Retrogradation—Digestibility Relationship of Selected Glutinous and Non-Glutinous Fresh and Stale Cooked Rice

Abstract

This study examined the retrogradation and digestibility relationship of fresh and stale cooked rice of three rice varieties: glutinous (TDK11) and non-glutinous (Doongara and floating rice). The effect of rice variety, degree of milling and retrogradation (staling) of cooked rice on the estimated glycaemic index was determined. Although high-glycaemic index values were obtained for fresh cooked rice of all varieties, staling rice at 4^oC for 24 h showed positive effect on floating rice only, yielding intermediate-glycaemic index. The effect of staling on retrogradation rates was corroborated by changes in x-ray diffraction peaks. The thermal and textural properties of rice samples showed higher pasting temperature, final viscosity, and hardness, and lower peak viscosity and adhesiveness for fresh cooked non-glutinous varieties, which were also significantly affected by degree of milling, in terms of hardness, after retrogradation.

Keywords:

• Digestibility
• Glycaemic index
• Amylose
• Staling
• Retrogradation
• Degree of milling

INTRODUCTION

Today’s consumer is well aware of the physiological aspects of the glycaemic index (GI). The GI is an intrinsic property of carbohydrates in foods, independent of other components of a meal, which defines the increment of blood glucose after the consumption, digestion, and absorption of an available carbohydrate.^[1] Several studies reflect the need of low-GI food, exhibiting important applications in diet formulation, with the aim of obtaining low-GI diets for the prevention or treatment of health disorders like diabetes and cancer, as well as body weight management.

Based on their GI values, carbohydrate-rich foods can be classified as low (GI = <45), medium (GI = 46–69), and high-GI (GI = >70).^[2] The GI value of a particular starchy food may be influenced by the analytical method,^[3] type of food, and the method and level of food processing.^[4] Rice (Oryza sativa L.) is a starchy food, vastly grown and consumed and found in the markets as milled rice or brown rice. The amylose content of milled rice varies from 0.8 to 37% among varieties^[5] and classifies as waxy (<2%), very low (2–12%), low (12–20%), intermediate (20–25%), and high (25–33%) amylose rice.^[6] GI values for rice have been reported to range between 48 and 109.^[7] However, classifying cooked rice as a high-, medium-, or low-GI food can represent a challenge, since it may be influenced by several factors, such as rice variety, amylose content, cooking method and time, processing, and food shape, affecting the amount of available carbohydrate and starch digestibility of cooked rice.^[5]

Processing of rice such as dehulling and milling, involves the removal of important constituents in the milling fraction that act as enzyme inhibitors or barriers that decrease starch digestibility^[8] and lowers GI. Therefore, preference toward consumption of brown rice is increasing, due to its higher nutritional profile and lower GI-value compared to that of milled rice.^[9] The degree of milling (DOM), determines the starch content of the milled grain and alters its amylose content.^[10]

Starch granules in rice are primarily located in the starchy endosperm of the grain. Each starch granule is composed by amylose and amylopectin, creating a helical and semi-crystalline structure.^[11] When starch molecules get in contact with water under cooking conditions, the crystalline structure gelatinizes, producing swollen and soluble granules,^[12] exposing starch for enzymatic hydrolysis, thus increasing starch digestibility.^[8] Gelatinization of starch forms a high shear resistance structure that intensifies upon cooling,^[13] resulting in a firm and viscoelastic gel with decreased solubility, due to a process called retrogradation of starch.^[14]

Retrogradation during cooling and storing of cooked rice, implicates the short- and long-rearrangement of amylose and amylopectin chains, respectively, into a more “crystalline” structure^[15] resulting in a type-3 resistant starch (RS), which is highly related to retrograded amylose.^[16] Hence, post-cooking storage (staling) of rice should reduce the GI value, due to induced retrogradation of starch, resulting in RS formation. The importance of retrogradation, therefore, relies on the production of RS that potentially reduces digestibility of cooked rice resulting in lower GI-values and associated health benefit like lower blood glucose responses. However, not much work has been done estimating the digestion rate of stale rice, and the influence of combined varietal and processing factors.

Therefore three rice varieties—TDK 11 (glutinous) and Doongara (DG) and floating rice (FR; non-glutinous) were selected for the study. Floating or deep water rice, grown predominantly in low-lying areas of Southeast Asia is of great agricultural importance^[17] but has been studied least among all rice types, especially for its GI and other quality characteristics. Hence, the main focus of this research was to study the role of retrogradation/staling on the in vitro digestibility of the three rice varieties subjected to different DOM. Additionally, the study measures and compares the amylose content, texture profile, pasting properties, degree of retrogradation, crystallinity (x-ray powder diffraction; XRD) and GI for all the three rice varieties at different DOM (0, 8–9, and 16–17%).

MATERIALS AND METHODS

Rice Samples

Three rice varieties

TDK11 from Laos, DG from Australia, and FR from Vietnamese origin were selected for this study. Each variety was dehusked with a Satake rice machine, and milled with a Satake mill to obtain 8–9% (D2) and 16–17% (D3) DOMs, respectively. Brown rice was considered 0% DOM (D1) in each variety. DOM was determined using Eq. (1).

(1)

Each variety was ground (<500 μm) at each DOM using a whole grain miller, in order to obtain the corresponding rice flour samples, which were used for rapid visco analyzer (RVA), differential scanning calorimetry (DSC), amylose content, and XRD, while whole grain samples were employed for GI determination and instrumental texture profile analysis (TPA).

Moisture Content

Moisture content of rice flour samples were determined by the standard vacuum-oven method.^[18]

Apparent Amylose Content (AAC)

The AAC of rice flour samples was determined by a colorimetric method.^[19] Amylose content of samples was calculated based on a standard curve over the range of 0 to 40% amylose, obtained by mixing different ratios of pure potato amylose (Sigma A-0512) and waxy maize amylopectin (Sigma S-9679).

Starch Hydrolysis Kinetics and GI Prediction

The method proposed by Goñi et al.^[20] for starch hydrolysis was adapted in this study. Two sets of samples were prepared for each treatment. The first set was analyzed immediately after cooking, while the second set was stored for 24 h at 4^oC to induce retrogradation, and then hydrolyzed. The rate of starch hydrolysis was measured at 30, 90, and 180 min. Glucose concentration was examined with a glucose oxidase-peroxidase kit (Sigma 510-A), and GI values for each treatment were predicted using the non-linear first-order kinetic model.^[20]

Crystallization Properties

The crystallization properties of cooked rice samples were determined using XRD with a Bruker Advance MK III X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) equipped with copper target and graphite monochromators, operating with voltage of 40 kV and current of 30 mA. Samples of rice were cooked at 95^oC with a water to rice ratio of 2:1 until the disappearance of white belly that symbolises complete cooking of rice. Two sets of samples were prepared for each treatment, as described in section 2.1.3. The fresh cooked samples were vacuum-oven dried at 37^oC for 12 h, immediately after cooking, and further ground. The second sets of samples were stored at 4^oC for 24 h after cooking, they were dried and ground in the same way as fresh cooked samples to obtain cooked rice flour. All the samples were analyzed for XRD. The scanning method proposed by Truong et al.^[21] was adopted. The degree of crystallinity (%DC) was calculated using XRD software DIFFRAC.SUITE TOPAS (Bruker Corporation, Germany).

Gelatinization and Retrogradation Properties

Thermal curves were obtained by scanning the rice flour from 0 to 100^oC at a heating rate of 15^oC/min with a DSC (DSC 1, STAR^e System, Mettler Toledo). Raw rice flour (4.0–4.5 mg) with 6 μL Mili-Q water were added into a 40 μL sealed aluminium pans. The samples were rescanned after 7 days of storage at 4^oC. Thermal transitions were expressed as T_o (onset temperature), T_p (peak gelatinization temperature), and T_c (concluding temperature). Gelatinization of starch was identified by the enthalpy (ΔH) obtained from the integrated area under the curve between T_o and T_c, and expressed as J/g. Same settings of DSC were used to scan cooked and stale (24 h at 4^oC) rice flour of each variety at 8–9% DOM. Sample preparation is in section 2.1.4.

Pasting Properties

The pasting properties of rice flour samples were evaluated using a RVA (Model RVA-4, Newport Scientific Pty Ltd, Australia), following the AACC method 61-02.01.^[22] Thermocline software helped to calculate pasting temperature (PT), peak viscosity (PV), trough viscosity (TV), final viscosity (FV), setback (SB=FV-PV), and breakdown (BD=PV-TV).

Instrumental TPA

TPA was performed on fresh and retrograded (4^oC for 24 h) cooked rice samples. The rice was cooked in the same way as in section 2.1.4. A texture analyzer (TA.XT Plus, Stable Micro system, Arrow Scientific Pty., Ltd) with 35 mm diameter cylinder aluminium probe was used to compress the rice to a 2 mm distance for 5 s, with pre-test and test speed of 1.0 mm/s and post-test speed of 5.0 mm. Parameters derived from test curves were hardness (HA) and adhesiveness (AD).

Data Analysis

All measurements were done in duplicates and the data presented is the mean. The effect of variety and DOM on each variable was evaluated by two-way analysis of variance (ANOVA), using Statistix 8.1 (Analytical Software^©, Tallahassee, Florida). One-way ANOVA and Tukey method were employed to determine significant differences between fresh and stale samples for estimated GI values and TPA parameters (overall 5% significance), using Minitab^® R16 (Minitab Inc, Chicago).

RESULTS AND DISCUSSION

Amylose Content

The results showed a significant difference (p < 0.05) in %AAC among the three rice varieties, ranging from 18.62–24.00 for FR, 13.85–16.82 for DG, and 4.17–4.68 for TDK11, confirming them as intermediate-, low-, and very low-amylose rice, respectively, as per the classification proposed by Juliano and International Rice Research.^[6] Rice varieties from different geographical areas^[7] and singular grain positions in the panicle^[23] may present different amylose content, varying from 0.8 to 37%.^[5]

The effect of DOM on the %AAC is presented in Fig. 1. No significant difference (p > 0.05) was found in %AAC for TDK11 when increasing the DOM, possibly due to the higher ratio of amylopectin to amylose present in glutinous rice cultivars, which limits the vacant space for the synthesis of amylose-chains throughout the starch granule.^[5] On the contrary, for DG, a significant difference (p < 0.05) was only found when increasing the DOM from D1 to D3 while for FR when increasing the DOM up to D2 (Table 1). These results agree with the findings of Itani et al.^[24] They found higher amylose contents in the fractions of the inner part of non-glutinous rice kernels, which may have contributed to an increase in the starch content due to a homogeneous ratio of amylose and starch content within a grain.

TABLE 1 Experimental results for moisture content (%MC), apparent amylose content (%AAC), estimated GI values, and pasting properties of selected rice varieties at different degree of milling (DOM)

				Estimated GI		Pasting properties
Variety	DOM (%)	MC* (%)	AAC (%)	0 h	24 h	PT (^oC)	PV (mPa-s)	TV (mPa-s)	BD (mPa-s)	FV (mPa-s)	SB (mPa-s)
Floating rice (FR)	0	11.86 ± 0.53	18.62 ± 1.1b	94.42 ± 2.62d	68.78± 0.28e	79.58 ± 0.60a	1678.5 ± 6.36e	1476 ± 2.83bc	202.5 ± 9.19h	3125 ± 11.31bc	1649 ± 8.49de
	8-9	11.14 ± 0.49	22.46 ± 1.1a	82.39 ± 1.83e	52.00 ± 1.03f	79.13 ± 0.18ab	2279 ± 82.02c	1855.5 ± 61.52a	423.5 ± 20.51g	3959 ± 113.14a	2103.5 ± 51.62a
	16-17	11.44 ± 0.72	24.00 ± 1.1 a	84.33 ± 1.70e	51.57 ± 0.13f	78.7 ± 0.64ab	2450 ± 25.46bc	1935 ± 72.12a	515 ± 46.67f	3756 ± 63.64a	1821 ± 8.49b
Doongara (DG)	0	11.02 ± 0.09	13.85 ± 0.7d	126.67 ± 3.21ab	105. 97 ±0.13c	77.08 ± 0.53bc	1870.5 ± 95.46d	1167.5 ± 103.94d	703 ± 8.49e	2957.5 ± 136.47c	1790 ± 32.53bc
	8-9	10.75 ± 0.00	15.88 ± 0.0cd	119.94 ± 1.94b	105.08 ± 1.88cd	77.15 ± 0.49bc	2365.5 ± 40.35bc	1494 ± 7.07b	871.5 ± 33.23d	3216.5 ± 6.36b	1722.5 ± 0.71cd
	16-17	11.33 ± 0.51	16.82 ± 0.4bc	128.61 ± 2.52a	101.98 ± 1.00d	75.18 ± 1.10c	2530.5 ± 19.09b	1527 ± 18.38b	1003.5 ± 0.71c	3121 ± 19.80bc	1594 ± 1.41e
TDK11	0	11.96 ± 0.12	4.17 ± 0.1e	105.97 ± 2.69c	111.61 ± 0.52b	68.05 ± 0.00d	1922.5 ± 13.44d	883 ± 2.83e	1039.5 ± 10.61c	1111 ± 7.07e	288 ± 4.24f
	8-9	13.03 ± 0.15	4.17 ± 0.1e	119.78 ± 0.25b	123.62 ± 0.13a	68.08 ± 0.04d	2490.5 ± 8.49b	1171 ± 2.83d	1319 ± 5.66b	1443.5 ± 0.71d	272.5 ± 3.54f
	16-17	12.54 ± 0.57	4.68 ± 0.1e	120.30 ± 0.74ab	125.27 ± 0.91a	67.7 ± 0.49d	2712.5 ± 6.36a	1290.5 ± 3.53cd	1422 ± 9.89a	1565 ± 4.24d	274.5 ± 0.71f

*%MC of rice flour samples.

ND: not determined; 0 h, 24 h: fresh and staled (4^oC for 24 h) cooked rice samples. Values sharing same letters are non-significantly (p > 0.05) different.

Figure 1 Effect of DOM on the apparent amylose content (AAC) of the selected rice varieties: TDK11, DG = Doongara, and FR = Floating rice.

Pasting Properties

PT refers to the temperature at which the initial rise in the viscosity of the suspension takes place, and is an indicator of the minimum temperature needed to cook rice.^[25] Overall PT was significantly different (p < 0.05) for the three selected rice varieties. From Table 1, an increase in PT was observed when increasing %AAC. This may be attributed to the compositional structure of starch granules differing between waxy and non-waxy rice varieties, as amylopectin contributes to starch swelling, while amylose inhibits it, maintaining the starch granule integrity during heating.^[25] This results in a smoother curve for PT in TDK11, compared to FR and DG, as shown in Fig. 2. In terms of combined effects, PT in TDK11 was significantly different (p < 0.05) from DG and FR at each DOM tested, with no significant effect (p > 0.05) of DOM within each variety, even though an apparent decrease in PT can be observed in Table 1.

Figure 2 RVA graphs: Viscosity (mPa-s) vs. Time (min) for selected rice varieties (TDK11, DG = Doongara, FR = Floating rice) at different DOM (D1 = 0%, D2 = 8–9%, D3 = 16–17.

PV is recorded when swelling and shear are balanced.^[26] The general trend of PV is presented in Table 1. An overall significant difference (p < 0.05) was found in PV due to the effect of variety, showing an increasing tendency with the decrease of %AAC. Waxy rice swells more than non-waxy varieties,^[25] providing greater thickness to the mixture due to a higher content of amylopectin, which does not associate or form chemical linkages as amylose.^[27] Similar results were also reported by Suwannaporn et al.^[28]

When considering the combined effects of variety and DOM, only D1 was significantly lower (p < 0.05) for all the three rice varieties, in comparison to the rest of treatments. However, when increasing the DOM to D2 and above, no significant difference (p > 0.05) was found within each variety, except for TDK11 (D3), which was significantly (p < 0.05) higher than TDK11 (D2) and the rest of treatments. Similarly, higher PV have been reported by increasing the DOM in medium- and long-grain rice,^[10] which was attributed to the loss of surface and total lipid content resulting from milling.

TV and BD

The composition of the paste after reaching PV, determines its ability to withstand constant heating and shear stress, which is represented by the BD viscosity,^[29] calculated as the difference between PV and TV, refer to Table 1. BD differed significantly (p < 0.05) among varieties, decreasing in inverse relationship with %AAC. In addition, a significant (p < 0.05) increase of BD viscosity was observed with the increase of DOM in each variety. Similar results have been described for other rice varieties.^[30] BD has been related to the firmness and amylose content of the paste,^[26] providing an indicator for rice processing conditions, since lower BD values in high-amylose varieties, reflect a higher stability of the starch granules.

FV and SB

The FV is commonly used to define the sample’s quality, indicating the behavior of the mixture after cooking and cooling.^[29] SB viscosity, which was calculated as the difference between FV and TV, refers to the re-association of starch during cooling, and has been correlated to the texture of various products.^[29] In this study, both FV and SB were significantly higher (p < 0.05) for FR, followed by DG and TDK11 (Table 1). This can be explained by the setting of amylose during cooling, with the formation of hydrogen cross-bonds among amylose, resulting in a gel network. While, amylopectin exhibits less propensity to reassociate, producing viscous pastes only.^[27]

With DOM from Table 1, significantly (p < 0.05) lower FV was observed for D1 within each variety, compared to D2. However, no consistent effect was observed at increasing DOMs for DG and FR, endorsing the findings reported by Perdon et al.^[10] for other non-glutinous rice varieties. Similar inconsistent effect was found for SB; except for TDK11, that showed no significant (p > 0.05) difference by increasing DOM, which may be attributed to the non-effect of DOM in the %AAC of TDK11, as shown in Fig. 1.

Estimated GI Values for Fresh Cooked Rice

Table 1 presents the GI values of freshly cooked rice of the three rice varieties that were significantly (p < 0.05) higher for DG, followed by TDK11 and FR. The results ranged from 119.94–128.62, 105.97–120.30, and 82.39–94.92 for DG, TDK11, and FR, respectively, which is considered as high-GI foods, as per Wong and Chung^[2] classification. The higher GI-values for DG compared to TDK11 was an unexpected result of this experiment. However, studies identifying DG as a medium-GI food have reported intermediate- to high- amylose contents,^[31,32] while low %AAC was determined in the DG sample used in this research, as reported in section 3.1.1. Even though it has been argued that higher content of amylose increases the resistance to digestion of starchy foods, inconsistent effect of amylose content on the GI values has been reported,^[11,21] implying that amylose content alone cannot be considered a sufficient predictor of GI. In fact, the digestibility of starch may be affected by factors that reduce the activity of hydrolytic enzymes and the exposure of starch substrates to such enzymes.^[33]

Estimated GI Values for Cooked Rice after Staling

The effect of induced retrogradation, after staling fresh cooked rice at 4^oC for 24 h, on the estimated GI value was also analysed and presented in Table 1. Overall, the retrograded GI value was significantly different among varieties (p < 0.05), showing an inverse relationship with %AAC. Rice varieties with higher amylose content, present increased retrogradation, which may be associated with the ability of amylose to readily reassociate into a more crystalline structure.^[25]

In terms of DOM from Table 1, a significant (p < 0.05) decrease of estimated GI in FR was observed when increasing DOM up to D2, with no effect observed with further increase in DOM. On the other hand, DG exhibited a significant (p < 0.05) decrease of estimated GI value only when increasing DOM from D1 to D3. This might be a consequence of the distribution of amylose in each variety, according to the DOM, as described in section 3.1.1. Retrograded amylose is associated with the formation of 3 type RS,^[8,16] reducing the digestibility of starch, and hence, resulting in lower estimated GI values, compared to that of fresh cooked rice.

The lower amylose to amylopectin ratio present in glutinous rice varieties, gives amylopectin an increased surface area, compared to amylose, exposing its branched chains to enzymatic activity.^[8] Since no significant increase of %AAC was detected in the inner part of TDK11 grains, a higher hydration and gelatinization rate in milled rice^[9] may explain the significant rise in the GI value of TDK11 when increasing DOM to D2, with no further significant impact in higher DOMs (p > 0.05), Table 1.

Comparison of GI Values between Fresh and Retrograded Cooked Rice

The estimated GI values for fresh and stored cooked rice is presented in Table 1. For the case of DG and FR, significant decrease of estimated GI values after staling of cooked rice was observed. Even though the estimated GI value decreased for both non-glutinous rice varieties, retrograded DG still falls into the classification of high- GI foods, while retrograded FR can be classified as medium GI- food. The different classifications given to each variety are related to the higher amylose content in FR, compared to DG, and of the higher estimated GI value obtained for DG in freshly cooked rice.

On the contrary, an increased estimated GI value was obtained after staling the glutinous rice variety (TDK11), compared to that obtained for the fresh cooked sample. Although this result was not expected, related literature has reported similar findings for glutinous, freshly cooked rice and instant rice with same staling conditions as used in this study.^[34] Furthermore, Chung et al.^[35] reported no reduction in estimated GI value for retrograded waxy rice starch up to 4 days of cold storage, suggesting an increased storage time under cooling conditions to achieve a significant reduction in the estimated GI value.

Gelatinization and Retrogradation Properties

The gelatinization transition temperatures: onset (T_o), peak (T_p), conclusion (T_c), and enthalpy of gelatinization (ΔH_gel) of raw rice flour samples are presented in Table 2. These properties are influenced by the molecular distribution of the crystalline region, which relates to amylopectin short chains distribution (degree of polymerization DP6-11).^[36] Overall, highest T_o and T_p were recorded for FR with intermediate %AAC, followed by DG and TDK11 with low- and very-low %AAC, respectively. FR had higher T_c than TDK11, while DG presented values in between, with no significant difference (p > 0.05) from the other two varieties. The lower gelatinization temperatures of glutinous TDK11 compared to non-glutinous rice varieties agreed with the lower PT obtained with RVA. Comparable results were obtained for waxy rice with shorter long branched chains.^[37] On the contrary, inverse trend was observed for ΔH_gel, since TDK11 had higher ΔH_gel than both non-glutinous varieties. The higher ΔH_gel recorded for TDK11 compared to non-glutinous rice varieties, represents a greater percentage of crystallinity of amylopectin in native starch of rice flour.

TABLE 2 Gelatinization, retrogradation, and degree of crystallinity (%DC) of selected flour of raw and cooked rice varieties

			Temperature (^oC)			Enthalpy (J/g)	Temperature (^oC)					DC (%)
	Samples	DOM (%)	To	Tp	Tc	ΔHgel	Tor	Tpr	Tcr	Enthalpy (J/g) ΔHr	%Ret	0 h	24 h
Raw samples	Floating rice (FR)	0	72.27 ± 0.06a	77.63 ± 0.15a	84.68 ± 0.35a	6.30 ± 0.11c	43.49 ± 0.14a	54.64 ± 0.52a	85.71 ± 0.88a	5.40 ± 0.04abc	85.71 ± 0.88a	ND	ND
		8-9	72.07 ± 0.15ab	77.09 ± 0.20ab	83.33 ± 0.06ab	6.41 ± 0.34c	43.58 ± 0.01a	54.61 ± 0.17a	90.39 ± 5.34a	5.79 ± 0.04ab	90.39 ± 5.34a	3.27	5.69
		16-17	71.52 ± 0.28b	76.73 ± 0.01b	83.79 ± 0.01ab	6.20 ± 0.01c	43.84 ± 0.12a	54.63 ± 0.16a	96.05 ± 0.79a	5.95 ± 0.04a	96.05 ± 0.79a	ND	ND
	Doongara (DG)	0	69.55 ± 0.13c	75.84 ± 0.19c	83.66 ± 0.59ab	6.58 ± 0.15c	41.46 ± 0.20ab	53.38 ± 0.18ab	84.81 ± 8.15a	5.57 ± 0.41ab	84.81 ± 8.15a	ND	ND
		8-9	68.86 ± 0.07d	75.35 ± 0.16cd	82.83 ± 0.13ab	6.39 ± 0.25c	42.54 ± 0.30ab	53.89 ± 0.17ab	85.41 ± 2.65a	5.45 ± 0.04ab	85.41 ± 2.65a	3.64	5.06
		16-17	68.95 ± 0.08d	74.97 ± 0.01d	82.52 ± 0.11ab	6.23 ± 0.18c	43.25 ± 0.25ab	53.87 ± 0.18ab	92.83 ± 4.11a	5.78 ± 0.09ab	92.83 ± 4.11a	ND	ND
	TDK11	0	63.55 ± 0.21e	71.85 ± 0.20e	82.89 ± 0.24ab	8.28 ± 0.18a	40.38 ± 1.22ab	51.29 ± 0.00c	58.51 ± 6.05b	4.85 ± 0.61bcd	58.51 ± 6.05b	ND	ND
		8-9	63.47 ± 0.06ef	71.47 ± 0.02ef	81.67 ± 0.21b	7.66 ± 0.20ab	41.41 ± 0.19ab	52.53 ± 0.33bc	56.36 ± 2.47b	4.32 ± 0.08d	56.36 ± 2.47b	0.00	0.00
		16-17	62.93 ± 0.11f	70.88 ± 0.20f	82.38 ± 1.60ab	7.33 ± 0.06b	39.81 ± 2.41b	51.14 ± 0.86c	59.91 ± 3.94b	4.39 ± 0.25cd	59.91 ± 3.94b	ND	ND
Cooked samples	Floating rice	8-9	NEP		NEP	48.98 ± 1.00a	59.92 ± 0.03b	67.46 ± 0.97a	3.33 ± 0.73a	-	0		4-5
	Doongara	8-9	NEP		NEP	48.45 ± 1.07a	58.27 ± 0.16a	64.93 ± 0.59a	2.26 ± 0.36a	-	0		4-5
	TDK11	8-9	NEP		NEP	NEP		NEP	-			0	0

Values sharing same letters are non-significantly (p > 0.05) different.

NEP: No enthalpy peak; 0 h, 24 h: fresh and staled (4^oC for 24 h) cooked rice samples.

Values sharing same letters are not-significantly different (p > 0.05).

In terms of interactive effect of variety and DOM, decreased gelatinization T_o and T_p were observed only when reaching D3 for the three varieties, except for DG, which presented decreased T_o beyond D2. With T_c, no significant difference (p > 0.5) was found within DOM in each variety. For ΔH, no difference was recorded within DOM for FR and DG, whereas lower ΔH_gel was obtained for TDK11 D3, compared to D1.

For the retrograded properties, lower transition temperatures and ΔH_r were obtained for all treatments, compared to those of gelatinization. Usually, ΔH_r is 60 to 80% lower than ΔH_gel, and transition temperatures range between 10 to 26°C less than those obtained for gelatinization,^[25] due to a change of the crystalline-amorphous form after retrogradation. However, only about 40% lower ΔH after retrogradation for TDK11 and 10% for FR and DG were obtained in this study, suggesting a possible experimental error. Though, 30 and 20% less ΔH have been reported after retrogradation under same storage conditions for other starches,^[37] attributable to variability of branch structures and chain length of amylopectin, as reported by the authors.

In this study, both non-glutinous rice varieties presented overall higher transition temperatures T_or, T_pr, T_cr, and ΔH_r than glutinous TDK11, indicating an increased crystallinity with time. No effect of DOM was observed within each variety. In general, these results agree with XRD patterns (Fig 3), showing increased crystallinity for non-glutinous rice varieties after 24 h of staling (discussed in section 3.4). No comparison can be done with TDK11, since a storage time longer than 24 h may be needed for glutinous varieties. The rate of retrogradation after 7 days of cold storage was higher for FR (85.71–96.05%) and DG (84.81–92.83%), compared to TDK11 (56.36–59.91%), with no significant (p > 0.05) difference between the non-glutinous rice varieties. This implies faster retrogradation of non-glutinous rice varieties, compared to TDK11 during storage.

Figure 3 XRD patterns of fresh and stored (4^oC for 24 h) cooked rice at 8–9% DOM: (a) FR, (b) DG, (c) TDK11.

%DC

The XRD patterns for fresh and stale cooked rice samples are presented in Fig. 3. The low intensity of some crystalline regions may be attributed to the formation of small crystallites.^[38] For the two non-glutinous rice varieties, two peaks were observed at 13° and 20° (2θ), with an additional peak at 7° (2θ) in DG. The occurrence of such peaks represents the V–type crystals of amylose-lipid complexes remaining after cooking, which tend to be higher for starches with higher amylose contents.^[39] Both non-glutinous rice varieties showed %DC of 0% and 4–5% for fresh and stale cooked samples, respectively, indicating the corresponding amorphous and recrystallized states. The small changes in %DC between fresh and stale cooked samples (see Table 2) agree with the non-effect of staling on the amylose-lipid complexes reported by Rewthong et al.^[34] In addition, the peaks at 5–6, 15, 17, 22, and 24° (2θ) that were observed in both non-glutinous varieties (more intense in FR) correspond to a B-type structure,^[40,41] which is typical of retrograded starch.^[38] No crystalline regions were observed for TDK11 at 0 h or after 24 h of staling. These results agreed with the DSC of staled TDK11, and explain the non-effect of 24 h staling in the reduction of estimated GI value and textural properties analyzed in this study.

In order to corroborate the crystalline properties of cooked and stale samples, DSC was performed and the results for cooked rice flour showed no enthalpy peaks for all varieties at 8–9% DOM, as presented in Table 2, indicating the amorphous state of the samples after cooking, where all the starch had been gelatinized. In addition, post-cooking holding time of 24 h at 4°C had no effect on retrogradation for TDK11, since no enthalpy peak was obtained, representing no recrystallization of starch. Whereas, for non-glutinous rice varieties, no significant differences (p > 0.05) were found for T_or, T_cr, and ΔH_r, although T_pr was higher for FR, compared to DG, denoting the recrystallized state of amylose after 24 h of cold storage similar to what was observed through XRD.

HA and AD of Fresh Cooked Rice

The results for TPA parameters are presented in Table 3. It is well-known that amylose determines the texture of cooked rice, having a positive correlation with HA, and negative correlation with AD. In this study, HA was only affected by varietal difference, with no effect of DOM. From Table 3, the HA of fresh cooked TDK11 rice was significantly (p < 0.05) lower than DG and FR, with no significant (p > 0.05) difference between the two later varieties. Other studies have also reported similar results.^[28] No significant (p > 0.05) difference was caused by effect of DOM in each variety, which can be attributed to the reduced size of bran layer present in brown rice, which is only about 0.19 to 0.39 mm.^[42] Although different studies have reported a decrease in HA by increasing DOM,^[30] no effect has also been reported by Saleh and Meullenet.^[43] Variation of results may be attributed to the effect of different cooking conditions employed in each experiment, and water content of cooked rice.^[44,45] In terms of AD, only varietal effect was found, being significantly (p < 0.05) higher for TDK11, compared to DG and FR, see Table 3. These results indicate that the AD behavior depends on the starch structure of each variety, while DOM only increases the proportion of starch, as mentioned by Perdon et al.^[10] In the case of TDK11, a significant (p < 0.05) increase of AD was found when increasing DOM from D1 to D3 only. While no significant (p > 0.05) difference was observed for DG and FR between varieties and within DOMs. Similar results for AD values were reported by Saleh and Meullenet.^[43]

TABLE 3 Texture profile analysis (TPA) parameters

Variety	DOM (%)	HA (0 h) (N)	HAr (24 h) (N)	AD (0 h) (N)	ADr (24 h) (N)
Floating rice (FR)	0	25.17 ± 0.78abc	36.12± 0.92bc	–0.04 ± 0.03a	–0.01 ± 0.002a
	8-9	29.88 ± 1.77a	39.74 ± 1.19b	–0.07 ± 0.002a	–0.03 ± 0.01a
	16-17	33.93 ± 0.93a	47.88 ± 1.57a	–0.08 ± 0.00a	–0.02 ± 0.003a
Doongara (DG)	0	23.64 ± 1.45abc	29.05 ± 0.45d	–0.07 ± 0.01a	–0.05 ± 0.01a
	8-9	27.17 ± 2.42ab	34.25 ± 2.53c	–0.13 ± 0.02a	–0.10 ± 0.01a
	16-17	25.11 ± 3.10abc	38.68 ± 1.04bc	–0.35 ± 0.03ab	–0.14 ± 0.08a
TDK11	0	15.15 ± 4.70c	11.76 ± 1.04e	–0.98 ± 0.35bc	–0.84 ± 0.31b
	8-9	18.00 ± 1.05bc	12.34 ± 0.44e	–1.73 ± 0.27cd	–1.59 ± 0.00c
	16-17	23.97 ± 4.54abc	16.23 ± 0.65e	–2.01 ± 0.40d	–1.86 ± 0.01c

Values sharing same letters are non-significantly (p > 0.05) different.

HA and AD of Retrograded Rice

As presented in Table 3, the HA after induced retrogradation (HAr) differed significantly (p < 0.05) among varieties, increasing proportionally with the %AAC. Hence, TDK11 presented the lowest HAr, followed by DG and FR. No significant (p > 0.05) effect was observed by increasing the DOM in TDK11; whereas increased HAr was recorded for DG and FR by increasing the DOM, due to a higher content of amylose present in the core of non-glutinous rice varieties, which retrogrades to form a gel network, increasing its firmness. AD after induced retrogradation (ADr) was still significantly (p < 0.05) higher for TDK11, compared to DG and FR. While no significant (p > 0.05) difference was observed for DG and FR between varieties and within DOMs, a significant (p < 0.05) increase of ADr was detected in TDK11 when increasing DOM up to D2, with no further effect (p >0.05) beyond D2.

Comparison of TPA between Fresh and Retrograded Cooked Rice

When comparing TPA parameters of fresh and stored samples from Table 3, no significant (p > 0.05) effect was found in HA and AD for TDK11 at any DOM; a significant (p < 0.05) increase in HA, and decrease in AD was observed for DG, only when increasing DOM to D3; whereas significant (p < 0.05) increased HA was observed for each DOM in FR, although AD was significantly (p < 0.05) reduced only at D3. These results can be explained due to higher rate of retrogradation occurring during low-temperature storage conditions of non-glutinous rice varieties,^[46] as confirmed in section 3.3. The increase %AAC in milled rice of FR and DG suggest greater formation of RS due to retrogradation, and hence increased HA. Retrogradation of starch during storage is less expected in glutinous rice varieties due to the absence of amylose. In addition, the decreased AD of non-glutinous milled rice after storage may be attributed to the retrogradation of leached solids (specially amylose), occurring within less than 1 day.^[47]

CONCLUSIONS

The AAC in non-glutinous rice varieties (FR and DG) increases with DOM. The three selected rice varieties, differing in the AAC are classified as high-GI foods when digested fresh, implying that amylose content alone cannot predict the GI value of cooked rice. However, after staling the rice for 24 h at 4^oC, only FR, containing intermediate AAC, yielded a lower GI value, from high to intermediate, suggesting a higher rate of retrogradation. In addition, stale milled non-glutinous rice with increased amylose content has lower starch digestibility, compared to brown rice. On the other hand, longer staling time may be required for glutinous rice varieties, such as TDK11, to obtain significant reduction on the estimated GI value. The AAC affects pasting properties and textural parameters, useful to predict the cooking conditions and textural properties of rice, respectively. Rice varieties with higher amylose content differed in quality, compared to glutinous rice varieties. Staling the cooked rice of intermediate and high amylose content under refrigerated conditions for 24 h may reduce its glycaemic response, albeit affecting its quality. Further analysis of amylose to amylopectin ratio, and amylopectin branching properties should be carried out for better understanding of gelatinization, retrogradation, and crystallization properties influencing starch hydrolysis and GI values of cooked rice.

ACKNOWLEDGMENTS

The authors would like to thank Mr. Malik Adil Nawaz for technical support.

FUNDING

The authors wish to thank the National Government of the Republic of Ecuador and its Secretariat of Higher Education, Science, Technology and Innovation (SENESCYT) for funding the scholarship.

Additional information

Funding

The authors wish to thank the National Government of the Republic of Ecuador and its Secretariat of Higher Education, Science, Technology and Innovation (SENESCYT) for funding the scholarship.