Characterization of Amylopectin Fine Structure and its Role on Pasting Properties of Starches in Rice (Oryza sativa L.)

Abstract

The characteristics of amylopectin fine structure in rice starches are still elusive and the effect of amylopectin fine structure on starch physicochemical properties remains to be determined. In this paper, the fine structure of amylopectin especially the ratio of A-chains to B-chains was investigated using the improved enzymatic method. The influences of amylopectin fine structure on pasting properties of 91 rice accessions were also analyzed. The statistics results showed that the fine structure of rice starches highly varied with cultivars and these accessions were representative in terms of rice quality. Further correlation studies suggested that the ratio of A-chains to B-chains was negatively correlated with setback viscosity and pasting temperature, while average chain length and average external chain length was positively correlated with them, which indicated that the length of external chain and proportion of long and short chain of amylopectin played an important role in determining the rice cooking quality and functionality.

Introduction

Rice (Oryza sativa L.) is a staple food and improvement of its quality especially eating and cooking quality is a major concern in current breeding programs. The cooking quality of the cereal grains, often evaluated as pasting characteristics, was largely determined by amylose content (AC) and the fine structure of amylopectin (Bergman et al., 2004; De Vries et al., 2016). Although the fine structure of amylopectin from diverse botanical sources has been previously reported (Annor et al., 2014; Kalinga et al., 2014), the characteristics of amylopectin fine structure in rice starches are still elusive. Accordingly, the effect of amylopectin fine structure on starch pasting properties remains to be clarified (Kubo et al., 2010; Suzuki et al., 2006; Yu et al., 2012).

It is well-known that the unit chains of amylopectin are broadly divided into A-, B- and C-chains (Hizukuri, 1986). The degree of multiple branching can be expressed as the ratio of A-chains to B-chains (A:B). Additionally, amylopectin includes external and internal chains (Manners, 1989). The average external chain length (ECL), i.e. the segments that extend from the outermost branch to the non-reducing end of the chains, and the average internal chain length (ICL), defined as the length of the chain segment between two branches. The differences in amylopectin structure were primarily due to the variation of chain length. Thus, fine structure of amylopectin is mainly characterized with A:B, average chain length (CL), ECL, ICL, and chain length distribution (Yao et al., 2004).

The chain length distribution of the amylopectin was analyzed in detail by high performance anion exchange chromatography (HPAEC) (Gayin et al., 2016a). Although HPAEC was widely applied and CL has been also analyzed recently (Kowittaya and Lumdubwong, 2014), it was not suitable to measure other structural parameters. In recent years, substantial progress has been made in investigating the fine structure of amylopectin using the highly purified amylolytic enzymes. The enzymatic method was further applied in investigating the fine structure of amylopectin in other botanical sources such as arrowhead starch and ginkgo starch (Wang et al., 2009; Ao et al., 2000). But until now, the method used in determining the amylopectin structure of rice starch has seldom been reported.

Therefore, the amylopectin fine structure in rice, especially the structure parameter A:B were determined using the improved enzymatic method. In addition, this study investigated the AC and pasting properties of rice starch based on a collection of 91 rice accessions, and analyzed the correlations between the fine structure of amylopectin and the pasting properties of rice starch. This result will facilitate to understand the effect of amylopectin fine structure on pasting properties of rice.

Materials and Methods

Rice materials and starch processing    In 2014, 91 rice accessions were planted in the experimental field of Jiangxi Agricultural University with conventional method in the rice growing season (Table S1). After harvest, the grains were air-dried to a moisture level of about 12%. The samples were dehulled on a Rice Milling Tester (Ssangyong Machinery Industry Co. Ltd, Japan) and fine rice flour was obtained by high-speed grinding using a whirlwind milling machine (FW80-1, Tianjin Taisite Instrument Co. Ltd, China). Rice starch was isolated using alkaline steeping and the starch was passed through a 100-mesh sieve and sealed prior to being used (Wang and Wang, 2004).

AC and pasting properties    AC of rice samples was analyzed using the iodine staining method (Bao et al., 2006). Pasting properties of normal rice starch were analyzed using a rapid visco-analyzer (RVA-Tec Master, Newport Scientific, Warriewood, Australia), according to the AACC method (AACC methods 61-02). Rice starch (3.0 g, 12% moisture) was weighed into an RVA aluminum canister, and 25 g of distilled water was added. The sample was first kept for 1.5 min at 50°C, heated to 95°C at 12°C/min, kept for 2.0 min at 95°C, cooled to 50°C at 12°C/min, and finally kept for 1.5 min at 50°C. Starch viscosity characteristics included the following original components: peak viscosity (PKV), hot paste viscosity (HPV), and cool paste viscosity (CPV). Three secondary parameters including breakdown (BDV) and setback (SBV) were calculated based on the original data: BDV = PKV - HPV, SBV = CPV - HPV. In addition, pasting temperature (temperature of the initial viscosity increase, PaT) and pasting time (time of the initial viscosity increase, PeT) was also recorded. The viscosity parameters were expressed in centipoises (cP). All these experiments were performed at least in triplicate. Analytical-grade chemicals were used in this study unless otherwise noted.

Amylopectin preparation    Amylopectin samples were prepared according to the method of He et al. (2010). About 0.5 g of rice starch was weighed to 50 mL centrifuge tube, added 1 mL of anhydrous ethanol and 15 mL 0.5 M NaOH were then added. The soaked sample was then heated in boiling water for 30 min until the solution became clear and transparent. After cooling, the starch sample was centrifuged at 4000 r/min for 20 min and the centrifuged solution was neutralized to neutral with 2 M HCl. Five mL of a 1:1 (v/v) n-butanol-isoamyl alcohol solution was added to mixture the and then heated in a boiling water bath for 10 min. After cooling to room temperature, the mixture was transferred into 2 ∼ 4°C refrigerator for standing overnight and centrifuged at 4000 r/min for 20 min when took out. The supernatant was divided into three layers and the lower latex solution was crude amylopectin solution. Five mL of a 1:1 (v/v) n-butanol-isoamyl alcohol solution was added to the mixture and then repeated the previous steps. Carefully sucked out of the latex-like supernatant and slowly adding 2 times the volume of absolute ethanol. The mixture was then transferred into 2 ∼ 4°C refrigerator for standing 2 h and centrifuged at 4000 r/min for 10 min. The starch sediment was dried in an oven and ground into powder to pass through a standard 100-esh sieve, and then the pure amylopectin starch powder was obtained.

Characterizing fine structure of amylopectin Blue value    The blue value (BV) was measured in a solution containing amylopectin (15 mg/100 mL), iodine (2 mg/100 mL), and potassium iodine (20 mg/100 mL), according to the method of Yu et al. (2012). The blue value was calculated with the absorbance measured at 680 nm, according to the following equation:   

Where Abs680 is the absorbance at 680 nm and C is the concentration of amylopectin in the solution in 1 mg/100 mL.

Production of β-limit dextrins and β-amylolysis    The β-limit dextrins of amylopectin were prepared using the method of Yuan et al. (1993). A 25 mL amylopectin dispersion (in 0.02 N sodium acetate buffer, pH 5.0) was prepared, including 500 mg of starch and 2500 U of β-amylase [EC 3.2.1.2, 968 U/mg, Sigma-Aldrich (Deisenhofen, Germany)]. The amylopectins were digested at 37°C for about 48 h. In the incubation process, the reducing power generated in the reaction liquid was constantly tested until it reached a certain value. Then the digest was heated in boiling water for 25 min to inactivate the enzyme. β-amylolysis was obtained by calculating the mole ratio of total carbohydrate to reducing ends of debranched amylopectin. Then the β-limit dextrins were precipitated by the addition of 3 vol of methanol. After having settled in an ice bath for 30 min, the β-limit dextrins precipitates were separated from the soluble maltose by centrifuging for 5 min at 6000 r/min. The β-limit dextrins were then washed three times with 75% methanol and air-dried.

Average chain length    The average chain length (CL) was determined by using pullulanase [EC 3.2.1.41, 20 U/mg, Sigma–Aldrich (Deisenhofen, Germany)]. The 22 mg of amylopectins were digested by 4 U pullulanase in 5 mL sodium acetate buffer (0.05 N, pH 5.0) at 37°C for about 24 h. The external (ECL) and internal chain lengths (ICL) were calculated according to the following equations of Lin et al. (2013):   

  

Debranching of amylopectin β-limit dextrins    The resulting β-limit dextrins were dispersed in 0.04 N sodium acetate buffer with PH 4.5; after having been heated in a boiling water bath for 5 min, the β-limit dextrins dispersion was prepared for 1 mg/mL. After cooling, the dispersion was incubated with 100 U/mL of the isoamylase [EC 3.2.1.68, 210 U/mg, Megazyme (Wicklow, Ireland)] solution at 37°C for 24 h. The digest was then heated in a boiling water bath for 30 min to inactivate the enzyme; after cooling, the maltose production was determined (C1). Then the dispersion was incubated with 1 U/mL of pullulanase solution at 37°C for another 24 h. The enzyme was inactivated by heating the digest in a boiling water bath for 30 min and the maltose production was determined again (C2). Each sample was digested on two separate occasions, and derived data presented were the mean for the two digestions. Finally, A:B could be calculated according to the following equations (Ao et al., 2000):   

  

Statistical analysis    One-sample t test and pearson correlation methods were carried out using SPSS software. Significant differences of the mean values were determined at p < 0.05. Each amylopectin structural measurement, starch chemical compositions and pasting properties were done at least in duplicate. The means of duplicated measurements were used for the analysis.

Results and Discussion

Characterization of starch physicochemical properties and fine structure of amylopectin    The starch physicochemical properties related to eating quality in 91 rice accessions were shown in Table 1. Significant variations among the rice accessions for the properties were observed. The majority of the traits among accessions exhibited a wide range of variations and the traits from the present study were in the same ranges as those in the literature (Chen et al., 2017; Houjyo et al., 2017). This suggested that the 91 accessions were representative in terms of rice grain quality in this study. The abundant diversity in pasting properties may allow the breeders to make effective selections for improvement of the cooking quality according to consumers' choice.

Table 1. Descriptive statistics of all parameters for rice

Traits	Parameters	Minimum	Maximum	Mean	SD
	AC (%)	2.20	26.48	14.28	5.95
Pasting properties	PKV	2404.00	5168.50	4043.83	667.07
	HPV	1346.00	3786.50	2517.60	446.61
	BDV	285.00	2714.00	1526.22	600.19
	CPV	1698.00	6788.00	4290.26	1051.32
	SBV	352.00	3336.00	1775.77	670.08
	PeT	3.87	6.97	5.76	0.58
	PaT	72.75	90.20	81.00	4.38
Amylopectin fine structure	BV	0.013	0.206	0.065	0.050
	β-amylolysis (%)	48.94	63.15	56.58	4.13
	CL	16.18	21.56	18.98	1.13
	ECL	10.07	15.23	12.74	1.27
	ICL	3.83	6.92	5.23	0.64
	A:B	1.01	2.27	1.48	0.22

AC, amylase content; PKV, peak viscosity; HPV, hot paste viscosity/ Minimum viscosity; BDV, breakdown viscosity; CPV, cool paste viscosity/ Final viscosity; SBV, setback, viscosity; PeT, peak time; PaT, pasting temperature; CL, average chain length; ECL, average exterior chain length; ICL, average internal chain length; A:B, ratio of A-chains to B-chains.

In the previous studies of other botanical sources starch, the enzymatic reaction conditions were different. In this study, the optimized method was applied to characterize the amylopectin fine structure in endosperm of these rice varieties and the fine structures of amylopectin fractionated from starches of all samples were also shown in Table 1. The BV was correlated with the affinity for iodine, ranging from 0.013 to 0.206. It was reported that the BV of amylose ranged from 0.8 to 1.2 while amylopectin ranged from 0.08 to 0.22 (Gilbert, 1964). The results of this study were within the range of amylopectins, which appeared to indicate that the amylopectins being tested were of higher purity. Furthermore, the β-amylolysis of the rice amylopectins, which generally reflected the length of the external chain, ranged from 48.94 to 63.15%, which appeared to indicate that the differences in fine structure existed on the rice accessions being tested. This range was consistent with what was previously reported for rice amylopectin and also comparable to the values reported for other plants (Laohaphatanaleart et al., 2009). In addition, the CL, ECL and ICL among the rice amylopectin samples were different across rice varieties, which were comparable to values reported for other A-type starches (Hizukuri, 1986; Zhu et al., 2011). It was worth noting that A:B of nonwaxy rice amylopectin and waxy rice amylopectin also appeared to be different, ranging from 1.01 to 1.76 and 1.77 to 2.27 respectively. Amylopectins from waxy varieties of cereals were frequently used in structural analysis. This was not accurate when the results were extrapolated to nonwaxy amylopectin without reservation. For example, amylopectin had an A:B of 2.6 for waxy sorghum and waxy corn while it was 1.7 for nonwaxy corn (Marshall and Whelan, 1974), suggesting that waxy amylopectin might have a different branching pattern with nonwaxy amylopectin. Furthermore, the A:B in this study was consistent with previously reported values using gel chromatography (Enevoldsen and Juliano, 1988; Umeki and Yamamoto, 1977), which was a complex method and could only roughly separate chains with different degree of polymerization in the debranched amylopectin but inapplicable in comparison of amylopectin fine structures (Hizukuri, 1986). Thus, the improved enzymatic method was suitable for the determination of amylopectin fine structure in rice and the results of structural analysis suggested that the fine structure of rice starch highly varied with cultivars.

Correlation analyses among different physicochemical properties    Pasting properties are important indicators in determining the application values of starches and affect quality of the end-use products (Bergman et al., 2004; Hung, 2007). The correlation coefficients between AC and pasting properties were summarized in Table 2. Interestingly, the significant correlations were found between almost any two parameters; and only the correlations between BDV and PeT did not reach the significant level. The result suggested that the pasting properties of 91 rice cultivars were interdependent, similar to the results by Zhao et al. (2012). AC was reported as the major factor that influenced the physicochemical properties of rice starch (Asaoka et al., 1985; Leng et al., 2014). In this study, the significant correlations were found between AC and any pasting parameters. AC was positively correlated with HPV, CPV, SBV, PeT and PaT, but negatively correlated with PKV and BDV. The correlations found here were consistent with the reports that the cooked rice from rice cultivar which had high AC was dry, fluffy, separate and hard, while cooked rice of low AC was glossy, soft and sticky (Bergman et al., 2004; Cameron and Wang, 2005; Ohtsubo et al., 1990).

Table 2. Correlation between physicochemical properties

Parameters	PKV	HPV	BDV	CPV	SBV	PeT	PaT
HPV	0.477**
BDV	0.756**	−0.214*
CPV	0.211*	0.913**	−0.445**
SBV	−0.399**	0.572**	−0.869**	0.812**
PeT	0.276**	0.625**	−0.158	0.699**	0.492**
PaT	−0.300	0.258*	−0.525**	0.478**	0.628**	0.474**
AC	−0.240**	0.665**	−0.548**	0.851**	0.827**	0.662**	0.451**

PKV, peak viscosity; HPV, hot paste viscosity/ Minimum viscosity; BDV, breakdown viscosity; CPV, cool paste viscosity/ Final viscosity; SBV, setback, viscosity; PeT, peak time; PaT, pasting temperature; AC, amylase content.

* and **  means the correlations are significant at p < 0.05 and p < 0.01levels, respectively.

Correlation analyses among fine structural parameters of amylopectin    The correlation coefficients among the characteristics of amylopectin fine structure of all samples were shown in Table 3. Correlation analysis showed that β-amylolysis limit was positively correlated with CL and ECL, negatively correlated with ICL and the A:B, which implied that the β-amylolysis of starch depended on not only the length of starch chains, but also the arrangement of starch chain and distribution of branch point. Additionally, the significant correlations were found between almost any two parameters, which were consistent with previously reports (Hanashiro et al., 2013; Kowittaya and Lumdubwong, 2014). The lower values of ICL, indicated that branch points of the amylopectin were close, i.e. the molecule is more compact. These results suggested that the parameters of amylopectin fine structure were mutually influenced and correlated with each other, and also had an effect in determining the pasting properties.

Table 3. Correlation between characteristics of amylopectin fine structure.

Parameters	β-Amylolysis (%)	CL	ECL	ICL
CL	0.591**
ECL	0.914**	0.866**
ICL	−0.77**	0.057	−0.450**
A:B	−0.293**	−0.342**	−0.363**	0.132

CL, average chain length; ECL, average exterior chain length; ICL, average internal chain length; A:B, ratio of A-chains to B-chains.

* and **  means the correlations are significant at p < 0.05 and p < 0.01 levels, respectively.

Correlation analyses between amylopectin structure and pasting parameters    To some extent, the starch pasting behavior determines rice cooking quality and functionality, since starch is the main component in rice (Hung et al., 2007). Six properties PKV, PaT, HPV, BDV, CPV and SBV were closely correlated with the final product quality and all of them had significant correlations with the fine structural features. The chain length (CL and ECL) and A:B are important parameters in fine structure of amylopectin, which would lead to the differences of starch crystalline properties, and result in different physical and chemistry properties of starch. Thus, Pearson correlation analysis was conducted to examine the effect of fine structural features on physicochemical properties. The correlation coefficients were shown in Table 4.

Table 4. Correlation between amylopectin structure and pasting properties.

Parameters	PKV	HPV	BDV	CPV	SBV	PeT	PaT
β-amylolysis (%)	−0.156*	0.188	−0.033	0.260*	0.282**	0.108	0.284**
CL	−0.176*	0.304**	−0.031	0.369**	0.377**	0.351**	0.364**
ECL	−0.177*	0.276**	−0.011	0.353**	0.370**	0.243*	0.364**
ICL	−0.028	−0.007	−0.027	−0.047*	−0.068	0.145	−0.086
A:B	0.233*	−0.321**	0.245*	−0.435**	−0.470**	−0.374**	−0.487**

PKV, peak viscosity; HPV, hot paste viscosity/ Minimum viscosity; BDV, breakdown viscosity; CPV, cool paste viscosity/ Final viscosity; SBV, setback, viscosity; PeT, peak time; PaT, pasting temperature; CL, average chain length; ECL, average exterior chain length; ICL, average internal chain length; A:B, ratio of A-chains to B-chains.

* and **  means the correlations are significant at p < 0.05 and p < 0.01levels, respectively.

The effect of structure on PKV    The RVA parameter PKV reflects water-binding capacity and the extent of swelling of starch granules, and is often correlated with rice quality since the swollen and collapsed starch granules affect the texture of products (Wani et al., 2012). In the present study, PKV showed a negative correlation with CL and ECL. This trend reflected the tendency of long-chain amylopectin to hold the integrity of starch granules during heating and shearing (Okuda, 2006). It was speculated that a higher proportion of long chains might help to maintain the gelatinized starch granule structure (Han and Hamaker, 2001). Therefore, long-chain amylopectin would assist in forming a stable crystal conformation of rice amylopectin, resulting in lower PKV and lower eating quality (Kubo et al., 2010). On the basis of the study of Ao et al. (2000), amorphous lamellae of starch contain branch points of the amylopectin side chain and possibly some amylase. As expected, PKV is observed to have a positive correlation with A:B. Obviously, the higher A:B of amylopectin (branch point of binding sites higher) and amorphous regions in the proportion of starch granules is, the easier the starch gelatinized. Thus, A:B is also a reflection of the difficulty of gelatinized starch.

The effect of structure on BDV    The RVA parameter BDV measured the starch paste resistance to heat and shear, related to the taste of rice (Mar et al., 2013). As far as we know, the double helical domains in granules were primarily formed by exterior chain. In the present study, BDV showed a positive correlation with A:B. Because long amylopectin chains hold the integrity of starch granules during heating and shearing and the interactions by the short branch chains were not strong enough to maintain the integrity of the swollen granules, so HPV became lower and then BDV became higher. This suggested that the more short A chains the amylopectin consisted, the greater the BDV and the more soft the rice. However, we could not find any significant relationship between BDV and the chain length of amylopectin (CL, ECL and ICL). It showed that the swelling of starch granules was not dependent on the average chain length, but dependent on the chain length of constituted chains.

The effect of structure on SBV    It was reported that the higher degree of retrogradation of rice starch corresponded to its higher SBV. SBV exhibits the tendency of starch pastes to retrograde, which is an index of starch retrogradation (Jangchud et al., 2004). In this study, A:B had significantly negative correlation with SBV, CL and ECL had significantly positive correlation with SBV. This may be due to amylopectins with higher percentage of long chain requiring more energy to dissolve the greater number of double helical linkages of retrograded amylopectin (He et al., 2010). Additionally, the external chain of amylopectin could re-associate faster than internal chain did and this recrystallization process could increase CPV, resulting in higher SBV (Silverio et al., 2000). Consequently, the rice starch which has different proportion of branch chain will result in different rice quality. The proportion of short chains higher, the SBV lower, the cultivar had a softer texture, the proportion of long chains higher, the SBV higher, the cultivar showed a harder texture after being cooked.

The effect of structure on PaT    The RVA parameter PaT, reflecting the crystalline stability, is the temperature at which the viscosities of the starch pastes begin to rise. It was reported that PaT of rice significantly positively correlated with cooking time (Gayin et al., 2016a). In this study, PaT was positively correlated with CL and ECL. This would be due to the fact that longer external chain could increase the crystalline stability by forming additional hydrogen bonds between glucan strands and double helices (Vamadevan et al., 2013). Wang et al. also have reported that the rice starch had a higher PaT, which might be attributed to its longer ECL and shorter ICL (Wang and Wang, 2002). Thus, starch with a longer branch CL tends to have a higher PaT. In addition, A:B had significantly negative correlation with PaT. Starch with relative higher amounts of very short amylopectin chain would thus have lower molecular and crystalline order and a non-optimized packing within the crystalline lamellae (Vandeputte and Delcour, 2004). So the rich starch with more short amylopectin chain, they would most likely have lower PaT, the rice would easier being cooked.

Thus, it was shown that pasting properties of starches were largely dependent on the fine structure of amylopectin and could be affected by a number of factors like the proportion of long and short chain of amylopectin and interactions among starch chains.

Conclusion

In this study, the fine structure of amylopectin was determined using the improved enzymatic method, and the AC and pasting properties of rice starch based on a collection of 91 rice accessions were also investigated. Intriguing correlations were found between the fine structure of amylopectin and the pasting properties, which indicated that the fine structure of amylopectin strongly affected the pasting properties and functional properties of rice starch. The correlation analysis showed that the A:B and both parameters (CL and ECL) primarily affected the pasting properties especially SBV and PaT, indicating that the length of external chain and proportion of long and short chain of amylopectin played an important role in determining the rice cooking quality and functionality. This new finding would facilitate the understanding of details amylopectin fine structure in rice and pave the way for an improved understanding of the role of amylopectin fine structure on rice quality, for molecular breeding for improvement of rice eating and cooking quality. In addition, the investigation of how the genes responsible for amylopectin synthesis influencing starch structure and how to use them in changing the starch structure in improving rice quality is also underway.

Acknowledgments    We appreciated Dr. Yangping Wen in providing valuable advice on statistical analysis. This work was supported by the Natural Science Foundation of Jiangxi Province, under Grant No. 20114BAB204007, the Project from Advantage of Science Technology Innovation Team of Jiangxi Province, under Grant No.GJJ11398; the Project from Advantage of Science Technology Innovation Team of Jiangxi Province, under Grant No. 20133BCB24005.

Abbreviations

AC

amylase content

PKV

peak viscosity

HPV

hot paste viscosity/ Minimum viscosity

BDV

breakdown viscosity

CPV

cool paste viscosity/ Final viscosity

SBV

setback, viscosity

PeT

peak time

PaT

pasting temperature

BV

blue value

CL

average chain length

ECL

average exterior chain length

ICL

average internal chain length

A:B

ratio of A-chains to B-chains

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