Use of Size‐Exclusion High‐Performance Liquid Chromatography for Wheat Quality Prediction in Ethiopia

Wheat quality testing facilities in Ethiopia are limited. The aim of this study was to determine whether size‐exclusion high‐performance liquid chromatography (SE‐HPLC) could be used in breeding programs for quality testing. Thirteen Ethiopian and two South African wheat cultivars were evaluated in two diverse environments for milling and dough characteristics. SE‐HPLC was done on the same samples. Across environments, both SDS‐soluble and SDS‐insoluble polymeric proteins significantly influenced important quality characteristics such as SDS‐sedimentation and mixograph development time. The large monomeric proteins, which are mainly gliadins, had a consistently significantly negative effect on quality. The increase of polymeric protein as opposed to monomeric protein led to improvement of quality characteristics. The SDS‐soluble and SDS‐insoluble polymeric proteins were equally important in quality prediction. The amount of polymeric proteins was significantly higher in the high‐protein environment. Despite a large environmental effect on most fractions, a large ratio of polymeric proteins to monomeric proteins (both SDS‐soluble and SDS‐insoluble) can be a good indicator of baking quality. SE‐HPLC is therefore an option to use in breeding programs in Ethiopia for quality evaluation.     

Isolation and Characterization of High‐Molecular‐Weight (HMW) Gliadins from Wheat Flour

The aim of this study was to isolate high‐molecular‐weight (HMW) gliadins from wheat flour and to characterize the protein components that contribute to HMW gliadins. Wheat flour Akteur was extracted with a modified Osborne procedure, and the fraction soluble in 60% ethanol (total gliadins) was separated by gel‐permeation HPLC, yielding three fractions, GP1–GP3. GP1 (21.5%) consisted of oligomeric HMW gliadins, GP2 (15.2%) of ω5‐gliadins, and GP3 (63.3%) of ω1,2‐, α‐, and γ‐gliadins. Two‐dimensional SDS‐PAGE of HMW gliadins showed that interchain disulfide bonds were present in HMW gliadins. The molecular mass distribution of HMW gliadins determined by gel‐permeation HPLC was in a range from 66,000 to 680,000 with an average degree of polymerization of 13. Reduced HMW gliadins were further separated by preparative reversed‐phase HPLC into four subfractions (RP1, RP2, RP3, and RP4), which were characterized by SDS‐PAGE and semiquantitative N‐terminal sequencing. HMW gliadins of the wheat flour Akteur contained all types of gluten proteins: 48% low‐molecular‐weight glutenin subunits, 18% γ‐gliadins, 13% α‐gliadins, 9% ω1,2‐gliadins, 8% HMW glutenin subunits, and 4% ω5‐gliadins. We postulate that the existence of HMW gliadins can be explained by the presence of terminators, which interrupt the polymerization of glutenin subunits during biosynthesis and lead to polymers of limited size (oligomers) that are still soluble in aqueous ethanol.     

Effects of Temperature,Sonication Time,and Power Settings on Size Distribution and Extractability of Total Wheat Flour Proteins as Determined by Size‐Exclusion High‐Performance Liquid Chromatography

The size‐exclusion (SE) HPLC profile of total protein extract obtained by sonication of flour samples at ambient temperature showed marked instability on reinjection. Instability was related to the presence of flour proteases that were inactivated by thermal treatment of flour samples at 60°C. Extraction of flour protein by sonication was a function of ultrasonic energy (sonication time × power product) delivered to flour sample. As protein solubility increased, the proportions of the earliest eluted SE‐HPLC fractions (F1 and F2) increased. Oversonication of proteins evidenced by a decrease in F1 amount at the benefit of F2 occurred below the sonication energy level needed for total protein extraction. Ultrasonic energy level was adjusted to allow total protein extraction while limiting oversonication. The sonication procedure was applied on 27 flour samples of contrasting dough strength to extract total proteins. Absolute amount of protein extractable by sonication, determined from SE‐HPLC area, was strongly correlated with flour protein content. Very significant and equivalent relationships were found between alveographic W index and absolute amount of either unextractable protein extract or F1 of SE‐HPLC profile from total protein extract.     

Rapid High‐Performance Liquid Chromatography Determination of Tocopherols and Tocotrienols in Cereals

A rapid normal‐phase high‐performance liquid chromatography (NP‐HPLC) method has been developed for rapid determination of the total content of tocopherols (T) and tocotrienols (T3) in cereals. The new method uses a one‐step extraction followed by NP‐HPLC coupled with a fluorescence detector. The new method provides a baseline separation of the critical peaks of beta‐tocotrienol (β‐T3) and gamma‐tocopherol (γ‐T) within a short time of analysis. The extraction step requires no saponification step or addition of antioxidant. Addition of butylated hydroxytoluene (BHT) had no effect on the stability of vitamin E during sampling. The recovery was 96–100%. The method is demonstrated by successful quantification of vitamin E in barley, wheat, and spelt grains, as well as wheat germ and flours from wheat, spelt, and rye.     

Formation of Enzyme‐Resistant Starch in Bread as Affected by High‐Amylose Wheat Flour Substitutions

High‐amylose wheat flour was used to substitute for normal wheat flour in breadmaking and formation of resistant starch (RS) in bread during storage was determined. Substitution with high‐amylose wheat flour (HAF) decreased peak and final viscosities, breakdown, and setback. Doughs with HAF substitutions were weaker and less elastic, and absorbed more water than those of the normal wheat flour. After baking, RS contents in breads with 10, 30, and 50% HAF substitutions were 1.6, 2.6, and 3.0% (db), respectively, higher than that of the control (0.9%, db). The levels of RS increased gradually during storage for one, three, and five days. With substitutions of 30 and 50% HAF, the total levels of dietary fiber (DF) and RS in bread after five days of storage were 15.5 and 16.8% (db), respectively, as compared to 13.0% (db) in bread from the normal wheat flour. The loaf volumes and appearances of bread crumbs made from HAF substitutions of 10 and 30% were not significantly different from those of the control, whereas the substitution with 50% HAF decreased loaf volume and resulted in inferior appearance of breadcrumbs. The firmness of breadcrumbs increased along with increase in the level of HAF substitutions after baking. During storage, the firmness of breadcrumb with 10% HAF substitutions was higher than that of the control, whereas breads with 30 and 50% HAF substitutions had similar firmness to the control. As a result, HAF might be used to substitute for up to 50% normal wheat flour to make bread with acceptable bread quality and significantly high amount of RS.     

Quantitative Determination of Gluten Protein Types in Wheat Flour by Reversed-Phase High-Performance Liquid Chromatography

A combined extraction-HPLC procedure was developed on a microscale to determine the amounts of the different gluten protein types (ω5-, ω1,2-, α- and γ-gliadins; high molecular weight [HMW] and low molecular weight [LMW] glutenin subunits) in wheat flour. After preextraction of albumins and globulins from flour (100 mg) with a salt solution (2 × 1.0 mL), extraction of gliadins was achieved with 60% aqueous ethanol (3 × 0.5 mL). Subsequently, the glutenin subunits were extracted under nitrogen and at 60°C with 50% aqueous 1-propanol containing Tris-HCl (0.05 mol/L, pH 7.5), urea (2 mol/L) and dithioerythritol (1%). The separation and quantitative determination of gliadins and glutenin subunits was then performed by reversed-phase HPLC on C₈ silica gel at 50°C using a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid. The flow rate was 1.0 mL/min, and the detection wavelength was 210 nm. Temperature and flow rate were modified for the quantitation of single underivatized HMW subunits. To determine the absolute amounts of protein types, different protein standards (gliadin, LMW and HMW subunits, bovine serum albumin) with known protein contents were compared to HPLC absorbance areas. The calibration curves were almost identical and linear over a broad range (20–220 μg). This extraction-HPLC procedure allows an accurate, reproducible, sensitive, and relatively fast quantitative determination of all gluten protein types in wheat flour, and can be applied to quality evaluation of cereals as raw materials or in processed products.     

Application of Protease and High‐Intensity Ultrasound in Corn Starch Isolation from Degermed Corn Flour

The conventional corn wet‐milling process requires a long steeping time and has environmental and health concerns from the use of SO₂. A recently proposed two‐stage enzymatic milling procedure with the first stage of water soaking and coarse grinding of corn and the second stage of incubating with enzymes has been shown to reduce the soaking time and possibly eliminate the need for SO₂ addition. This current work explored the applications of protease and high‐intensity ultrasound in the second stage of the two‐stage enzymatic milling for corn starch isolation to further shorten the process time without use SO₂. of The starch yield from sonication alone was 55.2–67.8% (starch db) as compared with 53.4% of the water‐only control with stirring for 1 hr and 71.1% of the conventional control with SO₂ and lactic acid steeping for 48 hr. Protease digestion alone for 2 hr was not effective (45.8–63.9% yield) in isolating corn starch, but the starch recovery was increased to 61.2–76.1% when protease was combined with sonication. The preferred combination was neutral protease digestion for 2 hr followed by sonication at 75% amplitude for 30 min. The results demonstrated that combinations of high‐intensity ultrasound and neutral protease could replace SO₂ and shorten the steeping time in the enzymatic wet‐milling process for corn starch isolation.     

Effect of High‐Molecular‐Weight Glutenin Subunit Allelic Composition on Wheat Flour Tortilla Quality

Wheat cultivars possessing quality attributes needed to produce optimum quality tortillas have not been identified. This study investigated the effect of variations in high‐molecular‐weight glutenin subunits encoded at the Glu‐1 loci (Glu‐A1, Glu‐B1, and Glu‐D1) on dough properties and tortilla quality. Flour protein profiles, dough texture, and tortilla physical quality attributes were evaluated. Deletion at Glu‐D1 resulted in reduced insoluble polymeric protein content of flour, reduced dough compression force, and large dough extensibility. These properties produced very large tortillas (181 mm diameter) compared with a control made with commercial tortilla wheat flour (161 mm). Presence of a 7 + 9 allelic pair at Glu‐B1 increased dough strength (largest compression force, reduced extensibility, and small‐diameter tortillas). Deletion at Glu‐A1 produced large tortillas (173 mm) but with unacceptable flexibility during storage (score <3.0 at day 16). In general, presence of 2* at Glu‐A1, in combination with 5 + 10 at Glu‐D1, produced small‐diameter tortillas that required large force to rupture (tough texture). Presence of 2 + 12 alleles instead of 5 + 10 at Glu‐D1 produced tortillas with a good compromise between diameter (>165 mm) and flexibility during storage (>3.0 at day 16). These allele combinations, along with deletion at Glu‐D1, show promise for tortilla wheat development.     

NIR Transmittance Estimation of Free Lipid Content and Its Glycolipid and Digalactosyldiglyceride Contents Using Wheat Flour Lipid Extracts

Free lipids (FL) in 72 hard winter wheat flours were extracted and dissolved in hexane. Absorbance (log 1/T) values were measured using a scanning spectrophotometer with a 2‐mm cuvette, and used to develop calibration models for estimating flour FL content and glycolipid (GL) and digalactosyldiglyceride (DGDG) contents. Fifty‐one calibration samples were selected based on the cutoff point of 0.3 of Mahalanobis distance, and the remaining twenty‐one samples were used for validation. The best model for the estimation of FL content showed coefficients of determination (R²) of 0.95 for the calibration set and R² = 0.89 for the validation set. Glycolipid contents could be estimated by a model which had R² = 0.87 for the calibration set and R² = 0.89 for the validation set. For DGDG, the best model showed R² = 0.94 for the calibration set and R² = 0.88 for the validation set.     

Gelatinization and Retrogradation Kinetics of High‐Fiber Wheat Flour Blends: A Calorimetric Approach

The impact of dietary fiber (DF) mixtures on dough thermal properties needs to be investigated when designing high‐fiber wheat bread. Effects of flour replacement at different levels (6–34%) by soluble (inuline [FN]), partially soluble (sugar beet [FX], pea cell wall [SW]), and insoluble (pea hull [EX]) DF on wheat dough thermal profiles have been investigated by simulating baking, cooling, and storage in differential scanning calorimetry (DSC) pans. In general, DF incorporation into water‐flour systems delayed endothermic transition temperatures for both gelatinization and retrogradation phenomena except for the peak temperature (T_p) of retrogradation. With some exception, the pattern of the enthalpy of amylopectin retrogradation was lower and slower (lower constant of proportion, k) over 10 days of storage in gelatinized hydrated flour‐fiber blends when compared with control without DF. FX, a partially soluble fiber, provided major effects on gelatinization (T_p decrease and ΔH increase) and retrogradation kinetics (the Avrami exponent, n, increase). Single presence of EX allowed a significant reduction in the Avrami exponent n leading to slower kinetics for amylopectin retrogradation when included in the blends.     

Relationship Between Soft Wheat Flour Physicochemical Composition and Cookie‐Making Performance

Nowadays in Argentina, cookies, crackers, and cakes are made of flour obtained from bread wheat with additives or enzymes that decrease the gluten strength but increase production costs. The present research work aims to study the relationship between flour physicochemical composition (particle size average [PSA], protein, damaged starch [DS], water soluble pentosans [WSP], total pentosans [TP], and gluten), alkaline water retention capacities behavior, solvent retention capacities profile (SRC) and cookie‐making performance in a set of 51 adapted soft wheat lines with diverse origin to identify better flour parameters for predicting cookie quality. Cookie factor (CF) values were 5.06–7.56. High and significant negative correlations between sucrose SRC (–0.68), water SRC (–0.65), carbonate SRC (–0.59), and CF were found, followed by lactic SRC that presented a low negative but significant correlation (r = –0.35). The flour components DS (r = –0.67), WSP (r = –0.49), and TP (r = –0.4) were negatively associated to CF. PSA showed a negative correlation with CF (r = –0.43). Protein and gluten were the flour components that affected cookie hardness, but no significant correlation were found with pentosan or DS content. A prediction equation for CF was developed. Sucrose SRC, PSA, and DS could be used to predict 68% of the variation in cookie diameter. The cluster analysis was conducted to assess differences in flour quality parameters among genotypes based on CF. Clusters 1 and 4 were typified by lower CF (5.70 and 5.23, respectively), higher DS, pentosan content, and SRC values. Cluster 2 with a relative good CF (6.47) and Cluster 3 with the best cookie quality, high CF (7.32) and low firmness, and the lowest DS, TP, WSP content, and sucrose SRC values.     

High‐Speed NIR Segregation of High‐ and Low‐Protein Single Wheat Seeds

Wheat breeders need a nondestructive method to rapidly sort high‐ or low‐protein single kernels from samples for their breeding programs. For this reason, a commercial color sorter equipped with near‐infrared filters was evaluated for its potential to sort high‐ and low‐protein single wheat kernels. Hard red winter and hard white wheat cultivars with protein content >12.5% (classed as high‐protein, 12% moisture basis) or < 11.5% (classed as low‐protein) were blended in proportions of 50:50 and 95:5 (or 5:95) mass. These wheat blends were sorted using five passes that removed 10% of the mass for each pass. The bulk protein content of accepted kernels (accepts) and rejected kernels (rejects) were measured for each pass. For 50:50 blends, the protein in the first‐pass rejects changed as much as 1%. For the accepts, each pass changed the protein content of accepts by ≈0.1%, depending on wheat blends. At most, two re‐sorts of accepts would be required to move 95:5 blends in the direction of the dominant protein content. The 95:5 and 50:50 blends approximate the low‐ and high‐protein mixture range of early generation wheat populations, and thus the sorter has potential to aid breeders in purifying samples for developing high‐ or low‐protein wheat. Results indicate that sorting was partly driven by color and vitreousness differences between high‐ and low‐protein fractions. Development of a new background specific for high‐ or low‐protein and fabrication of better optical filters for protein might help improve the sorter performance.     

Quantitative Characterization of Polar Lipids from Wheat Whole Meal,Flour, and Starch

Lipids constitute only a minor proportion of total flour components but the composition and structure of wheat flour polar lipids influence the end‐use quality of bread. So it is important to determine which specific lipid class and molecular species are present in wheat. Lipid profiling is the targeted, systematic characterization and analysis of lipids. The use of lipid profiling techniques to analyze grain‐based food has the potential to provide new insight into the functional relationships between a specific lipid species and its functionality. The objective of this study was to utilize lipid profiling techniques to quantitatively determine the polar lipid species present in whole wheat meal, flour, and starch. Two commonly grown wheat cultivars, Alpowa and Overley, were used in this study. Direct infusion electrospray ionization tandem mass spectrometry was used to identify and quantitatively determine 146 polar lipid species in wheat. The predominant polar lipid classes were digalactosyldiglycerols, monogalactosyldiglycerols, phosphatidylcholine, and lysophosphatidylcholine. ANOVA results concluded that the wheat fraction contributed a greater source of variation than did cultivar on total polar, total phospholipid, and total galactolipid contents. Wheat whole meal, flour, and surface starch contained greater concentrations of total galactolipids, whereas internal starch lipids contained greater concentrations of monoacyl phospholipids. This research provides evidence that lipid profiling will provide the ability to determine the functional relationships between specific lipid species and the impact on end‐use quality.     

Effects of Microencapsulated High‐Fat Powders on the Empirical and Fundamental Rheological Properties of Wheat Flour Doughs

Microencapsulated high‐fat powders are a healthy and convenient alternative to fats normally used in cereal‐based products. In powder form they are easier to use than block fat. Microencapsulation involves dispersion of the fat using homogenization. The globules are then fixed by spray‐drying. Empirical and fundamental rheological tests were conducted on doughs containing commercial vegetable fat and four microencapsulated high‐fat powders. The doughs were compared with a standard dough containing no fat. The powders contained 70% vegetable fat or milk fat. The encapsulating agent used was either sodium caseinate or whey protein concentrate (5–10%). Sucrose or lactose were also present in the powders (20–25%). The powders were manufactured at low‐ or high‐pressure homogenization. Farinograph and extensigraph tests were performed on all doughs. Dynamic oscillation tests were conducted in the linear visco‐elastic region of the dough. Addition of fat and microencapsulated high‐fat powders produced using low‐pressure homogenization reduced the complex modulus of the doughs. The results showed an increase in phase angle with incorporation of commercial fat and the microencapsulated high‐fat powders. Scanning electron microscopy was conducted to examine the effects of the additives on dough structure. This study demonstrated that microencapsulated high‐fat powders, especially powders produced using low‐pressure homogenization, had some beneficial effects on dough rheology when compared with doughs produced with commercial fat.     

Mixograph Responses of Gluten and Gluten‐Fortified Flour for Gluten Produced by Cold‐Ethanol or Water Displacement of Starch from Wheat Flour

The total protein of gluten obtained by the cold‐ethanol displacement of starch from developed wheat flour dough matches that made by water displacement, but functional properties revealed by mixing are altered. This report characterizes mixing properties in a 10‐g mixograph for cold‐ethanol‐processed wheat gluten concentrates (CE‐gluten) and those for the water‐process concentrates (W‐gluten). Gluten concentrates were produced at a laboratory scale using batter‐like technology: development with water as a batter, dispersion with the displacement fluid, and screening. The displacing fluid was water for W‐gluten and cold ethanol (≥70% vol, ‐12°C) for CE‐gluten. Both gluten types were freeze‐dried at ‐10°C and then milled. Mixograms were obtained for 1) straight gluten concentrates hydrated to absorptions of 123–234%, or 2) gluten blended with a low protein (9.2% protein) soft wheat flour to obtain up to 16.2% total protein. The mixograms for gluten or gluten‐fortified flour were qualitatively and quantitatively distinguishable. We found differences in the mixogram parameters that would lead to the conclusion of greater stability and strength for CE‐gluten than for W‐Gluten. Differences between the mixograms for these gluten types could be markedly exaggerated by increasing the amount of water to the 167–234% range. Mixograms for evaluation of gluten have not been previously reported in this hydration range. Mixograms for fortification suggest that less CE‐gluten than W‐gluten would be required for the same effect.     

Nutritional Profile of Whole‐Grain Soft Wheat Flour

Whole‐grain wheat flour is used in baking to increase fiber content and to provide vitamins from the bran layers of the kernel. We surveyed whole‐grain soft flour samples from North America to determine the nutritional profile using recently revised fiber quantification protocols, Codex 2009.1. Standard compositional and vitamin analyses were also included in the survey. Three separate studies were included in the survey: sampling of commercial whole‐grain soft wheat flour, a controlled study of two cultivars across three years and two locations, and a regional study of soft white and soft red grain from commercial grain production. The Codex method for fiber measurement estimated total fiber concentration in the commercial sampling at 15.1 g/100 g, dry weight basis (dwb). In the controlled research trial, the largest source of variation in total fiber concentration was attributed to year effects, followed by genotype effects. For the two locations used in this study, location effects on fiber concentration were significant but an order of magnitude less important than the year and genotype effects. The third study of regional variation within North America found limited variation for total fiber, with the resistant oligosaccharide fraction having the greatest variation in concentration. When all three studies were combined into a meta‐analysis, the average total fiber concentration was 14.8 g/100 g dwb. In the meta‐analysis, concentrations of folate, thiamin, riboflavin, niacin, and pyridoxine were lower than in previous summary reports. Vitamin E and pantothenic acid were the exceptions, with concentrations that were nearly identical to previous standard reports. Several other recent studies also point to current cultivars and production systems as producing lower concentrations of the essential vitamins than previously reported. The results suggest that vitamin concentrations in diets of populations using grain‐based diets from modern cereal‐production systems may require review to determine if previous assumptions of vitamin consumption are accurate.     

Separation of Water‐Soluble Proteins from Cereals by High‐Performance Capillary Electrophoresis (HPCE)

Most research concerning grain proteins has concentrated on the gluten storage proteins. The albumins and globulins are the water‐ and salt‐soluble proteins that contain biologically active enzymes and enzyme inhibitors. A free‐zone capillary electrophoresis method was developed to separate these proteins. Optimization included sample extraction method, capillary temperature, buffer composition, and additives. The optimal conditions for separation of these proteins was 50 μm i.d. × 27 cm (20 cm to detector) capillary at 10 kV (with a 0.17 min ramp‐up time) and 25°C. The optimum buffer was 50 mM sodium phosphate, pH 2.5 + 20% acetonitrile (v/v) (ACN) + 0.05% (w/v) hydroxypropylmethyl‐cellulose (HPMC) + 50 mM hexane sulfonic acid (HSA). Sample stability was an issue that was addressed by lyophilizing fresh extracts and redissolving in aqueous 50% ethylene glycol and 10% separation buffer. This method was successfully used in both wheat flour and whole meal samples. Comparisons were made of several wheats of different classes as well as several cereal grains. This methodology could be useful in screening cereal grains for important enzymes and their impact on end‐use quality such as food functionality, food coloration, and malting quality.     

Lipid Extraction Process on Texturized Soy Flour and Wheat Gluten Protein‐Protein Interactions in a Dough Matrix

Protein‐protein interactions between wheat flour and solvent‐extracted (SE) or nonsolvent extracted (NSE) texturized soy flours were compared. Doughs were prepared to contain varying ratios of texturized soy flour in combination with wheat flour. Sucrose esters (2.5%) were included in several formulations. Doughs were fractionated into soluble and insoluble fractions at pH 4.7 and pH 6.1. Fractions were dried, powdered, and analyzed using SDS‐PAGE and spectrophotometric techniques. Electrophoretic evaluation indicated interactions between wheat gluten proteins and texturized soy proteins in the absence of sucrose esters. Electrophoretic gels of the wheat‐soy flour mixtures maintained a characteristic soy protein band after acidification to the soy protein isoelectric point. Inclusion of sucrose esters increased the interaction. Texturization conferred effects similar to that of sucrose ester on both forms of lipid‐extracted soy. Sulfhydryl analyses using 7‐chloro‐4‐nitrobenzo‐2‐oxa‐4, 3‐diazole (NBD‐Cl) revealed no change in the relative amount of sulfhydryl groups present in doughs prepared from either the texturized soy flours or the doughs containing equal amounts of wheat starch. These data indicate that interactions between soy protein from texturized soy flours and wheat proteins are not covalent.