 peptide in blood 120 min after eating HPT KO rice tended to be lower than amyloid β40 after ingesting the cooked "Koshihikari" rice [26].

Recent epidemiological studies have suggested a link between
Alzheimer's disease (AD), and type 2 diabetes mellitus associated with insulin resistance [27][28][29]. Diabetes is a lifestyle disease, and its prevention and treatment are extremely important in improving longevity and quality of life. Low glycemic index (GI) foods inhibit rapid increases in blood glucose and insulin secretion after meals.
Because working memory is impaired by AD, the disease has spawned dynamic research investigating the influence of gammaaminobutyric acid (GABA) on working memory performance in AD sufferers [30]. It is well-recognized that the prevalence of dementia is higher in diabetic patients than in non-diabetic subjects [31], and Tokutake et al. [32] showed that there is a molecular link between AD and insulin signaling.
Molecules in rice with antioxidant activity include phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, γ-oryzanol, and phytic acid. Rice bran also contains a variety of functional substances, such as γ-oryzanol, ferulic acid, sterol, wax, ceramide, phytin, inositol, and protein [33]. In previous studies, black rice bran contained more oleic acid and anthocyanin and showed greater BACE1 inhibitory activity than "Koshihikari" (a traditional, premium polished rice). Jensen et al. [34] and Qureshi et al. [35] showed that an increased daily consumption of bran significantly reduced the risk of coronary heart disease and lowered total cholesterol, LDL cholesterol, and triglycerides in serum. Abe et al. [36] showed that rice components that included inositol hexaphosphate significantly inhibited Aβ production in neuroblastoma cells without causing cytotoxicity, suggesting that such foods might prevent AD.
Pigmented rice contains naturally occurring pigmented flavonoids known as anthocyanins. Positive health effects have been reported for the pigments in the bran layer of rice. For example, consumption of red and black rice by rabbits has been found to reduce their atherosclerotic plaque formation and increase antioxidants, ostensibly by increasing concentrations of high-density lipoprotein cholesterol and apolipoprotein A1 [37]. In previous studies, we found that a super-hard rice bread blended with black rice bran (SRBBB), which contained a high amount of resistant starch (RS), provided a strong inhibitory activity against β-secretase (BACE1) and acetylcholinesterase, even after heating. Furthermore, aged mice fed the SRBBB diet for 4 weeks showed lower amyloid β40 peptide in their blood than mice fed a commercial feed diet [38].
In this study, we sought to develop a novel food product that could reduce abrupt increases in postprandial blood glucose spikes after meals for the main purpose of the experimental study, and as a side effect, those of inhibit Aβ protein production after meals. We examined the effects of two blended rice blend diets and a traditional diet of standard polished rice on insulin sensitivity, concentration of LDL to HDL cholesterol ratio, and body fat percentage (BFP). Both blended diets were cooked after HPT pretreatment of rice grains.
One blended diet was comprised of super-hard rice blended with black unpolished rice (HPT KBH rice) and the other comprised of a β-glucan-enriched rice blend diet (HPT KWB rice). We used a 12-wk supplementation period with 1.6g RS/d, and with 2.9g β-glucan/d, compared with placebo, in 36 healthy subjects (slightly higher BGL) and assessed the results statistically.

Materials
We used four rice varieties and a mutant barley variety as food material. The ae-mutant rice cultivar "Chikushi-kona 85" was cultivated in 2017 in an experimental field at Fukuoka Agriculture and Forestry Research Center, Japan [9]. The high-dietary-fiber mutant rice cultivar "Goami2", developed in Korea, was provided to us by Dr. Hae Chune Choi in 2017. We purchased the high quality, premium, polished rice "Koshihikari" and the black rice

Preparation of Unpolished Rice Flour Samples and Polished Barley Grain Sample
We prepared unpolished rice flour with a cyclone mill (SFC-S1; Udy, Fort Collins, CO, USA) screened with 1 mm diameter pores.
Polished barley grain sample was prepared from barley seeds to be 70% yield ratio by barley pearling system 3RSB-10 (Hoden kogyo, Kyoto).

Moisture Content of Rice Flour
We measured the moisture content of the brown rice and cooked brown rice using the ovendrying method. Brown rice samples (2g) were dried for 1 h at 135°C, whereas cooked rice samples (2g) were dried for 3 h at 135 °C. Cooked rice samples were stored at −80 °C followed by lyophilizing the sample in a freeze dryer (FD-1; Tokyo Rikakikai Co., Ltd., Tokyo, Japan). Rice flour was prepared using a cyclone mill (SFC-SI; Udy Corp., Fort Collins, USA) screened with 1 mm diameter pores.

Measurement of Iodine Absorption Spectra
We measured the iodine absorption spectrum of milled rice with a Shimadzu UV-1800 spectrophotometer and estimated its apparent amylose content (AAC) using the iodine colorimetric method (as described by Juliano) [39]. The iodine absorption spectrum was analyzed [40] from 200 to 900 nm using a square cell with an inner dimension of 1 × 1cm. (The control was distilled water in the same-sized cell.) We measured absorbance at λmax

Glucose Content
To each flour sample, we added 1 mL of 60% ethanol to a tube with 0.1 g of brown rice flour and then rotated the tube at 20 °C for 1 h to extract glucose. The solution was then centrifuged at 1500 × g for 15 min, and the resulting supernatant was collected as a sample solution. We determined glucose content of each sample solution using the nicotinamide adenine dinucleotide phosphate enzyme assay method, measured with a glucose assay kit (Roche, Darmstadt, Germany).

Physical Properties of Boiled Rice Grains
We measured physical properties of cooked rice grains, based on 10 g of rice grains per sample, using a My Boy System Tensipresser (Taketomo Electric Co., Tokyo, Japan), according to the method described by Okadome et al. [41] The bulk measurements were taken six times, from which we calculated the mean for each sample.

Measurement of RS
RS of freeze-dried rice samples was measured according to the official method established by AOAC, using an RS assay kit (Mega-zyme Ltd., Wicklow, Ireland). Each sample (0.1g) was digested with pancreatin and amyloglucosidase at 37 °C for 6 h. Then, we measured glucose content using a spectrophotometer set at 510 nm.

Beta-Glucan Assay Method
We measured β-glucan in freeze-dried rice samples according to the official method of AOAC, using a β-glucan assay kit (Megazyme Ltd., Wicklow, Ireland). We measured β-glucan content by adding 0.2 mL of 50% ethanol to each rice flour sample (0.1 g) and then adding 5.0 mL of sodium phosphate buffer (20 mM, pH 6.5), stirring the tubes on a vortex mixer, and then incubating the tubes in a boiling water bath for approximately 2 min. Then, we removed the tubes and vigorously stirred them on a vortex mixer, heated the tubes at 50 °C for 5 min, and added 0.2 mL of lichenase (10 U) to each tube. We then capped the tubes, stirred, and incubated them at 50 °C for 1 h. We then added aliquot of 5.0 mL of sodium acetate buffer (200 mM, pH 4.0) and stirred the tubes on a vortex mixer.
The tube remained at room temperature for 5 min and then was centrifuged at 1000 × g for 10 min. The supernatant was removed, and 0.1 mL aliquots were transferred to three test tubes; to one we added 0.1 mL of sodium acetate buffer (50 mM, pH 4.0) (the reaction blank) and to the other two tubes (the reaction) we added 0.1 mL of β-glucosidase (0.2 U) in 50 mM acetate buffer (pH 4.0).
We then incubated the tubes at 50 °C for 10 min, added GOPOD reagent 3.0 mL to each tube, and incubated them at 50 °C for 20 min. We measured glucose content with a spectrophotometer set at 510 nm.

BACE1 Inhibitory Activity
We determined BACE1 inhibitory activity of freeze-dried rice samples using a BACE1 activity detection kit (Fluorescent; Sigma-Aldrich Co. LLC. Tokyo, Japan). To obtain the measurements, we

Measurement of Hydrophilic and Lipophilic Oxygen Radical Absorbance Capacity
We measured hydrophilic and lipophilic oxygen radical absorbance capacities (H-ORAC, L-ORAC) of rice samples as described by Prior et al. [42], Ito et al. [43], and Watanabe et al. [44,45]. For the hydrophilic antioxidant assay, we extracted freezedried rice samples (0.1g) with 10 mL of hexane at room temperature for 2 min and then centrifuged the extract at 3000 × g for 15 min.
The hexane layer was removed, and we repeated the process.
Residual hexane was evaporated using a water bath at 70 °C for 10 min, and then the residue was extracted with 10 mL of acetone/ water/acetic acid (70:29.5:0.5, v/v/v). Thereafter, the solution was sonicated (Ultrasonic cleaner 3510J-MTH: Branson Ultrasonics Co, Ltd. Kanagawa, Japan) at 37 °C for 15 min. The tube remained at room temperature for 15 min and then was centrifuged at 3000 × g for 15 min. The supernatant was removed and transferred to a volumetric flask and diluted to 25 mL total volume. For the lipophilic antioxidant assay, freeze-dried rice samples (0.1g) were extracted with 1 mL of hexane at room temperature for 2 min and then centrifuged at 3000 × g for 15 min. The hexane layer was collected, and the process was repeated. The hexane was evaporated using a water bath at 70 °C for 10 min.
We prepared Trolox calibration solutions (50, 25, 12.5, and 6.25 μmol/L in an assay buffer solution) to obtain a standard curve.
We used a 96-well-plate microplate (AS ONE Corporation Osaka, Japan) to read fluorescence of samples. We measured absorbance

Study Design
The study protocol for human test subjects was approved by

Statistical Analyses
All results were subjected to t-tests and Dunnett's test using Excel Statistics (ver. 6, Microsoft Corporation, Tokyo, Japan). A value of p< 0.1 was considered to show the tendency, and a value of p< 0.05 was considered to be statistically significant. Table 1 β-glucan can reduce serum concentrations of total and LDL cholesterol [46] but the mechanism behind this reduction is unclear. However, it is difficult to predict the cholesterol-lowering effect of any particular food product enriched with β-glucan. [46] The Food and Drug Administration (FDA) of USA has stated that daily intakes of 3 g or more of soluble fiber (β-glucan) in barley may reduce the risk of heart disease due to its ability to lower total and LDL cholesterol.  Test meal-1: "Koshihikari" unpolished rice, black unpolished rice "Okunomurasaki", and unpolished super-hard rice "Chikushikona 85", and polished super-hard rice "Goami 2" blended at a ratio of 2:4:2:2 and cooked after high-pressure-treatment (HPT).

Changes in Physical Findings and Blood Inspection Values of Three Groups of Test Subjects
Murao et al. [48]. showed that the LDL-to-HDL cholesterol  (Table 2). Table 2 summarizes the effects of the intervention on the measured anthropometric and serum biochemical parameters. During the trial, body weight and BMI did not show significant difference among the three groups. As a result, the HPT KBH and HPT KWB diets had no effect on total or LDL cholesterol after 12 weeks following the diet.

Physical Properties of Cooked Rice
Cooked grains of ae-mutant rice cultivars are hard and nonsticky because they lack the starchbranching enzyme IIb and contain more SLCs. Ae-mutant grains appear to provide promising health benefits, such as diabetes prevention [7,8]. The physical properties of cooked rice, measured in bulk using a Tensipresser, detected differences among rice varieties in hardness, toughness, degree of adhesion, and stickiness, all important indices when evaluating rice palatability [41], with toughness showing similar tendencies as hardness. "Chikushi-kona 85" rice was the hardest and toughest among all samples tested, but it was also the least sticky and least adhesive ( Therefore, HPT would be very beneficial to the rice industry due to the physical improvements HPT affords unpolished rice.
The hardness value of kernels was 4.8-fold higher in cooked HPT KBH rice than cooked rice of the control diet ("Koshihikari" polished rice). Toughness values were positively related to hardness, whereas adhesion was positively related to stickiness (Table 3b). However, the stickiness was 0.3-fold lower in cooked HPT KBH and HPT KWB rice than in cooked rice of the control diet.
The polished waxy barley contained starch that was 97%-100% amylopectin; [49], for that reason, the hardness and toughness for HPT KWB rice were similar to the control rice diet.   Means with the same letter are not significantly different (p<0.05). Control meal: "Koshihikari" polished cooked rice.

Measurement of Iodine Absorption Spectra
Starch properties measured by the iodine colorimetric method is shown in Table 4. AAC is an effective parameter for estimating the cooking or eating quality of rice grains. Because ae-mutants lack the starch-branching enzyme IIb, its amylopectin contains more longchain glucans than does ordinary indica and japonica rice grains. [50][51][52][53] AAC concentration values for ae-rice cultivars "Chikushikona 85" (28.5%) and "Goami2" (35.1%) were higher than those of typical japonica rice cultivar "Koshihikari" (12.1%) and black rice "Okunomurasaki" (12.1%). The high molecular weight amyloses in ae-mutant cultivars tend to have a higher value for λmax, Aλmax, New λmax, and Fb3 (the proportion of amylopectin with longer chains) (DP ≥37) [40], indicating that rice with high λmax/Aλmax ratios are palatable [40]. The λmax/Aλmax ratios for "Chikushikona 85" (1082.1) and "Goami2" (1049.9) were lower than ratios for "Koshihikari" (2189.3) and "Okunomurasaki" (2257.7). samples. It appears from these data that pretreating rice with HPP renders the starch component of rice grains more palatable. Table 4: Analyses of iodine absorption curve and glucose content of individual material rice by HPT and untreated rice.

Changes in Postprandial BGL, Insulin Levels, Area Under the Curve (AUC), And the Insulin Rise Curve (AUCi) In Humans
Panlasigui and Thompson [54] showed that unpolished rice is healthier and more beneficial than polished rice for diabetics and hyperglycemic individuals and that the glycemic area and GI were 35.4% lower in unpolished rice than in polished rice. Jung et al. [55] showed that ferulic acid might be beneficial in the treatment of type-2 diabetes because it regulates BGLs by elevating glucokinase activity and the production of glycogen in the liver. The BGL of test subjects 30 min after ingesting cooked HPT KBH (test meal-1) showed lower tendency after 12 weeks on the diets than that in subjects who consumed the control diet (p< 0.1), and that of cooked HPT KWB (test meal-2) was significantly lower than that of control diet (p< 0.01) ( Figure 1A), whereas AUC showed a similar tendency in response to diet after 30 and 60 min ( Figure 1B). After 12 weeks on treatment diets, insulin levels of test subjects that ingested cooked HPT KBH rice (test meal 1) were significantly lower at 120 min after the meal than insulin levels in subjects consuming the control diet (p < 0.05) (Figure 2A). Furthermore, after 12 weeks on their assigned diets, AUCi in test subjects at 90 and 120 min after ingesting the cooked HPT KWB (test meal 2) showed lower tendency than those in test subjects who ingested the control diet (p < 0.1, Figure 2B).     Table   7). In contrast, RS content was 0.8-fold lower in the cooked HPT KWB rice than in the cooked control rice diet. Means with the same letter are not significantly different (p<0.05).

RS Contents of Samples
Control meal: "Koshihikari" polished cooked rice.

Changes in BFP Values for Three Groups of Meals
Test subjects who consumed the HPT KWB rice blend diet had significantly lower BFP values than those who consumed the control diet (p < 0.05) after 12 weeks on the diet (Figure 3). Although HDL cholesterol level is a strong inverse predictor of cardiovascular events, it is not clear whether this association holds true when cholesterol is at a very low level [55,57]. Manninen et al. showed that the LDL-to-HDL cholesterol ratio was the best single predictor of cardiac events in a placebo group. This ratio, in combination with serum triglyceride level, indicates a high-risk for heart disease.

Measurement of Hydrophilic and Lipophilic Oxygen Radical Absorbance Capacity
Many dietary compounds have been proposed as being important antioxidants, and although there is credible evidence that vitamins E and C are important antioxidants, evidence is weaker for carotenoids and related plant pigments [62]. L-ORAC values of "Koshihikari" unpolished rice, "Okunomurasaki" unpolished rice, "Chikushi-kona 85" unpolished rice, and "Goami2" polished rice increased markedly after HPT (  (Table 7).

H-ORAC values trended in similar directions to L-ORAC values in
response to dietary treatment effects. Tsuda et al. [63] showed that anthocyanins extracted from purple corn prevented obesity and ameliorated hyperglycemia in mice. Moreover, Suzuki et al. [64].

Glucose Content and BACE1 Inhibitory Activity
Glucose concentrations of cooked rice samples were high in "Goami2" polished rice (0.284 g/L), "Haneumamochi" (0.230 g/L), and "Chikushi-kona 85" unpolished rice (0.180 g/L), whereas glucose content in "Koshihikari" unpolished rice (0.030 g/L) was very low (Table 4b). The glucose content remained almost identical between HPT (200 MPa 2 min) samples and untreated cooked rice (data not shown). However, glucose content was 2.3-fold higher (P < 0.05) in cooked HPT KWB rice (0.32 g/L) than in the cooked rice (0.14 g/L) of the control diet (Table 7). BACE1 is the first protease involved in the process of converting APP into Aβ in the brain. BACE1 inhibitory activity for the HPT KBH rice (19.0%), HPT KWB rice (5.0%), and control diet (0.0%), indicated that the BACE1 inhibitory activity of pigmented rice bran is much stronger than inhibitory activities in ordinary rice bran, as reported in our previous paper [38] (Table 7).

Conclusion
Because it has been reported that type-2 diabetes increases the risk of AD, we investigated whether Aβ generation could be a) The texture of cooked HPT KBH and HPT KWB rice diets became more palatable following treatment with HTP.
Test subjects who consumed these diets over the long term (12 weeks) had no medical emergencies; their safeties were not compromised, and they had no problem tolerating their assigned diets (determined from a questionnaire survey).  d) The BGL of test subjects 30 min after ingesting cooked HPT KBH (test meal-1) showed lower tendency after 12 weeks on the diets than that in subjects who consumed the control diet (p< 0.1), and that of cooked HPT KWB (test meal-2) was significantly lower than that of control diet (p< 0.01) .
e) After 12 weeks on their assigned diets, insulin levels in human test subjects at 120 min after ingesting cooked HPT KBH rice was significantly (P < 0.05) lower than insulin levels in subjects ingesting the control diet.
f) After 12 weeks on their assigned diets, AUCi in human test subjects was significantly lower (P < 0.05) at 90 and 120 min after ingesting the cooked HPT KWB diet than after ingesting the control diet.
g) The variation in Aβ42/40 ratio after 6 weeks was significantly higher (P < 0.04) for test subjects consuming the HPT KBH rice diet than that for subjects consuming the control diet.
The results of our long-term (12 week) human intervention study showed that ingesting 2.9 g/d of β-glucan in the HPT KWB rice weeks. We will continue this investigation on multiple-preventive rice products against diabetes and dementia by the combination of black rice and barley in the near future.