Slow digestion property of native cereal starches

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

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.     

Effect of enzymatic hydrolysis on native starch granule structure

Enzymatic digestion of six starches of different botanical origin was studied in real time by in situ time-resolved small-angle neutron scattering (SANS) and complemented by the analysis of native and digested material by X-ray diffraction, differential scanning calorimetry, small-angle X-ray scattering, and scanning electron microscopy with the aim of following changes in starch granule nanostructure during enzymatic digestion. This range of techniques enables coverage over five orders of length-scale, as is necessary for this hierarchically structured material. Starches studied varied in their digestibility and displayed structural differences in the course of enzymatic digestion. The use of time-resolved SANS showed that solvent-drying of digested residues does not induce any structural artifacts on the length scale followed by small-angle scattering. In the course of digestion, the lamellar peak intensity gradually decreased and low-q scattering increased. These trends were more substantial for A-type than for B-type starches. These observations were explained by preferential digestion of the amorphous growth rings. Hydrolysis of the semicrystalline growth rings was explained on the basis of a liquid-crystalline model for starch considering differences between A-type and B-type starches in the length and rigidity of amylopectin spacers and branches. As evidenced by differing morphologies of enzymatic attack among varieties, the existence of granular pores and channels and physical penetrability of the amorphous growth ring affect the accessibility of the enzyme to the substrate. The combined effects of the granule microstructure and the nanostructure of the growth rings influence the opportunity of the enzyme to access its substrate; as a consequence, these structures determine the enzymatic digestibility of granular starches more than the absolute physical densities of the amorphous growth rings and amorphous and crystalline regions of the semicrystalline growth rings.     

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.     

In depth study of a new highly efficient raw starch hydrolyzing α-amylase from Rhizomucor sp

A new α-amylase from Rhizomucor sp. (RA) was studied in detail due to its very efficient hydrolysis of raw starch granules at low temperature (32 °C). RA contains a starch binding domain (SBD) connected to the core amylase catalytic domain by a O-glycosylated linker. The mode of degradation of native maize starch granules and, in particular, the changes in the starch structure during the hydrolysis, was monitored for hydrolysis of raw starch at concentrations varying between 0.1 and 31%. RA was compared to porcine pancreatic α-amylase (PPA), which has been widely studied either on resistant starch or as a model enzyme in solid starch hydrolysis studies. RA is particularly efficient on native maize starch and release glucose only. The hydrolysis rate reaches 75% for a 31% starch solution and is complete at 0.1% starch concentration. The final hydrolysis rate was dependent on both starch concentration and enzyme amount applied. RA is also very efficient in hydrolyzing the crystalline domains in the maize starch granule. The major A-type crystalline structure is more rapidly degraded than amorphous domains in the first stages of hydrolysis. This is in agreement with the observed preferential hydrolysis of amylopectin, the starch constituent that forms the backbone of the crystalline part of the granule. Amylose-lipid complexes present in most cereal starches are degraded in a second stage, yielding amylose fragments that then reassociate into B-type crystalline structures, forming the final resistant fraction.     

Acid hydrolysis of native and annealed starches and branch-structure of their Naegeli dextrins

Eight commercial starches, including common corn, waxy corn, wheat, tapioca, potato, Hylon V, Hylon VII, and mung bean starch, were annealed by a multiple-step process, and their gelatinization characteristics were determined. Annealed starches had higher gelatinization temperatures, reduced gelatinization ranges, and increased gelatinization enthalpies than their native starches. The annealed starches with the highest gelatinization enthalpies were subjected to acid hydrolysis with 15.3% H2SO4, and Naegeli dextrins were prepared after 10 days' hydrolysis. Annealing increased the acid susceptibility of native starches in the first (rapid) and the second (slow) phases with potato starch showing the greatest and high amylose starches showing the least changes. Starches with a larger shift in onset gelatinization temperature also displayed a greater percent hydrolysis. The increase in susceptibility to acid hydrolysis was proposed to result from defective and porous structures that resulted after annealing. Although annealing perfected the crystalline structure, it also produced void space, which led to porous structures and possible starch granule defects. The molecular size distribution and chain length distribution of Naegeli dextrins of annealed and native starches were analyzed. The reorganization of the starch molecule during annealing occurred mainly within the crystalline lamellae. Imperfect double helices in the crystalline lamellae improved after annealing, and the branch linkages at the imperfect double helices became protected by the improved crystalline structure. Therefore, more long chains were observed in the Naegeli dextrins of annealed starches than in native starches.     

Structure of lintnerized starch is related to X-ray diffraction pattern and susceptibility to acid and enzyme hydrolysis of starch granules

Acid-resistant residues (lintnerized starches, Ls) were prepared from starches showing A-, B- and C- X-ray diffraction patterns. Ls retained the same X-ray crystalline type as their native counterparts with an improvement in diffraction intensity. Fluorophore-assisted capillary electrophoresis (FACE) study indicated that structural characteristics of Ls were associated with X-ray diffraction patterns. Double helices originated from linear chains with an approximate average degree of polymerisation (DP) 14, 16, and 15 would span the entire length of crystalline lamellae of A-, B-, and C-type starches, respectively. The proportion of singly branched materials (SB) with DP 25 protected in Ls was higher for A-type Ls (10-17%) than for B-type Ls (4-6%) and C-type Ls (8%). The structures of SB were similar in which branched chain (DP 13-15) was longer than main chain (DP 10-12). The structural characteristics of Ls are discussed in relation to acid and enzymatic degradations of starch granules.     

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

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

The effect of water content on the ordered/disordered structures in starches

(13)C cross-polarization magic angle spinning NMR has been used to study the ordered and disordered structures of starches with different water contents. The amorphous regions of starch have been shown to produce NMR patterns only if they are in a glassy state, the widths, positions, and areas of the peaks to some extent being dependent on the temperature and the water content of the starch. In the amorphous region, the peaks were all Gaussian in shape, while the peaks in the ordered regions had Lorentz profiles. Water contents in the range 10-50% did not influence the proportion of double helices in the starch. Decreasing the water content to 1-3%, however, resulted in a significant decrease in the proportion of double helices, the effect being greater in B- than in A-type starches. It is suggested that short-range order structures in starches (double helices) are stabilized by becoming part of long-range order structures (crystallites).     

Monitoring the crystallization of amylose–lipid complexes during maize starch melting by synchrotron x‐ray diffraction

Most starch granules exhibit a natural crystallinity, with different diffraction patterns according to their botanical origin: A‐type from cereals and B‐type from tubers. The V polymorph results essentially from the complexing of amylose with compounds such as iodine, alcohols, or lipids. The intensity and nature of phase transitions (annealing, melting, polymorphic transitions, recrystallization, etc.) induced by hydrothermal treatments in crystalline structures are related to temperature and water content. Despite its small concentration, the lipid phase present mainly in cereal starches has a large influence on starch properties, particularly in complexing amylose. The formation of Vh crystalline structures was observed by synchrotron x‐ray diffraction in native maize starch heated at intermediate and high moisture contents (between 19 and 80%). For the first time, the crystallization of amylose–lipid complexes was evidenced in situ by x‐ray diffraction without any preliminary cooling, at heating rates corresponding to the usual conditions for differential scanning calorimetry experiments. For higher water contents, the crystallization of Vh complexes clearly occurred at 110–115°C. For intermediate water contents, mixed A + Vh (or B + Vh for high amylose starch) diffraction diagrams were recorded. Two mechanisms can be involved in amylose complexing: the first relating to crystallization of the amylose and lipid released during starch gelatinization, and the second to crystalline packing of separate complexed amylose chains (amorphous complexes) present in native cereal starches. © 1999 John Wiley & Sons, Inc. Biopoly 50: 99–110, 1999     

Crystallinity and structuring role of water in native and recrystallized starches by C CP-MAS NMR spectroscopy: 1: Spectral decomposition

Amorphous, native, and recrystallized starches were studied by ¹³C CP-MAS NMR spectroscopy with respect to their behavior with hydration. The study of space groups, associated to crystalline polymorphs (B2 and P6₁ for A and B forms, respectively), provided decomposition rules for the spectral part due to crystalline phases. Moreover, the subtraction of a standard amorphous spectrum apparently showed the existence of three phases in native starches (amorphous, partially-ordered and crystalline) and only two in spherulitic crystals (partially-ordered and crystalline). The proportion of each phase was estimated at two different hydration levels. The amount of crystalline phase was compared to the degree of crystallinity as evaluated by wide angle X-ray scattering. The NMR spectral changes with hydration could be interpreted in terms of two complementary roles of water molecules, i.e. structuring and plasticizing.     

Granule-bound starch synthase I. A major enzyme involved in the biogenesis of B-crystallites in starch granules.

Starch defines a semicrystalline polymer made of two different polysaccharide fractions. The A- and B-type crystalline lattices define the distinct structures reported in cereal and tuber starches, respectively. Amylopectin, the major fraction of starch, is thought to be chiefly responsible for this semicrystalline organization while amylose is generally considered as an amorphous polymer with little or no impact on the overall crystalline organization. STA2 represents a Chlamydomonas reinhardtii gene required for both amylose biosynthesis and the presence of significant granule-bound starch synthase I (GBSSI) activity. We show that this locus encodes a 69 kDa starch synthase and report the organization of the corresponding STA2 locus. This enzyme displays a specific activity an order of magnitude higher than those reported for most vascular plants. This property enables us to report a detailed characterization of amylose synthesis both in vivo and in vitro. We show that GBSSI is capable of synthesizing a significant number of crystalline structures within starch. Quantifications of amount and type of crystals synthesized under these conditions show that GBSSI induces the formation of B-type crystals either in close association with pre-existing amorphous amylopectin or by crystallization of entirely de novo synthesized material.     

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

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

Susceptibility of annealed starches to hydrolysis by α-amylase and glucoamylase

The objective of this work was to determine if annealing altered the susceptibility of different starches to enzyme hydrolysis. Five commercial starches, including waxy corn, common corn, Hylon V, Hylon VII, and potato, were annealed by a multiple-step process, and their susceptibility to α-amylase and glucoamylase and the physicochemical properties of the hydrolyzed native and annealed starches were determined. During 36 h of enzyme hydrolysis, significant differences were noted between annealed starch and its native counterpart in the extent of α-amylolysis for Hylon V, Hylon VII, and potato, and in the extent of glucoamylolysis for potato. Waxy and common corn starches were hydrolyzed to a greater degree by both enzymes when compared with the other starches. The apparent amylose content of both native and annealed starches decreased during α-amylolysis for all starches, but increased for Hylon V, VII, and potato starches during glucoamylolysis. Most native and annealed starches exhibited comparable or increased peak gelatinization temperatures and comparable or decreased gelatinization enthalpy on hydrolysis with the exception of annealed potato starch, which showed a significant decrease in peak gelatinization temperature on hydrolysis. Annealed starches displayed significant higher peak gelatinization temperatures than their native counterparts. The intensity of main X-ray diffraction peaks of all starches decreased upon hydrolysis, and the changes were more evident for glucoamylase-hydrolyzed starches. The annealing process allowed for a greater accessibility of both enzymes to the amorphous as well as the crystalline regions to effect significant changes in gelatinization properties during enzyme hydrolysis.     

Effects of annealing on the polymorphic structure of starches from sweet potatoes (Ayamurasaki and Sunnyred cultivars) grown at various soil temperatures

Starches extracted from the sweet potato cultivars Sunnyred and Ayamurasaki grown at 15 or 33 degrees C (soil temperature) were annealed in excess water (3 mg starch/mL water) for different times (1, 4, 8 or 10h) at the temperatures 2-3 degrees K below the onset melting temperature. The structures of annealed starches, as well as their gelatinisation (melting) properties, were studied using high-sensitivity differential scanning calorimetry (HSDSC). In excess water, the single endothermic peak shifted to higher temperatures, while the melting (gelatinisation) enthalpy changed only very slightly, if any. The elevation of gelatinisation temperature was associated with increasing order/thickness of the crystalline lamellae. The only DSC endotherm identified in 0.6 M KCl for Sunnyred starch grown at 33 degrees C was attributed to A-type polymorphic structure. The multiple endothermic forms observed by DSC performed in 0.6M KCl for annealed starches from both cultivars grown at 15 degrees C provided evidence of a complex C-type (A- plus B-type) polymorphic structure of crystalline lamellae. The A:B-ratio of two polymorphic forms increased upon annealing due to partial transformation of B- to A-polymorph, which was time dependent. Long heating periods facilitated the maximal transformation of B- to A-polymorph associated with limited A:B ratio.     

Effects of structural imperfection on gelatinization characteristics of amylopectin starches with A- and B-type crystallinity

The aim of the present work was to investigate the effect of physical structures on the properties of starch granules. Starches with a high amylopectin content possessing A- and B-type crystallinity were chosen for the study. The gelatinization temperature decreased in the following order: maize (A) > potato (B) > wheat (A) > barley (A), which did not reflect a correlation with the type of crystallinity. Low values of gelatinization temperature were accompanied with high free surface energy of the crystallites. It is proposed that these data are caused by different types of imperfections in starch crystals. Annealing resulted in an enhancement of the gelatinization temperature and a decrease of the free surface energy of the crystallites for all starches reflecting a partial improvement of crystalline perfection. A limited acid hydrolysis (lintnerization) of the starches decreased the gelatinization temperature because of a partial disruption of the crystalline lamellae and an increase of the amount of defects on the edges of the crystallites. Annealing of the lintnerized starches improved the structure of maize and potato starch, giving them similar structural and physicochemical parameters, which was opposite the behavior of the annealed sample from wheat. The possible nature of removable and nonremovable defects inside the crystalline region of the starch granules is discussed. It is concluded that, besides the allomorphic A- and B-types of crystal packing, physical defects in the crystals possess a major impact on starch gelatinization.     

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.     

Recrystallisation behaviour of native and processed waxy maize starch in relation to the molecular characteristics

Molecular characteristics were determined for native waxy maize starch and maize starch modified in different way (by mechanical treatment or/and acid hydrolysis). Recrystallisation behaviour was studied. Methods used in this study were MALLS, HPAEC-PAD, NMR, DSC, SEM, light microscopy.

Five starch materials were subjected to storage under the same conditions in the presence of water (70 w/w%). Molecular weight, radius of gyration, initial crystallinity, and degree of polymerisation, degree of branching, chain length distribution profiles, were related to nucleation rate during the recrystallisation process, rate of recrystallisation, thermal stability and amount of obtained crystallinity. This allowed the following connections between the molecular characteristics and kinetic of recrystallisation to be proposed: Amylopectin molecular weight appeared to affect the number of starch crystallites formed and amount of crystallinity but not the stability of the rebuilt crystallites. The stability of rebuilt crystallites can be controlled by degree of polymerization, degree of branching and unit chain length distribution, characteristics which were similar for the starches.

A mixture of two starches, with and without crystalline structure in initial state but with molecular weight in same range, were stored and scanned in order to understand possible cocrystallisation effects.     

Slowly digestible waxy maize starch prepared by octenyl succinic anhydride esterification and heat-moisture treatment: glycemic response and mechanism

The mechanism and molecular structure of the slowly digestible waxy maize starch prepared by octenyl succinic anhydride (OSA) esterification and heat-moisture treatment were investigated. The in vitro Englyst test showed a proportion of 28.3% slowly digestible starch (SDS) when waxy maize starch was esterified with 3% OSA (starch weight based, and it is named OSA-starch), and a highest SDS content of 42.8% was obtained after OSA-starch (10% moisture) was further heated at 120 degrees C for 4 h (named HOSA-starch). The in vivo glycemic response of HOSA-starch, which showed a delayed appearance of blood glucose peak and a significant reduction (32.2%) of the peak glucose concentration, further confirmed its slow digestion property. Amylopectin debranching analysis revealed HOSA-starch had the highest resistance to debranching enzymes of isoamylase and pullulanase, and a simultaneous decrease of K m and V m (enzyme kinetics) was also shown when HOSA-starch was digested by either alpha-amylase or amyloglucosidase, indicating that the slow digestion of HOSA-starch resulted from an uncompetitive inhibition of enzyme activity during digestion. Size exclusion chromatography analysis of HOSA-starch showed fragmented amylopectin molecules with more nonreducing ends that are favorable for RS conversion to SDS by the action of amyloglucosidase in the Englyst test. Further solubility analysis indicates that the water-insolubility of HOSA-starch is caused by OSA-mediated cross-linking of amylopectin and the hydrophobic interaction between OSA-modified starch molecules. The water-insolubility of HOSA-starch would decrease its enzyme accessibility, and the digestion products with attached OSA molecules might also directly act as the uncompetitive inhibitor to reduce the enzyme activity leading to a slow digestion of HOSA-starch.