Pasting Properties of Various Waxy Rice Flours: Effect of α -Amylase Activity, Protein, and Amylopectin

Waxy rice has a long history of being cultivated and consumed in China. In this study, the eﬀect of diﬀerent factors including α -amylase activity, protein, and amylopectin structure on the pasting properties of four waxy rice varieties were investigated. Rice ﬂours were divided into four groups (Vietnam indica (VI), Jiangxi indica (JI), Anhui japonica (AJ), and Dongbei japonica (DJ) group) and treated with AgNO 3 solution, DL-dithiothreitol (DTT), or protease ( n � 3). Results suggested that both α -amylase activity and protein signiﬁcantly decrease the pasting viscosity of waxy rice ﬂours. Chain length distribution of amylopectin as measured by high performance ion exchange chromatography (HPAEC-PAD) showed that starch with a higher ratio of short chain leads to a higher pasting viscosity. X -Ray diﬀractograms showed that the crystal type of all the four varieties of rice starches were characteristic A-type. Relative crystallinity of each rice starch was further calculated, and higher crystallization resulted in a higher viscosity. Our study would provide a fundamental knowledge of the relationship between diﬀerent factors and waxy starch pasting properties, as well as be a reference for controlling the quality of waxy rice starch-based food products.


Introduction
Waxy (or glutinous) rice (Oryza sativa L.) has a long history of being cultivated and consumed in China. Traditional Chinese waxy rice-based foods include waxy rice ball, sweet green rice ball, waxy rice cake, nuomici, zongzi, etc., [1] while in the modern food industry, waxy rice is usually processed into flour before usage and consumption. e physicochemical properties of the flours especially pasting properties guarantee the appealing texture of its food products.
Many factors could influence the pasting properties of waxy rice flour, such as the endogenous α-amylase activity, proteins, and amylopectin structure [2].
ere is an agreement that endogenous α-amylase strongly impacts the pasting properties of grain flours [3,4], and the in-activation of α-amylase by adding silver nitrate (AgNO 3 ) would enhance their viscosities [4]. However, there were controversial opinions for proteins. Some reported that protein increased the pasting viscosity of rice flour [5,6], while others only discovered a weak correlation between proteins and rice flour. us, more evidence should be documented in this area. Most starches consist of 20-30% amylose and 70-80% amylopectin, ratios vary with the botanical sources [7]. Comparing with that, waxy rice starch contains almost 100% of amylopectin that makes it crucial in its pasting characteristics [8][9][10] and therefore could serve as a good model to study the relationship between amylopectin structure and its pasting properties [2]. e molecular structure of amylopectin has the same basic structure as amylose, while considerably contains more α- (1,6)-branches and shorter chains that contribute to a three-dimensional complex structure. e chain-length distributions were reported to be a main controller of its pasting properties [11]. Although there are a number of studies regarding the relationship between amylopectin chain-length distribution and pasting properties, conflicting results were obtained. Some studies reported that higher proportion of shorter chain (DP [6][7][8][9][10][11][12] and lower proportion of long chain (DP > 37) indicating a higher peak pasting viscosity [12][13][14], while others found out that peak viscosity is positively correlated with the amount of amylopectin medium and long chains. In this aspect, more studies should be focused on chain-length distribution and pasting viscosity in the future.
To our knowledge, the effect of α-amylase activity, protein, and amylopectin structure were often investigated separately in most studies, regarding the relationship between waxy rice starch and its pasting properties. e present work investigated the effect of three factors at the same time, with emphasis on the underlying reason for the difference of pasting properties among different cultivars from North and South China. In addition, the present study was the first to compare the pasting viscosities among the four varieties simultaneously.
us, our study would not only provide a fundamental knowledge of the association between various factors and waxy starch pasting properties but also be a reference for quality control of waxy rice starchbased food to meet the desired texture.

Materials.
Four representative high production waxy rice cultivars from North or South China were selected: Vietnam indica (VI); Jiangxi indica (JI); Anhui japonica (AJ); and Dongbei japonica (DJ). Rice samples were provided by Henan Huangguo Grains Industry Co., Ltd. (Henan, China). All reagents and chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) or Sigma-Aldrich (Bornem, Belgium) unless specified otherwise.

Preparation of Waxy Rice
Flour. Waxy rice flour was prepared by a wet-milling method according to the method suggested by Tong et al [15]. Briefly, rice samples were cleaned and washed three times to remove impurities and then soaked in distilled water at room temperature for 4 h before put into a colloid mill (JM-L50, Shanghai Nuoni Light Industrial Machinery Co. Ltd, Shanghai). e rice slurry was then vacuum filtered and dried in an air-dry oven at 40°C for 12 h, and then grounded, and passed through a 100-mesh screen to obtain the dried rice flour. e flour samples were packed and stored at 4°C for further use.

Composition Analyses of Waxy Rice Flour.
Starch content was determined by PRC National Standard (GB 5009.9-2016) and measured by a Futura continuous flow analyzer (AMS Alliance, France). Crude protein content was estimated by determination of nitrogen (N) content using a Vario EL element analyzer (Elementar, German), and then, N values were converted to protein content by N × 5.95. Crude ash, crude lipid, and moisture content were determined by Chinese Standard GB/T5009.4-2010, GB/ T5009.6-2003 and GB/T5009.3-2010, respectively. All the components were converted to dry base contents based on water content.

Rice Starch Isolation.
Waxy rice starch was isolated by an alkaline method according to the study by Mu et al. [16] with slight modifications. e rice flour was soaked in 0.085 mol/ L NaOH solution with a material to liquid ratio of 1 : 5 g/ml, stirred at 40°C for 4 h, and then centrifuged at 1580 × g for 15 min. e dark yellow supernatant was scraped from the sediment and discarded, and then the sediment was washed with distilled water and adjusted to pH 7.0 with 1 M HCl before being centrifuged for 15 min. e procedure was repeated twice until the sediment was pure white and then lyophilized to dryness to obtain high purity rice starch.

2.5.
Determination of α-Amylase Activity. A colorimetric method as described by Li [17] was hired to determine α-amylase activity. Maltose was used for the standard curve, and 3,5-dinitrosalicylic acid (DNS) was used as a coloring agent. OD values were measured at 540 nm. e α-amylase activity of each waxy rice flours was calculated according to OD values and the maltose standard curve and expressed as micrograms per Gram per minute (mg/(g·min)).

Rapid Viscosity Analysis (RVA).
e pasting properties of the selected four rice flours with and without 0.085 g/L AgNO 3 or 0.77 g/L DL-dithiothreitol (DTT) solution or protease were tested in triplicate with a Rapid Visco Analyzer (RVA) (Newport Scientific, Narrabeen, Australia), using std1 temperature programming according to ICC standard No. 162 and AACC66-21. In addition, pasting properties of rice starches were also tested.

Differential Scanning Calorimetry.
e thermal properties of waxy rice flours and starch were measured by the differential scanning calorimetry (DSC) method using DSC 8500 (Perkin-Elmer Inc., CT, USA). ree milligram waxy rice flour or starch powder (on a dry basis) was accurately weighed and placed into a DSC aluminum sample pan. Distilled water was added to obtain the emulsion with a powder/water ratio of 1 : 3 (w/w). e DSC pans were sealed, equilibrated for 24 h, and then heated from 20 to 100°C at a rate of 10°C/min. e gas flow rate of nitrogen was 40 mL/min. An empty pan was used as a reference.

X-Ray
Diffraction. X-Ray diffractograms were obtained with a D/Max-2200 X-ray diffractometer (Righku Denki Co., Tokyo, Japan) with Cu Kα radiation at 40 kV and 50 mA. e diffractograms were scanned between 5°and 60°(2θ) at a rate of 1.2°/min. Relative crystallinity was estimated by the ratio of the peak areas to the total diffractogram area as described by Chen et al. [18].

Chain-Length Distribution of Amylopectin.
e branch chain-length distribution of amylopectin was measured by high-performance ion exchange chromatography (HPAEC) equipped with a pulse amperometric detector (PAD) as described by Blennow et al [19]. Starch samples were gelatinized and then debranched by isoamylase. e debranched amylopectin solution was filtered through a PVDF membrane filter (0.45 μm) before being injected into the HPAEC-PAD system. Eluents A (0.5 M sodium hydroxide), B (0.5 M sodium acetate), and C (distilled water) were operated at a flow rate of 1 mL/ min. e separation gradient of eluents was as follows: 0 min, 20% A, 10% B, and 70% C; 0-40 min, linear gradient to 20% A and 80% B.

Statistical Analysis.
Each experiment was done in triplicate. Results were expressed as average ± standard deviation (SD). Analysis of one-way ANOVA with Duncan's multiple range test and Pearson's correlation were performed using SPSS software (version 19.0, Statistical Package for the Social Sciences Inc., Chicago, USA). A value of p < 0.05 was considered to be statistically significant.

Composition of Rice Flours.
Total starch, protein, lipid, moisture, and ash contents of the four varieties from North and South China are listed in Table 1. Moisture content was higher in japonica rice than that in indica rice. Japonica rice contains more starch than indica, especially Dongbei japonica (DJ). e protein and starch contents of the four varieties were around 7% and 90%, respectively, which were in accordance with other findings [20,21]. e moisture content of japonica rice was significant higher than that of indica rice (Table 1), which is in line with the findings of Ayabe et al. [22]. Both protein and lipid contents were the highest in Jiangxi indica (JI) among the four cultivars. Ash content in DJ was the highest followed by JI.
ese results indicate that the compositions of the rice may varies with their varieties and growing environment [23].

Gelatinization Temperatures of Waxy Rice Flour and
Starches.
e gelatinization temperatures (T 0 ) as determined by DSC measurement are shown in Table 2. e T 0 value for each sample was in the range of 55-75°C, which was in accordance with other findings [24]. e gelatinization temperatures for the same variety treated by distilled water, AgNO 3 , or DTT seemed not changed according to Table 2, indicating that α-amylase or protein would not affect its T 0 values. In the meantime, the T 0 values of starches from the four varieties were lower than that of their flours, which could be explained by their different starch contents (88-91% in the flours) [24]. Besides, a certain amount of the starch might be damaged during the extraction process that lead to a lower T 0 value [25]. Moreover, as shown in Table 2, the indica rice exhibits significant higher T 0 values in their flours and starches than those of japonica rice because of the different branch chain-length distributions in their amylopectin structures (data are listed in Table 3 and discussed in section 3.5). Higher amount of short chain (DP6-12) and lower amount of long chain (DP > 37) would resulted in a lower gelatinization temperature [12].

Effect of α-Amylase on Pasting Properties of Waxy Rice
Flours. AgNO 3 could serve as an inhibitor for α-amylase to verify the presence of endogenous amylase activity in flours and characterize the pasting properties of flours without enzyme interference [2]. In this study, the pasting properties of different waxy rice flours treated with AgNO 3 were tested, and the flours treated with distilled water were used as the control. Results are shown in Figure 1(a) and are summarized in Table 2. In the meantime, α-amylase activity was measured and results are shown in Table 1. e peak, trough, and final viscosity of all the four varieties showed an increase with AgNO 3 treatment when compared with water treatment (Figure 1(a) and Table 2), suggesting that the endogenous α-amylase of the four varieties decreased their viscosities. Meanwhile, the largest increasement of viscosity was showed in AJ (Figure 1(a)), which was found to possess the highest α-amylase activity among the four varieties (Table 1), indicating that higher activity of α-amylase may contribute to lower viscosity of AJ. is result was in accordance with the findings of Zhu et al. [2], Noda et al. [4], and Cheng et al. [26], since α-amylase catalyzes the endohydrolysis of α-1, 4 glucan bonds and plays a major role during the degradation of native starch granules. Waxy rice flours seem to be sensitive to α-amylase breakdown, even a few internal cleavages of amylopectin would lead to a reduction in molecular weight and result in a lower viscosity [3]. e apparent difference in viscosities observed in the flours with and without enzyme inactivation underscores the effect of amylase activity on the waxy rice flour pasting profile.
To investigate the relationship between α-amylase activity, composition contents of flours, and pasting viscosities, correlation coefficients were further calculated. Significant inverse correlation was observed between α-amylase activity and pasting viscosities including peak (r � −0.958), trough (r � −0.967), and final (r � −0.957) viscosities at p < 0.05, which confirmed the effect of α-amylase activity on the pasting viscosity of waxy rice flours. However, no significant correlation was found between composition contents and pasting viscosities, indicating that the compositions of the waxy rice flour seemed to be not associated with its gelatinization characteristics.

Effect of Protein on Pasting Properties of Waxy Rice
Flours. To investigate the effect of protein on pasting properties of waxy rice flours, DTT or protease was used in our study. Results are shown in Figure 1(b). For DTT treatment, all the flours showed a decrease in their viscosities, among which the peak viscosity of JI decreased most, followed by AJ and DJ. e starch granules break down easily in the absence of rigidity caused by disulfide-bound protein polymers that surround the native granules. DTTcould cleave disulfide bonds between cysteines in the proteins and diminish the carbohydrate binding activity of starch with proteins, leading to an increase of starch to be released from the proteins. e released starch would correspondently get hydrolyzed by the endogenous α-amylase that further resulted in the decrease of viscosity in waxy rice flours. According to Figure 1(b), the decrease of viscosities varied in the four varieties, indicate that other than protein, amylase activities, or other factors may play an additional role. e present study did not find obvious associations between α-amylase activities and the varied decrease of viscosities in the DTT treated flours (Table 2 and Figure 1(b)), which seemed not in consistence with the findings of Zhu et al. [2]. As they reported that α-amylase activity positively influences the decrease of viscosity in the DTT treatment, to clear out the influence of amylase activity and further study the effect of proteins on flours pasting properties, the flours were treated with and without protease in the presence of AgNO 3 ; results are shown in Figure 2. e pasting viscosities were increased in the four varieties in the protease-treated group (Figure 2 left) when compared with the non-protease-treated group (Figure 2 right), indicating that protein had an inhibitory effect on the pasting properties of waxy rice flour. DTTor protease, either by disulfide bond disruption or protein removal, was reported to share similar effects on the pasting properties of waxy rice flours [5].

Effect of Starch and Its Amylopectin Structure on Pasting
Properties of Waxy Rice Flours. Different waxy rice starches showed different pasting properties (Table 2). DJ    Journal of Food Quality  possessed the highest peak viscosity, followed by AJ. e peak viscosity was significantly higher in japonica than indica rice (p > 0.05). Although not statistically significant, the trough viscosity was still higher in japonica than indica rice. For final viscosities and setback, JI was the highest while VI was the lowest. e difference of pasting properties among various waxy rice starches might due to the difference in their structures [12,[27][28][29], such as their branch chain length and the degree of crystallinity in the amylopectin. In our study, the branch chain-length distribution of amylopectin was measured by high-performance ion exchange chromatography (HPAEC); meanwhile, the degree of crystallinity was estimated by the ratio of the peak areas to the total diffractogram area as described by Chen et al. [18]. e X-ray diffraction patterns are presented in Figure 3, and the relative crystallinity of the four waxy rice starches is summarized in Table 3. e waxy rice starches displayed the typical A-type polymorphic form with an unresolved peak at 17°and 18°and individual peaks at 15°, 20°, and 23°in the X-ray diffractograms (Figure 3). e relative crystallinity (Table 3) calculated from X-ray diffraction pattern ranged from 34.5% (JI) to 38.9% (DJ), which was slightly lower than that previously reported [30,31]. is might due to different cultivars of waxy rice were used. Despite that, the amylopectin chain-length distribution also had an impact on relative crystallinity [32]. According to Table 3, japonica rice possessed a higher relative crystallinity than indica rice. e relative crystallinity of DJ was the highest, meanwhile that of JI was the lowest. e relative crystallinity of each starches increased with the amounts of DP 6-12 (Table 3), because short chains (DP < 10) in amylopectin resulted in a decrease in the stability of double helix that leads to a lower value of relative crystallinity [30]. Moreover, the relative amounts of DP 13-24 increased with relative crystallinity confirmed that the semicrystalline nature of starch was ascribed to amylopectin double helices in the crystalline lamellae of the starch granule structure, which was in line with the studies of G. E. Vanderputte et al. [32]. A higher degree of crystallization in waxy starch probably reflects more closely packed crystal structures [3] which may lead to higher viscosities (Table 2). e branch chain-length distribution of amylopectin as measured by HPAEC-PAD are shown in Figure 3. e lengths of the amylopectin chains were reported to have a major impact on rice starch properties [33]. e chain of amylopectin was classified into A chain (DP 6-12), B1 chain (DP 13-24), B2 chain (DP 25-36), and B3+ chain (DP > 37) according to previous research studies [32][33][34]. e pattern of area percentages of amylopectin chains showed a bimodal distribution; DPs of the four varieties of waxy rice starches ranged from 6 to 80 ( Figure 3). All the four varieties displayed two distinct groups with DP 6-33 and DP 34-62 in the chromatogram were observed. Specifically, there were very few short chains of DP6 and a gradual increase in chains of DP 7-9, with a maximum peak at DP 12 and a shoulder at DP 15 in the DP 6-33 group, meanwhile the DP 34-62 group showed a peak at DP 40. ese results were in accordance with previous research that A-type starches had its first peak at DP 12-14 and second peak at DP 41-51 [7]. Among the four varieties, JI possessed the lowest proportion of DP 6-12 chains (27.60 ± 0.23%), indicating that there were fewer short chain A chains in JI starch. DJ possessed the largest DP 6-12 (A chains) and lowest DP > 37 (B3 + chains) proportions, respectively, suggesting the average chain length of DJ was shorter. ese results were in accordance with the findings that indica rice possessed more long chains (DP > 37) than japonica rice [13]. Waxy rice starch with a higher proportion of shorter chain (DP 6-12) and lower proportion of long chain (DP > 37), for example DJ, indicate a higher peak pasting viscosity (Tables 2 and 4) [12][13][14].

Conclusions
In conclusion, the present study showed that the pasting viscosities of the four waxy rice flours could be increased by adding AgNO 3 , while decreased by their own protein.
In addition, the crystal type of the four varieties of rice starches is characteristic A-type. Higher degree of crystallization with a more short chain in the amylopectin structure would lead to higher viscosities. Taken together, the rice protein, α-amylase activity, and amylopectin structure were the major causes of the different pasting properties among various varieties of waxy rice flours. Our study would provide a fundamental knowledge of the mechanism for the discrepancy of different pasting properties of waxy rice flour, transferring this fundamental knowledge to food production by controlling the viscosity of the starch from waxy rice flour-based food products to maintain an appealing texture and meet the requirements of consumers.

Data Availability
All the data regarding this study are presented in this article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.