Morphological, Molecular Structural and Physicochemical Characterization of Starch Granules Formed in Endosperm of Rice with Ectopic Overexpression of α-Amylase.

The objective of this study was to characterize the endosperm starch in rice that ectopically overexpressed the α-amylase. Transgenic rice plants, transformed with cauliflower mosaic virus 35S promoter driven AmyI-1 (35S::AmyI-1) and AmyII-4 (35S::AmyII-4), and 10 kDa prolamin promoter driven AmyI-1 (P10::AmyI-1), were cultivated under standard conditions (23 °C, 12 h in the dark/ 26 °C, 12 h in the light), and brown grains were subsequently harvested. Each grain displayed characteristic chalkiness, while electron microanalyzer (EPMA)-SEM images disclosed numerous small pits on the surface of the starch granules, attributable to α-amylase activity. Fluorescence labeling and capillary electrophoresis analysis of starch chain length distribution revealed no significant alterations in the starches of 35S::AmyI-1 and 35S::AmyII-4 transgenic rice compared to the wild-type. Conversely, the extremely short α-glucan chains (DP 2-8) exhibited a dramatic increase in the P10::AmyI-1 starch. Rapid visco-analyzer analysis also identified variations in the chain length distribution of P10::AmyI-1 starch, manifesting as changes in viscosity. Moreover, 1H-NMR analysis uncovered dynamic modifications in the molecular structure of starch in rice grain transformed with P10::AmyI-1, which was found to possess unprecedented structural characteristics.

Effects of Hard Water Boiling on Chalky Rice in Terms of Texture Improvement and Ca Fortification.

In the present paper, we investigated the characteristics of chalky rice grains generated by ripening under high temperature and compared them with whole grains. We evaluated 14 unpolished Japonica rice grains harvested in Japan in 2021, and these samples (original grains) were divided into two groups (a whole grain group and a chalky grain one). We found that not only activities of endogenous amylase and proteinase, but also cell wall-degrading enzymes, such as xylanase and cellulase, changed markedly between chalky grains and whole grains. Using rice grains blended with 30% of chalky grains as the material, we compared the sugar and mineral contents and textural properties of the rice grains soaked and boiled in either ordinary water or hard water, such as Evian or Contrex. It was shown that xylanase, in addition to amylase and proteinase, may play an important role in changing the texture of the boiled chalky rice grains. For the sake of preventing the above-mentioned deterioration in the texture of boiled grains of chalky rice, we tried to use hard water, such as Evian or Contrex, to soak and cook the chalky rice grains. It was shown that the hard water was useful for the prevention of texture deterioration of the boiled rice grains due to inhibition of the activities of endogenous hydrolytic enzymes, such as α-amylase, ß-amylase, proteinase, and xylanase. Furthermore, we found that the hard water was useful in increasing the calcium absorption through the meal by 2.6 to 16.5 times.

Palatability and Bio-Functionality of Chalky Grains Generated by High-Temperature Ripening and Development of Formulae for Estimating the Degree of Damage Using a Rapid Visco Analyzer of Japonica Unpolished Rice.

Global warming inhibits grain filling in rice and leads to chalky grains, which are damaged in physical and cooking qualities. In the present paper, we evaluated 54 Japonica unpolished rice grains harvested in Japan in 2020, and these samples (original grains) were divided to two groups (whole grains and chalky grains). Using rice grains of 100% whole grains or those blended with 30% of chalky grains, we measured contents of sugars and amino acids, and textural properties of boiled rice grains. It was shown that the α-amylase activity and proteinase activity of raw chalky rice were significantly higher than those of whole rice grains, which led to the significant increase of low-molecular-weight sugars and free amino acids after boiling. Furthermore, hardness and toughness of the boiled rice grains were decreased markedly by blending chalky grains. The ratio of α-amylase activity of chalky grains to that of whole grains was shown to be a useful indicator for damage degree by high-temperature ripening. It became possible to estimate the degree of high-temperature damage of rice grains based on only the pasting properties of unpolished rice.

Effect of heat-moisture treated brown rice crackers on postprandial flow-mediated dilation in adults with mild endothelial dysfunction.

Background: Endothelial dysfunction is an early pathophysiological feature and independent predictor of a poor prognosis in most forms of cardiovascular disease. We evaluated the effect of brown rice crackers (BR-C) on endothelial function. Methods: Effect of heat-moisture treated (HMT) -BR-C on postprandial flow-mediated dilation (FMD) in adults with mild endothelial dysfunction was compared with that of BR-C and white rice crackers (WR-C) in 12 adults with mild endothelial dysfunction (less than 7.0% of FMD) by a randomized, single-blind, three-treatment three-period crossover trial (UMIN 000034898). Since we considered that the FMD increase was associated with the treatment of HMT-BR-C, we examined the effect of three possible factors: postprandial glucose levels, polyphenol content, and polyphenol release from the food matrix. Results: Mean pre-intake baseline FMD values of HMT-BR-C, BR-C, and WR-C were 4.9%, 5.1%, and 4.9%, respectively, and those values 1 h post-intake were 6.3%, 5.1%, and 4.8%, respectively. There was no difference in intergroup comparisons of FMD using Dunnett's multiple comparison test. There was a significant increase in FMD only in HMT-BR-C in intragroup comparisons (P = 0.042 by paired-t test). In comparison with BR-C, no significant difference was noted in the postprandial glucose level nor in the content of total polyphenols and ferulic acid derivatives in HMT-BR-C. However, the 70% ethanol extracted from HMT-BR-C contained a significantly larger amount of free and bound ferulic acids than from BR-C. Conclusion: HMT-BR-C intake increased the postprandial FMD response.

Black rice bran intake reduces phosphorylated tau levels and enhances insulin signaling in the brain of aged normal mice.

This study reports that black rice bran (BRB) intake for 50-52 consecutive weeks (∼12 months) reduces tau phosphorylation with a concomitant activation of insulin signaling and subsequent PI3K/AKT pathway in the brain of aged normal mice. BRB holds promise for preventing the formation of neurofibrillary tangles consisting of hyperphosphorylated tau, a pathological hallmark of Alzheimer's disease.

Possibility for Prevention of Type 2 Diabetes Mellitus and Dementia Using Three Kinds of Brown Rice Blends after High-Pressure Treatment.

As it has been reported that type 2 diabetes mellitus increases the risk of Alzheimer's disease, we investigated how to prevent type 2 diabetes and dementia using biofunctional boiled rice. We adopted unpolished super-hard rice (SHBR) for diabetes and wax-free unpolished black rice (WFBBR) for dementia and blended those with ordinary non-polished rice (KBR) (blending ratio 4:4:2), adding 2.5% waxy black rice bran (WBB) and 0.3% rice oil after high-pressure treatment (HPT) (WFBSK) to improve its palatability. This boiled rice is rich in dietary fiber, anthocyanin, free ferulic acid and ß-secretase inhibitory activity. A randomized, parallel-group comparison study was conducted for 12 weeks with 24 subjects, using Cognitrax to evaluate their cognitive function primarily. Furthermore, as the secondary purpose, we performed a single-dose test for postprandial blood glucose and insulin secretion at the end of the human intervention test. After 12 weeks, consumers of the WFBSK rice exhibited significant improvement in language memory by cognitive test battery compared with those who consumed the control white rice (p < 0.05). Moreover, subjects who consumed the WFBSK rice had lower insulin secretion levels than those who consumed the control polished rice (p < 0.05).

Evaluation of Hardness and Retrogradation of Cooked Rice Based on Its Pasting Properties Using a Novel RVA Testing.

With rice being one of the most important crops worldwide, rapid and objective quality evaluation methods based on physicochemical measurements of rice are necessary. We compared the pasting properties of various rice samples using three different heating and cooling programs (maximum temperatures were 93, 120, and 140 °C, respectively) in a newly developed high-temperature-type Rapid Visco Analyzer (RVA , RVA 4800). Furthermore, we investigated the relationship between the different pasting properties measured by the three programs, with starch microstructure measured by iodine scanning analysis, the physical properties of the cooked rice measured by a Tensipresser after 2 h at 25 °C or after 24 h at 6 °C, and prolamin ratio measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The consistency value (final viscosity-minimum viscosity) yielded by a new program of maintenance for 2 min at 120 °C using RVA 4800 had a higher positive correlation with retrograded surface hardness H1(R) (r = 0.92), retrograded overall hardness H2(R) (r = 0.90), and the absorbance at λmax (Aλmax) of cooked rice (r = 0.88) and resistant starch (r = 0.80) than those by the conventional program at 93 °C. We developed estimation formulae for H1(R) for various kinds of rice, of which the determination coefficient was 0.86. It led to an easy and rapid assay method for the cooking properties of the various rice samples.

Characteristics of Physicochemical Properties of Chalky Grains of Japonica Rice Generated by High Temperature during Ripening.

Global warming has caused devastating damage to starch biosynthesis, which has led to the increase in chalky grains of rice. This study was conducted to characterize the qualities of chalky rice grains and to develop the estimation formulae for their quality damage degree. We evaluated the chalkiness of 40 Japonica rice samples harvested in 2019, in Japan. Seven samples with a high ratio of chalky rice grains were selected and divided into two groups (whole grain and chalky grain). As a results of the various physicochemical measurements, it was shown that the surface layer hardness (H1) of cooked rice grains from chalky grains was significantly lower, and their overall hardness was significantly lower than those from the whole grains. In addition, α- and ß-amylase activities, and sugar contents of the chalky rice grains were significantly higher than those of the whole rice grains. The developed estimation formula for the degree of retrogradation of H1 based on the α-amylase activities and pasting properties, showed correlation coefficients of 0.84 and 0.81 in the calibration and validation tests, respectively. This result presents the formula that could be used to estimate and to characterize the cooking properties of the rice samples ripened under high temperature.

Development of a novel formula for estimating the amylose content of starch using japonica milled rice flours based on the iodine absorption curve.

The apparent amylose content (AAC) is usually measured by an iodine colorimetric method using the starch as a sample. Nevertheless, it is time-consuming and labor-intensive to prepare rice starch. Therefore, we compared two methods, starch method and milled rice flour method. The former AACs were higher than the latter and correlated with pasting properties, physical properties and sensory scores better than the latter. Therefore, we developed a novel formula for estimating the AACs of starches from japonica rice cultivars using milled rice flours instead of using rice starch. The correlation coefficients of the new formula were 0.89 for calibration and 0.94 for validation, which showed that this equation can be applied to unknown japonica rice samples, and will lead to easy, rapid, and accurate iodine method to determine rice AACs using milled rice flours instead of starch. Abbreviations: AAC: apparent amylose content; RS: resistant starch; SLC: super-long chain; CD: chain length distribution; RVA: Rapid Visco Analyzer; SB: setback; BD: breakdown; Max.vis: maximum viscosity; Min.vis: minimum viscosity; Pt: pasting temperature; Cons: consistency; Final.vis: Final viscosity; SB/Cons: setback/consistency; Max/Min: maximum viscosity/minimum viscosity; Max/Fin: maximum viscosity/final viscosity.

Physicochemical measurements of Japonica rice cultivars in Heilongjiang Province.

We here characterized 27 japonica rice cultivars grown in Heilongjiang province and evaluated the relationship among their iodine absorption curve, physical properties, and ratio of 13 kDa prolamin. We developed the novel estimation formulae for ratio of 13 kDa prolamin and overall hardness (H2) with the use of Aλmax and λmax.

Effects of different heat-moisture treatments on the physicochemical properties of brown rice flour.

We evaluated the effect of heat-moisture treatment (HMT) on the main chemical components, physical properties, and enzyme activities of two types of brown rice flour: high-amylose Koshinokaori and normal-quality Koshiibuki. Five different HMTs using brown rice (moisture content was 12.0%) were assessed: 0.1 MPa/120 °C for 5 or 10 min, 0.2 MPa/134 °C for 5 or 10 min and 0.3 MPa/144 °C for 10 min. HMT, decreased the α-amylase and lipase activities, and fat acidity, and slightly increased the dietary fiber and resistant starch levels. After 2 months' storage at 35 °C, rice samples that were treated with 0.2 MPa/134 °C or 0.3 MPa/144 °C for 10 min had a lower fat acidity than untreated samples, which would be useful for long-term storage and export of rice flour. And HMT exhibited inhibition of retrogradation in the pasting and physical properties, which is profitable to promote the qualities of the rice products.

Effects of super-hard rice bread blended with black rice bran on amyloid ß peptide production and abrupt increase in postprandial blood glucose levels in mice.

Alzheimer's disease and type 2 diabetes are very serious diseases with the latter having been suggested to cause the former. We prepared super-hard rice bread blended with black rice bran (SRBBB), which contained a high amount of resistant starch that showed strong inhibitory activities against ß-secretase and acetylcholinesterase even after heating. Black rice bran showed greater ß-secretase inhibitory activity (3.6-fold) than Koshihikari rice. The bran contained more oleic acid and anthocyanin, meaning that it is potentially a biofunctional food with a high antioxidant capacity. Furthermore, aged mice, which were fed a SRBBB diet for four weeks, showed lower amyloid ß 40 peptide in the blood than mice fed a commercial diet (p < 0.01). Additionally, their initial blood glucose levels (BGLs) after 12 weeks of being fed SRBBB were significantly lower than those in the control group. Taken together, our results indicate SRBBB shows promise for inhibiting not only amyloid ß production, but also abrupt increases in postprandial BGLs.

Comparison of eating quality and physicochemical properties between Japanese and Chinese rice cultivars.

In this study, we evaluated 16 Japanese and Chinese rice cultivars in terms of their main chemical components, iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch content, physical properties, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and enzyme activity. Based on these quality evaluations, we concluded that Chinese rice varieties are characterized by a high protein and the grain texture after cooking has high hardness and low stickiness. In a previous study, we developed a novel formula for estimating AAC based on the iodine absorption curve. The validation test showed a determination coefficient of 0.996 for estimating AAC of Chinese rice cultivars as unknown samples. In the present study, we developed a novel formulae for estimating the balance degree of the surface layer of cooked rice (A3/A1: a ratio of workload of stickiness and hardness) based on the iodine absorption curve obtained using milled rice.

Development of formulae for estimating amylose content and resistant starch content based on the pasting properties measured by RVA of Japonica polished rice and starch.

We searched for the easy and simple method to measure the novel indicators which reflect not only AAC, but also (RS) based on pasting properties using RVA. Novel indexes such as SB/Con and Max/Fin (Maximum viscosity/Minimum viscosity) ratios had a very high correlation with proportion of intermediate and long chains of amylopectin; Fb1+2+3 (DP â‰§ 13). In Japonica polished rice, estimation formulae for AAC and RS content were developed using novel indexes based on pasting properties by RVA, and these equations showed determination coefficients of 0.89 and 0.80 for calibration and 0.71 and 0.75 for validation test. We developed the estimation formulae for AAC and RS content for Japonica starch samples. These equations showed determination coefficients of 0.86 and 1.00 for calibration and 0.76 and 0.83 for validation test, which showed that these equations can be applied to the unknown rice samples.

Measurement of resistant starch content in cooked rice and analysis of gelatinization and retrogradation characteristics.

Digestion-resistant starch (RS) has many physiologic functions. The RS content is measured by enzymatically degrading flour samples according to the method of the Association of Official Analytical Chemists. Experiments have been performed with wheat, corn, and other grains, but there are no data for cooked rice grains in the form ingested by humans. Thus, we investigated a method to measure RS that is suitable for cooked rice grains using rice cultivars that are reported to differentially increase postprandial blood glucose in humans. Using a method for cooking individual rice grains and optimized enzyme reaction conditions, we established an RS measurement method. We also found that the amylopectin crystal condition affects the RS content measured using our method.

Development of formulae for estimating amylose content, amylopectin chain length distribution, and resistant starch content based on the iodine absorption curve of rice starch.

Not only amylose but also amylopectin greatly affects the gelatinization properties of rice starch and the quality of cooked rice grains. We here characterized the starches of 32 rice cultivars and evaluated the relationship between their iodine absorption curve, apparent amylose content (AAC), pasting property, resistant starch (RS) content, and chain length distribution of amylopectin. We found that the iodine absorption curve differed among the various sample rice cultivars. Using the wavelength at which absorbance becomes maximum on iodine staining of starch (λmax), we propose a novel index, "new λmax" (AAC/(λmax of sample rice starches-λmax of glutinous rice starch)). We developed the novel estimation formulae for AAC, RS contents, and amylopectin fractions with the use of λmax and "new λmax." These formulae would lead to the improved method for estimating starch properties using an easy and rapid iodine colorimetric method.

Improved palatability and bio-functionality of super-hard rice by soaking in a barley-koji miso suspension.

Cooked grains of ae rice cultivars are too hard and non-sticky due to the presence of long-chain amylopectin, and ae rice cultivars are therefore called ``super-hard rice'' and cannot be used as table rice. However, they are promising in terms of their bio-functionality such as preventing diabetes. Miso (soybean paste) is a yeast-fermented food, made from steamed soybeans, salt, and inoculated cereals known as koji, made from rice, barley, or soybeans.We investigated the effects of soaking ae mutant rice cultivars in a miso suspension. Their chemical components, physical properties, and enzyme activities were measured under different conditions (milled rice before or after soaking in a 5% barley-koji miso suspension). Rice grains cooked after soaking in the miso suspension were less hard and more sticky than those cooked after soaking in water. Rice grains cooked after soaking in a 5% barley-koji miso suspension maintained high amounts of resistant starch and dietary fiber, and were fortified with polyphenols and isoflavones. Palatable and bio-functional ae rice could therefore be produced by cooking after soaking in a 5% barley-koji miso suspension.

A novel method for the preparation of template DNA for PCR from beer to detect materials and to develop DNA markers to evaluate the quality of beer.

We developed a method for the preparation of template DNAs for polymerase chain reaction (PCR) from beer. We improved the method by (i) lyophilizing and pulverizing the beer to concentrate DNAs, (ii) decomposition of polysaccharides and proteins so as not to inhibit DNA extraction by the use of heat-resistant amylase and proteinase K, (iii) separation of template DNA by purification using 70% EtOH extraction and isopropyl alcohol precipitation, and (iv) the use of magnetic beads to purify DNA. We developed suitable 7 STS (sequence-tagged site) primers related to beer quality for PCR, and it proved possible to identify 16 dominant malting barley cultivars and 22 kinds of beers. To digitize the results of PCR, discriminative DNA bands were binarized as 0 (disappeared) or 1 (appeared) and subjected to multiple regression analysis. Estimation formulae for the quality of beer were developed using the above-mentioned independent variables based on the results of PCR against dependent variables related to the qualities of beer, including foam stability, bitterness, sourness and astringency. These equations showed multiple regression coefficients of 0.93, 0.82, 0.87, and 0.87 for calibration.

Prediction and structural equation model of sertraline treatment response in Japanese patients with major depressive disorder.

OBJECTIVE: The aim of this study was to extract the factors possibly associated with sertraline treatment response and elucidate their interactions and extent of influence. METHODS: Demographic state, stress state, personality, and eight genetic polymorphisms at baseline and clinical symptoms at baseline and 8 weeks were analyzed and examined by logistic regression and a structural equation model in sertraline treatment study of 96 Japanese patients with major depressive disorder. RESULTS: Non-responders were associated with higher scores of harm avoidance in Temperament and Character Inventory, higher scores (≥24) of 17-item Hamilton Rating Scale for Depression at baseline, recurrence, and 12/12 genotype of the serotonin transporter variable number of tandem repeat polymorphism in intron 2 (5HTTSTin2). When we calculated the response index using four factors extracted, the mean response index value of non-responders was significantly higher than that of responders. The symptoms at baseline, personality, recurrence, and polymorphism of 5HTTSTin2 showed significantly direct and positive influences on the symptoms at 8 weeks in our final structural equation model with a good model fit. CONCLUSION: Considering the combination of four factors extracted may be useful for predicting a worse response to sertraline treatment and selecting different treatment other than sertraline.

Search for cell-wall-degrading enzymes of world-wide rice grains by PCR and their effects on the palatability of rice.

Such rice cultivars as Japonica, Japonica-Indica hybrid, Javanica and Indica, were evaluated for their main chemical components (amylose content and protein content), pasting property of rice flour (consistency), physical property of the cooked rice grains (adhesion, L3), and enzyme activities (cellulase and xylanase). The amylose content, cellulase activity and xylanase activity showed significant positive or negative correlation with the pasting property (consistency) of rice flour (r = 0.89, r = 0.58, r = 0.70, respectively) and with the physical property of the cooked rice grains (adhesion, L3: r = -0.51, r = -0.61, r = -0.71, respectively) at the level of 1%. Endogenous xylanase and cellulase played important roles to determine the texture of the cooked rice grains similarly to the amylose content. Part of the DNA sequences of the α-glucosidase gene differed among the Japonica, Japonica-Indica hybrid and Indica subspecies. We found discriminative DNA bands appearing by PCR, corresponding to 1,4-ß-xylanase and endo-1,4-ß-glucanase 13 in the case of Indica rice, Indica-Japonica hybrid rice, and Javanica rice (non-Japonica subspecies). The equation for estimating the physical property (adhesion) of cooked rice grains by PCR was improved by adding novel primers related to the cell-wall-degrading enzymes.