The effects of amylose content on the molecular size of amylose, and on the distribution of amylopectin chain length in maize starches

The amylose to amylopectin ratios in six maize starch samples of differing amylose contents were measured by enzymatic debranching, followed by high performance size exclusion chromatography (HPSEC). The molecular size of amyloses, estimated by -log K_wav, shows progressive decrease with the increase in amylose content in maize starches. The gel permeation chromatographs of the corresponding amylopectins, debranched with isoamylase, showed bimodal distributions containing long and short chains. The average chain length of amylopectin has a correlation with amylose content. The correlation coefficients between amylose content and average chain length, long chain length, weight ratio and the mole ratio of long and short chain length, were 0.97, 0.92, 0.96, 0.94 respectively. The maize starch with the highest amylose content has the lowest amylose molecular size and the longest chains, with a high ratio of long to short chains in its amylopectin fraction. Comparing the values of amylose content determined by HPSEC of starch or debranched starch with those of the iodinecomplex method, we conclude that long chains of amylopectin in high amylose starches contribute significantly to apparent amylose content.     

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.     

Internal structure and physicochemical properties of corn starches as revealed by chemical surface gelatinization

The organization of amylose and amylopectin within starch granules is still not well elucidated. This study investigates the radial distribution of amylose and amylopectin in different corn starches varying in amylose content (waxy corn starch (WC), common corn starch (CC), and 50% and 70% amylose corn starches (AMC)). Corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The gelatinized surface starch and remaining granules were characterized for amylose content, amylopectin chain-length distribution, thermal properties, swelling power (SP), and water solubility index (WSI). Except for the outmost 10% layer, the amylose content in CC increased slightly with increasing surface removal. In contrast, amylose was more concentrated at the periphery than at the core for 50% and 70% AMC. The proportion of amylopectin A chains generally decreased while that of B1 chains generally increased with increasing surface removal for all corn starches. The gelatinization enthalpy usually decreased, except for 70% AMC, whereas the retrogradation enthalpy relatively remained unchanged for CC but increased for WC, 50% and 70% AMC with increasing surface removal. The SP and WSI increased with increasing surface removal for all corn starches, with WC showing a significant increase in SP after the removal of the outmost 10% layer. The results of this study indicated that there were similarities and differences in the distribution of amylose and amylopectin chains along the radial location of corn starch granules with varying amylose contents. More amylose-lipid complex and amylopectin long chains were present at the periphery than at the core for amylose-containing corn starches.     

The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

Acetyl substitution patterns of amylose and amylopectin populations in cowpea starch modified with acetic anhydride and vinyl acetate

To study the effect of reagent type on the distribution pattern of acetyl groups in acetylated cowpea starch, amylose and amylopectin populations were isolated from the starch granules after modification to a low degree of substitution (DS < 0.1) with acetic anhydride and vinyl acetate, respectively. Slowly reacting reagent vinyl acetate resulted in higher DS values for the amylopectin populations when compared to the rapidly reacting reagent acetic anhydride. The two reagents had similar effects on the acetylation level of amylose, suggesting that the amorphous regions of granules were easily accessible for both reagents. The acetyl substitution patterns were analyzed by enzymatic degradation followed by characterization of the obtained fragments using chromatographic and mass spectrometric techniques. The distributions of acetyl groups along the amylose and amylopectin chains were more clustered for modification with vinyl acetate as compared with modification with acetic anhydride. Between the two acetylation types, pronounced differences in the acetyl substitution patterns were observed for the large fragments obtained after -amylase digestion; only slight differences were exhibited for the small fragments obtained by exhaustive enzymatic digestion of amylose and amylopectin populations.     

Some recent observations on the retrogradation of amylose

Starch is the major storage polysaccharide of higher plants where it occurs as water insoluble granules. Two component polymers may be extracted from the starch granules, namely amylose and amylopectin. Both polymers are based on chains on α-1,4-linked d-glucose. However, whereas amylose is an essentially linear polymer, amylopectin is highly branched with branches linked at the 6-position.     

Characterization of Molecular Structure of Starch Granules in Suspension-cultured Cells from Ipomoea cordatotriloba Denn.

The molecular structure of starch granules formed in suspension-cultured cells of Ipomoea cordatotriloba Denn. was characterized by its chain length distribution, which was compared to those of the starches from the root and leaf of the original plant. The cultured cell starches were spherical and had a very small granule size (about 2 μm). The debranched starches roughly separated into three fractions during gel-permeation chromatography, and the fractions were defined as Fr.1, 2, and 3, respectively. The chain length distribution of the debranched cultured cell starch showed that the high molecular weight fraction (Fr.1), referred to as an amylose fraction, was much less than those of the root and leaf starches. The ratio of the two lower fractions (Fr.3/Fr.2) of the cultured cell starch, which was mainly derived from unit chains of amylopectin, was greatest among the starches, suggesting that the amylopectin from the cultured cell starch has much shorter unit chains. By X-ray diffraction analysis, it was found that both cultured cell and leaf starch granules have low crystallinity.     

Structural and thermodynamic properties of rice starches with different genetic background Part 2. Defectiveness of different supramolecular structures in starch granules

High-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray scattering (SAXS), light (LM) and scanning electronic (SEM) microscopy techniques were used to study the defectiveness of different supramolecular structures in starches extracted from 11 Thai cultivars of rice differing in level of amylose and amylopectin defects in starch crystalline lamellae. Despite differences in chain-length distribution of amylopectin macromolecules and amylose level in starches, the invariance in the sizes of crystalline lamellae, amylopectin clusters and granules was established. The combined analysis of DSC, SAXS, LM and SEM data for native starches, as well as the comparison of the thermodynamic data for native and annealed starches, allowed to determine the structure of defects and the localization of amylose chains in crystalline and amorphous lamellae, defectiveness of lamellae, clusters and granules. It was shown that amylose “tie chains”, amylose–lipid complexes located in crystalline lamellae, defective ends of double helical chains dangling from crystallites inside amorphous lamellae (“dangling” chains), as well as amylopectin chains with DP 6–12 and 25–36 could be considered as defects. Their accumulation can lead to a formation of remnant granules. The changes observed in the structure of amylopectin chains and amylose content in starches are reflected in the interconnected alterations of structural organization on the lamellar, cluster and granule levels.     

The influence of amylose on starch granule structure

Starch granules are principally composed of the two glucose polymers amylose and amylopectin. Native starch granules typically contain around 20% amylose and 80% amylopectin. However, it is possible to breed plants that produce starch with very different amylose and amylopectin contents. At present, the precise structural roles played by these two polymers are incompletely understood. In this study, small-angle X-ray scattering techniques have been applied to investigate the effect of varying amylose content on the internal structure of maize, barley and pea starch species. The results suggest that amylose disrupts the structural order within the amylopectin crystallites.     

Physico-chemical and degradative properties of in-planta re-structured potato starch

Starch re-structured directly in potato tubers by antisense suppression of starch branching enzyme (SBE), granule bound starch synthase (GBSS) or glucan water dikinase (GWD) genes was studied with the aim at disclosing the effects on resulting physico-chemical and enzyme degradative properties. The starches were selected to provide a combined system with specific and extensive alterations in amylose and covalently esterified glucose-6-phosphate (G6P) contents. As an effect of the altered chemical composition of the starches their hydrothermal characteristics varied significantly. Despite of the extreme alterations in phosphate content, the amylose content had a major affect on swelling power, enthalpy for starch gelatinization and pasting parameters as assessed by Rapid Visco Analysis (RVA). However, a combined influence of the starch phosphate and long glucan chains as represented by high amylose or long amylopectin chain length was indicated by their positive correlation to the final viscosity and set back (RVA) demonstrating the formation of a highly hydrated and gel-forming system during re-structuring of the starch pastes. Clear inverse correlations between glucoamylase-catalyzed digestibility and amylopectin chain length and starch phosphate and lack of such correlation with amylose content indicates a combined structuring role of the phosphate groups and amylopectin chains on the starch glucan matrix.     

The priming of amylose synthesis in Arabidopsis leaves

We investigated the mechanism of amylose synthesis in Arabidopsis leaves using (14)C-labeling techniques. First, we tested the hypothesis that short malto-oligosaccharides (MOS) may act as primers for granule-bound starch synthase I. We found increased amylose synthesis in isolated starch granules supplied with ADP[(14)C]glucose (ADP[(14)C]Glc) and MOS compared with granules supplied with ADP[(14)C]Glc but no MOS. Furthermore, using a MOS-accumulating mutant (dpe1), we found that more amylose was synthesized than in the wild type, correlating with the amount of MOS in vivo. When wild-type and mutant plants were tested in conditions where both lines had similar MOS contents, no difference in amylose synthesis was observed. We also tested the hypothesis that branches of amylopectin might serve as the primers for granule-bound starch synthase I. In this model, elongated branches of amylopectin are subsequently cleaved to form amylose. We conducted pulse-chase experiments, supplying a pulse of ADP[(14)C]Glc to isolated starch granules or (14)CO(2) to intact plants, followed by a chase period in unlabeled substrate. We detected no transfer of label from the amylopectin fraction to the amylose fraction of starch either in isolated starch granules or in intact leaves, despite varying the time course of the experiments and using a mutant line (sex4) in which high-amylose starch is synthesized. We therefore find no evidence for amylopectin-primed amylose synthesis in Arabidopsis. We propose that MOS are the primers for amylose synthesis in Arabidopsis leaves.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

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.     

Effect of amylose deposition on potato tuber starch granule architecture and dynamics as studied by lintnerization

The effect of amylose deposition on the amylopectin crystalline lamellar organization in potato starch granules was studied by mild acid, so‐called lintnerization, of potato tuber starch transgenically engineered to deposit different levels of amylose. The starch granules were subjected to lintnerization at different temperatures (25, 35, and 45°C) and to two levels of solubilization, ～ 45 and 80%. The rate of the lintnerization increased with temperature but was suppressed by amylose. The molecular size of the lintner dextrins increased with temperature, but this effect was suppressed by the presence of amylose. At high temperatures and low‐amylose content, the degree of branches was high with the concomitant increase in size in the dextrins. A portion of the branches was resistant to debranching enzymes possibly due to specific structural formations. The effects of temperature suggested a unique granular architecture of potato starch, and a model showing the dependence of temperature on the dynamic arrangement of amylopectin and amylose in the crystalline and amorphous lamellae for the potato starch is suggested. © 2012 Wiley Periodicals, Inc.     

Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure

The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.     

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.     

Chemical and physical properties of ginkgo (Ginkgo biloba) starch

Starch isolated from mature Ginkgo biloba seeds and commercial normal maize starches were subjected to α-amylolysis and acid hydrolysis. Ginkgo starch was more resistant to pancreatic α-amylase hydrolysis than the normal maize starch. The chain length distribution of debranched amylopectin of the starches was analyzed by using high performance anion-exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector. The chain length distribution of ginkgo amylopectin showed higher amounts of both short and long chains compared to maize starch. Naegeli dextrins of the starches prepared by extensive acid hydrolysis over 12 days demonstrated that ginkgo starch was more susceptible than normal maize to acid hydrolysis. Ginkgo dextrins also demonstrate a lower concentration of singly branched chains than maize dextrins, and unlike maize dextrin, debranched ginkgo shows no multiple branched chains. The ginkgo starch displayed a C-type X-ray diffraction pattern, compared to an A-type pattern for maize. Ginkgo starch and maize starch contained 24.0 and 17.6% absolute amylose contents, respectively.     

Development of a low glycemic maize starch: preparation and characterization

A low glycemic index starch was developed by partial alpha-amylase treatment, and its fine structure responsible for slowly digestible and resistant properties was investigated. Different digestion rates were obtained for gelatinized, retrograded starch by varying the enzyme dosage and reaction time. Analysis by high performance size-exclusion chromatography (HPSEC) coupled with multiangle laser-light scattering indicated that the molecular weighs of amylopectin and amylose were reduced during the digestion, to less than 100 kDa. A debranched chain length study using high performance anion-exchange chromatography equipped with an amyloglucosidase reactor and a pulsed amperometric detector and HPSEC revealed that short chains of amylopectin and noncrystalline amylose were rapidly digested, while DPn 121 chains showed resistance, followed by DPn 46 chains. X-ray diffraction analysis revealed that the crystalline structure in the treated starches survived cooking. These starches not only have slowly digestible and resistant character, but also retain some branched structure for adequate functionality.     

Pathway of cytosolic starch synthesis in the model glaucophyte Cyanophora paradoxa

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.     

Nature of Lysolecithin in Rice Grains

Problems of the interaction between starch and lysolecithin are treated in this article. The endosperm of rice grains was fractionated and it was found that lysolecithin was associated with starch granules. The association was not a mere adsorption, but rather a more firm combination. It was demonstrated that lysolecithin had an ability to combine with starch in aqueous solution, and to cause amylose to precipitate, but not amylopectin. The lysolecithin-amylose complex was isolated, and it was shown that lysolecithin is most certainly included in the helicoidal structure of amylose chain. A possible role of lysolecithin during the course of the starch formation in rice grains is discussed.