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.     

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.     

The distribution of water in native starch granules—a multinuclear NMR study

The microscopic distribution and dynamic state of water in native potato, maize and pea starch granules are investigated with NMR relaxometry and diffusometry. Besides extra-granular water, three water populations can be identified inside native potato starch granules. These are assigned to water in the amorphous growth rings; water in the semi-crystalline lamellae and “channel water”, which is located in the hexagonal channels within the B-type amylopectin crystals. The first two water populations are orientationally disordered and exchange with each other on a millisecond timescale at 290 K. NMR diffusometry shows that the water in packed granule beds is undergoing translational diffusion in a 2-dimensional space, either in thin layers between granules and/or in amorphous growth rings within the granules. The “channel water” is uniquely characterised by a 1 kHz deuterium doublet splitting and is in slow exchange with water in the other compartments on the NMR timescale. In the smaller maize granules all intra-granular water populations are in fast exchange and there is no evidence for “channel water” in the A-type crystal lattice. The NMR water proton and deuterium data for pea starch are consistent with a composite A and B-type crystal structure.     

Determination of the polymorphic composition of smooth pea starch

A method for estimating the proportions of ‘A’ and ‘B’ polymorphs comprising a sample of ‘C’ type starch is proposed which uses established experimental techniques with commercially available spreadsheet and X-ray analysis software. Waxy maize, potato and smooth pea starches were used to provide X-ray diffraction patterns characteristic of the ‘A’, ‘B’ and ‘C’ starch polymorphs. Samples of amorphous starches were also prepared. The method initially involved subtraction of the amorphous phase and instrumental background from the X-ray diffraction patterns of each starch sample using the spreadsheet program, Lotus 1-2-3. The remainder of the pattern, representing the crystalline portion of the starch sample, was then analysed by profile fitting to elucidate the positions and areas of individual diffraction peaks. The ratio of the total peak area to the areas under peaks characteristic of ‘A’ and ‘B’ type starches, respectively, were used to calculate the relative proportions of these polymorphs in smooth pea starch. These proportions were found to be 56±3% ‘A’ polymorph to 44±3% ‘B’ polymorph. A ‘C’ type pattern was constructed by using Lotus 1-2-3 to combine diffraction patterns from the crystalline portions of ‘A’ and ‘B’ type starches in the proportions given above. Polymorph patterns were obtained by manipulation of the diffraction patterns from the crystalline portions of starches using Lotus 1-2-3. An ‘A’ type pattern was obtained by subtraction of a ‘B’ type pattern from that of a ‘C’ type. Similarly, a ‘B’ type pattern was obtained by subtraction of an ‘A’ type pattern from that of a ‘C’ type.     

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.     

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.     

Physicochemical properties of endosperm and pericarp starches during maize development

Endosperm starch and pericarp starch were isolated from maize (B73) kernels at different developmental stages. Starch granules, with small size (2–4 μm diameter), were first observed in the endosperm on 5 days after pollination (DAP). The size of endosperm-starch granules remained similar until 12DAP, but the number increased extensively. A substantial increase in granule size was observed from 14DAP (diameter 4–7 μm) to 30DAP (diameter10–23 μm). The size of starch granules on 30DAP is similar to that of the mature and dried endosperm-starch granules harvested on 45DAP. The starch content of the endosperm was little before 12DAP (less than 2%) and increased rapidly from 10.7% on 14DAP to 88.9% on 30DAP. The amylose content of the endosperm starch increased from 9.2% on 14DAP to 24.2% on 30DAP and 24.4% on 45DAP (mature and dried). The average amylopectin branch chain-length of the endosperm amylopectin increased from DP23.6 on 10DAP to DP26.9 on14DAP and then decreased to DP25.4 on 30DAP and DP24.9 on 45DAP. The onset gelatinization temperature of the endosperm starch increased from 61.3 °C on 8DAP to 69.0 °C on 14DAP and then decreased to 62.8 °C on 45DAP. The results indicated that the structure of endosperm starch was not synthesized consistently through the maturation of kernel. The pericarp starch, however, showed similar granule size, starch content, amylose content, amylopectin structure and thermal properties at different developmental stages of the kernel.     

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.     

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.     

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.     

A new proposed sweet potato starch granule structure--pomegranate concept

There are two competing concepts about organization of starch granule, fibrillar concept (or amylopectin clustering concept) and blocklet concept. A new micrograph of gelatinized sweet potato starch mixed with lactose might combine the two concepts and recover the mysterious structure of starch granule. Here we propose a possible granule structure of sweet potato starch by analyzing its gelatinization micrographs mixed with different carbohydrates. As the structure of pomegranate, out-layer of granule is equivalent to skin of pomegranate, blocklets are same to garnet of pomegranate, the amylopectin clusters with one reducing end at hilum, equivalent to primary body of pomegranate, constitute the basic structure of granule, in the special parts of the clusters, lots of blocklets form and increase, very like the born of garnet of pomegranate. At the same time double helix appears in blocklets, the arrangements of blocklets, same as garnet in pomegranate, form the semi-crystalline growth rings. The crystal type of starch is determined by arrangement of blocklet on it, hexagonal for A type, monoclinic for B type.     

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.     

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.     

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 heat–moisture treatment on the structure and physicochemical properties of tuber and root starches

Starch from tubers potato (Solanum tuberosum), taro (Alocassia indica), new cocoyam (Xanthosoma sagitifolium), true yam (Dioscorea alata), and root cassava, (Manihot esculenta) crops was isolated and its morphology, composition and physicochemical properties were investigated before and after heat–moisture treatment (HMT) (100 °C, for 10 h at a moisture content of 30%). Native starch granules were round to oval to polygonal with smooth surfaces. The granule size (diameter) ranged from 3.0 to 110 μm.The total amylose content ranged from 22.4 to 29.3%, of which 10.1–15.5% was complexed by native lipid. The phosphorus content ranged from 0.01 to 0.1%. The X-ray pattern of potato and true yam was of the ‘B’-type. Whereas, that of new cocoyam and taro was of the ‘A’-type. Cassava exhibited a mixed ‘A+B’-type X-ray pattern. The relative crystallinity, swelling factor (SF), amylose leaching (AML), gelatinization temperature range and the enthalpy of gelatinization of the native starches ranged from 30 to 46, 22 to 54, 5 to 23%, 13 to 19 °C and 12 to 18 J/g, respectively. Susceptibility of native starches towards hydrolysis by 2.2N HCl and porcine pancreatic -amylase were 60–86% (after 12 days), and 4–62% (after 72 h), respectively. Retrogradation was most pronounced in the B-type starches. Granule morphology remained unchanged after HMT. The X-ray pattern of the B-type starches was altered (B→A+B) on HMT. However, that of the other starches remained unchanged. HMT decreased SF, AML, gelatinization enthalpy and susceptibility towards acid hydrolysis, but increased gelatinization temperatures and enzyme susceptibility. Extent of retrogradation and relative crystallinity decreased on HMT of true yam and potato starches, but remained unchanged in the other starches. The foregoing data showed that changes in physicochemical properties on HMT are influenced by the interplay of crystallite disruption, starch chain associations and disruption of double helices in the amorphous regions.     

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.     

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.     

Hydrolysis of concentrated raw starch: A new very efficient α-amylase from Anoxybacillus flavothermus

A new α-amylase from Anoxybacillus flavothermus (AFA) was found to be effective in hydrolyzing raw starch in production of glucose syrup at temperatures below the starch gelatinization temperature. AFA is very efficient, leading to 77% hydrolysis of a 31% raw starch suspension. The final hydrolysis degree is reached in 2-3 h at starch concentrations lower than 15% and 8-24 h at higher concentrations. AFA is also very efficient in hydrolyzing the crystalline domains in the starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in agreement with the observed preferential hydrolysis of amylopectin. Amylose-lipid complexes are degraded in a second step, yielding amylose fragments which then re-associate into B-type crystalline structures forming the final α-amylase resistant fraction. The mode of action of AFA and the factors limiting complete hydrolysis are discussed in details.     

A Parameterized Model of Amylopectin Synthesis Provides Key Insights into the Synthesis of Granular Starch

A core set of genes involved in starch synthesis has been defined by genetic studies, but the complexity of starch biosynthesis has frustrated attempts to elucidate the precise functional roles of the enzymes encoded. The chain-length distribution (CLD) of amylopectin in cereal endosperm is modeled here on the basis that the CLD is produced by concerted actions of three enzyme types: starch synthases, branching and debranching enzymes, including their respective isoforms. The model, together with fitting to experiment, provides four key insights. (1) To generate crystalline starch, defined restrictions on particular ratios of enzymatic activities apply. (2) An independent confirmation of the conclusion, previously reached solely from genetic studies, of the absolute requirement for debranching enzyme in crystalline amylopectin synthesis. (3) The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. (4) The model provides a means by which a small number of key parameters defining the core enzymatic activities can be derived from the amylopectin CLD, providing the basis for focusing studies on the enzymatic requirements for generating starches of a particular structure. The modeling approach provides both a new tool to accelerate efforts to understand granular starch biosynthesis and a basis for focusing efforts to manipulate starch structure and functionality using a series of testable predictions based on a robust mechanistic framework.     

Influence of fiber on the phase transformations in the starch-water system

High-sensitivity, temperature-controlled DSC measurements at a low heating rate and creation of differential DSC traces scaled with respect to the reference material (completely dehydrated starch or completely dehydrated fiber, or their respective blends) permitted investigation of the influence of fiber on phase transformations in the wheat-starch-water system in the course of thermal gelatinization. Thermal effects associated with water interactions over the temperature range from 283 to 384 K under atmospheric pressure were determined. These thermal effects and previous structural studies permit us to make the following observations: (1) The main endothermic transition associated with melting of the crystalline part of the starch granule followed by a helix-coil transition in amylopectin occurs over the temperature range 319-333 K independent of the water and fiber contents. Adding fiber causes that transition to disappear both in the native blends and in water suspensions at low water contents. After adding more water and heating, recrystallization is observed and the transition reappears. (2) The fiber content has practically no influence on the slow exothermic transformation, which follows melting and helix-coil transition in amylopectin, proving that the slow transformation has a specific chemical character. In this reaction, the free ends of the unwound helices of amylopectin reassociate with parts of amylopectin molecules other than their original helix duplex partner, forming physical junctions and creating more general amorphous hydrogen bonded associations. (3) The high-temperature transition and small, but reproducible, distortions on the peaks of the main endothermic transition for water contents near 70-80 wt % are associated with smectic and nematic transitions, respectively. These are significantly influenced by the fiber content; higher fiber content causes an almost complete disappearance of these transitions. (4) The slow exothermic effect appearing almost from the very beginning of the heating in the starch-water system, associated with softening and uptake of water in the amorphous growth rings of the starch granule, is significantly hindered by added fiber.