Study of Internal Structure of Waxy Wheat Starch Granules by KI/I2 Solution

Wheat starch granules were separated from waxy wheat flour by the acetic acid (pH 3.5) fractionation technique. Waxy wheat starch samples were stained red‐brown using KI/I₂ solution. Subsequently, the concentration of the KI/I₂ solution was increased to >2.5% KI and 1.0% I₂. Then the appearance of the waxy wheat starch granules quickly became ghostlike. The internal structure of the ghost could be observed. This structure consisted of two parts with different colors: a red‐brown central part and a slightly pinkish surrounding part. Sonication of the ghost at 20 kHz for 20 sec separated the central part from the surrounding part. Each of the separated parts were subjected to gel‐filtration chromatography, and their chromatography profiles indicated only amylopectin and no amylose. The central and surrounding parts had molecular weights of 54.3 × 10⁵ and 23.8 × 10⁶ from Berry plots, respectively.     

Oil Binding of Heat‐Treated Waxy Wheat Starch Granules and Starch Granule Ghosts Produced with Concentrated KI and I2

Dry waxy wheat starch granules were heat‐treated at 120°C for 5 hr, and then shaken vigorously in a biphasic system of oil and water. Non‐heat‐ treated starch remained in the aqueous phase, whereas the heat‐treated starch granules showed a strong oil‐binding ability that was lost by trypsin treatment. This result showed that the starch granule surface protein changed from hydrophilic to hydrophobic due to the heat treatment. The presence of starch granule surface protein was ascertained by staining with fluorescamine and fluorescence microscopic observation. Heat‐treated waxy wheat starch granules were incubated with a 25% KI/10% I₂ (w/v) solution, which produced “ghosts” (exterior and interior) structures. The exteriors stained red‐brown, whereas the interiors stained black‐brown. Sonication (20 kHz for 255 sec) followed by centrifugation separated the structures, which were then shaken vigorously in an oil and water system. Only the exterior ghosts exhibited a remarkable emulsification property, which disappeared after trypsin treatment. The ghosts from unheated control granules did not show emulsification. The presence of protein in the exterior ghost fraction was further substantiated by fluorescamine treatment. No protein was detectable in the interior fraction with this dye. From these results, we suggest that the ghost fraction of the waxy wheat starch contained the starch granule surface protein that was made hydrophobic by heat treatment. Also, the nature of the induced emulsification property of the exterior fraction (ghosts) and the oil‐binding ability of the heat‐treated waxy wheat starch granules coincided. Both were due to the hydrophobic nature of the same starch granule surface protein, which showed that the ghosts were the swollen form of the outer region of the waxy starch granule.     

Role of Starch Granule Characteristics (volume fraction,rigidity, and fractal dimension) on Rheology of Starch Dispersions With and Without Amylose

The link between rheological behavior and morphological‐structural characteristics of gelatinized starch granules has been studied in two starch dispersions (SDs): a cross‐linked waxy maize (CLWM), and tapioca starch, a tuber starch with 19.3 % amylose. Based on the power law relationship between the elastic modulus and volume fraction of the granules predicted by scaling theory, fractal dimension values were obtained for CLWM starch D = 2.81, and tapioca starch D = 2.79, suggesting that both starch granules have highly convoluted surfaces. However, the preexponential coefficient (G′_⊘=1) for CLWM SDs was an order of magnitude greater than that for tapioca SDs, in the same range of volume fractions. The G′_⊘=1 was mainly dependent on the granule rigidity, and the amylose content in the continuous phase played only a minor role in the rheological behavior.     

Starch Granule Size Distribution from Seven Wheat Cultivars Under Different Water Regimes

Seven wheat cultivars with different starch contents were used as materials to investigate the distribution of grain starch granule size under irrigated and rainfed conditions. In mature grains, the diameter of starch granules was 0.37–52.6 μm, and the percent volume distribution showed a two‐peak curve with the mean particle diameter of 5 (B‐type) and 25 μm (A‐type) at each peak. The volume percentages of A‐ and B‐types were 52.7–65.5% and 34.5–47.3%, respectively. A two‐peak curve is also shown in percent surface area distribution of starch granules, but only one peak in percent number. Both irrigated and rainfed conditions had a significant effect on the starch granule size distribution of the seven cultivars. As compared with irrigated treatment, rainfed treatment affected the distribution of starch granules in grains of all cultivars through increasing the volume percentage and surface area percentage of 2–9.8 and <9.8 μm starch granules and decreasing those of >9.8 and >18.8 μm starch granules. The soil water deficit also decreased the contents of amylose and starch in grains and increased protein content, indicating that different water regimes had an evident effect on grain quality. According to correlation coefficients (r), the contents of amylose, starch, and protein in grains was significantly correlated with the volume percentage of starch granules with different diameter ranges.     

Effect of Starch Granule Size Distribution and Elevated Amylose Content on Durum Dough Rheology and Spaghetti Cooking Quality

To obtain an indication of the effect of increasing the starch amylose content above normal levels (27–74%) and increasing the percentage of B‐type starch granules (11–60%) on durum dough properties and the quality of the spaghetti derived from these doughs, the reconstitution approach was used. Reconstituted flours were prepared from a common Wollaroi gluten, solubles and tailings fraction combined with starches containing varying B‐granule contents, or with starches from maize with varying amylose content. An increased B‐granule content increased farinograph water absorption. Cooked spaghetti firmness was highest with B‐type granules at 32–44% (volume percentage basis), which is ≈10–15% higher than normally found in durum starch. Increasing the amylose content in the starch caused the dough to be more extensible, increased spaghetti firmness, and decreased water absorption with optimum quality of amylose at 32–44%. The information indicates there would be benefit in producing durum wheats with slightly elevated B‐granule and amylose contents.     

Soft Wheat Starch Pasting Behavior in Relation to A‐ and B‐type Granule Content and Composition

Flours of two soft wheat cultivars were fractionated into native, prime, tailing, A‐, and B‐type starch fractions. Starch fractions of each cultivar were characterized with respect to A/B‐type granule ratio, amylose content, phosphorus level (lysophospholipid), and pasting properties to investigate factors related to wheat starch pasting behavior. While both cultivars exhibited similar starch characteristics, a range of A‐type (5.7– 97.9%, db) and B‐type granule (2.1–94.3%, db) contents were observed across the five starch fractions. Though starch fractions displayed only subtle mean differences (<1%) in total amylose, they exhibited a range of mean phosphorus (446–540 μg/g), apparent amylose (18.7–23%), and lipid‐complexed amylose (2.8–7.5%) values, which were significantly correlated with their respective A‐ and B‐type granule contents. A‐type (compared with B‐type) granules exhibited lower levels of phosphorus, lipid‐complexed amylose, and apparent amylose, though variability for the latter was primarily attributed to starch lipid content. While starch phosphorus and lipid‐complexed amylose contents exhibited negative correlation with fraction pasting attributes, they did not adequately account for starch fraction pasting behavior, which was best explained by the A/B‐type granule ratio. Fraction A‐type granule content was positively correlated with starch pasting attributes, which might suggest that granule size itself could contribute to wheat starch pasting behavior.     

Resistant Starch Content Is Related to Granule Size in Barley

Physical properties of resistant starch (RS) were examined in a range of barley genotypes to determine the contribution of starch and seed physical characteristics to the RS component. Thirty‐three barley genotypes were studied, which varied significantly in their RS, amylose, and starch contents and grain yield. From 33 genotypes, 13 exhibiting high RS were selected for detailed physicochemical analysis of starch. In high‐RS varieties, granule size and number were unimodal, compared with normal starches from a reference genotype, which showed a bimodal distribution. Principal component analysis (PCA) showed that a higher content of granules <15 µm was positively correlated with RS and amylose content, whereas the proportion of granules 15–45 µm was negatively correlated with the RS and amylose contents. Physical fractionation of starches by centrifugation into different population sizes demonstrated that size alone is not an accurate indicator of the population of A‐type and B‐type granules within a given genotype. PCA also showed that large 15–45 µm granules were positively correlated with seed thickness and that thousand grain weight was positively correlated with seed width. High‐RS and high‐amylose genotypes showed variation in overall yield and starch content, with some genotypes showing yield comparable to the reference genotype. Analysis of amylopectin chain length distribution showed that high amylose or RS content was not associated with a higher proportion of amylopectin long chains when compared with either waxy or reference (normal) barley genotypes. This study highlights useful markers for screening barley genotypes with favorable starch characteristics.     

Granule‐Bound Starch Synthase I (GBSSI) in Quinoa (Chenopodium quinoa Willd.) and Its Relationship to Amylose Content

The amylose concentration in starch from 16 quinoa (Chenopodium quinoa Willd.) genotypes grown under identical conditions was 4–20%. Based on the amylose content, a selection of six genotypes was made. Starch granule‐bound proteins were extracted from six genotypes and analyzed using denaturing gel electrophoresis. Two major polypeptides with apparent molecular masses of 56 and 62 kDa were present in all genotypes. Both were identified as granule‐bound starch synthase I (GBSSI) using immunoblot analysis and internal peptide sequencing. The content of the two GBSSI isoforms in starch granules from the six genotypes, as determined by densiometry of the peptide bands, was positively correlated with the concentration of amylose in starch from mature seed. Starch synthase activity in developing seed was positively correlated to starch concentration in seed and amylose concentration in starch during seed development.     

Bread Quality of Spelt Wheat and Its Starch

Flours from five spelt cultivars grown over three years were evaluated as to their breadbaking quality and isolated starch properties. The starch properties included amylose contents, gelatinization temperatures (differential scanning calorimetry), granule size distributions, and pasting properties. Milled flour showed highly variable protein content and was higher than hard winter wheat, with short dough‐mix times indicating weak gluten. High protein cultivars gave good crumb scores, some of which surpassed the HRW baking control. Loaf volume was correlated to protein and all spelt cultivars were at least 9–51% lower than the HRW control. Isolated starch properties revealed an increase in amylose in the spelt starches of 2–21% over the hard red winter wheat (HRW) control. Negative correlations were observed for the large A‐type granules to bread crumb score, amylose level, and final pasting viscosity for cultivars grown in year 1999 and to pasting temperature in 1998 samples. Positive correlations were found for the small B‐ and C‐type granules relative to crumb score, loaf volume, amylose, and RVA final pasting viscosity for cultivars grown in 1999, and to RVA pasting temperature for samples grown in 1998. The environmental impact on spelt properties seemed to have a greater effect than genetic control.     

Susceptibility of Waxy Starch Granules to Mechanical Damage

Starch samples isolated from wheat flour that represented four possible waxy states (0, 1, 2, and 3‐gene waxy) were subjected to crushing loads under both dry and wet conditions. Calibrated loads of 0.5–20 kg were applied to the starch samples and the percentage of damaged granules was visually determined. Under dry crushing conditions, starches containing amylose (0, 1, and 2‐gene waxy) had between 1% (5‐kg load) to 3% (15‐ and 20‐kg load) damaged granules, whereas waxy starch (3‐ gene waxy; <1% amylose) began rupturing at 0.5‐kg load (3.5% damaged granules) and had 13% damaged granules when ≥10‐kg load was applied. Under wet crushing conditions, normal and partial waxy starch (0, 1, and 2‐gene waxy) showed little difference in percentage of damaged granules when compared to the results of dry crushing. Waxy starch (3‐gene waxy), however, showed substantially increased numbers of damaged granules: 12% damaged granules at 0.5‐kg load, rising to 55% damaged granules at 15‐kg load. The results indicate that waxy starch granules are less resistant to mechanical damage than normal starch granules. Furthermore, blends of normal and waxy wheats or wheat flours intended to have a particular amylose‐amylopectin ratio will be a complex system with unique processing and formulation considerations and opportunities.     

Physicochemical and Enzyme Characterization of Small and Large Starch Granules Isolated from Two Maize Cultivars

In this study, some morphological, physicochemical, and the initial characterization of the starch granule enzymes of blue and white maizes of small and large granules are described. Starch was isolated from blue and white maizes, and the small and large granules were separated. The efficiency of separation was higher in large granules than small of the blue maize starch. The amylose content was slightly greater (1.3%) in large granules than smaller in both starches studied. No differences in the average gelatinization temperature were found between large and small granules, but the enthalpy of gelatinization value was higher in the small granules. The electrophoretic pattern showed that the granule‐bound starch synthase (GBSSI) had higher expression in large than small granules and that explain the higher amylose content in the former granules. The differences showed in the starch biosynthesis enzymes in small and large granules might explain partially the physicochemical and functional properties of maize.     

Why do gelatinized starch granules not dissolve completely? Roles for amylose, protein, and lipid in granule "ghost" integrity

After gelatinization in water, starch granules persist in swollen hydrated forms known as ghosts. Three potential mechanisms for ghost formation are tested. Proteins and lipids on the granule surface are found to be a determinant of ghost robustness, but not ghost formation. Proteins inside pre-made maize or wheat starch ghosts are degraded extensively by proteases without any apparent change in ghost properties, making an internal protein cross-linking mechanism unlikely. Waxy maize mutants with a range of amylose contents have ghost integrities that correlate with (low) apparent amylose levels. It is hypothesized that ghost formation is due to cross-linking of polysaccharide chains within swollen granules, most likely involving double helices formed from polymer chains that become free to move following heat-induced granule swelling. The size and robustness of granule ghosts is proposed to be determined by the relative rates of swelling and cross-linking, modulated by surface non-polysaccharide components.     

Effect of Partial Gelatinization and Lipid Addition on α‐Amylolysis of Barley Starch Granules

The effect of partial gelatinization with and without lipid addition on the granular structure and on α‐amylolysis of large barley starch granules was studied. The extent of hydrolysis was monitored by measuring the amount of soluble carbohydrates and the amount of total and free amylose and lipids in the insoluble residue. Similarly to the α‐amylolysis of native large barley starch granules, lipid‐complexed amylose (LAM) appeared to be more resistant than free amylose and amylopectin. Partial gelatinization changed the hydrolysis pattern of large barley starch granules; the pinholes typical of α‐amylase‐treated large barley starch granules could not be seen. Lipid addition during partial gelatinization decreased the formation of soluble carbohydrates during α‐amylolysis. Also free amylose remained in the granule residues and mostly amylopectin hydrolyzed into soluble carbohydrates. These findings indicate that lysophospholipid (LPL) complexation with amylose occurred either during pretreatment or after hydrolysis, and free amylose was now part of otherwise complexed molecules instead of being separate molecules. Partial gelatinization caused the granules to swell somewhat less during heating 2% starch‐water suspensions up to 90°C, and lipid addition prevented the swelling completely. α‐Amylolysis changed the microstructure of heated suspensions. No typical twisting of the granules was seen, although the extent of swelling appeared to be similar to the reference starch. The granules with added LPL were partly fragmented after hydrolysis.     

Differences in Starch Granule Composition and Structure Influence In Vitro Enzymatic Hydrolysis of Grain Meal and Extracted Starch in Two Classes of Canadian Wheat (Triticum aestivum L.)

Starch granule composition and amylopectin structure affect starch digestibility, an important factor influencing wheat grain utilization for human food consumption. Six bread wheat cultivars with four belonging to the Canada Western Red Spring (CWRS) and two Canada Prairie Spring Red (CPSR) market classes were analyzed for the relationship between their grain constituents and in vitro enzymatic hydrolysis of starch. CPSR cultivars had higher starch and amylose concentrations compared with CWRS cultivars, which had a higher protein concentration. Starch granule size distribution did not differ among the genotypes, except AC Foremost, which had significantly (P < 0.05) higher volume percent of B‐type starch granules (≈15%) and lower volume percent of A‐type starch granules (≈9%) compared with other cultivars. Fluorophore‐assisted capillary electrophoresis revealed a lower content of R‐IV (DP 15–18, ≈6%) and a higher content of R‐VII (DP 37–45, ≈7%) chains in the CPSR cultivars compared with the CWRS cultivars. Starch in vitro enzymatic hydrolysis showed that compared with CWRS cultivars, the two CPSR cultivars had reduced amounts of readily digestible starch and higher amounts of slowly digestible starch and resistant starch. Consequently, the two CPSR cultivars also showed lower hydrolysis indexes in grain meal as well as extracted starch. CPSR cultivars, with higher starch and amylose concentrations, as well as a higher content of long chains of amylopectin, showed a reduced starch in vitro enzymatic hydrolysis rate.     

Physicochemical Properties and In Vitro Starch Digestibility of Cooked Rice from Commercially Available Cultivars in Canada

The influence of amylose content, cooking, and storage on starch structure, thermal behaviors, pasting properties, and rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) in different commercial rice cultivars was investigated. Long grain rice with high‐amylose content had a higher gelatinization temperature and a lower gelatinization enthalpy than the other rice cultivars with intermediate amylose content (Arborio and Calrose) and waxy type (glutinous). The intensity ratio of 1047/1022 cm^–1 determined by Fourier Transform Infrared (FT‐IR), which indicated the ordered structure in starch granules, was the highest in glutinous and the lowest in long grain. Results from Rapid ViscoAnalyser (RVA) showed that the rice cultivar with higher amylose content had lower peak viscosity and breakdown, but higher pasting temperature, setback, and final viscosity. The RDS content was 28.1, 38.6, 41.5, and 57.5% in long grain, Arborio, Calrose, and glutinous rice, respectively, which was inversely related to amylose content. However, the SDS and RS contents were positively correlated with amylose content. During storage of cooked rice, long grain showed a continuous increase in pasting viscosity, while glutinous exhibited the sharp cold‐water swelling peak. The retrogradation rate was greater in rice cultivars with high amylose content. The ratio of 1047/1022 cm^–1 was substantially decreased by cooking and then increased during storage of cooked rice due to the crystalline structure, newly formed by retrogradation. Storage of cooked rice decreased RDS content and increased SDS content in all rice cultivars. However, no increase in RS content during storage was observed. The enthalpy for retrogradation and the intensity ratio 1047/1022 cm^–1 during storage were correlated negatively with RDS and positively with SDS (P ≤ 0.01).     

Associations of Starch Gel Hardness,Granule Size,Waxy Allelic Expression,Thermal Pasting,Milling Quality,and Kernel Texture of 12 Soft Wheat Cultivars

Starches were isolated from 12 soft wheat (Triticum aestivum L.) cultivars and were characterized for waxy (Wx) allelic expression, thermal pasting characteristics, and starch granule size. Gels were produced from the thermally degraded starches and were evaluated using large deformation rheological measurements. Data were compared with cultivar kernel texture, milling characteristics, starch chemical analyses, and flour pasting characteristics. Larger flour yields were produced from cultivars that had larger starch granules. Flour yield also was correlated with lower amylose content and greater starch content. Harder starch gels were correlated with higher levels of amylose content and softer kernel texture. The cultivar Fillmore, which had a partial waxy mutation at the B locus, produced the highest peak pasting viscosity and the lowest gel hardness. Softer textured wheats had greater lipid‐complexed amylose and starch phosphorus contents and had less total starch content. Among these wheats of the soft market class, softer textured wheats had larger starch granules and harder textured wheats had smaller starch granules. In part, this may explain why soft wheats vary in texture. The smaller granules have larger surface area available for noncovalent bonding with the endosperm protein matrix and they also may pack more efficiently, producing harder endosperm.     

Morphological Changes of Granules of Different Starches by Surface Gelatinization with Calcium Chloride

Native starch granules of 11 selected cultivars (potato, waxy potato, sweet potato, normal maize, high‐amylose maize, waxy maize, wheat, normal barley, high‐amylose barley, waxy barley, and rice) were treated with a calcium chloride solution (4M) for surface gelatinization. The surface‐gelatinized starch granules were investigated using light microscopy and scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). In general, those starches with larger granule sizes required longer treatment time to complete the gelatinization. The salt solution treatment of starch was monitored by light microscopy and stopped when the outer layer of the granule was gelatinized. The surface gelatinized starch granules were studied using scanning electron microscopy. On the basis of the gelatinization pattern from calcium chloride treatments, the starches could be divided into three groups: 1) starches with evenly gelatinized granule surface, such as normal potato, waxy potato, sweet potato, maize, and high‐amylose maize; 2) starches with salt gelatinization concentrated on specific sites of the granule (i.e., equatorial groove), such as wheat, barley, and high‐amylose barley; and 3) starches that, after surface gelatinization, can no longer be separated to individual granules for SEM studies, such as waxy barley, waxy maize, and normal rice. The morphology of the surface gelatinized starch resembled that of enzyme‐hydrolyzed starch granules.     

Comparison of Flour Starch Properties in Half‐Sib Families of Opaque‐2 Maize (Zea mays L.) from Argentina

Since the discovery of the o2 mutation in maize, many studies have reported the characterization of the protein quality of opaque‐2 genotypes. However, few have reported the properties of their starch. The objective of this study was to characterize flour starch properties of 12 half‐sib families of opaque‐2 maize from Argentina. Chemical composition and thermal and pasting properties of whole grain flour were determined. Nonopaque genotypes were used as a control. Starch content of opaque‐2 genotypes did not show significant differences compared with nonopaque genotypes, yet amylose content was significantly lower. A high variability in pasting and thermal properties was observed in genotypes. Opaque samples showed a significantly higher peak viscosity and a lower pasting temperature compared with nonopaque samples, probably owing to larger and less compact starch granules in the floury endosperm. The higher the gelatinization enthalpy of opaque‐2 genotypes was, the lower the amylose content in relation to nonopaque varieties. Two retrogradation endotherms were observed in DSC analysis: one corresponding to amylopectin crystallization and the other to melting of amylose‐lipid complex. Both enthalpies were considered total starch retrogradation (ΔH _RT). A wide range of variation was obtained in ΔH _RT in opaque‐2 genotypes, but no significant differences between opaque and nonopaque genotypes were observed. The differences in starch properties found in this study would make it possible to identify opaque‐2 families with particular characteristics for the development of starchy food items adapted to specific processing traits.     

Starch and By‐Products from a Laboratory‐Scale Barley Starch Isolation Procedure

Starch was isolated from three different barleys with normal, highamylose, or high‐amylopectin (waxy) starch. The laboratory‐scale starch isolation procedure included crushing of grains, steeping, wet milling, and sequential filtration and washing with water and alkali, respectively. Yield and content of starch, protein, and dietary fiber, including β‐glucan, were analyzed in isolated starch and in the by‐products obtained. Starch yield was 25–34%, and this fraction contained 96% starch, 0.2–0.3% protein, and 0.1% ash. Most of the remaining starch was found in the coarse material removed by filtration after wet milling, especially for the high‐amylose barley, and in the starch tailings. Microscopy studies showed that isolated starch contained mostly A‐granules and the starch tailings contained mostly B‐granules. Protein concentration was highest in the alkali‐soluble fraction (54%), whereas dietary fiber concentration was highest in the material removed by filtration after alkali treatment for the normal and waxy barleys (55%). The β‐glucan content was especially high for the waxy barley in this fraction (26%). The study thus showed that it was possible to enrich chemical constituents in the by‐products but that there were large differences between barleys. This result indicates a need for modifications in the isolation procedures for different barleys to obtain high yields of starch and different by‐products. Valuable by‐products enriched in β‐glucan or protein, for example, may render starch production more profitable.     

Investigation of Digestibility In Vitro and Physicochemical Properties of A‐ and B‐Type Starch from Soft and Hard Wheat Flour

In this study, the functional properties of A‐ and B‐type wheat starch granules from two commercial wheat flours were investigated for digestibility in vitro, chemical composition (e.g., amylose, protein, and ash content), gelatinization, retrogradation, and pasting properties. The branch chain length and chain length distribution of these A‐ and B‐type wheat starch granules were also determined using high‐performance anion exchange chromatography (HPAEC). Wheat starches with different granular sizes not only had different degrees of enzymatic hydrolysis and thermal and pasting properties, but also different molecular characteristics. Different amylose content, protein content, and branch chain length of amylopectin in A‐ and B‐type wheat starch granules could also be the major factors besides granular size for different digestibility and other functional properties of starch. The data indicate that different wheat cultivars with different proportion of A‐ and B‐type granular starch could result in different digestibility in wheat products.