Characteristics of Granular Cold‐Water Gelling Starches of Cereal Grains and Legumes Prepared Using Liquid Ammonia and Ethanol

Starches of wheat, corn, smooth and wrinkled peas, and chickpeas were modified to a free‐flowing powder of granular cold‐water gelling (GCWG) starch using liquid ammonia and ethanol at 23°C and atmospheric pressure. Amylose content of starches was 26.3% in wheat, 27.1% in corn, 35.4% in chickpeas, 43.2% in smooth peas, and 79.9% in wrinkled peas. The modified starches remained in granular form with an increased number of grooves and fissures on the surface of the granules compared with native starch, while the crystallinity was mostly lost, as shown by X‐ray diffractograms and DSC endothermic enthalpies. Pasting viscosity of modified starches at 23°C was 171 BU and 305 BU in wheat and corn, respectively, and much higher in legume starches, ranging from 545 BU to 814 BU. Viscosities of modified legume starches at 23°C were at least twice as high as those of native starches determined at 92.5°C. Swelling power of modified starches at 23°C ranged from 8.7 g/g to 15.3 g/g, while swelling power of native starches heated to 92.5°C ranged from 4.8 g/g to 16.0 g/g. GCWG starches exhibited higher dextrose equivalent (DE) values of enzymatic hydrolysis, ranging from 25.2 to 27.0 compared with native starches (1.5–2.9). Modified starches from wheat, corn, smooth peas, and chickpeas formed weak gels without heat treatment and experienced no changes in gel hardness during storage, while native starch gels formed by heat treatment showed an increase in hardness by 1.1–7.5 N during 96 hr of storage at 4°C.     

Evaluation of Liquid Nitrogen Freeze Drying and Ethanol Dehydration as Methods to Preserve Partially Cooked Starch and Masa Systems

Preservation of starch structure/properties, including structures formed during partial or complete cooking, are important when the impact of processing conditions is being studied. Two preservation techniques used to study changes in starch during thermal‐mechanical processing are commonly cited in the literature: 1) rapid freezing followed by lyophilization, and 2) a dehydration procedure using alcohols. A comparative determination on how these methods affect various starch structures has not been widely reported. Corn starch samples were collected from the Rapid Visco‐Analyser (RVA) at 3 min (swollen granules, 30°C), at the top of the pasting peak (gelatinized granules, 95°C), at the bottom of the trough (dispersed polymers, 95°C), and a completed RVA sample stored for 120 hr at 4°C (retrograded starch). Samples of masa were obtained by nixtamalizing corn. Differential scanning calorimetry (DSC) endotherms of starch and masa, and X‐ray diffraction (XRD) patterns of masa were evaluated after being preserved by alcohol‐ or freeze‐drying. No significant differences (P > 0.05) between methods were found for onset, end, and peak temperatures (°C), enthalpy (J/g) and % relative crystallinity in any of the samples analyzed. Liquid nitrogen freeze‐drying and ethanol dehydration are both effective methods of preserving various starch systems for structural changes detectible by DSC and XRD; freeze‐drying is generally less expensive and time‐consuming.     

Enzymatic Hydrolysis of Barley Starch Lipids

The efficiency of phospholipase and lipase preparations in the hydrolysis of lysophospholipids of native and gelatinized barley starch was examined. The degree of hydrolysis was analyzed by determination of the amount of released fatty acids by an enzymatic method. Thermal and structural properties of the enzyme-treated starch were studied by differential scanning calorimetry and light microscopy. Lysophospholipids of the gelatinized barley starch were easily hydrolyzed, in contrast to the lipids of the granular starch. The maximum degree of hydrolysis achieved for the gelatinized starch was 80% and for the native starch ≈20%. Gelatinization enthalpies and micrographs indicated that even though the amount of the released fatty acids from the native starch was small, formation of free fatty acids inhibited swelling and gelatinization of starch granules.     

Resistant Starch Escaped from Ethanol Production: Evidence from Confocal Laser Scanning Microscopy of Distiller's Dried Grains with Solubles (DDGS)

The origin of resistant starch (RS) in distiller's dried grains with solubles (DDGS) of triticale, wheat, barley, and corn from dry‐grind ethanol production was studied. A considerable portion of starch (up to 18% in DDGS) escaped from either granular starch hydrolysis or conventional jet‐cooking and fermentation processes. Confocal laser scanning microscopy revealed that some starch granules were still encapsulated in cells of grain kernel or embedded in protein matrix after milling and were thus physically inaccessible to amylases (type RS1). The crystalline structures of native starch granules were not completely degraded by amylases, retaining the skeletal structures in residual starch during granular starch hydrolysis or leaving residue granules and fragments with layered structures after jet‐cooking followed by the liquefaction and saccharification process, indicating the presence of RS2. Moreover, retrograded starch molecules (mainly amylose) as RS3, complexes of starch with other nonfermentable components as RS4, and starch–lipid complexes as RS5 were also present in DDGS. In general, the RS that escaped from the granular starch hydrolysis process was mainly RS1 and RS2, whereas that from the jet‐cooking process contained all types of RS (RS1 to RS5). Thus, the starch conversion efficiency and ethanol yield could be potentially affected by the presence of various RS in DDGS.     

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.     

Detection of Proteins in Starch Granule Channels

Proteins were detected in channels of commercial starches of normal maize, waxy maize, sorghum, and wheat through labeling with a protein‐specific dye and examination using confocal laser scanning microscopy (CLSM). The dye, specifically 3‐(4‐carboxybenzoyl)quinoline‐2‐carboxaldehyde (CBQCA), fluoresces only after it reacts with primary amines in proteins, and CLSM detects fluorescence‐labeled protein distribution in an optical section of a starch granule while it is still in an intact state. Starch granules in thin sections of maize kernels also had channel proteins, indicating that proteins are native to the channels and not artifacts of isolation. Incubation of maize starch with protease (thermolysin) removed channel proteins, showing that channels are open to the external environment. SDS‐PAGE analysis of total protein from gelatinized commercial waxy maize starch revealed two major proteins of about M_r 38,000 and 40,000, both of which disappeared after thermolysin digestion of raw starch. Commercial waxy maize starch granule surface and channel proteins were extracted by SDS‐PAGE sample buffer without gelatinization of the granules. The major M_r 40,000 band was identified by MALDI‐TOF‐MS and N‐terminal sequence analysis as brittle‐1 (bt1) protein.     

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.     

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.     

Granule Bound Starch Synthase I (GBSSI) Gene Effects Related to Soft Wheat Flour/Starch Characteristics and Properties

Eight soft spring wheat (Triticum aestivum L.) genotypes representing the four granule bound starch synthase I (GBSSI) classes were evaluated with respect to flour/starch characteristics and pasting behaviors. Native starch was isolated from genotype straight‐grade flours (94.8–98.1% of starch recovered) to approximate the starch populations of the parent flours. As anticipated, amylose characteristics varied among the genotypes according to GBSSI class and accounted for the primary compositional difference between genotypes. Total (TAM), apparent (AAM), and lipid‐complexed (LAM) amylose contents ranged from 1.0–25.5 g, 0.7–20.4 g, and 0.3–5.6 g/100 g of native starch, respectively, and gradually decreased with the progressive loss of active Wx alleles. In addition, genotype flour total starch (FTS) and A‐type starch granule contents, which ranged from 81.7–87.6 g/100 g of flour (db) and 61.6–76.8 g/100 g of native starch (db), respectively, generally decreased with an increase in waxy character in parallel with amylose characteristics, as likely secondary effects of Wx gene dosage. Though amylose characteristics predominantly accounted for the majority of genotype flour pasting properties, FTS content and ratios of A‐ to B‐type granules also exhibited significant influence. Thus, loss of one or more Wx genes appeared to induce measurable secondary effects on starch characteristics and properties.     

Association of Starch Granule Proteins with Starch Ghosts and Remnants Revealed by Confocal Laser Scanning Microscopy

Starch suspensions (0.25%) were gelatinized to 70 and 100°C, and starch ghosts (defined as gelatinized starch granule envelopes after the majority of internal starch polymers have been released) and remnants were collected by centrifugation and washed with water. Protein was revealed in isolated gelatinized normal starch ghosts using confocal laser scanning microscopy and a protein‐specific dye that fluoresces only after reaction with primary amines in protein. This technique eliminates background interference from residual dye. Observation of fluorescent‐labeled protein in the starch ghosts at different optical depths of field revealed that protein was concentrated in the envelopes of swollen, gelatinized potato, maize, and wheat starch ghosts. Only traces of protein were found in gelatinized starch granule remnants of waxy maize and amylose‐free potato starches after they were heated to 100°C, indicating that the proteins observed in gelatinized normal maize starch were largely granule‐bound starch synthase (GBSS). Moreover, fragility of the gelatinized waxy and amylose‐free starch granule remnants might be caused in part by the lack of GBSS. Gel electrophoresis of proteins in starch ghosts confirmed that GBSS in potato and maize was tightly associated with the starch ghosts. The study provides a structural explanation for a role of granule‐associated proteins in maintaining the integrity of starch ghosts and remnant structures, and their consequent effect on paste rheology.     

Effects of Varying Weight Ratios of Large and Small Wheat Starch Granules on Experimental Straight‐Dough Bread

One commercial bread wheat flour with medium strength (11.3% protein content, 14% mb) was fractionated into starch, gluten, and water solubles by hand‐washing. The starch fraction was separated further into large and small granules by repeated sedimentation. Large (10–40 μm diameter) and small (1–15 μm diameter) starch fractions were examined. Flour fractions were reconstituted to original levels in the flour using composites of varying weight percentages of starch granules: 0% small granules (100% large granules), 30, 60, and 100% (0% large granules). A modified straight‐dough method was used in an experimental baking test. Crumb grain and texture were significantly affected. The bread made from the reconstituted flour with 30% small granules and 70% large granules starch had the highest crumb grain score (4.0, subjective method), the highest peak fineness value (1,029), and the second‐highest elongation ratio (1.55). Inferior crumb grain scores and low fineness and elongation ratios were observed in breads made from flours with starch fractions with 100% small granules or 100% large granules. As the proportion of small granules increased in the reconstituted flour, it yielded bread with softer texture that was better maintained than the bread made from the reconstituted reference flour during storage.     

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.     

Starch Participation in Durum Dough Linear Viscoelastic Properties

The contribution of starch to dough rheological properties has been largely overshadowed by the role of gluten, receiving much less attention in comparison. The influence of starch granule surface properties on durum wheat dough linear viscoelasticity was investigated, and surface interactions between starch granules and gluten were assessed using a model system. Proportions of starch were substituted in dough on a volume basis with an inert filler (glass powder) with a similar particle size range. The doughs were subjected to dynamic and creep measurements. Dough linear viscoelastic properties were weakened on substitution of starch with glass powder at ≤50% substitution, inferring a reduction in adhesion at the matrix‐filler (starch and glass powder) interface with declining proportions of starch granules. Surface modification of starch granules or glass powder altered dough rheological properties, confirming the importance of starch granule surface characteristics and the nature of protein‐starch bonding on durum dough linear viscoelastic behavior.     

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.     

Synthesis and Characterization of Starch Acetates with High Substitution

Acetylation of high‐amylose (70%) maize starch to high degree of substitution (DS) was studied by reacting starch with acetic anhydride using 50% aqueous NaOH as the catalyst. DS increased with increasing reaction times and increasing ratios of acetic anhydride to starch. Reaction efficiency (RE) increased with longer reaction times and decreased with increases in the ratios of acetic anhydride to starch for extended reaction times. Increasing the amount of NaOH increased both DS and RE. A series of starch acetates with DS values of 0.57–2.23 were prepared and their crystalline structures, chemical structures, thermal stability, and morphological properties were investigated. After acetylation, and as DS increased from 0.57 to 2.23, the crystalline structures of starch steadily disappeared. The carbonyl group's peak at 1,740 cm^‐1 appeared in the FTIR spectra. The intensity of this peak increased with a decrease in the peak intensity of the hydroxyl groups at 3,000‐3,600 cm^‐1, indicating that the hydroxyl groups on starch were replaced by the acetyl groups. Thermal stability of starch acetates increased. The smooth surface of the starch granules became rough with acetylation. Further acetylation led to the loss of the starch granules and the formation of beehive‐ and fibrous‐like structures.     

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.     

Cross‐Linked Resistant Starch: Preparation and Properties

Resistant starches (RS) were prepared by phosphorylation of wheat, waxy wheat, corn, waxy corn, high‐amylose corn, oat, rice, tapioca, mung bean, banana, and potato starches in aqueous slurry (≈33% starch solids, w/w) with 1–19% (starch basis) of a 99:1 (w/w) mixture of sodium trimetaphosphate (STMP) and sodium tripolyphosphate (STPP) at pH 10.5–12.3 and 25–70°C for 0.5–24 hr with sodium sulfate or sodium chloride at 0–20% (starch basis). The RS₄ products contain ≤100% dietary fiber when assayed with the total dietary fiber method of the Association of Official Analytical Chemists (AOAC). In vitro digestion of four RS₄ wheat starches showed they contained 13–22% slowly digestible starch (SDS) and 36–66% RS. However after gelatinization, RS levels fell by 7–25% of ungelatinized levels, while SDS levels remained nearly the same. The cross‐linked RS₄ starches were distinguished from native starches by elevated phosphorus levels, low swelling powers (≈3g/g) at 95°C, insolubilities (<1%) in 1M potassium hydroxide or 95% dimethyl sulfoxide, and increased temperatures and decreased enthalpies of gelatinization measured by differential scanning calorimetry.     

Ethanol Fermentation of Starch from Field Peas

Field peas (Pisum sativum) were evaluated as a potential feedstock for ethanol production. Ground peas were dry‐milled and separated into starch, protein, and fibrous fractions by air classification. Starch‐enriched fractions prepared from whole peas and dehulled peas contained 73.7% wt and 77.8% wt starch, respectively, a nearly two‐fold enrichment compared with whole peas. The fractions were liquefied and saccharified using industrial α‐amylase and glucoamylase at recommended enzyme loadings. A final ethanol concentration of 11.0% (w/v) was obtained in 48–52 hr, with yields of 0.43–0.48 g of ethanol/g of glucose. Starch present in whole ground peas was also saccharified and fermented, with 97% of the starch fermented when an autoclaving step was included in the liquefaction stage.     

Gelatinizing,Pasting, and Gelling Properties of Potato and Amaranth Starch Mixtures

Physicochemical properties of mixtures of native potato and native amaranth (Amaranthus cruentus), heat‐moisture treated (HMT) potato and heat‐moisture treated amaranth, cross‐linked potato and cross‐linked amaranth, native potato and heat‐moisture treated amaranth, and heat‐moisture treated potato, and native amaranth were tested at different ratios. Two peaks were noticed in the pasting curves when large differences of swelling factor and amylose leaching existed between individual components in the mixture. It seems that amylose leaching from one starch in a mixture may affect the swelling and much of the granular break down of the other. The mixtures showed stabilities in hot pastes that were higher than the less stable components in a mixture. Some mixtures such as HMT potato and native amaranth showed very specific nonadditive pasting behavior. Mixing 10% of native amaranth to HMT potato starch caused a large reduction of peak viscosity and cold paste viscosity, resulting in a very soft gel. In the differential scanning calorimeter, each component of a mixture gelatinized independently, showing two peaks corresponding to the individual components. When transition temperatures of both components were similar in DSC, the result was a single endotherm. Dramatic changes of pasting and subsequent gel properties resulted when thermal transition of the two components occurred in the same temperature range. Retrogradation enthalpies as measured by DSC were between the two individual components in all tested mixtures.     

Enzymatic method for measuring starch gelatinization in dry products in situ

An enzymatic method based on hydrolysis of starch by amyloglucosidase and measurement of d-glucose released by glucose oxidase-peroxidase was developed to measure both gelatinized starch and hydrolyzable starch in situ of dried starchy products. Efforts focused on the development of sample handling steps (particle size reduction of dry samples followed by a unique mechanical resolubilization step) prior to the enzymatic hydrolysis using native and fully gelatinized flours of corn and rice. The new steps, when optimized, were able to maximize resolubilization of gelatinized/retrograded starch while minimizing solubilization of native starch in dried samples, thus effectively addressing issues of insusceptibility of retrograded starch and susceptibility of native starch to enzymatic attacks and eliminating the need to isolate starch from dry samples before using an enzymatic method. Various factors affecting these and other steps were also investigated, with the objectives to simplify the procedures and reduce errors. Results are expressed as the percentage of the total starch content. The proposed method, verified by measuring mixed samples of native and fully gelatinized flours of five grain species (corn, rice, barley, oat, and wheat) at different ratios, is simple, accurate, and reliable, with a relative standard deviation of less than 5%.