Physicochemical properties and amylopectin fine structures of A- and B-type granules of waxy and normal soft wheat starch

Journal of Cereal Science 51 (2010) 256–264

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Physicochemical properties and amylopectin fine structures of A- and B-type granules of waxy and normal soft wheat starch Hyun-Seok Kim 1, Kerry C. Huber* School of Food Science, University of Idaho, P.O. Box 442312, Moscow, ID 83844-2312, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 April 2009 Received in revised form 21 November 2009 Accepted 30 November 2009

This work fractionated waxy and normal wheat starches into highly purified A- and B-type granule fractions, which were representative of native granule populations within parent native wheat starches, to accurately assess starch characteristics and properties of the two granule types. Wheat starch A- and B-type granules possessed different morphologies, granule specific surface area measurements, compositions, relative crystallinities, amylopectin branch chain distributions, and physical properties (swelling, gelatinization, and pasting behaviors). Within a genotype, total and apparent amylose contents were greater for A-type granules, while lipid-complexed amylose and phospholipid contents were greater for B-type granules. B-type (relative to A-type) granules within a given genotype possessed a greater abundance of short amylopectin branch chains (DPn < 13) and a lesser proportion of intermediate (DPn 13–33) and long (DPn > 33) branch chains, contributing to their lower relative crystallinities. Variation in amylose and phospholipid characteristics appeared to account for observed differences in swelling, gelatinization, and pasting properties between waxy and normal wheat starch fractions of a common granule type. However, starch granule swelling and gelatinization property differences between A- and B-type granules within a given genotype were most consistently explained by their differential amylopectin chain-length distributions. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Waxy and normal wheat starch A-type and B-type Granule size Amylopectin structure

1. Introduction Endosperm of mature wheat (Triticum aestivum L.) grain contains at least two distinct starch granule populations, commonly referred to as A-type and B-type granules. A-type granules are larger (10–38 mm) in size and disc- or lenticular-shaped, while B-type granules are smaller (<10 mm) and possess a spherical or angular morphology (Kim and Huber, 2008; Peng et al., 1999). Furthermore, wheat starch A- and B-type granules possess differing amylose (AM) and lipid-complexed amylose (LAM) contents; amylopectin (AP) chain-length distributions; relative crystallinities; microstructures (e.g., surface pores, channels, cavities); swelling behaviors; gelatinization properties; and pasting/rheological characteristics (Ao and Jane, 2007; Bertolini et al., 2003; Chiotelli and Le Meste, 2002; Eliasson and Karlsson, 1983; Geera et al., 2006a; Kim and Huber, 2008; Peng et al., 1999; Sahlstro¨m et al., 2003; Salman et al., 2009; Shinde et al., 2003;

* Corresponding author. Tel.: þ1 208 885 4661; fax: þ1 208 885 2567. E-mail address: [email protected] (K.C. Huber). 1 Current address: Whistler Center for Carbohydrate Research, Department of Food Science, Purdue University, 745 Agricultural Mall Drive, West Lafayette, IN 47907-2009, USA. 0733-5210/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2009.11.015

Soulaka and Morrison, 1985a,b; Vermeylen et al., 2005). However, there are yet inconsistent reports regarding AP chain-length distributions, relative granule crystallinities, and swelling/gelatinization properties of wheat starch A- and B-type granules. Much of this confusion is likely attributable to the wide range of A- and Btype granule fraction purities achieved in published experiments. Only a minority of published reports have documented granule fraction purities (Bertolini et al., 2003; Geera et al., 2006a; Kim and Huber, 2008; Salman et al., 2009; Shinde et al., 2003), which range from 89 to 99% and 66–92% for A- and B-type granule fractions, respectively. For the majority of published studies, the issue of granule fraction purity has not been directly addressed. Nevertheless, the ratio of A- to B-type granules impacts wheat starch characteristics and properties. Shinde et al. (2003) and Soh et al. (2006) reported that increases in B-type granule content altered AM and phospholipid contents, as well as decreased wheat starch paste peak and final viscosities. Thus, purities of both A- and B-type granule fractions are vital to investigating their respective characteristics and properties. The primary goal of this study was to assess and contrast A- and B-type granule starch characteristics and properties for both waxy and normal wheat starch using highly purified granule fractions. Specific research objectives were to: 1) fractionate native waxy and normal wheat starch into highly purified A- and B-type granule

H.-S. Kim, K.C. Huber / Journal of Cereal Science 51 (2010) 256–264

fractions, 2) characterize purified A- and B-type granule fractions in regard to morphology, chemical composition, AP fine structure, and relative crystallinity, and 3) relate A- and B-type granule swelling, gelatinization, and pasting properties to their respective starch characteristics.

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ethanol, recovered on a Bu¨chner funnel, and allowed to air-dry. Granule size distributions and purities of the final purified starch A(Isolate III) and B-type (Isolate IV) granule fractions were monitored using an Accusizer model 780 with SW 788 Windows software (Particle Sizing Systems, Santa Barbara, CA, USA) as described by Geera et al. (2006b).

2. Experimental 2.3. Scanning electron microscopy (SEM) 2.1. Starch sources and chemicals Grain of waxy (IDO 630) and wild-type (Jubilee) soft wheat lines, obtained from the University of Idaho Aberdeen Research and Extension Center (Aberdeen, ID, USA), was the source of all flour and starch used in this study. Wheat grain was milled to straightgrade flour (Method 26–31, AACC, 2000), and starch was isolated from obtained flours via the adapted protein-digestion scheme outlined by Kim and Huber (2008). All reagents and chemicals used in all experiments were at minimum of analytical grade. 2.2. Fractionation of wheat starch into A- and B-type granule populations Isolated wheat starch was separated into A- and B-type starch granule fractions using a combination of methods: sedimentation in both deionized water and 80% (w/v) aqueous sucrose solution, as well as microsieving (Kim and Huber, 2008; Shinde et al., 2003; Soulaka and Morrison, 1985a). Native starch (25 g, d.b.) was suspended in deionized water (900 ml, containing 0.02% sodium azide) by stirring for 10 min in a tall beaker (1000 ml, 7.6 cm in diameter, 18 cm depth). The starch suspension was allowed to settle 140 min at ambient temperature (22  C), permitting larger granules to sediment to the bottom of the beaker. Following the sedimentation period, the top layer of supernatant (z500 ml), containing Btype starch granules, was carefully pipetted from the beaker and set aside. This sedimentation procedure (comprising replenishment of fresh deionized water [500 ml] to the beaker, sedimentation [140 min], and collection of supernatant [z500 ml]) was repeated four additional times. The five collected supernatants obtained from all sedimentation procedures were pooled to comprise Isolate I (i.e., B-type granule fraction). Residual supernatant (z400 ml) and starch sediment remaining at the bottom of the beaker following the final sedimentation procedure were each transferred to separate beakers and set aside (Isolates II and III, respectively). To further purify Isolate II, starch granules were passed over a 10-mm precision sieve (ATM Co., Milwaukee, WI) with mechanical vibration to yield a purified B-type starch granule fraction (sieve ‘‘throughs’’), which was combined with Isolate I (previously set aside in an earlier step) to yield a final purified B-type granule fraction (Isolate IV). Sieve ‘‘overs’’ (i.e., a mixture of A- and B-type granules) were discarded. Isolate IV, which consisted of purified B-type granules, was collected by centrifugation (3500 g, 20 min), re-suspended in absolute ethanol, recovered on a Bu¨chner funnel, and allowed to air-dry. Isolate III, obtained earlier from the final sedimentation procedure, was further refined to obtain a purified A-type granule fraction. Starch granules were suspended in 80% (w/v) aqueous sucrose solution (180 ml) within a 250 ml centrifuge tube using a Wrist Action Shaker (Model 75, Burrell Co., Pittsburgh, PA, USA). Shaking was continued for 20 min, after which the starch suspension was centrifuged (2000 g, 3 min), and the supernatant was carefully discarded. The tube was replenished with fresh sucrose solution (180 ml), and the aforementioned refining procedure was repeated a total of five times. The final starch pellet obtained after the last refining step in 80% (w/w) sucrose solution was washed three times with deionized water (200 ml per wash), re-suspended in absolute

Waxy and normal starch A- and B-type granules were mounted onto aluminum stubs using double-sided carbon tape, and coated with a 20 nm layer of gold:palladium (60:40). Starch granules were visualized with a field emission scanning electron microscope (SEM; SUPRAÔ 35VP, Carl Zeiss Microimaging, Inc, Thornwood, NY, USA) at an accelerating voltage of 3 kV. 2.4. Granule specific surface area Specific surface areas of waxy and normal starch A- and B-type granule fractions were measured using an ASAP 2010 Surface Area Analyzer (Micromeritics, Norcross, GA, USA), as described by Fortuna et al. (2000), except for the use of a modified degassing scheme. Starch (1.0 g, d.b.) was weighed into a tared sample tube, and degassed at 90  C for 24 h under vacuum (600 mm Hg). After degassing, starch was weighed again to determine specimen mass, and was placed on the surface area analyzer, which determines specific surface area based on physical sorption of high purity nitrogen gas at liquid nitrogen temperature (77.3 K). For each starch sample, a nitrogen adsorption isotherm, drawn through 19 predetermined experimental points, was constructed in duplicate (linear correlation coefficients >0.99). Specific surface area was calculated from the equilibrium adsorption isotherm, and expressed as the mean value of duplicate measurements. 2.5. Compositional analyses Total (TAM), apparent (AAM), and lipid-complexed (LAM) AM contents of waxy and normal starch A- and B-type granule fractions were determined using the colorimetric method outlined by Morrison and Laignelet (1983). Starch protein contents were estimated according to Method 46–30 (AACC, 2000) by nitrogen combustion (%N  5.7). Starch lysophospholipid contents were approximated by starch phosphorus levels determined via inductively coupled plasma-atomic emission spectroscopy (ICP-AES) (Anderson, 1996). 2.6. X-ray diffraction Powder X-ray diffraction (XRD) patterns of waxy and normal starch A- and B-type granule fractions (14.8% moisture content) were obtained using an X-ray diffractometer (Siemens D5000, Bruker, Madison, WI, USA) as described by Cheetham and Tao (1998). Relative crystallinity was calculated as the percent (%) ratio of the sum of total crystalline peak areas to that of the total diffractogram (sum of total crystalline and amorphous peak areas). 2.7. Amylopectin fine structure Waxy and normal starch A- and B-type granule fractions were solubilized using an alkaline-microwave dissolution method (Kim et al., 2006). Starch (100 mg, d.b.) was suspended in 6 M urea (0.5 ml) and 1 M KOH (10 ml) within a 25 ml glass tube, and vortexed mildly. The suspension was transferred to a 90 ml Teflon$PFA (perfluoroalkoxy teflon) jar (Savillex, Minnetonka, MN), and heated for 35 s in a 2450 MHz microwave oven (Model AR732, Emerson Radio Co., North Bergen, NJ). After cooling to ambient temperature,

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starch solution was neutralized (pH 7.0–7.6) with 4 M HCl. Resultant starch solution (6 ml) was mixed with absolute ethanol (30 ml) to precipitate starch molecules, which were then collected by centrifugation (3000 g, 20 min). Precipitated starch molecules were washed once with 85% (v/v) aqueous ethanol, recovered by centrifugation (3000 g, 20 min), and allowed to air-dry for 24 h. For debranching, dried starch precipitate was suspended in boiling water (9 ml), and stirred for 30 min in a boiling water bath to achieve solubilization. After cooling to ambient temperature, the aqueous starch solution was combined with 40 mM sodium acetate buffer (1 ml, pH 4.0) containing 0.2% sodium azide, followed by addition of isoamylase (EC 3.2.1.68; 1000 U/ml; Megazyme International Ireland Ltd, Wicklow, Ireland) suspension (12.5 ml, 2.5 ml/ 10 mg of starch). Resultant starch solution was incubated at 40  C for 24 h, followed by inactivation of isoamylase in boiling water (15 min). To remove impurities (e.g., residual starch granule-associate proteins, isoamylase, salts, etc.), debranched starch solution was mixed with an ion-exchange resin (1 g, IONAC NM-60 Hþ/OH form, J. T. Baker, Phillipsburg, NJ, USA) (Wang and Wang, 2000), and shaken for 1 min on a Wrist Action Shaker (Model 75, Burrell Co., Pittsburgh, PA). Purified debranched starch solution was recovered by passage through a 5.0 mm syringe filter (National Scientific Co., Duluth, GA, USA) prior to analysis on an intermediate-pressure size-exclusion chromatography (IPSEC) system. Final debranched starch solution was injected onto an IPSEC system (Waters Corp., Milford, MA, USA) comprised of a model 1525 binary HPLC pump, Rheodyne 7725i manual sample injector with a 200 mL sample loop, and model 2410 refractive index (RI) detector (operating at an internal temperature of 30  C) (Kim and Huber, 2007). Separation of debranched AP chains was achieved using two Tricorn 10/300 columns packed with Superdex 75 (Mr 3  103 to 7  104) and 30 (Mr w 1  104) prep grade gels (Amersham Biosciences, Piscataway, NJ, USA), respectively, operated in series at ambient temperature. The mobile phase consisted of filtered, deionized water containing 0.02% (w/v) sodium azide that had been de-aerated offline by passage through a 0.2 mm membrane filter, followed by sonication for 20 min. Flow rate for the IPSEC system was set at 0.4 ml/min. For all IPSEC experiments, the dissolution procedure was conducted twice for each sample, and chromatograms for each debranched starch granule fraction were depicted as mean values of duplicate RI signals. Peaks and corresponding peak areas were automatically identified and calculated by Breeze HPLC system software (Waters Co., Milford, MA, USA). Proportions of various starch molecular fractions (AM; AP branch chain fractions I, II, and III) were calculated as the percent (%) ratio of the peak area representing each individual fraction to that of the total chromatogram. For molecular weight analyses, both void and total volumes of the two serial columns were determined with blue dextran (Pharmacia, Mr 2  106) and glucose (180 g/mol), respectively. Pullulan (Mw 47,300, 22,800, 11,800, and 5900; Shodex Standard P82, JM Science, Inc, NY, USA) and dextran (Mn 55,500 and 1010; Pharmacosmos A/S, Roervangsvej, Denmark) standards were used to calculate molecular weights of fractionated amylopectin chains. Weight-average molecular weights (Mw) of pullulan standards were converted to number-average molecular weights (Mn) using the Mw/Mn ratios defined by the supplier. A standard curve was prepared based on a semi-logarithmic plot of standard molecular weights (Mn) versus partition coefficients (Kav) at maximum RI signals for pullulan and dextran standards. The linear regression coefficient between log Mn and Kav was 0.99. Number-average degree of polymerization (DPn) for AP chains of each separated peak, except for that of AM, was calculated by dividing the Mn of AP branch chains by the anhydroglucose molecular weight (162 g/mol).

2.8. Swelling, gelatinization, and pasting properties Swelling factors for waxy and normal starch A- and B-type granule fractions were assessed using a blue dextran exclusion method (Tester and Morrison, 1990). Gelatinization properties were analyzed using a DSC 2920 thermal analyzer (TA Instruments, Newcastle, DE, USA) as described by Geera et al. (2006a). Pasting characteristics were determined as outlined by Batey et al. (1997) using the Rapid Visco Analyzer (RVA) (Newport Scientific, NSW, Australia). 2.9. Statistical analyses All analyses of starch characteristics and properties consisted of at least duplicate measurements for each starch granule fraction. Experimental data were analyzed using Analysis of Variance (ANOVA), and expressed as mean values  standard deviations. A least square difference (LSD) test was conducted to examine significant differences among experimental mean values (a < 0.05). All statistical computations and analyses were performed with SAS version 9.1 for Windows (SAS Institute, Cary, NC, USA). 3. Results and discussion Multiple studies have reported the influence of granule type on both wheat flour and/or native wheat starch properties, as well as on wheat-based product qualities (Park et al., 2005; Sahlstro¨m et al., 2003; Shinde et al., 2003; Soh et al., 2006; Soulaka and Morrison, 1985b). The scope of this study is confined to the characterization of purified A- and B-type granule fractions of waxy and normal wheat starches to better understand the basis for their respective physicochemical characteristics and properties. 3.1. Morphological characteristics and fraction purities of wheat starch A- and B-type granules A-type granules possessed the typical disc-like or lenticular shape, while B-type granules exhibited both spherical and/or irregular morphologies (Fig. 1). For the two genotypes, A-type granules exhibited average diameters ranging from 20.9  0.1 to 21.9  0.2 mm, whereas B-type granules possessed average diameters of 6.2  0.2 to 6.3  0.2 mm (Table 1). These values were consistent with those reported by Kim and Huber (2008) (mean granule diameter ranges of 22.5–22.8 mm and 6.4–6.8 mm for A- and B-type granules, respectively). However, no differences between waxy and normal B-type granule dimensions were observed, as previously noted by Salman et al. (2009). Fraction purities of waxy and normal A-type starch granule populations (assuming a 10 mm cut-off for differentiation of A- and B-type granules) determined by particle size analysis were 99.6  0.1 and 99.7  0.0, respectively (volume or weight basis). These measurements coincided with visual SEM observations (Fig. 1A and D) that A-type starch granule fractions were not contaminated by smaller B-type starch granules. For B-type granule populations of waxy and normal wheat starch, fraction purities determined via particle size were 94.5  0.1 and 94.1  1.1, respectively. As observed by SEM, B-type starch granule fractions (Fig. 1B and E) contained occasional small, disc-like A-type granules, though no contaminating A-type granules larger than 10 mm were observed (Kim and Huber, 2008). A- and B-type starch granule fraction purities determined via particle size analysis did not differ significantly between the two genotypes. Further, purified granule fractions for waxy and normal wheat starch possessed higher purities than those previously reported (Bertolini et al., 2003; Geera et al., 2006a,b; Salman et al., 2009; Shinde et al., 2003).

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Fig. 1. SEM micrographs (A, B, D, and E) and granule size distribution profiles (C and F) of isolated A-type (A and D) and B-type (B and E) granule fractions of waxy (A–C) and normal (D–F) wheat starch (Scale bars ¼ 10 mm).

Granule size distribution profiles for fractionated A- and B-type starches were further contrasted to those of their native (unfractionated) parent starches. Profiles of purified A- and B-type starch granule fractions (Fig. 1C and F, light gray and dark gray shaded areas, respectively) appeared to match those of their respective native starches (Fig. 1C and F, thin black lines). The only exception was a small population of granules (ranging from 10 to 14 mm in the native populations) that was not represented in isolated A-type granule fractions (Fig. 1C and F, black shaded areas), depicting starch granules lost during fractionation. However, these starch losses constituted less than 5% of the total native starch by weight. Average diameters of fractionated A- and B-type granule populations (determined by particle size analysis) closely approximated those within native starch granule size distributions. Thus, purified A- and B-type starch granule fractions were demonstrated to be reasonably representative of native A- and B-type starch granule populations within parent wheat starches. Waxy A- and B-type starch granule fractions possessed specific surface areas of 0.9  0.0 and 2.8  0.2 m2/g, respectively, while values for normal A- and B-type starch granule fractions were 0.8  0.1 and 2.3  0.2 m2/g, respectively (Table 1). As anticipated, B-type starch granule fractions exhibited approximately three-fold greater specific surface areas than A-type starch granule fractions, irrespective of genotype. Soulaka and Morrison (1985a) and Sahlstro¨m et al. (2003) calculated specific surface areas of wheat starch A- and B-type granules from granule mean volumes, based on the assumption that all wheat starch granules are spherical. Calculated specific surface area values (0.33 and 0.97 m2/ml for A- and B-type granules, respectively) reported by Soulaka and Morrison (1985a) were much lower than those measured via the nitrogen adsorption and desorption isotherm in the present study. Values, based on nitrogen absorption, not only account for outer surfaces of granules, but also inner granule surfaces (e.g., those of potential channels and cavities opening to the granule exterior), which were observed by Kim and Huber (2008). In contrast, specific surface area estimates of Soulaka and Morrison (1985a) and Sahlstro¨m et al. (2003) account only for external granule specific surface areas.

Nevertheless, their calculated B/A-type granule specific surface area ratios (2.9–3.0) are very close to those determined in the present study (3.1 and 3.2 for normal and waxy starches, respectively). Fortuna et al. (2000) also determined specific surface areas for large and small wheat starch granule fractions via the same instrument used in the present study (methods differed only in regard to degassing procedures). While a greater specific surface area value was obtained for their small granule fraction (1.02 m2/g) relative to that of their large granule fraction (0.62 m2/g), these reported values were much lower than those obtained in the present study. This discrepancy is likely explained by the low fraction purity (z60%, weight basis) reported for their small granule fraction. In summary, A- and B-type granule fractions of waxy wheat starch possessed slightly greater specific surface areas than their respective normal wheat starch counterparts (Table 1), in spite of the fact that average granule diameters hardly differed between like-granule types of the two genotypes (Table 1). Differences in surface area measurements noted between waxy and normal starch granule fractions could reflect differences in inner granule specific surface areas, possibly implying differences in channel and/or cavity dimensions or frequencies for like-granule types of the two genotypes. 3.2. Chemical composition of wheat starch A- and B-type granules Compositional attributes (e.g., TAM, AAM, LAM, phosphorus, protein) for A- and B-type granule fractions of waxy and normal wheat starch are depicted in Table 1. For both genotypes, A-type granule fractions possessed higher TAM and AAM contents than B-type granule fractions, in agreement with the majority of previous reports (Ao and Jane, 2007; Bertolini et al., 2003; Geera et al., 2006a; Peng et al., 1999; Shinde et al., 2003; Soulaka and Morrison, 1985a). LAM contents were higher in B-type than in A-type granule fractions for normal wheat starch, while the opposite trend was observed for waxy wheat starch granule fractions (Table 1). Similar observations have been also noted by others (Bertolini et al., 2003;

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Table 1 Meana values for morphological and compositional characteristics, relative cystallinities, and debranched starch chain-length distributions for A- and B-type granule fractions of waxy and normal wheat starch. Properties

Waxy (IDO630)

Normal (Jubilee)

A-type

A-type

Morphology 20.9 Mean granule diameter (mm)b Specific surface 0.9 area (m2/g)c Chemical composition TAM (%)d 1.5 AAM (%)d 0.7 LAM (%)d 0.8 Phosphorus 90.5 e (ppm) Protein (%)f 0.2 Relative crystallinity 35.2 (%)g IPSECh (debranched starch) 2.3 AMi peak (%)j APi branch chain j peak (%) Fraction Ik 19.1 Fraction IIk 33.1 Fraction IIIk 47.8

B-type

B-type

 0.1(b)

6.2  0.2(c)

21.9  0.2(a)

6.3  0.2(c)

 0.0(c)

2.8  0.2(a)

0.8  0.1(d)

2.3  0.2(b)

   

0.1(c) 0.0 0.0(c) 0.0 0.1(c) 0.0 2.1(d) 240.0

   

0.0(c) 25.6 0.0(d) 20.3 0.0(d) 5.3 0.0(c) 470.0

   

0.2(a) 21.0 0.1(a) 14.4 0.1(b) 6.6 14.1(b) 585.0

   

0.1(b) 0.1(b) 0.1(a) 7.1(a)

 0.1(a)  0.3(a)

0.5  0.0(b) 32.8  0.2(b)

0.2  0.0(a) 27.2  0.6(c)

0.5  0.1(b) 23.6  0.4(d)

 0.1(c)

0.8  0.1(d)

24.9  0.3(a)

19.5  0.5(b)

 0.6(a)  1.2(a)  0.6(c)

16.1  0.1(d) 28.6  1.1(c) 55.3  1.2(a)

17.7  0.4(b) 34.2  0.6(a) 48.1  0.2(c)

16.8  0.4(c) 31.9  0.0(b) 51.3  0.4(b)

a

Mean value of two measurements; values within a row sharing a common letter (in parenthesis) are not significantly different (p < 0.05). b Determined via particle size analysis. c Determined using a ASAP 2001 Surface Area Analyzer. d TAM, total amylose; AAM, apparent amylose; LAM, lipid-complexed amylose. e Approximation of starch phospholipid content. f Estimation of protein content defined as the percent (%) ratio of the protein weight within the isolated starch granule fractions. g Defined as the percent ratio (%) of the total area of the crystalline regions to that of the total diffractogram (crystalline þ amorphous regions). h IPSEC, intermediate-pressure size-exclusion chromatography. i AP, amylopectin; AM, amylose. j Defined as the percent ratio of each fraction peak area to the total peak area of the chromatogram. k Classified based on IPSEC chromatograms for debranched amylopectin chains as defined in Fig. 2; Fraction I, DPn > 33; Fraction II, DPn 13–33; Fraction III, DPn < 13.

Geera et al., 2006a). For phosphorus content, which approximates granule lysophospholipid (LPL) levels, B-type granule fractions, regardless of genotype, consistently possessed higher phosphorus levels compared to A-type granule fractions (Table 1), in accordance with previous reports for normal wheat starch (Shinde et al., 2003; Soulaka and Morrison, 1985a). However, for waxy starch, Bertolini et al. (2003) and Geera et al. (2006a) both reported higher phosphorus contents for A-type granules relative to the B-type starch granules. This subtle discrepancy likely stems from heterogeneity among waxy wheat genotypes (both previous studies utilized a common waxy wheat cultivar, different from that used in this study). In addition, waxy starch A-type and B-type granule fractions possessed much lower phosphorus contents than their corresponding normal starch granule fractions (Table 1). Protein contents were lower for A-type (relative to B-type) starch granule fractions regardless of genotype, and did not differ between like-granule fractions of the two genotypes. 3.3. Relative crystallinity of wheat starch A- and B-type granules Powder X-ray diffraction patterns exhibited major peaks at a d-spacing of 0.59, 0.52, 0.49, 0.44, and 0.39 nm for all wheat starch granule fractions, indicative of an A crystalline packing arrangement (Zobel, 1988). Waxy starch A- and B-type granule fractions possessed greater relative crystallinities than their normal starch

counterparts (Table 1), which is in agreement with prior trends observed between waxy and normal native wheat starches (Fujita et al., 1998). Within a genotype, A-type starch granule fractions possessed greater relative crystallinities than B-type granule fractions (Table 1), consistent with reports of Chiotelli and Le Meste (2002). However, Ao and Jane (2007) observed smaller granules of wheat starch to possess greater relative crystallinities than larger granules. Further, they reported very low average diameters (2 mm) for small granule fractions. Thus, their isolated small granule fraction may have represented only a subpopulation of the total B-type granule fraction within the parent native wheat starch. Sahal and Jackson (1996) fractionated corn starch granules according to size, and reported that small granule fractions possessed greater relative crystallinities than large granule fractions. While it is not known whether relative crystallinity varies among wheat starch B-type granule subpopulations (i.e., for B- and C-type granule fractions), the noted discrepancy between literature reports regarding A- and B-type granule relative crystallinities might be due in part to differing granule size distributions for wheat starch small granule fractions analyzed in various studies. 3.4. Amylopectin fine structure of wheat starch A- and B-type granules IPSEC chromatograms were divided into four fractions, based on order of elution: AM (data not shown) and Fractions I, II, and III (corresponding to AP branch chains). AM peaks were excluded from chromatograms (Fig. 2) to facilitate accurate comparison of AP branch chain proportions among genotype/granule type combinations. Nevertheless, based on original chromatogram peak areas that included AM peaks, proportions of AM within the A- and Btype granule fractions of waxy and normal wheat starch (Table 1) were compatible with the TAM contents determined colorimetrically (Table 1). Fractions I (DPn > 33), II (DPn 13–33), and III (DPn < 13) were designated as long (B3 and longer), intermediate (B1 and B2), and short (A) AP branch chains, respectively (Table 1), based on the classification scheme of Hanashiro et al. (1996). Weighted averages for number-average degrees of polymerization (DPn) of long, intermediate, and short AP branch chain fractions ranged from 52 to 54, 19–20, and 8, respectively, across all A- and Btype granule fractions. For waxy wheat starch, A-type granule fractions possessed greater proportions of intermediate (Fraction II) and long (Fraction I) AP branch chains compared to B-type granule fractions (Fig. 2A and Table 1). In addition, short (Fraction III) AP branch chains were more prevalent in B-type granule fractions than A-type granule fractions (Table 1). In the chromatogram (Fig. 2A), the Fraction III peak for the B-type (relative to the A-type) granule fraction was shifted toward a later elution volume, implying the presence of shorter branch chains. Moreover, the greater abundance of short (Fraction III) AP branch chains for B-type (relative to A-type) granules explains in part their lower relative crystallinities (Table 1). Salman et al. (2009) also reported slightly higher proportions of short AP branch chains (DP 6–12) and slightly lower proportions of long chains (DP 25–36) for waxy wheat starch B-type (relative to Atype) granules (though no statistical differentiation of chain fractions was included). However, they did not note any differences between long AP branch chains (DP > 36) for the two granule types or the shift of the B-type (relative to A-type) short chain fraction (DP 2–12) to a shorter DP, both of which were observed in the present study. Similar to waxy wheat starch, proportions of normal wheat starch intermediate (Fraction II) and long (Fraction I) AP branch chains were greater in A-type relative to B-type granules, while B-type granules exhibited greater relative abundance of short

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Table 2 Meana swelling factors for waxy and normal wheat starch A- and B-type granules at temperatures ranging from 30 to 90  C. Temperature ( C) Waxy (IDO630) A-type 30 40 50 60 70 80 90

1.4 1.8 2.0 4.2 13.8 38.3 27.3

      

Normal (Jubilee) B-type

4.1  0.1(c/F) 0.3(b/EF) 6.7  0.2(b/E) 5.1  0.2(b/D) 8.3  (b/C) 0.1 26.6  0.1(a/A) N/Db (a/B) 1.9 N/Db

A-type 0.7(a/C) 3.2(a/C) 1.1(a/C) 0.2(a/B) 0.8(a/A)

1.2 1.9 2.1 3.5 5.5 11.7 17.3

      

B-type 0.1(c/F) 2.5  0.1(b/E) 4.1  0.1(b/E) 4.5  0.0(c/D) 4.2  0.5(d/C) 7.6  0.2(c/B) 16.5  0.3(c/A) 24.1 

0.5(b/E) 0.3(a/D) 1.4(a/D) 0.0(b/D) 0.7(c/C) 1.1(b/B) 0.5(b/A)

a Mean value of two measurements; values within a row sharing a common lower-case letter (in parenthesis) are not significantly different (p < 0.05); values within a column sharing a common upper-case letter (in parenthesis) are not significantly different (p < 0.05). b N/D, not determined.

Fig. 2. Amylopectin branch chain-length profiles for A- and B-type granules of waxy (A) and normal (B) wheat starch.

(Fraction II) AP branch chains (Table 1). No shift in chain-length distribution peak profiles was observed between normal starch Aand B-type granule fractions (Fig. 2B) (in contrast to that observed for waxy starch). These results are in partial agreement with previous reports by Ao and Jane (2007), Liu et al. (2007), Sahlstro¨m et al. (2003), Salman et al. (2009), and Vermeylen et al. (2005), all of whom observed greater proportions of short AP branch chains (DPn 6–12) in B-type relative to A-type granule fractions of normal wheat starch cultivars. Only Liu et al. (2007) and Salman et al. (2009) reported trends in A- and B-type granule intermediate and long branch chains similar to those of the present study. Unresolved differences in AP chain-length distributions among the various published reports likely arise due to inherent heterogeneity between genotypes, varied granule fraction purities, and varied starch dissolution schemes. 3.5. Swelling properties of wheat starch A- and B-type granules Swelling factors for A-type granules of waxy and normal genotypes did not differ over the range of 30–50  C (Table 2). Significant differences in swelling factors between A-type starch granule fractions of the two genotypes were only observed beginning at 60  C, above which temperature the waxy (relative to the normal) A-type granule fraction exhibited a much greater degree of swelling. A substantial decline in swelling was observed for waxy

A-type granules at 90  C, due to rupture of swollen (gelatinized) starch granules (Table 2). For B-type granules, greater swelling factors were observed for waxy relative to normal starch granule fractions for all temperatures investigated, though swelling factors could not be determined for waxy B-type granules beyond 70  C, due to granule gelatinization and rupture (Table 2). In short, differential swelling properties for waxy and normal starches of a given granule fraction (A-type or B-type) were likely attributable to differences in AM and LAM contents (Table 1), all of which restrict swelling of starch granules during heating (Morrison et al., 1993; Tester and Morrison, 1990). Within a genotype, swelling factors for B-type granule fractions exceeded those of A-type granule fractions at all temperatures investigated (Table 2). For waxy wheat starch, B-type granules possessed lower TAM, AAM, and LAM contents (but higher phosphorus contents) than A-type granules (Table 1), which is generally consistent with higher swelling capacities for B-type granules. However, normal wheat starch B-type (relative to A-type) granules exhibited greater LAM and phosphorus contents (but a lower TAM content), which together might be expected to adversely impact swelling properties (yet swelling factors for normal B-type granules consistently exceeded those of normal A-type granules). Thus, greater swelling capacities of B-type relative to A-type starch granules of a given genotype are not solely explained on the basis of TAM, AAM, LAM, or lysophopholipid contents. AP fine structure provides a more consistent explanation for differential swelling properties of wheat starch A- and B-type granule fractions. Srichuwong et al. (2005) noted that a deficiency of short AP branch chains (DP < 13) coupled with an increase in intermediate AP branch chains (DP 13–24) generally improved molecular packing and enhanced stability of granule crystalline regions, leading to restricted granule swelling. In the present study, B-type granules of both waxy and normal genotypes possessed greater proportions of short AP branch chains (DPn < 13) and lesser proportions of intermediate AP branch chains (DPn 13–33) compared to their respective A-type granule fractions (Table 1), both of which are consistent with higher swelling capacities for B-type (relative to A-type) granules. Thus, the differential swelling properties of A- and B-type granules of a given genotype are more consistently explained by noted differences in AP fine structure rather than by differences in amylose-related characteristics. 3.6. Gelatinization properties of wheat starch A- and B-type granules Gelatinization properties of A- and B-type granule fractions of waxy and normal wheat starch are presented in Table 3. Waxy wheat starch A- and B-type granule fractions generally exhibited

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Table 3 Meana gelatinization and pasting attributes of A- and B-type granules of waxy and normal wheat starch. Properties

Waxy (IDO630) A-type

Gelatinization Tob Tpb Tcb DHb

Normal (Jubilee) B-type

A-type

B-type

   

0.1(a) 0.0(b) 0.0(b) 0.3(a)

54.7 66.6 79.5 13.3

   

0.1(c) 0.1(a) 0.3(a) 0.5(b)

56.2 61.0 71.1 13.6

   

0.1(b) 0.0(d) 0.1(c) 0.4(b)

54.6 63.7 76.0 11.4

   

0.1(c) 0.3(c) 0.5(b) 0.2(c)

Pasting viscosity (RVU) Peak 455.6  Trough 114.2  Breakdown 341.5  Final 180.1  Setback 65.9 

0.4(a) 1.1(c) 1.5(a) 0.1(c) 1.0(c)

227.5 77.0 150.5 110.1 33.2

    

0.4(c) 1.9(d) 1.6(b) 1.6(d) 0.4(d)

338.6 198.6 140.0 395.1 196.6

    

1.1(b) 1.8(a) 0.8(c) 1.0(a) 2.8(a)

215.8 171.2 44.6 282.7 111.5

    

1.8(d) 2.4(b) 0.6(d) 4.5(b) 6.9(b)

57.0 64.5 75.4 15.9

a Mean value of two measurements; values within a row sharing a common letter (in parenthesis) are not significantly different (p < 0.05). b Gelatinization onset, peak, and completion temperatures ( C) are denoted by To, Tp, and Tc, respectively; gelatinization enthalpy is abbreviated DH.

higher gelatinization temperatures (Tp, Tc) and greater gelatinization enthalpies (DH) compared to their corresponding granule counterparts within normal starch, though gelatinization onset temperatures (To) did not differ between B-type granule fractions of the two genotypes (Table 3). These results are consistent with trends reported for native starches of waxy and normal genotypes (Geera et al., 2006a; Sasaki et al., 2000). Higher gelatinization temperatures and enthalpies for waxy (relative to normal) starch fractions of a given granule type are likely attributable to greater relative starch crystallinities (Table 1), which are positively correlated with gelatinization properties (Fujita et al., 1998). Within a genotype, B-type starch granule fractions consistently exhibited higher gelatinization peak (Tp) and completion (Tc) temperatures, while A-type starch granule fractions exhibited higher gelatinization onset (To) temperatures and enthalpies (DH) (Table 3). These results are in good agreement with previous reports (Chiotelli and Le Meste, 2002; Eliasson and Karlsson, 1983; Geera et al., 2006a; Liu et al., 2007; Peng et al., 1999; Soulaka and Morrison, 1985a). The lower gelatinization onset (To) temperature observed for B-type (relative to A-type) starch granules of a given genotype is in harmony with their higher relative swelling factors (Table 2) and greater proportions of short (DPn < 13) AP branch chains (Table 1). Tester and Morrison (1990) noted that gelatinization enthalpy is associated with overall degree of starch granule crystallinity, while gelatinization temperatures are indicative of the degree of crystallite perfection and stability. Within a genotype, lower gelatinization onset (To) temperatures, higher gelatinization completion (Tc) temperatures, broader gelatinization temperature ranges (Tc–To), and lower gelatinization enthalpies for B-type (relative to A-type) granules together infer a wide range of crystallite perfection (quality) within small starch granule fractions (Chiotelli and Le Meste, 2002; Geera et al., 2006a). Lower gelatinization enthalpies for B-type (relative to A-type) granules of waxy and normal B-type starch granule fractions are consistent with their lower relative crystallinities (Table 1). 3.7. Pasting properties of wheat starch A- and B-type granules Pasting profiles of waxy and normal starch granule fractions are shown in Fig. 3A and B, respectively, while specific pasting characteristic values obtained from pasting profiles are depicted in Table 3. Waxy A- and B-type starch granule fractions exhibited higher peak and breakdown viscosities relative to their respective normal starch granule fractions, while the reverse was true for trough, final, and setback viscosity attributes (Table 3). Variations in

Fig. 3. Pasting profiles for A- and B-type granules of waxy (A) and normal (B) wheat starch.

pasting characteristics for waxy and normal wheat starches of a given granule type are likely attributable to differences in TAM, AAM, and LAM contents, compatible with previous trends reported for pasting properties of waxy and normal native wheat starches (Geera et al., 2006b; Zeng et al., 1997). Within a genotype, A-type (relative to B-type) granule fractions exhibited higher viscosities at practically all points of the pasting profile (Fig. 3A and B), and thus exhibited higher peak, trough, final, breakdown, and setback viscosities compared to B-type starch granule fractions (Table 3). Reduced pasting viscosities of normal starch B-type (relative to A-type) granule fractions generally has been attributed to their greater LAM and phosphorus contents, resulting in restricted swelling of granules (Ao and Jane, 2007; Sahlstro¨m et al., 2003; Shinde et al., 2003). However, this explanation does not account for similarly observed pasting differences between waxy starch A- and B-type granules. As similarly reported by Geera et al. (2006a), waxy B-type granules, which possess lower AM levels, reduced LAM contents, and greater swelling factors than waxy A-type granules (Tables 1 and 2), still fail to develop pasting viscosities comparable to those of waxy A-type granules (Fig. 3). Therefore, starch compositional characteristics alone do not consistently explain pasting property differences between wheat starch A- and B-type granules. Another potential factor contributing to pasting viscosity differences between A- and B-type granules may be related to swollen granule volumes (Geera et al., 2006a). Swollen A-type (relative to B-type) starch granules exhibit relatively greater

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volumes, due to their larger dimensions, though B-type starch granules exhibit greater swelling factors (Table 2). Thus, swollen A-type starch granules are presumed to occupy a greater volume fraction of a paste than swollen B-type granules (assuming equivalent starch concentrations by weight) (Wong and Lelievre, 1981, 1982), contributing a greater degree of friction and viscosity (Srichuwong et al., 2005). Shinde et al. (2003) and Soh et al. (2006) revealed that wheat starch peak and final pasting viscosities were inversely related to B-type granule contents, though Ao and Jane (2007) only observed this phenomenon for wheat starches possessing up to 30% (w/w) B-type granules. However, pasting experiments of Ao and Jane (2007) were conducted at lower than normal starch solids concentrations (8% versus 10%, w/v), resulting in relatively lower pasting viscosities. At reduced starch concentration, RVA pastes likely did not possess a critical concentration of swollen granules necessary to detect pasting differences among wheat starches containing B-type granule contents in excess of 30% (w/w) (paste peak viscosities were less than 100 RVU). In short, the factor of swollen granule size or volume remains an important consideration in understanding wheat starch rheological behavior. 4. Conclusions This work fractionated native wheat starch of both waxy and normal genotypes into highly purified A- and B-type granule populations, which were representative of native A- and B-type granule populations within parent wheat starches (based on granule size distributions). Despite similar mean granule size measurements for waxy and normal wheat starch fractions of a given granule type, waxy granule fractions possessed greater specific surface areas than those of normal starch, potentially implying greater intragranule porosity. Comparing waxy and normal starch fractions of a given granule type, differences in AM levels (TAM, AAM, and LAM), phospholipid contents, and relative crystallinities provided explanation for their differential swelling, gelatinization, and pasting properties. Conversely, these same starch characteristics did not provide consistent explanation for swelling, gelatinization, or pasting property differences of A- and B-type granules within a specific starch genotype (most notably, waxy). Rather, these differences appeared to be better explained by variations in AP branch chain-length distributions and relative crystallinities of the two granule types (B-type granules possessed a greater abundance of short and a decreased proportion of intermediate/long AP branch chains). In addition, waxy B-type (relative to A-type) granules possessed not only a greater abundance of short AP branch chains (similar to normal starch), but also possessed an overall A-chain population of reduced chain-length. Pasting property differences between A- and B-type granules of a common genotype further emphasized the importance of granule size (or swollen granule size/volume) in wheat starch pasting viscosity development. In short, the use of highly purified and representative A- and B-type granule fractions was key to elucidating relationships between their starch characteristics and properties. These noted differences in starch characteristics and properties among the two genotypes and/or granule types are further anticipated to aid explanation of wheat starch A- and B-type granule reactivities for production of modified wheat starch. Acknowledgements We acknowledge the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service for financial support of this study (Grant No. 2004-35503-14128).

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