Molecular and Morphological Aspects of Annealing-Induced

Article pubs.acs.org/Biomac

Molecular and Morphological Aspects of Annealing-Induced Stabilization of Starch Crystallites Sara V. Gomand,*,† Lieve Lamberts,‡ Cedric J. Gommes,§ Richard G. F. Visser,∥ Jan A. Delcour,† and Bart Goderis⊥ †

Laboratory of Food Chemistry and Biochemistry, and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium ‡ S.A. Citrique Belge N.V., Pastorijstraat 249, B-3300 Tienen, Belgium § Department of Chemical Engineering, University of Liège, allée du 6 Août 3, B-4000 Liège, Belgium ∥ Laboratory of Plant Breeding, Department of Plant Sciences, Wageningen University and Research Centre, PO Box 386, NL-6700 AJ Wageningen, The Netherlands ⊥ Polymer Chemistry and Materials, Chemistry Department, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium S Supporting Information *

ABSTRACT: A unique series of potato (mutant) starches with highly different amylopectin/amylose (AP/AM) ratios was annealed in excess water at stepwise increasing temperatures to increase the starch melting (or gelatinization) temperatures in aqueous suspensions. Small-angle X-ray scattering (SAXS) experiments revealed that the lamellar starch crystals gain stability upon annealing via thickening for high-AM starch, whereas the crystal surface energy decreases for AM-free starch. In starches with intermediate AP/AM ratio, both mechanisms occur, but the surface energy reduction mechanism prevails. Crystal thickening seems to be associated with the cocrystallization of AM with AP, leading to very disordered nanomorphologies for which a new SAXS data interpretation scheme needed to be developed. Annealing affects neither the crystal internal structure nor the spherulitic morphology on a micrometer length scale.

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INTRODUCTION Starch generally consists of the quasi-linear α-(1,4)-glucose polymer, amylose (AM, 18−33%), and the highly branched α(1,4)-glucose polymer with ca. 5% α-(1,6)-bonds, amylopectin (AP, 67−82%).1 However, starches containing even higher AM contents exist as well, as do pure AP starches. The latter are generally referred to as AM-free or waxy starches. The AM and AP molecules are organized at different levels: the intertwining of AP branches into stiff double helices (∼0.1 to 2.0 nm) and their sidewise packing into crystalline lamellae, semicrystalline layer stacks composed of alternating crystalline lamellae and amorphous layers (∼9 nm), stacks arranged into blocklets (20− 400 nm), the presence of growth rings (120−400 nm), and finally the presence of granules (1−110 μm).1 AM usually does not contribute to the crystalline fraction. Several packing modes can be observed, leading to distinguishable A-, B-, or C-type diffraction patterns when examining starches with wide-angle Xray diffraction (WAXD).2 Heating a starch−water suspension above the gelatinization temperature induces crystal melting (gelatinization), the endothermic heat of which can readily be recorded. Two hydrothermal treatments of starch granules can be distinguished: heat-moisture treatment (HMT) and annealing.3 In both treatments, samples are incubated for a given time at an increased temperature close to the gelatinization temperature, © 2012 American Chemical Society

leading to modified physicochemical properties without disrupting the granular structure. HMT occurs at low moisture levels (60%).3 HMT leads to a broadening of the gelatinization temperature range (Tc−To) and occasionally to an altered crystal structure.4 In contrast, annealing leads to progressively narrower Tc−To the closer the incubation temperature is to the onset temperature of gelatinization (To).5 Annealing can also involve a two-step or multistep increase in the temperature during incubation, whereby starch properties are changed to a larger extent.3 Annealing can occur unintentionally as part of food processing or industrial starch isolation but can also be applied to obtain starches with new functionalities. The label-free character of this physical treatment is an asset over the use of chemically modified starches. It has been difficult to define what happens to the internal structure of starch granules in response to annealing. Mostly, an improvement of the crystalline order is mentioned6,7 together with few if any changes to the granular morphology. There is no clear view on what happens at the lamellar level. The Received: January 13, 2012 Revised: March 27, 2012 Published: March 30, 2012 1361

dx.doi.org/10.1021/bm3000748 | Biomacromolecules 2012, 13, 1361−1370

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incubated in a sealed container for 24 h. In literature, annealing is often applied at a temperature 3 to 4% below the endothermic peak temperature of gelatinization (Tp), with Tp expressed in Kelvin and measured by differential scanning calorimetry (DSC) (cf. infra). However, annealing temperatures selected according to this criterion occasionally occurred within the gelatinization endotherm of some of our samples. Therefore, to avoid excessive crystal melting, annealing temperatures were chosen at a temperature exactly before the start of the DSC endotherm. Following incubation, starches were Büchnerfiltered and dried at room temperature for 24 h. These starches are referred to as one-step annealed starches. Two-step annealed starches were prepared by continuing the first incubation for another 24 h, with the second annealing temperature determined based on the thermal properties of the one-step annealed starches. Isolation of the two-step annealed starches was similar to that of the one-step annealed starches. Three-step annealed starches were prepared by incubation of the twostep annealed starches in a similar way. Table S1 in the Supporting Information contains the annealing temperatures for the different materials. Gelatinization Properties. DSC measurements were carried out with a Q1000 DSC (TA Instruments, DE) according to Gomand et al.18 DSC was carried out with a Q1000 DSC (TA Instruments, New Castle, DE). Starch (4.00−6.00 mg) was accurately weighed in an aluminum pan (Perkin-Elmer, Waltham, MA), and deionized water was added (1:3 w/w starch dm:water). The pans were hermetically sealed and equilibrated for at least 20 min. The pans were heated from 20 to 130 °C at 4 °C/min. An empty pan was used as a reference, and indium was used as a calibration sample. Analyses were performed at least in triplicate. Onset, peak, and conclusion temperatures (To, Tp, and Tc) and ΔH were determined using TA Universal Analysis software. Statistical analyses were conducted using the Statistical Analysis System software 8.1 (SAS Institute, Cary, NC). Temperature was expressed in degrees Celsius and enthalpy in joules per gram. Granule Damage. The granule damage degree of native and annealed starches was measured according to the damaged starch assay (Megazyme, AACC method 76-31). All measurements were done at least in triplicate and expressed on dm basis. Crystallinity and Crystal Structure. WAXD experiments using Cu Kα radiation (wavelength, λ = 1.54 Å) were performed as previously described by Gomand et al.18 Starches were first equilibrated for 48 h in a humidifier to a moisture content of 20 ± 2%. A highly crystalline B-type and an amorphous starch were used to calculate the relative crystallinity (RC) according to Wakelin and coworkers.21 Differences between two measurements were 0. The disorder would increase further if also the amorphous layer thickness would have been varied. There are, however, limits to increasing the paracrystalline disorder. If for Gaussian distributions the ratio σA/lA or σC/lC exceeds 0.4, then part of the layer thickness distribution becomes negative,38 which is physically impossible. 1364


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( ) ⎤⎥ S(q) exp(−q σ ) + D

qlA ⎡ CαS(ρC − ρA )2 ⎢ 2sin 2 I(q) = ⎢ q 4πq2 ⎢⎣

2

⎥ ⎥⎦

2 2

(1)

The parameter C in eq 1 depends on the intensity of the incoming X-ray beam and the irradiated sample volume, D accounts for the background due to local electron density fluctuations within the layers, and αS represents the volume fraction of layer stacks.34 The factor between square brackets is the form factor of the monodisperse amorphous layers with thickness, lA. S(q) is the interference function associated with the paracrystalline positioning of the amorphous layers and is given by S(q) =

2 1 − exp( −q2σLP ) 2 2 1 + exp( −q2σLP ) − 2 exp( −0.5q2σLP ) cos(qL P)

(2)

where σLP is the standard deviation of LP. Note that in the present case, the LP distribution is fully defined via the lC distribution, given that the lA distribution is monodisperse. For a fully ordered stack, with eq 1 in the limiting case of σLP = 0, one would obtain extremely sharp reflections in I(q) at q values that are multiples of 2π/LP, and CF(x) would display triangles with peaks at integer multiples of LP and a width at the basis equal to 2lA. The blue I(q) and CF(x) curves corresponding to the blue ρ(x) profile in Figure 5 are of that type. The CF(x) triangular peaks do not fade with increasing distance, x, in contrast with when a distribution in LP would be allowed (green curves with σLP = 20 Å). In that case, the triangles in CF(x) progressively smear out with increasing x and tend toward zero at large x. The effect in I(q) is that the reflections broaden, that higher order peaks progressively fade away, and that the maximum of the first-order peak slightly shifts to smaller q values. These effects intensify with increasing σLP. Note that it is impossible to display a representative ρ(x) function for paracrystalline layer stacks because the x range in Figure 5 is too small to cover the entire lC distribution realistically. Modeling of the morphology in section B of Stack 3 in Figure 4 is less obvious because the one dimensionality is lost. The thesis is defended that the 1-D ρ(x) profile depicted red in Figure 5 captures the most important morphological features of section-B-like morphologies. This ρ(x) profile can be interpreted as the result of averaging all of the profiles present within the irradiated volume and (arbitrarily) starting form the center of the amorphous layers. At the (small) length scale of the amorphous layer, independent of whether it is present between the layers or at the lateral crystal borders, the 1-D layer like character is preserved. Therefore, this part of the ρ(x) profile should be identical to that of truly 1-D systems. Upon increasing the distance from the amorphous−crystalline interface, the electron density of an average situation evolves slowly to the average electron density of the system rather than staying in the crystalline phase for the full thickness of the neighboring crystalline layer (as would be so for a 1-D system). Because, in Figure 5, ρC and ρA were set equal to 1 and 0, the red sigmoidal profile directly reflects the probability of finding crystalline material at a given distance x > 0.5lA. At high x values, this probability is equal to the crystallinity within the semicrystalline regions; in Figure 5, ϕL = 0.75. Equation 3

Figure 5. Series of model ρ(x) profiles with the corresponding CF(x) and I(q) functions. All curves are based on D = 0, CαS = 1, ρC = 1, ρA = 0, LP = 100 Å, lA = 25 Å, and σ = 0 Å. The blue curves represent layer stacks with monodisperse crystalline and amorphous layers. The green curves correspond to paracrystalline stacks in which only the crystalline layer thickness is varied according to a Gaussian distribution, that is, σLP = 20 Å. No ρ(x) profile can be depicted for this paracrystalline situation, as explained in the text. The red and black curves correspond to highly disordered systems of which the average electron density around a given amorphous layer evolves from the crystalline density directly to the system average density (0.75). The transition to the average density for the red curve is smoother (σL = 25 Å) than that for the black (σL = 0 Å).

Even with the model beyond its reasonable limits, it is not possible to describe the haps SAXS patterns depicted in Figure 3, likely because the disorder is of another nature, such as depicted in stack 3 of Figure 4. Here disorder has been introduced by limiting the lateral size of the lamellar crystallites. Section A of stack 3 still very much resembles a 1-D layer stack, whereas in section B the 1-D character is lost because electron density variations are not restricted to the stacking direction. Obviously, the disorder would be even larger if in addition to the lateral size the layer thickness had been varied in stack 3. Modeling morphologies such as represented by stack 3 in Figure 4 for the purpose of extracting useful information form SAXS data is by no means trivial. A convenient approach turned out to be a combination of two models to, respectively, account for the A and B typical sections. To limit the total number of fitting parameters, we simplified the 1-D crystalline−amorphous paracrystalline model (of which the corresponding SAXS equation can be found in the Supporting Information), which in the end will account for the A-typical sections, by assuming that lA is monodisperse, that is, σA = 0. In that case, the equation of the corresponding SAXS powder patterns reduces to 1365


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Figure 6. SAXS patterns of native and three-step annealed haps1 and haps2. Experimental data (open circles) and fit of the data with eq 7 (full line through the experimental data) with the paracrystalline (full lines outside the experimental data) and disordered stack (dashed curves) contributions.

translates such ρ(x) profiles into the corresponding SAXS I(q) curve. I(q) =

CαS 4πq2

heights equal to (ρC − ⟨ρ⟩) and (ρC − ρA), respectively. A smooth transition − as, for example, for the red profile in Figure 5 − is conveniently introduced by convoluting the LP block profile with a Gaussian with standard deviation σL. The Fourier transform of the resulting ρ(x) − ⟨ρ⟩ profile equals F(q), as given by eq 4. The red curves in Figure 5 have been calculated with σL at its theoretical maximum value, that is, (LP − lA)/3. Higher σL values do not leave ρ(x) in the range of the amorphous layer (−0.5LA < x < 0.5LA) intact. The corresponding red CF(x) function displays only one very weak and broad maximum at x = LP, after which it immediately damps to zero. The maximum in I(q) has shifted dramatically to smaller q values, and higher order reflections are completely absent. Note that for this model, the value I(q = 0) equals zero! This unusual feature is characteristic of so-called hyperuniform structures.39 Hyperuniformity in the case of two-phase random heterogeneous media refers at the absence of infinite-wavelength volume fraction fluctuations, the crystallinity in the present case. Recently, the notion of hyperuniformity has been useful in describing the structure of different jammed particle packings.40,41 Note that the position of the first side maximum of CF(x) for the extreme case when σL = (LP − lA)/3 is representative for LP and that the CF(x) minimum coincides with that of the paracrystalline model curves. Both CF(x) features are commonly used to calculate ϕL from CF(x) as, for example, discussed in Strobl and Schneider42 and Goderis et al.34 It thus seems that these methods can be used irrespective of whether the morphology is paracrystalline (A section in Figure 4) or highly disordered (B section in Figure 4). However, if σL < (LP − lA)/3, then the mentioned CF(x) features are drifting away and therefore loose their relation with LP and ϕL, as is clearly

[F(q)]2 exp(−q2σ 2) + D (3)

with

( qL2 ) exp(−0.5q2σ 2) P

2 sin F(q) = (ρC − ⟨ρ⟩)

L

q

qlA 2

( )

2 sin − (ρC − ρA )

q

(4)

where ⟨ρ⟩ is the local average electron density within the semicrystalline regions given by ⟨ρ⟩ = ϕLρC + (1 − ϕL)ρA

(5)

2

The factor 1/4πq in eq 3 (just like in eq 1) converts the scattering of the average 1-D profile to the scattering of a powder of such profiles. F(q) represents the scattered wave function of the underlying average 1-D electron density profile, ρ(x), which can be computed via the inverse Fourier transformation of F(q) ρ(x) = ⟨ρ⟩ +

1 2π

∫0

∞

2F(q) cos(qx) dq

(6)

The black curves in Figure 5 represent the case where σL equals zero. Although this is rather artificial, it helps explain the origin of eq 3 and the meaning of σL. The electron density profile relevant to SAXS is ρ(x) − ⟨ρ⟩ rather than ρ(x). This ρ(x) − ⟨ρ⟩ profile for the situation represented by the black curves in Figure 5 can be constructed from the difference of two block-like ρ(x) profiles with widths equal to LP and lA and 1366


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illustrated for the situation where σL = 0 (black CF(x) curve in Figure 5). As to avoid complications, the haps SAXS patterns were analyzed by direct fitting rather than via the CF(x) approach. Because the envisaged morphology contains both paracrystalline and highly disordered sections (stack 3 in Figure 4) a linear combination of eqs 1 and 3 was used to analyze the haps SAXS patterns

(0.67), whereas that of haps2 was in between that of amfps and wtps (0.76). Both haps samples had a larger lA than the other types (between 25 and 30 Å). The crystal thickness for native haps1 was remarkably small (59 Å), whereas that of haps2 was rather large (79 Å). Through stepwise annealing, wtps showed only a very moderate increase in lC at the expense of lA. Amfps did not show variations in lA, lC, LP, or ϕL. Haps showed a pronounced increase in lC (up to 112 Å for haps2) and LP. The parameter lA increased slightly for haps2 and remained constant for haps1 (except for three-step annealed haps1). Understanding Annealing-Induced Crystal Stabilization. Annealing increases the gelatinization temperatures. This has often been interpreted to be a result of increased crystal stability. WAXD data revealed that for all samples the crystal structure and packing density remained unaffected by annealing. Consequently, the often cited annealing-induced crystal perfection needs to be ascribed to other factors. To pinpoint the underlying reason for the stability increase during annealing, it is convenient to plot the melting point, Tm (here put equal to Tp), as a function of 1/lC, in line with the Gibbs− Thomson equation for lamellar crystallites

⎡ CαS ⎢ I(q) = ⎢(1 − β)[F(q)]2 + β(ρC − ρA )2 2 4πq ⎢ ⎣ qlA ⎡ ⎢ 2 sin 2 ⎢ q ⎢⎣

⎤

( ) ⎤⎥ S(q)⎥⎥ exp(−q σ ) + D 2

⎥ ⎥⎦

2 2

⎥ ⎦

(7)

where β is the fraction that can be described as a paracrystalline layer stack. During fitting, the product CαS/4π was lumped together in a single parameter, C′. The densities ρC and ρA were fixed, respectively, at 1 and 0 with deviations from the real densities being incorporated in C′. Transition zones are not involved, and thus σ = 0. Accordingly, the fit involves seven parameters: C′, D, β, LP, lA, σL, and σLP, of which only the last five actually influence the shape of I(q). Fitting with the paracrystalline model (and assuming distributions in both the crystalline and amorphous layers) involves four shapedetermining parameters (with σ = 0). The crystalline layer thickness is calculated as lC = LP − lA and ϕL = lC/LP. Assigning different LP and lA values to the paracrystalline and disordered fractions would unnecessarily increase the number of parameters because it was found that the fitting quality did not improve upon doing so. This observation may indicate that paracrystalline and highly disordered sections are actually coexisting in common stacks with one characteristic local crystallinity. Figure 6 illustrates the relative shares (paracrystalline stacks and disordered stacks) to the SAXS fitting with eq 7 of haps1 and haps2 (native and three-step annealed). The ordered share, β, of native haps1 decreased form 0.44 to 0.36 as a result of three-step annealing and for haps2 β decreased from 0.37 to 0.29. For the fitting of haps2, σL needed to be constrained to its maximum value (LP − lA)/3. Furthermore, the ratio σLP/LP for haps2 exceeded 0.5. (For haps1, this value is close to 0.3.) It has been previously mentioned that σLP/LP should remain below 0.4. Given that the scattering patterns are dominated by the disordered fraction, it is believed that the resulting morphological parameters are nevertheless accurate. Influence of Annealing on the Lamellar Semicrystalline Morphology. In the Supporting Information, all SAXSbased morphological parameters are available in Table S4. Because of the uncertainty in σC for the wtps and amfps samples, only the ϕL, lA, lC, and LP values extracted from CF(x) are included. For the haps samples, the reported data are based on a fitting with eq 7. The most salient observations can be summarized as follows. Prior to annealing, wtps showed a ϕL value of 0.8, which is in accordance with Sanderson and coworkers43 and slightly higher than the amfps ϕL values (typically 0.76). The lA (17 Å) and lC (67 Å) values of wtps were, respectively, smaller and larger than in the amfps samples and also typically for B-type potato starches.43 The local crystallinity ϕL for haps1 was rather low

⎛ 2γ ⎞ Tm = Tm0⎜1 − ⎟ ΔHlC ⎠ ⎝

(8)

Equation 8 expresses that the melting temperature (Tm) depends on lC as well as on the equilibrium melting point of the system (T0m), the energy of the lamellar top surface (γ), and ΔH. Both γ and ΔH reflect the crystal quality in terms of surface quality (lower γ) and internal packing (higher ΔH). In the present analysis, local differences or changes in water concentration are neglected. Because WAXD revealed no changes in internal packing, ΔH can, to a first approximation, be considered to be equal for all samples in the present case. This leaves a reduction of γ or an increase in lC as possible reasons for crystal stabilization. Figure 7 displays such a Gibbs−Thomson plot (Tp of Table S2 as a function of 1/lC from Table S4 of the Supporting Information). It allows distinguishing clearly the three samples classes. Only the haps samples exhibit a linear relation between Tm and 1/lC, as highlighted by the linear regression to the corresponding data points. Although the scatter in the data

Figure 7. Gibbs−Thomson plot, relating the average crystalline layer thickness (lC) of the different samples to their corresponding melting peak temperature (Tm). The full line represents a linear regression through the haps data. The dashed lines are the upper and lower 95% confidence limits. 1367


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This surface activity is in line with Genkina and coworkers,9 Kiseleva and coworkers,15 and Tester and coworkers,23,48 who postulated that no new double helices are formed during annealing but that optimization of the double-helix packing is obtained at the crystal borders. Some minor crystal thickening seems to also be involved during three-step annealing for wtps3. The fact that equal melting points can be obtained for wtps as for amfps although the wtps crystal thickness is larger demonstrates that the crystal surface quality of wtps is poorer; that is, γ is higher. Literature describes that AM (amenable to crystal thickening) and AP are intermingled within the native potato granule, whereas they occur separately in the granules of cereal starches.24 This difference is suggested to be one of the reasons for the higher impact of annealing (larger shifts of the gelatinization temperatures) on potato than on cereal starches.24 Another mentioned factor is the high molecular weight of potato starch AM.24,49 Influence of Annealing on the Entire Gelatinization Behavior. According to literature, the “weakest” crystallites have the lowest melting temperature and have a higher susceptibility to crystal perfection during annealing.50,25 The decrease in Tc − To3 together with the increase in gelatinization temperatures suggests that the crystallites with the lowest stability (gelatinization temperatures) indeed improve preferentially. The previous paragraph clearly points at two different stabilization processes: a reduction of γ (which, as mentioned above, can reflect any stabilization process that leaves lC intact) and an increase of lC. For amfps and wtps, “weak crystals” may tentatively be translated into “crystals with a high γ”. For amfps, γ is already relatively low and annealing therefore has less impact on the gelatinization behavior. Mü h rbeck and Svensson51 observed an important shift and narrowing of the gelatinization endotherm when annealing wtps with lower phosphate ester group substitution. However, this trend was not confirmed in the present case. Gomand and coworkers18 measured the glucose-6-phosphate ester levels, and although wtps2 and amfps2 have a low phosphate ester substitution (9.8 and 8.6 nmol/mg starch, respectively), they do not exhibit a higher annealing susceptibility compared with wtps1, wtps3, and amfps1 that have higher phosphate ester levels (16.9, 14.4, and 21.4 nmol/mg starch, respectively). Some authors observed for wtps unchanged ΔH with annealing,29 whereas others reported a small increase.24,52 No additional crystallinity or double helices were generated by annealing amfps and wtps, as can be seen from their constant ΔH values. These results are also in line with previous 13C nuclear magnetic resonance results.15,53 Reductions of γ may easily happen without altering the sample crystallinity or ΔH. Note that amfps displayed a lower ϕL but higher ΔH than did wtps. ΔH, however, reflects the total amount of crystals in the granules, to be compared with the product αSϕL rather than with ϕL. ϕL represents the local crystallinity, only valid within the semicrystalline layer stacks. The increase in ΔH of haps2 is in line with the results of Knutson54 for amylomaize starch, with an AM content of 50 and 70%. Comparing the results with those for normal and waxy maize starches allowed us to attribute this to the interaction of AM and AP. 13C Nuclear magnetic resonance previously confirmed an increased doublehelical content of annealed high-AM maize starches.44 Our SAXS measurements revealed an increase in lC and ϕL for haps samples, which suggests the creation of longer double helices.

points is considerable, it can be concluded that the main mechanism for crystal stabilization in the haps case is crystal thickening. The linear trend furthermore indicates that no changes in crystal surface quality (γ) are involved. One can calculate T0m, that is, the melting point expected for crystals with an infinite crystal thickness, from the intercept of the linear regression. On the basis of our data for haps, T0m is between 131 and 157 °C. This melting point is relevant only to starch−water systems at the water concentration used here. Note that the mean crystal thickness for haps2 as a result of extensive annealing equals 112 Å, which requires sequences of at least 32 glucose units (six glucose units in a double-helix span 21 Å). The linear regression furthermore predicts 210 Å thick crystals at To for haps2 after 3 annealing steps (To = 126 °C, see Table S2 of the Supporting Information). Such crystals are the most stable ones encountered in the present study and require at least 60 glucose units in a row. The notion at least refers to the possibility of chain tilting, which, if relevant, would require an even higher number of glucose units for spanning the lamellar thickness. Such a high number of glucose units points to participation of AM in the crystals. Another argument for AM being partially crystalline in (at least) haps2 stems is the fact that this samples crystallinity equals 21%, whereas its AP content amounts to only 22%. Not all APs can be in the crystalline state simply because the AP branching points cannot be accommodated within crystalline zones. Therefore, AM necessarily has to contribute to the crystalline fraction. Our SAXS analysis reveals a high degree of disorder. The cocrystallization of AM with AP side chains likely contributes to this effect.14 Similarly, for high-AM maize starches, it is suggested that AM participates at least partially in the double helices.44 Tm of the amfps samples does not depend on lC. All Tm increases can therefore be attributed to reductions in γ. Recently, relaxation processes at the crystal surface leading to a decreasing γ have been coined as the final stabilization step in the crystallization of synthetic polymers.45 The exact nature of these processes is ill-defined but may involve the relaxation of conformationally constrained chain fragments at the crystal borders or the migration of chain entanglements or branching points away from the crystal−amorphous interface toward the noncrystalline layers. In the context of the side-chain liquidcrystalline model of starch, such a stabilization may be driven by the entropy gain of the backbone and side chains rather than by pure surface energy issues.46 Vermeylen et al.10 argued that a merging of the block-like mosaics of the lamellae may also contribute to the annealing-induced increase in the gelatinization temperatures. Any of these processes increase the crystal stability without markedly affecting the crystal thickness, which in the formalism of eq 8 is cast into a decrease in γ. Clearly, the haps crystals have a lower γ than those of amfps because haps crystals with an lC equal to that of the amfps samples are expected (95% confidence limits) to melt somewhere in the range between 80 and 88 °C. Obviously, the AP branching points prevent crystal thickening of amfps, whereas this process is readily accomplished by a reshuffling of the linear AM chains in the haps crystals. Such a reshuffling of polymeric chains through solid lamellar crystals has been reported in synthetic polymers, and the associated process is referred to as chain sliding diffusion.47 The trend for the wtps samples essentially follows that of the amfps samples, with the lC values being thicker from the start: the crystal stability predominantly increases by a reduction of γ. 1368


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CONCLUSIONS The AM content as well as multistep annealing processes influence the semicrystalline morphology of starch, which in turn affects the gelatinization properties of aqueous starch suspensions. Prior to annealing, all studied starches displayed a B-type WAXD diffraction pattern with rather broad and weak reflections. Haps samples have lower crystallinities and less perfect crystals than amfps and wtps. The SAXS patterns of amfps and wtps are typical for paracrystalline stacks of alternating crystalline and amorphous layers, whereas the morphology of haps seemed to be due to a combination of paracrystalline and highly disordered layer stacks. A new approach was developed to extract morphological information from the uncommon haps SAXS patterns. The lA and lC values of wtps were, respectively, smaller and larger than in the amfps samples but in both cases typically for B-type potato starches. Both haps samples had a larger lA than the other types. The crystal thickness, lC, for haps1 was remarkably small, whereas that of haps2 was rather large. The fraction of semicrystalline regions that can be modeled as paracrystalline layers stacks is larger for haps1 (0.44) than for haps2 (0.37), indicating that the stacking disorder increases with increasing AM content. Irrespective of the starch type, stepwise annealing increases and narrows the DSC-based gelatinization temperature ranges without affecting ΔH. Annealing thus increases the crystal stability, whereas the amount of crystalline material is not affected. WAXD revealed that for all samples the B-type crystal structure and packing density remain identical. Amfps and wtps did not or hardly show any changes in the SAXS based lA, lC, LP, or ϕL values. In contrast, haps showed a pronounced increase in lC and LP concomitantly to an increase in the stacking disorder. To pinpoint the underlying reason for the crystal stabilization during annealing, the SAXS lC data evolution was analyzed in terms of the Gibbs−Thomson equation for lamellar crystallites, which consists of plotting the melting point (Tp) as a function of 1/lC. The haps samples exhibited a linear relation between Tp and 1/lC, revealing that the main mechanism for crystal stabilization in the haps case is crystal thickening. The strictly linear trend furthermore indicates that no changes in crystal surface quality are involved. The analysis further forecasts the melting point of crystals with an infinite crystal thickness to be between 131 and 157 °C at the water concentration used. The analysis also reveals that the amfps and wtps crystal stability increases through annealing by a reduction of γ. The wtps crystals thicken only a little in a three-step annealing procedure. For amfps, γ is already relatively low prior to annealing, which explains the moderate impact of annealing on the crystal stability and hence the gelatinization behavior. The exact nature of the reduction in γ is unknown but may involve the relaxation of conformationally constrained chain fragments at the crystal borders or the migration of chain entanglements or branching points away from the crystal−amorphous interface toward the noncrystalline layers. In principle, also a merging of the blocklike mosaics of the lamellae may contribute to the annealinginduced increase in the gelatinization temperatures.

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and by making use of linear correlation functions; an overview of the potato starches with the corresponding AM contents, enzyme suppressions of the potatoes and selected annealing temperatures; all To, Tp, Tc, Tc−To, and ΔH values of native and annealed starches; starch damage degrees (%) of the native and annealed starches; and all SAXS-based morphological parameters. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+32)-16-321582. Fax: (+32)-16-321997. E-mail: sara. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Steven Cabus, Joke Putseys, and Luc Van Den Ende for technical assistance. The ‘Instituut voor aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen’ (IWT, Brussels, Belgium) is thanked for financial support. C.J.G. is supported by the Funds for Scientific Research (F.R.S.FNRS, Belgium) through a research associate position. This study was partially carried out within the framework of research projects GOA/03/10 financed by the Research Fund K.U. Leuven. It is also part of the Methusalem programme “Food for the Future” at the K.U. Leuven. We thank FWO Vlaanderen for supporting the DUBBLE project. J.A.D. is W.K. Kellogg Chair in Cereal Science and Nutrition at the K.U. Leuven.

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S Supporting Information *

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