Article pubs.acs.org/JAFC

Different Structural Properties of High-Amylose Maize Starch Fractions Varying in Granule Size Canhui Cai,†,‡ Lingshang Lin,†,‡ Jianmin Man,†,‡ Lingxiao Zhao,†,‡ Zhifeng Wang,# and Cunxu Wei*,†,‡ †

Key Laboratories of Crop Genetics and Physiology of the Jiangsu Province and Plant Functional Genomics of the Ministry of Education, ‡Co-Innovation Center for Modern Production Technology of Grain Crops, and #Testing Center, Yangzhou University, Yangzhou 225009, China S Supporting Information *

ABSTRACT: Large-, medium-, and small-sized granules were separated from normal and high-amylose maize starches using a glycerol centrifugation method. The different-sized fractions of normal maize starch showed similar molecular weight distribution, crystal structure, long- and short-range ordered structure, and lamellar structure of starch, but the different-sized fractions of high-amylose maize starch showed markedly different structural properties. The amylose content, iodine blue value, amylopectin long branch-chain, and IR ratio of 1045/1022 cm−1 significantly increased with decrease of granule size, but the amylopectin short branch-chain and branching degree, relative crystallinity, IR ratio of 1022/995 cm−1, and peak intensity of lamellar structure markedly decreased with decrease of granule size for high-amylose maize starch. The large-sized granules of high-amylose maize starch were A-type crystallinity, native and medium-sized granules of high-amylose maize starch were CAtype crystallinity, and small-sized granules of high-amylose maize starch were C-type crystallinity, indicating that C-type starch might contain A-type starch granules. KEYWORDS: maize, high-amylose starch, granule size, structural property, molecular weight distribution, crystal structure

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INTRODUCTION In higher plants, starch consists of two main components, mainly linear amylose and highly branched amylopectin, and exists as discrete semicrystalline granules with varying sizes (1− 100 μm), shapes (spherical, lenticular, polyhedral, and irregular), and size distributions (unimodal and bimodal).1 Starches from wheat, barley, and triticale have a bimodal granule size distribution.2 Specifically for wheat, there is one population of large lenticular-shaped granules ranging in size from about 10 to 40 μm and another population of small spherical granules ranging from about 1 to 10 μm. These large and small granules significantly differ in their chemical compositions, functional properties, and molecular structures and also have different end uses.2−5 Starches from most other plant species, such as maize and potato, have only a unimodal size distribution, covering a wide range of granule sizes. Some unimodal size distribution starches have been separated into large-, medium-, and small-sized fractions.6−10 Larger granules contain more amylose,6−8 have higher peak, trough, and final pasting viscosities,9 and show lower gelatinization temperature than smaller granules.10 However, the branch-chain length distribution of amylopectin is similar for granules of different sizes.6,7 The above structural property studies of different-sized granules can provide insights into the role that granule size plays in determining functional properties and uses of starches.6 However, these studies are mainly focused on waxy and normal crop starches, particularly bimodal starches.2−10 No work has been done to relate the effect of granule size to structural properties in high-amylose starches. High-amylose starches show significantly different physicochemical properties from normal starches, have a high level of resistant starch content, © XXXX American Chemical Society

and are of interest because of their potential health benefits.11−20 Many high-amylose crop starches contain different morphology granules, covering a wide range of granule sizes.12,13,16−19,21 For example, elongated granules, hollow granules, and aggregate granules appear in high-amylose maize,12 wheat,16,18 and rice starches,13,19 respectively. The structure difference of individual granules in high-amylose starch has been in situ observed using some microscopy techniques.21−25 However, the structural properties of different morphology and sized granules have not been determined in high-amylose starches. This might be due to the practical difficulty of separating different-sized granules from highamylose starches. In this study, large-, medium-, and small-sized granules were separated from normal and high-amylose maize starches. The molecular weight distribution of different-sized fractions was investigated using gel permeation chromatography (GPC), and crystal structure was studied using multiple physical techniques, including X-ray powder diffraction (XRD), attenuated total reflectance Fourier transform infrared (ATR-FTIR), solid-state 13 C cross-polarization magic-angle spinning nuclear magnetic resonance (13C CP/MAS NMR), and small-angle X-ray scattering (SAXS). The objective of this study was to investigate the difference of structural properties of highamylose maize starch fractions varying in granule size. The results could add to our understanding of structural properties of different-sized granules from high-amylose starch and would Received: August 11, 2014 Revised: November 6, 2014 Accepted: November 13, 2014

A

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Glycerol Centrifugation. The starches were separated into different-sized fractions by glycerol centrifugation according to the method of Peng et al.28 with some modifications (Supporting Information Figure S5). Starch suspension (2.5%, w/v; 40 mL) in 80% glycerol was centrifuged at 100g for 5 min. The supernatant was removed to a beaker. The pellet was suspended with 40 mL of 80% glycerol and centrifuged five times to obtain starch precipitate that constituted the large-sized fraction. The supernatants were pooled and centrifuged at 5000g for 10 min. The resulting starch pellet was suspended with 40 mL of 60% glycerol and centrifuged at 100g for 5 min. The supernatant was removed to a beaker. The pellet was suspended with 40 mL of 60% glycerol and centrifuged five times to obtain starch precipitate that constituted the medium-sized fraction. The supernatants were pooled and centrifuged at 5000g for 10 min. The resulting starch pellet comprised the small-sized fraction. Finally, the starch fractions were washed and dried as described above for microsieving. Morphology Observation. The micromorphology of starch granules was viewed with an Olympus BX53 light microscope according to the method described by Cai et al.22 For scanning electron microscope (SEM) observation, starch granules were suspended in anhydrous ethanol. Twenty microliters of the starch− ethanol suspension was applied to an aluminum stub using doublesided adhesive tape, and the starch was coated with gold using a sputter coater. Starch samples were observed and photographed using an environmental SEM (ESEM XL-30, Philips, The Netherlands). Granule Size Analysis. Granule size analysis was carried out using a laser diffraction particle size analyzer (Mastersizer 2000, Malvern, UK). The starches were suspended in distilled water and stirred at 2000 rpm. The obscuration in all measurements was >10%. Particle size is defined in terms of the 10th percentile [d(0.1)], median [d(0.5)], 90th percentile [d(0.9)], surface-weighted mean [D(3,2)], and volume-weighted mean [D(4,3)]. Apparent Amylose Content (AC) Determination. Apparent AC was determined using an iodine colorimetric method as described by Man et al.15 Molecular Weight Distribution Analysis. Starch was deproteinized with protease and sodium bisulfite and debranched with isoamylase according to the methods of Tran et al.29 and Li et al.30 The molecular weight distribution of debranched starch was analyzed using a PL-GPC 220 high-temperature chromatograph (Agilent Technologies UK Limited, Shropshire, UK) with three columns (PL110-6100, 6300, 6525) and a differential refractive index detector according to the method of Cai et al.31 Crystal Structure Analysis. The crystal structure of starch was analyzed on an XRD (D8, Bruker, Germany) spectroscope. The XRD analysis and determination of the relative crystallinity were carried out following the method described by Man et al.15 Before measurements, all of the specimens were stored in a moist chamber where a saturated solution of NaCl maintained a constant humidity (relative humidity = 75%) for 1 week. Short-Range Ordered Structure Analysis. Short-range ordered structure of starch was analyzed on a Varian 7 000 FTIR spectrometer with a DTGS detector equipped with an ATR single-reflectance cell containing a germanium crystal (45° incidence angle) (PIKE Technologies, USA) as previously described by Man et al.15 Original spectra were corrected by subtraction of the baseline in the region from 1200 to 800 cm−1 before deconvolution was applied using Resolutions Pro. The assumed line shape was Lorentzian with a halfwidth of 19 cm−1 and a resolution enhancement factor of 1.9. Intensity measurements at 1045, 1022, and 995 cm−1 were performed on the deconvoluted spectra by recording the height of the absorbance bands from the baseline using Adobe Photoshop 7.0 image software. Solid-State 13C CP/MAS NMR Analysis. High-resolution solidstate 13C CP/MAS NMR analysis of starch was carried out at B0 = 9.4T on a Bruker AVANCE III 400 WB spectrometer as described previously by Cai et al.31 Amorphous starch was prepared by gelatinizing native starch following the method of Atichokudomchai et al.32 The spectrum of amorphous starch was matched to the intensity of native starch at 84 ppm and subtracted to produce the

be useful for various applications of high-amylose starches in the food and nonfood industries.

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MATERIALS AND METHODS

Materials. Native normal maize starch (S4126) (NS) and highamylose maize starch (S4180) (HS) were purchased from SigmaAldrich. Separation of Large-, Medium-, and Small-Sized Fractions. The native normal and high-amylose maize starches were separated into large-, medium-, and small-sized fractions using five methods (Supporting Information Figures S1−S5). The main procedure of the method is briefly explained below. Microsieving. The starches were separated into different-sized fractions by microsieving according to the method of Kaur et al.9 with some modifications (Supporting Information Figure S1). Starch suspension (0.2%, w/v) in distilled water was passed sequentially through 10 and 5 μm sieves. The granules retained on the 10 μm sieve were collected as the large-sized fraction. The filtrate that passed through the 10 μm sieve was screened further through the 5 μm sieve to obtain a filtrate containing the small-sized fraction. The starch retained on the 5 μm sieve was collected as the medium-sized fraction. The collected starch fractions were centrifuged and washed in distilled water and twice in anhydrous ethanol and then dried at 40 °C for 2 days, ground into powders in a mortar with pestle, and passed through a 100 mesh sieve. Extreme Sedimentation. The starches were separated into different-sized fractions by extreme sedimentation according to the method of Ao and Jane2 with some modifications (Supporting Information Figure S2). Starch suspension (0.2%, w/v; 150 mL) in distilled water was allowed to settle for 0.5 h in a graduated cylinder (250 mL). The supernatant was decanted to a glass beaker. The starch pellet was made up to 150 mL with distilled water and mixed and then was allowed to settle for 0.5 h to obtain the supernatant and starch precipitate again. The process was repeated 10 times. The starch precipitate was collected as the large-sized fraction. The suspensions were pooled and centrifuged at 5000g for 10 min. The resulting precipitate was suspended to 150 mL in the cylinder and allowed to settle for 1 h. The supernatant was decanted to another cylinder and made up to 150 mL with distilled water and was mixed and allowed to settle for 1 h to obtain the supernatant and starch precipitate again. The process was repeated 10 times. The starch in the supernatant was collected as the small-sized fraction. The starch precipitate during the separation of small-sized granules was pooled and collected as the medium-sized fraction. Finally, the starch fractions were washed and dried as described above for microsieving. Differential Sedimentation. The starches were separated into different-sized fractions by differential sedimentation according to the method of Takeda et al.26 with some modifications (Supporting Information Figure S3). Starch suspension (0.2%, w/v; 150 mL) in distilled water was allowed to settle in 250 mL cylinders for 0.5 and 2 h. The fractions of 0.5 h precipitate, 0.5−2 h precipitate, and 2 h supernatant were collected as large-, medium-, and small-sized granules, respectively. The fractionation was repeated 10 times on each fraction to be refined. Finally, the starch fractions were washed and dried as described above for microsieving. Differential Supernatant. The starches were separated into different-sized fractions by differential supernatant according to the method of Dhital et al.27 with some modifications (Supporting Information Figure S4). Starch suspension (0.2%, w/v) in distilled water was allowed to settle in a cylinder for 2 and 0.5 h. After the desired time, the top 10 cm of supernatant was pipetted to a beaker. The remaining suspension in the cylinder was then made up to the original volume with distilled water and mixed and then was allowed to settle for collecting supernatant. The pipetting supernatant was repeated 10 times. The starches in the supernatants of 2 and 0.5 h settlement were collected as small- and medium-sized fractions, respectively. The starch precipitate of 0.5 h settlement was collected as large-sized fraction. Finally, the starch fractions were washed and dried as described above for microsieving. B

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ordered subspectrum. The quantitative analyses of single-helix, doublehelix, and amorphous conformational features within starch were carried out according to the method described by Tan et al.33 SAXS Analysis. SAXS measurement of starch was performed according to the method of Yuryev et al.34 with some modifications. Starch was dispersed in an excess of distilled water to form slurries. SAXS measurement was obtained using a Bruker NanoStar SAXS instrument equipped with Vantec 2000 detector and pinhole collimation for point focus geometry. The X-ray source was a copper rotating anode (0.1 mm filament) operating at 50 kV and 30 W, fitted with cross-coupled Göbel mirrors, resulting in Cu Kα radiation wavelength of 1.5418 Å. The optics and sample chamber were under vacuum to minimize air scattering. During X-ray exposure, the starch slurries were kept in sealed cells to prevent dehydration. SAXS data sets were analyzed using DIFFRACplus NanoFit software. Parameters of the SAXS spectrum were determined according to the simple graphical method.34 Statistical Analysis. The data reported in all tables are mean values and standard deviation. Analysis of variance (ANOVA) by Tukey’s test (p < 0.05) was evaluated using the SPSS 16.0 Statistical Software Program.

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RESULTS AND DISCUSSION Morphology of Large-, Medium-, and Small-Sized Starch Fractions. Figures S6 and S7 in the Supporting Information show the micromorphologies of native starches and their different-sized fractions. For native normal maize starch, most of the granules were polygonal with large size, and some granules were spherical with small size (Figure S6). For native high-amylose maize starch, some elongated starch granules were also observed except polygonal granules with large size and spherical granules with small size (Figure S7). It is very important to establish the size separation methods that give narrow granule size fractions for investigating the structural properties of different-sized starch granules. In this study, five procedures, including microsieving, extreme sedimentation, differential sedimentation, differential supernatant, and glycerol centrifugation separation, have been used to separate large-, middle-, and small-sized fractions from normal and highamylose maize starches. Microsieving could not completely separate large-, medium-, and small-sized fractions. Some medium- and small-sized granules were retained in the largesized fraction. Some large-sized granules passed through the screen and were present with medium- and small-sized granules in the filtrate (Figures S6 and S7). Some literature also reports that microsieving can not give a homogeneous size granule population.28 Sedimentation in a water column separates different-sized granules on the basis of the difference in sedimentation rates. Different sedimentation procedures have been reported in refs 2, 26, and 27. In this study, extreme sedimentation, differential sedimentation, and differential supernatant separation methods were compared (Figures S6 and S7). The results showed that the extreme sedimentation and differential supernatant separation methods were better than differential sedimentation for giving homogeneous size granules. Because of the low density of water, the difference among sedimentation rates of different-sized granules in water is not large enough to separate the different-sized granules. In addition, it takes a very long time to obtain enough small-sized granules by extreme sedimentation and differential supernatant separation methods. Because glycerol solution has higher density than water, the difference among sedimentation rates of different-sized granules in glycerol is large enough to separate the different-sized granules. Thus, in the present study, the glycerol centrifugations through two glycerol solutions (80

Figure 1. SEM photographs of native (A, E) and large- (B, F), medium- (C, G), and small-sized (D, H) fractions of normal (A−D) and high-amylose (E−H) maize starches separated by glycerol centrifugation method. Scale bar = 20 μm.

and 60%, v/v) were used to purify large-, medium-, and smallsized granules from native normal and high-amylose maize starches. The granule morphology and sizes were significantly different in different-sized fractions separated by glycerol centrifugation (Figures S6 and S7). SEM was used to complement the light microscope images in an effort to reveal details about the granule surface that could not be distinguished by light microscope (Figure 1). The surfaces of the granules from native and large-, medium-, and small-sized fractions of normal and high-amylose maize starches were smooth. The native starches from normal and highamylose maizes consisted of mixed populations of large, medium, and small granules. The large and medium granules were mainly polyhedral or irregular in shape, whereas the small granules were mainly spherical or ellipsoidal. In addition, some elongated granules were also observed in the small-sized fraction of high-amylose starch. Size of Large-, Medium-, and Small-Sized Starch Fractions. The laser diffraction instrument for granule size measurement provides derived outputs of a volume distribution, standard mean diameters [D(3,2) and D(4,3)], and distribution information [d(0.1), d(0.5), and d(0.9)]. The C

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13.86 17.72 13.84 8.40

± ± ± ± 0.01c 0.01d 0.00b 0.01a

d(0.5)

b

20.38 25.11 20.99 14.00

± ± ± ± 0.02b 0.02d 0.01c 0.03a

d(0.9)

b

normal maize (μm) 7.49 17.11 13.16 7.79

± ± ± ± 0.01a 0.01d 0.00c 0.01b

D(3,2)

c

13.88 18.38 14.54 9.03

± ± ± ± 0.01b 0.01d 0.01c 0.01a

D(4,3)

c

6.60 13.96 8.55 4.26

± ± ± ± 0.01b 0.00d 0.01c 0.00a

d(0.1)

b

13.22 19.75 14.36 8.15

± ± ± ± 0.01b 0.00d 0.03c 0.01a

d(0.5)

b

22.53 28.01 21.36 13.70

± ± ± ± 0.02c 0.01d 0.08b 0.02a

d(0.9)b

high-amylose maize (μm) 7.70 19.07 7.64 5.45

± ± ± ± 0.00c 0.00d 0.01b 0.00a

D(3,2)c 13.83 20.48 14.41 8.52

± ± ± ±

0.01b 0.00d 0.04c 0.01a

D(4,3)c

D

30.9 31.4 31.0 30.3

± ± ± ±

MC 0.4a 0.7a 0.5a 0.8a

b

normal maize (%) 30.9 31.7 29.8 29.3

± ± ± ±

MD 0.4bc 0.4c 0.7ab 0.5a

b

30.9 31.9 31.2 29.7

± ± ± ± 0.4b 0.4c 0.3bc 0.4a

MEb

56.0 45.5 56.3 65.5

± ± ± ±

0.4b 0.8a 1.0b 0.8c

MAb

56.0 32.1 46.5 68.5

± ± ± ±

0.4c 0.9a 0.9b 0.8d

MBb

56.0 31.6 46.5 58.1

± ± ± ±

0.4c 0.9a 0.8b 0.7d

MCb

high-amylose maize (%) 56.0 32.6 48.9 75.3

± ± ± ±

0.4c 0.9a 0.9b 1.0d

M Db

56.0 33.2 50.5 74.1

± ± ± ±

0.4c 0.4a 0.9b 0.3d

MEb

Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (P < 0.05). bMA, MB, MC, MD, and ME indicate the separation methods of microsieving, extreme sedimentation, differential sedimentation, differential supernatant, and glycerol centrifugation, respectively.

a

0.4ab 0.5c 0.7bc 0.3a

± ± ± ±

30.9 32.4 31.4 29.8

native large-sized medium-sized small-sized

0.4ab 0.4b 0.6a 0.4a

± ± ± ±

fraction

30.9 31.5 30.1 29.8

MBb

MAb

Table 2. Apparent Amylose Contents of Native and Large-, Medium-, and Small-Sized Fractions of Normal and High-Amylose Maize Starchesa

a

Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (P < 0.05). bd(0.1), d(0.5), and d(0.9) are the granule sizes at which 10, 50, and 90% of all the granules by volume are smaller, respectively. cD(3,2) is the surface area-weighted mean diameter. D(4,3) is the volume-weighted mean diameter.

0.01b 0.01d 0.00c 0.00a

± ± ± ±

native large-sized medium-sized small-sized

8.30 12.54 9.12 5.02

d(0.1)

fraction

b

Table 1. Diameters of Native and Large-, Medium-, and Small-Sized Fractions of Normal and High-Amylose Maize Starches Separated by Glycerol Centrifugation Methoda

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Figure 2. Absorbance spectra of starches in the I2/KI solution (A, B) and GPC chromatograms of isoamylase-debranched starches (C, D) from normal (A, C) and high-amylose (B, D) maize starches separated by glycerol centrifugation method. NS, NS-L, NS-M, and NS-S: native and large-, medium-, and small-sized fractions of normal maize starch. HS, HS-L, HS-M, and HS-S: native and large-, medium-, and small-sized fractions of highamylose maize starch.

On the basis of the morphologies (Supporting Information Figures S6 and S7), ACs (Table 2), and purified efficiency of different-sized fractions separated by five methods, the separation effectiveness of glycerol centrifugation method was the best among five methods. In this study, structural properties of starches were investigated in the different-sized fractions separated by glycerol centrifugation method. Iodine Absorption Spectra of Large-, Medium-, and Small-Sized Starch Fractions. The absorption spectra of iodine−starch complex are shown in Figure 2A,B. The absorption spectra were similar among native and large-, medium-, and small-sized starch fractions of normal maize (Figure 2A), but significantly different among native and large-, medium-, and small-sized starch fractions of high-amylose maize (Figure 2B). The maximum absorption wavelength was similar among different-sized fractions of normal and highamylose maize starches. The iodine blue value slightly decreased with decrease of granule size for normal maize starch, but markedly increased with decrease of granule size for high-amylose starch (Table 3). Molecular Weight Distribution of Large-, Medium-, and Small-Sized Starch Fractions. The molecular weight distributions of isoamylase-debranched starches as determined by GPC are shown in Figure 2C,D. A trimodal distribution of low, middle, and high molecular weight peaks, designated peak 1, peak 2, and peak 3, respectively, was observed. Peaks 1 and 2 consist of short (A and short B chains) and long (long B chains) branch-chains of amylopectin, respectively. The area ratio of peak 1 to peak 2 might be used as an index of the extent of branching of amylopectin; the higher the ratio, the higher the branching degree.35 Peak 3 includes amylose.36 The GPC

volume distributions of different-sized fractions of normal and high-amylose maize starches are presented in Figure S8 in the Supporting Information. Native starches were unimodal distribution and could be separated into large-, medium-, and small-sized fractions. The granule size range of high-amylose maize starch was broader than that of normal maize starch, especially for native starch. An overlap between the fractions could be seen. The results of granule size measurements are listed in Table 1. The d(0.5), D(3,2), and D(4,3) values of large-, medium-, and small-sized fractions were reduced from 17.72 to 13.84 and 8.40 μm, from 17.11 to 13.16 and 7.79 μm, and from 18.38 to 14.54 and 9.03 μm, respectively, for normal maize starch and from 19.75 to 14.36 and 8.15 μm, from 19.07 to 7.64 and 5.45 μm, and from 20.48 to 14.41 and 8.52 μm, respectively, for high-amylose maize starch. Apparent AC of Large-, Medium-, and Small-Sized Starch Fractions. Apparent ACs of native and large-, medium-, and small-sized fractions of normal and high-amylose maize starches were determined using the iodine colorimetric method and are shown in Table 2. The results of five separation methods all showed that the apparent AC slightly decreased with decrease of granule size for normal maize starch, but markedly increased with decrease of granule size for highamylose starch. For normal unimodal starch, the decrease in AC with decrease of granule size has been previously reported.6,8−10 However, the significant increase of AC with decrease of granule size was not reported in previous studies. For high-amylose maize starch, it is noteworthy that the largesized granules had only about 32% apparent AC and the smallsized granules had about 75% apparent AC for differential supernatant and glycerol centrifugation separation methods. E

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Data are means ± standard deviations, n = 3 for iodine blue values and =2 for GPC parameters. Values in the same column with different letters are significantly different (P < 0.05). bIodine blue values are the absorbance at 680 nm of starch−iodine complex. cGPC parameters of isoamylase-debranched starches. The peak 1/peak 2 ratio is determined according to the area ratio of peak 1 and peak 2.

parameters are summarized in Table 3. The AC determined on the basis of iodine and starch affinity is described as apparent AC, which overestimates AC if there are branched molecules with long side chains that bind iodine.37 The present results showed that the AC determined by GPC (Table 3) was significantly lower than the apparent AC obtained by iodine colorimetric method (Table 2), which was in agreement with the report of Shi et al.37 and indicated that the intermediate chain and amylopectin long branch-chain could bind iodine to increase the value of apparent AC. The study of Dhital et al.6 showed that the molecular weight distributions of fully branched normal maize starch fractions were almost similar, although their apparent ACs were different. In the present study, the small-sized fraction of normal starch also showed significantly lower apparent AC than the large-sized fraction, but the ACs and amylopectin branching degree determined by GPC had no significant difference among different-sized fractions of normal maize starch. The results agreed with the conclusion of Dhital et al.6 that the lower apparent AC in the smaller normal starch granules was predominantly due to the higher amount of lipids in these granules that might have complexed with amylose and thereby reduced the amount of free amylose available to form a complex with iodine. For highamylose starch, the AC determined by GPC was markedly lower than the apparent AC, the branching degree of highamylose starch was also markedly lower than that of normal starch. These results indicated that high-amylose starch contained high levels of intermediate chain and amylopectin long branch-chain. The different-sized fractions of high-amylose starch had a significant increase in the amylopectin long branchchain and AC and a marked decrease in the amylopectin short branch-chain and branching degree with the decrease of granule size, indicating that the different-sized fractions of high-amylose starch had significantly different molecular structures. However, it is noteworthy that the molecular structure of large-sized fraction of high-amylose starch was similar to that of normal starch. XRD Spectra of Large-, Medium-, and Small-Sized Starch Fractions. Starches can be classified into A-, B-, and Ctype crystallinity on the basis of their XRD spectra. C-type starch is a mixture of both A- and B-type polymorphs and can be further classified to CA-type (closer to A-type), C-type, and CB-type (closer to B-type) according to the proportion of Atype and B-type polymorphs.38 The XRD patterns of native and large-, medium-, and small-sized fractions of normal maize starches are shown in Figure 3A. They all showed strong reflection at about 15° and 23° 2θ, and an unresolved doublet at 17° and 18° 2θ, which resembled the typical A-type XRD pattern of normal cereal starches,38 indicating that the nature of individual crystallites is similar in all normal maize granules regardless of the granule size. This result was in agreement with previous papers.2,6 Native high-amylose maize starch showed strong reflection at about 15°, 17°, and 23° 2θ, a small peak at about 5.6° 2θ, and a shoulder peak at about 18° 2θ (Figure 3B). The peak at 5.6° 2θ was characteristic of B-type crystallinity, and the shoulder peak of 18° 2θ was indicative of the A-type polymorph, which suggested that the starch consisted of A- and B-type polymorphs, with the A-type polymorph being the main component. Therefore, native high-amylose maize starch was CA-type crystallinity. For the large-sized fraction of highamylose maize starch, the peak at 5.6° 2θ disappeared, the peaks at 17° and 18° 2θ became an unresolved doublet, and the

a

0.1b 0.4c 0.2b 0.0a ± ± ± ± 1.9 3.6 2.3 0.9 0.2a 0.0a 0.0a 0.0a ± ± ± ± 3.5 3.6 3.6 3.8 1.3c 0.6a 0.4b 0.7d ± ± ± ± 35.6 27.9 31.5 49.1 0.1b 1.6a 1.2b 0.5c ± ± ± ± 22.6 15.8 20.7 26.5 1.4b 1.1d 1.6c 0.2a ± ± ± ± 41.9 56.4 47.9 24.5 0.5a 0.4a 1.1a 0.8a ± ± ± ± 27.5 27.8 25.2 26.3 0.6a 0.1a 0.2a 0.1a ± ± ± ± 16.0 15.8 16.3 15.4 1.1a 0.3a 0.8a 0.7a ± ± ± ± 0.355 0.367 0.354 0.334

0.006b 0.009b 0.006b 0.010a

0.520 0.367 0.476 0.636

0.002c 0.006a 0.005b 0.006d ± ± ± ± ± ± ± ± native large-sized medium-sized small-sized

56.6 56.5 58.6 58.4

high-amylose maize normal maize peak 3 peak 2 peak 1 peak 3 peak 2 peak 1 high-amylose maize normal maize fraction

peak 1/peak 2c peak area of high-amylose maizec (%) peak area of normal maizec (%) iodine blue valueb

Table 3. Iodine Blue Values and GPC Parameters of Native and Large-, Medium-, and Small-Sized Fractions of Normal and High-Amylose Maize Starches Separated by Glycerol Centrifugation Methoda

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Figure 3. XRD spectra (A, B) and ATR-FTIR deconvoluted spectra (C, D) of native and large-, medium-, and small-sized fractions of normal (A, C) and high-amylose (B, D) maize starches separated by glycerol centrifugation method. NS, NS-L, NS-M, and NS-S: native and large-, medium-, and small-sized fractions of normal maize starch. HS, HS-L, HS-M, and HS-S: native and large-, medium-, and small-sized fractions of high-amylose maize starch.

Table 4. Relative Crystallinities and IR Ratios of Native and Large-, Medium-, and Small-Sized Fractions of Normal and HighAmylose Maize Starches Separated by Glycerol Centrifugation Methoda 1045/1022 (cm−1)

relative crystallinity (%) fraction native large-sized medium-sized small-sized a

1022/995 (cm−1)

normal maize

high-amylose maize

normal maize

high-amylose maize

normal maize

high-amylose maize

± ± ± ±

19.33 ± 0.63b 22.26 ± 0.97c 19.61 ± 1.02b 16.36 ± 0.97a

0.60 ± 0.01b 0.61 ± 0.01b 0.61 ± 0.01b 0.53 ± 0.00a

0.68 ± 0.00b 0.65 ± 0.01a 0.67 ± 0.00b 0.70 ± 0.01c

0.91 ± 0.01b 0.84 ± 0.01a 0.93 ± 0.01c 0.95 ± 0.01d

0.80 ± 0.01c 0.88 ± 0.00d 0.76 ± 0.01b 0.64 ± 0.01a

22.31 21.49 22.25 21.42

0.77a 0.96a 0.71a 0.91a

Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (P < 0.05).

peaks at 15° and 23° 2θ appeared to sharpen, indicating that the large-sized fraction of high-amylose maize starch was typical A-type crystallinity. The spectrum of the medium-sized fraction of high-amylose maize starch was very similar to that of native high-amylose maize and exhibited a CA-type crystallinity. However, the shoulder peak at 18° 2θ disappeared and the peak at 23° 2θ became broad in the small-sized fraction of highamylose maize starch, exhibiting a typical C-type crystallinity. Most high-amylose starches are B-type crystallinity,12,14 but some C-type starches have also been reported in high-amylose cereal crops.20,38 Cheetham and Tao38 thought that the crystal type of maize starch could be varied from A- to B- via C-type when AC increased; the transition occurred at about 40% AC. In the present study, the AC of native high-amylose maize starch determined by GPC was about 35.6%. The result was in agreement with the report of Cheetham and Tao38 that maize starch with about 40% AC had C-type crystallinity. C-type starch contains A- and B-type allomorphs. To date, the literature reports that A- and B-type allomorphs are distributed in the same granule. It is never reported that A- and B-type

polymorphs are distributed in different granules; that is, C-type starches consist of A-type and B-type granules. In the present study, the results of C-type native starch and A-type large-sized starch from high-amylose maize indicated that C-type starches could contain some A-type granules. Table 4 shows the relative crystallinities of starches, which were calculated from the XRD patterns. For normal maize starch, the crystallinities did not show significant difference among different-sized fractions. For high-amylose maize starch, the crystallinities significantly decreased with the decrease of granule size. This result was consistent with the findings of Cheetham and Tao38 that relative crystallinity was negatively correlated with AC. ATR-FTIR Spectra of Large-, Medium-, and SmallSized Starch Fractions. The ATR-FTIR spectrum of starch is sensitive to the short-range ordered structure, defined as the double-helical order, as opposed to the long-range ordered structure related to the packing of double helices.39 The variations in spectra among different starches can be interpreted in terms of the short-range ordered structure present in the G

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Figure 4. 13C CP/MAS NMR original spectra (A, B) and ordered subspectra (C, D) of native and large-, medium-, and small-sized fractions of normal (A, C) and high-amylose (B, D) maize starches separated by glycerol centrifugation method. AS: amorphous starch. NS, NS-L, NS-M, and NS-S: native and large-, medium-, and small-sized fractions of normal maize starch. HS, HS-L, HS-M, and HS-S: native and large-, medium-, and small-sized fractions of high-amylose maize starch.

according to data reported in the literature.32,40 The C1 resonance, which gives information about both the crystalline nature as well as the noncrystalline (but rigid) chains, has been used to examine the structure of different types of starches. The multiplicity of C1 resonance corresponds to the packing type of the starch granule. A-type starch, which has three nonidentical sugar residues, shows a triplet at the C1 region, whereas B-type starch, which has two nonidentical sugar residues, shows a doublet.32 In general, typical C-type starch has an inconspicuous triplet or doublet at the C1 region. However, CA-type starch shows a triplet at the C1 region and CB-type starch shows a doublet at the C1 region.40 The C1 resonances of native and large-, medium-, and smallsized fractions of normal maize starch occurred as triplets at about 99.5, 100.5, and 101.5 ppm (Figure 4A), those of native and large-sized fractions of high-amylose maize starch showed an inconspicuous triplet, and those of medium- and small-sized fractions of high-amylose maize starch showed an inconspicuous doublet (Figure 4B). Panels C and D of Figure 4 show the ordered subspectra of starches. In this analysis, the standard amorphous starch spectrum was subtracted from the test spectrum until there was no residual intensity at 84 ppm (a region of the spectrum with intensity due solely to amorphous conformations).33 The ordered subspectra were similar to the original spectra, but they had clearer crystalline peaks of C1 resonances than those in the original spectra. The clear triplets were observed in the spectra of different-sized fractions of normal maize starch and native and large-sized fractions of high-amylose maize starch, and the clear doublet was observed in the spectra of medium- and small-sized fractions of highamylose maize starch. These results, combined with the results from the XRD analysis, further confirmed that the different-

external region of starch granule. The intensity of absorbance at 1045, 1022, and 995 cm−1 is sensitive to changes in starch conformation. The bands at 1045 and 1022 cm−1 are associated with ordered/crystalline and amorphous regions in starch, respectively. The ratio of absorbance 1045/1022 cm−1 is used to quantify the degree of order, and that of 1022/995 cm−1 can be used as a measure of the proportion of amorphous to ordered carbohydrate structure in the starch.39 The ATR-FTIR spectra of native and large-, medium-, and small-sized fractions of normal and high-amylose maize starches in the 1200−900 cm−1 region are shown in Figure 3C,D. The ratios for 1045/1022 and 1022/995 cm−1 of starches from normal and high-amylose maizes are shown in Table 4. On the basis of both the spectra and calculated data, the ATR-FTIR characteristics of different-sized fractions of normal maize starch were similar, but high-amylose maize starch had significantly different ATR-FTIR characteristics among different-sized fractions. The ATR-FTIR characteristics of the largesized fraction of high-amylose maize starch was much closer to those of different-sized fractions of normal maize starch. It is noteworthy that the ratio of 1022/995 cm−1 significantly increased with the decrease of granule size for normal maize starch, but markedly decreased for high-amylose maize starch. These results indicated that the different-sized fractions of normal maize starch and large-sized granules of high-amylose maize starch had similar short-range ordered structure, whereas granule size significantly affected the short-range ordered structure of high-amylose maize starch. 13 C CP/MAS NMR Spectra of Large-, Medium-, and Small-Sized Starch Fractions. The solid-state 13C CP/MAS NMR patterns for starches are shown in Figure 4. The resonances at different parts per million were assigned H

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Data are means ± standard deviations, n = 3 for the proportions of single-helix, double-helix, and amorphous conformations and = 2 for SAXS parameters. Values in the same column with different letters are significantly different (P < 0.05). bRelative proportions of single-helix, double-helix, and amorphous conformations are determined by 13C CP/MAS NMR. cImax, peak intensity; D, lamellar distance (Bragg spacing).

sized fractions of normal maize starch and the large-sized fraction of high-amylose maize starch were all A-type crystallinity, native and medium-sized fractions of high-amylose maize starch were CA-type crystallinity, and the small-sized fraction was C-type crystallinity. The ordered subspectra were peak-fitted. A combination (50/50) of Lorentzian and Gaussian profiles gave an acceptable fit (data not shown). The proportions of single-helix, doublehelix, and amorphous components are listed in Table 5 and showed similarity among different-sized fractions of normal maize starch but some difference among different-sized fractions of high-amylose maize starch. The amylopectin side chains can form two types of helices in starch granule. Helices that are packed in the short-range order are defined as the double-helical order, and helices that are packed in long-range order are related to the packing of double helices forming crystallinity. The double helices in the short- and long-range distance can both be detected by 13C CP/MAS NMR, but only the double helices in the long-range distance can be detected by XRD.32 Therefore, the double-helix content of starch was generally higher than its relative crystallinity. SAXS Spectra of Large-, Medium-, and Small-Sized Starch Fractions. A comparison of the SAXS patterns of the starches is shown in Figure 5. The well-resolved main scattering peak around scattering vector (q0) of about 0.06 Å−1 is thought to arise from the periodic arrangement of alternating crystalline and amorphous lamellae of amylopectin and corresponds to the lamellar repeat distance or Bragg spacing. The location of the peak depends on the size of lamella and may differ among starches from different plants, whereas the peak area or intensity depends mainly on the degree of order in semicrystalline regions.41 As shown in Figure 5, all starches were scaled to equal intensity at high q (q = 0.2 Å−1) to account for variations in sample concentrations, according to the method of Sanderson et al.42 The SAXS patterns were at the same relative scale and, therefore, were directly comparable. Native and large-, medium-, and small-sized fractions of normal and highamylose maize starches all showed the most defined lamellar peak centered at 0.063 Å−1, which corresponded to a lamellar repeat distance of 10.0 nm. However, the lamellar peak intensity showed significant difference among these starch fractions, especially for high-amylose maize starch (Table 5). The extremely low lamellar peak intensity of small-sized fraction of high-amylose starch likely indicated a severely disrupted or nonexistent lamellar structure compared to that of large- and medium-sized fractions. Yuryev et al.34 reported that an increase in AC in wheat starches was accompanied by decreased peak scattering intensity, which resulted from accumulation of amylose tie-chains with increasing AC in the granules. The starch fractions from high-amylose maize showed significantly lower peak intensities with decrease of granule size, which might result from the elevated AC (Tables 2 and 3). In conclusion, the structural properties of native and large-, medium-, and small-sized fractions of normal and high-amylose maize starches separated by glycerol centrifugation method were investigated to reveal whether structural properties differed among the different-sized fractions of the starches. The results showed that the granule sizes had no significant effect on structural properties of normal starch, but the different-sized fractions of high-amylose maize starch showed markedly different structural properties. The small-sized granules of high-amylose starch had significantly higher AC, amylopectin long branch-chain, and IR ratio of 1045/1022

a

0.0a 0.0a 0.0a 0.0a ± ± ± ± 10.0 10.0 10.0 10.0 5.8b 0.3c 18.6b 1.4a ± ± ± ± 171.3 224.1 181.2 74.6 0.9a 0.9a 0.7a 0.7b ± ± ± ± 63.6 61.2 63.3 66.2 0.7b 0.9b 0.6b 1.0a ± ± ± ± 30.8 32.0 30.2 26.7 0.5a 0.6a 0.2a 0.9a ± ± ± ± 5.7 6.8 6.5 7.2 0.0a 0.0a 0.0a 0.0a ± ± ± ± 10.0 10.0 10.0 10.0 0.0b 7.0c 0.6bc 1.9a ± ± ± ± 200.7 215.0 205.8 187.9 0.8a 0.9a 1.1a 0.9a ± ± ± ± 6.2 6.1 6.3 6.9

0.2a 0.2a 1.0a 0.3a

32.5 31.7 33.7 32.9

0.5a 0.6a 1.0a 0.7a ± ± ± ± ± ± ± ± native large-sized medium-sized small-sized

61.2 62.2 60.0 60.2

Dc (nm) Imaxc (counts) amorphousb (%)

high-amylose maize

double helixb (%) single helixb (%) Dc (nm) Imaxc (counts) normal maize

amorphousb (%) double helixb (%) single helixb (%) fraction

Table 5. Proportions of Single-Helix, Double-Helix, and Amorphous Conformations and SAXS Parameters of Native and Large-, Medium-, and Small-Sized Fractions of Normal and High-Amylose Maize Starches Separated by Glycerol Centrifugation Methoda

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Figure 5. SAXS patterns of native and large-, medium-, and small-sized fractions of normal (A) and high-amylose (B) maize starches separated by glycerol centrifugation method. NS, NS-L, NS-M, and NS-S: native and large-, medium-, and small-sized fractions of normal maize starch. HS, HS-L, HS-M, and HS-S: native and large-, medium-, and small-sized fractions of high-amylose maize starch.

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cm−1, lower amylopectin short branch-chain and branching degree, relative crystallinity, IR ratio of 1022/995 cm−1, and peak intensities of lamellar structure than the large-sized granules. The differences in crystal structure among the different-sized fractions indicated that C-type starch might contain A-type starch granules.

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ABBREVIATIONS USED AC, amylose content; ATR-FTIR, attenuated total reflectance Fourier transform infrared; 13C CP/MAS NMR, solid-state 13C cross-polarization magic-angle spinning nuclear magnetic resonance; GPC, gel permeation chromatography; SAXS, small-angle X-ray scattering; SEM, scanning electron microscope; XRD, X-ray powder diffraction

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ASSOCIATED CONTENT

S Supporting Information *

Schematic diagrams of the microsieving separation, the extreme sedimentation separation, the differential sedimentation separation, the differential supernatant separation, and the glycerol centrifugation separation of starch granules (Figures S1−S5); micrographs of different-sized fractions of normal and highamylose maize starches separated by different methods (Figures S6 and S7); granule size distribution of native and large-, medium-, and small-sized fractions of normal and high-amylose maize starches separated by glycerol centrifugation method (Figure S8). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(C.W.) Mail: College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China. Phone: +86 514 87997217. E-mail: [email protected]. Funding

This study was financially supported by grants from the National Natural Science Foundation of China (31270221), the Talent Project of Yangzhou University, the Innovation Program for Graduates of Jiangsu Province (CXZZ13_0895), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Robert G. Gilbert (Huazhong University of Science and Technology, China) and Prof. Chengjun Zhu (Wuhan Ausinorigin High Tech Co., Ltd., China) for kindly providing technical assistance in GPC analysis. J

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K

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