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

Abstract

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

Graphical abstract

Formula for estimating the resistant starch.

Key words:

• apparent amylose content
• resistant starch
• pasting property
• rice
• amylopectin

Starch is composed of two kinds of essential α-glucans that have distinctive structure. Amylose is small, linear, and slightly branched molecules, whereas amylopectin is large, highly branched molecule. Particularly, amylose is one of the components of rice starch that greatly affects the quality and gelatinization properties of cooked rice.¹⁾ Low-amylose rice generally becomes soft and sticky after cooking, whereas high-amylose rice becomes hard with fluffy separated grains.²⁾ The group of high-amylose starches includes two types of rice starches with similar AAC but different super-long chains (SLC) contents of amylopectin.^3,4) The structure of amylopectin in the starch granule has been described using a cluster model.^5–7) Nakamura et al. classified the starches of 129 rice varieties cultivated in Asia into two types, L and S, based on the differences in the chain length of the amylopectin cluster.⁸⁾ Rice starches contain 0–30% of amylose, of which contents among varieties of rice varies with the ambient temperatures during development of rice grains. The starches of rice grown at low temperature had significantly higher amylose content than that of rice grown at high temperatures.^9–13) Inouchi et al.⁹⁾ and Hirano et al.¹⁴⁾ showed a high positive correlation between the amounts of waxy (Wx) protein and the SLC contents of starch.

The most widely used method for amylose determination is a colorimetric assay where iodine binds with amylose to produce a blue-purple color, which is measured spectrophotometrically at a single wavelength (620 nm).¹⁵⁾ Amylopectin also has a color reaction with iodine, which interfering with the direct measurement of the color generated by the amylose–iodine complex. Igarashi et al.¹⁶⁾ used an automatic analyzer, and developed an estimation equation from an iodine absorption spectrum ranging from 400 to 900 nm setting 600 nm as a bordering wavelength. Nakamura et al.¹⁷⁾ showed that wavelength at which absorbance becomes maximum on iodine staining of starch (λ_max) and absorbance at λ_max (Aλ_max) were used for estimating AAC (not used standard curve).

Several researchers reported the development of high-resistant starch (RS) rice^18,19) as well as high-amylose and high-dietary fiber rice²⁰⁾ through physical and chemical mutations. The glycemic effect of foods depends on numerous factors, such as the structures of amylose and amylopectin structure.^21,22) However, the cooked grains of ae mutant rice cultivars are too hard and non-sticky because ae mutant rice cultivars lack starch-branching enzyme Ⅱb and the presence of SLC. They are promising in terms of their bio-functionality such as diabetes prevention.^23–29) RS may be included within the term “fiber” on the nutrition labels used in some countries but not in others.^30,31) There are several kinds of method of measurement for determining the RS contents.^32,33) RS differs depending on the different processing methods. It is necessary to develop the novel and simple method for measuring RS.

Physicochemical properties of the starches were often evaluated as pasting characteristics using an RVA. High-amylose rice cultivars were reported to have a higher final vis. than low-amylose cultivars, where the final vis. is related to the degree of starch retrogradation after cooling.^3,34) For many years, the SB, defined by subtracting the viscosity at the trough from the final vis., has been related to the firmness and the amylose content of the rice, and BD, defined by subtracting the viscosity at the trough from the viscosity at the peak, has been related, too.³⁵⁾

We here characterized the Japonica polished rice and starch; evaluated the relationship between their pasting properties, AAC and RS. The improvements, which we performed for analyzing the pasting properties measured by RVA and, the novel estimation formulae, which we developed in the present paper against the AAC and RS, would lead to an easy and rapid method for clarifying starch characteristics.

Materials and methods

Materials

Japonica rice cultivar (Niigata103go; a–e, Niigata104go; a–g, Koshihikari; a–d, Todorokiwase, Nourin1go, Nourin6go, Nourin22go, Moritawase, Asahi, Rikuu20go, Rikuu132go, Nourin6go, Nourin8go, Ginbouzu, Ginbouzu-bansei, Kamenoo; a and b, Senichi, Hounenwase, Koshiibuki, Nihonkai, Jyoshu, Koshijiwase, Yoneyama, Senshuraku) were cultivated at the Niigata Prefectural Agricultural Research Institute in 2014. Japonica rice cultivars (Tsuyahime; a and b, Yumepirika; a and b, Sagabiyori; a–c, Genkitsukushi, Hitomebore; a–c, Akitakomachi; a and b), Nanatsuboshi, Kirara397) were purchased from a local market. The small letters show difference of rice-growing district.

Ae mutant rice (EM10, EM189, EM72, EM129, and EM16) and Wx mutant rice (EM21), Japonica rice (Kinmaze) were cultivated in an experimental field of the Kyushu University in 2011. The ae mutant rice (Hokurikukona243go) was cultivated in an experimental field at the Hokuriku Research Center in the Central Agricultural Research Center, Joetsu in 2011. Glutinous rice (Koganemochi and Hakuchomochi) were cultivated at the Niigata Prefectural Agricultural Research Institute in 2011.

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) 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.^36,37)

Amylose content

Amylose content of the polished rice and starch (treated with alkaline solution; dry matter 0.1 g) were estimated by the iodine colorimetric method of Juliano.¹²⁾ Potato amylose (Type III, Sigma Chemical Co., St. Louis, MO) and waxy rice starch (fat and proteins removed from waxy rice) were used as standard amylose and standard amylopectin, respectively, for amylose determination.

Measurement of pasting properties of rice flours

The pasting properties of starch rice flours (dry matter 3.5 g) were measured using RVA (model Super 4; New-port Scientific Pty Ltd., Warriewood, Australia). A programmed heating and cooling cycle was followed, as described by Toyoshima et al.³⁸⁾

We adopted three novel parameters, Max/Min, Max/Fin, and SB/Con, among the pasting properties by RVA measurement. Max/Min means the ratio of the maximum viscosity, which corresponds to the peak viscosity on gelatinization of starch, to the minimum viscosity, which corresponds to the lowest viscosity due to the broken-down starch granules by stirring. It seems that the rice of which Max/Min is high is palatable because its starch is sticky and soft. Max/Fin means the ratio of the maximum viscosity, which corresponds to the peak viscosity on gelatinization of starch, to the final viscosity, which corresponds to the rebound viscosity due to the retrograded starch granules by lowering of temperature. It seems that the rice of which Max/Fin is high is palatable because its starch is sticky and resistant to retrogradation. SB/Con means the ratio of the difference between final viscosity and maximum viscosity, which corresponds to the difference between retrogradation and gelatinization, to the difference between final viscosity and minimum viscosity, which corresponds to the retrogradation degree of starch granules. It seems that the rice of which SB/Con is high is unpalatable because its starch is easy to retrograde.

Measurement of RS

RS in the starch rice flour was measured according to the AOAC method using an RS assay kit (Megazyme, Wicklow, Ireland). Each sample (100 mg) was digested with pancreatin and amyloglucosidase at 37 °C for 6 h, and glucose was measured using a spectrophotometer at 510 nm.

Statistical analyses

All results, including the significance of regression coefficients, were statistically analyzed using the t test and a one-way ANOVA, the method of Tukey, and Excel Statistics (ver. 2006, Microsoft Corp., Tokyo, Japan). The predictor equations were determined by the stepwise method of multiple regression analysis. As predictor variable of conventional indexes using an RVA; Max. vis., Min. vis., Final vis., BD, SB Pt, and Cons and those of novel indexes; SB/Con ratio, Max/Min ratio, and Max/Fin ratio.

Results and discussion

AAC of Japonica polished rice

The molecular structures of many starches, including amylose molecular sizes and amylopectin branch chain lengths, have been reported.^39–41) AAC has been used a good parameter for estimating the cooking or eating qualities of rice grains, and the iodine colorimetric method at 620 nm for AAC measurement was developed by Juliano.¹⁶⁾ Eating or cooking quality of rice is related to the amylose content and the fine structure of amylopectin.⁴²⁾ The AAC contents are higher than the actual amylose contents because of long-chain amylopectin binding with iodine.³⁹⁾

The structure and properties of endosperm starches are well known to change in rice plants grown in different environments. The amylose content has been reported to be significantly affected by the temperature during the grain-filling period.

Table 1 shows that the AAC of Sagabiyori (average: 16.47%), Tsuyahime (average: 16.3%), and Niigata103go (average: 15.9%) were of intermediate values. Nourin6go (14.5%), Ginbouzu (14.4%), Nourin22go (14.4%), Nourin8go (14.4%), and Asahi (14.3%) were of low values, while Koshihikari (average: 13.7%), Genkitsukushi (12.8%), Hitomebore (average: 12.7%), Moritawase (12.4%), Nanatsuboshi (12.4%), Rikuu20go (12.3%), Yumepirika (average: 12.3%), Todorokiwase (12.1%), Akitakomachi (average: 12.0%), and Niigata104go (average: 11.9%) were of a very low values. As a result, AAC of Japonica polished rice were 11.2–17.6 (%). Koshihikari and Tsuyahime showed rather high values for coefficient of variation based on different growing district. From this result, it was considered that these cultivars were often influenced by numerous stresses from the environment. In case of similar AAC in various rice cultivars, the difference in RS content tends to reflect amylose molecular sizes (the length of the glucan chain; molecular size of amylose, and SLC), as shown in the relationship between Akitakomachi-a (AAC; 12.1%, RS; 2.0%) and Akitakomachi-b (AAC; 12.0%, RS; 1.3%), the relationship between Nourin6go (AAC; 14.5%, RS; 1.1%) and Ginbouzu (AAC; 14.4%, RS; 0.6%). As a result, in case of similar AAC (AAC contains a lot of amylose and a little SLC in amylopectin) in various rice cultivars, the difference in RS content are presumed to be related to the SLC in amylopectin.

Table 1. Amylose contents and resistant contents of Japonica polished rice.

		Amylose (%)	SD	RS (%)	SD
A	Niigata103go-a	15.4^a	0.3	1^a	0
	Niigata103go-b	15.8^a	0.1	1^a	0
	Niigata103go-c	16.5^b	0.2	1.1^a	0
	Niigata103go-d	15.1^a	0.2	1.1^a	0
	Niigata103go-e	16.8^b	0.2	0.9^a	0
	Niigata104go-a	11.2^c	0.2	2.2^b	0
	Niigata104go-b	12.5^d	0.1	1.8^c	0
	Niigata104go-c	12^d	0.1	1.9^c	0
	Niigata104go-d	11.9^d	0.1	2.1^b	0
	Niigata104go-e	12.3^d	0.2	1.7^c	0.1
	Niigata104go-f	11.4^c	0.2	2^b	0
	Niigata104go-g	12.3^d	0.1	1.6^c	0
	Koshihikari-a	12.5^d	0	1.7^c	0
	Koshihikari-b	15.1^a	0.2	1.4^d	0
	Koshihikari-c	15.2^a	0	1.3^d	0
	Tsuyahime-a	15.4^a	0.1	1.3^d	0
	Tsuyahime-b	17.1^e	0.1	1.3^d	0
	Yumepirika-a	12.1^ｄ	0.1	0.9^a	0
	Yumepirika-b	12.5^d	0.2	0.9^a	0
	Sagabiyori-a	17.6^e	0.1	0.5^e	0
	Sagabiyori-b	17.3^e	0.1	0.7^e	0
B	Sagabiyori-c	15.3^a	0.4	0.7^e	0.1
	Genkitsukushi	12.8^d	0.3	1.7^c	0
	Koshihikari-d	12.1^d	0.2	1.9^c	0
	Hitomebore-a	12.8^d	0.2	1.9^c	0
	Hitomebore-b	12.6^d	0.2	1.7^c	0.1
	Hitomebore-c	12.6^d	0.1	1.7^c	0
	Akitakomachi-a	12.1^d	0.1	2^b	0.1
	Akitakomachi-b	12^d	0.1	1.3^d	0
	Nanatsuboshi	12.4^d	0.2	1.4^d	0
	Tdorokiwase	12.1^d	0.3	1.9^c	0
	Nourin22go	14.4^d	0.1	0.8^a	0
	Moritawase	12.4^d	0.4	1.8^c	0
	Asahi	14.3^f	0.5	0.7^a	0
	Rikuu20go	12.3^d	0.3	1.8^c	0
	Nourin6go	14.5^f	0.2	1.1^a	0
	Nourin8go	14^f	0.1	0.8^a	0
	Ginbouzu	14.4^f	0.4	0.6^e	0

Notes: (A) Samples for calibration. (B) Samples for validation. Small letters show the difference of rice-growing district. Means with same letter are not significantly different (p < 0.05). Values are average of triplicate measurements.

RS contents of Japonica polished rice

Yang et al.¹⁸⁾ reported about the mutant rice that are rich in RS. The Japonica rice cultivars had significantly lower the RS contents than the indica rice and japonica-indica hybrid rice cultivars with similar amylose contents.⁴³⁾ Two crystalline structures of starch have been identified (A and B type), which contain differing proportions of amylopectin. A-type starches are found in cereals, whereas B-type starches are found in tubers and amylose-rich starches.⁴⁴⁾ The resistance of starch to digestion is influenced by the nature of the association between starch polymers, with higher amylose levels in the starch being associated with slower digestibility rates.⁴⁵⁾ In general, starches rich in amylose are naturally more resistant to digestion and more susceptible to retrograde, while SLC in amylopectin behaved in a manner similar to amylose by restricting starch swelling.⁴⁶⁾ Goni et al. investigated resistant-type starches are not digested by enzymes, they are fermented in the large intestine, the fermentation of which results in the production of short-chain fatty acids⁴⁷⁾ and amylopectin retrogradation can significantly increase the amount of RS.⁴⁸⁾ RS contents are important as they can yield foods with greater nutritional quality. Table 1 shows that the RS content of Japonica polished rice were 0.5–2.2 (%).

Pasting properties of Japonica polished rice flour

The pasting properties also influence the rice eating quality; therefore, it is useful to conduct a gelatinization test as a quality assay for rice. In the present paper, the SB (Final vis. −Max. vis.) and the Cons (Final vis. −Mini. vis.) were calculated by the method of Toyoshima.^38,49)

Table 2 shows that the Max. vis. of polished Japonica rice were 322.5–424.8 RVU, those of Mini. vis. were 144.8–237.0 RVU, those of BD were 161.1–262.5 RVU, those of Final vis. were 256.2–365.4 RVU, those of Pt were 64.2–70.3 °C, those of SB were −35.0 to −140.1 RVU, and those of Consistency were 100.1–148.3 RVU. A novel index SB/Con ratios were −0.3 to −1.2, those of Max/Min ratios were 1.8–2.7, and those of Max/Fin ratios were 1.1–1.5 (data not shown). In previous studies, we have found that the Japonica-Indica hybrid rice and high-amylose Japonica rice cultivars showed higher Pt, Max. vis., Final vis., and BD compared with the ae mutant rice cultivars and Japonica rice cultivars.¹⁷⁾ Miles et al. found that, in dilute gels, the amylopectin crystallinity was reversible lower than 100 °C, whereas it was not the case for the amylose where the temperature was higher than 100 °C. They suggested that the smaller amylopectin chains were attached as clusters onto the longer chains; however, into-chain association could only occur over DP 15 chains before being interrupted by a branch point, as opposed to amylose.⁵⁰⁾ The proportion of short chains (DP ≦ 12) of amylopectin correlated negatively with the peak temperature of gelatinization of the rice starches.⁴⁰⁾ Okuda et al. showed that the pasting temperature of milled rice as measured by RVA correlated well with the ratio of short-to-long chain amylopectin and with enzyme digestibility.³⁵⁾

Table 2. Pasting properties of Japonica polished rice.

		Maxi			Mini			Break.d			Final		SD	Setback			Past.t			Consis			Setback/			Max			Max/
		(RVU)		SD	(RVU)		SD	(RVU)		SD	(RVU)			(RVU)		SD	℃		SD	(RVU)		SD	consis		SD	Mini		SD	Final		SD
A	Niigata103-a	356.8	a	0.4	155.5	a	0.1	201.3	a		268.0	a	1.4	−88.8	a	0.9	64.6	a	2.6	112.5	a	1.5	−0.8	a	0.0	2.3	a	0.0	1.3	a	0.0
	Niigata103-b	347.5	a	0.5	150.1	a	4.8	197.4	a	4.3	261.0	a	0.7	−86.5	a	0.2	65.7	a	0.0	110.9	a	4.1	−0.8	a	0.1	2.3	a	0.0	1.3	a	0.0
	Niigata103-c	350.3	a	2.5	166.0	a	2.4	184.3	a	0.2	278.4	a	3.1	−71.8	b	0.6	65.8	a	0.1	112.4	a	0.8	−0.6	b	0.0	2.1	a	0.0	1.3	a	0.0
	Niigata103-d	363.0	a	3.3	162.5	a	3.6	200.5	a	6.9	277.6	a	0.5	−85.4	a	3.8	66.5	a	0.0	115.1	a	3.1	−0.7	a	0.1	2.2	a	0.0	1.3	a	0.0
	Niigata103-e	322.7	b	0.8	174.3	a	1.1	148.4	b	1.8	303.1	b	1.2	−19.6	c	2.0	65.0	a	0.1	128.8	b	0.2	−0.2	c	0.0	1.9	b	0.0	1.1	b	0.0
	Niigata104-a	359.5	a	0.1	151.1	a	0.7	208.4	a	0.8	256.2	a	0.8	−103.3	d	0.7	69.3	b	0.0	105.1	a	1.5	−1.0	d	0.0	2.4	a	0.0	1.4	c	0.0
	Niigata104-b	356.3	a	7.0	144.8	a	1.8	211.5	a	5.2	252.9	a	1.6	−103.3	d	5.4	68.6	b	0.0	108.2	a	0.1	−1.0	d	0.1	2.5	a	0.0	1.4	c	0.0
	Niigata104-c	363.1	a	1.8	153.3	a	3.3	209.8	a	1.5	267.0	a	3.7	−96.1	a	1.9	68.6	b	0.0	113.8	a	0.4	−0.8	a	0.0	2.4	a	0.0	1.4	c	0.0
	Niigata104-d	384.8	a	1.1	169.3	a	1.2	215.6	a	0.1	281.6	a	1.2	−103.3	d	0.1	67.9	b	0.5	112.3	a	0.0	−0.9	a	0.0	2.3	a	0.0	1.4	c	0.0
	Niigata104-e	347.6	a	3.1	149.1	a	0.9	198.5	a	4.0	261.3	a	2.9	−86.3	a	6.1	67.9	b	0.5	112.3	a	2.1	−0.8	a	0.1	2.3	a	0.0	1.3	a	0.0
	Niigata104-f	367.5	a	0.1	178.8	a	0.7	188.8	a	0.6	279.3	a	2.1	−88.3	a	1.9	67.9	b	0.0	100.5	c	1.4	−0.9	a	0.1	2.1	a	0.0	1.3	a	0.0
	Niigata104-g	358.3	a	2.7	157.6	a	5.4	200.8	a	2.8	265.8	a	3.3	−92.5	a	0.7	67.9	b	0.1	108.3	a	2.1	−0.9	a	0.0	2.3	a	0.0	1.3	a	0.0
	Koshi-a	377.3	a	1.4	160.4	a	2.2	216.8	a	3.5	277.6	a	3.6	−99.7	d	2.2	68.6	b	1.0	117.2	a	5.8	−0.9	a	0.1	2.4	a	0.0	1.4	c	0.0
	Koshi-b	384.2	a	2.8	168.1	a	1.5	216.1	a	1.2	290.1	a	0.7	−94.1	a	2.1	67.9	b	0.0	122.0	b	0.8	−0.8	a	0.0	2.3	a	0.0	1.3	a	0.0
	Koshi-c	368.5	a	2.2	169.3	a	1.4	199.2	a	0.8	293.8	a	1.5	−74.7	b	0.7	67.8	b	0.1	124.5	b	0.1	−0.6	b	0.0	2.2	a	0.0	1.3	a	0.0
	Tsuya-a	378.6	a	0.3	183.9	a	5.9	194.8	a	5.7	311.0	b	2.1	−67.6	b	1.8	66.1	a	0.5	127.1	b	3.8	−0.5	b	0.1	2.1	a	0.0	1.2	d	0.0
	Tsuya-b	370.6	a	2.1	201.5	b	3.0	169.1	c	0.9	327.3	b	3.2	−43.3	e	1.1	66.4	a	0.1	125.8	b	0.2	−0.3	c	0.0	1.8	b	0.0	1.1	b	0.0
	Yumepi-a	366.0	a	0.7	145.5	c	1.6	220.5	a	0.9	244.7	a	2.9	−121.3	d	2.2	66.8	a	0.5	99.1	c	1.4	−1.2	d	0.1	2.5	a	0.0	1.5	c	0.0
	Yumepi-b	364.0	a	2.4	154.2	a	0.7	209.8	a	1.8	255.3	a	0.5	−108.6	d	2.9	67.1	a	0.0	101.1	c	1.1	−1.1	d	0.1	2.4	a	0.0	1.4	c	0.0
	Sagabi-a	322.5	b	2.6	153.6	b	1.3	168.9	c	1.3	287.2	a	1.2	−35.3	e	1.4	64.2	a	0.0	133.6	d	0.1	−0.3	c	0.0	2.1	a	0.0	1.1	b	0.0
	Sagabi-b	327.9	a	2.1	166.8	a	1.1	161.1	c	3.1	292.8	a	2.0	−35.0	e	4.1	65.0	a	0.1	126.1	b	0.9	−0.3	c	0.1	2.0	a	0.0	1.1	b	0.0
B	Sagabii-c	364.3	a	1.2	161.5	a	6.0	202.9	a	5.0	289.0	a	5.8	−75.4	b	4.8	64.3	a	0.0	127.5	b	0.3	−0.6	b	0.1	0.4	c	0.0	1.3	a	0.0
	Genkitsukui	418.1	c	12.7	165.2	a	12.2	252.9	d	4.5	278.0	a	7.9	−140.1	f	5.3	68.1	b	0.4	112.9	a	4.6	−1.2	d	0.1	0.4	c	0.0	1.5	e	0.0
	Koshi-d	424.8	c	3.4	166.7	a	2.5	258.1	d	5.8	290.4	a	1.1	−134.4	f	4.5	69.3	b	0.1	123.7	b	1.4	−1.1	d	0.1	0.4	c	0.0	1.5	e	0.0
	Hitome-a	392.3	c	10.1	166.0	a	4.2	226.3	a	5.9	293.4	a	1.5	−98.9	d	8.5	68.3	b	0.5	127.4	b	2.7	−0.8	a	0.1	0.4	c	0.0	1.3	a	0.0
	Hitome-b	414.0	c	2.5	161.7	a	6.3	252.3	d	3.8	286.9	a	4.2	−127.2	d	1.7	67.5	b	0.5	125.2	b	2.1	−1.0	d	0.1	0.4	c	0.0	1.4	c	0.0
	Hitome-c	419.2	c	10.3	165.6	a	4.7	253.6	d	5.5	290.7	a	4.0	−128.5	d	6.3	68.5	b	0.0	125.1	b	0.8	−1.0	d	0.1	0.4	c	0.0	1.4	c	0.0
	Akitako-a	400.6	c	11.4	157.9	a	3.5	242.7	d	8.7	286.8	a	1.9	−113.8	d	10.0	68.8	b	0.4	128.9	b	1.6	−0.9	a	0.1	0.4	c	0.0	1.4	c	0.0
	Akitako-b	418.1	c	6.2	161.8	a	3.0	256.3	d	3.1	289.7	a	1.4	−128.3	d	4.8	68.6	b	0.0	127.9	b	1.7	−1.0	d	0.1	0.4	c	0.0	1.4	c	0.0
	Nanatsu-a	372.7	a	4.5	147.3	c	10.4	225.5	a	7.0	268.2	a	7.9	−104.5	d	4.5	67.5	b	0.4	120.9	b	2.6	−0.9	a	0.1	0.4	c	0.0	1.4	c	0.0
	Tdoroki	423.4	c	3.5	172.8	a	3.1	250.6	d	5.6	306.9	b	3.5	−116.4	d	6.1	68.8	b	0.4	134.2	d	0.5	−0.9	a	0.1	0.4	c	0.0	1.4	c	0.0
	Nourin22	358.8	a	2.6	150.2	a	2.1	208.5	a	3.1	283.3	a	2.8	−75.4	b	2.8	66.0	a	0.4	133.1	d	1.1	−0.6	b	0.1	2.4	a	0.0	1.3	a	0.0
	Moritawase	426.8	c	2.7	237.0	b	3.6	189.8	a	3.5	365.4	b	4.0	−61.3	b	2.4	68.9	b	0.4	128.4	b	2.1	−0.5	b	0.1	1.8	b	0.0	1.2	d	0.0
	Asahi	355.4	a	12.2	171.9	a	10.2	183.5	a	7.8	316.5	b	4.7	−38.9	e	11.5	66.5	a	0.1	144.6	e	6.7	−0.3	c	0.1	2.1	a	0.0	1.1	b	0.0
	Rikuu20	417.8	c	3.6	155.2	a	1.2	262.5	d	2.6	279.7	a	1.8	−138.1	f	2.1	70.3	b	0.4	124.5	b	0.6	−1.1	d	0.0	2.7	d	0.0	1.5	e	0.0
	Nourin6	376.6	a	3.1	162.8	a	4.1	213.8	a	5.6	304.5	b	5.9	−72.1	b	7.2	67.4	b	0.4	141.7	e	1.7	−0.5	b	0.1	2.3	a	0.0	1.2	d	0.0
	Nourin8	379.0	a	1.9	172.0	a	3.9	207.0	a	5.3	317.0	b	4.0	−62.0	b	5.6	65.0	a	0.7	145.0	e	0.8	−0.4	c	0.1	2.2	a	0.0	1.2	d	0.0
	Ginbouzu	384.1	a	2.1	161.9	a	1.4	222.2	a	3.5	302.2	b	2.5	−81.9	b	4.6	65.7	a	0.0	140.3	e	1.1	−0.6	b	0.1	2.4	a	0.0	1.3	a	0.0

Notes: (A) Samples for calibration. (B) Samples for validation.

Means with same letter are not significantly different (p < 0.05).

Values are average of triplicate measurements.

Correlation between AAC, RS, and pasting properties of 38 Japonica polished rice samples

AAC showed a positive correlation with SB (r = 0.76) and SB/Con ratios (r = 0.76) at p < 0.01 and negative correlation with Pt (r = −0.80), Max/Fin ratios (r = −0.75), BD (r = −0.67), Max/Min ratios (r = −0.62), and Maxi. vis. (r = −0.57) at p < 0.01. The novel indexes, such as SB/Con and Max/Fin ratios, had higher correlations with AAC than Max. vis., Min. vis., BD, and Cons of conventional indexes. Moreover, RS showed a positive correlation with Pt (r = 0.86), Max/Fin ratios (r = 0.56), Max. vis. (r = 0.54), BD (r = 0.49) at p < 0.01 and negative correlation with SB (r = −0.60) and SB/Con ratios (r = −0.59) at p < 0.01. The novel indexes, such as SB/Con and Max/Fin ratios, had higher correlations with RS than conventional indexes, such as Max. vis., Min. vis., BD, and Cons (data not shown).

Formula for estimating the AAC based on the pasting properties of Japonica polished rice

Fig. 1(A) shows the formula developed for estimating the AAC based on the novel index by the pasting properties of Japonica polished rice. The equation had a determination coefficient (R²) of 0.89 based on the calibration. The following formula for estimating the AAC was obtained using 21 samples for calibration:

Fig. 1. Formula for estimating the amylose content based on the pasting properties of Japonica polished rice and application of the formula to unknown samples.

Notes: The equation had determination coefficients (R²) of 0.89 based on the calibration. (A) Estimation formula; amylose content (%) = −0.84 × Pt + 37.76 × SB/Con −13.09 × Max/Mini + 103.92 × Max/Fin −8.02. (1) Niigata103go-a; (2) Niigata103go-b; (3) Niigata103go-c; (4) Niigata103go-d; (5) Niigata103go-e; (6) Niigata104go-a; (7) Niigata104go-b; (8) Niigata104go-c; (9) Niigata104go-d; (10) Niigata104go-e; (11) Niigata104go-f; (12) Niigata104go-g; (13) Koshihikari-a; (14) Koshihikari-b; (15) Koshihikari-c; (16) Tsuyahime-a; (17) Tsuyahime-b; (18) Yumepirika-a; (19) Yumepirika-b; (20) Sagabiyori-a; (21) Sagabiyori-b. (B): Application of the formula to unknown samples; (1) Sagabiyori-c; (2) Genkitsukushi; (3) Koshihikari-d; (4) Hitomebore-a; (5) Hitomebore-b; (6) Hitomebore-c; (7) Akitakomachi-a; (8) Akitakomachi-b; (9) Nanatsuboshi; (10) Todorokiwase; (11) Nourin22go; (12) Moritawase; (13) Asahi; (14) Rikuu20go; (15) Nourin6go; (16) Nourin8go; (17) Ginbouzu. (B) show that a determination coefficients (R²) of 0.71 was obtained with the application of the estimation formula to 17 unknown samples. Therefore, the validation test showed that the equation can be applied to unknown samples.

Fig. 1(B) shows that a determination coefficient (R²) of 0.71 was obtained with the application of the above-mentioned formula to 17 unknown samples. Thus, the validation test showed that the equation can be applied well to unknown samples, which means that it became possible to estimate AAC by the simple and easy measurement of pasting properties of Japonica polished rice by RVA.

On the contrary, it was shown that the formula developed for estimating the AAC based on the pasting property values of conventional indexes using RVA (data not shown) cannot be applied to the estimation of AAC widely.

The equation had a determination coefficient (R²) of 0.87 for calibration and 0.30 for validation test, which showed this equation cannot be applied well to unknown samples (data not shown).

Formula for estimating the RS content based on the pasting properties of Japonica polished rice

The RS content was used as response variables and the parameters from analysis of the novel index of the pasting properties were used as predictor variables in the multiple regression analyses. Fig. 2(A) shows the formula developed for estimating the RS content based on the novel index of the pasting properties of Japonica polished rice.

Fig. 2. Formula for estimating the resistant starch content based on the pasting properties of Japonica polished rice and application of the formula to unknown samples.

Notes: The equation had determination coefficients (R²) of 0.80 based on the calibration. (A) Estimation formula; resistant starch content (%) = 0.31 × Pt + 0.24 × SB/Con −1.20 × Max/Mini + 1.93 × Max/Fin −18.87. (1) Niigata103go-a; (2) Niigata103go-b; (3) Niigata103go-c; (4) Niigata103go-d; (5) Niigata103go-e; (6) Niigata104go-a; (7) Niigata104go-b; (8) Niigata104go-c; (9) Niigata104go-d; (10) Niigata104go-e; (11) Niigata104go-f; (12) Niigata104go-g; (13) Koshihikari-a; (14) Koshihikari-b; (15) Koshihikari-c; (16) Tsuyahime-a; (17) Tsuyahime-b; (18) Yumepirika-a; (19) Yumepirika-b; (20) Sagabiyori-a; (21) Sagabiyori-b. (B):Application of the formula to unknown samples; (1) Sagabiyori-c; (2) Genkitsukushi; (3) Koshihikari-d; (4) Hitomebore-a; (5) Hitomebore-b; (6) Hitomebore-c; (7) Akitakomachi-a; (8) Akitakomachi-b; (9) Nanatsuboshi; (10) Todorokiwase; (11) Nourin22go; (12) Moritawase; (13) Asahi; (14) Rikuu20go; (15) Nourin6go; (16) Nourin8go; (17) Ginbouzu. (B) show that a determination coefficients (R²) of 0.75 was obtained with the application of the estimation formula to 17 unknown samples. Therefore, the validation test showed that the equation can be applied to unknown samples.

The equation has a determination coefficient (R²) of 0.80 based on the calibration. The following formula for estimating the RS content was obtained using 21 samples for calibration. Fig. 2(B) shows that a determination coefficient (R²) of 0.75 was obtained with the application of the above-mentioned formula to 17 unknown samples. Thus, the validation test showed that the equation can be applied well to unknown samples, which means that it became possible to estimate RS by the simple and easy measurement of pasting properties of Japonica polished rice by RVA.

Although the pasting properties measured by RVA are reported to be correlated with amylose,^8,38) chain length of amylopectin,⁴⁶⁾ we propose, in the present paper, that they are also useful to estimate RS contents of rice grains.

On the contrary, it was shown that the formula developed for estimating the RS based on the pasting property values of conventional indexes using RVA cannot be applied to the estimation of RS widely.

The equation had a determination coefficient (R²) of 0.79 for calibration and 0.55 for validation test, which showed this equation cannot be applied well to unknown samples (data not shown).

Correlation between amylose contents, RS, and pasting properties of 23 Japonica starch samples

Japonica starch samples were Nourin1go, Rikuu132go, Kamenoo4go-a, Nourin8go, Ginbouzu-bansei, Senichi, Hounenwase, Koshiibuki, Nihonkai, Koshihikari, Kirara397, Moritawase, Rikuu20go, Nourin22go, Nourin6go, Jyoshu, Koshijiwase, Todorokiwase, Yoneyama, Senshuraku, Hitomebore, Kamenoo4go-b, and Yumepirika. As shown in Table 3, amylose content is significantly correlated with Max/Fin ratios (r = −0.90) at p < 0.01, and RS is significantly correlated with Max/Min ratios (r = −0.91), BD (r = −0.59), Min. vis. (r = 0.73), Final vis. (r = 0.60), SB (r = 0.52), Max/Fin ratios (r = 0.60), and AAC (r = −0.60) at p < 0.01. A novel index of the ratios of Max/Fin and Max/Min had a higher correlation with AAC and RS content than conventional indexes using RVA.

Table 3. Correlation between pasting properties, amylose contents and resistant starch contents of Japonica starch samples.

	Maxi. vis	Mini. vis	Break.down	Final.vis	Setback2	Pasting temp	Consis-tency	Setback2/Consis	Maxi./Mini.	Maxi./Final	Amylose	RS
Maxi. vis.	1.00
Mini. vis	0.20	1.00
Break.d	0.61^**	−0.65^**	1.00
Final.vis	0.18	0.94**	−0.62^**	1.00
Setback2	−0.50^*	0.70**	−0.95^**	0.76^**	1.00
Pasting.t	−0.32	0.06	−0.30	−0.01	0.21	1.00
Consistency	0.07	0.42^*	−0.29	0.70^**	0.57^**	−0.14	1.00
Setback2/Consis	−0.28	0.63^**	−0.73^**	0.81^**	0.90^**	0.09	0.86^**	1.00
Max. vis/Mini. vis	0.09	−0.83^**	0.74^**	−0.76^**	−0.73^**	−0.11	−0.29	−0.55^**	1.00
Max. vis/Final.vis	0.17	0.01	0.12	−0.19	−0.28	0.08	−0.54^**	−0.50^*	−0.36	1.00
Amylose	−0.29	−0.09	−0.15	0.05	0.24	−0.13	0.33	0.38	0.38	−0.90^**	1.00
RS	0.00	0.73^**	−0.59^**	0.60^**	0.52^*	0.18	0.05	0.30	−0.91^**	0.60^**	−0.60^**	1.00

Notes: Correlation significant at 5% * or 1% ** by t-test.

Storage of rice influences property changes, namely the swelling of starch granule, pasting properties, and thermal properties of rice.⁵¹⁾ The viscosity of rice flour increases dramatically after storage for several months of milled rice as this change depends on storage temperature and duration.^52,53) The Max. vis., Mini. vis., Final vis., and BD values of all samples increased noticeably with the increase of storage time.⁵²⁾ Yasumatsu et al. showed that the increase in amounts of free fatty acids during rice storage resulted in the increase in maximum viscosity of amylogram.⁵⁴⁾

The present study was undertaken to clarify the influences of starch structure and retrogradation on Japonica starch samples in terms of pasting properties by RVA.

AAC of Japonica starch samples were 12.1–20.5 (%) and those of RS content were 0.2–2.0 (%). In case of similar AAC in various rice cultivars, the difference in RS content tends to reflect amylose molecular sizes, as shown in the relationship between Kirara397 (AAC; 16.0%, RS; 1.9%) and Nourin1go (AAC; 17.0%, RS; 0.2%) (Supplemental Table 1). As a result, in case of similar AAC (AAC contains a lot of amylose and a little SLC in amylopectin) in various rice cultivars, the difference in RS content are presumed to be related to SLC in amylopectin.

Max. vis. of Japonica starch samples were 421.5–538.6 RVU, those of Mini. vis. were 99.8–247.8 RVU, those of BD were 289.5–433.5 RVU, those of Final vis. were 191.9–385.0 RVU, those of Pt were 67.5–72.2 °C, those of SB were −152.3 to −322.0 RVU, and those of Cons were 91.3–151.9 RVU. A novel index of SB/Con ratios were −1.1 to −3.1, those of Max/Min ratios were 1.4–5.2 and those of Max/Fin ratios were 1.7–4.0. Among 20 Japonica rice cultivars, Max. vis. and BD of Japonica rice starch samples were increased to 1.29 or 1.65 times compared with Japonica polished rice, on the contrary, Min. vis. and Final vis. of Japonica starch samples were decreased to 0.77 or 0.89 times compared with Japonica polished rice (data not shown).

Formula for estimating the AAC content based on the pasting properties of Japonica rice starch samples

Fig. 3(A) shows the formula developed for estimating the AAC based on the novel index of the pasting properties of Japonica starch samples.

Fig. 3. Formula for estimating the amylose content based on the pasting properties of Japonica starch samples and application of the formula to unknown samples.

Notes: The equation had determination coefficients (R²) of 0.86 based on the calibration. (A) Estimation formula; amylose content (%) = −1.46 × SB/Con – 0.81 × Max/Fin – 3.76 × Max/Mini + 26.72. (1) Nourin1go; (2) Rikuu132go; (3) Kamenoo4go; (4) Nourin8go; (5) Ginbouzu-bansei; (6) Senichi; (7) Hounenwase; (8) Koshiibuki; (9) Nihonkai; (10) Koshihikari; (11) Kirara397. (B) Application of the formula to unknown samples; (1) Moritawase; (2) Rikuu20go; (3) Nourin22go; (4) Nourin6go; (5) Jyoshu; (6) Koshijiwase; (7) Todorokiwase (8) Yoneyama; (9) Senshuraku; (10) Hitomebore; (11) Kamenoo4go; (12) Yumepirika. (B) show that a determination coefficients (R²) of 0.76 was obtained with the application of the estimation formula to 12 unknown samples. Therefore, the validation test showed that the equation can be applied to unknown samples.

The equation had a determination coefficient (R²) of 0.86 based on the calibration. The following formula for estimating the AAC was obtained using 11 samples.

Fig. 3(B) shows that a determination coefficient (R²) of 0.76 was obtained with the application of the above-mentioned formula to 12 unknown samples.

Thus, the validation test showed that the equation can be applied well to unknown samples, which means that it became possible to estimate AAC of Japonica rice simply and easily based on pasting properties by RVA.

On the contrary, it was shown that the formula developed for estimating the AAC based on the pasting property values of conventional indexes using RVA cannot be applied to the estimation of AAC widely.

The equation had a determination coefficient (R²) of 0.37 for calibration and 0.16 for validation test, which showed this equation cannot be applied well to unknown samples (data not shown).

Formula for estimating the RS content based on the pasting properties of Japonica starch samples

The RS content was used as response variables and the parameters from analysis of the novel index of the pasting properties were used as predictor variables in the multiple regression analyses. Fig. 4(A) shows the formula developed for estimating the RS content based on the novel index of the pasting properties of Japonica rice starch samples.

Fig. 4. Formula for estimating the resistant starch content based on the pasting properties of Japonica starch samples and application of the formula to unknown samples.

Notes: The equation had determination coefficients (R²) of 1.00 based on the calibration. (A) Estimation formula; resistant starch content (%) = 0.21 × SB/Con −0.27 × Max/Fin + 0.71 × Max/Mini + 0.46. (1) Nourin1go; (2) Rikuu132go; (3) Kamenoo4go; (4) Nourin8go; (5) Ginbouzu-bansei; (6) Senichi; (7) Hounenwase; (8) Koshiibuki; (9) Nihonkai; (10) Koshihikari; (11) Kirara397. (B) Application of the formula to unknown samples; (1) Moritawase; (2) Rikuu20go; (3) Nourin22go; (4) Nourin6go; (5) Jyoshu; (6) Koshijiwase; (7) Todorokiwase (8) Yoneyama; (9) Senshuraku; (10) Hitomebore; (11) Kamenoo4go; (12) Yumepirika. (B) show that a determination coefficients (R²) of 0.83 was obtained with the application of the estimation formula to 12 unknown samples. Therefore, the validation test showed that the equation can applied to unknown samples.

The equation has a determination coefficient (R²) of 1.00 based on the calibration. The following formula for estimating the RS content was obtained using 11 samples for calibration:

Fig. 4(B) shows that a determination coefficients (R²) of 0.83 was obtained with the application of the above-mentioned formula to 12 unknown samples Thus, the validation test showed that the equation can be applied well to unknown samples, which means that it became possible to estimate RS of Japonica rice starch simply and easily based on pasting properties by RVA.

On the contrary, it was shown that the formula developed for estimating the RS based on the pasting property values of conventional indexes using RVA cannot be used to the estimation of RS widely.

The equation had a determination coefficients (R²) of 0.72 for calibration and 0.16 for validation test, which showed this equation cannot be applied well to unknown samples (data not shown).

Correlation between DP, AAC, RS contents, and pasting properties using an RVA

In addition to amylose, amylopectin chain length distribution affects the eating or cooking qualities of rice.^3,4,9,11,13) The segregation pattern profile for amylopectin chain length demonstrated the enrichment of short chains (DP ≦ 11) and the depletion of intermediate-size chains (12 ≦ DP ≦ 24) in Japonica rice compared with indica rice, and starch granules that contained amylopectin with longer chains were more resistant to gelatinization.⁴⁰⁾ The waxy rice used to produce soft cakes, such as Hakuchomochi, contained more short chains and fewer long chains than that used to produce hard cakes, such as Koganemochi.⁵⁵⁾ In previous studies, we showed that the proportion of short chains was much lower in the ae mutant rice cultivars than the wild-type rice. In contrast, the proportion of longer chains was higher in the ae mutant rice cultivars than the wild-type rice. The present study was undertaken to clarify the influences of starch structure and retrogradation on ae mutant rice (EM10, Hokurikukona243, EM189, EM72, EM129, EM21, and EM16), Japonica rice (Kinmaze), and glutinous rice (Koganemochi and Hakuchomochi) of starch under a novel index included pasting properties using RVA. As shown in Table 4, RS contents had a positive correlation with Fb₃ (DP ≧ 37) (r = 0.98), Fb₁₊₂₊₃ (DP ≧ 13) (r = 0.98), SB/Con ratios (r = 0.96), AAC (r = 0.94), Cons (r = 0.94), and Final.vis. (r = 0.91) at p < 0.01 and negative correlation with Max/Fin ratios (r = −0.95), Fa (DP ≦ 12) (r = −0.91), and Max/Min ratios (r = −0.88), at p < 0.01, which means that RS content was affected higher with the Fb₃ and Fb₁₊₂₊₃ (ratio of long and intermediate-size chains of amylopectin) than Fa (proportion of short chains) and AAC. Moreover, a novel index of the ratios of SB/Con and Max/Fin had higher correlations with RS content than conventional indexes using an RVA, because Fb₁₊₂₊₃ had a higher correlation with SB/Con (r = 0.99) and Max/Fin ratios (r = −0.98) than Cons (r = 0.94), AAC (r = 0.92), Final vis. (r = 0.91), SB (r = 0.85), and Pt (r = 0.76). AAC showed a negative correlation with Fa (r = −0.94) and positive correlation with Fb₃ (r = 0.98), Fb₁₊₂₊₃ (r = 0.92), Final vis. (r = 0.93), Cons (r = 0.91), Min. vis. (r = 0.90), and SB/Con ratios (r = 0.87) at p < 0.01. A novel index of Max/Fin (r = −0.84) ratios had higher correlations with AAC than Pt (r = 0.80) and SB (r = 0.68) of conventional indexes using RVA. Inouchi et al. reported that SLC contents were 1–2% for Koshihikari, 5–7% for Hoshiyutaka, and 13–16% for Yumetoiro, and they showed that a highly positive relationship was observed between the SLC contents and consistency; therefore, SLC in amylopectin seems to have a great effect on the consistency of starch.⁵⁶⁾

Table 4. Correlation between pasting properties, amylose contents, resistant starch contents, and degree of polymerization of 10 rice cultivars starch samples.

	Maxi. vis.	Mini. vis.	Break down	Final vis	Set back2	Pasting temp	Consistency	Consis/Break.d	Max/Minu	Set/Con	Max/Final	Amylose	RS	fa	fb1	fb2	fb3	fb1+2+3	fb1/fa
Max. vis.	1.00
Mini. vis.	0.51	1.00
Breakdown (BD)	0.71^*	−0.25	1.00
Final.vis	0.47	0.98^**	−0.27	1.00
Setback (SB)	−0.31	0.64^*	−0.88^**	0.69^*	1.00
Pasting.temp (Pt)	0.30	0.71^*	−0.25	0.69	0.50	1.00
Consistency (Cons)	0.41	0.91^**	−0.29	0.97^**	0.71^*	0.63^*	1.00
Con/BD	−0.22	0.59	−0.74^*	0.65^*	0.89^**	0.69^*	0.69^*	1.00
Max/Minu	0.11	−0.78^**	0.77^**	−0.78^**	−0.94^**	−0.72^*	−0.75^*	−0.85^**	1.00
SB/Con	−0.03	0.81^**	−0.70^*	0.86^**	0.95^**	0.67^*	0.87^**	0.90^**	−0.95^**	1.00
Max/Fin	0.07	−0.79^**	0.73^*	−0.83^**	−0.96^**	−0.70^*	−0.84^**	−0.91^**	0.98^**	−0.99^**	1.00
Amylose	0.39	0.90^**	−0.30	0.93^**	0.68^*	0.80^**	0.91^**	0.79^**	−0.76^*	0.87^**	−0.84^**	1.00
RS	0.16	0.85^**	−0.52	0.91^**	0.85^**	0.76^*	0.94^**	0.88^**	−0.88^**	0.96^**	−0.95^**	0.94^**	1.00
fa	−0.35	−0.89^**	0.33	−0.89^**	−0.67^*	−0.92^**	−0.85^**	−0.76^*	0.83^**	−0.84^**	0.85^**	−0.94^**	−0.91^**	1.00
fb1	−0.65	−0.58	−0.25	−0.63	−0.15	−0.68^*	−0.66^*	−0.39	0.28	−0.42	0.38	−0.73^*	−0.62^**	0.71^*	1.00
fb2	−0.43	0.40	−0.81^**	0.46	0.85^**	0.40	0.51	0.77^**	−0.78^**	0.82^**	−0.82^**	0.55	0.69^*	−0.53	−0.13	1.00
fb3	0.30	0.89^**	−0.40	0.93^**	0.76^**	0.80^**	0.94^**	0.84^**	−0.83^**	0.92^**	−0.90^**	0.98^**	0.98^**	−0.94^**	−0.71^*	0.62	1.00
fb1+2+3	0.06	0.85^**	−0.63	0.89^**	0.91^**	0.72^*	0.89^**	0.90^**	−0.93^**	0.99^**	−0.98^**	0.92^**	0.98^**	−0.87^**	−0.48	0.78	0.96	1.00
fb1/fa	0.20	0.85^**	−0.47	0.84^**	0.74^*	0.91^**	0.79^**	0.83^**	−0.88^**	0.88^**	−0.88^**	0.92^**	0.90^**	−0.98^**	−0.59	0.59	0.92	0.90	1.00

Notes: Correlation significant at 5% * or 1% ** by t test.

We will try to improve the estimation formulae by the adoption of novel method in statistics and to elucidate the relationship between the physical properties or the palatability of the cooked rice grains and the results of RVA analyses which we reported in the present paper in the next report.

Conclusions

(1)	We searched for the easy and simple method to measure the novel indicators which reflect not only AAC, but also RS based on pasting properties using RVA to Japonica polished rice and starch.
(2)	In Japonica polished rice, estimation formula for AAC and RS content were developed using pasting properties of the novel index using RVA, and these equation showed determination coefficients (R2) of 0.89 and 0.80 for calibration and validation test, which showed that these equations can be applied to the unknown rice samples, because the multiple regression coefficients were 0.71 and 0.75.
(3)	In Japonica rice starch samples, estimation formula for AAC and RS content were developed using pasting properties of the novel index using RVA, and these equation showed determination coefficients (R2) of 0.86 and 1.00 for calibration and validation test showed that these equations can be applied to the unknown rice samples, because the determination coefficients (R2) were 0.76 and 0.83.
(4)	We developed the novel index of the ratios of SB/Con and Max/Fin had higher correlations with RS content than conventional indexes using RVA, because Fb1+2+3 (DP≧13) had a very high correlation with SB/Con (r = 0.99) and Max/Fin (r = −0.98) ratios.
(5)	Novel estimation formulae for AAC and RS contents on the basis of the novel index of pasting properties, which would enable us to evaluate the characteristics of various kinds of Japonica polished rice and starch using an easy and rapid RVA analysis.

Authors’ contributions

SN and KO designed the experiments; SN performed the experiments; SN, JK, and KK analyzed the data; SN and KO wrote the manuscript.

Supplemental materials

The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2015.1088373.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by Research Project of “Strategic Innovation Program”, Council of the Cabinet Japan.

Acknowledgment

We thank to the Niigata Prefectural Agricultural Research Institute for the gift of rice samples. We express our gratitude to Professor Hikaru Satoh for his great help.