Amylopectin Molecular Structure Reflected in Macromolecular

Biomacromolecules 2004, 5, 1775-1786

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Amylopectin Molecular Structure Reflected in Macromolecular Organization of Granular Starch Rudi Vermeylen,*,† Bart Goderis,‡ Harry Reynaers,‡ and Jan A. Delcour† Laboratory of Food Chemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium, and Laboratory of Macromolecular Structural Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium Received February 11, 2004; Revised Manuscript Received June 10, 2004

For lintners with negligible amylose retrogradation, crystallinity related inversely to starch amylose content and, irrespective of starch source, incomplete removal of amorphous material was shown. The latter was more pronounced for B-type than for A-type starches. The two predominant lintner populations, with modal degrees of polymerization (DP) of 13-15 and 23-27, were best resolved for amylose-deficient and A-type starches. Results indicate a more specific hydrolysis of amorphous lamellae in such starches. Small-angle X-ray scattering showed a more intense 9-nm scattering peak for native amylose-deficient A-type starches than for their regular or B-type analogues. The experimental evidence indicates a lower contrasting density within the “crystalline” shells of the latter starches. A higher density in the amorphous lamellae, envisaged by the lamellar helical model, explains the relative acid resistance of linear amylopectin chains with DP > 20, observed in lintners of B-type starches. Because amylopectin chain length distributions were similar for regular and amylose-deficient starches of the same crystal type, we deduce that the more dense (and ordered) packing of double helices into lamellar structures in amylose-deficient starches is due to a different amylopectin branching pattern. Introduction Starch, the major plant storage polysaccharide, is deposited in semicrystalline granular form. From a molecular perspective, it mainly consists of two types of R-D-glucose homopolymers, that is, amylose and amylopectin. Amylose is an essentially linear molecule with on average 2-12 × 103 R-(1-4) linked glucose units.1 Amylopectin is much larger (0.4-35 × 106 glucose units)2 and has a clusterlike organization.3,4 Clusters are built up by R-(1-4) linked glucan chains tied together by R-(1-6) linkages. Chains within a single cluster have a modal degree of polymerization (DP) of 1418, while longer chains with an average DP of 45-55 are envisaged to interconnect multiple clusters. In addition to these “normal” amylopectin chains, extra-long chains (DP ∼ 1000) occur in regular cereal starches.1 Apart from the carbohydrate moiety, a small level of lipids and proteins (both less than 1%) are present either inside the granule or at its surface. The proteins in starch granules include some of the enzymes involved in its synthesis. In regular starches, granule bound starch synthase (GBSS), which is responsible for the synthesis of amylose, is one of the most abundant of these granule bound enzymes.5 Enzymic and acid hydrolyses revealed the presence of alternating, readily degradable, and more resistant shells (120-400 nm) in the starch granule.6 * To whom correspondence should be addressed. Phone: +32.(0)16.92.16.34. Fax: +32.(0)16.32.19.97. E-mail: rudi.vermeylen@ agr.kuleuven.ac.be. † Laboratory of Food Chemistry, Katholieke Universiteit Leuven. ‡ Laboratory of Macromolecular Structural Chemistry, Katholieke Universiteit Leuven.

The more resistant shell is considered to be more “crystalline” than the rapidly degradable “amorphous” shell.6,7 GBSS and its reaction product amylose are largely confined to the more amorphous shell.8,9 On a smaller structural level, smallangle X-ray scattering (SAXS) of hydrated granular starch revealed a characteristic low-angle scattering peak, which resulted from alternating lamellae of loosely and densely packed material with a repeat distance of 9-11 nm.10-12 Lamellae were found in the “crystalline” shell, where they roughly parallel the shell boundaries6 or are organized in super-helices with lamellae twisted into helical structures.13,14 Wide-angle X-ray diffraction (WAXD) of native starches displayed three types of spectra. Two of them (A- and B-type) were ascribed to a different crystalline packing of double helices, with A-type crystallites being denser and less hydrated. The third diffraction pattern (C-type) was attributed to the joint presence of A- and B-crystallites.15 Combined chromatographic and enzymic analysis has been used to relate starch crystallinity and molecular structure of starch polymers.3,16-18 For starches with regular or low amylose contents, it was hypothesized that short external amylopectin chains would intertwine to form double helices and that double helices belonging to the same cluster would be arranged such that either the A- or B-crystalline unit cell is formed. Individual crystallites are thought to be aligned in high density “crystalline” lamellae, while amylopectin branching points would reside in the low density “amorphous” lamellae.3,7,11 Hence, it is accepted that the amylopectin fraction of granular starches accounts for its crystallinity.19 Furthermore, starches with somewhat longer

10.1021/bm0499132 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/22/2004

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amylopectin branches show B-type diffraction behavior, while shorter branch lengths coincide with A-type crystallinity.4,20 Moreover, on top of the differences in chain length distributions, starches with A- and B-type crystallinities may also have a different branching pattern.21,22 From the above, it is clear that starch exhibits a complex structure with ordered features at different organization levels. Insights on the impact of amylopectin structure on starch crystallinity have greatly advanced. However, the influence of molecular architecture on lamellar organization is less documented. It was recently established that amylopectin behaves as a side-chain liquid-crystalline polymer, with amylopectin double helices acting as mesogens coupled to the molecular backbone via single-stranded chains, called flexible spacers.23-25 Depending on the length of both mesogens and flexible spacers, and the solvation of the latter, mesogens would be pulled in or out of the lamellar register. Although this line of thinking renders a valuable framework to link molecular structure to lamellar order, to the best of our knowledge, it has not been tested rigorously. We, therefore, aimed to relate molecular structure, crystallinity, and lamellar organization. To that end, amylopectin chain length distributions and X-ray scattering behaviors (WAXD and SAXS) were determined for some cereal, root, legume, and tuber starches. Starches were extensively hydrolyzed, and the branched nature and crystallinities of the acid-resistant fractions were assessed. Experimental Section Materials. Waxy (wx) maize starch and high-amylopectin (HAP) potato starch were from Amylum (Aalst, Belgium). Tapioca, pea, and high-amylose (HAM) maize starch were from Remy Industries (Wijgmaal, Belgium), Cosucra (Warcoing, Belgium), and Cargill-Cerestar (Vilvoorde, Belgium), respectively. These starches were used as received. Regular maize starch was isolated using a batchwise laboratory steeping procedure.26 Wheat and potato starches were isolated according to Morrison and coauthors.27 Apparent Amylose Content (AAC). AAC was determined colorimetrically after defatting of the starches with aqueous methanol (85% v/v) for 30 min at 60 °C.28 Calibration was with potato amylose (Calbiochem, Darmstadt, Germany). Analyses were performed in triplicate, with standard errors smaller than 5%. Acid Hydrolysis. Partial hydrolysis of granular starch with 2.2 N hydrochloric acid was performed as described earlier.29 After 30 days, the solubilized carbohydrate in the centrifuged supernatant (1500 g, 15 min) was determined by the phenol sulfuric acid method.30 Aqueous suspensions of residues were brought to pH 6-7 with 0.1 N sodium hydroxide and then extensively washed with deionized water. After a final centrifugation step as above, the residues (lintnerized starches) were air-dried at room temperature. Branch Chain Length Distributions of Native and Lintnerized Starches. Debranching of native starch was essentially as described by Vandeputte and co-workers.31 Modifications included solubilization of starch (10.0 mg) in 5.0 mL of deionized water by heating for 10 min at 120 °C

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followed by 50 min at 100 °C. Isoamylase dosage was as described,31 and after 24 h of incubation at 37 °C, the samples were boiled for 10 min, and half of the original isoamylase dose was added after cooling. After a second incubation (48 h, 37 °C), the samples were boiled (10 min) and stored at -18 °C until further analysis. For lintnerized starches, debranching conditions were identical, but samples (3.0 mg lintner/1.0 mL 5 mM aqueous sodium acetate) were initially solubilized by heating for 30 min at 100 °C. Immediately prior to analysis, debranched samples were boiled (15 min), and to 1000 µL of the cooled solutions was added 250 µL of 1.5 M sodium hydroxide. After vortexing, samples were filtered through a 0.2 µm membrane filter and analyzed. Chain length distributions of debranched starches and lintners were determined in duplicate with high performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) as described by Jacobs and coworkers.29 Separation was with a linear gradient of 150 mM sodium hydroxide (eluent A) and 75 mM sodium hydroxide containing 500 mM sodium acetate (eluent B). Individual peak areas in the chromatogram were corrected for molar PAD detector responses.32 Where branched dextrins were present, chains were classified according to their “apparent DP”. Branching tends to reduce the retention time,33 and from DP 12 on, branched material eluted as a shoulder of the main peak originating from a shorter linear chain. For consistency reasons, over the whole elution range linear chains of DP “n” and branched chains of DP “n + 1” were grouped and represented as chains with an “apparent degree of polymerization” (DPap) “n”. For quantitative comparison on chain populations of completely linear (isoamylase debranched starch) and partially branched structures (lintners, isoamylase debranched lintners), use was made of the DP and DPap classifications, respectively. Chain Length Distributions of Lintnerized Starches. Lintners (3.0 mg) were suspended in 1.0 mL of 5 mM sodium acetate solution and heated for 30 min at 100 °C. Following cooling, 250 µL 1.5 M sodium hydroxide was added to 500 µL of the lintner solution. After vortexing, samples were filtered through a 0.2-µm membrane and analyzed by HPAEC-PAD. Instrument parameters were as above, but eluents were 150 mM sodium hydroxide (eluent A) and 150 mM sodium hydroxide containing 500 mM sodium acetate (eluent B). Data handling was as above. Analyses were performed in duplicate. Crystallinity. Native and lintnerized starches were equilibrated above a saturated sodium chloride solution at 23 °C, and powder WAXD spectra were measured as described earlier.31 Data were normalized to equal total scattering in the 6.5-33.0° 2θ range (with 2θ the diffraction angle obtained with a Cu KR radiation source). The relative levels of amorphous, A-type, and B-type material in starches and lintners were estimated on the basis of comparison with reference spectra corresponding to the amorphous state and the A- and B-type crystalline states.34 For the HAM maize starch, the presence of crystalline amylose-lipid complexes was complied with by including the V-reference spectrum in the procedure. After subtraction of an amorphous halo,

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Macromolecular Organization of Starch Granules Table 1. Structural Characteristics of Native Starches HPAEC sample wx maize maize wheat HAM maize tapioca pea HAP potato potato

WAXD

SAXS

AAC(%)

DP 9-24a (mol %)

A (%)b

B (%)b

Qap (a.u.)c

D (nm)d

1.0 28.8 37.0 nde 29.8 47.4 6.3 14.0

77.4 78.3 78.6 nde nde 74.7 71.5 72.0

47 34 32 0f 44 20 0 0

4 1 4 21f 4 16 38 43

1.68 0.99 0.72 0.87 1.06 0.81 0.98 0.50

10.3 10.2 9.9 -g 9.8 10.7 8.8 9.0

a Fraction of chains with DP 9-24. b A-type, B-type crystallinity, according to A/B/amorphous best-fit procedure. c “Approximate invariant” intensity integrated from 0.03 to 0.20 nm-1. d Lamellar repeat distance. e Not determined. f A-type, B-type crystallinity, according to A/B/V/ amorphous best-fit procedure: on top of B-type crystallinity, 7% V-type crystallinity was detected. g Not detectable.

spectra of wx maize and HAM maize lintners, prepared as above, were used as A- and B-type reference spectra, respectively. Highly crystalline amylose-glycerol monostearate complexes, obtained according to Gelders et al.,35 delivered the V-type reference spectrum. Lamellar Organization. SAXS experiments were conducted on a Kratky camera, using Ni-filtered Cu KR irradiation (wavelength λ ) 1.54 Å) generated by a Rigaku rotating anode device (Tokyo, Japan) operating at 40 kV and 100 mA. Scattering in the range s 0.02-0.70 nm-1 [with s ) 2 sin(θ)/λ, the modulus of the scattering vector] was detected with a one-dimensional Braun position-sensitive proportional detector. Samples were prepared by centrifuging (10 000 tpm, 10 min) aqueous starch suspensions (∼30% starch). The resulting pellet was smeared in a 1-mm-thick aluminum sample holder sealed with aluminum foil, to minimize moisture loss. At the end of the SAXS measurement, the moisture content of the starch pastes was 4143% for all starches. Parasitic scattering was removed by subtracting the scattering of an empty sample holder. Additionally, a constant liquid scattering term, equal for all recordings, was subtracted. Scattered intensity ˜I(s) resulting from starch interacting with the slit collimated primary beam was desmeared to point collimation I(s) using the computer program TOPAS.36 The lamellar repeat distance (D) was calculated as the Bragg spacing at the scattering peak intensity: D ) 1/sp, with sp being the modulus of the scattering vector at peak intensity. The mean square fluctuation of electron density in the stacked lamellae was approximated by integration of the smeared intensities in the range s 0.03-0.20 nm-1: Qap ) ∫I˜(s)s ds, with Qap being the “approximate” invariant. Results and Discussion Native Starches. AACs. Table 1 shows the AACs of the starches. Wx maize had the lowest starch-iodine absorbance, effectively indicating the absence of amylose. HAP potato starch had a somewhat higher AAC. Light microscopy of the iodine-stained HAP potato starch (not shown) revealed that most granules in the sample were effectively free of amylose. However, a non-negligible fraction of the granules

showed a blue core, while a few granules stained blue entirely. A staining pattern characteristic for GBSS-antisense starches37 contaminated with some regular granules was, thus, obtained. AAC of regular potato starch was markedly higher than that of HAP potato starch but lower than the values reported earlier (18-21%).38 AAC of regular maize starch was in agreement with data on cereal starches (25-29%).38 Wheat starch displayed substantially higher AAC (37%), and it is unclear whether this is caused by extra-long amylopectin chains1,39 or genuine amylose. AAC of tapioca starch equally exceeded the range usually reported (14-24%),40 while the amylose content of the pea starch sample (47%) concurred with the upper limit reported for smooth pea starch (49%).41 Amylopectin Branch Chain Distribution. Molar chain length distributions of isoamylase debranched starches are depicted in Figure 1. Short chains of cereal starches displayed a peak at DP 11-12. HPAEC profiles of wx maize and maize starches showed small differences that are probably due to variation in the genetic background of the samples. Inouchi and co-workers42 studied starch chain length distribution of several wx mutants and their regular counterparts in the same maize background and found no systematic differences, contradicting earlier results of Hizukuri20 on nonisogenic lines. Wheat starch has considerably more short chains (DP < 12) and less intermediate chains (DP 13-24) than maize starch. Pea and tuber starches all showed a dip in population density at DP 8, while their peak DP was 12-13, higher than for cereal starches. No differences were found between the chain length distributions of regular and HAP potato starches. As a whole, our findings on short and intermediate amylopectin chains corroborate earlier HPAEC results.43-49 In literature, on column enzymic conversion of oligosaccharides to glucose enabled the detection of a second main peak at DP 41-48 for cereal44 and pea46 starches. Without enzymic conversion and by applying molar detector response correction, the second peak of these starches was reduced to a nondistinct tail at high DPs (>40; Figure 1). However, the regular and amylose-deficient potato starches displayed marked but broad chain populations at approximately DP 45. This is in line with earlier reports where the second peak was centered at DP 48-52.43-45 Crystallinity of NatiVe Starches. Figure 2 shows diffractograms of the cereal (a-d) and root, legume, and tuber (e,f) starches. Table 1 lists semiquantitative information about the relative abundance of amorphous and crystalline phases. Wx maize, maize, and wheat starches displayed typical A-type diffraction patterns with strong reflections at 15.1, 17.2, 18.1, and 23.2° 2θ. As reported, wx mutant starch is more crystalline than regular cereal starch.31,50,53 Furthermore, the wheat starch exhibits a weak diffraction maximum at 5.6° 2θ, and the 17.2° 2θ peak is somewhat more intense than its 18.1° 2θ neighbor. Both features indicate some B-type crystalline structures. In general, regular cereal starches contain only the A-allomorph,52 but rye, wheat, and maize starches have been reported to contain (small levels of) B-crystallites.38,53 The HAM maize starch showed diffraction maxima typical for B-type crystals (5.6, 15.0, 17.2, 22.4, and 24.1° 2θ). The additional peak at 20.0° 2θ may indicate crystalline amylose-lipid complexes and was observed

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Figure 1. Chain length distributions of isoamylase debranched cereal starches [A; wx maize, maize, and wheat starches] and legume and tuber starches [B; pea, HAP potato, and potato starches].

Figure 2. WAXD pattern of native starches: wx maize (a), maize (b), wheat (c), HAM maize (d), tapioca (e), pea (f), HAP potato (g), and potato (h) starches. Data were normalized to equal areas for diffraction angles from 6.5 to 33.0 and shifted along the ordinate for clarity.

earlier for starches of several maize genotypes, such as dull, sugary, and amylose extender.52 The tapioca starch clearly exhibited A-type crystallinity. Pure A-type as well as mixed A/B-type crystallinities40,54 have been reported. As expected, the powder diffractogram of the pea starch yielded a mixed A/B pattern.46,55,56 Both HAP and regular potato starches rendered typical B-type diffraction patterns (Figure 2), which concurs with earlier reports.45,57 However, in contrast to cereal starches (cf. supra), crystallinity was somewhat higher for the regular potato starch. Preliminary microfocus WAXD on GBSS-antisense potato starch revealed a B-type crystalline unit cell in the granule periphery and no reflections in the center.58 For regular potato starch, B-type crystals were found throughout the granule.59,60 This indicates that the core of the GBSS-antisense granule is either inherently totally amorphous or that some crystallites liable to X-ray beam damage are present.58 An amorphous core seems to be in line with the lower crystallinity obtained from conventional powder WAXD data. However, because imperfect or small

B-crystallites might be more sensitive to X-ray beam damage than normal B-crystallites, their presence cannot be ruled out. Lamellar Organization of NatiVe Starches. As a consequence of the slit collimated X-ray beam, the characteristic scattering peak at approximately s ) 0.1 nm-1 was smeared to lower angles. Nevertheless, smeared SAXS patterns (Figure 3) indicated clear differences in scattering behavior of various starches. For cereal starches, scattering patterns were comparable for wx maize, maize, and wheat starches, although scattered intensities at low angles decreased markedly in that order. In accordance with literature data,10,11,61 the scattering peak at 0.1 nm-1 was much less pronounced for HAM maize starch. Literature SAXS and electron microscopy data on starches with even higher amylose contents (wrinkled pea starch) failed to discern features characteristic for stacked lamellae10 suggesting that highamylose starches do not show clear-cut lamellar structures. In this study, we noticed that scattering at 0.3-0.5 nm-1 does not fall back to the same base level in high-amylose starch as in the other starches. This might indicate that (more) small-scale structural heterogeneities add to the observed scattering pattern. At about 0.6 nm-1, a faint scattering excess was observed, which corresponds to the 5.6° 2θ peak observed in WAXD measurements. Regular and HAP potato starches equally displayed a scattering peak at 0.63 nm-1. However, these peaks were more distinct than for the HAM maize starch. Root, legume, and tuber starches alike showed the 0.1 nm-1 scattering peak. As for maize starches, the amylose-deficient potato starch exhibited a much stronger scattering at s ) 0.1 nm-1 than its regular counterpart. For GBSS-antisense and control transformant potato starches, similar observations were made earlier.49,58 It can be assumed that starch in a first approximation scatters as a two-phase lamellar stacked system, where the small-angle scattering in the experimentally assessed window is dominated by the alternating amorphous and crystalline lamellae in the “crystalline” shells of the starch granules. This approach is justified because the crystalline shells in


Macromolecular Organization of Starch Granules

Figure 3. SAXS pattern for native cereal starches [A; wx maize, maize, wheat, and HAM maize starches] and root, legume, and tuber starches [B; tapioca, pea, HAP potato, and potato starches]. The constant liquid scattering background is indicated with an arrow. Table 2. Characteristics of Lintnerized (2.2 N Hydrochloric Acid, 35 °C, 30 Days) Starches lintner WAXD sourcea wx maize (93) maize (87) wheat (91) HAM maize (62) tapioca (89) pea (81) HAP potato (90) potato (85)

A

(%)b 85 55 52 0 72 43 33 0

debranched lintner HPAEC

B

(%)b 0 4 26 64 21 28 33 54

HPAEC

DPMax-1c

DPMax-2d

F1/ F2e

DPMax-L1f

FL1/ FL2g

13 13 15 ndh 13 14 15 14

24 23 23 ndh 24 23 27 -i

2.1 1.8 1.4 ndh 1.6 2.2 3.0 2.3

14 14 14 ndh ndh 14 15 15

22.6 5.6 2.8 ndh ndh 3.2 6.9 4.2

a Parent starch from whom lintners were derived, degree of acid hydrolysis between parentheses. b A-type, B-type crystallinity, according to A/B/ amorphous best-fit procedure. c DPap at which the PAD is maximal for DPap 20. e Ratio of number of chains with DPap wheat, for the cereal starches, and

tapioca > HAP potato > pea > regular potato, for the remainder. The effect of nonuniformity in lamellar stacks on invariant values of starches is presumably marginal, because starch with reported heterogeneity on crystalline and lamellar levels (HAP potato)58 displayed a Qap far higher than starch whose heterogeneity on both levels is not documented (regular potato). SAXS intensities and Qap values will, hence, be influenced by a combination of the three factors indicated above. Desmeared scattering patterns yielded lamellar repeat distances in the range 8.8-10.7 nm (Table 1). Results were comparable to those by Sterling,10 Oostergetel and van Bruggen11 and Jacobs and co-workers.65 Jenkins and coauthors61,66 used a para-crystalline model11 in which onedimensional stacked lamellae were assumed and statistical variation in layer thickness was brought into account. In general, this method leads to slightly lower average repeat distances than Bragg spacings62 as corroborated for starches from various botanical origins.61,66 Lintnerized Starches. Crystallinity of Lintnerized Starches. Hydrolysis with hydrochloric acid (2.2 N, 35 °C, 30 days) produced “lintnerized starches”. Table 2 lists the extents of hydrolysis of the various starches. Values are in agreement with literature data for prolonged lintnerization.29,34,54

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Figure 4. WAXD pattern for lintners obtained from wx maize (a), maize (b), wheat (c), HAM maize (d), tapioca (e), pea (f), HAP potato (g), and potato (h) starches. Data were normalized to equal areas for diffraction angles from 6.5 to 33.0 and shifted along the ordinate for clarity.

WAXD patterns of lintnerized starches are depicted in Figure 4, and semiquantitative estimates of the amorphous and A-type, B-type crystalline phases are displayed in Table 2. Lintnerization (93% hydrolysis) increased crystallinity of the wx maize starch (from 51 to 85%). For regular cereal starches, lintnerization increased crystallinity as well, which concurs with literature.16,51,67,68 Also in agreement with these reports are the observations that hydrolysis did not markedly change the crystal type of maize starch, while the wheat lintner displayed a far greater level of B-crystallites than the parent starch16,68 (Figure 4; the 5.6 and 17.2° 2θ peaks have higher intensities, the 23° 2θ peak is broader). Although the relatively amorphous HAM maize starch was hydrolyzed less efficiently than the other starches, its lintner displayed a clear B-type diffraction pattern, resembling that of potato starch. The relatively weak peak at 20° 2θ indicated that crystalline amylose-lipid complexes detected in the native starch were destroyed upon hydrolysis. Others have suggested that, before gradually being lost during the later stages of lintnerization,34,51 V-type crystallinity may develop during the first phase of hydrolysis.51 Lintnerization of tapioca starch dramatically increased crystallinity (Table 2) and triggered the appearance of an appreciable fraction of B-crystallites. Pea lintner showed higher crystallinity than its starting material,18 but the ratio of A- to B-crystallites slightly increased after partial hydrolysis (Tables 1 and 2). The HAP potato starch lintner was clearly more crystalline than the parent starch. In contrast to native starch, it displayed a mixed A/B-diffraction pattern with comparable levels of both crystal types. For regular potato starch, lintnerization did not impact the diffraction pattern type3,68 (Figure 4), and crystallinity was increased relatively little compared to that of the other starches. Overall, mild acid hydrolysis markedly increased starch crystallinity, reinforcing the general view that less dense, amorphous domains within the starch granule are preferentially hydrolyzed. However, the lintner crystallinities varied strongly (54-93%) and did not approach 100%, indicating

Vermeylen et al.

the presence of acid-resistant amorphous domains. The nature of these relatively acid resistant amorphous fractions in the various starches will be discussed in General Discussion. As mentioned earlier,67 along with etching of acid labile material, rearrangements during hydrolysis need to be considered. For wheat and tapioca starches not only total crystallinity but also the level of B-crystallites increased markedly. In literature, HAM maize starch mutants were found to be highly susceptible to acid degradation when Arather than B-type crystals were present.34,69 A “selective” hydrolysis of A-type crystals may have enriched the small level of B-type crystals in the native wheat and tapioca starches. Our results suggest that the amorphous fraction, which seems to be more acid resistant in B-type starches (Table 2; wx maize vs HAP potato, regular maize vs regular potato), is (in part) responsible for the lower acid degradability of B-type starches. Furthermore, Morrison and coworkers67 suggested that amylose during the early stages of lintnerization is hydrolyzed into chains that retrograde into double helices with B-type crystallinity. In this study, wheat lintner displayed partial B-type crystallinity and, after debranching, had a relatively large proportion of long chains (DP 21-40), which corroborates the involvement of amylose. The crystallinity of HAM maize starch markedly increased, despite its comparatively low degree of hydrolysis, indicating also that amorphous amylose or long-chained amylopectin became incorporated into crystallites during partial hydrolysis. This is consistent with earlier observations that lintners of high-amylose pea56 and maize34 mutant starches have a large proportion of long chains, even after debranching. Pea and HAP potato lintners displayed a limited and vast enrichment in A-type crystallites, respectively. Recently, amylopectin has been suggested to behave as a side-chain liquid-crystalline polymer,24 and the decoupling of individual double helices from the amylopectin backbone by acid hydrolysis would allow their rearrangement, not only by relief of the entropy driven effects exerted by the amylopectin backbone23 but also by canceling the influence of internal amylopectin chain length. Computer simulations on pairs of double helices linked to one another through an internal R-(1-6) linkage indicated that internal chain lengths determine the lateral distance between the double helices in stable conformations.70 Because the lateral distance between the various parallel double helices in A- and B-type crystalline unit cells shows some differences,71 crystal type is influenced by internal chain length as well. Removal of covalent linkages between double helices would, thus, enable a switch from A- to B-type crystallinity or vice versa. Because short linear maltooligosaccharides tend to crystallize into an A-type crystalline register,72 it is foreseen that, for shorter amylopectin double helices (resulting from shorter external chains), a recrystallization from B- to A-type would occur during partial acid hydrolysis. Furthermore, HPAEC analysis revealed (cf. infra) that the chain lengths of double helices in the lintners were smaller than the thickness generally assumed for crystalline lamellae. The grazing of double helices in the transition layer at the edge of the lamellae would equally favor A-type crystallinity. For native pea starch, the granule core exhibited B-type crystallinity, while



Figure 5. Chromatograms of lintners (A) and isoamylase debranched lintners (B) obtained from wx maize (a), maize (b), wheat (c), pea (d), HAP potato (e), and potato (f) starch.

at the periphery A-type crystals were found.15 The above reasoning suggests that B-crystallites with intermediate chain length (possibly positioned at the verge of A- and Bcrystalline domains within a single granule) diverge to A-type crystal packing upon hydrolysis. For pea starch lintners, the increase in A-type crystallinity was smaller than for their HAP potato counterparts. This may indicate some amylose retrogradation and, hence, the formation of B-type crystallites during lintnerization of the former. Furthermore, it was anticipated that the core of HAP potato granules consists in part of B-type crystals with reduced resistance toward X-ray beam damage (cf. supra). Such reduced resistance may be caused by a shorter double helix length. The crystal type would, hence, incline toward the A-type, a propensity that would be strengthened by mild acid hydrolysis. Our WAXD results on lintnerized HAP potato starch are, thus, in line with this thinking. Branch Chain Profile of Lintnerized Starches. In general, HPAEC analyses of lintnerized starches displayed two main populations, with peak maxima at DPap 13-15 and DPap 2327 (Figure 5A). Lintners of cereal starches showed additional shoulders (DPap 35) and peaks (DPap 50 and ∼ 65). This concurs with earlier results on acid-resistant regular maize starch fractions21,56 and wheat lintner.29 Tapioca lintner (chromatogram not shown), similar to lintners of regular cereal starches, displayed two main populations (DPap 13 and 24) and additional shoulders at higher elution volumes. In agreement with Bogracheva et al.56 and McPherson and Jane,45 pea and potato lintners had the two main populations but did not display the additional shoulders. In HPAEC-PAD chromatograms (Figure 5A), small peaks (DP < 12) and peak shoulders (DP > 12) were detected between main peaks. These were observed before for mild acid hydrolyzed starches21,29,45,56 and probably originate from branched products.73 Isoamylase debranching of mild acid hydrolyzed starch residues failed to eliminate

the branched material completely in the present (Figure 5B) and earlier studies.21,45,56 Resolution of the main peaks, with peak bases approaching the common baseline, however, improved markedly, leading others21,45,56 and ourselves to assume that interpretation would not be clouded by the minority fraction of branched material. All isoamylase debranched lintners displayed a single main population at DPap 14-15 (Figure 5B). This was most obvious for both amylose-deficient starches (wx maize, HAP potato). For starches with regular amylose contents, the second population in nondebranched lintners was only largely reduced, as observed earlier.21,45,56 For lintners of wx maize, regular maize, wheat, pea, HAP potato, and regular potato starches, statistical correlations between chain length distributions before and after isoamylase debranching and crystallinity were worked out. For the nondebranched lintners, strong correlations (p < 0.05) were found between DPap 9-11 and A-type and DPap 16-20 and B-type crystallinities. Furthermore, after debranching, DPap 11-13 and DPap 18-22 were significantly (p < 0.05) correlated with A- and B-type diffraction patterns, respectively. These results confirm that shorter double helices are preferentially organized in the A-type register.72 The lack of correlation with either crystal type for chains of intermediate length (DPap 12-15 and 14-17, respectively) probably indicates that, in the heterogeneous set of lintners studied here, these chains are involved in both A- and B-type crystallites. For the nondebranched lintners, an additional weak correlation (p < 0.10) was found between DPap 2426 and the A-type allomorph, validating that chains with acid resistant R-(1-6) linkages contribute to crystallinity. Results indicated that the first population (F1; DPap 5-20) with modal DPap 13-15 in lintnerized starches corresponds to linear chains, probably originating from double helices formed by two such chains. The modal DPap of this fraction

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Figure 6. Schematic representation of crystalline structures in native (A) and lintnerized (B) starches with large (upper panel) and small (lower panel) density difference between amorphous and crystalline lamellae. Electron density profiles of stacked lamellae are indicated on the left.

was markedly smaller than the DP 18-21 necessary to bridge crystalline lamellae.44 Interactions both within and between double helices have been proposed to hamper acid hydrolysis.51 This implies that the high-density core of crystalline lamellae is most resistant and that frayed ends of these crystalline structures (single-stranded amylopectin chains or double helices protruding the “crystalline” core) make up a “transition layer” that could be hydrolyzed during lintnerization. The second population (F2; DPap 21-30) largely disappeared upon debranching and, thus, consists for the most part of singly branched chains with the R-(1-6) linkage protected from acid hydrolysis. In the parent starch, corresponding branched structures are believed to make up double helices with branching points within the crystalline lamellae.16-18 However, the relatively limited crystallinity increase upon lintnerization (cf. supra) indicated that the amorphous material was not completely hydrolyzed. The contribution of “amorphous” domains in the lintner population is expected to be larger for starches with smaller density contrasts between amorphous and crystalline phases: the probability of forming stabilizing carbohydrate-carbohydrate interactions in both phases would differ less, and hydrogen ions would furthermore diffuse less preferentially into the amorphous lamellae. Consequently, the lintnerized material might in part consist of (1) long B-chains, connecting amylopectin clusters positioned in consecutive crystalline lamellae, and (2) branch points, located in the amorphous lamellae or transition layers, connecting chains belonging to a single amylopectin cluster (Figure 6). General Discussion We here combine knowledge on molecular, crystalline, and lamellar organization of starches with a focus on how presumed difference between amylopectin structure in (1) regular and amylose-deficient starches and (2) A- and B-type starches may affect crystallite alignment in the crystalline shell. Amylose-Deficient and Regular Starches. It may seem peculiar that starches mainly differing in GBSS activity would have a different crystalline organization. After all,

Vermeylen et al.

GBSS is primarily responsible for amylose synthesis, and amylopectin accounts for starch crystallinity. Amylose and amylopectin synthesis is, however, not completely segregated, and indications that pathways for biosynthesis of both starch polymers are mutually affected are quite numerous. Not only is the same primary substrate (adenosine 5′diphosphate-glucose) used for the synthesis of both polymers, but also some enzymes mainly involved in the synthesis of one of the starch polymers seem to have side activity in the synthetic pathway of the other. In vitro experiments revealed that GBSS elongates amylopectin chains.74 Furthermore, GBSS activity in cereal starches but not in regular potato starch49 was related to the presence of extra-long amylopectin chains.39 In addition, branching enzymes may interfere with amylose synthesis: an appreciable level (25-48 mole %) of amylose in regular cereal starches is slightly branched,1 and hot-water-soluble potato amylose is even more branched than its cereal analogues.75 Whatever the exact cause, molecular weight of amylopectin is inversely related to GBSS activity in regular and wx cereal starches.2 In the past decade, some integrated views on amylopectin synthesis have been proposed.76-78 They all emphasize the subtle interplay between starch synthases and branching and debranching enzymes. It seems plausible that GBSS, by altering the glucose flux into the amylopectin pathway, or amylose, by (temporarily) distracting amylopectin-synthesizing enzymes, may alter this subtle interplay and, hence, disturb amylopectin synthesis. Structural differences between amylopectins of starches with regular or reduced GBSS activity would be small, however, because the chain length distributions of maize starches42 and their β-limit dextrins79 both did not show marked differences in their short branch range after debranching. Figure 7 presents a schematic view on amylopectin molecular and macromolecular structure in amylose-deficient and regular starches. In line with the above, microfocus WAXD suggested structural differences between regular and GBSS-antisense starches.58,60 Although these experiments await further confirmation, powder WAXD data on lintnerized starches in this paper (Figure 4) affirm structural differences between HAP and regular potato starches. While one may suggest that amylose directly affects crystalline properties in the core of GBSS-antisense potato starch granules,58 in transgenic potatoes GBSS synthesizes amylose deep within the granule matrix at a site where amylopectin synthesis and macromolecular ordering (intertwining of outer chains, packing of double helices) is probably already completed before the beginning of amylose synthesis.80 Heterogeneity of intragranular crystal structure in GBSS-antisense potato starch must, thus, reflect the spatially varying amylopectin structure within single granules. Other experimental indications for the difference in amylopectin structural units between regular and amylosedeficient starches are propounded by SAXS. Qap values of amylose-deficient starches (wx maize and HAP potato) were higher than those of the respective regular counterparts (maize and potato). Amylose has been hypothesized to disturb lamellar organization in regular starches78 and, hence, to provoke the differences in electron density profiles of


Figure 7. Schematic summary of the macromolecular organization of amylopectins in amylose-deficient and regular and A- and B-type starches. Straight vertical lines represent R-(1-4) linked glucose units organized in rigid double helices. Wobbled lines indicate R-(1-4) linked glucose units in flexible chains (mobile backbone, flexible spacers, or dangling chains). Horizontal tie lines represent R-(1-6) linkages. Electron density profiles of stacked lamellae are indicated, with c and a the dimensions of the crystalline and amorphous lamella in the two-phase approximation, respectively. Amylose-deficient starches display larger density differences between amorphous and crystalline lamellae, with broader transition layers for the regular starches. A-type starches show larger density contrast within stacked lamellae with presumably thinner transition layers than B-type starches. B-type starches have longer double helices. Relatively higher densities in the amorphous lamellae may be due to (i) increased level of flexible chains (regular A-type, amylose-deficient B-type), (ii) increased level of double helices (regular A-type starches, and especially regular B-type starches, where the longer double helices may frustrate optimal stacking in crystalline lamellae).

regular and amylose-deficient starches appearing from SAXS. However, amylose resides predominantly in the amorphous and not in the crystalline shells where the stacks are located.9 Consequently, differences in scattering behavior should be explained on the basis of inherent differences in the amylopectin structural units. According to the definition of the invariant, the level of stacked lamellae may influence SAXS intensities. Large differences in amylopectin content and total crystallinity might suggest this parameter to be important for differentiating regular and wx maize starches. Regular and HAP potato starches on the other hand showed relatively limited differences in genuine amylopectin and total crystallinity contents, and the level of stacked lamellae cannot account for the varying scattering intensities. It is, hence, doubtful that the difference in the levels of stacks in regular and amylosedeficient starches can give a universal explanation for the higher SAXS intensities of the latter. Sophisticated model fitting techniques, however, indicated that crystalline lamellae were both smaller and denser in amylose-deficient than in regular maize starch.61 The present study complements this view on the lamellar organization of maize starches with data on lintnerized starches. For wx maize lintner, F1 and F2 were both relatively monodisperse, indicating homogeneous crystal thickness. Lintners of regular cereal starches have less distinguishable F1 and F2 populations, while F2 was larger than for wx maize lintner. The former may reflect a more heterogeneous thickness of crystallites or lamellae. The latter might indicate directly that regular cereal starches have more double helices with R-(16) branch points within the crystalline lamella than amylosedeficient starches. However, similar results are expected for


mildly hydrolyzed starches with lower density differences between amorphous and crystalline lamellae (Figure 6). The differences in crystallinities of the lintners (85 vs 59%) may indeed mirror the larger density contrast between crystalline and amorphous domains in the native wx maize starch. Furthermore, debranched lintners of regular maize starch showed a smaller fraction of shorter (FL1; DPap 5-20) and a larger fraction of longer (FL2; DPap 21-40) chains than its wx counterpart. In principle, the FL2 fraction can in part be attributed to amylose retrograded during partial hydrolysis.67 Lintners of both maize starches displayed A-type crystallinity, making retrogradation of long, nonbranched chains less conceivable. The FL2 fraction in regular maize lintner would, thus, originate from B-chains connecting amylopectin clusters in consecutive crystalline lamellae. In view of their Qap values, a similar difference in density contrast between amorphous and crystalline lamellae may be assumed for regular and HAP potato starches. Analogous to maize lintners, HAP potato lintner displayed better resolved F1 and F2 populations than its regular counterpart (Figure 5A), while its crystallinity was higher than that of regular potato lintner (66 vs 54%). Moreover, lintner derived from amylose-deficient potato starch showed a smaller FL2 fraction after debranching. The latter may result from (a more pronounced) retrogradation of amylose in the regular potato lintner. However, the AAC of regular maize starch is twice that of the regular potato starch while no signs of amylose retrogradation were found in the former (cf. supra). Hence, a more prominent role of amylose in potato lintners is not expected. Although differences between potato lintners were smaller, a similar reasoning as for maize lintners suggests that amylose-deficient potato starch has a larger density contrast between amorphous and crystalline lamellae than its regular analogue. Both for A- and B-type starches, acid hydrolysis and SAXS experiments, thus, independently point toward the larger density difference between alternating crystalline and amorphous lamellae in amylose-deficient starches (Figure 7). According to the definition of the invariant, higher intensities at small angles may also indicate relatively thinner crystalline lamellae for amylose-deficient starches. As already mentioned, this has been inferred also from modeling a threephase para-crystalline model to the scattering profile of wx and regular maize starch.61 Although the scattering pattern of GBSS-antisense potato starch was not amenable to modeling,58 the large tendency toward A-type crystallinity of HAP potato lintner suggests a shorter double helix length in the corresponding native starch (cf. “Crystallinity of Lintnerized Starches”), which may indeed indicate thinner crystalline lamellae. The modal DPap of FL1 was independent of the amylose content of the maize or potato starch (Table 2), indicating equal thickness of the high-density cores of the crystalline lamellae. To be consistent with SAXS data, this implicates thicker transition layers for the regular starch (Figure 7). Results, thus, indicate more compact crystalline lamellae that contrast more strongly with the amorphous ones in amylose-deficient starches. The molecular background of

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density profile differences between the various starches originates from a different positioning of branching points, because amylopectin chain length distributions of amylosedeficient and regular starches are similar (Figure 1). In Figure 7, further differences in the arrangements of maize and potato amylopectin (either regular or amylose-deficient) were deduced from the different chain length profiles of both starch types (cf. infra). First, however, the effect of differences in amylopectin chain length will be discussed for starches with almost exclusive A-type crystallinity. Regular A-type Starches. Wheat and maize starches differed in amylopectin chain length distributions, with, for wheat, a larger fraction of short (DP 9-12) chains. However, the starches had similar crystallinities (Table 1). Hence, crystallinity in cereal starches seems to be only hampered by the presence of even shorter chains as revealed by the negative correlation between chains of DP 6-9 and crystallinity for homogeneous A-type crystalline rice starches. 31 At low angles, however, we found more intense scattering peaks (and higher Qap values) for regular maize than for wheat starch. Chains of DP 18-20 span the full crystalline region, and the presence of a large population of short chains (DP 9-12), too short to span the entire crystalline lamella, may have marked effects. Such chains would fill the crystalline lamella less efficiently, decreasing its density and, hence, lowering the scattering at low angles. Wheat lintner had a larger F2 population than regular maize lintner (Figure 5A), but in contrast to maize starches, no information on the density contrast between amorphous and crystalline lamellae in the native starches could be deduced from hydrolysis experiments. The larger FL2 fraction and an appreciable level of B-type crystallites (Table 2) indicated that long linear chains retrograded during lintnerization of wheat starch. A- and B-type Starches. Maize starches (A-type) scattered more intensely at low angles (s < 0.2 nm-1) than potato starches (B-type). Moreover, for regular tapioca, pea, and potato starches, the scattered intensities in the low-angle region coincidently decreased with the ratio of A- to B-crystallites, while Qap did not relate to total crystallinity or amylopectin contents. A first reason for the lower SAXS intensities of B-type starches may be the larger local crystallinity in the “crystalline” shell. Acid hydrolysis indeed showed that modal DPap of lintners, which is taken as a measure of the thickness of the high-density core of the crystalline lamella, is larger for B-type starches (Table 2), while their Bragg repeat distances (Table 1) are shorter. Results may also indicate larger density contrasts between amorphous and crystalline lamellae in A-type starches, however. This might originate from a higher density in the amorphous lamellae of B-type starches,81 as well as from a higher electron density of the crystalline lamellae in A-type starches. The latter may in part ensue from the higher density of the A-type crystalline unit cell.71 Interestingly, A-type starches had a larger population of chains with DP 9-24 (Table 1) than B-type starches. These chains are likely to positively impact the lamellar order (that is, the electron density contrast between lamellae). Very short chains (DP 6-9), on the other hand, may have a deleterious effect on

Vermeylen et al.

the density of the crystalline lamellae by hampering the packing of individual double helices into crystallites.31 The effects of DP 6-9 in B-type starches on the lamellar organization would, hence, be more drastic than the DP 9-12 chains in wheat starch (cf. supra). Chains exceeding the dimensions of a single amylopectin cluster (DP >24) may frustrate double helix packing in the crystalline lamellae or increase carbohydrate density in the amorphous lamellae (Figure 7), also resulting in a less intense small-angle scattering. The difference in density contrast between amorphous and crystalline lamellae in regular and amylose-deficient starches of the same crystal type was reflected in the lintner structure if no apparent rearrangements took place during acid hydrolysis (cf. supra). A similar difference between A- and B-type starches was inferred from SAXS, and the structures of acid resistant fractions obtained from both starch types were affected accordingly. F1 and F2 in lintners derived from B-type starches (Figure 5A; HAP and regular potato) were indeed less distinguishable than those in A-type starches (wx and regular maize). Lintners are, moreover, not completely crystalline, and hydrolysis of the amorphous fraction seems to proceed less readily in B-type starches (Table 2). This may result from the incomplete etching of amorphous lamellae in the latter as explained earlier (Figure 6). Lintnerized B-type starches displayed a comparatively smaller F2 population than A-type starches: F1/F2 was larger. This seems in contradiction with the anticipated lower density contrast in these starches. However, the fraction of long linear chains (FL2) in the lintners was relatively large, clearly indicating less branching of the acid-resistant residues of B-type starches than those of A-type starches. The latter can directly result from the lower branching density in B-type starches.22 Moreover, the absence of additional peaks (DPap > 30) in lintners of B-type starches (Figure 5A) may reflect its smaller cluster dimensions.22 The difference in the levels of F1 and F2 in A- and B-type starches has led researchers to conclude that R-(1-6) linkages are more confined to the amorphous lamellae in B-type than in A-type starches.21 In doing so, however, they have neglected the partially amorphous character of starch lintners. That B-type starch with its small amylopectin clusters22 would be organized in nicely ordered lamellae21 is intuitively hard to reconcile with the recently proposed bent lamellar structure,82 formed by splayed double helices. From the above, the picture of B-type starches with less compact crystalline lamellae, which moreover do not contrast as much with the amorphous lamella as in A-type starches, emerges. The composition of the amorphous lamella and, consequently, the nature of the “acid-resistant” amorphous material in potato lintners can be discussed on the basis of chain length distribution after isoamylase debranching. As indicated above, the FL2 fraction is relatively large for lintnerized B-type starches, and amylose retrogradation is believed to be of minor importance (cf. supra). The long (DP 30), acid-resistant chains are, hence, thought to originate from the long B-chain population that is quite large in native B-type starches (Figure 1). Crystalline lamellae with a corresponding thickness of 10.5 nm are presumably not


present, and the relative acid resistance of the long B-chains should be attributed to the higher density in the amorphous lamellae of B-starches.81 The density in the amorphous lamellae is presumed constant throughout the sample81 or may fluctuate. The latter was suggested by the simultaneous existence of individual crystalline lamellae and stacked semicrystalline structures in lintnerized wx maize starch.83 Analogous phenomena might come into play for potato starches, and the presence of long chains after mild acid hydrolysis can also be interpreted in the light of the helical lamellar model.12 In potato starches, lamellae are twisted into super-helical structures that partially interpenetrate one another: crystalline lamellae of a super-helix are, thus, partially located between two crystalline lamellae originating from successive turns of a neighboring super-helix.12 For long amylopectin chains, it would, hence, be possible to run from the crystalline lamella of a first super-helix into a crystalline lamella of a second super-helix, without passing a genuine amorphous layer. This would warrant the relatively high resistance of long amylopectin chains toward mild acid hydrolysis. Conclusions For both A- and B-type starches, lintners of regular and amylose-deficient starches showed markedly different HPAEC chromatograms, indicating that either the acid-resistant domains of these starches are organized differently or that dissimilar structural rearrangements took place during partial acid hydrolysis of both starch types. The former hypothesis was validated by SAXS measurements on regular and amylose-deficient native starches, which revealed systematically more intense 9-nm scattering peaks for the latter. Furthermore, isoamylase debranching of native starches did not reveal important differences between amylopectin chain lengths of regular amylopectin and amylopectin synthesized in plant tissues devoid of GBSS activity. Differences in the amylopectin structure deduced from lintnerized starches probably result from a different positioning of R-(1-6) linkages between A- and B-chains of amylopectins of regular and amylose-deficient starches. GBSS activity, thus, seems to have a similar influence on the amylopectin structure of A- and B-type starches. However, starches with differing crystal lattice showed dissimilarities in other structural aspects. Amylopectin branch length distribution profiles showed larger proportions of very short (DP 24) chains for B-type starches. These chains may add defects to crystallites or can frustrate double helix packing and, hence, prevent their optimal alignment in the crystalline lamellae. Furthermore, lintners of cereal starches, for whom WAXD did not suggest amylose retrogradation, had a smaller fraction of long linear acidresistant chains than potato starches. This indicates that long linear chains (DP ∼30), derived from either amylose or amylopectin, were more protected from acid hydrolysis in potato starches. If these chains would originate from long B-chains, which are relatively abundant in tuber starches, this can be envisaged in the light of the lamellar helical model suggested previously for potato starch. In this view, local


high-density zones in the amorphous lamella would render long amylopectin chains less susceptible to hydrolysis. From the whole of the experimental results emerges, hence, that B-type starches display a less pronounced lamellar organization than A-type starches. Acknowledgment. R.V. wishes to acknowledge the “instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen” (IWT, Brussels, Belgium) for the receipt of a scholarship. B.G. is a postdoctoral fellow of the Fund for Scientific Research-Flanders (FWO-Vlaanderen, Brussels, Belgium). B.G., H.R., and J.A.D. thank the FWO-Flanders for continuous support and equipment. Greet Gelders (Laboratory of Food Chemistry, K.U.Leuven, Leuven, Belgium) is thanked for the preparation of the high crystalline V-type reference material. Overall technical assistance by Luc Van den Ende and Hilde Van den Broeck (Laboratory of Food Chemistry, K.U.Leuven, Leuven, Belgium) is gratefully appreciated. References and Notes (1) Hizukuri, S. In Carbohydrates in Food; Eliasson, A. C., Ed.; Marcel Dekker: New York, 1996; pp 347-429. (2) Yoo, S. H.; Jane, J. Carbohydr. Polym. 2002, 49, 307-314. (3) Robin, J. P.; Mercier, C.; Charbonnie`re, R.; Guilbot, A. Cereal Chem. 1974, 51, 389-406. (4) Hizukuri, S. Carbohydr. Res. 1986, 147, 342-347. (5) Baldwin, P. M. Starch/Staerke 2001, 53, 475-503. (6) French, D. In Starch Chemistry and Technology; Whistler, R. L., BeMiller, J. N., Paschall, J. F., Eds.; Academic Press: San Diego, CA, 1984; pp 183-247. (7) Jenkins, P. J.; Cameron, R. E.; Donald, A. M.; Bras, W.; Derbyshire, G. E.; Mant, G. R.; Ryan, A. J. J. Polym. Sci., Part B: Polym. Phys. 1994, 32, 1579-1583. (8) Han, X. Z.; Hamaker, B. R. J. Cereal Sci. 2002, 35, 109-116. (9) Blennow, A.; Hansen, M.; Schulz, A.; Jørgensen, K.; Donald, A. M.; Sanderson, J. J. Struct. Biol. 2003, 143, 229-241. (10) Sterling, C. J. Polym. Sci. 1962, 56, S10-S12. (11) Oostergetel, G. T.; van Bruggen, E. F. J. Starch/Staerke 1989, 41, 331-335. (12) Cameron, R. E.; Donald, A. M. Polymer 1992, 33, 2628-2636. (13) Oostergetel, G. T.; van Bruggen, E. F. J. Carbohydr. Polym. 1993, 21, 7-12. (14) Waigh, T. A.; Donald, A. M.; Heidelbach, F.; Riekel, C.; Gidley, M. J. Biopolymers 1999, 49, 91-105. (15) Buleón, A.; Ge´rard, C.; Riekel, C.; Vuong, R.; Chanzy, H. Macromolecules 1998, 31, 6605-6610. (16) Robin, J. P.; Mercier, C.; Duprat, F.; Charbonnie`re, R.; Guilbot, A. Starch/Staerke 1975, 27, 36-45. (17) Watanabe, T.; French, D. Carbohydr. Res. 1980, 84, 115-123. (18) Biliaderis, C. G.; Grant, D. R.; Vose, J. R. Cereal Chem. 1981, 58, 502-507. (19) Zobel, H. F. Starch/Staerke 1988, 40, 44-50. (20) Hizukuri, S. Carbohydr. Res. 1985, 141, 295-306. (21) Jane, J. L.; Wong, K. S.; McPherson, A. E. Carbohydr. Res. 1997, 300, 219-227. (22) Ge´rard, C.; Planchot, V.; Colonna, P.; Bertoft, E. Carbohydr. Res. 2000, 326, 130-144. (23) Waigh, T. A.; Perry, P.; Riekel, C.; Gidley, M. J.; Donald, A. M. Macromolecules 1998, 31, 7980-7984. (24) Waigh, T. A.; Kato, K. L.; Donald, A. M.; Gidley, M. J.; Clarke, C. J.; Riekel, C. Starch/Staerke 2000, 52, 450-460. (25) Perry, P. A.; Donald, A. M. Carbohydr. Polym. 2002, 49, 155-165. (26) Steinke, J. D.; Johnson, L. A. Cereal Chem. 1991, 68, 7-12. (27) Morrison, W. R.; Milligan, T. P.; Azudin, M. N. J. Cereal Sci. 1984, 2, 257-271. (28) Chrastil, J. Carbohydr. Res. 1987, 159, 154-158. (29) Jacobs, H.; Eerlingen, R. C.; Rouseu, N.; Colonna, P.; Delcour, J. A. Carbohydr. Res. 1998, 308, 359-371. (30) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Anal. Chem. 1956, 28, 350-356. (31) Vandeputte, G. E.; Vermeylen, R.; Geeroms, J.; Delcour, J. A. J. Cereal Sci. 2003, 38, 43-52.

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