Article pubs.acs.org/JAFC

Different Structures of Heterogeneous Starch Granules from HighAmylose Rice Jianmin Man,†,‡ Lingshang Lin,†,‡ Zhifeng Wang,§ Youping Wang,†,‡ Qiaoquan Liu,*,†,‡ 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 ABSTRACT: High-amylose cereal starches usually have heterogeneous starch granules in morphological structure. In the present study, the polygonal, aggregate, elongated, and hollow starch granules were separated from different regions of the kernels of high-amylose rice, and their structures were investigated. The results showed that the polygonal starch granules had low amylose content and high short branch-chain and branching degree of amylopectin, and exhibited A-type crystallinity. The aggregate starch granules had high long branch-chain of amylopectin, relative crystallinity, and double helix content, and exhibited C-type crystallinity. The elongated starch granules had high amylose content and low branching degree of amylopectin and relative crystallinity, and exhibited C-type crystallinity. The hollow starch granules had very high amylose content, proportion of amorphous conformation, and amylose−lipid complex, and very low branch-chain of amylopectin, branching degree of amylopectin, and double helix content, and exhibited no crystallinity. The different structures of heterogeneous starch granules from high-amylose rice resulted in significantly different thermal properties. KEYWORDS: high-amylose rice, heterogeneous starch granule, molecular weight distribution, crystalline structure, lamellar structure, thermal property

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INTRODUCTION In higher plants, starch consists of two main components, linear amylose and highly branched amylopectin, and exists as discrete semicrystalline granules with varying sizes (1−100 μm), shapes (spherical, lenticular, polygonal, and irregular), and size distributions (unimodal and bimodal).1 Starches from wheat, barley, and triticale have a bimodal granule size distribution.2 The large lenticular granules and the small spherical granules significantly differ in their chemical compositions, functional properties, and molecular structures, and also have different end uses.2−4 Starches from most other plant species, such as maize and potato, have only a unimodal size distribution, and cover a wide range of granule sizes. Some unimodal size distribution starches have been separated into large-, medium-, and smallsize fractions.5−7 Larger granules contain more amylose,5 have higher peak, trough, and final pasting viscosities,6 and show lower gelatinization temperature than smaller granules.7 High-amylose starches have a high level of resistant starch content, and they are of interest because of their potential health benefits.8−14 Some high-amylose cereal crop lines have been developed using mutation or transgenic breeding approaches.8−14 High-amylose cereal starches always show markedly different morphologies compared with waxy and normal starches.8−12 For example, high-amylose maize starch contains some elongated starch granules;8 high-amylose wheat and barley starches have some sickle-shaped starch granules with hollow interiors;9−11 and high-amylose rice mutant Goami 2 (previously known as Suweon 464) starch consists of two populations: large voluminous bodies consisting of tightly packed small subgranules and individual granules.12 Recently, the different morphology granules in high-amylose starches © 2014 American Chemical Society

have been in situ observed using light microscopy, polarizing microscopy, hot stage microscopy, scanning electron microscopy, transmission electron microscopy, confocal laser scanning microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, and Raman microscopy, and they exhibit significantly different structures.15−20 However, the structures of different morphology granules in high-amylose starches have not been determined and analyzed using chemical and spectroscopic methods. This might be due to the practical difficulty of separating different morphology granules from high-amylose starches. A transgenic resistant starch rice line (TRS) has been developed by antisense RNA inhibition of both starch branching enzyme I (SBE I) and SBE IIb in our laboratory, which yields a starch with an amylose content (AC) of about 60%.13,21 TRS kernels are rich in resistant starch and have shown significant potential to improve the health of the large bowel in rats.13 Results from the microstructure and ultrastructure studies have revealed that TRS starch granules are significantly heterogeneous in morphological structure, and can be classified into four types: polygonal, aggregate, elongated, and hollow starch granules according to their morphologies and Maltese crosses.16 The polygonal starch granules are mainly distributed in the central region of the endosperm, the hollow starch granules in the surrounding subaleurone layer, and the aggregate and elongated starch granules in the middle region Received: Revised: Accepted: Published: 11254

August 20, 2014 October 27, 2014 October 27, 2014 October 27, 2014 dx.doi.org/10.1021/jf503999r | J. Agric. Food Chem. 2014, 62, 11254−11263

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Separation of TRS Elongated and Aggregate Starch Granules. The middle regions between the central region and the surrounding subaleurone layer of the kernels were cut with a stainless razor blade and used to isolate the crude starch as total starch isolation from kernels. The crude starch was suspended in deionized water and centrifuged at 100g for 5 min. The precipitate was repeatedly suspended and centrifuged ten times. After that, the precipitate was suspended in 30% (v/v) glycerol aqueous solution and centrifuged at 100g for 5 min. The supernatant was collected, and the precipitate was repeatedly suspended and centrifuged ten times. The collected supernatant was centrifuged at 5000g for 10 min, and the starch precipitate was washed with deionized water three times, dehydrated, and dried to obtain the TRS elongated starch granules. The precipitate obtained from starch suspension in 30% glycerol was again suspended with 40 mL of deionized water in a 50 mL centrifuge tube and settled for 30 min. The supernatant was discarded, and the starch precipitate was repeatedly suspended and settled ten times. Finally, the starch precipitate was dehydrated and dried to obtain the TRS aggregate starch granules. Morphology Observation. A starch suspension (1%, w/v) was prepared with 50% glycerol aqueous solution. Ten microliters of starch suspension was placed on the microscope slide and covered with a coverslip. The starch granule morphology and Maltese cross were viewed under an Olympus BX 53 polarizing microscope equipped with a CCD camera. Measurements of Iodine Absorption Spectrum, Iodine Blue Value, and Amylose Content. Iodine absorption spectrum, iodine blue value, and amylose content of starch were determined according to the method of Man et al.26 with some modifications. Ten milligrams of dry starch was dispersed in 5 mL of dimethyl sulfoxide (DMSO) containing 10% 6.0 M urea. Dissolution was obtained by incubating the mixture at 95 °C for 1 h with intermittent vortexing. A 1.0 mL aliquot of the starch−DMSO solution was then placed in a 50 mL volumetric flask along with 45 mL of deionized water, and 1.0 mL of iodine solution (0.2% I2 and 2% KI, w/v) was added. The mixture was made up to 50 mL with deionized water, mixed immediately, and placed in darkness for 20 min. Control solution was made in the same way but without sample. All samples were scanned from 400 to 900 nm with a spectrophotometer (Ultrospec 6300 pro, Amershan Biosciences). The λmax was the wavelength (in nm) at which the absorbance was the highest over the range of the wavelengths. Iodine blue value was measured at 680 nm under the above experimental conditions. Apparent amylose content (AAC) was evaluated from absorbance at 620 nm. The recorded values were converted to percent of amylose by reference to a standard curve prepared with amylopectin from corn (Sigma-Aldrich 10120) and amylose from potato. The experiments were performed in triplicate. Gel-Permeation Chromatography (GPC) Analysis. Starch was deproteinized with protease and sodium bisulfite, and debranched with isoamylase following the methods of Tran et al.27 and Li et al.28 The molecular weight distribution of debranched starch was analyzed using a PL-GPC 220 high temperature chromatograph (Agilent Technologies UK Limited, Shropshire, U.K.) with three columns (PL110-6100, 6300, 6525) and a differential refractive index detector according to the method of Cai et al.29 The areas of peaks were integrated, and their relative peak area was expressed as a percentage of the total area of all peaks. The experiments were performed two times. X-ray Powder Diffraction (XRD) Analysis. XRD analysis of starch was carried out on an XRD (D8, Bruker, Germany) as described by Wei et al.30 All the specimens were stored in a desiccator where a saturated solution of NaCl maintained a constant humidity for 1 week before measurements. The relative crystallinity (%) was measured following the method of Wei et al.30 A smooth curve that connected peak baselines was computer-plotted on the diffractogram. The areas of peaks at 5.6°, 15°, 17°, 18°, 20°, and 23° at 2θ above the smooth curve were taken as the crystalline portion. The ratio of diffraction peak area to total diffraction area over the diffraction angle 4−30° at 2θ was taken as the relative crystallinity. The relative crystallinity was quantitatively analyzed three times.

between the central region and the surrounding subaleurone layer.22 However, it is not clear whether these heterogeneous starch granules have different structures. In this study, heterogeneous starch granules were separated from the kernels of TRS, and their structures were investigated. The objective of this study was to compare the structures of heterogeneous starch granules from high-amylose rice. The results could add to our understanding of the structures of different morphology starch granules from high-amylose cereal crops.

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MATERIALS AND METHODS

Plant Materials. An indica rice cultivar Te-qing (TQ) and its transgenic resistant starch rice line (TRS) with high amylose content were used in this study. TRS was generated from TQ after transgenic inhibition of both SBE I and SBE IIb through an antisense RNA technique and was homozygous for the transgene.13 TQ and TRS were cultivated in the transgenic close experiment field of Yangzhou University, Yangzhou, China. Mature kernels were used to isolate starches. Total Starch Isolation. Total starches were isolated from TQ and TRS mature kernels according to the method of Wei et al.21 with some modifications. Brown kernels were steeped in deionized water overnight at 4 °C, and homogenized with ice-cold water in a home blender. The homogenate was squeezed through five layers of cotton cloth. The residue was homogenized and squeezed twice more in a mortar with a pestle to facilitate the release of starch granules. The combined extract was filtered with 150, 75, and 38 μm sieves and centrifuged at 5000g for 10 min. The yellow gel-like layer on top of the packed white starch precipitate was carefully scraped off and discarded. The process of centrifugation separation was repeated several times until no dirty material existed. The precipitated starch was dehydrated with anhydrous ethanol two times, dried at 40 °C, ground into powders, and passed through a 38 μm sieve. Separation of Heterogeneous Starch Granules. TRS polygonal, aggregate, elongated, and hollow starch granules were separated from the different regions of the mature kernels referring to the methods of Ao et al.,2 Peng et al.,23 Naguleswarana et al.,24 and Dhital et al.,25 and combining with the regional distribution of heterogeneous starch granules in endosperm. The detailed separation methods were as described below. Separation of TRS Polygonal Starch Granules. The central regions of the kernels were cut with a stainless razor blade, and used to isolate the crude starch as total starch isolation from kernels. The crude starch was suspended in deionized water and centrifuged at 100g for 5 min. The precipitate was repeatedly suspended and centrifuged ten times. Finally, the precipitate was again suspended with 40 mL of deionized water in a 50 mL centrifuge tube and settled for 1 h. The upper 30 mL of starch suspension was collected, and the residual starch suspension was repeatedly made up to 40 mL with deionized water and settled ten times. After that, the collected starch suspension was centrifuged at 5000g for 10 min, and the starch precipitate was dehydrated and dried to obtain the TRS polygonal starch granules. Separation of TRS Hollow Starch Granules. The kernels were steeped in deionized water overnight at 4 °C. The peripheral regions (about 0.3 mm thickness) of kernels were cut with a stainless razor blade and used to isolate the crude starch as total starch isolation from kernels. The crude starch was suspended in 50% (v/v) glycerol aqueous solution and centrifuged at 100g for 5 min. The supernatant was collected, and the precipitate was repeatedly suspended and centrifuged ten times. After that, the collected supernatant was centrifuged at 5000g for 10 min to obtain the starch precipitate. The starch precipitate was suspended in 30% (v/v) glycerol aqueous solution and centrifuged at 100g for 5 min. The starch precipitate was repeatedly suspended and centrifuged ten times. Finally, the starch precipitate was washed with deionized water three times to remove the glycerol, dehydrated, and dried to obtain the TRS hollow starch granules. 11255

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Figure 1. Microphotographs of starch granules under normal light (A−F) and polarizing light (a−f). (A, a), TQ total starch granules; (B, b), TRS total starch granules; (C, c), TRS polygonal starch granules; (D, d), TRS aggregate starch granules; (E, e), TRS elongated starch granules; (F, f), TRS hollow starch granules. Scale bar = 20 μm. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Analysis. ATR-FTIR analysis of starch was carried out on a Varian 7000 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 Wei et al.30 The spectra were quantitatively analyzed three times. Solid-State 13C Cross-Polarization Magic-Angle Spinning Nuclear Magnetic Resonance (13C CP/MAS NMR) Analysis. High-resolution solid-state 13C CP/MAS NMR analysis of starch was carried out at B0 = 9.4 T on a Bruker AVANCE III 400 WB spectrometer as previously described by Man et al.31 Amorphous starch was prepared by gelatinizing native starch following the method of Wei et al.30 The spectrum of amorphous starch was matched to the intensity of native starch at 84 ppm and subtracted to produce the ordered subspectrum. The 13C CP/MAS NMR original spectrum and ordered subspectrum were peak fitted by using PeakFit version 4.12. The quantitative analyses of double-helix, single-helix, and amorphous conformational features within starch were carried out according to the method described by Tan et al.32 The spectra were quantitatively analyzed three times. Small-Angle X-ray Scattering (SAXS) Analysis. SAXS measurement of starch was carried out on a Bruker NanoStar SAXS instrument equipped with Vantec 2000 detector and pinhole collimation for point focus geometry as previously described by Cai et al.33 SAXS data sets were analyzed using DIFFRACplus NanoFit software. Parameters of the SAXS spectrum were determined according to the simple graphical method.33 The experiments were performed two times.

Differential Scanning Calorimetry (DSC) Analysis. The thermal properties of starch were determined using DSC (200-F3, Netzsch, Germany). Three milligrams of starch was mixed with 9 μL of deionized water and hermetically sealed in an aluminum pan overnight at 4 °C. After equilibrating for 1 h at room temperature, the sample was scanned against a blank (empty pan) at a heating rate of 10 °C/min from 25 to 130 °C. The major parameters of the DSC profile were described as onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), gelatinization range (ΔT), and gelatinization enthalpy (ΔH). The experiments were performed two times. Statistical Analysis. The data reported in all of the tables were mean values and standard deviation. One-way analysis of variance (ANOVA) and Tukey’s test (p < 0.05) were evaluated using the SPSS 16.0 Statistical Software Program.

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RESULTS AND DISCUSSION Separations and Morphologies of TRS Heterogeneous Starch Granules. TRS starch contained heterogeneous starch granules.16 These heterogeneous granules were distributed in different regions of the endosperm.22 According to the distribution regions of heterogeneous starch granules, we separated these heterogeneous starch granules using different methods in this study. Figure 1 showed the morphology and the separation effectiveness of heterogeneous starch granules. TQ total starch granules isolated from kernels were homogeneous in morphological structure, showed polygonal 11256

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Figure 2. Absorbance spectra of starch−iodine complex (A) and GPC profiles of isoamylase-debranched starches (B). TQ, TRS, TRS-p, TRS-a, TRS-e, and TRS-h are TQ total starch, TRS total starch, TRS polygonal starch granules, TRS aggregate starch granules, TRS elongated starch granules, and TRS hollow starch granules, respectively.

Table 1. Iodine Absorbance, Amylose Content, and Molecular Weight Distribution of Starcha peak aread (%) starches TQ total starch TRS total starch TRS polygonal granule TRS aggregate granule TRS elongated granule TRS hollow granule

λmaxb (nm) 597.3 608.0 601.3 607.3 608.7 618.7

± ± ± ± ± ±

1.5 0.0 0.6 0.6 1.2 0.6

BVb a c b c c d

0.294 0.650 0.438 0.579 0.681 0.893

± ± ± ± ± ±

0.007 0.001 0.001 0.007 0.001 0.002

AACc (%) a d b c e f

25.5 60.0 39.8 52.6 62.6 80.2

± ± ± ± ± ±

0.6 0.1 0.1 0.5 0.1 0.2

peak 1

a d b c e f

55.3 20.9 37.0 34.2 20.3 7.7

± ± ± ± ± ±

0.2 0.8 0.8 0.1 0.2 0.3

peak 2 e b d c b a

19.4 23.9 27.1 28.9 22.1 16.5

± ± ± ± ± ±

0.5 0.2 0.1 0.0 1.1 0.2

peak1/peak2d

peak 3 b d e f c a

25.3 55.2 35.9 36.9 57.5 75.8

± ± ± ± ± ±

0.3 0.6 0.7 0.0 0.9 0.5

a c b b d e

2.84 0.88 1.36 1.18 0.92 0.47

± ± ± ± ± ±

0.08 0.04 0.04 0.00 0.06 0.01

e b d c b a

a Data are means ± standard deviations, n = 3 for λmax, BV, and AAC, n = 2 for peak area and peak1/peak2. Values in the same column with different letters are significantly different (P < 0.05). bMaximum absorption wavelength (λmax) and the absorbance at 680 nm (iodine blue value, BV) of starch−iodine complex. cApparent amylose content (AAC) determined by iodine colorimetric method from the absorbance at 620 nm. dGPC parameters of isoamylase-debranched starch. Peak1/peak2 is the area ratio of peak 1 and peak 2.

subgranules, and was therefore designated as aggregate starch. At the early stage of TRS aggregate starch development, starch subgranules individually form and develop in one amyloplast. At the middle stage of starch development, adjacent subgranules start fusion. At the late stage of starch development, the subgranules located at the periphery of the aggregate starch fuse together and form a continuous outer band or wall.34 Starch granule is considered as elongated granule when the ratio of long axis and short axis is larger than 1.3.8,35 TRS elongated starch granules contained some small spherical granules. The spherical granule had one typical Maltese cross, and the elongated granule consisted of several small granules and showed very weak Maltese cross (Figures 1E, 1e). The elongated starch granules in high-amylose maize are formed from fusion of several small granules.35 TRS hollow starch granules were similar to TRS aggregate starch granule in surface shape, but their interiors lacked subgranules and had no birefringence (Figures 1F, 1f). The hollow starch granule has been reported in high-amylose wheat and barley endosperm,9−11 but its formation is not unclear until now. Among the four types of heterogeneous starch granules, the purity of aggregate starch granules was the highest (Figures 1D, 1d), but the polygonal starch granules contained some small spherical and elongated starch granules (Figures 1C, 1c), the elongated starch granules had some polygonal and small aggregate starch granules (Figures 1E, 1e), and the hollow starch granules contaminated some small aggregate starch granules (Figures 1F, 1f).

morphology, and had a typical Maltese cross in one granule (Figures 1A, 1a). Normal rice starch is compound starch, which means that many starch granules individually form and develop in one amyloplast. These separate starch granules (in fact they are subgranules of compound starch) are freely released from the amyloplast when starch is isolated from mature endosperm.16,34 TRS total starch granules isolated from kernels were significantly heterogeneous in morphological structures and birefringent patterns (Figures 1B, 1b). These heterogeneous starch granules could be classified into four types: polygonal, aggregate, elongated, and hollow starch granules according to their morphological structures and birefringent patterns. TRS polygonal starch granules showed polygonal morphology, had a typical Maltese cross in one granule, and were essentially similar to the TQ polygonal starch granule (Figures 1 C, 1c). According to the morphological structure of TRS polygonal starch granule, we thought that it was compound starch. TRS aggregate starch granules consisted of many subgranules, showed large voluminous, nonangular rounded bodies, and had many Maltese crosses in one granule. In fact, each subgranule of aggregate starch had only one Maltese cross. There was a thick band or wall encircling the entire exterior of the aggregate starch granule (Figures 1D, 1d). The morphological structure of TRS aggregate starch had been reported in high-amylose rice by Kim et al.12 and Wei et al.,16 and the exterior band or wall prevented the release of subgranules during starch isolation.12,16 That meant that the isolated starch was still the aggregate body of many 11257

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Figure 3. XRD spectra (A), ATR-FTIR spectra (B), and 13C CP/MAS NMR original spectra (C) and ordered subspectra (D) of starches. AS is amorphous starch. TQ, TRS, TRS-p, TRS-a, TRS-e, and TRS-h are TQ total starch, TRS total starch, TRS polygonal starch granules, TRS aggregate starch granules, TRS elongated starch granules, and TRS hollow starch granules, respectively.

Iodine Absorption Spectra and Amylose Contents of TRS Heterogeneous Starch Granules. The iodine absorption spectra of TQ total starch, TRS total starch, and TRS heterogeneous starch granules are presented in Figure 2A, and they showed significant difference. The maximum absorption wavelength (λmax), iodine blue value (BV), and AAC of starch were summarized in Table 1. The λmax, BV, and AAC of starches had significantly positive relationships for each other. The correlation coefficients were 0.989, 0.983, and 0.999 between λmax and BV, λmax and AAC, and BV and AAC, respectively (P < 0.001). The AAC of TRS total starch was 60.0%, and was markedly higher than that of TQ total starch (25.5%), which was in agreement with the previous report.21 TRS heterogeneous starch granules showed significantly different AAC. The hollow starch granules had the highest AAC (80.2%), and the polygonal starch granules showed the lowest AAC (39.8%). The significantly different AACs of TRS heterogeneous starch granules indicated that these heterogeneous starch granules significantly differed in not only the morphology but also the structure. Molecular Weight Distributions of TRS Heterogeneous Starch Granules. The molecular weight distributions of isoamylase-debranched starches as determined by GPC are shown in Figure 2B. A trimodal distribution of low, middle, and high molecular weight peaks, designated peak 1, peak 2, and peak 3, respectively, was observed. Peak 1 and peak 2 consist of

short (A and short B chains) and long (long B chains) branchchains 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.36 Peak 3 mainly includes amylose.37 The GPC parameters are summarized in Table 1. The AC determined based on iodine and starch affinity is described as AAC, which overestimates AC if there are branched molecules with long side chains that bind iodine.38 The present results showed that the ACs of TRS total starch and TRS heterogeneous starch granules determined by GPC were significantly lower than AACs obtained by iodine colorimetric method (Table 1), which was in agreement with the report of Shi et al.38 and indicated that the intermediate chain and amylopectin long branch-chain could bind iodine to increase the value of AAC. In the present study, the correlation coefficient between AAC obtained by iodine colorimetric method and AC determined by GPC was 0.963 (P < 0.002), indicating that the AAC and AC had a significantly positive relationship though the AC was lower than the AAC. The molecular weight distributions and the branching degrees were significantly different among TRS heterogeneous starch granules, indicating that these heterogeneous starch granules had different molecular structures. XRD Patterns of TRS Heterogeneous Starch Granules. Native starches can be divided into A-, B-, and C-type crystallinity according to their XRD patterns.39 XRD patterns of 11258

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Table 2. Relative Crystallinity, IR Ratio, and the Proportions of Single Helix, Double Helix, and Amorphous Conformation of Starcha IR ratioc starches TQ total starch TRS total starch TRS polygonal granule TRS aggregate granule TRS elongated granule TRS hollow granule

b

rel crystallinity (%) 25.6 14.2 18.9 20.1 11.8 1.1

± ± ± ± ± ±

0.1 0.2 0.5 0.6 0.4 0.0

f c d e b a

−1

1045/1022 cm 0.57 0.62 0.57 0.74 0.57 0.46

± ± ± ± ± ±

0.02 0.02 0.02 0.02 0.02 0.03

rel proportiond (%)

1022/995 cm

b b b c b a

0.99 0.74 0.98 0.67 0.77 1.02

± ± ± ± ± ±

0.00 0.01 0.00 0.02 0.01 0.00

−1

d b d a c e

single helix 5.6 4.6 4.7 6.9 6.2 6.6

± ± ± ± ± ±

0.2 0.2 0.2 0.2 0.3 0.3

b a a d c cd

double helix 40.8 28.9 30.9 35.4 26.7 8.0

± ± ± ± ± ±

1.4 1.1 1.2 1.1 1.2 0.4

e bc c d b a

amorphous conformation 53.7 66.5 64.4 57.6 67.0 85.4

± ± ± ± ± ±

1.6 1.3 1.4 1.3 1.5 0.7

a c c b c d

Data are means ± standard deviations, n = 3. Values in the same column with different letters are significantly different (P < 0.05). bRelative crystallinity determined by XRD. cIR ratio determined by ATR-FTIR. dRelative proportion determined by 13C CP/MAS NMR. a

of TQ total starch, TRS total starch, and TRS heterogeneous starch granules are shown in Figure 3C. The resonances at different ppm were assigned according to the data reported in the literature.42,43 The C1 resonance, which gives information about both the crystalline nature and the noncrystalline (but rigid) chains, has been used to examine the crystallinity of starches. A-type starch is a triplet at the C1 region; B-type starch shows a doublet. C-type starch has an inconspicuous triplet or doublet according to the proportions of A- and B-type allomorph.42,43 Amorphous starch has one peak at 103 ppm, which arises from the amorphous domain for C1, and the peak intensity is positively relative with AC.32 TQ total starch had a clear triplet at 99.5, 100.5, and 101.5 ppm, and TRS total starch showed an inconspicuous doublet at the C1 region and a strong peak at 103 ppm, indicating that TQ total starch had A-type crystallinity, and TRS total starch had C-type crystallinity and high AC. This result was in agreement with our previous report.21 TRS polygonal starch granules showed a triplet at the C1 region, TRS aggregate and elongated starch granules had an inconspicuous doublet, and TRS hollow starch granules exhibited no peak except the amorphous peak at 103 ppm. These results indicated that TRS polygonal starch granules had A-type crystallinity, TRS aggregate and elongated starch granules had C-type crystallinity, and TRS hollow starch granules had no crystallinity, and were in agreement with the XRD patterns of TRS heterogeneous starch granules. Figure 3D shows 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).32 The ordered subspectra were similar to the original spectra, but they had clearer crystalline peaks of C1 resonances than the original spectra. 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 helixes, double helixes, and amorphous components are listed in Table 2, and showed significant difference among TRS heterogeneous starch granules. 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. That means that the double helices at a shortrange level would be prerequisite for occurrence of the crystallinity but the crystallinity would not necessarily be present when short-range order exists.41 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

TQ total starch, TRS total starch, and TRS heterogeneous starch granules are presented in Figure 3A, and their relative crystallinities are shown in Table 2. TQ total starch showed strong reflection at about 15° and 23° 2θ and an unresolved doublet at 17° and 18° 2θ, indicating that it had a typical A-type XRD pattern, and was similar to that of normal cereal starches.39 TRS total starch showed strong reflection at about 15°, 17°, and 23° 2θ and a small peak at about 5.6° 2θ, indicating that TRS total starch was C-type crystallinity, which was in agreement with our previous report.21 It was very interesting that TRS heterogeneous starch granules had different XRD patterns. The polygonal starch granules exhibited A-type crystallinity, the aggregate and elongated starch granules showed C-type crystallinity, and the hollow starch granules had no crystallinity. The different crystalline structures might result from the different ACs. Cheetham and Tao39 thought that the crystalline type of maize starch could be varied from A- to B- via C-type when AC increased. The TQ total starch had the highest relative crystallinity, which was in agreement with the lowest amylose content. The elongated starch granules had very low crystalline peak intensities, resulting in low relative crystallinity. The hollow starch granules did not show birefringence under polarizing light, which was in agreement with an absence of crystallinity measured by XRD. The absence of crystallinity has also been reported in wheat hollow starch granules.40 ATR-FTIR Spectra of TRS Heterogeneous Starch Granules. The ATR-FTIR spectrum of starch is sensitive to the short-range ordered structure. The variations in spectra among different starches can be interpreted in terms of the short-range ordered structure present in the external region of starch granule.41 The ATR-FTIR spectra of TQ total starch, TRS total starch, and TRS heterogeneous starch granules in the 1200−900 cm−1 region are shown in Figure 3B. 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.41 The ratios for 1045/1022 and 1022/995 cm−1 of TQ total starch, TRS total starch, and TRS heterogeneous starch granules are shown in Table 2. On the basis of both the spectra and calculated data, the ATR-FTIR characteristics were significantly different among TRS heterogeneous starch granules, indicating that TRS heterogeneous starch granules had different short-range ordered structures in the external region of granule. 13 C CP/MAS NMR Spectra of TRS Heterogeneous Starch Granules. The solid-state 13C CP/MAS NMR spectra 11259

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distance can be detected by XRD.42 Therefore, the double helix content of starch was generally higher than its relative crystallinity, especially for high-amylose starch (Table 2). In the present study, the double helix contents of the elongated and hollow starch granules were markedly higher than their relative crystallinities, indicating that most of the double helices did not form the crystallinity in these starches, which was in agreement with their very high amylose contents (Table 1). SAXS Spectra of TRS Heterogeneous Starch Granules. A comparison of the SAXS spectra of starches is shown in Figure 4. All starches were scaled to equal intensity at high q (q

detected in TRS hollow starch granules, which might result from the high AC and was in agreement with the finding that no crystallinity was detected by XRD and NMR. In native starches, the intensity of the scattering peak decreases with increasing AC, which might result from the decrease in the electron density difference between crystalline and amorphous regions of the lamellae.45 This in turn may be caused by several factors: cocrystallization of amylose macromolecules with amylopectin side chains within crystalline lamellae, accumulation of amylose chains oriented transverse to the lamellar stack within amorphous lamellae, and accumulation of amylose tie-chains inside both crystalline and amorphous lamellae.45,46 Thermal Properties of TRS Heterogeneous Starch Granules. DSC thermograms of TQ total starch, TRS total starch, and TRS heterogeneous starch granules are shown in Figure 5, and the thermal properties are summarized in Table 4.

Figure 4. SAXS patterns of starches. TQ, TRS, TRS-p, TRS-a, TRS-e, and TRS-h are TQ total starch, TRS total starch, TRS polygonal starch granules, TRS aggregate starch granules, TRS elongated starch granules, and TRS hollow starch granules, respectively. Figure 5. DSC thermograms of starches. TQ, TRS, TRS-p, TRS-a, TRS-e, and TRS-h are TQ total starch, TRS total starch, TRS polygonal starch granules, TRS aggregate starch granules, TRS elongated starch granules, and TRS hollow starch granules, respectively.

= 0.2 Å−1) to account for variations in sample concentrations.44 The SAXS patterns were at the same relative scale and therefore directly comparable. The well-resolved main scattering peak around scattering vector (q0) of about 0.07 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 peak intensity depends mainly on the degree of order in semicrystalline regions.45 The parameters of the SAXS peak were determined using the simple graphical method33 and are presented in Table 3. The peak position and Bragg spacing were not significantly different among TQ total starch, TRS total starch, and TRS heterogeneous starch granules. However, the scattering peak intensity and peak full width at halfmaximum were significantly different among TRS heterogeneous starch granules. The scattering peak could not be

TRS total starch had significantly higher gelatinization temperature and lower gelatinization enthalpy than TQ total starch, which might result from the high AC in TRS total starch and was in agreement with our previous report.47 TRS heterogeneous starch granules showed significantly different thermal properties. TQ total starch, TRS polygonal starch granules, and TRS aggregate starch granules had one clear peak with Tp from 73.6 to 76.2 °C. TRS total starch and TRS elongated starch granules had two peaks with Tp at about 76 and 98 °C, but the first peak was very weak in TRS elongated

Table 3. SAXS Parameters of Starcha starches TQ total starch TRS total starch TRS polygonal granule TRS aggregate granule TRS elongated granule TRS hollow granule

Imaxb (counts) 187.4 53.7 65.7 109.4 16.4 −c

± ± ± ± ±

0.0 d 0.0 b 11.5 b 2.7 c 0.3 a

Smaxb (Å−1) 0.068 0.068 0.068 0.067 0.067

± 0.001 ± 0.001 ± 0.001 ± 0.001 ± 0.001 −

ΔSb (Å−1) a a a a a

0.017 0.015 0.015 0.016 0.011 −

± ± ± ± ±

0.000 0.000 0.000 0.001 0.000

Db (nm) c b b c a

9.3 9.3 9.3 9.4 9.4 −

± ± ± ± ±

0.1 0.1 0.1 0.1 0.1

a a a a a

Data are means ± standard deviations, n = 2. Values in the same column with different letters are significantly different (P < 0.05). bImax, peak intensity; Smax, peak position; ΔS, peak full width at half-maximum; D, Bragg spacing (2π/Smax). cData are not detected. a

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0.3 0.3 0.3 0.1 0.1 ± ± ± ± ±

Tc (°C)

78.9 83.6 84.8 86.9 78.2 − a c b bc bc 0.0 0.4 0.4 0.1 0.1

(°C)

± ± ± ± ± 73.6 76.3 75.2 76.2 75.4 − a c b bc d 0.3 0.1 0.1 0.2 0.3 ± ± ± ± ± 66.9 69.1 68.3 68.5 70.6 −

(°C) starches

TQ total starch TRS total starch TRS polygonal granule TRS aggregate granule TRS elongated granule TRS hollow granule

Tpb Tob

Table 4. DSC Parameters of Starcha

Article

starch granules. TRS hollow starch granules had one clear peak with Tp at 98 °C. After immediate rescan of the gelatinized TRS total starch, elongated starch, and hollow starch, the peaks with Tp at about 76 °C disappeared, but the peaks with Tp at about 98 °C still existed on rescanned DSC thermograms (data not shown). The peaks with Tp at about 98 °C disappeared after removal of lipids from high-amylose maize starch.48 Therefore, the peak with Tp from 73.6 to 76.3 °C was due to the dissociation of double-helical crystallites of the amylopectin molecules, and the peaks with Tp at about 98 °C corresponded to the melting of the amorphous amylose−lipid complex, which had a lower thermal transition temperature than the crystalline amylose−lipid complex (Tp, about 120 °C).48 In the present study, no V-type crystalline pattern was found in TQ total starch, TRS total starch, and TRS heterogeneous starch granules. Therefore, the peaks with Tp at 98 °C were the melting of the amorphous amylose−lipid complex. It was noteworthy that TQ total starch, TRS polygonal granules, and TRS aggregate granules also had very weak amylose−lipid dissociation peak for their low amylose contents. This result was in agreement with XRD patterns that the amylose−lipid complex diffraction peak of 2θ 20° was very prominent in TRS total starch, TRS elongated starch granules, and TRS hollow starch granules, especially for TRS hollow starch granules. The low relative crystallinity and double helix content and weak birefringence in TRS elongated starch granules were in agreement with the weak DSC peak of melting double-helical crystallites of the amylopectin molecules. The very low relative crystallinity and double helix content, no birefringence, and very high proportion of amorphous conformation in TRS hollow starch granules were also in agreement with the only DSC peak of melting the amorphous amylose−lipid complex. Though TRS total starch had similar double helix content to TRS elongated granules, their DSC parameters were significantly different. The main reason might be attributed to the difference in amylose content, granule morphology and size distribution, crystalline type, and molecular ordered structure in short- and long-range distance.49 In addition, TRS total starch was a mixture of polygonal, aggregate, elongated, and hollow granules, which added the complication of analyzing DSC parameters for TRS total starch. In conclusion, TRS polygonal, aggregate, elongated, and hollow starch granules were separated from the different regions of the mature kernels of high-amylose rice. These heterogeneous starch granules were essentially different in structures, including morphology, birefringence, iodine maximum absorption wavelength, iodine blue value, AC, molecular weight distribution, crystalline type, relative crystallinity, shortrange ordered structure, relative proportions of single helix, double helix, and amorphous conformation, and scattering peak intensity and peak full width at half-maximum of SAXS. The different structures of TRS heterogeneous starch granules resulted in significantly different thermal properties.

a Data are means ± standard deviations, n = 2. Values in the same column with different letters are significantly different (P < 0.05). bTo, onset temperature; Tp, peak temperature; Tc, conclusion temperature; ΔT, gelatinization range; ΔH, enthalpy of gelatinization. cData are not detected.

ΔH (J/g) ΔT (°C)

− 12.8 ± 0.4 a − − 15.1 ± 0.4 b 13.7 ± 0.5 ab

Tc (°C)

− 103.2 ± 0.4 a − − 105.2 ± 0.3 b 103.9 ± 0.1 a

Tp (°C)

− 97.6 ± 0.4 a − − 97.9 ± 0.3 a 97.7 ± 0.2 a

To (°C)

−c 90.4 ± 0.1 a − − 90.1 ± 0.1 a 90.3 ± 0.4 a d b c c a 0.2 0.3 0.1 0.2 0.1 ± ± ± ± ±

ΔH (J/g)

12.4 2.8 8.9 8.9 0.4 − b c d e a 0.6 0.1 0.4 0.1 0.2 ± ± ± ± ±

ΔT (°C)

b b b

peak 1

b c d e a

12.0 14.5 16.6 18.4 7.6 −

peak 2

− 1.6 ± 0.4 a − − 3.5 ± 0.3 b 3.2 ± 0.1 b

Journal of Agricultural and Food Chemistry

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AUTHOR INFORMATION

Corresponding Authors

*C.W.: College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, China. Tel: +86 514 87997217. E-mail: [email protected]. *Q.L.: Agricultural College, Yangzhou University, Yangzhou 225009, China. Tel: +86 514 87996648. E-mail: qqliu@yzu. edu.cn. 11261

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Funding

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors 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 of GPC analysis.

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ABBREVIATIONS USED AC, amylose content; AAC, apparent amylose content; ATRFTIR, attenuated total reflectance Fourier transform infrared; 13 C CP/MAS NMR, solid-state 13C cross-polarization magicangle spinning nuclear magnetic resonance; DSC, differential scanning calorimetry; GPC, gel-permeation chromatography; SAXS, small-angle X-ray scattering; TQ, Te-qing (wild type rice cultivar); TRS, transgenic resistant starch rice line; XRD, X-ray powder diffraction

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