Pasting properties of stored rice with ascorbic acid before or after storage

ABSTRACT

Ascorbic acid was applied to the rice before or after storage, and the effects on pasting properties were investigated. Ascorbic acid pretreatment accelerated rice aging during storage, which may be attributed to ascorbic acid as a pro-oxidant agent. Meanwhile, ascorbic acid post-treatment improved the pasting properties of pretreated-aged rice due to the increased volume fraction of larger particles near 250 μm in gelatinized aged rice, and this characteristic was confirmed by microscopy observation. Therefore, ascorbic acid is useful to regulate the pasting properties of stored rice over a range of storage periods for various uses.

KEYWORDS:

• Rice aging
• Pasting properties
• Accelerated aging
• Relieving aging
• Quality control

Introduction

Rice aging is a problem that is still not fully elucidated in cereal science field. To study the mechanisms of rice aging, aged rice needs to be prepared using a storage procedure. However, it takes a long time for new rice to become aged rice, even with high-temperature storage. Therefore, accelerated aging is usually needed to obtain experimental material for aging research. Another demand for accelerated aging is the acceptance of eating quality of the cooked rice for certain regional peoples. South Asian populations strongly dislike freshly harvested rice and prefer aged rice.^[1–5] It is even believed that new rice does not cook well, is not digested well, and gives rise to digestive problems.^[6] In addition, accelerated aging of rice by chemical methods is an alternative route for physical modification of cereal starch into resistant starch or slowly digestible starch, in light of contemporary concerns about type 2 diabetes and metabolic syndrome.^[7,8]

At the same time, the consumers in northeast Asia have an equally hearty dislike of aged rice.^[5,9] It is said that aging yields cooked rice with an undesirable hard and dry texture and a “stale” flavor.^[10,11] Moreover, rice flour is one of the most suitable cereal flour for gluten-free bakery products necessary in the diet of celiac patients.^[12] Therefore, controlling the characteristics of rice flour might be an important method to improve the quality of rice cake or bread.^[13] Because aging profoundly affects consumer acceptance and the processing behavior of rice, further research to understand this phenomenon is necessary. Therefore, methods to accelerate, prevent, delay, relieve, and preferably reverse the aging of rice must to be developed. In addition, this is an entry point for further understanding of the mechanisms of rice aging.

Several hypotheses have been proposed for the mechanism of rice aging, including enzyme action, starch changes, fat oxidation, changes in nonstarch polysaccharides and cell walls, and protein changes.^[14] However, an increasing amount of research supports the hypothesis of protein changes. Disulfide cross-linking of glutelin in rice was reported to be the main reason for rice aging.^[15–17] Oxidation of proteins during rice storage, especially of sulfhydryl group to disulfide, is closely correlated to aging changes.^[15,18] Changes in albumin and globulin during rice aging were demonstrated to be the main causes of changes in pasting properties.^[19] Protein oxidation and changes in interactions with starch during rice aging have also been characterized by infrared and Raman spectroscopies.^[20] Therefore, any factors that could cause protein changes would likely regulate the pasting properties of rice. Ascorbic acid is a food-grade additive, environmentally “green”, safe, and cheap. Ascorbic acid is a reducing agent and a pro-oxidant in vitro when its concentration falls below a certain level.^[21,22] In general, ascorbic acid can have a pro-oxidant role in aqueous media when its concentration is below 10^–5 mol ⋅ L^−1.[23] At a concentration of ascorbic acid higher than 10^–3 mol ⋅ L⁻¹, it plays an antioxidant role.^[23] The dual roles for ascorbic acid are typical in aqueous media.^[22,24] However, to our knowledge, no literature has been reported related to the dual roles of ascorbic acid on rice aging. In this paper, the dual roles of ascorbic acid on the pasting properties of stored rice and its mechanism were investigated, facilitating an enhanced understanding of the essence of rice aging, and providing a new method to regulate the pasting properties of stored rice rapidly and conveniently.

Materials and methods

Materials

Freshly milled non-waxy japonica rice, Oryza sativa L. cultivar Wuyujing 3, grown in Xuyu County, Jiangsu Province, China, was used in this work. The chemical composition of rice starting material was moisture 14.08 ± 0.08%, starch 77.10 ± 0.69%, protein 6.69 ± 0.04%, and lipid 0.79 ± 0.05%. Ascorbic acid, dithiothreitol, and N-ethylmaleimide (NEMI) of analytical grade were purchased from the Sino Pharm Chemical Reagent Co. Ltd. (Shanghai, China).

Ascorbic acid pretreatment of new rice and storage aging

Freshly milled rice was pretreated by spraying an ascorbic acid solution of 2.5 mol ⋅ L⁻¹ (25 mL solution/kg rice), according to the equivalent concentration of pretreatment^[25] and maximum water uptake volume of rice grain based on the trial. The pretreated rice was dried at room temperature until the moisture content was 14.5%, filled into sealed, tin foil-coiled glass bottles, and stored at 37°C in a thermostatically controlled incubator for 12 months.^[26,27] Rice pretreated with an equal amount of distilled water and stored at 37°C was used as the control. The pretreated-aged rice was sampled every four months to determine pasting properties.

Ascorbic acid post-treatment of aged rice

Non-pretreated or pretreated-aged rice was subjected to post-treatment with ascorbic acid, dithiothreitol, and/or NEMI for further investigation. Ascorbic acid post-treatment was performed while the pasting properties of aged rice were determined; ascorbic acid solution was used instead of distilled water in the process of gelatinization.^[28] The concentrations of ascorbic acid solutions used for post-treatment were 5, 10, 20, and 50 mmol·L⁻¹. Freshly milled rice stored at 4°C for 12 months in a refrigerator was considered as fresh rice. Other post-treatments, including dithiothreitol and NEMI alone or combined with ascorbic acid, were used in a similar manner as ascorbic acid.

Determination of carbonyl group content

The defatted rice samples were extracted with fourfold (v/w) of 0.1 mol⋅L⁻¹ NaOH at 30 ℃ for 1 h and centrifuged at 4000 g for 20 min. The extraction was repeated three times. The supernatants were combined and precipitated at the isoelectric point. After adjustment of the pH to 7.0, protein samples were freeze-dried and stored at 4 ℃ until use. Carbonyl group content was determined as described by Levine et al.^[29]

Determination of pasting properties

After storage, both bottles of rice stored at 4°C and 37°C were placed in ambient temperature (25°C) for 48 h to balance the sample temperature. Rice samples (20 g) were ground using a Cyclone Sample Mill (Tester Corporation, Tianjin, China) for 20 s and passed through a 0.45-mm sieve screen immediately prior to testing. The pasting properties were determined using a Rapid Visco Analyzer (RVA) (Newport Scientific, Warriewood, Australia) following the AACC 61-02.^[30] The selected characteristic parameters of pasting properties were similar to those described elsewhere^[31]: peak viscosity (PV), holding strength (HS), breakdown (BD = PV – HS), final viscosity (FV), setback (SB = FV – PV), and pasting temperature (PT).

Cooking and texture measurement

Fresh rice, aged rice, and pretreated-aged rice were used as samples. Unwashed 10-g samples of rice were soaked in 16 mL of distilled water for 1 h in an aluminum cup covered with aluminum foil and then cooked as described by Ohno et al.^[32] The hardness (H) and stickiness (S) of the individual cooked rice grains were then measured using 90% deformation with a Texture Analyzer (TA.XTC; Bosin Tech, Shanghai). The key parameter, stickiness/hardness (S/H) ratio, was calculated.^[32] More than 30 cooked rice grains of each type were measured. The measurement conditions were as follows: load cell, max. 20 kgf; plunger, 50-mm diameter; plunger and stage, aluminum; bite speed, 2 mm/s; sample temperature at measurement, 25°C. ^[32]

Particle size analysis

The particle size distribution of rice flour samples was measured in triplicate using a laser light scattering-based particle size analyzer (Mastersizer 2000, Malvern Instruments Ltd., Malvern, UK). Rice flour samples were sieved through 120 and 140 meshes (105–125 μm). A 2% suspension dispersed in distilled water or 50 mmol·L⁻¹ ascorbic acid solution was heated at 75°C for 5 min. The samples were suspended in distilled water and sonicated in an ultrasonic bath (Ney, 50/60 Hz, 175W, CA, USA) for particle size analysis. The samples were stirred at 1800 rpm during measurement. The refractive indices of water and starch were taken as 1.330 and 1.5295, respectively, with an absorption of 0.1 for starch particles.^[33]

Light microscopy observations

Microscopy observation was performed according to the method of Guo et al.^[34] with slight modification. Rice flour particles (105–125 μm) dispersed in distilled water or 50 mmol·L⁻¹ ascorbic acid solution (2.5%, w/w) were heated in a water bath at 75°C or 95°C for 5 min in a quiescent state after complete dispersal using a vortex for 1 min and kept at 50°C until use. The suspensions (10 μL) were dispersed onto a microscope slide and observed using a light microscope (LW200CA, Cewei Photoelectricity Technology Co., Ltd, Shanghai, China) equipped with a combined digital camera (Nikon COOLPIX 5400, Tokyo, Japan) at room temperature. Photomicrograms of a calibration slide at the same magnification were used as a reference for size determination.

Statistical analysis

Pasting properties were determined at least in triplicate for each sample. Data were analyzed by analysis of variance by Duncan’s multiple-range tests at the significance level p < 0.05, and the mean values with standard deviations (in Tables 1 and 2 and Fig. 1) or mean values (in Figs. 2–5) were reported. All statistical analyses were performed using SAS Software Version 8.0 (SAS Institute Inc., Cary, NC, USA).

Table 1. Textural parameters of cooked rice.

Sample	Hardness (N)	Stickiness (N)	S/H ratio*
New rice	14.79 ± 1.25c	4.82 ± 0.47a	0.33 ± 0.05a
Aged rice	20.01 ± 3.24b	2.24 ± 0.68b	0.11 ± 0.04b
Pretreated-aged rice	23.41 ± 4.07a	0.36 ± 0.15c	0.02 ± 0.01c

* S/H ratio represents the ratio of stickiness and hardness. Different letters in the same column indicate significant differences (p<0.01).

Table 2. Effects of ascorbic acid post-treatment on pasting properties of pretreated-aged rice.

Sample and post-treatment	PV (cP)	BD (cP)	SB (cP)	PT (°C)
Fresh rice	3898 ± 17b	1874 ± 27b	−272 ± 31e	68.27 ± 0.12f
Pretreated-aged rice	2089 ± 13f	−1 ± 3f	2191 ± 20a	89.72 ± 0.33a
Ascorbic acid post-treatment of pretreated-aged rice
5 mmol⋅L^−1	2720 ± 43e	553 ± 31e	1374 ± 20b	87.53 ± 0.42b
10 mmol⋅L^−1	3011 ± 118d	882 ± 114d	952 ± 129c	86.67 ± 0.38c
20 mmol⋅L^−1	3652 ± 38c	1596 ± 23c	34 ± 32d	85.48 ± 0.88d
50 mmol⋅L^−1	4809 ± 42a	2807 ± 29a	−1475 ± 38f	81.48 ± 0.33e

PV represents peak viscosity, BD represents breakdown, SB represents setback, and PT represents pasting temperature. The pretreated-aged rice refers to the rice pretreated by ascorbic acid before storage and then stored at 37°C for 12 months. Different letters in the same column indicate significant differences (p < 0.05).

Figure 1. Effect of ascorbic acid pretreatment on the selected RVA parameters of pretreated rice during storage. (A) PV (peak viscosity), (B) BD (breakdown), (C) SB (setback) and (D) PT (pasting temperature). Vc represents ascorbic acid. Different letters indicate significant differences (p < 0.05).

Figure 2. Effect of ascorbic acid post-treatment on pasting properties of aged rice. Vc represents ascorbic acid, and DTT represents dithiothreitol.

Figure 3. Effect of ascorbic acid post-treatment on pasting properties of pretreated-aged rice. Vc represents ascorbic acid, and DTT represents dithiothreitol. The pretreated-aged rice refers to the rice pretreated by ascorbic acid before storage and then stored at 37°C for 12 months.

Figure 4. Effect of ascorbic acid post-treatment on disulfide cross-linking of aged rice in gelatinization process. Vc represents ascorbic acid, and NEMI represents N-ethylmaleimide.

Figure 5. Particle size analysis of rice flour particles heated at 75°C for 5 min. Fd: new rice heated in distilled water; Ad: aged rice heated in distilled water; PAd: pretreated-aged rice heated in distilled water; Aa: aged rice heated in 50 mmol·L⁻¹ ascorbic acid solution; PAa: pretreated-aged rice heated in 50 mmol·L⁻¹ ascorbic acid solution.

Results

Effects of ascorbic acid pretreatment on pasting properties and texture

The RVA characteristic parameters of pretreated rice by ascorbic acid during storage are shown in Fig. 1. The PV of pretreated rice decreased steadily with prolonged storage and was lower than the control (Fig. 1A). Interestingly, the PV of the control was higher at 4 months than 0 months^[35], but this was not true for pretreated rice. The differences in PV between the pretreated rice and the control at 4, 8, and 12 months were greater than at 0 month, and the largest difference was observed at 4 months. For BD, there was no significant difference at 0 month between pretreated rice and the control (Fig. 1B). With increased storage, the difference in BD at 4 months was the most obvious, and those at 8 and 12 months were most similar. After storage, the BD of pretreated aged rice was lower than the control.

The SB of pretreated rice was lower at 0 month than the control, but the opposite was true with storage duration up to 4 or 8 months (Fig. 1C). The difference in SB between pretreated rice and the control decreased at 8 months compared to 4 months and was eliminated when the storage duration was 12 months. Before storage, PT showed no significant difference between pretreated rice and the control (Fig. 1D). However, PT significantly increased with greater storage duration. PT of the pretreated rice was significantly higher than the control after storage, and the most obvious difference appeared at 4 months. The textural parameters of fresh rice, aged rice, and pretreated-aged rice are listed in Table 1. The data showed that hardness increased obviously (p < 0.01), while in contrast, stickiness and S/H decreased significantly (p < 0.01), in the order fresh rice, aged rice, and pretreated-aged rice.

Changes in carbonyl group content after rice storage

Carbonyl group content is an index of protein oxidation. Carbonyl group content of proteins in fresh rice, aged rice, and pretreated-aged rice was 0.173 ± 0.02, 0.222 ± 0.07, and 0.240 ± 0.05 nmol/mg protein, respectively. Aged rice has higher carbonyl group content than fresh rice, indicating the oxidation of proteins with rice aging. Ascorbic acid pretreatment obviously increased the carbonyl group content of pretreated-aged rice above that of normally aged rice (p < 0.01).

Effects of ascorbic acid post-treatment on pasting properties

Ascorbic acid applied post-treatment obviously improved the pasting properties of the non-pretreated aged rice; in particular, when the concentration of ascorbic acid in post-treatment was 10 mmol·L⁻¹, PV of aged rice exceeded that of fresh rice or dithiothreitol post-treatment (Fig. 2). Ascorbic acid pretreatment deepened the degree of rice aging during storage (Fig. 1). The improved effect of ascorbic acid on deeply aged rice was unclear, and therefore, the effects of ascorbic acid post-treatment on pasting properties of the pretreated-aged rice were investigated (Table 2). PV and BD increased with the increasing concentration of ascorbic acid solution from 0 to 50 mmol·L⁻¹, and the change displayed a positive dose–effect relationship. When the pretreated-aged rice was post-treated with 50 mmol·L⁻¹ ascorbic acid, its PV and BD significantly exceeded the corresponding values for fresh rice. However, SB and PT decreased with an increasing concentration of ascorbic acid solution, and the trend showed a positive dose–effect relationship. The SB of the aged rice post-treated with 50 mmol·L⁻¹ ascorbic acid was lower than that of fresh rice, although the PT was still higher for fresh rice.

Regulation role of ascorbic acid in pretreatment or post-treatment

Dithiothreitol is known to improve the pasting properties of aged rice. To explore the nature of pretreated-aged rice, the effect of dithiothreitol on pasting properties of the pretreated-aged rice was determined. Dithiothreitol increased PV and BD, decreased SB, and reduced initial gelatinization time (Fig. 3), with the same pattern in non-pretreated aged rice.^[34] Therefore, it was believed that there was no obvious difference between non-pretreated and pretreated aged rice. However, it is apparent that ascorbic acid pretreatment deepened the aging degree of rice over an equivalent storage period (Fig. 1).

Ascorbic acid applied post-treatment enhanced the swelling of aged rice^[34], but the result still need to be demonstrated. Here, we evaluated the effect of ascorbic acid on disulfide cross-linking in aged rice via blocking of the sulfhydryl group by NEMI. As shown in Fig. 4, NEMI decreased all parameters of PV, HS, and FV in the aged rice, possibly due to depression of disulfide cross-linking in the gelatinization process. However, mixed treatments of ascorbic acid and NEMI still significantly improved the pasting properties of aged rice. There was no observable difference in pasting profiles between the ascorbic acid post-treatments alone and combined with NEMI. This suggested that the blocking role of NEMI on sulfhydryl groups became ineffective to paste viscosity due to the presence of ascorbic acid in the gelatinization of aged rice. Therefore, the roles of ascorbic acid in post-treatment may include prevention of disulfide cross-link formation.

Particle size analysis showed that the gelatinized new rice particles have three peaks located near 9/100/800 μm (Fig. 5). Particle size distribution peaks for the gelatinized aged rice and pretreated-aged rice were near 9/220 μm and 9/250 μm, respectively. Ascorbic acid post-treatment on non-pretreated and pretreated aged rice had little effect on peak positions at 9 μm and within 100–250 μm, but peak intensity (volume fractions in peak position) changed obviously. The peak intensity at the 9 μm position decreased and that within the 100–250 μm increased gradually, in the order new, aged, and pretreated-aged rice (Fig. 5). Meantime, ascorbic acid post-treatment also amplified the amplitude of variation. In addition, microscopy observation also showed that ascorbic acid post-treatment promoted the swelling of rice flour particles in size and quantity (Fig. 6). The edges of the particles post-treated by ascorbic acid are irregular, rough, and hairy (Fig. 6B), with more fragments (Figs. 6B and Figs. 6D) and clefts (Fig. 6F).

Figure 6. Light microscopy observations of rice flour particles of pretreated-aged rice. (A) Heated at 75°C in distilled water (40×), (B) heated at 75°C in 50 mmol·L⁻¹ ascorbic acid solution (40×), (C) heated at 75°C in distilled water (100×), (D) heated at 75°C in 50 mmol·L⁻¹ ascorbic acid solution (100×), (E) heated at 95°C in distilled water (100×), and (F) heated at 95°C in 50 mmol·L⁻¹ ascorbic acid solution (100×). Swelling point (arrow), separating gap (dotted arrow), and separated fragment (arrowhead).

Discussion

Ascorbic acid pretreatment of rice before storage depressed PV and BD and enhanced SB and PT compared to the control. Both the decreased PV or BD and the increased SB or PT are characteristic of rice aging.^{[2,5,9,34,36,37]} Increased hardness and decreased stickiness and S/H were found by Ohno and Zhou after rice aging.^[25,26] Our results showed that ascorbic acid pretreatment enhanced the hardness of pretreated-aged rice and decreased the stickiness and S/H ratio compared with aged rice without pretreatment (Table 1), showing that ascorbic acid pretreatment accelerated rice aging during storage. Dithiothreitol could also improve the pasting properties of the pretreated-aged rice (Fig. 2). This indicates that ascorbic acid pretreatment only played a role in accelerating rice aging. Aging has been repeatedly found to be strongly related (positively) to temperature^[18,38,39], but no available literature was reported involving rice aging accelerated by ascorbic acid. However, in our research, pretreatments with other reducing agents, such as dithiothreitol or tea polyphenol, retarded rice aging (data not listed). Ascorbic acid likely accelerates rice aging by acting as a pro-oxidant during storage. The higher temperature (37°C) and the longer storage period could oxidize ascorbic acid, reducing its concentration in rice and acting as a pro-oxidant. Ascorbic acid likely became a pro-oxidant agent at a definite time point during storage and thereafter accelerated the aging of rice.

Whatever depression or enhancement to the four selected pasting parameters, the most obvious difference appeared at 4 months. Therefore, the time point at which ascorbic acid serves as a pro-oxidant should be less than 4 months. The accelerating role of ascorbic acid on rice aging nearly disappeared after 8 months, which may be attributed to the depletion of substances that could be oxidized in rice. However, the degree of aging was deepened in pretreated-aged rice and resulted in a lower PV and BD and a higher PT. The most likely oxidized substance that influences pasting properties in aged rice should be non-starch constituents, especially proteins.^[32,39,40] Albumin and globulin were demonstrated to be the largest contributors to aging changes in pasting properties^[19], and protein oxidations during rice aging have been characterized by infrared and Raman spectroscopies.^[20] Protein oxidation during rice aging includes the sulfhydryl group to disulfide, sulfone, or sulfoxide.^[15,16,20] Therefore, a deepened aging of rice was attributed to a pro-oxidant effect of ascorbic acid on proteins. Ascorbic acid pretreatment significantly increased the carbonyl group content of protein from pretreated-aged rice compared to that from aged rice without pretreatment, which illustrates the pro-oxidant role of ascorbic acid.

However, ascorbic acid post-treatment increased the PV and BD of pretreated-aged rice and depressed the increase in SB and PT (Table 2). Except for PT, ascorbic acid solution of 50 mmol·L⁻¹ restored the selected characteristic parameters of pretreated-aged rice excessively compared to fresh rice, suggesting that ascorbic acid improved the pasting properties of the pretreated-aged rice. The aging of rice appears to be reversible in this sense. This agreed with the result reported previously: that ascorbic acid significantly improved the pasting properties of non-pretreated aged rice. ^[34] The pasting property improvement was disclosed by particle size analysis, which shows that ascorbic acid increased the volume fraction of larger particles (centered at 250 μm, Figs. 5 and 6). This is in accordance with the previous results where ascorbic acid increased water absorption and leaching of soluble solid^[34] since swelling and water absorption properties are well known to be related to the pasting and gelatinization of starch.^[41] Among four types of rice proteins, prolamin and glutelin are tightly bound surface and/or integral components of starch granules, with strong disulfide and/or hydrophobic bonding.^[42] A protein network formed by disulfide bonding could act as a barrier to the swelling of starch granules.^[14] Ascorbic acid, a small molecule, easily permeates the gap between starch granules. The roles of ascorbic acid on rice proteins could be attributed to hindering the denaturation cross-linking of proteins on the surface and in the interior of starch granules during gelatinization as a reducing agent. Protease treatment on aged rice produced a similar RVA curve to new rice, which was attributed to the elimination of protein barriers on starch granule swelling.^[43] Ascorbic acid was considered to relieve protein barriers through interfering denaturation, and thus increased swelling of starch granules. It must be pointed out that the pasting properties of aged rice improved by ascorbic acid still differed from new rice in essence because the latter included a greater volume fraction of smaller particles centered at 9 μm, which separated from original particles (105–125 μm) of rice material. Therefore, rice aging is considered irreversible since starch granules in rice are more difficult to separate after aging.

The improvement of pasting properties by ascorbic acid post-treatment showed the antioxidant capacity of ascorbic acid based on the similar behavior to other reducing agent, such as 2-mercaptoethanol.^[18] However, a related study in which ascorbic acid prevents the formation of disulfide bond is lacking. NEMI decreased rice paste viscosity due to blocking of sulfhydryl group in the gelatinization of aged rice, indicating that formation of disulfide cross-linking contributed to rice paste viscosity. In the presence of ascorbic acid, the effect of NEMI on pasting viscosity of aged rice was eliminated (Fig. 4), suggesting that ascorbic acid perhaps inhibited the formation of disulfide bonds. This suggests that a large amount of disulfide cross-linking would be formed even in conventional gelatinization of aged rice. Furthermore, the obvious difference in NEMI effects on the pasting profiles of aged rice between with and without ascorbic acid (Fig. 4) indicated that swelling contributed more to rice paste viscosity than disulfide cross-linking. Compared to fresh rice, the reduced formation of disulfide cross-linking in the gelatinization of aged rice should not be the major reason for rice aging. On the contrary, the prevention of disulfide cross-linking is likely the reason that dithiothreitol or ascorbic acid increases swelling degree of aged rice.

Conclusion

Ascorbic acid, as a pro-oxidant, accelerated aging of pretreated rice during storage; however, it also improved the pasting properties of non-pretreated or pretreated aged rice as a reducing agent by increasing the volume fraction of larger particles. Particle size analysis and microscopic observation showed that the essence of rice aging is that starch granules are difficult to separate in the gelatinization, and this trend is irreversible. This helps to disclose the mechanism of rice aging, control the speed of rice aging, and improve the eating quality of cooked rice and rice products for consumers with different requirements. The underlying reasons why starch granules are difficult to separate should be researched further.

Additional information

Funding

We are very thankful to the National Natural Science Foundation of China (No. 31671784) and the Special Fund for Agro-scientific Research in the Public Interest (No. 201303088) for their financial support of this study.