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.


Introduction
Rice is one of the most important crops in the world, together with wheat and corn. Rice is used as table rice and for various food processing applications, such as sake (rice wine), rice snacks, and rice cake. The many rice cultivars grown in the world vary greatly in their cooking, sensory, and processing quality [1]. Rice-based foods have also been diversified, and convenient products, such as frozen cooked rice, extruded pregerminated brown rice, retort-pouch cooked rice, and aseptic cooked rice, have become increasingly available [2,3]. It, therefore, seems important and meaningful to develop a novel and simple method to evaluate the quality of rice products. As described by Juliano, rice quality evaluation includes eating quality assays for sensory evaluation and aroma testing as well as physical property measurements using, e.g., Instron, Texturometer, or Tensipresser, which are highlighted as indirect methods, in addition to amylose content, starch gelatinization temperature, gel consistency, amylography, and protein content determination [4]. Bhattacharya et al. classified the world's 177 rice germplasms into 8 types (type I to type VIII) based on amylose, gel consistency, and Brabender relative breakdown [1]. Ohtsubo et al. reported on the quality assay of rice using traditional and novel tools, such as the measurements of amylose, physical properties, and pasting properties [5]. Bergman et al. reviewed rice end-use quality analysis, such as Apparent amylose content (AAC) and RVA analysis [1]. As starch properties, physical properties, and pasting properties of rice are flours. Furthermore, we developed novel estimation formulae for oleic and linoleic acid contents based on the pasting properties of brown rice flours using an RVA, making it possible to predict easily and rapidly the nutritive and biofunctional characteristics of material rice [29].
To evaluate pasting properties, the behavior of rice starch at temperatures higher than 100 • C should be examined to elucidate the changes in rice quality after cooking and processing, such as extrusion cooking [30] or superheated moisture treatment [31]. In the present paper, we compare the pasting properties of various rice samples using three different types of heating and cooling programs on a newly developed high-temperaturetype RVA (RVA 4800). We tried to differentiate the gelatinization and retrogradation properties of various rice starches more clearly by measuring the pasting properties at higher temperatures than the ordinary temperature used for a conventional RVA, 93 • C. Furthermore, we investigated the relationship between pasting properties and starch molecular structure, physical properties of cooked rice grains, and the prolamin ratio. Making use of RVA analysis, we were challenged to estimate the physical properties and retrogradation degree of the rice samples.
Ae mutant rice (Goami 2 and Dodam) were cultivated by Gyeonggi-do Agricultural Research and Extension Services in 2019 in Korea. Niigata 129gou was cultivated at the Niigata Prefectural Agricultural Research Institute in 2019.

Preparation of Polished White Rice Samples
Brown rice was polished using an experimental friction-type rice milling machine (Yamamotoseisakusyo, Co. Ltd., Tendoh, Japan) to obtain a milling yield (yield after polishing) of 90-91%. White rice flour was prepared using a cyclone mill (SFC-S1; Udy, Fort Collins, CO, USA) with a screen of 1 mm diameter pores.

Preparation of Starch Granules
Starch granules were prepared from polished rice flour using the cold alkaline method [32].

Iodine Absorption Spectrum
The iodine absorption spectrum of alkali-treated rice starch and cooked rice flour was measured using a UV-1800 spectrophotometer (Shimadzu Co. Ltd., Kyoto, Japan). The cooked rice samples were stored in a freezer at −80 • C. Subsequently, each sample was lyophilized using a freeze dryer (FD-1, Eyela, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The AACs of alkali-treated rice starch and cooked rice flours were estimated using Juliano's iodine colorimetric method [7]. The iodine absorption spectrum was analyzed from 200 to 900 nm using a square cell with a 1 × 1 cm inner dimension [14]. The absorbance was measured at 620 nm (following Juliano's method); we also measured peak wavelength on iodine staining (λ max ), the molecular size of amylose and super-long chains (SLCs) of amylopectin, and absorbance at λ max (Aλ max ).

Pasting Properties
The pasting properties of milled rice flours from 17 rice cultivars were measured using an RVA (model Super 4; Newport Scientific Pty Ltd., Warriewood, Australia). Each sample (3.5 g based on 14% moisture content) was suspended in 25 mL of water. The measurement conditions were as follows: 1 min of heating at 50 • C, 4 min of heating from 50 to 93 • C, maintenance for 7 min at 93 • C, 4 min of cooling from 93 to 50 • C, and 3 min at 50 • C. The programmed heating and cooling cycle followed that of Toyoshima et al. [33].
The second program conditions were as follows: 1 min of heating at 50 • C, 6.5 min of heating from 50 to 120 • C, maintenance for 2 min at 120 • C, 6.5 min of cooling from 120 to 50 • C, and 3 min at 50 • C.
The third program conditions were as follows: 1 min of heating at 50 • C, 8.3 min of heating from 50 to 140 • C, maintenance for 3.3 min at 140 • C, 8.3 min of cooling from 140 to 50 • C, and 3 min at 50 • C.

Measurement of RS
The RS of cooked rice flour was measured according to the Association of Official Analytical Collaboration International (AOAC) method (2002.02), with a slight modification, using an RS assay kit (Megazyme, Ltd., Wicklow, Ireland). Each sample (100 mg; dry matter) was digested with pancreatin and amyloglucosidase at 37 • C for 6 h, and the glucose content was measured using a spectrophotometer at 510 nm.

Physical Properties of Cooked Rice Grains
For standard samples, milled rice (10 g) was added with 14 g of distilled water (1.4 times, w/w; standard moisture content, 13.5%; coefficient (gross water volume/dry matter weight): 1.77, calculated for each sample) in an aluminum cup. After soaking for 1 h, the samples were cooked in an electric rice cooker (National SR-SW182). The cooked rice samples were kept in the vessel at 25 • C for 2 h and then used to obtain measurements. The hardness and stickiness of the cooked rice grains were measured using a Tensipresser (My Boy System, Taketomo Electric Co., Tokyo, Japan) with the individual grain method in low compression (25%) and high compression (90%) tests [18]. The average of each parameter was calculated by measuring 20 individual grains.
As a staling test for cooked rice, the cooked samples were stored at 6 • C for 24 h and measured again with a Tensipresser according to the previously described method in low-compression (25%) and high-compression (90%) tests [21].
We defined retrograded hardness as the hardness of rice after storage at 6 • C for 24 h and the retrogradation degree as the hardness of rice after storage at 6 • C for 24 h divided by the hardness of rice after storage at 25 • C for 2 h.
We considered the hardness of the surface layer (=H1) and hardness of the overall layer (=H2), the stickiness of the surface layer (=S1), the stickiness of the overall layer (=S2), and the adhesion of the surface layer (=L3). Balance H1 (=S1/H1) means the ratio of stickiness to hardness of the surface layer, Balance H2 (=S2/H2) means the ratio of stickiness to hardness of the overall layer, Balance A1 (=A3/A1) means the ratio of adhesive work to hardness work of the surface layer, and Balance A2 (=A6/A4) means the ratio of adhesive work to hardness work of the overall layer.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Protein was extracted from milled rice flour samples (0.5 g) by shaking with 2 mL of buffer A (50 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol) at 37 • C for 30 min and then centrifuging for 5 min at 3000× g. The supernatant (1 mL) was diluted with an equal volume of sample buffer (0.125 M Tris-HCl pH6.8, 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% bromophenol blue) and mixed well, followed by heating for 2 min at 100 • C. In total, 10 µg of extracted protein was loaded into each lane. SDS-PAGE was conducted with a 12% polyacrylamide gel, according to our previous report [21]. The values were calculated based on the intensities of various spots on the gel after SDS-PAGE analysis using the densitograph software library (CS Analyzer ver 3.0, ATTO Co., Tokyo, Japan).

Statistical Analyses
All results, including the significance of regression coefficients, were statistically analyzed using Student's t-test, one-way analysis of variance, and Tukey's test using Excel Statistics (ver. 2006; Microsoft Corp., Tokyo, Japan). Statistical analysis using an exponential function for RS was performed by XLSTAT 2021 for Microsoft Excel 2016.

Iodine Absorption Spectrum
Amylose is one component of rice starch that greatly affects the quality and gelatinization properties of cooked rice [1,[8][9][10][34][35][36]. The amylose content of rice is controlled mainly by the wx gene (encoding granule-bound starch synthase I, GBSS I) [37][38][39]. Another starch component, amylopectin, is synthesized through the concerted reactions of starch synthase, starch branching enzyme, and debranching enzyme [40,41]. Umemoto et al. [42] showed that varietal differences in amylopectin chain-length distribution are regulated by functional variations in starch synthase (SSIIa). AAC comprises a large amount of amylose and a small amount of super long chain (SLC) amylopectin. Previous reports have described the molecular structures of several starches, including amylose and amylopectin branch chain lengths [43]. Starches in rice cultivars grown under low temperatures have significantly higher amylose content and lower SLC amylopectin content than cultivars grown under high temperatures [44,45]. Taira et al. [46] reported that the lipid content and fatty acid composition of rice are affected by daily mean temperature during ripening.
Igarashi et al. [47] reported a positive correlation between absorbance at λ max and AAC. Inouchi et al. [48] and Jideani et al. [49] showed that the SLC content of starch can be estimated based on λ max and the blue value of purified amylopectin.
Among the waxy rice, those suitable for producing soft cake, such as Hakuchomochi, have more short chains and fewer long chains than those used for hard cake, such as Koganemochi. Varietal differences are found in the amylopectin structure [50]. The hardening process in the manufacture of rice cakes is the most important process because it is The Aλ max values of ae mutant rice cultivars tend to be higher than those of other cultivars because the others contain SLC. It reveals that Aλ max reflects not only the properties of amylose but also the effect of the amylopectin chain length; we selected Aλ max as an indicator for the microstructure of starch [14].
Among the waxy rice, those suitable for producing soft cake, such as Hakuchomochi, have more short chains and fewer long chains than those used for hard cake, such as Koganemochi. Varietal differences are found in the amylopectin structure [50]. The hardening process in the manufacture of rice cakes is the most important process because it is time-consuming. For this reason, Koganemochi is considered the best waxy rice cultivar in Japan [51]. In the present study, we investigate the characterization of new glutinous rice cultivars and search for a novel way to maintain the hardness of starch even after cooking. In the present paper, we analyze the iodine absorption curve of both starch and cooked rice. Table 1 shows that the absorbance values at 620 nm (representing AAC) of cooked ae mutant rice (0.466-0.551; mean, 0.498) were higher than those of other rice cultivars.
The λ max of cooked japonica-indica hybrid rice was higher than those of other rice cultivars.
The Aλ max values of the cooked ae mutant were higher than those of other rice cultivars. After cooking, the Aλ max values tended to be lower when compared to starch, as shown in Figure 1.
The Fb 3 (DP ≥ 37) ratios of cooked ae mutant rice were higher than those of other rice cultivars. Several studies have characterized the process of rice cake hardening [52][53][54]. The difference in amylopectin chain-length distribution causes differences in the rice cake hardening property [55]. The molar ratio of short chains to long chains of the amylopectin unit seemed to be one of the useful evaluation/selection indexes for the breeding of waxy rice [51].
In a comparison of iodine colorimetric parameters of starch and cooked rice, those of cooked rice tend to be less than those of starch [56]. This seems to be due to the decomposition of starch molecules during cooking. The degree of decomposition (lowering the ratio of the parameters of cooked rice to those of starch) is higher for glutinous and low-amylose rice than high-amylose rice and ae mutant rice.
In the present paper, we clarify the microstructures of rice starches using our novel and easy iodine method, the results of which will be useful to estimate processing suitability and the degree of retrogradation of various kinds of rice samples.

Pasting Properties
Pasting properties are a useful quality indicator because they affect the eating quality of rice [1,4,5]. The final viscosities (Fin. vis) of high-amylose rice cultivars have been shown to be higher than those of low-amylose cultivars, and Fin. vis is related to the degree of starch retrogradation during cooling [1,5]. A highly positive relationship was observed between SLC content and the consistency (Cons, Fin. vis-Mini. vis) of viscosity; therefore, SLCs in amylopectin appear to have a great effect on the Cons of starch [1]. When the temperature rises above the gelatinization temperature (GT) of the sample, starch granules begin to swell, and the viscosity increases. For waxy rice products, low-GT samples are preferred for desserts and rice cakes [57]. Takeda and Hizukuri [58] showed that amylose gelatinizes at higher temperatures than amylopectin. The resistance of amylose against gelatinization is considered an important factor in the gelatinization behavior of starch.
Here, we evaluate the relationship between the pasting properties by comparing three different programs: Program 1, maintenance for 7 min at 93 • C; Program 2, maintenance for 2 min at 120 • C; and Program 3, maintenance for 3.3 min at 140 • C using an RVA for 17 rice samples ( Figure 2 and Table 2). We used the same total time for the two programs (Program 1 and Program 2) because rapidity is indispensable for the RVA so as not to take more time even though a higher maximum temperature is set except for Program 3 (maintenance temperature is 140 °C).
As shown in Figure 2, the pasting properties of rice markedly change depending on the heating and cooling programs. Additionally, different groups of rice cultivars show various behavior for the different programs. The soft-type rice group, such as glutinous rice and japonica rice, showed higher maximum viscosities than final viscosities; on the contrary, the hard-type rice group, such as indica rice and ae mutant rice, showed higher final viscosities than maximum viscosities. Almost all rice groups showed the highest viscosities in Program 1 and the lowest viscosities in Program 3; intermediate viscosities were shown in Program 2. Nevertheless, only the ae mutant rice group showed the highest final viscosities in Program 2. Table 2 shows that for Program 1, the Max. vis of indica low-amylose rice (485.2 RVU) was higher than those of japonica (379.0-411.9 RVU; mean, 396.    We used the same total time for the two programs (Program 1 and Program 2) because rapidity is indispensable for the RVA so as not to take more time even though a higher maximum temperature is set except for Program 3 (maintenance temperature is 140 • C).
As shown in Figure 2, the pasting properties of rice markedly change depending on the heating and cooling programs. Additionally, different groups of rice cultivars show various behavior for the different programs. The soft-type rice group, such as glutinous rice and japonica rice, showed higher maximum viscosities than final viscosities; on the contrary, the hard-type rice group, such as indica rice and ae mutant rice, showed higher final viscosities than maximum viscosities. Almost all rice groups showed the highest viscosities in Program 1 and the lowest viscosities in Program 3; intermediate viscosities were shown in Program 2. Nevertheless, only the ae mutant rice group showed the highest final viscosities in Program 2. Table 2 shows that for Program 1, the Max. vis of indica low-amylose rice (485.2 RVU) was higher than those of japonica . vis tended to decrease as the maintenance temperature rose, as shown in Figure 2.
As shown in Figure 3, the second program (maintenance temperature 120 • C) is most suitable for differentiating the pasting properties of the various kinds of rice cultivars, from waxy to ae mutant rice. In the case of the conventional program (Program 1), the differentiation of final viscosity, the index for the degree of retrogradation, was not enough. Indica rice and risotto rice showed almost the same values, as did japonica, aromatic, and ae rice. Similarly, the final viscosities of aromatic, japonica, japonica-indica, and ae mutant rice showed almost the same values. We estimate that 93 • C is not high enough for hard-type rice to be completely gelatinized and 140 • C is a little too high, which leads to the inhibition of differentiation among the various rice starches.
from waxy to ae mutant rice. In the case of the conventional program (Program 1), the differentiation of final viscosity, the index for the degree of retrogradation, was not enough. Indica rice and risotto rice showed almost the same values, as did japonica, aromatic, and ae rice. Similarly, the final viscosities of aromatic, japonica, japonica-indica, and ae mutant rice showed almost the same values. We estimate that 93 °C is not high enough for hard-type rice to be completely gelatinized and 140 °C is a little too high, which leads to the inhibition of differentiation among the various rice starches. The Fin. vis obtained with Program 1 for indica rice (411.8 RVU) was higher than those of other rice cultivars. Fin. vis in Programs 2 and 3 tended to decrease as the maintenance temperature rose in the samples, excluding ae mutant rice. The group of high-amylose starches includes two types of rice starches with similar apparent amylose content (AAC) but different super-long chain (SLC) content of amylopectin from ae mutant rice [59,60]. Fin. vis becomes higher by retrogradation of amylose. We estimated that the hydrogen bonds in the SLC of ae mutant rice were partially uncoupled at 120 °C, and the coupling was furthered by Program 3, which resulted in the lowest value. We estimated that the hydrogen bonds of the SLC of ae mutant rice were partially dissociated at 120 °C and almost completely dissociated at 140 °C.
The Cons result from Program 2 for ae mutant rice and glutinous rice was higher than those from Programs 1 and 3. Among the 17 rice samples, ae mutant rice showed the highest values and glutinous rice showed the lowest values, which agreed well with the phenomena that the cooked rice from ae rice retrogrades most and those from glutinous rice retrogrades least.
Among the glutinous rice samples, the Cons value and the Fin. vis. value of Koganemochi in Program 2 were markedly higher than those of Hakuchomochi, which agreed well with the retrogradation degree of waxy rice cake, as reported by Igarashi et al. [51].
The Cons values from the program at 120 °C for glutinous rice (21.6-52.3 RVU; mean, 32.5 RVU) were higher than those from the programs at 140 °C (21.3-52.5 RVU; mean, 30.7 RVU) and 93 °C (16.0-47.7 RVU; mean, 27.0 RVU). Glutinous rice showed the lowest value The Fin. vis obtained with Program 1 for indica rice (411.8 RVU) was higher than those of other rice cultivars. Fin. vis in Programs 2 and 3 tended to decrease as the maintenance temperature rose in the samples, excluding ae mutant rice. The group of high-amylose starches includes two types of rice starches with similar apparent amylose content (AAC) but different super-long chain (SLC) content of amylopectin from ae mutant rice [59,60]. Fin. vis becomes higher by retrogradation of amylose. We estimated that the hydrogen bonds in the SLC of ae mutant rice were partially uncoupled at 120 • C, and the coupling was furthered by Program 3, which resulted in the lowest value. We estimated that the hydrogen bonds of the SLC of ae mutant rice were partially dissociated at 120 • C and almost completely dissociated at 140 • C.
The Cons result from Program 2 for ae mutant rice and glutinous rice was higher than those from Programs 1 and 3. Among the 17 rice samples, ae mutant rice showed the highest values and glutinous rice showed the lowest values, which agreed well with the phenomena that the cooked rice from ae rice retrogrades most and those from glutinous rice retrogrades least.
Among the glutinous rice samples, the Cons value and the Fin. vis. value of Koganemochi in Program 2 were markedly higher than those of Hakuchomochi, which agreed well with the retrogradation degree of waxy rice cake, as reported by Igarashi et al. [51].
The Cons values from the program at 120 • C for glutinous rice (21.6-52.3 RVU; mean, 32.5 RVU) were higher than those from the programs at 140 of the programs at 120 °C (223.2 RVU) and 140 °C (147.3 RVU). Indica rice showed the second-highest value among the 17 samples.
In this program, the Cons of japonica-indica hybrid rice (172.5 RVU) was higher than that of japonica (117.8-203.0 RVU; mean, 149.6 RVU), japonica low-amylose (121.5 RVU), and indica low-amylose (119.6 RVU) rice. Programs 2 and 3 showed a similar Cons trend, with the Mini. vis values decreasing as the maintenance temperature increases, as shown in Figure 4.  Table 3 shows the physical properties of cooked rice grains, evaluated by low-compression (25%) and high-compression (90%) methods [18] using a Tensipresser.   Table 3 shows the physical properties of cooked rice grains, evaluated by low-compression (25%) and high-compression (90%) methods [18] using a Tensipresser.  Table 3 shows that the hardness of the surface layer (H1) of ae mutant rice was higher than those of other rice cultivars. The hardness of the overall layer (H2) of ae mutant rice was higher than those of the other rice cultivars.

Physical Properties of Cooked Rice Grains
The stickiness of the surface layer (S1, sticky grains reveal high absolute values) of japonica rice was higher than those of the other rice cultivars, whereas the stickiness of the overall layer (S2) of japonica low-amylose rice was higher than those of the other rice cultivars.
The values for Balance H1, H2, A1, and A2 are important indices in evaluating the palatability of rice [18]. Balance A1 of glutinous rice was higher than those of the other rice cultivars, and the values of Balance H1, Balance H2, and Balance A2 showed a similar tendency.
Japonica low-amylose, indica low-amylose, japonica, and glutinous rice cultivars show soft and sticky eating qualities due to their physical properties of low hardness and high stickiness, while indica, japonica-indica hybrid, and ae mutant rice cultivars, with high amylose content, exhibit a texture of high hardness and low stickiness.
Low-amylose rice was found to be a stale-resistant rice cultivar, as many researchers have previously reported the staling characteristics of cooked low-amylose rice [61][62][63]. In the present investigation, the staling test for cooked rice was carried out by staling at 6 • C for 24 h. As shown in Table S1, the retrograded hardness of the surface layer H1(R) of ae mutant rice was higher than those of the other rice cultivars. Similarly, the retrograded hardness of the overall layer H2(R) of ae mutant rice was higher than those of the other rice cultivars.
As a result of the staling test of cooked rice, ae mutant and indica rice showed very high hardness after staling, while glutinous, japonica low-amylose, and indica low-amylose rice showed low hardness and high stickiness (Table S1).
The The H2(RD) of ae mutant rice was higher than those of the other rice cultivars; moreover, the H1(RD) of Koganemochi rice (1.21 times) was higher than that of Hakuchomochi rice (0.93 times). The retrogradation degree of hardness of ae mutant rice and indica rice showed very high values, while those of glutinous rice showed very low values.
As Umemoto et al. [64] showed, the variation in SS IIa (starch synthase IIa gene) affects eating quality after the storage of cooked rice at 5 • C; high-amylose and high-SLC rice cultivars seem to retrograde more markedly than low-amylose rice cultivars.
As shown in Figures 4 and 5, the RS contents of ae mutant rice (9.39-10.94%; mean, 10.28%) were higher than those of japonica-indica hybrid (2.34%), indica rice (1.75%), japonica (0.48-1.81%; mean, 0.88%), indica low-amylose (0.39%), japonica low-amylose (0.29%), and glutinous (0.04-0.06%; mean, 0.05%) rice cultivars. Ae mutant rice grains had markedly higher amounts of RS than the other rice cultivars even after cooking. Yang et al. [66] reported that mutant rice is rich in RS. The japonica rice cultivars had significantly lower RS contents than the indica rice and japonica-indica hybrids with similar amylose content [14]. In general, starches rich in amylose are naturally more resistant to digestion and more susceptible to retrogradation, while the SLCs in amylopectin behave in a manner similar to amylose by restricting starch swelling [67,68]. The japonica rice cultivars had significantly lower RS contents than indica and japonica-indica hybrid rice cultivars with similar amylose content [14,69,70].
Two crystalline structures of starch (A and B types) that contain different proportions of amylopectin have been identified. A-type starches are found in cereals, whereas B-type starches are found in tubers and amylose-rich starches [71].

SDS PAGE
Protein is the second-most abundant constituent of milled rice after starch. The physical properties of cooked rice grains are affected not only by starch but also by protein contents and their composition. Rice seed storage proteins consist mainly of glutelins and prolamins, such as 13 kDa prolamin [78,79]. The higher the protein content, the harder and less sticky the rice upon cooking [80].
The protein content in rice grains is influenced by weather conditions [81]. Protein production also tends to increase with higher levels of nitrogenous fertilizer at any planting density [82]. As shown in Figure S1 In general, starches rich in amylose are naturally more resistant to digestion and more susceptible to retrogradation, while the SLCs in amylopectin behave in a manner similar to amylose by restricting starch swelling [67,68]. The japonica rice cultivars had significantly lower RS contents than indica and japonica-indica hybrid rice cultivars with similar amylose content [14,69,70].
Two crystalline structures of starch (A and B types) that contain different proportions of amylopectin have been identified. A-type starches are found in cereals, whereas B-type starches are found in tubers and amylose-rich starches [71].

SDS PAGE
Protein is the second-most abundant constituent of milled rice after starch. The physical properties of cooked rice grains are affected not only by starch but also by protein contents and their composition. Rice seed storage proteins consist mainly of glutelins and prolamins, such as 13 kDa prolamin [78,79]. The higher the protein content, the harder and less sticky the rice upon cooking [80].
The protein content in rice grains is influenced by weather conditions [81]. Protein production also tends to increase with higher levels of nitrogenous fertilizer at any planting density [82]. As shown in Figure S1

Correlations between the Pasting Properties of Rice from the Three Programs with the Results of Cooked Rice Characterization
The pasting properties also influence the rice eating quality; therefore, it is useful to conduct tests of gelatinization properties in a quality assay for rice. AAC contains high levels of amylose and low levels of SLC in amylopectin. Generally, low-amylose rice becomes soft and sticky upon cooking, while high amylose rice becomes hard and separated [83]. H1(R) showed a positive correlation with Cons for the programs at 93 • C (r = 0.53; p < 0.05), 120 • C (r = 0.92; p < 0.01), and 140 • C (r = 0.67; p < 0.01). Furthermore, H2(R) showed a similar trend.
H2(RD) showed a positive correlation with Cons for the program at 93 • C (r = 0.47), 120 • C (r = 0.72; p < 0.01), and 140 • C (r = 0.56; p < 0.01). Furthermore, S2(RD) showed a negative correlation with Cons for the program at 93 • C (r = −0.57; p < 0.05), 120 • C (r = −0.92; p < 0.01), and 140 • C (r = 0.72; p < 0.01), as shown in Table 4. As shown in Table 3, the second program (120 • C) showed the highest correlation with the physical properties of cooked rice grains and starch microstructure. In a previous study, we reported that pasting properties are correlated to the texture of cooked rice grains [28]. These consistency values from Programs 2 and 3 using an RVA enable us to easily and rapidly evaluate the physical property of various kinds of cooked rice. Table 4. Correlation between the pasting properties of the three programs with the results of the physical parameters of cooked rice, iodine analysis, RS content, and prolamin ratio of 17 rice cultivars.
The Aλ max of cooked rice showed a positive correlation with Cons for Program 1 (r = 0.50; p < 0.05), Program 2 (r = 0.88; p < 0.01), and Program 3 (r = 0.61; p < 0.01). Furthermore, the Aλ max of starch showed a similar trend, as shown in Table 4. In our previous study, we reported that pasting properties are correlated to the AAC of various kinds of starch or milled rice [14,28]. AAC is higher than the actual amylose content because of the long-chain amylopectin binding with iodine. SLC in amylopectin appears to have a great effect on the consistency of starch, as reported in our previous study [14].
The RS of cooked rice showed a positive correlation with Cons for Program 2 (r = 0.80; p < 0.01), as shown in Table 4. Cons for Program 2 using an RVA enables us to evaluate the RS of various kinds of cooked rice by an easy and rapid RVA analysis.
As shown in Figure 6A, Cons for Program 2 showed a high correlation with Aλmax of cooked rice by linear regression analysis; R 2 was 0.77. Additionally, based upon the estimation of Aλmax, RS can be estimated by the analysis using an exponential function, as shown in Figure 6B; the estimation formula is revealed below. The 13 kDa prolamin ratio showed a positive correlation with Cons for Program 1 (r = 0.74; p < 0.01), Program 2 (r = 0.51; p < 0.05), and Program 3 (r = 0.62; p < 0.01). It is well known that rice with high protein content shows inferior palatability. Matsui et al. [78] showed that the final viscosity and consistency of near-isogenic line pairs for the low glutelin content1 (Lgc1) gene locus were significantly higher in low-glutelin lines; moreover, surface stickiness was significantly decreased in the low-glutelin lines.
In the present investigation, the Cons values for a new Program 2 using RVA had a higher correlation with the retrogradation of hardness, stickiness, and RS values of various kinds of cooked rice than the conventional program at 93 °C.
We also investigated the correlation of pasting property with starch molecular structure and physical property using narrow-range japonica low-amylose rice cultivars for commercial use in Japan. Differently from the wide range of various rice samples, from waxy rice to ae mutant rice samples, Aλmax, an indicator for the microstructure of starch by iodine scanning analysis, did not show a significant correlation; however, λmax showed a significant correlation with pasting property (Cons; r = 0.45, p < 0.05) and hardness of the cooked rice grains (r = 0.46, p < 0.05) in the case of narrow-range japonica rice cultivars for commercial use in Japan. The reason for this difference is that SLC differs markedly for the wide range of rice samples, from waxy rice to ae-mutant rice; on the contrary, narrow-range japonica low-amylose rice, such as Japanese rice for commercial use, differs in amylose, mainly in content (λmax), but does not differ in the SLC of amylopectin (Aλmax).

Formulae for Estimating the Retrograded Hardness H1(R) of Various Kinds of Cooked Rice Based on the Program at 120 °C Using an RVA
In our previous paper, we developed a novel estimation formula for the balance degree of the surface layer (A3/A1) based on the iodine absorption curve of milled rice [56].
The retrogradation of starch is responsible for the hardening of processed cooked rice, rice cakes, and crackers [84]. Figure 7 shows the formula developed to estimate H1(R) based on the pasting properties of various kinds of rice samples using the novel Program 2. The equation had a determination coefficient (R 2 ) of 0.86 based on the calibration. The following formula for estimating H1(R) was obtained using 17 varieties of rice for calibra- According to the abovementioned equation, the mean squared error is 1.38 and the RMSE (root mean squared error) is 1.18.
It is possible to estimate RS using Aλmax as a parameter without using the laborconsuming enzyme method.
The 13 kDa prolamin ratio showed a positive correlation with Cons for Program 1 (r = 0.74; p < 0.01), Program 2 (r = 0.51; p < 0.05), and Program 3 (r = 0.62; p < 0.01). It is well known that rice with high protein content shows inferior palatability. Matsui et al. [78] showed that the final viscosity and consistency of near-isogenic line pairs for the low glutelin content1 (Lgc1) gene locus were significantly higher in low-glutelin lines; moreover, surface stickiness was significantly decreased in the low-glutelin lines.
In the present investigation, the Cons values for a new Program 2 using RVA had a higher correlation with the retrogradation of hardness, stickiness, and RS values of various kinds of cooked rice than the conventional program at 93 • C.
We also investigated the correlation of pasting property with starch molecular structure and physical property using narrow-range japonica low-amylose rice cultivars for commercial use in Japan. Differently from the wide range of various rice samples, from waxy rice to ae mutant rice samples, Aλ max , an indicator for the microstructure of starch by iodine scanning analysis, did not show a significant correlation; however, λ max showed a significant correlation with pasting property (Cons; r = 0.45, p < 0.05) and hardness of the cooked rice grains (r = 0.46, p < 0.05) in the case of narrow-range japonica rice cultivars for commercial use in Japan. The reason for this difference is that SLC differs markedly for the wide range of rice samples, from waxy rice to ae-mutant rice; on the contrary, narrow-range japonica low-amylose rice, such as Japanese rice for commercial use, differs in amylose, mainly in content (λ max ), but does not differ in the SLC of amylopectin (Aλ max ).

Formulae for Estimating the Retrograded Hardness H1(R) of Various Kinds of Cooked Rice
Based on the Program at 120 • C Using an RVA In our previous paper, we developed a novel estimation formula for the balance degree of the surface layer (A3/A1) based on the iodine absorption curve of milled rice [56].
The retrogradation of starch is responsible for the hardening of processed cooked rice, rice cakes, and crackers [84]. Figure 7 shows the formula developed to estimate H1(R) based on the pasting properties of various kinds of rice samples using the novel Program 2. where Pt is the pasting temperature. This formula will enable us to rapidly and easily estimate the H1(R) of various kinds of cooked rice based on the novel Program 2 using an RVA. where Pt is the pasting temperature. This formula will enable us to rapidly and easily estimate the H1(R) of various kinds of cooked rice based on the novel Program 2 using an RVA.

Formulae for Estimating H1(RD) of Cooked Glutinous Rice Based on the Program at 120 °C Using an RVA
In measuring the pasting property of glutinous rice with an RVA, copper sulfate is usually used to prevent the effects of endogenous amylase activity. Copper sulfate is a deleterious substance; therefore, Sugiura et al. [85] used an RVA for measuring rice cake hardness with a 3% sodium chloride solution. Several studies related to the assessment of rice cake hardening have been reported [50,52,53].
The hardness of glutinous rice is important in order to improve working efficiency in the manufacture of rice cakes and crackers. Okamoto and Nemoto [53] tried to establish a method to estimate rice cake hardness of upland rice using an RVA with a 400-ppm solution of copper sulfate. Gelatinization temperature is related to the hardening speed of rice cake, and glutinous rice cultivars with higher gelatinization temperatures are more suitable for rice cake processing [54,84,86]. Figure 8 shows the formula developed for estimating H1(RD) using the pasting properties of glutinous rice based on the novel program at 120 °C. The equation had a determination coefficient (R 2 ) of 0.95 based on the calibration. The following formula for estimating H1(RD) was obtained using seven glutinous rice varieties for calibration ( where SB represents a setback. This formula enables us to evaluate the H1(RD) of cooked rice or rice cake made from glutinous rice, based on the novel Program 2 using an RVA.  In measuring the pasting property of glutinous rice with an RVA, copper sulfate is usually used to prevent the effects of endogenous amylase activity. Copper sulfate is a deleterious substance; therefore, Sugiura et al. [85] used an RVA for measuring rice cake hardness with a 3% sodium chloride solution. Several studies related to the assessment of rice cake hardening have been reported [50,52,53].
The hardness of glutinous rice is important in order to improve working efficiency in the manufacture of rice cakes and crackers. Okamoto and Nemoto [53] tried to establish a method to estimate rice cake hardness of upland rice using an RVA with a 400-ppm solution of copper sulfate. Gelatinization temperature is related to the hardening speed of rice cake, and glutinous rice cultivars with higher gelatinization temperatures are more suitable for rice cake processing [54,84,86]. Figure 8 shows the formula developed for estimating H1(RD) using the pasting properties of glutinous rice based on the novel program at 120 • C. The equation had a determination coefficient (R 2 ) of 0.95 based on the calibration. The following formula for estimating H1(RD) was obtained using seven glutinous rice varieties for calibration (1, Benika; 2, Shihou; 3, Hakuchomochi; 4, Koganemochi; 5, Himenomochi; 6, Kinunohada; 7, Kitayukimochi): H1(RD) = 0.012 × Mini. vis − 0.079 × Pt + 0.007 × SB − 6.155 where SB represents a setback. This formula enables us to evaluate the H1(RD) of cooked rice or rice cake made from glutinous rice, based on the novel Program 2 using an RVA. In the present investigation, we have proposed an easy and rapid method to estimate rice quality using iodine colorimetric analysis, texture measurements, and pasting properties by a new-type RVA.
We have reported the novel estimation formulae to estimate the fatty acid composition of brown rice using RVA analysis [29]. RVA and other physicochemical measurements will be useful to estimate the biofunctional properties of rice in the near future.
By the combination of physicochemical measurements, sensory tests, and genotyping, the ability to estimate or classify rice quality towards diversified consumer needs in the future is improved.

Conclusions
We have made improvements to analyzing pasting properties in a new program using RVA 4800, and novel estimation formulae were developed in this paper for estimates of the retrograded hardness of cooked rice H1(R) and the degree of retrogradation H1(RD), which has led to an easy and rapid evaluation of the cooking qualities of various rice samples. The following are the main features: 1. Among the three kinds of RVA programs, Program 2 (120 °C) showed the highest correlations with starch microstructure (Aλmax), RS, physical properties, and degree of retrogradation of the cooked rice grains. 2. The novel Program 2 (120 °C) showed high determination coefficients for hardness and the degree of retrogradation of cooked rice grains. 3. The novel RVA Program 2 enables us to easily and rapidly estimate the cooking and processing characteristics of various kinds of rice cultivars.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: SDS-PAGE analysis of proteins extracted from 17 rice samples; Table S1: Physical properties of cooked rice after 24 h at 6 °C.  In the present investigation, we have proposed an easy and rapid method to estimate rice quality using iodine colorimetric analysis, texture measurements, and pasting properties by a new-type RVA.
We have reported the novel estimation formulae to estimate the fatty acid composition of brown rice using RVA analysis [29]. RVA and other physicochemical measurements will be useful to estimate the biofunctional properties of rice in the near future.
By the combination of physicochemical measurements, sensory tests, and genotyping, the ability to estimate or classify rice quality towards diversified consumer needs in the future is improved.

Conclusions
We have made improvements to analyzing pasting properties in a new program using RVA 4800, and novel estimation formulae were developed in this paper for estimates of the retrograded hardness of cooked rice H1(R) and the degree of retrogradation H1(RD), which has led to an easy and rapid evaluation of the cooking qualities of various rice samples. The following are the main features:

1.
Among the three kinds of RVA programs, Program 2 (120 • C) showed the highest correlations with starch microstructure (Aλ max ), RS, physical properties, and degree of retrogradation of the cooked rice grains.

2.
The novel Program 2 (120 • C) showed high determination coefficients for hardness and the degree of retrogradation of cooked rice grains.

3.
The novel RVA Program 2 enables us to easily and rapidly estimate the cooking and processing characteristics of various kinds of rice cultivars.