Molecular arrangement in blocklets and starch granule architecture

The main hypotheses and the data regarding the starch granule structure and behaviour were gathered and considered comprehensively in this paper. The starch molecules such as amylopectin, amylose and intermediate materials, the non-starch molecules such as bound phosphates and lipids, and the crystal dimensions etc. their roles were demonstrated in the architectures of blocklet and granular ultrastructure. A normal blocklet is mainly constructed by the crystalline and amorphous lamellaes that are formed with the clusters of amylopectin molecule(s). The reducing terminal of the amylopectins in the blocklets may be toward an equal course. However, the defective blocklet production may be due to the participation of lower branching molecules such as amylose and intermediate materials. From the viewpoint of the physicochemical properties of the starch granules, the blocklet of two types may be arranged into the two formations, heterogeneous shell and homogenous shell. The amylopectin plays main role in blocklet architecture, while the other component is important in contributing to the strength and flexibility of starch granule.     

Atomic force microscopy of pea starch granules: granule architecture of wild-type parent, r and rb single mutants, and the rrb double mutant

AFM studies have been made of the internal structure of pea starch granules. The data obtained provides support for the blocklet model of starch granule structure (Carbohydr. Polym. 32 (1997) 177-191). The granules consist of hard blocklets dispersed in a softer matrix material. High-resolution images have yielded new insights into the detailed structure of growth rings within the granules. The blocklet structure is continuous throughout the granule and the growth rings originate from localised defects in blocklet production distributed around the surface of spheroidal shells within the granules. A mutation at the rb locus did not lead to significant changes in granule architecture. However, a mutation at the r locus led to loss of growth rings and changed blocklet structure. For this mutant the blocklets were distributed within a harder matrix material. This novel composite arrangement was used to explain why the granules had internal fissures and also changes in gelatinisation behaviour. It is suggested that the matrix material is the amylose component of the granule and that both amylose and amylopectin are present within the r mutant starch granules in a partially-crystalline form. Intermediate changes in granule architecture have been observed for the double mutant rrb.     

Potato starch granule nanostructure studied by high resolution non-contact AFM

Surface studies at ambient conditions of potato starch granules subjected to multiple freezing and thawing, performed by a high resolution non-contact atomic force microscopy (nc-AFM), revealed some details of the starch granule nanostructure. After the treatment, a significant separation and a chain-like organisation of the granule surface elements have been observed. An accurate analysis of the granule surface nanostructure with a single amylopectine cluster resolution could be carried out. The oblong nodules of approximately 20-50 nm in diameter have been observed at the surface of the potato starch granules. The same size particles were precipitated by ethanol from gelatinized potato starch suspensions. They were also detected at the surface of oat and wheat starch granules. After multiple freezing and thawing, the eroded potato granule surface revealed a lamellar structure of its interior. The 30-40 nm inter-lamellar distances were estimated by means of nc-AFM. These findings fit previously proposed dimensions of the structural elements in the crystalline region of the starch granule. The observed surface sub-particles might correspond to the single amylopectine side chain clusters bundled into larger blocklets packed in the lamellae within the starch granule. The results supported the blocklet model of the starch granule structure.     

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 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.     

Structural processes during starch granule hydration by synchrotron radiation microdiffraction

Starch granule hydration has been examined on the level of a single potato starch granule by static and dynamic synchrotron radiation (SR) microdiffraction techniques. A cryofrozen, hydrated granule was mapped through a 5 microm SR-beam in order to investigate its internal organization. The edge of the granule showed fiber texture scattering due to radially oriented amylopectin helices. The variation of fiber texture across the granule center supports the model of concentric shells. The crystalline phase appears, however, to increase strongly toward the granule center due to a random amylopectin fraction, which could be related to crystallization of short-range ordered amylopectin during hydration. During gelatinization, the shell structure breaks down and remaining fiber-textured amylopectin domains belong probably to the swollen starch granule envelope. Hydration of a granule was initiated by a microdrop generator and followed in situ by SR-microdiffraction. A fast hydration process with a half time of about 7 s seems to reflect the porous nature of starch granules. The size of the hydrated domains suggests that this process is limited to the level of amylopectin side chain clusters. Longer hydration times are assumed to involve remaining short-range ordered amylopectin and results in larger domains.     

Retrograded starches as potential anodes in lithium-ion rechargeable batteries

Retrograded starch is a crystal formed by starch molecules with hydrogen bonds. Many literatures have reported its physicochemical character, but its crystal structure is so far unclear. As we isolate amylose and amylopectin from retrograded maize, sweet potato and potato starches in 4.0M KOH solutions and make them retrograde alone in neutral solution (adjusted by HCl) to form crystal, a new phenomenon appears, crystals of KCl do not appear in retrograded potato amylose, potato amylopectin, and maize amylose, indicating that those crystals may absorb K(+) and (or) Cl(-), and those ions probably act with aldehyde of starch or hydroxy of fatty acid attached in starch, such characteristic may make retrograded starches replace graphite as anode with high-capacity in lithium-ion rechargeable batteries.     

Kinetic analysis of glucoamylase-catalyzed hydrolysis of starch granules from various botanical sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k(0)) were determined to be 23.3+/-4.4, 14.8+/-6.0, 6.2+/-1.8, 7.1+/-4.1, 4.6+/-3.0, and 1.6+/-0.6 s(-1) for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k(0) was largely influenced by the type of starch granules. A comparison of the k(0)-values in relation to the crystalline structure of the starch granules suggested that k(0) increases as the crystalline structure becomes dense.     

Internal structure of the starch granule revealed by AFM

Atomic force microscopy images of sectioned native corn starch granules show evidence of the well-known radial organisation of the starch macromolecules, with the less-ordered hilum region near to the centre. Native granules show blocks 400-500 nm in size that span the growth rings. Lintnerised starch granules, where a mild acid hydrolysis has been used to remove the amorphous and less crystalline parts of the granule, clearly show smaller 'blocklets' within the rings approximately 10-30 nm in size. This level of organisation within the growth rings corresponds to the blocklet or superhelix structures that have been proposed in the literature for the association or clustering of amylopectin helices. Mechanical property imaging techniques have provided enhanced contrast to view this morphology, and shown the deformability of the starch structure under contact mode imaging conditions.     

Gel formation in mixtures of high amylopectin potato starch and potato starch

The effects of starch granules on the rheological behaviour of gels of native potato and high amylopectin potato (HAPP) starches have been studied with small deformation oscillatory rheometry. The influence of granule remnants on the rheological properties of samples treated at 90 °C was evident when compared with samples treated at 140 °C, where no granule remnants were found. The presence of amylose in native potato starch gave to stronger network formation since potato starch gave higher moduli values than HAPP, after both 90 and 140 °C treatments. In addition, amylose may have strengthened the network of HAPP because higher moduli values were obtained when native potato starch was added to the system. The moduli values of the mixtures also increased with increasing polysaccharide concentration in the system, which is due to an increment in the polysaccharide chain contacts and entanglements. Finally, it was found that a mixture of commercial amylose from potato starch and HAPP resulted in lower values of G′ compared to native potato starch. This indicates that the source of amylose is important for the properties in a blend with native amylopectin.     

Kinetic Analysis of Glucoamylase-Catalyzed Hydrolysis of Starch Granules from Various Botanical Sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k ₀) were determined to be 23.3±4.4, 14.8±6.0, 6.2±1.8, 7.1±4.1, 4.6±3.0, and 1.6±0.6 s^?1 for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k ₀ was largely influenced by the type of starch granules. A comparison of the k ₀-values in relation to the crystalline structure of the starch granules suggested that k ₀ increases as the crystalline structure becomes dense.     

Microscopy of starch: evidence of a new level of granule organization

Considerable information on starch granule structure may be gathered from a review of published data. Evidence from a range of different (predominantly microscopic) techniques is compared and discussed, allowing the presence of a level of starch granule organization between that of the amylopectin lamellae and the large ‘growth rings’ to be deduced. This structural level of the granule involves the organization of the amylopectin lamellae into effectively spherical ‘blocklets’ which range in diameter from 20 to 500 nm depending on starch botanical type and their location in the granule. The presence of short, radial ‘channels’ of amorphous material within starch granules from some starch varieties is confirmed. The organization and structure of the crystalline and amorphous amylopectin lamellae is also discussed. Consideration of the information regarding starch granule structure and organization to date has significant implications on the internal architecture of the starch granule, and it is evident that the presence of the blockets and amorphous channels play a role in both the resistance of starch to enzymic attack and the structure of the semi-crystalline shells.     

Fractionation of a commercial cellulase preparation from Penicillium funiculosum and its use in the purification of a water soluble α-glucan from milled barley

A commercial enzyme preparation from a selected strain of Penicillium funiculosum has been partially purified using a single stage chromatofocusing fractionation to produce an amylase-free mixture of hydrolytic enzymes. This mixture has been used to remove the non-starch polysaccharides from aqueous extracts of laboratory milled barley. The structure of the resulting purified α-glucan fraction has been examined by gel filtration before and after enzymic debranching and by iodine staining. The mild damage caused to the starch within the barley kernel releases a low molecular weight amylopectin molecule with no detectable amylose in the product. In this respect the product is different from that produced after severe, direct grinding of the purified barley starch where some amylose appears in the water soluble extract. Although the small amount of α-glucan is not of any quantitative industrial significance in itself, it does reflect the extent and type of physical damage which is taking place in the starch granule. The model proposed to explain these results - a starch granule with a solid amylose/amylopectin core but with a number of patches of protruding amylopectin clusters - may have important implications in an industrial context.     

Atomic force microscopy of pea starch: origins of image contrast

Atomic force microscopy (AFM) has been used to image the internal structure of pea starch granules. Starch granules were encased in a nonpenetrating matrix of rapid-set Araldite. Images were obtained of the internal structure of starch exposed by cutting the face of the block and of starch in sections collected on water. These images have been obtained without staining, or either chemical or enzymatic treatment of the granule. It has been demonstrated that contrast in the AFM images is due to localized absorption of water within specific regions of the exposed fragments of the starch granules. These regions swell, becoming "softer" and higher than surrounding regions. The images obtained confirm the "blocklet model" of starch granule architecture. By using topographic, error signal and force modulation imaging modes on samples of the wild-type pea starch and the high amylose r near-isogenic mutant, it has been possible to demonstrate differing structures within granules of different origin. These architectural changes provide a basis for explaining the changed appearance and functionality of the r mutant. The growth-ring structure of the granule is suggested to arise from localized "defects" in blocklet distribution within the granule. It is proposed that these defects are partially crystalline regions devoid of amylose.     

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.     

Amylopectin aggregation as a function of starch phosphate content studied by size exclusion chromatography and on-line refractive index and light scattering

Starches with a natural 65-fold span in covalently bound phosphate content were prepared from five different crops including sorghum, cassava, three potato varieties and an exotic ginger plant, Curcuma zedoaria, with extreme starch phosphate content. These starches were subjected to size exclusion chromatography with refractive index detection (SEC/RI). A simple and rapid method for starch solubilisation was used. The conditions during solubilisation (2 M NaOH) and separation (10 mM NaOH, 50°C) were such as enabling >94% recovery of the starch without detectable degradation. The aggregation properties of the starch was investigated using on line refractive index/multi angle laser light scattering (RI/MALLS) detection. Three major regions in the SEC profile were identified, consisting of large amylopectin aggregates, amylopectin particles with radius of gyration (R_g) of approx 200 nm (400 nm blocklets) and amylose. A procedure for correction of light scattering signals spread over the SEC profile as a result of aggregate tailing was developed. The significance of the relative amounts of these three molecular species on standard starch pasting parameters, as measured by a Rapid Visco Analyzer (RVA), was investigated. Starches with a high amount of amylopectin aggregates showed high peak viscosities. Moreover, very high amounts of starch bound phosphate or amylose appears to suppress the content of large aggregates resulting in low viscosity.     

Preparation and characterization of soluble starches having different molecular sizes and composition,by acid hydrolysis in different alcohols

Potato and waxy-maize starches were separately modified for 1 h at 65° with 0.36% hydrochloric acid in methanol, ethanol, 2-propanol, and 1-butanol. All of the modified starches were readily soluble in hot water, to give crystal-clear solutions up to a concentration of at least 20% (w/v). The modified granules were studied by light-microscopy and iodine-iodide staining. All of the modified starches retained their granule appearance, although with various degrees of damage that progressively increased from methanol to 1-butanol. Both hydrolysis and alcoholysis occurred, but to different extents in the different alcohols. The highest proportion of alcoholysis occurred in methanol where 50% of the resulting molecules were glycosides, the lowest in 1-butanol where 6% were glycosides. The number-average molecular weights of the modified starches also progressively decreased from 126,670 for the methanol-modified waxy-maize starch to 4,750 for the 1-butanol-modified potato starch. The methanol- and ethanol-modified potato starches were fractionated into amylose and amylopectin components. The 2-propanol- and 1-butanol-modified potato starches gave only an amylopectin component. The amylose components were characterized by gel-permeation chromatography on Bio-Gel A-5m, and the amylopectin components, on Bio-Gels A-150m and A-0.5m. The molecular sizes of the amylose and amylopectin components progressively decreased from methanol- to 1-butanol-modified starches. Furthermore, the polymodal composition of the amylopectin component was decreased to give a more homogeneous product. Waxy-maize starch was modified in methanol and 2-propanol and gave products that were of lower molecular size and more homogeneous than the polymodal native starch. It is shown that the differential effect of the different alcohols on the modification of the starch granules is produced by effecting different concentrations of acid inside the granule, where hydrolysis occurs in the 10–12% of water contained in the granule. It is postulated that 2-propanol and 1-butanol dissolve the double-helical, crystalline regions in the starch granule to give different types of products under otherwise identical conditions of modification.     

Granule size affects the acetyl substitution on amylopectin populations in potato and sweet potato starches

Specific enzymatic degradation in combination with chromatographic and spectrometric techniques was used to understand acetyl group distribution over the amylopectin populations of differently sized granule fractions from potato and sweet potato starches. The hydrolysates obtained after -amylase, ß-amylase, pullulanase, and the combination of pullulanase, -amylase and amyloglucosidase treatment were investigated by high-performance size-exclusion chromatography (HPSEC), high-performance anion-exchange chromatography (HPAEC) and Maldi-Tof-MS (Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry). The acetyl groups were found to be located near the branching point, in the external chain and in the internal chain regions. The acetyl group distributions were different for amylopectin from different granule size fractions. Higher DP (degree of polymerization) fragments were present in the digests of acetylated amylopectin populations of the small size granule starches. Our studies confirmed that acetyl groups were unevenly distributed over the amylopectin populations.     

Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.     

Production of very-high-amylose potato starch by inhibition of SBE A and B

High-amylose starch is in great demand by the starch industry for its unique functional properties. However, very few high-amylose crop varieties are commercially available. In this paper we describe the generation of very-high-amylose potato starch by genetic modification. We achieved this by simultaneously inhibiting two isoforms of starch branching enzyme to below 1% of the wild-type activities. Starch granule morphology and composition were noticeably altered. Normal, high-molecular-weight amylopectin was absent, whereas the amylose content was increased to levels comparable to the highest commercially available maize starches. In addition, the phosphorus content of the starch was increased more than fivefold. This unique starch, with its high amylose, low amylopectin, and high phosphorus levels, offers novel properties for food and industrial applications.