Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach

Starch granule types from a variety of botanical sources were selected to represent differences in crystalline polymorph, amylose and phosphate content, and amylopectin chain length distribution. Equimolar labeling of starch molecules with the fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was used to construct a detailed map of the distribution of amylose and amylopectin within the granule by confocal laser scanning microscopy (CLSM) analysis. Medium- and high-resolution scanning electron microscopy (SEM) were used to provide detailed images of granule surface structures. By using a combined surface and internal imaging approach, interpretations of a number of previous structural observations is presented. In particular, internal images of high amylose maize and potato suggest that multiple initiations of new granules are responsible for the compound or elongated structures observed in these starches. CLSM optical sections of rice granules revealed an apparent altered distribution of amylose in relation to the proposed growth ring structure, hinting at a novel mechanism of starch molecule deposition. Well-described granule features, such as equatorial grooves, channels, cracks, and growth rings were documented and related to both the internal and external observations. A new method for probing the phosphate distribution in native granules was developed using a phosphate-binding fluorescent dye and CLSM.     

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.     

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.     

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.     

A review of cellular structure,starch, and texture qualities of processed potatoes

Certain combined characteristics of cellular structure and starch properties provide distinctions between varieties of potatoes and bear strong relation to their culinary qualities. Larger tissue cells and larger average starch granules are associated with mealiness. Smaller cells and starch granules characterize the less mealy and “waxy” varieties. Similarly, the same general relationships hold for the varietal characteristics of high vs. low solids and high vs. low starch contents. Within a variety, proportionately larger numbers of large starch granules are associated with tubers of high specific gravity, and more smaller granules, with low specific gravity. There also is a distinct reduction in percent of small granules during storage of tubers. Differences in starch granule size are accompanied by differences in amylose and amylopectin. Small granules contain less amylose and gel at higher temperatures than do the larger starch granules. Amylose content likewise appears to be a varietal characteristic. These variations in amylose content reflect fundamental differences in the properties of the starch gels formed when different varieties of potatoes are cooked. Likewise, there are similar distinctions between the starches within different tissue zones of individual tubers. Cell size also varies characteristically within different tuber regions. Starch gel properties may be manipulated during processing by such treatments as precooking-heating, chilling, freezing, and thawing. These treatments provide some measure of control of textural quality in the finished product. Additives such as stearates or glycerides complex readily with amylose and also influence gel properties and texture in processed potato products. Sucrose accumulated during tuber storage also may increase gel strength and influence texture. Varietal differences in cell structure and in starch granule size and composition offer opportunities for genetic exploitation. The merits of special processing for texture control vs. development of varieties for specific processed product qualities are briefly discussed.     

Heterogeneity of lotus rhizome starch granules as revealed by α-amylase degradation

Lotus (Nelumbo nucifera Gaertn.) rhizome starch granules have an elongated oval shape with the hilum located at one end. The morphologic characteristics were used as a direction anchor to study the heterogeneity of molecular organization of starch granules using microscopy before and after partial digestion by bacterial α-amylase (Bacillus sp.) The partially digested granule showed a single, big eroded hole at the end distant from the hilum. The enzyme-attacked end was revealed to be the loosely packed end and to be the weak point for enzyme hydrolysis. The α-amylase hydrolyzed the loosely packed central part of the granule faster than the densely packed periphery, and left an empty shell with a fish-bone-like tunnel inside. The periphery was more resistant to amylase hydrolysis and had strong birefringence. For the whole starch granule, the selectivity of α-amylase hydrolysis was low for the crystalline and amorphous regions and for amylose and amylopectin molecules. This study elucidated that the molecular organization of lotus rhizome starch granules was heterogeneous.     

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.     

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.     

Size distributions of amylose and amylopectin solubilized from corn starch granules

Size distributions of extracts derived from starch were investigated to aid in elucidating structure-function relationships of these polymers in water. Starch granules derived from waxy maize and amylomaize VII were dissolved in water by microwave heating in a high pressure vessel. Transmission electron microscopy of starch deposited from dilute solution and rotary shadowed with platinum, revealed that amylopectin imaged from waxy maize could be broadly classified as about 28% circular space filling patches containing branched clusters and 72% asymmetric linear containing branched clusters. Lengths of asymmetric linear amylopectin components ranged from about 37 to 980 nm whereas the diameter of circular amylopectin components ranged from about 44 to 200 nm. Although the starch in amylomaize VII is about 70% amylose, its narrow asymmetric structure when visualized by microscopy enabled us to image amylose even though amylopectin was present. Lengths of components ranged from about 46 to 254 nm. After smoothing and curve fitting, we found that all size distributions investigated could be treated as if they were multimodal in nature. The most abundant amylose component had a linear density of 8.2 × 10³ molar mass units/ nm. This value could be explained if amylose had an aggregation number of about 5.9.     

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.     

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.     

Role of granule-bound starch synthase in determination of amylopectin structure and starch granule morphology in potato.

Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.     

New Starch Phenotypes Produced by TILLING in Barley

Barley grain starch is formed by amylose and amylopectin in a 1∶3 ratio, and is packed into granules of different dimensions. The distribution of granule dimension is bimodal, with a majority of small spherical B-granules and a smaller amount of large discoidal A-granules containing the majority of the starch. Starch granules are semi-crystalline structures with characteristic X-ray diffraction patterns. Distinct features of starch granules are controlled by different enzymes and are relevant for nutritional value or industrial applications. Here, the Targeting-Induced Local Lesions IN Genomes (TILLING) approach was applied on the barley TILLMore TILLING population to identify 29 new alleles in five genes related to starch metabolism known to be expressed in the endosperm during grain filling: BMY1 (Beta-amylase 1), GBSSI (Granule Bound Starch Synthase I), LDA1 (Limit Dextrinase 1), SSI (Starch Synthase I), SSIIa (Starch Synthase IIa). Reserve starch of nine M3 mutant lines carrying missense or nonsense mutations was analysed for granule size, crystallinity and amylose/amylopectin content. Seven mutant lines presented starches with different features in respect to the wild-type: (i) a mutant line with a missense mutation in GBSSI showed a 4-fold reduced amylose/amylopectin ratio; (ii) a missense mutations in SSI resulted in 2-fold increase in A:B granule ratio; (iii) a nonsense mutation in SSIIa was associated with shrunken seeds with a 2-fold increased amylose/amylopectin ratio and different type of crystal packing in the granule; (iv) the remaining four missense mutations suggested a role of LDA1 in granule initiation, and of SSIIa in determining the size of A-granules. We demonstrate the feasibility of the TILLING approach to identify new alleles in genes related to starch metabolism in barley. Based on their novel physicochemical properties, some of the identified new mutations may have nutritional and/or industrial applications.     

Damaged starch characterisation by ultracentrifugation

The relative molecular size distributions of a selection of starches (waxy maize, pea and maize) that had received differing amounts of damage from ball milling (as quantified by susceptibility to alpha-amylase) were compared using analytical ultracentrifugation. Starch samples were solubilised in 90% dimethyl sulfoxide, and relative size distributions were determined in terms of the apparent distribution of sedimentation coefficients g*(s) versus s(20,w). For comparison purposes, the sedimentation coefficients were normalised to standard conditions of density and viscosity of water at 20 degrees C, and measurements were made with a standard starch loading concentration of 8 mg/mL. The modal molecular size of the native unmilled alpha-glucans were found to be approximately 50S, 51S and 79S for the waxy maize, pea and maize amylopectin molecules, respectively, whilst the pea and maize amylose modal molecular sizes were approximately 14S and approximately 12S, respectively. As the amount of damaged starch increased, the amylopectin molecules were eventually fragmented, and several components appeared, with the smallest fractions approaching the sedimentation coefficient values of amylose. For the waxy maize starch, the 50S material (amylopectin) was gradually converted to 14S, and the degradation process included the appearance of 24S material. For the pea starch, the situation was more complicated than the waxy maize due to the presence of amylose. As the amylopectin molecules (51S) were depolymerised by damage within this starch, low-molecular-weight fragments added to the proportion of the amylose fraction (14S)--although tending towards the high-molecular-weight region of this fraction. As normal maize starch was progressively damaged, a greater number of fragments appeared to be generated compared to the other two starches. Here, the 79S amylopectin peak (native starch) was gradually converted into 61 and 46S material and eventually to 11S material with a molecular size comparable to amylose. Amylose did not appear to be degraded, implying that all the damage was focused on the amylopectin fraction in all three cases. Specific differences in the damage profiles for the pea and maize starches may reflect the effect of lipid-complexed amylose in the maize starch.     

The roles of amylose and amylopectin in the gelation and retrogradation of starch

The retrogradation of starch gels has been studied by using X-ray diffraction, differential scanning calorimetry, and measurements of the shear modulus. Starch gels were considered as composites containing gelatinised granules embedded in an amylose matrix. The short-term development of gel structure and crystallinity in starch gels was found to be dominated by irreversible (T <100°) gelation and crystallisation within the amylose matrix. Long-term increases in the modulus of starch gels were linked to a reversible crystallisation, involving amylopectin, within the granules on storage. It was considered that the crystallisation resulted in an increase in the rigidity of the granules and thus enhanced their reinforcement of the amylose matrix.     

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.     

Molecular and granular characteristics of corn starch modified by HCl-methanol at different temperatures

Native corn starch was hydrolyzed with 0.36% HCl in methanol at 25 and 45 °C for periods of time up to 240 h. The action of acid penetration and hydrolysis was investigated by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), high-performance anion-exchange chromatography (HPAEC) and high-performance size-exclusion chromatography (HPSEC) equipped with viscometry, right-angle laser light scattering (RALLS) and refractive index (RI) detectors. Corn starch hydrolyzed at 45 °C for 240 h showed strong intensity of APTS (8-amino-1,3,6-pyrenetrisulfonic acid) fluorescence and sharp growth ring structure. Exocorrosion over the surface of corn starch was only observed on the corn starch hydrolyzed at 25 °C for 240 h and observed on all corn starch hydrolyzed at 45 °C. The M_w and R_h of acid-hydrolyzed corn starch decreased with increasing the degree of hydrolysis. The acid hydrolysis rate in methanol of corn starch was mainly dependent on the temperature, which dominated the penetration efficiency of acid.     

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.     

Solvent effects on starch dissolution and gelatinization

The disruption of starch granular structure during dissolution in varying concentrations of N-methyl morpholine N-oxide (NMMO) has been studied using three maize starches with varying ratios of amylose and amylopectin. Behavior in NMMO has been characterized by differential scanning calorimetry (DSC), microscopy, rapid viscosity analysis (RVA), and rheometry. Exothermic transitions were observed for the three starches in both 78 and 70% NMMO; the transition changed to an endotherm at 60 and 50% NMMO. Consistent with DSC, hot stage microscopy showed that starch granules dissolved at NMMO concentrations of 78 and 70%, whereas in 60 and 50% NMMO, gelatinization behavior similar to that found for starch in water was observed. Mechanical spectroscopy revealed the dominant viscous behavior (G″ > G') of starch at NMMO concentrations of 70 and 78% and more elastic behavior (G' > G″) at lower concentrations. Starch solutions in 78% NMMO obey the Cox-Merz rule, suggesting that the solutions are homogeneous on a molecular level.     

In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria.

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.