Origin of defects in assembled supramolecular structures of sweet potato starches with different amylopectin chain-length distribution

A combined approach of fluorophore-assisted capillary electrophoresis (FACEL), high-sensitivity differential scanning calorimetry (DSC), wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), and light (LM) and scanning electron microscopy (SEM) was applied to study the effects of changes in amylopectin chain-length distribution on the assembly structures of sweet potato starches with similar amylose levels. It was shown that unlike ordinary sweet potato starch, starch extracted from Quick Sweet cultivar of sweet potato had anomalous high level of amylopectin chains with a degree of polymerization (DP) 6–12. Joint analysis of the obtained data revealed that amylopectin chains with DP 10–24 are, apparently, the dominant material for the formation of supramolecular structures in starch granules. In contrast, amylopectin chains with DP < 10 facilitated the formation of defects within crystalline lamellae. An increase in relative content of amylopectin chains with DP < 10 is accompanied by the correlated structural alterations manifested at all levels of starch granule organization (crystalline lamellae, amylopectin clusters, semi-crystalline growth rings, and granule morphology). Thus, the short amylopectin chains with DP < 10 were considered as an origin of the defectiveness in starch supramolecular structures.     

Structural and thermodynamic properties of rice starches with different genetic background Part 1. Differentiation of amylopectin and amylose defects

A combined DSC - HPAEC-PAD approach, gel permeation chromatography and mild long-term acidic hydrolysis were employed to study the effects of amylopectin chain-length distributional and amylose defects on the assembly structures of amylopectin (crystalline lamellae, amylopectin clusters) in A-type polymorphic starches extracted from 11 Thai cultivars of rice with different amylose level. Joint analysis of the data allowed determining the contributions of different populations of amylopectin chains to the thermodynamic melting parameters of crystalline lamellae. It was shown that amylopectin chains with DP 6-12 and 25or=37 could be related to chains stabilizing these structures. The total effect of amylose and amylopectin defects can be described by means of Thomson-Gibbs' equation. The increase of defects in the assembly structures is accompanied by rise of the rates of acidic hydrolysis of both amorphous and crystalline parts in starches.     

Structural and thermodynamic properties of starches extracted from GBSS and GWD suppressed potato lines

A combined DSC–SAXS approach was employed to study the effects of amylose and phosphate esters on the assembly structures of amylopectin in B-type polymorphic potato tuber starches. Amylose and phosphate levels in the starches were specifically engineered by antisense suppression of the granule bound starch synthase (GBSS) and the glucan water dikinase (GWD), respectively. Joint analysis of the SAXS and DSC data for the engineered starches revealed that the sizes of amylopectin clusters, thickness of crystalline lamellae and the polymorphous structure type remained unchanged. However, differences were found in the structural organization of amylopectin clusters reflected in localization of amylose within these supramolecular structures. Additionally, data for annealed starches shows that investigated potato starches possess different types of amylopectin defects. The relationship between structure of investigated potato starches and their thermodynamic properties was recognized.     

Annealing of normal and mutant wheat starches. LM, SEM, DSC, and SAXS studies

Structure and thermodynamic properties of native and annealed wheat starches with different amylose content (from 1.5% to 39.5%) have been studied by high-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray diffraction (SAXS), light (LM), and scanning electron microscopy (SEM). Starch morphology, the values of the melting cooperative unit, the thickness of crystalline lamellae and the size of amylopectin clusters as well as thermodynamic parameters characterizing surface of the face side in starch crystals were determined. Some suppositions based on different physical approaches are used for a discussion of the results concerning structural reorganization of starches on different levels of macromolecular organization.     

Structural parameters of amylopectin clusters and semi-crystalline growth rings in wheat starches with different amylose content

Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) were used to investigate the internal structure of wheat starch granules with different amylose content. Different approaches were used for treatment (interpretation) of SAXS data to assess the values of structural parameters of amylopectin clusters and the size of crystalline and amorphous lamella in different wheat starches. The average values of the semi-crystalline growth rings thickness in starches have been determined and the relationship between structural characteristics and thermodynamic melting parameters is discussed.     

Structural characterization of wheat starch granules differing in amylose content and functional characteristics

Small-angle X-ray scattering (SAXS) together with several complementary techniques, such as differential scanning calorimetry and X-ray diffraction, have been employed to investigate the structural features that give diverse functional properties to wheat starches (Triticum aestivum L.) within a narrow range of enriched amylose content (36–43%). For these starches, which come from a heterogeneous genetic background, SAXS analysis of duplicate samples enabled structural information to be obtained about their lamellar architecture where differences in lamellar spacing among samples were only several tenths of nanometer. The SAXS analysis of these wheat starches with increased amylose content has shown that amylose accumulates in both crystalline and amorphous parts of the lamella. Using waxy starch as a distinctive comparison with the other samples confirmed a general trend of increasing amylose content being linked with the accumulation of defects within crystalline lamellae. We conclude that amylose content directly influences the architecture of semi-crystalline lamellae, whereas thermodynamic and functional properties are brought about by the interplay of amylose content and amylopectin architecture.     

Amylopectin molecular structure reflected in macromolecular organization of granular starch

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.     

Use of (13)c MAS NMR to study domain structure and dynamics of polysaccharides in the native starch granules

To investigate the domain structure and dynamics of polysaccharides in the native starch granules, a variety of high resolution, solid-state (13)C NMR techniques have been applied to all three (A-, B-, and C-) types of starch with different water content. Both single-pulse-excitation magic-angle-spinning (SPEMAS) and cross-polarization-magic-angle-spinning (CPMAS) methods have been employed together with the PRISE (proton relaxation induced spectral-editing) techniques to distinguish polysaccharide fractions in different domains and having distinct dynamics. It has been found that, for all three types of dry starch granules, there are two sets of NMR signals corresponding to two distinct ordered polysaccharides. Hydration leads to substantial mobilization of the polysaccharides in the amorphous regions, but no fundamental changes in the rigidity of the polysaccharides in the crystalline (double) helices. Full hydration also leads to limited mobility changes to the polysaccharides in the amorphous lamellae (branching zone) within the amylopectin clusters and in the gaps between the arrays of the amylopectin clusters. Under magic-angle spinning, proton relaxation-time measurements showed a single component for T(1), two components for T(1rho), and three components for T(2). PRISE experiments permitted the neat separation of the (13)C resonances of polysaccharides in the crystalline lamellae from those in the amorphous lamellae and the amylose in the gaps between amylopectin clusters. It has been found that the long (1)H T(1rho) component ( approximately 30 ms) is associated with polysaccharides in the crystalline lamellae in the form of double helices, whereas the short T(1rho) component (2-4 ms) is associated with amylose in the gaps between amylopectin clusters. The short (1)H T(2) component ( approximately 14 micros) is associated with polysaccharides in the crystalline lamellae; the intermediate component (300-400 micros) is associated with polysaccharides in the amorphous lamellae and amylose in the gaps between amylopectin clusters. The long T(2) component is associated with both mobile starch protons and the residue water protons.     

Distribution of methyl substituents in amylose and amylopectin from methylated potato starches

Granular potato starches were methylated in aqueous suspension with dimethyl sulfate to molar substitution (MS) values up to 0.29. Fractions containing mainly amylose or amylopectin were obtained after aqueous leaching of the derivatised starch granules. Amylopectin in these fractions was precipitated with Concanavalin A to separate it from amylose. Amylose remained in solution and was enzymatically converted into D-glucose for quantification, thereby taking into account the decreased digestibility due to the presence of methyl substituents. It was found that the MS of amylose was 1.6-1.9 times higher than that of amylopectin in methylated starch granules. The distributions of methyl substituents in trimers and tetramers, prepared from amylose- or amylopectin-enriched fractions, were determined by FAB mass spectrometry and compared with the outcome of a statistically random distribution. It turned out that substituents in amylopectin were distributed heterogeneously, whereas substitution of amylose was almost random. The results are rationalised on the basis of an organised framework that is built up from amylopectin side chains. The crystalline lamellae are less accessible for substitution than amorphous branching points and amylose.     

Starches from different botanical sources I: Contribution of amylopectin fine structure to thermal properties and enzyme digestibility

Fifteen starches from different botanical sources were selected to study the influence of structural features on thermal properties and enzyme digestibility. Morphological appearance, X-ray diffraction pattern, apparent amylose content, unit-chain length distribution of amylopectin, thermal properties and enzyme digestibility of starch varied with botanical source. It was demonstrated that the distribution of unit-chains of amylopectin significantly correlated with functional properties of the starches. Gelatinization temperature of native and retrograded starches decreased and increased with a relative abundance of unit-chains with an approximate degree of polymerization (DP) of 8–12 and 16–26, respectively (P<0.01). Similar unit-chain lengths also affected the enzyme digestibility of starch granules (P<0.01).     

Change of granular and molecular structures of waxy maize and potato starches after treated in alcohols with or without hydrochloric acid

Starches from waxy maize and potato were treated in methanol and 2-propanol either with or without 0.36% hydrochloric acid at 65 °C for 1 h. The granule morphology, molecular structure and pasting properties of the starches were determined and the effects of treatments on the granule and molecular structures of starch were investigated. Starch treated in alcohols without acid showed loss of native order through the hilum of granules, and no obvious molecular degradation was found. However, acid–alcohol treated starch showed many cracks inside granules, and both waxy maize and potato starches showed obvious molecular degradation after treated. Furthermore, the amylose chains and long chains of amylopectin of starch were more easily degraded with acid–alcohol treatment. The pasting viscosity of acid–alcohol treated starches were also obviously less than that of their counterpart native starch and starch after alcohol treatment. The extent of degradation of molecules and the decrease of pasting viscosity on potato starch after acid–alcohol treated were more obvious than that of waxy maize starch. The result indicates that the degradation preferentially occur in the amorphous region when starch treated by acid–alcohol, and the degradation of starch molecules enhances the amorphous excretion and the occurrence of cracks inside the granules.     

A comparative study of edible canna (Canna edulis) starch from different cultivars. Part II. Molecular structure of amylose and amylopectin

Molecular structures of starches isolated from Japanese-green, Thai-green and Thai-purple cultivars of edible canna (Canna edulis Ker) were investigated. The absolute amylose content ranged from 19 to 25%. Degrees of polymerization (DPn) values of amylose determined by fluorescence-labeling method were 1590 for Thai-purple, 1620 for Japanese-green and 1650 for Thai-green cultivars. Mole% of branched fraction of amyloses from edible canna starches examined by a HPLC system after β-amylolysis of labeled amyloses was 13–16%. Branch chain-length distributions of amylopectin analyzed by HPSEC after debranching with isoamylase, followed by fluorescence-labeling of unit chain, showed bimodal distribution with the DPn range of 25–28. The amylopectin of edible canna starches contained high amounts of organic phosphorus (391–420 ppm). The distribution profile of phosphorylated chains, separated from non-phosphorylated chains by DEAE-Sephadex A-50 chromatography, indicated that the phosphate groups were located mostly in long B-chains of amylopectin molecules.     

Locations of hypochlorite oxidation in corn starches varying in amylose content

The general oxidation mechanism by hypochlorite on starch has been well studied, but the information on the distribution of the oxidation sites within starch granules is limited. This study investigated the locations where the oxidation occurred within corn starch granules varying in amylose content, including waxy corn starch (WC), common corn starch (CC), and 50% and 70% high-amylose corn starch (AMC). Oxidized corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The surface-gelatinized remaining granules were separated and studied for structural characteristics including carboxyl content, amylose content, amylopectin chain-length distribution, thermal properties, and swelling properties. Oxidation occurred mostly at the amorphous lamellae. More carboxyl groups were found at the periphery than at the core of starch granules, which was more pronounced in oxidized 70% AMC. More amylose depolymerization from oxidation occurred at the periphery of CC. For WC and CC, amylopectin long chains (>DP 36) were more prone to depolymerization by oxidation. The gelatinization properties as measured by differential scanning calorimetry also supported the changes in amylopectin fine structure from oxidation. Oxidized starches swelled to a greater extent than their unmodified counterparts at all levels of surface removal. This study demonstrates that the locations of oxidation and physicochemical properties of oxidized starches are affected by the molecular arrangement within starch granules.     

Variation in crystalline type with amylose content in maize starch granules: an X-ray powder diffraction study

Comparative studies of native maize starches with different amylose contents were carried out using X-ray powder diffraction. The results show a transition of crystalline type from A through C to B, accompanying a decrease in degree of crystallinity from 41.8% to 17.2% across a range of apparent amylose content from 0% to 84%. Hydration induces an increase in degree of granule crystallinity, but does not change the transition of crystal type. Progressively from A-type to C-type, crystallinity decreases rapidly with an increase in amylose content. From C-type to B-type, overall crystallinity decreases more slowly. The crystal type is strongly dependent on amylose content and on average chain length of the respective amylopectin. Waxy A-types have an average chain length of about 20, while in high amylose B-types this rises to ≈35. The proportion of short chains (10–13 glucose units) appears to affect crystal type significantly. Some V-type material was detected at high amylose levels. The proportion of this increased after prolonged exposure of the granules to iodine vapour. Implications for the arrangement of starch components in the granule are discussed.     

New insights on the mechanism of acid degradation of pea starch

The degradation of pea starch granules by acid hydrolysis has been investigated using a range of chemical and structural methods, namely through measuring changes in amylose content by both the iodine binding and concanavalin A precipitation methods, along with small angle X-ray scattering (SAXS), wide angle X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM). The relative crystallinity, intensity of the lamellar peak and the low-q scattering increased during the initial stages of acid hydrolysis, indicating early degradation of the amorphous regions (growth rings and lamellae). In the first 2 days of hydrolysis, there was a rapid decline in amylose content, a concomitant loss of precipitability of amylopectin by concanavalin A, and damage to the surface and internal granular structures was evident. These observations are consistent with both amylose and amylopectin being located on the surface of the granules and attacked simultaneously in the early stages of acid hydrolysis. The results are also consistent with amylose being more concentrated at the core of the granules. More extensive hydrolysis resulted in the simultaneous disruption of amorphous and crystalline regions, which was indicated by a decrease in lamellar peak intensity, decrease in interhelix peak intensity and no further increase in crystallinity. These results provide new insights into the organization of starch granules.     

Structural basis for the slow digestion property of native cereal starches

Native cereal starches are ideal slowly digestible starches (SDS), and the structural basis for their slow digestion property was investigated. The shape, size, surface pores and channels, and degree of crystallinity of starch granules were not related to the proportion of SDS, while semicrystalline structure was critical to the slow digestion property as evidenced by loss of SDS after cooking. The high proportion of SDS in cereal starches, as compared to potato starch, was related to their A-type crystalline structure with a lower degree of perfection as indicated by a higher amount of shortest A chains with a degree of polymerization (DP) of 5-10. The A-type amorphous lamellae, an important component of crystalline regions of native cereal starches, also affect the amount of SDS as shown by a reduction of SDS in lintnerized maize starches. These observations demonstrate that the supramolecular A-type crystalline structure, including the distribution and perfection of crystalline regions (both crystalline and amorphous lamellae), determines the slow digestion property of native cereal starches.     

Hydrothermal treatment and iodine binding provide insights into the organization of glucan chains within the semi‐crystalline lamellae of corn starch granules

The importance of glucan chains that pass through both the amorphous and crystalline lamellae (tie chains) in the organization of corn starch granules was studied using heat‐moisture treatment (HMT), annealing (ANN), and iodine binding. Molecular structural analysis showed that hylon starches (HV, HVII, and HVIII) contained higher proportion of intermediate glucan chains (HVIII > HVII > HV) than normal corn (CN) starch. Wide angle X‐ray scattering revealed that on HMT, the extent of polymorphic transition in hylon starches decreased with increasing proportion of intermediate and long chains. Iodine treated hylon starches exhibited increased order in the V‐type polymorphism as evidenced by the intense peak at 20° 2θ and the strong reflection intensity at 7.5° 2θ and the extent of the change depended on the type of hylon starch. DSC results showed that the gelatinization enthalpy of CN and waxy corn starch (CW) remained unchanged after ANN. However, hylon starches showed a significant increase in enthalpy with more distinct endotherms after ANN. It can be concluded that tie chains influence the organization of crystalline lamellae in amylose extender mutant starches. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 871–885, 2014.     

Characterization of barley starches of waxy, normal, and high amylose varieties

Four varieties of barley starches, W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier, were isolated from barley seeds. Apparent and absolute amylose contents, molecular size distributions of amylose and amylopectin, amylopectin branch-chain-length distributions, and Naegeli dextrin structures of the starches were analyzed. W.B. Merlin amylopectin had the longest detectable chain length of DP 67, whereas glacier, high amylose glacier and high amylose hull-less glacier amylopectins had the longest detectable chain length of DP 82, 79, and 78, respectively. All the four starches displayed a substantially reduced proportion of chains at DP 18–21. Amylopectins of high amylose varieties did not show significantly larger proportions of long chains than that of normal and waxy barley starch. Onset gelatinization temperatures of all four barley starches ranged from 55.0 to 56.5°C. Absolute amylose contents of W.B. Merlin, glacier, high amylose glacier, and high amylose hull-less glacier were 9.1, 29.5, 44.7, and 43.4%, respectively; phospholipid contents were 0.36, 0.78, 0.79, and 0.97%, respectively.     

A photographic approach to the possible mechanism of retrogradation of sweet potato starch

Although the subject of starch retrogradation has been studied for about 20 years, the mechanism of starch retrogradation seems not yet to be completely established. In this paper, the possible retrogradation mechanism of sweet potato starch was postulated from four optical micrographs at the stages of melting of the starch granules, autoclaving treatment and aging. The possible process of retrogradation consists of three stages. Firstly, starch granules was swelled and melted with loss of X-ray crystallinity and formation of both crystalline and amorphous lamellae; secondly, in crystalline lamellae, amylopectin began to form nucleation when they were autoclaved; finally, the nucleus grew up to great rod-like crystals as the result of congregating of amylose on plates which were composed of and prolongated by amylopectin.     

Slow digestion property of native cereal starches

The slow digestion property of native cereal starches, represented by normal maize starch, was investigated. The in vitro Englyst test showed that 53.0% of the maize starch is slowly digestible starch (SDS), and scanning electron microscopy (SEM) revealed that SDS starts from an increase of pore size until almost complete fragmentation of starch granules. However, similar amounts of SDS ( approximately 50%) were shown for partially digested fragmented starch residuals, which would normally be considered resistant to digestion based on the Englyst assay. Molecularly, both amylopectin (AP) and amylose (AM) contributed to the amount of SDS as evidenced by a similar ratio of AP to AM at different digestion times. Consistently, similar degrees of crystallinity, comparable gelatinization behavior, and similar debranched profiles of starch residuals following different digestion times indicated that the crystalline and amorphous regions of starch granules were evenly digested through a mechanism of side-by-side digestion of concentric layers of semicrystalline shells of native starch granules.