Development and Utilization of Reflectance Confocal Laser Scanning Microscopy to Locate Reaction Sites in Modified Starch Granules

A new method of locating reaction sites within modified starch granules was developed and applied. The method involves converting anionic groups introduced into starch granules into their silver salt form, reducing the silver cations to silver atoms, and locating the silver atoms by means of reflectance confocal laser scanning microscopy. The method was tested on three types of starch (normal maize, waxy maize, potato) containing two types of derivatizing groups (mono‐ and distarch phosphate ester groups and the 2‐hydroxy‐3‐sulfonylpropyl ether group). The method also revealed anionic sites in native granules, presumably due to proteins, ionic lipids, or native phosphate ester groups.     

Packing Characteristics of Starch Granules

The rheological properties of granular materials and dispersions of solid particles in fluids are dependent on the packing characteristics of the particles. Maximum packing fractions (Φ_m) have been measured for corn, wheat, rice, potato, and amaranth starches, in the dry state and dispersed in either ethanol or hexane, using a tapping method. The observed maximum packing fraction increases with tapping time to a constant value. Values measured for dry starches were lower than those measured in liquids and reflect the effects of granule shape and intergranular friction. Values measured in fluids for potato, corn, and wheat starches were all similar in magnitude, and in the range of values (0.58–0.63) for random loose packing and random close packing of monodisperse spheres. Values for amaranth and rice starches were significantly lower due to agglomeration and clumping of individual granules. Blends of corn and potato starches show a slight enhancement of packing, with some Φ_m values greater than potato starch, consistent with data for bimodal blends of spheres. Blends of rice and potato starches displayed enhanced packing above ideal mixing but did not exceed the packing fraction of the potato starch. Knowledge of starch packing fractions is required for fundamental understanding of the rheological properties of granular starch‐filled materials and important for predicting processing characteristics.     

Surface Derivatization of Corn Starch Granules

Commercial corn starch, treated with stearoyl chloride in carbon tetrachloride and triethylamine, is surface-derivatized without noticeable swelling. Granules that are reacted with chloroacetic acid under similar conditions undergo surface etherification, visibly identified by their color binding with fuchsin. Interestingly, the dye experiment indicates surface-derivatization as well as derivatization of the granule central interior, suggesting reagent penetration to the granule interior through pores and derivatization of a lower density porous center. Esterification of corn starch granules treated with glucoamylase have five times greater esterification than normal corn starch granules. Placed in water, these and palmitoylated granules have a fatty feel to the fingers. Stearoylated corn and amaranth starches showed indications for use as fat replacers in frozen desserts.     

Enthalpic Transitions in Native Starch Granules

Crystalline properties of native starch granule fractions that varied in apparent density and size were investigated using differential scanning calorimetry (DSC). Endotherms obtained at 80% hydration showed significant variations in enthalpy between the six fractions. Typical bi-phasic endotherms exhibiting significant variation in start temperature were obtained for the six fractions at 50% hydration. However, on annealing at 50°C/50% hydration for 48 hr, all fractions showed a single endotherm without any significant variability in endotherm characteristics. At 10% hydration, the six fractions exhibited single high-temperature endotherms with significant differences in their peak temperatures. It was observed that mechanically damaging starch, resulted in the disappearance of any enthalpic transition. These observations could not be satisfactorily explained on the basis of prevailing concepts about DSC enthalpic transitions.     

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.     

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.     

Accessibility of Starch Granules to Fatty Acyl Amides

Microscopic techniques were used to determine accessible regions in various starches under different swelling conditions. Fatty acyl chlorides of increasing chain length (C₆–C₁₆) were reacted with 2‐aminopyridine to produce the corresponding fatty acyl amides. Starch granules (common corn, waxy maize, potato) were treated with the series of fluorescent amides under a variety of conditions: anhydrous (hot pyridine), aqueous (no heat), and aqueous alkali (49°C, with and without swelling‐inhibiting salts). Isolated granules were then viewed by confocal laser scanning microscopy to determine reagent analog penetration. All populations of granules were heterogeneous with respect to fatty acyl amide penetration, but general patterns could be discerned. Observations also indicated that the area surrounding the hilum was more easily penetrated than was the rest of the granule matrix. No substantial differences in penetration of the fluorescent fatty acyl amides as the chain length increased (C₆–C₁₆ was observed in hot pyridine‐swollen common corn starch granules. Common corn and potato starch granules swollen in room temperature water showed cutoffs for granular exclusion at C₁₄ and C₁₂, respectively. Common corn, waxy maize, and potato starch granules treated under industrial etherification conditions (heat, pH ≈ 11, swelling‐inhibiting salts) were less accessible to C₆, C₈, and C₁₀ fluorescent amides when sodium citrate was present than when sodium sulfate was used, and less accessible in either case than in water alone or in hot anhydrous pyridine. However, appreciable differences between inhibition by sodium sulfate and sodium citrate were not observed in every case.     

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.     

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.     

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.     

Role of Starch Granules in Controlling Expansion of Dough During Baking

The role of starch granules in the expansion of doughs during baking was investigated using artificial flours made from dry vital wheat gluten and wheat starch, potato starch, or tapioca starch. The three starches were selected because of their diverse gelatinization properties. Baking tests on flour from tapioca starch gave the largest loaf volume and the most extensive postbaking shrinkage. Potato starch flour gave the smallest volume and the least shrinkage. Amylograph test data, dough expansion under decreased pressure, progress of expansion during baking, and scanning electron microscopy revealed the role starch granules play in ideal baking conditions. Starch granules should not gelatinize early in the baking cycle as potato starch does but should gelatinize later in the baking cycle as wheat starch does. This prevents early setting of the dough which inhibits expansion. Starch granules should not disrupt and fuse together during gelatinization as tapioca starch does, forming an impermeable gas membrane. Granules should gelatinize individually as wheat starch does, causing a disruption of cell membranes which prevents shrinkage of the loaf during cooling after baking.     

Effects of Various Surfactants on Rheological Properties of Maize Starch Granules

In this study, the formation of complexes between surfactants and the helical chains of amylopectins was confirmed. Nonionic surfactants with hydrophobic and hydrophilic groups of appropriate size and chemical structure enhanced the swelling and gelatinization processes of starch granules. Hydrophobic groups form complexes with the amylose and linear chains of amylopectin by becoming inserted into the hydrophobic inner area of the helical structures. The hydrophilic groups help the approach of the hydrophobic groups into the hydrated molecular chains and thus aid the formation of the complex. Among the anionic surfactants tested, SDS and sodium n‐decyl benzenesulfate caused maximum swelling and gelatinization peaks. The average length of the amylopectin exterior chains is almost the same as that of the hydrophobic chains of SDS (16.9 Å) and of sodium decyl benzenesulfate (18.2 Å). This suggests that these anionic surfactants form rigid complexes with the exterior of the amylopectin by fitting their hydrophobic chains to the hydrophobic inside of the helical structures of these short exterior chains. This process was clarified by NMR analysis and by a decrease in the complex with the addition of iodine. The hydrophobic alkyl chains of anionic and cationic surfactants fix to the edge of the starch molecular chains by forming inclusion complexes with the helical chains of the amylopectin. Cationic ions interact with the starch molecular chains, causing a negative charge that results in a more rapid and efficient swelling of the starch granules. A decrease in setback value occurs due to the inhibition of rearrangement among the starch molecular chains. With SDS, the complex molecular chains become more extensively developed through the repulsion effects of the anionic ions resulting in a larger swelling power and gelatinization peak.     

Visualization of Channels and Cavities of Corn and Sorghum Starch Granules

Evidence is presented that corn (maize) and sorghum starch granules have channels that connect a central cavity to the external environment. A mutant sorghum starch with numerous, large surface pores was treated with a methanolic solution of merbromin and sectioned. Light, fluorescence, and compositional backscattering electron microscopy revealed channels connecting an internal cavity to the external surface in most granules. Cavities and channels could also be seen in whole corn and sorghum starch granules treated with merbromin in methanol and viewed by fluorescence microscopy. Treatment of sorghum starch granules with an aqueous solution of merbromin revealed that the molecule penetrated the granule matrix under even slightly swelling conditions. Light microscopy showed cavities in unstained, whole, dry corn and sorghum starch granules mounted in immersion oil.     

α-Amylolysis of Large Barley Starch Granules

The α-amylolysis of large (volume average 16 μm) barley starch granules was studied by measuring the amount of carbohydrates solubilizing during hydrolysis, and the changes in morphology and molecular structure of the granule residues by scanning electron microscopy, particlesize analysis, size-exclusion chromatography, X-ray diffraction, and differential scanning calorimetry. X-ray diffraction showed that, in the earlier stages of α-amylolysis, both amorphous and crystalline parts of the granules were equally solubilized. More extensive hydrolysis caused a gradual decrease in A-type crystallinity and degradation of the granular structure. Scanning electron microscopy revealed that hydrolysis proceeded through pinholes, and pitted and partially hollow granule residues were formed. The lipid-complexed amylose was less susceptible to α-amylolysis than free amylose and amylopectin. Lipid-complexed amylose started leaching out of the granule residues only after half of the starch had solubilized due to the α-amylase treatment. Even though scanning electron microscopy indicated that there were intact granules left throughout the hydrolysis, the results obtained suggested that α-amylolysis of large barley starch granules proceeded rather evenly among the granules.     

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.     

Isolation of Small Starch Granules and Determination of Their Fat Mimic Characteristics

Small starch granules from wheat and amaranth were isolated and examined by light microscopy (wheat), scanning electron microscopy (wheat and amaranth), and rapid viscosity (RVA) analysis. The isolated wheat and amaranth starches were incorporated into reduced‐fat frozen dessert mixes (2%, w/w) and the frozen desserts were characterized by sensory analysis for smoothness, creaminess, and preference. The results of the sensory analysis suggest that up to 50% of the fat in a frozen dessert may be replaced when substituted by these starches at the 2% level (w/w).     

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     

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.