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.     

Fragmentation of Waxy Rice Starch Granules by Enzymatic Hydrolysis

Small starch particles were prepared by hydrolyzing waxy rice starch using α‐amylase and then ultrasonicating in ethanol. Differential scanning calorimetry (DSC) revealed that a mild hydrolysis for 3 hr increased the melting enthalpy of the starch, which might indicate that the hydrolysis was selective in the amorphous regions. Later, at 6–24 hr, the hydrolysis rate was reduced, with gradual decreases in DSC melting enthalpy, indicating that the crystalline regions were eroded simultaneously. X‐ray diffraction patterns revealed the same trend as the DSC results. Average diameter of starch granules or particles was decreased dramatically in both volume‐ and number‐based measurements (5.94→1.64 μm, and 0.45→0.18 μm, respectively) during the early stage of rapid hydrolysis (up to 3 hr). Native waxy rice starch exhibited a particle size distribution with a major peak at 5.6 μm. After hydrolysis for 3 hr, the volume distribution of starch granules changed to two major size peaks at 0.5 and 3.6 μm. The starch fragment of 0.5 μm was assumed to consist of crystalline blocklets. With excessive hydrolysis (24 hr) or ultrasonication, however, starch particle diameter was increased, indicating that the particles might be swollen or aggregated into clusters.     

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.     

Molecular Structure and Organization of Starch Granules from Developing Wheat Endosperm

Wheat starches isolated from seeds harvested between 7 and 49 days after anthesis (DAA) were fractionated into large (>8 μm) and small (<8 μm) granules and studied for starch structure and architecture. Starch granules at 7 DAA possessed unimodal size distribution, whereas it was bimodal at later maturity stages. The apparent amylose fraction of starch granules at early maturity (7 and 14 DAA) consisted of intermediate‐type materials, whereas starch at later maturity stages (28 and 49 DAA) contained branched amylose. Wide‐angle X‐ray scattering (WAXS) revealed a well‐developed polymorphic structure already at 7 DAA. Although the presence of a small proportion of B‐type crystallites mixed with A‐type crystallites was observed in the X‐ray diffractogram of starches at early maturation (7 and 14 DAA), it was masked by the A‐type crystallites at later maturity stages. However, the large granules had a higher proportion of B‐type crystallites and lower relative crystallinity (RC) than their small‐granule counterpart. The iodine absorption properties of the starch granules demonstrated different levels of mobility of the starch polymers at different stages of maturity and the mobility of more glucan polymers in the large granule population compared with the small granules at the same maturity stage. Iodine did not change the characteristic A‐type crystalline pattern of starch, but it increased RC. Changes in peak width at half height based on WAXS data further suggested the possible interaction of iodine with amylopectin intercluster chain segments and branch chains in formation of inclusion complexes.     

Enzymatic and Acidic Hydrolysis of Cationized Waxy Maize Starch Granules

A series of wet‐cationized starch granules from waxy maize with different degrees of substitution (DS) were solubilized with either 2.2M HCl (lintnerization) or with the α‐amylase of Bacillus amyloliquefaciens. The maximum rate of the enzymatic hydrolysis occurred in starches with intermediate DS. It appeared that the cationic substituents interfered with the binding to the active site of the enzyme at high levels of substitution. The DS remained fairly constant in the granular residues after the enzymatic attack. The rate of the acidic hydrolysis increased with increasing DS but the final level of solubilization slightly decreased. The DS of the residual starch material decreased to 40% of the original level, showing that a large part of the cationic groups was found within the amorphous parts of the granules. A dry‐cationized sample with a high DS was also treated with the acid and lost a major part of its substituents at low levels of lintnerization. Probably most of the substituents were associated with the surface and channels of these granules. The cationized starches possessed branches that were resistant to isoamylase attack and the samples also contained β‐amylolysis resistant dextrins. The proportion of resistant dextrins in the granular residues decreased after lintnerization, but remained constant after the enzymatic hydrolysis.     

Composition and Reactivity of A‐ and B‐type Starch Granules of Normal,Partial Waxy,and Waxy Wheat

Wheat has great potential to make inroads into starch markets with the advent of partial waxy and waxy starches of diverse composition and properties. The majority of isolated starch utilized in food applications is chemically modified to improve starch properties according to the intended use. Therefore, it is critical to understand factors that affect wheat starch reactivity. This work investigated the relative reactivities of normal, partial waxy, and waxy wheat starches and their respective A‐ and B‐type starch granule fractions. Native starch isolated from four closely related soft wheat lines (normal, partial waxy, and full waxy) was modified through 1) substitution (propylene oxide analog) and 2) cross‐linking (phosphorus oxychloride) reactions to generate both types of modified starch products for each wheat line. Characterization of the unmodified starch fractions confirmed compositional differences among the cultivars and their respective granule types. In cross‐linking reactions, B‐type granules were slightly more reacted than A‐type granules for all cultivars, while the waxy starch generally exhibited higher reactivity compared with normal and partial waxy starches. For the substituted starches, no differences in reactivity were observed among the cultivars or between the two granule types.     

Study of Three-Dimensional Structure of Wheat Starch Granules Stained with Remazolbrilliant Blue Dye and Extracted with Aqueous Sodium Dodecyl Sulfate and Mercaptoethanol Solution

Wheat starch granules were obtained from soft wheat flour by acetic acid fractionation (pH 3.5), and the starch was stained by reaction with Remazolbrilliant blue (RBB) dye. RBB-stained starch was extracted with 1% sodium dodecyl sulfate (SDS) and 1% 2-mercaptoethanol (ME) for 14.5 hr at room temperature. This extraction step was repeated five times (extracts 1–5). SDS-ME extracts were subjected to size-exclusion column chromatography, and comparisons of their profiles for specific absorbance at 650 nm (A₆₅₀) and carbohydrates were made. After high molecular weight (HMW) carbohydrates on the starch granule surface were extracted, HMW carbohydrates inside the granule appeared to be extracted. Finally, low molecular weight (LMW) carbohydrates near the granule surface were extracted. Phase-contrast light microscopy of the treated starch granules showed that all granules became transparent. Two different interior structures were observed. Scanning electron microscopy indicated that the granule was split into two parts at the equatorial groove. The interior of the granule showed two different areas: a central hole area and the surrounding stratified area. Extraction beyond five times with the same solvent dissolved the weak part of the granule structure and left two types of skeletal structures. The appearance of the skeletal structure of the granule surface was different from the appearance of interior structures.     

Structure of Lysophosphatidylglyceryl-Remazolbrilliant Blue (LPG-RBB) from the Surface of Dyed Wheat Starch Granules

Wheat starch granule surface was covalently stained with Remazolbrilliant blue-R dye (RBB) and then extracted with 1% SDS containing 1% 2-mercaptoethanol (2-ME) at room temperature for 14.5 hr. The extracted blue-staining material (A₆₅₀) separated into two fractions. Low molecular weight (LMW) material was further purified by Sephadex G-75 size-exclusion chromatography and thin-layer chromatography. Infrared and nuclear magnetic resonance (¹H-NMR and ¹³C-NMR) spectroscopy indicated that the structure of the purified LMW material was 18-O-(6-lysophosphatidylglyceryl)-RBB.     

In Vitro Binding of Puroindolines to Wheat Starch Granules

Puroindoline (pin) preparations made from flours of hard and soft wheats contained a mixture of pin‐a, 0.19/0.53 α‐amylase inhibitor, and purothionins. Starch granule preparations from the same cultivars were treated with proteinase to remove surface proteins and incubated with solutions of the pin preparations. Binding of pin‐a and purothionins but not the 0.19/0.53 inhibitor was observed with no apparent differences between the behavior of the pin preparations or starch granule preparations from hard or soft types. No binding was observed when several other proteins (bovine serum albumin, total albumins, a commercial preparation of wheat α‐amylase inhibitors, and barley β‐amylase) were incubated with the starch granules under the same conditions, indicating that in vitro binding can be used to study specific starch granule and protein interactions.     

Effect of Small and Large Wheat Starch Granules on Thermomechanical Behavior of Starch

The physicochemical properties of small‐ and large‐granule wheat starches were investigated to reveal whether gelatinization properties and rheological behavior differ between size classes of wheat starch. All samples contained 60% water (w/w, wb). The starch granule size and shape were examined by scanning electron microscopy in the separated A‐ and B‐type granule populations and in the whole wheat starch granule population. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) analyses were performed in parallel with rheological measurements using dynamic mechanical thermal analysis (DMTA) to relate the viscoelastic changes to modifications in dynamic properties of aqueous solutions and structural disorganization of starch. The small (B‐type) granules had slightly higher gelatinization temperature and lower gelatinization enthalpy than did the large (A‐type) granules. Also, B‐type granules had higher enthalpy for the amylose‐lipid complex transition. Moreover, our results suggested that small granules have higher affinity for water at room temperature. It seems that there is a less ordered arrangement of the polysaccharide chains in the smaller granules when compared with the larger ones. These differences in functional properties of small and large granules suggested that the granule size distribution is an important parameter in the baking process.     

Heat-Induced Fragmentation of the Maize Waxy Protein During Protein Extraction from Starch Granules

The starch granule of maize contains a characteristic set of tightly bound polypeptides. Granule-associated polypeptides are typically extracted from starch granules by heating starch granule suspensions at 90–100°C in a detergent such as SDS. Solubilized proteins are recovered by centrifugation and analyzed by gel electrophoresis. Previously identified tightly bound granule intrinsic proteins consist of the 85-kDa starch-branching enzyme IIb, the 76-kDa starch synthase I, and the 60-kD waxy (Wx) protein, also known as granule-bound starch synthase I. However, SDS extracts from starch granules of maize also contain a cluster of proteins ranging in mass between 47 and 32 kDa In this study, we analyzed this group of granule-associated proteins and found that each was recognized by the Wx antibody. A 15 amino acid N-terminal sequence from the 47-kDa polypeptide was identical to the predicted N-terminus of the Wx protein. Further analysis revealed that each immunoreactive polypeptide between 47 and 32 kDa was a heat-induced fragmentation product of the Wx protein. Conditions for the extraction of granule proteins were evaluated. Our results demonstrate that granule proteins are effectively released by mild extraction (10-min incubation at 72°C). Relative to the Wx protein, starch synthase I and starch branching enzyme IIb were less susceptible to thermal fragmentation. These results demonstrate that the 85-, 76-, and 60-kDa polypeptides are authentic granule-intrinsic proteins, and that the majority of polypeptides between 47 and 32 kDa are artifacts of high-temperature granule extraction procedures.     

Presence of a Minor Amylose Fraction in the Central Portion of Waxy Wheat Starch Granules and Its Contribution to Granular Stability

When wheat starch granules containing various amounts of amylose (2.1–25%) were stained with 25% KI/10% I₂ solution, the granules largely changed to ghost structures below ≈5.0% amylose. The ghost showed a typical double structure: a black‐brown central portion and a red‐brown surrounding portion. The proportion of the black‐brown central portion in the ghost was strongly correlated to the amylose content (%) in the starch, that is, the black‐brown central portion decreased with a decrease in amylose. This suggests that amylose is possibly present in the black‐brown central portion. Sonication (20 kHz) followed by centrifugation of the ghost separated the black‐brown central portion from the red‐brown surrounding portion, and the amylose content in each portion was determined. The results indicated that the amylose content in the black‐brown central portion was 6.9%, whereas in the surrounding portion, it was only 1.0%. Furthermore, the central and surrounding portions were subjected to Sepharose CL‐2B gel‐filtration column chromatography and the presence of amylose could only be observed in the black‐brown central portion.     

Impact of Waxy,Partial Waxy,and Wildtype Wheat Starch Fraction Properties on Hearth Bread Characteristics

Thirteen different wheat cultivars were selected to represent GBSS mutations: three each of wildtype, axnull, and bxnull, and two each of 2xnull and waxy. Starch and A‐ and B‐granules were purified from wheat flour. Hearth bread loaves were produced from the flours using a small‐scale baking method. A‐granules purified from wildtype and partial waxy (axnull, bxnull, and 2xnull) starches have significantly higher gelatinization enthalpy and peak viscosity compared with B‐granules. A‐ and B‐granules from waxy starch do not differ in gelatinization, pasting, and gelation properties. A‐ and B‐granules from waxy starch have the highest enthalpy, peak temperature, peak viscosity, breakdown, and lowest pasting peak time and pasting temperature compared with A‐ and B‐granules from partial waxy and wildtype starch. Waxy wheat flour has much higher water absorption compared with partial waxy and wildtype flour. No significant difference in hearth bread baking performance was observed between wildype and partial waxy wheat flour. Waxy wheat flour produced hearth bread with significantly lower form ratio, weight, a more open pore structure, and a bad overall appearance. Baking with waxy, partial waxy, and wildtype wheat flour had no significant effect on loaf volume.     

Separation and Characterization of A- and B-Type Starch Granules in Wheat Endosperm

Mature wheat (Triticum aestivum L.) endosperm contains two types of starch granules: large A-type and small B-type. Two methods, microsieving or centrifugal sedimentation through aqueous solutions of sucrose, maltose, or Percoll were used to separate A- and B-type starch granules. Microsieving could not completely separate the two types of starch granules, while centrifuging through maltose and sucrose solutions gave a homogenous population for B-type starch granules only. Centrifuging through two Percoll solutions (70 and 100%, v/v) produced purified populations of both the A- and B-type starch granules. Analysis of starch granule size distribution in the purified A- and B-type granule populations and in the whole-starch granule population obtained directly from wheat endosperm confirmed that the purified A- and B-type starch granule populations represented their counterparts in mature wheat endosperm. Centrifugations through two Percoll solutions were used to purify A- and B-type starch granule populations from six wheat cultivars. The amylose concentrations and gelatinization properties of these populations were analyzed. All of the A-type starch granules contained higher amylose concentrations and had higher gelatinization enthalpies than did B-type starch granules. Although A- and B-type starch granules started to gelatinize at a similar temperature, B-type starch granules had higher gelatinization peak and completion temperatures than did A-type starch granules     

Biochemical Characterization of the Wheat Waxy A Protein and Its Effect on Starch Properties

Granule bound starch synthase1 (GBSS1) is a key enzyme in amylose biosynthesis and is encoded by the A, B and D GBSS1 wx loci in wheat. Wheat lines with mutations at the three GBSS1 loci have been identified. We have characterized and compared the grain starch of CDCW6 wheat line (null B and D for GBSS1) with PI235238 (null A and B for GBSS1), waxy (null A, B and D for GBSS1), and AC Reed (wild type wheat) grain starches. The grain starch of waxy, CDCW6, PI235238, and AC Reed lines contained ≈0, 12, 23, and 25% amylose (w/w), respectively. Waxy, partially waxy, and wild wheat grain starches showed significant differences in onset and peak transition temperatures as determined by differential scanning calorimetric analysis. Grain starches extracted from waxy, CDCW6, and PI235238 also had higher enthalpy of gelatinization values than did wild wheat starch. X-ray diffraction analysis revealed the highest crystallinity for starch extracted from waxy wheat, followed by CDCW6. The starch produced from the CDCW6 line may find special food and industrial applications because of its relatively low amylose concentration.     

Effect of Wheat Starch Structure on Swelling Power

Starches were isolated from the endosperm of 12 wheat samples with a wide swelling power range in the wholemeal. Starch amylose content (24.8–34.2%) correlated negatively with the swelling power of isolated starch (18.3–26.9), but starch lipid content showed no such correlation. Higher proportions of long chains (DP ≥ 35) in amylopectins contributed to increased starch swelling. Native starch gelatinization temperatures and enthalpy measured by differential scanning calorimetry correlated positively with swelling power, which also correlated significantly with the regelatinization enthalpy of retrograded starches stored at 5°C for two and four weeks.     

Fine Structures of Amylose and Amylopectin from Large,Medium, and Small Waxy Barley Starch Granules

Amylose and amylopectin were prepared from large, medium, and small granule starches of classified waxy barley flour, and their fine structures were investigated. The amylose content had a wide distribution range (≈1.4–9.4%). Number‐average degrees of polymerization (DP_n) of the amyloses were similar among the samples (≈1,200–1,300). But number of chains per molecule (NC) decreased from the surface to the center (≈6–10 chains). DP_n of the amylopectins varied from 4,657 to 14,604; decreased in the order of large, medium, and small granules in same fractions of the grain; and increased from the surface layer to the center. Longest chains (LC) were not found in any of the amylopectin molecules. The large amylopectin molecule had more long chains and fewer A chains than the small molecule. The amylose content had definite effects on the transition temperature range and crystal formation of the starch granules. There were positive correlations between DP_n of the amylopectin and relative crystallinity (γ = +0.69) and enthalpy value (γ = +0.80), respectively. These findings may help to elucidate biosynthesis mechanism of starch.     

Jet Cooking of Waxy Maize Starch: Solution Rheology and Molecular Weight Degradation of Amylopectin

The effect of processing conditions in an excess steam jet cooker on the degradation of waxy maize starch was studied. The temperature of the steam, the flow rate of the starch slurry, and the concentration of starch were determined to influence the extent of degradation. The viscosity of concentrated solutions of the jet‐cooked product and the intrinsic viscosity of dilute solutions were used as measures of the extent of molecular degradation. The viscosity decreased at higher reaction temperatures, and at higher team‐to‐starch ratios. Multiple passes through the jet cooker decreased the viscosity dramatically for the first two passes, but little additional change was observed for further passes. The results show that mechanical and thermal degradation effects are both important in the jet cooking of waxy maize starch, although the primary effect is due to mechanical degradation.     

Physicochemical and Thermal Characteristics of Starch Isolated from a Waxy Wheat Genotype Exhibiting Partial Expression of Wx Proteins

A unique wheat genotype carrying waxy‐type allelic composition at the Wx loci, Gunji‐1, was developed, and its starch properties were evaluated in comparison to parental waxy and wild‐type wheat varieties. Gunji‐1 was null in all three of the Wx genes but exhibited a lower level of Wx proteins than the wild‐type. Starch amylose content and cold water retention capacity were 10.1 and 70.5% for Gunji‐1, 4.2 and 76.6% for waxy, and 27.9 and 65.0% for wild‐type, respectively. No significant differences were observed in microstructure, granule size distribution, and X‐ray diffractograms of the starch granules isolated from Gunji‐1 compared with those of waxy and wild‐type wheat varieties. Starch pasting peak, breakdown, and setback viscosities and peak temperature of Gunji‐1 were intermediate between waxy and wild‐type wheat. In starch gel hardness, Gunji‐1 (1.1 N) was more similar to waxy wheat (0.5 N) than to the wild‐type variety (17.6 N). Swelling power, swelling volume, paste transmittance during storage, and gelatinization enthalpy of Gunji‐1 were lower than those of waxy wheat but greater than those of wild‐type wheat. Retrogradation of starch stored for one week at 4°C expressed with DSC endothermic enthalpy was absent in the waxy wheat variety, whereas Gunji‐1 exhibited both retrogradation of amylopectin and amylose‐lipid complex melting similar to the wild‐type parent, even though enthalpies of Gunji‐1 were much smaller than the wild‐type parent.