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.


Introduction
Rice (Oryza sativa L.) is one of the most important crops in the world. Global warming is the most serious environmental issue, and high-temperature stress in rice ripening periods causes a decrease in not only grain yield but also quality by generating chalky grains [1]. Nakata et al. [2] showed that the high temperatures accelerating the expression of starch-decomposing α-amylases during ripening is determinative for grain chalkiness. The elucidation of the mechanisms behind grain chalking under high-temperature stress in ripening is indispensable to developing a strategy for preventing the generation of chalky grains in the rice-cultivating region to produce tasty and high-quality rice despite climate warming [3]. In the 1990s, an evasion of high-temperature damage by changing the cultivating period or by using the agronomical method such as deep-water cultivation of rice was attempted. In the 2000s, the mechanisms of high-temperature damage and the location of the related genes to the damage were investigated using a quantitative trait locus (QTL) analysis and a proteome analysis. For example, the metabolism of starch and proteins, oxidative/reductive homeostasis, transcriptive control, mechanisms for prevention, and signal transduction were reported [4,5]. Mitsui et al. [6], Asaoka et al. [7], and Ahmed et al. [8] reported that high temperatures cause the down-regulation of genes for starch synthases and the up-regulation of α-amylases genes, which leads to decreases in the amylose content in endosperm starches of Japonica and Basmati rice cultivars. Nakata et al. [2] identified

Ratios of Whole (Head) and Chalky Rice Grains
We classified the unpolished rice grains into whole grains and chalky ones using an experimental grain inspector (Grain Quality Inspector RGQ120; SATAKE, Corp., Higashihiroshima, Japan) and separated the whole grains and chalky ones visually from 54 rice samples [13].

Preparation of Three Kinds of Brown Rice Flours
Three kinds of unpolished rice flours were from 54 Japonica rice samples, which were pre-fractionated original brown rice grains. Concretely, we divided each original rice sample to two groups manually, chalky grains and whole grains, based on the apparent chalkiness. We fractionated chalky grains visually in condition of more than 50% chalkiness based on national inspection standards of agricultural products, Japan. These rice grains were pulverized using a cyclone mill (SFC-S1; UDY, Corp., Fort Collins, CO, USA).

Measurement of the Moisture Content of Rice Flour
The moisture content of the unpolished rice flours was measured using an oven-drying method with slight modification by drying 2 g flour samples for 1 h at 135 • C [14].

Preparation of Starch Granules
Starch granules were purified from whole and chalky unpolished rice flours using cold alkaline solution according to the method reported by Yamamoto et al. [15].
Milled rice flour (2.0 g) was suspended in 30 mL of a 0.1% sodium hydroxide solution and vibrated by a water bath at 0 • C for 3 h at 100 min −1 (100 rpm). The suspension was then centrifuged for 5 min at 1500× g, the supernatant layer was removed, and the precipitate was suspended by adding 40 mL of distilled water. The solution was then vibrated by a water bath at 0 • C for 0.5 h at 100 vibrations min −1 . After centrifugation for 5 min at 1500× g and removal of the supernatant layer, the process was repeated three times. A check of pH neutrality was then conducted using pH test paper. After the precipitate was re-suspended in 30 mL of 60% ethanol solution, it was vibrated by a water bath at 0 • C for 0.5 h with reciprocating motions (100 turns/min). After centrifugation for 5 min at 1500× g, the supernatant layer was removed and the precipitate was suspended by adding 30 mL of an acetone solution and vibrated by a water bath at 0 • C for 0.5 h at 100 vibrations min −1 . After centrifugation for 5 min at 1500× g, the supernatant layer was removed and the precipitate was dried at room temperature.

Pasting Properties
The pasting properties of pre-fractionated original unpolished rice flours, fractionated whole unpolished rice flours, or the fractionated chalky unpolished rice flours from 54 Japonica rice samples were measured using a Rapid Visco Analyzer (RVA) (model Super 4; Newport Scientific Pty Ltd., Warriewood, Australia) [16,17].

Iodine Absorption Spectrum
The iodine absorption spectrum of alkali-treated whole and chalky starch flours was measured using a Shimadzu UV-1800 spectrophotometer. The AACs of alkali-treated rice starch were measured using the iodine colorimetric method of Juliano [18][19][20].

α-Amylase Activity
α-amylase activity of the whole and chalky unpolished rice flours was determined by the enzyme kit (Megazyme International Ireland, Ltd., Wicklow, Ireland).

Polishing and Boiling of Rice Samples
We prepared polished rice (milling yield of 90-91%) using an experimental frictiontype rice milling machine (Yamamotoseisakusyo Co., Yamagata, Japan). Then, 10 g of the polished rice grains of 100% whole rice grains (whole rice) and 30% chalky grains blended rice (blend rice) were boiled according to the method of our previous report [13].
, and Seitennohekireki (Aomori) rice were used as rice samples.

Measurements of Textural Properties of Boiled Rice Grains
Textural properties of the samples (10 g) were measured by the bulk measurement using a Tensipresser (My Boy System, Taketomo Electric Co., Tokyo, Japan) according to the method described by Odahara et al. [21]. The bulk measurements were repeated five times, and the mean value was calculated.

Measurement of D-Glucose, Maltose and Saccharose Contents
The cooked rice flour sample was prepared by pulverization after lyophilization. D-glucose of each sample (0.1 g) was extracted by shaking with 1 mL of 60% ethanol at room temperature for 1 h and measured by the enzyme assay method (F-kit, Roche/R-Biopharm AG., Darmstadt, Germany).

Measurement of L-Glutamic Acid
The cooked rice flour sample was prepared by pulverization after lyophilization. L-glutamic acid was extracted from each sample (0.1 g) by shaking with 1 mL of 60% ethanol at room temperature for 1 h and then measured by the enzyme assay method (F-kit, Roche/R-Biopharm AG., Darmstadt, Germany).

Measurement of L-Amino Acid
The cooked rice flour sample was prepared by pulverization after lyophilization. Each sample (0.1 g) was extracted by shaking with 1 mL of 10 mM PBS buffer (pH 7.0) at 4 • C for 1 h and then centrifuged for 15 min at 3000× g. After that, the supernatant of the extraction solution (50 µL) was measured by (50 µL) of reaction mix buffer (0.5 µL fluorometric probe, 0.1 µL horseradish peroxidase catalyzes, 1.7 µL L-Amino acid Oxidase, and 47.7 µL × 1 Assay B) at 37 • C for 2 h using L-Amino Assay kit (Fluorometric, CELL BIOLABS, INC., San Diego, CA, USA). The sample L-Amino Acid concentrations were determined by comparison with a known L-Alanine standard.

Analysis of Rice Protein Composition (SDS-PAGE)
The proteins were analyzed using SDS-PAGE described in the report using 12% polyacrylamide gel [13]. We used ATTO densitograph software library (CS Analyzer ver. 3.0, ATTO CORPORATION, Toyko, Japan) to calculate the intensities of various spots on the gel after SDS-PAGE.

Protease Activity
Protease activities of the whole and chalky unpolished rice flours were determined by the Amplite TM Universal fluorometric protease activity assay kit Green Fluorescence (AAT Bioquest, Inc., Sunnyvale, CA, USA). For activity measurements, proteases were extracted from Koshihikari brown rice flour (0.5 g) with 2 mL of extraction buffer (20 mM Tris-HCl, Foods 2022, 11, 3422 5 of 21 pH 6.8, 50 mM NaCl, 5 mM CaCl 2 ) at 5 • C for 16 h. After centrifugation for 15 min at 3000× g, the supernatant of the extraction solution was subjected to lyophilization. These freeze-dried samples (0.009 g) were dissolved in 180 µL of de-ionized water, and those solutions (50 µL) and substrates (casein labeled with a fluorescent dye) (50 µL) were mixed and incubated at 37 • C for 50 min in a 96-well solid black microplate, and protease activity was determined by the fluorometric method (Grating Based Multimode Reader SH-9000: Corona Electric Co, Ltd., Hitachinaka, Japan). The experiments were repeated three times.

Statistical Analyses
We used Excel Statics (ver. 2006; Microsoft Corp., Tokyo, Japan) for the statistical analysis of the significance of regression coefficients using Student's t-test, one-way analysis of variance, and Tukey's test. Additionally, the method of Tukey's multiple comparison was statistically analyzed using Excel NAG Statistics add in 2.0 (The Numerical Algorithms Group Ltd., Tokyo, Japan).

Ratio of Whole Rice Grains in 54 Japonica Rice Samples in 2020
According to the report of the Japan Meteorological Agency, there were many extremely hot days with a temperature of more than 35 • C in 2020. The ratios of the whole unpolished rice grains in low-amylose Japonica rice (12.1-49.9%; mean, 30.4%) were lower than those in 35 ordinary Japonica rice (5.6-83.2%; mean, 58.0%) and those in 15 premium rice, Koshihikari (44.3-65.0%; mean, 56.0%) (data not shown). Those of rice samples were damaged by a minimum temperature of higher than 25 • C from evening to next morning in August 2020.

Pasting Properties
Rapid Visco Analyzer (RVA) is commonly used for the evaluation of physicochemical properties of the starches as pasting characteristics [12,22]. Starch is essentially composed of amylose and amylopectin. Starch is composed of amylose and amylopectin. The former is small and linear molecule, whereas the latter is a large and highly branched one in the form of amylose-lipid complexes (ALCs) [23,24]. In our previous paper, we found that it is possible to estimate the fatty acid composition based upon the pasting properties measured by an RVA [25].
Pasting properties are useful quality indicators because they affect the eating quality of rice [9,12,22]. The final viscosity (Fin. vis) and consistency (Cons) are related to the degree of starch retrogradation during cooling [24]. In the previous study, we found a novel index of the ratios of setback/consistency (SB/Cons) and Max. vis/Fin. vis (Max/Fin), which positively or negatively correlated with the proportion of intermediate-and long-chains of amylopectin (Fb 1+2+3 (DP ≥ 13)) [17]. Table 1 and Supplemental Tables S1-S3 show that the Fin. vis and Cons of low-amylose Japonica rice were significantly lower than premium and ordinary Japonica rice.
Supplemental Tables S1-S3 shows that the Max. vis (maximum viscosity) of chalky rice grains of premium Japonica rice, ordinary Japonica rice, and low-amylose Japonica rice were significantly lower than those of whole rice grains. The pasting properties of ordinary Japonica rice showed a similar tendency as whole rice grains, whereas those of original rice grains of premium Japonica rice showed a little higher than that of whole rice grains, while those of low-amylose Japonica rice of original rice showed a little lower than that of whole rice grains. As shown in Figure 1, almost all chalky rice grains were significantly lower than those of whole rice grains at Fin. vis (p < 0.01 **) and Cons (p < 0.05 *). rice were significantly lower than those of whole rice grains. The pasting properties of ordinary Japonica rice showed a similar tendency as whole rice grains, whereas those of original rice grains of premium Japonica rice showed a little higher than that of whole rice grains, while those of low-amylose Japonica rice of original rice showed a little lower than that of whole rice grains. As shown in Figure 1, almost all chalky rice grains were significantly lower than those of whole rice grains at Fin. vis (p < 0.01 **) and Cons (p < 0.05 *). In the previous study, we developed a novel estimation formula for linoleic acid, oleic acid contents, and a ratio of omega-6 fatty acids to omega-3 fatty acids (n-6/n-3) based upon the pasting properties of Japonica brown rice cultivars [25,26]. Figure 2 shows that the whole rice grains contained less linoleic acid than the chalky rice grains significantly (p < 0.01), and Supplemental Figure S1 shows n-6/n-3 values of chalky rice grains were significantly higher than those of whole rice grains (p < 0.01). ANOVA showed significant difference (p < 0.01) by comparing all 54 chalky grains and whole grains.  In the previous study, we developed a novel estimation formula for linoleic acid, oleic acid contents, and a ratio of omega-6 fatty acids to omega-3 fatty acids (n-6/n-3) based upon the pasting properties of Japonica brown rice cultivars [25,26]. Figure 2 shows that the whole rice grains contained less linoleic acid than the chalky rice grains significantly (p < 0.01), and Supplemental Figure S1 shows n-6/n-3 values of chalky rice grains were significantly higher than those of whole rice grains (p < 0.01). ANOVA showed significant difference (p < 0.01) by comparing all 54 chalky grains and whole grains.
Simopoulos et al. [27] showed that a low n-6/n-3 ratio exert suppressive effects to pathogenesis of several diseases, whereas Western diets showed an excessive amount of ones. According to this report [27] and our results, it seems that the fatty acid composition in brown Japonica rice were deteriorated by high temperatures during ripening.
Supplemental Figure S1 shows that the high-temperature ripening affects not only eating/processing qualities but also the bio-functionality through the change in fatty acid compositions.
Taira et al. [28] reported that lipid content and fatty acid composition of rice were affected by the temperature during ripening. The dissociation temperature of ALCs increased with an increase in chain length of the fatty acids. Nevertheless, dissociation enthalpy was practically independent of chain length [29,30].
As a result, it seems that pasting properties and fatty acid of almost all Japonica rice samples were affected by high temperatures during ripening, and degree of damage shows varietal differences. It is estimated that low-amylose japonica rice is more susceptible to high-temperature damage than premium and ordinary japonica rice cultivars. Simopoulos et al. [27] showed that a low n-6/n-3 ratio exert suppressive effects to pathogenesis of several diseases, whereas Western diets showed an excessive amount of ones. According to this report [27] and our results, it seems that the fatty acid composition in brown Japonica rice were deteriorated by high temperatures during ripening.
Supplemental Figure S1 shows that the high-temperature ripening affects not only eating/processing qualities but also the bio-functionality through the change in fatty acid compositions.
Taira et al. [28] reported that lipid content and fatty acid composition of rice were affected by the temperature during ripening. The dissociation temperature of ALCs increased with an increase in chain length of the fatty acids. Nevertheless, dissociation enthalpy was practically independent of chain length [29,30].
As a result, it seems that pasting properties and fatty acid of almost all Japonica rice samples were affected by high temperatures during ripening, and degree of damage shows varietal differences. It is estimated that low-amylose japonica rice is more susceptible to high-temperature damage than premium and ordinary japonica rice cultivars.

Iodine Absorption Spectrum
It has been reported that most genes are markedly influenced by high temperature during the ripening of rice grains, either up-regulated or down-regulated [31].
It was reported that the high-temperature-ripened grains contained decreased levels of amylose and long chain-enriched amylopectin, which might arose from the repressed expression of granule bound starch synthase (GBSS) and branching enzymeⅡb (BEⅡb), respectively [32]. Low-amylose rice generally becomes soft and sticky after cooking, whereas high-amylose rice becomes hard with fluffy separated grains [33,34]. The starches in the rice grains grown under low temperature have higher amylose content and lower SLC (super-long chains) amylopectin content [35]. Inouch et al. [36] showed that the SLC content of starch can be estimated on the basis of λmax and the blue value of purified

Iodine Absorption Spectrum
It has been reported that most genes are markedly influenced by high temperature during the ripening of rice grains, either up-regulated or down-regulated [31].
It was reported that the high-temperature-ripened grains contained decreased levels of amylose and long chain-enriched amylopectin, which might arose from the repressed expression of granule bound starch synthase (GBSS) and branching enzymeIIb (BEIIb), respectively [32]. Low-amylose rice generally becomes soft and sticky after cooking, whereas high-amylose rice becomes hard with fluffy separated grains [33,34]. The starches in the rice grains grown under low temperature have higher amylose content and lower SLC (super-long chains) amylopectin content [35]. Inouch et al. [36] showed that the SLC content of starch can be estimated on the basis of λ max and the blue value of purified amylopectin. Furthermore, Igarashi et al. [37] reported a positive correlation between absorbance at λ max and apparent amylose content (AAC). Table 2 and Supplemental Tables S4 and S5 show that the absorbance values around 620 nm (AAC) of the chalky rice grains of low-amylose Japonica rice were lower than those of whole rice grains. As a result, whole rice grains showed higher AAC values than chalky grains significantly (p < 0.05). Moreover, λ max and Aλ max values of chalky rice grains in low-amylose rice were lower than those of whole rice grains similarly with AAC. It seems that starch synthase activities were lower and amylase activities were higher in the chalky grains than those in the whole grains in low-amylose rice group.
In the previous study, we developed the λ max /AAC ratios as novel index for the degree of damage by high temperature [13]. The λ max /A λmax ratios of the chalky rice grains of premium Japonica rice were significantly higher than those of whole rice grains.
It seems that AACs of low-amylose rice samples were affected and lowered by high temperature during ripening. The ratios of AACs of whole grains to those of chalky rice grains in the case of low-amylose Japonica rice (1.13 ± 0.0) were higher than those of premium Japonica rice (1.07 ± 0.1) and ordinary Japonica rice (1.05 ± 0.1). AAC, λ max , A λmax , and Fb 3 were measured, and the ratios of A λmax to AAC or λ max to A λmax were calculated. In Table 2, data of premium Japonica rice Koshihikari (n = 15), ordinary Japonica rice (n = 35), and low-amylose Japonica rice (n = 4) are shown in the same lanes. Additionally, difference between whole grains and chalky grains in the same column were compared. Different letters (a, b) mean that whole and chalky grains in each same rice samples are significantly different. Abbreviation: AAC, apparent amylose content; λmax, peak wavelength on iodine staining; Aλmax, absorbance at λmax; Fb 3 , proportion of long chains in amylopectin (DP ≥ 37)%; Values are shown as mean ± standard deviation.
It was reported that the high-temperature damage for low-amylose rice is accelerated by alleles located at dull 1~5, and those of five dull loci, which leads to lowering the amylose content [38][39][40]; moreover, these low-amylose rice cultivars have both genes of Wx, which causes stronger effects by high temperature during the ripening period than ordinary non-glutinous rice cultivars [32].

α-Amylase Activity
Nakata et al. [2] showed that the promoter activity of most α-amylase genes was elevated at high temperature. Mitsui [3] and Yamakawa et al. [41,42] reported that αamylase is a key factor in grain chalkiness using transgenic studies of ectopic overexpression and suppression of α-amylase. As shown in Figure 3, the α-amylase activities of chalky unpolished rice grains of premium Japonica rice Koshihikari, ordinary Japonica rice, and low-amylose Japonica rice were significantly higher than those of whole rice grains. As a result, whole unpolished rice grains were shown to have significantly lower α-amylase activities than chalky unpolished rice grains. The ratios of α-amylase activities of chalky unpolished rice grains to whole unpolished rice grains in low-amylose rice (1.61 ± 0.1) was higher than those of premium rice Koshihikari (1.41 ± 0.2) and ordinary Japonica rice cultivars (1.32 ± 0.3). Chalky unpolished rice grains showed markedly higher α-amylase activities (1.3-1.7 times) than whole unpolished rice grains in 54 Japonica rice in 2020 (data not shown).
Our results show that α-amylase activity of chalky unpolished rice grains are higher The ratios of α-amylase activities of chalky unpolished rice grains to whole unpolished rice grains in low-amylose rice (1.61 ± 0.1) was higher than those of premium rice Koshi-hikari (1.41 ± 0.2) and ordinary Japonica rice cultivars (1.32 ± 0.3). Chalky unpolished rice grains showed markedly higher α-amylase activities (1.3-1.7 times) than whole unpolished rice grains in 54 Japonica rice in 2020 (data not shown).
Our results show that α-amylase activity of chalky unpolished rice grains are higher than those of whole unpolished rice grains in accordance with the reports by other researchers [3,42,43]. Additionally, it was found that the tendency is stronger in the lowamylose rice group.

Correlation between Pasting Properties of Original Rice and Ratios of α-Amylase Activity of Chalky Grains to Those of Whole Rice Grains
The global warming rates of 1.5 • C and 2 • C may be exceeded during the 21st century [44].
In a previous study, we found an novel index for RS content, the ratios of Max/Mini and Max/Fin, which have stronger negative correlations than the conventional indexes reported using an RVA [16].
Hakata et al. [45] proposed that the suppression of α-amylase genes is a potential strategy for ameliorating grain damage from global warming. One of the reasons why α-amylase is very important may be that α-glucosidase is predominantly localized in the inner endosperm [46], whereas α-amylase is localized mainly in the outer layers.
The α-amylase activities of chalky unpolished rice grains were much higher than those of whole unpolished rice grains in 54 Japonica rice in 2020. Particularly, low-amylose rice and premium rice Koshihikari in Niigata showed very high values.
The whole and chalky grains have the same genes, and those of ratios of α-amylase activites of chalky unpolished grains to those of whole ones were shown as an index of degree of damage by high-temperature ripening [6] because the chalky unpolished rice grains of almost all Japonica rice were significantly higher than those of whole unpolished grains. Table 3 shows that the ratios of α-amylase activities of chalky unpolished grains to those of whole ones of 50 japonica rice except low-amylose rice showed negative correlations with Mini.vis (r = −0.51; p < 0.01), Fin. vis (r = −0.60; p < 0.01), and Cons (r = −0.45; p < 0.01) and a positive correlation with Max/Mini (r = 0.35; p < 0.05) of pasting properties of (before dividing to two groups) original 50 Japonica rice.
To summarize, the ratios of α-amylase activites of chalky unpolished grains to those of whole ones of almost all Japonica rice except low-amylose rice showed a high correlation with pasting properties of original Japonica rice. In our previous paper, we reported that the pasting properties, measured by the program at 120 • C using an RVA, were useful to estimate the retrogradation degree of hardness of the boiled rice grains [47]. In the present paper, we developed a novel estimation formula for the degree of high-temperature damage based on only the pasting properties by an RVA. Figure 4A shows the formula for estimating the ratios of α-amylase activities of chalky unpolished grains to those of whole ones of 24 original Japonica brown rice based on the pasting properties of original Japonica brown rice using an RVA. In our previous paper, we reported that the pasting properties, measured by the program at 120 °C using an RVA, were useful to estimate the retrogradation degree of hardness of the boiled rice grains [47]. In the present paper, we developed a novel estimation formula for the degree of high-temperature damage based on only the pasting properties by an RVA. Figure 4A shows the formula for estimating the ratios of α-amylase activities of chalky unpolished grains to those of whole ones of 24 original Japonica brown rice based on the pasting properties of original Japonica brown rice using an RVA.
where C, chalky grains; W, whole grains; and ratios of α-amylase activities (C/W), ratios of α-amylase activities of chalky grains to those of whole ones. Figure 4B shows that a correlation coefficient (r) of 0.68 was obtained with the application of the abovementioned formula for the validation test using 24 unknown samples.
Thus, the validation test showed that the equation can be applied to unknown samples. In the whole and chalky unpolished rice grains with the same genotype, the chalky unpolished rice grains of almost all Japonica rice have significantly higher αamylase activities than the whole unpolished grains on high-temperature ripening. Therefore, α-amylase activities could be a good index for the degree of high-temperature damage. As a result, it became possible for us to estimate the degree of damage by hightemperature ripening, using only the pasting properties of original (mixture of whole and chalky grain) Japonica unpolished rice except low-amylose samples because the enhancement of α-amylase activities had been reported to be a good index for hightemperature damage [6,13].  The equation had a correlation coefficient (r) of 0.74 in the calibration. The following formula for estimating the ratios of α-amylase activities of chalky grains to those of wholes of 24 original brown Japonica rice.
Ratios of α-amylase activities (C/W) = −0.01 × Fin. vis + 0.33 × Max/Mini + 2.69. (2) where C, chalky grains; W, whole grains; and ratios of α-amylase activities (C/W), ratios of α-amylase activities of chalky grains to those of whole ones. Figure 4B shows that a correlation coefficient (r) of 0.68 was obtained with the application of the abovementioned formula for the validation test using 24 unknown samples.
Thus, the validation test showed that the equation can be applied to unknown samples. In the whole and chalky unpolished rice grains with the same genotype, the chalky unpolished rice grains of almost all Japonica rice have significantly higher α-amylase activities than the whole unpolished grains on high-temperature ripening. Therefore, α-amylase activities could be a good index for the degree of high-temperature damage. As a result, it became possible for us to estimate the degree of damage by high-temperature ripening, using only the pasting properties of original (mixture of whole and chalky grain) Japonica unpolished rice except low-amylose samples because the enhancement of α-amylase activities had been reported to be a good index for high-temperature damage [6,13].

Textural Properties of Boiled Rice Grains
In our previous report, boiled rice grains from the chalky grains showed lower hardness and higher retrogradation degree after boiling compared with the whole grains [13,19].
In the recent commercial market, rice grains containing about 30% of chalky rice are graded as low class and have prices lower than whole rice grains. Markedly, damaged rice samples in our 54 rice samples contained about 30% of chalky rice.
In this study, we measured the physical properties of the boiled rice of whole rice (100% whole grains) and blend rice (30% chalky grains blended ones) of 19 Japonica rice by the bulk measurement (10 g) with a Tensipresser. Although we used single grain method in our previous papers, we adopted "bulk method" in order to clarify the effect of blending the chalky rice grains. The value of Hardness is indicated by the height and that of Toughness is area for continuous progressive compression in Tensipresser [48].
As shown in Supplemental Tables S6 and S7, the stickiness of blended boiled rice (0.0242-0.0364 × 10 5 N/cm 2 ; mean, 0.0282) were a little lower than those of whole boiled rice (0.0239-0.0341 × 10 5 N/cm 2 ; mean, 0.0300 × 10 5 N/cm 2 ), and those of adhesion showed a similar tendency. The stickiness and adhesion of blended boiled rice were lower (0.94~0.98 times) than those of whole boiled rice grains.
We ascertained that blended rice grains showed a little lower hardness and stickiness than whole rice grains after boiling, which means the physical properties of blended boiled rice are inferior to whole rice grains in terms of eating quality. This means that the practical or commercial rice grains (about 30% chalky rice blend) in the market may be inferior to the un-damaged rice grains in terms of textural quality.
As shown in Supplemental Table S8, the stickiness of blended boiled rice showed a positive correlation with α-amylase activity of chalky grains (r = 0.81, p < 0.01). Moreover, the hardness of blended boiled rice showed a positive correlation with the total oligo saccharides (r = 0.50, p < 0.05), saccharose (r = 0.58, p < 0.05), and maltose (r = 0.56, p < 0.05) of the blended rice. It means that the acceleration of amylase affects rice quality markedly in the case of high-temperature ripening. As shown in Supplemental Tables S6 and S7, the stickiness of blended boiled rice (0.0242-0.0364 × 10 5 N/cm 2 ; mean, 0.0282) were a little lower than those of whole boiled rice (0.0239-0.0341 × 10 5 N/cm 2 ; mean, 0.0300 × 10 5 N/cm 2 ), and those of adhesion showed a similar tendency. The stickiness and adhesion of blended boiled rice were lower (0.94⁓0.98 times) than those of whole boiled rice grains.
We ascertained that blended rice grains showed a little lower hardness and stickiness than whole rice grains after boiling, which means the physical properties of blended boiled rice are inferior to whole rice grains in terms of eating quality. This means that the practical or commercial rice grains (about 30% chalky rice blend) in the market may be inferior to the un-damaged rice grains in terms of textural quality.
As shown in Supplemental Table S8, the stickiness of blended boiled rice showed a positive correlation with α-amylase activity of chalky grains (r = 0.81, p < 0.01). Moreover, the hardness of blended boiled rice showed a positive correlation with the total oligo saccharides (r = 0.50, p < 0.05), saccharose (r = 0.58, p < 0.05), and maltose (r = 0.56, p < 0.05) of the blended rice. It means that the acceleration of amylase affects rice quality markedly in the case of high-temperature ripening.

D-Glucose, Maltose, and Saccharose Contents in Boiled Rice Grains
Awazuhara et al. [49] showed that the thermal dependency and stability of enzymes producing reducing sugar were different between outer endosperm and inner endosperm of rice. The amounts of reducing sugars were involved by multiple amylase actions, and those ones showed largest increases at 40-60 °C in during boiling [50].
In this study, we measured the sugar contents of the boiled rice of whole grain rice and blended grain rice of 19 Japonica rice samples by UV absorption measurement using the enzymatic method.

D-Glucose, Maltose, and Saccharose Contents in Boiled Rice Grains
Awazuhara et al. [49] showed that the thermal dependency and stability of enzymes producing reducing sugar were different between outer endosperm and inner endosperm of rice. The amounts of reducing sugars were involved by multiple amylase actions, and those ones showed largest increases at 40-60 • C in during boiling [50].
In this study, we measured the sugar contents of the boiled rice of whole grain rice and blended grain rice of 19 Japonica rice samples by UV absorption measurement using the enzymatic method.
As shown in Supplemental Table S8, the saccharose contents of whole grains boiled rice showed a positive correlation with the hardness of whole grains ones (r = 0.57, p < 0.05).
The total oligo saccharides of blended boiled rice (0.662-0.848%; mean = 0.725%) were significantly higher than those of whole boiled rice (0.621-0.755%; mean = 0.678%), at p < 0.01 as shown in Figure 6. As a result, the sugar contents of the boiled rice of blended rice were 1.1 times higher than those of whole grains rice. The reason blended boiled rice contains more total oligo saccharides than whole grains could be due to the higher activities of multiple amylases and lower activities of starch synthesizing enzymes [13]. It was confirmed that the sweetness component of boiled rice was increased by blended of 30% chalky grains.
As shown in Supplemental Table S8, the total oligo saccharides of whole grains boiled rice showed a positive correlation with the hardness (r = 0.56, p < 0.05) and toughness (r = 0.52, p < 0.05) of whole grains boiled ones. were 1.1 times higher than those of whole grains rice. The reason blended boiled rice contains more total oligo saccharides than whole grains could be due to the higher activities of multiple amylases and lower activities of starch synthesizing enzymes [13]. It was confirmed that the sweetness component of boiled rice was increased by blended of 30% chalky grains.
As shown in Supplemental Table S8, the total oligo saccharides of whole grains boiled rice showed a positive correlation with the hardness (r = 0.56, p < 0.05) and toughness (r = 0.52, p < 0.05) of whole grains boiled ones. Figure 6. Total oligo saccharides of 100 % whole rice boiled grains and 30 % chalky rice blended grains in 19 kinds of Japonica rice in 2020. Different letters (a, b) mean that whole and 30% chalky grains in each same rice samples are significantly different. ** Correlation is significant at 1% by the method of Tukey's multiple comparison.

Difference in L-Glutamic Acid
The amino group metastasizes to α-ketoglutaric acid; after that, α-keto acid is produced. Finally, those of all amino groups were collected to glutamic acid. Moreover, the glutamic acid is one of the umami (delicious taste) components.
Generally, the protease activities of germinated cereal seeds are activated. Abe et al. [51] has found an endo-type proteolytic enzyme of the cysteine proteinase class from germinating rice seeds. Doi et al. [52,53] showed that germinating rice contained three carboxypeptidases or carboxypeptidase-like enzymes. Moreover, Tashiro et al. [54] showed that the seeds of corn, foxtail millet, barnyard millet, wheat, barley, and bran of rice have proteinase inhibitor activities.
It was presumed that the protease activity of chalky grains is higher than whole grains. It was confirmed that the Umami component of boiled rice was increased by blending 30% chalky grains.  Figure 6. Total oligo saccharides of 100% whole rice boiled grains and 30% chalky rice blended grains in 19 kinds of Japonica rice in 2020. Different letters (a, b) mean that whole and 30% chalky grains in each same rice samples are significantly different. ** Correlation is significant at 1% by the method of Tukey's multiple comparison.

Difference in L-Glutamic Acid
The amino group metastasizes to α-ketoglutaric acid; after that, α-keto acid is produced. Finally, those of all amino groups were collected to glutamic acid. Moreover, the glutamic acid is one of the umami (delicious taste) components.
Generally, the protease activities of germinated cereal seeds are activated. Abe et al. [51] has found an endo-type proteolytic enzyme of the cysteine proteinase class from germinating rice seeds. Doi et al. [52,53] showed that germinating rice contained three carboxypeptidases or carboxypeptidase-like enzymes. Moreover, Tashiro et al. [54] showed that the seeds of corn, foxtail millet, barnyard millet, wheat, barley, and bran of rice have proteinase inhibitor activities.

Difference in L-Amino Acids
Tamura et al. [55] showed that the amino acid contents of aspartic acid and glutamic acid are more abundant in the outer than in the inner layers, and those amino acids increased in the cooking water during soaking and increased in the rice grains in the temperature range of 80-100 °C during cooking [50]. Matsuzaki et al. [56] reported the It was presumed that the protease activity of chalky grains is higher than whole grains. It was confirmed that the Umami component of boiled rice was increased by blending 30% chalky grains.

Difference in L-Amino Acids
Tamura et al. [55] showed that the amino acid contents of aspartic acid and glutamic acid are more abundant in the outer than in the inner layers, and those amino acids increased in the cooking water during soaking and increased in the rice grains in the temperature range of 80-100 • C during cooking [50]. Matsuzaki et al. [56] reported the correlation of the glutamic acid and aspartic acid contents with the eating quality of boiled rice, and those of a low level of free amino acid showed a similar tendency in the Japonica and Indica rice cultivars.
As shown in Supplemental Table S8, the L-amino acids in the boiled rice of whole grains showed a positive correlation with the total oligo saccharides (r = 0.62, p < 0.01) of whole grains boiled ones.
In the present research, we found that not only starch-related enzymes and sugars but also amino acid contents change markedly in the case of chalky rice grains.

Difference in L-Amino Acids
Tamura et al. [55] showed that the amino acid contents of aspartic acid and glutamic acid are more abundant in the outer than in the inner layers, and those amino acids increased in the cooking water during soaking and increased in the rice grains in the temperature range of 80-100 °C during cooking [50]. Matsuzaki et al. [56] reported the correlation of the glutamic acid and aspartic acid contents with the eating quality of boiled rice, and those of a low level of free amino acid showed a similar tendency in the Japonica and Indica rice cultivars.
As shown in Figure 8, the L-amino acid content in the boiled rice of the blended rice (797.2-1455.1 RFU (530/590 nm); mean = 984.9 RFU (530/590 nm)) was significantly higher than that of whole boiled rice (707.7-1121.1 RFU (530/590 nm); mean = 881.4 RFU (530/590 nm)), at p < 0.05.  Figure 8. L-amino acid of 100% whole rice boiled grains and 30% chalky blended grains in 19 kinds of Japonica rice in 2020. Different letters (a, b) mean that whole and 30% chalky grains in each same rice samples are significantly different. * Correlation is significant at 5% by the Tukey's multiple comparison method.

SDS-PAGE of Rice Proteins
The weather conditions influence the protein content in rice grains [57]. It was reported that prolamin contents showed a positive correlation with the hardness of boiled rice grains [2,3,57]. We reported that the 13 kDa prolamin ratios of chalky rice grains were lower than those of whole rice grains [13], and Yamakawa et al. [58] reported a similar tendency.
As shown in Figure 9, the ratios of chalky grains to whole grains in terms of the intensities of the total residual protein bands were 2.00 ± 0.08 after 16 h, 1.65 ± 0.02 after 6 h, and 1.21 ± 0.07 after 1 h for soaking in a buffer solution. As a result, it seems that the protease activity of chalky grains is higher than whole ones. Although many researchers reported that α-amylase activity increases markedly under the high-temperature ripening of rice, there are few reports on the increase in protease activities in chalky rice grains generated under high-temperature ripening. As L-amino acids increase and residual proteins after soaking decrease in the chalky rice grains, we think that high-temperature ripening affects not only starch-related enzymes but also protein-related enzymes in rice grains.
reported that prolamin contents showed a positive correlation with the hardness of boiled rice grains [2,3,57]. We reported that the 13 kDa prolamin ratios of chalky rice grains were lower than those of whole rice grains [13], and Yamakawa et al. [58] reported a similar tendency.
As shown in Figure 9, the ratios of chalky grains to whole grains in terms of the intensities of the total residual protein bands were 2.00 ± 0.08 after 16 h, 1.65 ± 0.02 after 6 h, and 1.21 ± 0.07 after 1 h for soaking in a buffer solution. As a result, it seems that the protease activity of chalky grains is higher than whole ones. Although many researchers reported that α-amylase activity increases markedly under the high-temperature ripening of rice, there are few reports on the increase in protease activities in chalky rice grains generated under high-temperature ripening. As L-amino acids increase and residual proteins after soaking decrease in the chalky rice grains, we think that high-temperature ripening affects not only starch-related enzymes but also protein-related enzymes in rice grains. Figure 9. SDS-PAGE analysis of residual proteins extracted from hydrolyzed whole and chalky brown rice flour. 1, hydrolyzed chalky brown rice (Koshihikari) at 37 °C for 16 h; 2, hydrolyzed whole brown rice (Koshihikari) at 37 °C for 16 h; 3, hydrolyzed chalky brown rice (Koshihikari) at 37 °C for 6 h; 4, hydrolyzed whole brown rice (Koshihikari) at 37 °C for 6 h; 5, hydrolyzed chalky brown rice (Koshihikari) at 37 °C for 1 h; and 6, hydrolyzed whole brown rice (Koshihikari) at 37 °C for 1 h. Chalky brown rice grains are expressed in circled numbers; a, gluterin precursor; b, glutelin α-subunit; c, α-globulin; d, glutelin β-subunit; e-g, prolamin.

Protease Activity
From the above results in Section 3.11, we measured the protease activities of the whole and chalky unpolished rice by the universal fluorimetricp assay kit. As shown in Figure 10, the protease activities of chalky unpolished grains of rice flour (mean = 1193.0 RFU) were significantly higher (p < 0.01) than those of whole unpolished grains (mean =

Protease Activity
From the above results in Section 3.11, we measured the protease activities of the whole and chalky unpolished rice by the universal fluorimetricp assay kit. As shown in Figure 10, the protease activities of chalky unpolished grains of rice flour (mean = 1193.0 RFU) were significantly higher (p < 0.01) than those of whole unpolished grains (mean = 1075.8 RFU). It was presumed that the neutral protease activities of chalky unpolished grains is higher than whole ones because we used buffer on neutral pH, although both of whole and chalky unpolished grains did not show protease activities in the acidic buffer (pH = 3.0). 1075.8 RFU). It was presumed that the neutral protease activities of chalky unpolished grains is higher than whole ones because we used buffer on neutral pH, although both of whole and chalky unpolished grains did not show protease activities in the acidic buffer (pH = 3.0). Figure 10. Proteinase activities of unpolished rice flours prepared from whole or chalky unpolished rice grains premium rice Koshihikari. Different letters (a, b) mean that whole and chalky grains in each same rice samples are significantly different. ** Correlation is significant at 1% by the method of Tukey's multiple comparison.
As we described in Section 3.11, there are few reports on the protease activation under the high-temperature ripening. As we ascertained the increase in protease activities in the chalky unpolished grains, it seems very interesting that not only starch-related enzymes but also protein-related enzymes are activated under the high-temperature ripening of rice grains.
It was reported that protease activities in cereals are enhanced during the germination period similarly to α-amylase activity [59]. Our results reveal that not only  Figure 10. Proteinase activities of unpolished rice flours prepared from whole or chalky unpolished rice grains premium rice Koshihikari. Different letters (a, b) mean that whole and chalky grains in each same rice samples are significantly different. ** Correlation is significant at 1% by the method of Tukey's multiple comparison.
As we described in Section 3.11, there are few reports on the protease activation under the high-temperature ripening. As we ascertained the increase in protease activities in the chalky unpolished grains, it seems very interesting that not only starch-related enzymes but also protein-related enzymes are activated under the high-temperature ripening of rice grains.
It was reported that protease activities in cereals are enhanced during the germination period similarly to α-amylase activity [59]. Our results reveal that not only α-amylase but also protease are activated by high-temperature ripening. It was reported that the gene expression of gibberellin is closely related with the activation of α-amylase and protease activities [60,61]. Our results are in accordance with these reports at the points of activations of these hydrolytic enzymes by high temperature during ripening.

Conclusions
Global warming impairs grain filling in rice and leads to chalky-appearing grains, which were damaged in their physicochemical and cooking qualities. In the present paper, we evaluated 54 Japonica brown rice grains harvested in Japan in 2020 when it was extraordinary hot during the summer all over Japan from meteorological observation of Japan Meteorological Agency, and these samples (original grains) were divided, manually based on the apparent chalkiness, into two groups (whole grains and chalky grains). The chalky rice grains showed lower values of Max.vis., Mini.vis., BD, Fin. vis, and Cons of pasting properties than the whole rice grains, and their AAC showed a similar tendency, while those of α-amylase activities, protease activities, linoleic acid, oligo saccharide, amino acids, and n-6/n-3 ratio of polyunsaturated fatty acid showed higher than those of whole rice grains. Additionally, we developed a novel estimation formula for the damage degree of rice grains ripened under high temperatures using an RVA.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/foods11213422/s1, Figure S1: Comparison between ratio of omega-6/omega-3 (n-6/n-3) of chalky and whole rice grains of 54 Japonica brown rice samples in 2020. Table S1: Pasting properties of whole grains in 54 Japonica brown rice in 2020. Table S2: Pasting properties of chalky grains in 54 Japonica brown rice in 2020. Table S3: Pasting properties of original brown rice samples in 54 Japonica brown rice in 2020. Table S4: Analysis of iodine absorption parameters of whole rice grains in 54 Japonica rice in 2020. Table S5: Analysis of iodine absorption parameters of chalky rice grains in 54 Japonica rice in 2020. Table S6: Physical properties of boiled rice of 100% whole rice grains in 19 kinds of Japonica rice in 2020. Table S7: Physical properties of boiled rice of 30% chalky rice blended rice grains in 19 kinds of Japonica rice in 2020. Table S8: Correlation between whole and chalky rice grains with the results of physical parameters of boiled rice, iodine analysis, pasting properties, α-amylase activities, sugar contents, L-glutamic acid, and L-amino acid of 19 kinds of Japonica rice in 2020. Table S9: Sugar contents of boiled rice with 100% whole rice grains and 30% chalky rice blended grains in 19 kinds of Japonica rice in 2020.

Data Availability Statement:
The datasets generated for this study are available on request to the corresponding author.

Conflicts of Interest:
Although Mr. Junji Katsura and Mr. Yasuhlro Maruyama belong to a company, NSP Ltd., they participated this research work as scientists, for example, designing of this research, performing of the experiments as described in the paper. The authors declare no conflict of interest.