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Causal Relations between Structural Features of Amylopectin, a Semicrystalline Hyperbranched Polymer Torsten Witt†,‡ and Robert G. Gilbert*,†,‡ †

Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, China, 430030 Centre for Nutrition and Food Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Queensland 4072, Australia

‡

S Supporting Information *

ABSTRACT: The relationships were determined between molecular properties of amylopectin, a hyperbranched glucose polymer and the major component of starch, and higher-level structures in native starch (double helices, crystallinity and crystalline−amorphous lamellae). Parameters from NMR, differential scanning calorimetry, and size exclusion chromatography of β-limit dextrins of a series of waxy starches, together with literature data, gave information on relationships between the structure of the interior of the amylopectin molecule and crystallinity. The structure of internal B chains (those with one or more branches) of amylopectin influences both crystalline properties and crystallinity. More B chains produce larger crystalline−amorphous lamellae, probably by expanding the amorphous lamellae, while larger B chains increase the ability of annealing to increase order through greater mobility for the chains to rearrange at higher temperatures. This study brings into question the common assumption that A chains (unbranched chains) are necessarily smaller than B chains.

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INTRODUCTION Starch is a homopolymer of glucose that is usually divided into two macromolecules with different molecular structures: amylose and amylopectin. Amylose is typically present in a smaller amount, has only a few long-chain branches, and has molecular weight ∼104−106. Amylopectin is significantly larger, with a molecular weight of 107−108, contains a large number of short branches, and is responsible for many of the structural features of interest in native starch. The glucose monomers are attached to one another via an α-(1 → 4) bond to form the linear chains while branch points attach another starch chain via an α-(1 → 6) bond. In native starch, the branches aggregate to form either single or double helices. Single helices are typically produced by the long linear amylose molecules that are in a complex with a variety of small molecules,1 while double helices are produced by shorter amylopectin chains. The helices can aggregate further to produce three different types of crystallites; V-type crystals are produced by the single helical amylose, while the double helical starches can form two different crystallites, A- and B-types. A-type crystals exhibit a monoclinic unit cell while B-type starches exhibit a hexagonal unit cell. A third designation, C-type, is given to starches with a mixture of both A- and B-type crystallites. The A- and B-type starch crystallites can also take part in the production of alternating crystalline and amorphous lamellae. Amylopectin has a complex, infinitely hierarchical structure.2 A common method of obtaining information about the amylopectin structure is to debranch it, using the debranching © XXXX American Chemical Society

enzymes isoamylase or pullulanase, then measuring the relative proportion of the resulting linear chains. The most convenient method to do this is to plot the number distribution of the chains as a function of the branch length (the chain-length distribution, CLD), optimally as a log plot.3 This number distribution, “Level 1” of starch structure, can then be parametrized, in the past typically by division into different empirical categories, the most popular of which is the Hanashiro method;4 it is now preferable to use biosynthetic modeling to provide mechanism-based parameters,5−7 the code for which is publicly available.6 The CLD of amylopectin is one of the many pieces of information about amylopectin structure. Another way is to divide its fully branched (“Level 2”) structure into three types of chains based upon the branching they take part in, as follows. There is a single C-chain of the amylopectin molecule which contains the only free reducing end and some number of branch points; B chains are connected via the reducing end to another starch chain and have one or more branches upon them; A chains are those chains which are attached via the reducing end to another starch chain and exhibit no branch points along their length. The analysis of this structure can be undertaken using β-amylase to enzymatically hydrolyze maltose from the nonreducing ends of the amylopectin molecule.8,9 When the digestion is allowed to progress to completion, all Received: March 7, 2014 Revised: June 8, 2014

A

dx.doi.org/10.1021/bm500353e | Biomacromolecules XXXX, XXX, XXX−XXX

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angle spinning (CP/MAS) nuclear magnetic resonance (NMR) provides data about the double helices present in starch which are involved predominately in crystallites, but which may be embedded in the amorphous areas of the starch granule.18 The third commonly used probe of starch crystalline material is differential scanning calorimetry (DSC), which measures the amount of energy required to reduce the order present in the starch sample. It can also be used to determine the crystalline stability, by observing the onset and peak melting and conclusion temperatures of the starch crystals, and the relative amount of crystallinity present in different starches.19 It can also be used in conjunction with an annealing process to observe the number of crystalline defects that are present in a starch, as represented by the differences in enthalpy before and after annealing removes the defects associated with double helices that are out of register, and by unordered portions of chains extending from double helices which can produce longer double helices.20−22 While all of these starch structural levels have been explored, there is limited information on the manner in which these structures interact with one another to produce the granules within native starch. Indeed there is significant uncertainty to what extent the biological properties of the starch biosynthesis affect these interactions and to what extent they influence the crystalline and lamellar properties. Some work has been done to show that there is a link between the chain length distributions of amylopectin with the type of crystallite produced,23 the stability of those crystallites,24 and changes in the size of the crystalline−amorphous lamellae, as discussed in our previous work.7 Figure 2 shows a representation of what is known about the interrelationship between the synthesis conditions and the different structural levels of starch. The relationships that are marked as unknown, namely, those between molecular parameters and crystallinity and between crystallinity and crystalline−amorphous lamellae, are the topic of this study.

chains exterior to branch points are reduced to their minimum size, a size which depends on whether they are an A or B chain. A chains are reduced to either a maltose or maltotriose, while B chains are reduced to a chain length equal to the number of monomers before the branch point closest to the nonreducing end of the B chain with an additional one or two glucose monomers. The difference in the number of glucose units remaining is related to whether the initial chain had a degree of polymerization (DP, symbol X) that was odd or even.8 After the production of these β-limit dextrins is complete, a debranching enzyme can be used to produce a distribution that shows the proportion of A and B chains, the ratio of DP 3 and 4 chains compared to all other chains, as well as providing information about the CLD of the B chains that are interior to the branch points within amylopectin. A representation of this process is provided in Figure 1.

Figure 1. Diagram representing the removal of nonreducing end maltose via β-amylase digestion, resulting in the A and B chains separating into two populations after debranching with Isoamylase.

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METHODS AND MATERIALS

The variety of waxy starch samples used here were the same purified waxy starches as those produced in the previous work7 and are presented in the Supporting Information in Table SI-1 for completeness. Differential Scanning Calorimetry. DSC experiments were performed on a TA Instruments (New Castle, DE, USA) Q2000. Five milligrams of starch and 20 mg of water were added to a T0 aluminum hermetic DSC pan (TA Instruments, Switzerland) and sealed. The experiments were run from 303 to 373 K with a temperature increase of 2 K/min; the analysis was performed using the TA Universal Analysis software (TA Instruments, New Castle, DE, USA), giving the onset temperature, TO, peak temperature, TP, enthalpy ΔH, and the distribution of temperatures TD = TP − TO. Annealed samples were made from 100 mg of the native starch samples added to ∼2 mL of water and annealed by keeping at 10 K below their onset temperature for 8 h. After annealing, the starches were freeze-dried, reanalyzed via DSC, and stored for further analysis. This temperature was selected to minimize any loss of crystallinity through gelatinization. A preliminary test was undertaken on starches which were annealed at both 5 and 10 K below TO; it was found that starches annealed at 5 K below TO displayed a loss of enthalpy compared to those annealed at 10 K below TO. All analyses were performed at least in triplicate, with up to six replicates performed for some samples; an example of the DSC patterns and parameters is given in Figure 3. 13 C CP/MAS NMR. 13C CP/MAS NMR experiments were performed on a Bruker Avance III 300 MHz NMR spectrometer using the same experimental parameters as Tan et al., with the exception that the number of scans was reduced to 1000 per sample.18 Approximately 200−300 mg of dry starch, with a moisture content of 10−11% for all samples, was packed into a 4 mm cylindrical, PSZ

The size distribution of the whole molecule is another Level2 structural quantity; however, there are significant technical limitations on the examination of the whole amylopectin molecules.10−12 The current best method to measure this, size exclusion chromatography (SEC), struggles with molecules as large as amylopectin for a variety of reasons. First, the columns currently available for separation may not fully separate the largest amylopectin molecules; second, there are significant shear forces present within columns which cause shear scission, reducing the largest amylopectin molecules to a smaller stable size.13 These two issues can be difficult to distinguish from one another and will both result in a distorted size distribution being produced. While field-flow fractionation can overcome the shearscission problem, there are other problems which arise for applying this technique to native starch.12,14 The last significant problem is that there are no standards available for molecules of amylopectin’s size, so calibration using the universal calibration principle is unreliable for larger sizes of amylopectin.15 SEC is however sufficient to examine amylopectin structures that have been reduced significantly in size, such as those that have undergone exhaustive β-amylase digestion. There are many methods to observe the crystalline (“Level 3”) structure of starch, all of which provide slightly different average information about the structure of the crystallites. The most commonly used is X-ray diffraction, which can measure the type and relative amount of crystallinity.16,17,13 Cross polarized magic B


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Figure 2. Diagram showing the known relationships between different levels of starch structure. Unknown relationships, indicated with a question mark, are tested in this study.

Figure 3. Example of the native and annealed waxy sorghum starch with definitions of the parameters TO, TP, TD, and ΔH. Size Exclusion Chromatography. Samples were prepared by dissolving 1 mg of whole βDA or 2 mg of debranched βDA in 1 mL of a 0.5% LiBr/DMSO solution overnight at 80 °C with shaking. The debranched βDAs were separated using a PSS (Mainz, Germany) GRAM precolumn, Gram 1000, and Gram 30 columns to provide better separation between the maltosyl and trimaltosyl chains and the solvent peak; the whole-molecule starches and βDAs were run together on a PSS Gram precolumn, PSS GRAM 30000, and PSS Gram 1000 columns to compare them directly. Universal calibration and data manipulation with relation to the SEC differential refractive index (DRI) signal were conducted as detailed elsewhere,13,15,26 including the relation between elution volume, hydrodynamic volume Vh (and the corresponding hydrodynamic radius Rh), and DP of linear chains (following SEC characterization after debranching). The standards used for universal calibration were PSS (Mainz, Germany) pullulan standards that varied in molecular weight from 342 to 2 350 000. The linear debranched βDAs were converted to a number distribution, Nde(X), while the whole molecule βDAs are left in terms of their weight distributions, w(log Rh). Each βDA sample was run in duplicate for the SEC experiments and parametrization. The debranched distributions of the βDAs were parametrized as follows. The proportion (the number of chains of a group as a ratio of the total number of chains) of A chains (those that are represented by

(partially stabilized zirconium oxide) rotor with a KelF end-cap. The native and annealed starches were analyzed and values obtained for the proportion of double helices and single helices of the starches, as well as the difference between the native and annealed starches. Duplicate experiments were run for the annealed samples while only a single experiment was performed for the native starches. β-Amylase Digestion. The β-amylase digested amylopectins (βDAs) were produced by gelatinizing 50 mg of the waxy starch samples in 5 mL of water in a boiling water bath for 20 min. This was allowed to cool to room temperature, and a 10 mM sodium acetate buffer, which had been adjusted to pH 4.5 using acetic acid, plus β-amylase from barley (Sigma, A7130−10KU) were added to produce a 0.02 mg/mL solution. Five milliliters of the β-amylase solution was added to the starch gelatinized at room temperature, and then the mixture was incubated at 40 °C for 3 h. This time was sufficient to produce the β-limit dextrin, as no change in SEC size distribution was observed from 2 to 24 h of digestion. After incubation, the enzymes were deactivated by boiling again for 20 min, and the solution was then allowed to cool to room temperature. The βDAs were separated from maltose by precipitation: 15 mL of ethanol was added followed by centrifugation at 4000 g for 10 min.25 The debranching of the βDAs was accomplished using isoamylase with the method of Hasjim et al.25 C


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Figure 4. Weight distribution example of the features that are parametrized. shoulder with a DP of ∼2.5−5 nm, short B chains (those that were represented by the first large peak) and long B chains (those that were represented by a second peak larger than the first main peak); additionally, the DP of the maximum of the weight distribution for the short B chains and long B chains was observed. The average DP of the short B chains was calculated using the number distribution as previously.7 An example of the division is shown on the βDA in Figure 4. The whole-molecule βDAs were parametrized by calculating the average Rh of the peak that was observed, using eq 1, and recording the Rh of the maximum.15

off to form a new branch by the branching enzyme in set 2. Graphical illustrations of the mechanisms have been given by Wu et al.5,6,27 The correlations between these parameters were found in the previous work and will only be mentioned, and briefly explained, when directly relevant to the current work. Statistical significance is defined as a p-value ≤0.05. The following parameters are contributed from this experimental work: native TO, native TM, native TD, native ΔH, annealed TO, annealed TM, annealed TD, annealed ΔH, difference in TO, difference in TM, difference in TD, difference in ΔH, numberaverage DP X̅ n of short B chains, proportion of A chains, proportion of short B chains, proportion of long B chains, maximum DP of short B chains, maximum DP of long chains, whole βDA R̅ n, whole βDA maximum Rh, proportions of native double helices, native single helices, annealed double helices, annealed single helices, difference in double helices, and difference in single helices.

∞

log(R̅ h) =

∫−∞ w(log Vh)d log R h ∞ w(log Vh) d log R h

∫−∞

log R h

(1)

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Statistics. Pearson correlation coefficients were used to check for linear relationships between the different parameters for the 11 different waxy starches. The results of this work was analyzed in conjunction with the results from the previous work,7 which examined the molecular and lamellar properties of these starches and the manner in which they correlated. The following parameters are from that work: average repeat distance, half width at half-maximum of lamellae, proportion of A- or/B-type crystals, proportion of V-type crystallites, fractions of DP 6−12, DP 13−24, DP 25−36, DP ≥ 37, degree of branching, proportion of amylose, and the following parameters for expressing the CLD in terms of the mechanistic model of Wu et al.:5,6,27 β1, β2, h(ii/i), and Xmin2. In brief, these parameters are as follows. The model assumes that the CLD is controlled by various enzyme sets (one each of one type of starch branching enzyme, one type of debranching enzyme, and one type of starch synthase), acting either independently or together. β1 and β2 are the ratio of the activities of starch branching enzyme to that of starch synthase, for enzyme sets 1 and 2; set 1 largely controls chains confined to a single lamella and set 2 to those spanning two lamellae. The quantity h(ii/i) gives the relative contribution of each enzyme set to the overall CLD. Xmin2 is the minimum number of monomer units that can be broken

RESULTS AND DISCUSSION Internal Molecular Structure of Amylopectin. The results for the size of the native whole molecule amylopectin samples are unreliable due to the presence of significant problems of sample recovery: loss between injection and analysis. The measured recovery of whole amylopectin molecules is not repeatable. Further, shear scission in the SEC cannot be avoided. Nevertheless, semiquantitative inferences can be drawn: thus there is a significant range of sizes represented with the different samples; there are significant numbers of molecules with a hydrodynamic radius of ∼40−50 nm, although the majority of molecules are somewhat smaller. The whole-molecule βDA distributions in Figure 5 show a maximum at Rh ∼ 3 nm and are monomodal, with the exception of the starch Waxiro which is bimodal. The Waxiro starches were digested with β-amylase a second time, now for a longer period. This treatment did not remove the second peak, D


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Figure 5. SEC weight distribution of whole molecule β-amylase digested amylopectins.

Figure 6. SEC weight distribution of debranched β-amylase digested starches.

render inaccurate the value of the DP.28 This is supported by the results for the pullulan standard with a molar mass of 342 which corresponds to two glucose units which are connected via α-(1 → 4) or α-(1 → 6) bonds in a ratio of 2:1. Therefore, the majority of molecules will be maltose, and after undergoing the same data manipulation as the debranched starch samples displayed a peak DP of 2.7. The band broadening and Mark− Houwink relationship affects all of the samples similarly, so the results are semiquantitative. That implies that the values determined can be compared readily to one another, but comparisons to values for the proportion of A chains as determined by different methods is problematic. All other chains are the interior B chains, either the short B chains or the long B chains, seen in Figure 5. The short B chains are present in all of the starches and are therefore required for the production of amylopectin. The short B chains, which are interior to the branch points, vary in their maxima. The

indicating that the multimodal peaks are not due to undigested amylopectin. The debranched β-amylase digested samples always displayed a large peak with a DP of 10−20, together with a small peak or shoulder at a DP ∼ 3−4 glucose units. Some of the distributions also displayed a second peak at DP 100−200, which does not correspond to branches commonly found in amylopectin. These features can all be seen in Figure 6. The small chains at DP 3−4 can be assigned to the outer A chains of amylopectin; these starch chains contain no other branch points, and are therefore digested to the DP 2 or 3 limit dextrin.8 The shift from the expected DP 2−3 to the range of DP 2.5−5 that is observed is due to a combination of factors. One such factor is band broadening, which will lead to an increase in the width of the distribution.28 At these small sizes, there may also be a breakdown of the Mark−Houwink relation (used to convert hydrodynamic radius to DP), which would E


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Table 1. Structural Parameters Relating to the β-Amylase Digested Waxy Starches and the Helices of the Native and Annealed Starches double helical structures within waxy starches

debranched β-amylase digested starch

starch EH-6 Eliane F97 2−1 KiaryoMochi Mazaca Pulut-Siding ShizukiMochi W64A Waxiro Waxy Sorghum YRW4

average Rh (nm) of whole molecule β-amylase digested starch 2.75 2.84 3.17 2.91

± ± ± ±

0.01 0.08 0.06 0.01

average degree of polymerization of short B chains 8.66 6.62 9.41 6.66

± ± ± ±

0.89 1.29 0 0.15

proportion of A chains 40.0 49.7 47.2 47.3

± ± ± ±

2.05 0.99 0 0.18

proportion of short B chains 58.2 50.3 50.2 51.6

± ± ± ±

1.74 0.99 0 0.09

maximum DP of short B chains

native double helices

± ± ± ±

0.75 2.19 0 0.18

33.3 32.7 25.7 32.2

32.9 35.0 31.9 34.7

9.81 13.46 10.89 10.52

annealed double helices

difference in double helices

± ± ± ±

1.83 0.41 0.61 0.48

−0.34 2.33 6.14 2.52

3.07 ± 0.01 5.18 ± 2.98 3.14 ± 0.01

6.78 ± 0.36 6.37 ± 0.02 6.37 ± 0.02

43.3 ± 0.53 48.2 ± 0.53 49.1 ± 0.38

56.7 ± 0.53 51.8 ± 0.53 50.2 ± 0.29

12.01 ± 0.83 11.69 ± 0.16 10.75 ± 0.08

34.9 17.3 35.1

37.5 ± 1.10 18.5 ± 1.37 28.3 ± 1.07

2.61 1.28 −6.81

3.06 ± 0.03 5.33 ± 0.06 3.09 ± 0.04

7.65 ± 1.41 13.66 ± 1.51 7.69 ± 0.28

38.2 ± 0.28 34.5 ± 0.39 47.6 ± 0.82

59.9 ± 0.72 56.1 ± 1.62 51.1 ± 0.67

11.32 ± 0.72 8.83 ± 1.45 10.84 ± 0.18

25.9 26.7 31.5

29.8 ± 0.36 30.2 ± 1.51 33.2 ± 3.09

3.89 3.55 1.64

3.08 ± 0.2

6.53 ± 0.18

51.2 ± 0.36

48.8 ± 0.36

11.3 ± 0.43

30.2

28.9 ± 0.94

−1.33

distributions of the short B chains are similar to each other, and are monomodal. Those starches that display the long B chains display a significant difference between their distributions; Waxiro displays the greatest number of long B chains while Eliane, Pulut−Siding, and Shizuki−Mochi display no long B chains. Even in the case of Waxiro, the long B chains are relatively few: they are a small fraction of the number of chains although they are a larger fraction of the total weight of the starch sample. These are either extra-long amylopectin chains or some of the small amount of amylose that is present that displays a branch point near the nonreducing end. This is supported by the starch with the greatest amount of amylose, Waxiro, having the greatest number of long B chains. Another interesting feature of the long B chains is the repeated bimodal distribution of waxy sorghum, W64A and F97 2−1, which suggests that there are two differently branched populations of amylose (as observed in amylose CLDs29−31) if amylose is the source of the long B chains. The amount of each of these categories as a proportion of the total amount of amylopectin chains, as well as the position of the maximum short and long B chains, is displayed in Table 1. The proportion of A chains and short B chains are linked, and will be correlated negatively, as they make up essentially all of the total starch in the debranched βDA sample, so the number of the A chains will inversely mirror any change in the number of short B chains, with the long B chains responsible for only 0−3% of all starches except Waxiro. A similar, albeit more complex, technique was used by Bertoft et al. to determine that the ratio of the number of A to B chains in cereal starches is 1.0, while it is 1.2−1.3 in noncereal starches.9 The current results for the various starches show a different range of ratios from 0.6−1 for the A to B chains; as mentioned earlier the semiquantitative results of the proportion of A chains from SEC may not be quantitatively comparable to values using HPAEC, as done by Bertoft et al. Both systems have inaccuracies: band-broadening and conversion from size to DP for SEC, and mass bias and limited range of DP for HPAEC. Double Helical Properties. The values for the proportion of double helices before and after annealing, as well as the difference between them, are given in Table 1. The measure-

ments of fractions of single helices were found to be unreliable, with errors not dissimilar to that of the very small amount of single helical material present; consequently this quantity has been disregarded for the rest of the analysis, although it can be found in the Supporting Information for completeness. Crystal Stability and the Influence of Crystalline Defects. The onset temperature and enthalpy increased to varying degrees after annealing for all starches. The peak temperature increased in some starches and was unchanged in others. Some starches displayed a lower average peak temperature after annealing than before, but change in the magnitude of these peak temperatures was similar to the error related to the reproducibility of these experiments. Thus, the average peak temperature of these starches, F97 2−1, ShizukiMochi, and waxy sorghum, can be considered indistinguishable before and after annealing. These values are displayed in Table 2. Onset and peak temperature were used in the statistical analysis, but were always found to correlate with one another, probably due to the similarity in the gelatinization peak for all of the starches. That is, the difference between the shape and range of the gelatinization curve of the starches was less than the difference between their onset temperatures. Therefore, only TP will be referred to in the Discussion. Correlations between Structural Parameters. The nature of Pearson correlations is that they are heavily influenced by outlying values, so while the correlations between all starches have been observed, it is also important to observe whether there are any samples which are consistently outliers. The ability of a ranking correlation method, such as Spearman correlations, to overcome these outliers for this type of system was tested in our previous paper.7 The ranking correlations exhibited the same problems with outliers as the Pearson correlations in the previous paper, so Spearman correlations were not used here. There are two outlier samples with the inclusion of all the parameters obtained in this and the previous paper:7 the B-type potato starch, Eliane, and the barley starch, Waxiro. As noted, the β-amylase digestion of the Waxiro starch produced a very different bimodal βDA whole-molecule structure, which caused false positive correlations about the β-amylase digested starches when including Waxiro in the statistical analysis. The correlations relating to chain length F


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Table 2. DSC Analysis of Native and Annealed Starches native starch TO (K) EH-6 Eliane F97 2−1 Kiaryo-Mochi Mazaca Pulut-Siding Shizukimochi W64A Waxiro Waxy Sorghum YRW4

332.9 335.6 343.3 334.5 336.1 333.3 330.6

± ± ± ± ± ± ±

0.37 0.58 0.33 0.18 0.73 0.44 0.3

TP (K) 338.6 341.0 348.2 339.9 343.1 339.2 336.4

± ± ± ± ± ± ±

0.25 0.08 0.45 0.32 0.73 0.23 0.41

annealed starch ΔH

ΔT (K)

± ± ± ± ± ± ±

0.28 0.15 0.27 0.17 0.01 0.42 0.15

5.75 5.4 4.91 5.44 7.0 5.9 5.75

336.1 339.6 344.4 336.3 340.0 336.3 331.3

5.7 14.9 5.0 7.0 12.2 3.0 3.9

TO (K) ± ± ± ± ± ± ±

TP (K)

0.08 0.11 0.03 0.21 0.15 0.11 0.24

338.6 342.2 348.0 340.8 344.8 340.4 336.3

± ± ± ± ± ± ±

0.46 0.23 0.2 0.12 0.19 0.2 0.46

change caused by annealing ΔH

ΔT (K)

TO (K)

TP (K)

ΔH

ΔT (K)

± ± ± ± ± ± ±

0.51 0.51 1.05 0.8 0.46 0.28 0.2

2.52 2.57 3.69 4.52 4.78 4.07 5.0

3.2 4. 1.1 1.8 3.9 3.0 0.7

−0.1 1.2 −0.2 0.9 1.7 1.2 −0.05

7.3 2.2 4.6 4.3 1.1 1.5 4.1

−3.23 −2.84 −1.22 −0.92 −2.22 −1.84 −0.75

13.0 17.1 9.7 11.2 13.3 4.5 8.0

341.6 ± 0.69 332.2 ± 0.55 344.1 ± 0.31

345.8 ± 0.56 337.5 ± 0.68 348.8 ± 0.75

3.5 ± 0.35 6.5 ± 0.33 6.9 ± 0.14

4.29 5.26 4.66

343.1 ± 0.05 337.5 ± 0.5 346.0 ± 0.07

346.8 ± 0.37 339.5 ± 1.16 348.1 ± 0.05

6.2 ± 0.37 9.0 ± 0.18 12.3 ± 0.76

3.65 1.99 2.14

1.6 5.3 1.9

0.9 2.0 −0.7

2.7 2.5 5.3

−0.64 −3.27 −2.52

332.2 ± 0.44

337.9 ± 0.78

6.0 ± 0.14

5.74

335.4 ± 0.17

339.2 ± 0.32

9.8 ± 0.7

3.76

3.2

1.2

3.8

−1.98

the shoulder, indicating that when the interior of the amylopectin molecule is smaller, the shoulder size decreases. There is a common belief that the first linear portion of the CLD represent chains which terminate after interacting in a single cluster or lamella, and the second linear portion interacts with more than one cluster35,36 or lamella.37,38 The shoulder is, then, those chains which take part in a single crystalline lamella region and the amorphous lamella region. The present results potentially show support for this, provided the proportion of B chains is not related to the crystallite properties that are formed in these native starches. This will be examined in greater detail later. Another comment can be made on the common belief that A chains are necessarily smaller than B chains, as put forward by Hizukuri and used by others.4,23,39 Aside from the single correlation already mentioned, the proportion of B chains and the range of shoulder-region chains, there are no correlations between the CLD when using either the Hanashiro empirical parametrization4 or the Wu-Gilbert mechanism-based parametrization5,6 and the CLDs of the A and B chains. So there is no evidence that the A chains are more likely to be short chains than any other chains. Indeed, the observed correlation shows a preference, not uniqueness; the number of B chains in the sample are too numerous to be accounted for solely by the chains that represent the shoulder. Analysis of the crystallites within the starch samples is helpful to produce an idea of how the molecular parameters affect the crystalline−amorphous lamellae. If the molecular parameters affect the crystalline lamellae specifically, then it would be expected that this would be observed in the crystalline parameters. The difference in enthalpy, a measure of the increase in the ordered structures, whether they are double helices or crystallites, caused by gentle annealing is correlated positively with the degree of branching (essentially the reciprocal of X̅ n), while both of these parameters correlate negatively with the maximum of the distribution of short B chains. The maximum of the short B chains is a measure of the size of the chains in the short B chain region, something which is reinforced by its positive correlation with the X̅ n (negative correlation with degree of branching) of these chains. The reason for the lack of correlation between the X̅ n of short B chains and degree of branching is not obvious, but may be due to the overlap of the short B chain and A chain regions. The correlation between the maximum of the short B chains in the weight distribution and degree of branching implies that the

were drastically altered with the inclusion of the B-type potato starch Eliane. Presumably, this is because the B-type crystallites are packed in a different manner than those of the A-type starches, and thus the relationship between the molecules and aggregated structures may be different. For this reason, the 9 starches EH-6, F97 2-1, Kiaryo-Mochi, Mazaca, Pulut Siding, Shizuki-Mochi, W64A, Waxy Sorghum, and YRW4 were used in the statistical analysis. The statistical results have been separated into the crystalline results correlating with the molecular results from the previous paper in Table 3 and the crystalline parameters correlating with βDA parameters results in Table 4. Although all parameters described in the experimental section were considered, the tables only include those parameters which displayed correlations. As noted in the previous paper, the average repeat distance as well as the HWHM (half width at half-maximum, the distribution of sizes of crystalline−amorphous lamellae) are linked by the observation that the increase in the average repeat distance is caused by increases in the number of larger crystalline−amorphous lamellae produced; therefore both parameters are simply referred to as lamellar size.7 A simple model, that of Blazek et al.,32 that requires few assumptions was used to produce information on the lamellar size of the waxy starches as the more complex methods based on paracrystalline structure produced33,34 results with significant uncertainty. Lamellar size correlates negatively with the proportion of A chains and correlates positively with the proportion of B chains. That is, when there are more chains which are interior to a branch point (interior chains) compared to those that are exterior to a branch point (exterior chains), the size of the lamellae of starch will increase. The proportion of A chains and B chains is also related to the parameter Xmin2, which largely controls the length of the shoulder region of the native debranched number distribution (see Figure 7 for a graphical representation of the CLD parametrizations used), the B chains correlating positively, and the A chains correlating negatively. This allows the following interpretation: the greater the proportion of B chains present, the longer the shoulder region of the native CLD and the larger the lamellae. The average DP of the short B chains correlates positively with the proportion of A chains and negatively with the proportion of B chains, indicating that, as there is an increase in the size of the interior chains, it is likely that there will be fewer B chains. The average DP of the short B chains correlates negatively with the size of G


−0.78 (0.01) 0.88 (0.0)

native TD

−0.75 (0.02)

−0.75 (0.02) −0.85 (0.0) −0.72 (0.03)

0.66 (0.05)

0.91 (0.0)

native ΔH

−0.66 (0.05)

difference TM

0.79 (0.01)

H

Native TM Native ΔH Native TD Difference TM Difference ΔH Debranched βDA X̅ n Proportion of A chains Proportion of long B chains Deb Short B-Chain max Whole βDA Avg Native double helices

−0.78 (0.01)

difference ΔH

debranched βDA X̅ n

0.86 (0.0)

−0.87 (0.0) −0.98 (0.0)

proportion of A proportion of short chains B chains

proportion of A chains

−0.78 (0.01)

−0.7 (0.03)

0.84 (0.0)

−0.76 (0.02) 0.7 (0.04) 0.68 (0.04)

0.76 (0.02)

−0.82 (0.01)

0.77 (0.02)

0.76 (0.02)

−0.86 (0.0) 0.78 (0.01)

0.76 (0.02)

annealed double helices

0.82 (0.01)

native double annealed helices double helices

difference double helices

deb long B-chain max

native double helices

−0.95 (0.0)

deb short B-chain max

whole βDA max

0.78 (0.01)

proportion of long B chains

deb long B-chain max

0.8 (0.01)

deb short Bchain max

−0.77 (0.02) −0.81 (0.01)

−0.72 (0.03)

proportion of Short B chains

−0.81 (0.01) 0.88 (0.0) −0.74 (0.02) 0.83 (0.01)

proportion of long B chains

difference ΔH

Table 4. Correlations between βDA Parameters and Crystallite Parameters

Avg. repeat distance HWHM Proportion of crystallinity DP 6−12 DP 13−24 DP 37+ Degree of Branching β2 Xmin1 Xmin2 Proportion of Amylose

native TM

debranched βDA X̅ n

Table 3. Correlations between CLD, Crystalline−Amorphous Lamella and βDA Parameters, Crystallite Parameters

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Figure 7. Example of the parametrizations obtained in previous paper and used in this one. The red refers to the common Hanashiro empirical parametrization47 while the green and black refer to the Wu-Gilbert biosynthesis-based parametrization.5

the annealing was attributed to an increase in the electron density difference of the crystalline and amorphous lamellae, rather than a change in the size of the lamellae.42,43 The peak temperatures and onset temperatures correlate positively with one another in both the native and annealed samples, indicating no difference between the distributions of the melting starches. The value of TD is more appropriate to describe the distribution of melting temperatures, and so the onset temperature will no longer be referred to. The native thermal and double helical parameters correlated positively with all of the corresponding annealed parameters; for example, the native enthalpy correlates positively with annealed enthalpy, indicating that the property most relevant to the final properties of the annealed starches is the initial properties of the native starches. DP 13−24 chains were positively correlated with native and annealed TM while DP 6−12 chains correlated negatively with TM. This is similar to the result seen by Koroteeva et al., who found the same correlation between the melting temperature and the long and short chains.24 Additionally Koroteeva et al. found a correlation between the short- and medium-length chains with the total enthalpy of the starch melt, which was not observed in this work; rather a correlation with the longest chains, DP 37+, was observed. The correlation of the DP 37+ chains that was observed is related to the production of crystallites, as noted in the previous work,7 which are directly related to an increase in the amount of order in the starch. The value of Xmin1 correlated negatively with the native TD, indicating that starches with a greater range of chain lengths involved in the formation of the maxima seen in the starch CLD display a narrower distribution of melting temperatures. It has been noted that a polydipserse set of linear chains leads to irregular crystallite formation,44 and that smaller chains can cocrystallize in the presence of longer chains.45 The increase in the range of sizes taken by these maxima chains may then produce more irregular crystals, resulting in a broader range of melting enthalpies. However, the small amount of variation of Xmin1, DP 8 or 9 in all samples, means that these results must be treated with care. The proportion of long B chains is somewhat troublesome, as they are found only in some of the starches; when using all of the starches in the correlation, the long B chains were found to

interior of the amylopectin is as significant a cause of the degree of branching as the exterior. That is, the proportion of A chains does not correlate with the degree of branching, showing that it is not just an increase in the number of small A chain branches that are added to the exterior of the molecule that drives high degrees of branching. Any effect of the branch distribution on the crystalline defects is unlikely to occur in the crystallites, as the branches are unable to aggregate into double helices. It seems more likely either that the branches prevent the alignment of double helices into the most well aligned crystallites or that the CLD leads to the production of more dangling chains which extend beyond the native double helix. The nature of this rearrangement to produce a higher enthalpy can be tested by the 13C CP/MAS NMR experiments; if the difference in the proportion of double helices before and after annealing is related to the difference in enthalpy then this is strong evidence for the production of longer double helices rather than the realignment of the double helices. There was, however, no such correlation observed between the difference in enthalpy and the difference in double helices. The errors seen in the production of the annealed NMR results (Table 1) are of a similar size for five of the starches; the other six had observable differences in double helices before and after annealing. Hence, the evidence points to most, if not all, of the crystalline defects being due to poorly aligned double helices; however, more accurate experiments on a much larger sample size may prove this incorrect. It is not clear whether the misalignment of the double helices is a function of the amount of branching or if the amount of branching allows this misalignment to be more easily removed. However, if there are fewer of the larger short B chains in the interior of the molecule, it would suggest that there is less flexibility afforded to the helices, and this may lead to the production of more misalignment during biosynthesis. A similar observation by Vamadevan et al.40,41 noted that the parameter IB-CL (“interblock chain length”, a measure of the distance between branch points) correlated positively with enthalpy change after annealing. They suggest either a lengthening of double helices or increasing alignment of helices to explain this; the current results support the realignment hypothesis. The lack of correlation between the crystalline parameters of annealed starches and changes in the size of the crystalline− amorphous lamellae has been observed previously. The effect of I


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correlate positively with the parameter β2 and negatively with the native TD. However, when a statistical analysis is only performed on the 6 samples which display the long B chains, there is instead a positive correlation with β2 and a negative correlation with β1; a visual inspection of the correlation confirms that those starches without the long B chains are clearly not in a grouping with those that have the long B chains. The 7 starches with long B-chains coincide with those starches which contain the greatest amount of amylose, found in Table SI-1 of the Supporting Information. The largest number of long B-chains is present in the barley starch Waxiro, which contains approximately 10% amylose, so their source seems likely to be the amylose found in these starches. Due to the bimodal nature of the correlations when considering all of the starches, and the small sample size when considering the starches which contain long B-chains, an in depth discussion is not presented here. The plots relating to the correlations of only starches which contain long B-chains can be found in the SI. A brief discussion of both sample groups is also provided in the Supporting Information. The value of TD correlates negatively with the proportion of amylose. Hence as the proportion of amylose increases, the distribution of temperatures at which the crystallites melt becomes narrower. This correlation between amylose and melting temperature has been observed in nonwaxy starches a number of times.19,46 The difference in the specifics of the results may be due to the larger differences in the proportion of amylose, approximately 1.5−40% in the previous work, and 0.3−2.4% amylose content in the current work. The whole βDAs maximum Rh correlates positively with the average DP of the short B chains: as the chains in the interior of the amylopectin increase in size, so does the total size of the βDA. An amylopectin that contains a greater number of chains than another amylopectin molecule, but had an otherwise identical branching structure, would result in a larger βDA. Therefore, the correlation implies that the number of internal chains between the different βDAs, and by extension, the different whole amylopectin, is similar. The whole βDA average Rh is correlated negatively with the proportion of double helices before and after annealing. It is not entirely clear why the whole size of the interior βDA would be related to the proportion of double helices; a speculative explanation is that the larger average size of the interior chains results in a smaller average chain length of the exterior chains which are capable of forming the double helices.

dependence of the A or B chains on any particular chain length in the CLD, except the shoulder region. It is speculated that the size of the whole β-limit amylopectin is related via a similarity in the number of internal chains to the average size of the interior B chains. It is also speculated that the relationship between the X̅ n of the interior B chains and the degree of branching indicates that the main determinant of branching is not related solely to the addition of A chains, but also to the production of more B chains; this may have some relevance to ideas about the biosynthesis of the branching structure.

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ASSOCIATED CONTENT

S Supporting Information *

Tables that contain values for the single helical and long B chain parameters as well as figures that compare the proportion of long B chains with both β1 and β2. Additionally, the correlations between β1, β2 and Xmin2 and the long B chain parameters are presented. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Fax +61 7 3365 1188. Telephone +61 7 3365 4809. E-mail address: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the Australian Institute of Nuclear Science and Engineering for a PGRA grant (T.W.). The authors would also like to thank CSIRO Plant Industry, DPI Yanco Agricultural Institute, DEEDI, and MARDI for providing samples.

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REFERENCES

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CONCLUSIONS The CLD has previously been shown to affect the structure of the crystalline−amorphous lamellae of native starch. This study has shown that there is a link between the CLD of amylopectin and the structure of the whole amylopectin molecule, and these factors together influence the size of the crystalline−amorphous lamellae. Specifically, the shoulder region of the CLD represents a large number of B chains which determine the interior structure of the amylopectin; increases in the number of these chains causes an increase in the size of the C−A lamellae by increasing the size of the amorphous region of the lamellae. The size of these interior chains was also found to be relevant to the amount of crystalline defects present in the crystalline structure, which are probably caused by the shorter interior chains producing a more rigid structure, preventing the double helices from aligning perfectly. The study also shows that the common belief that A chains are represented by the DP 6−12 chains is questionable, and there is no significant J


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