Procedure for Obtaining Stable Protein Extracts of Cereal Flour and Whole Meal for Size‐Exclusion HPLC Analysis

Experiments were conducted to determine the extent of instability of size‐exclusion HPLC extracts prepared from flour, semolina, or whole meal. Procedures to obtain stable extracts were investigated. Whole meal extracts of durum wheat and triticale were the most unstable samples, whereas bread wheat showed smaller changes. Samples prepared from crushed maturing grains, especially during early stages, were greatly influenced by the instability process. By using protease inhibitors, evidence was obtained that endogenous proteases were the source of the instability. Reproducible peak 1 (polymeric protein) results were obtained for a period of at least 72 hr after extract preparation if protein extracts were heated for 2 min at 80°C in a water bath immediately after filtration into the sample vials and before SE‐HPLC analysis. This treatment is a viable solution to avoid sample instability in whole meal and developing grain extracts, particularly when large sample sets are prepared for automatic injection into the HPLC.     

Rapid Size‐Exclusion Chromatography Analysis of Molecular Size Distribution for Wheat Endosperm Protein

A quick method for the separation of the main size classes of wheat endosperm proteins using size‐exclusion HPLC is presented. Separations achieved in a 10‐min run showed high correlation with the reference method of our laboratory (35‐min) using the same type of column. The clear separation obtained allows a quick analytical determination of important parameters such as the proportion of the main classes of wheat endosperm protein, the glutenin‐to‐gliadin ratio, and the percentage of unextractable (SDS‐soluble with sonication) polymeric protein, all of which have been closely correlated with breadmaking quality parameters. The improved method offers the advantages of better utilization of valuable resources such as HPLC equipment, quicker analysis of large sample sets, and collection of eluted fractions.     

Impact of Cultivar and Environment on Size Characteristics of Wheat Proteins Using Asymmetrical Flow Field‐Flow Fractionation and Multi‐Angle Laser Light Scattering

The use of multi‐angle laser light scattering (MALLS) in conjunction with asymmetrical flow field‐flow fractionation (A‐FFFF) was investigated for the determination of the molecular weight distribution (MWD) of wheat proteins. The wheat flour proteins were dissolved by sonication in 0.1M sodium phosphate (pH 6.9) containing 2% SDS. The results presented make it evident that efficient separation and size characterization of monomeric (M < 10⁵ g/mol) and polymeric protein (10⁵ ≤ M < 10⁸ g/mol) wheat proteins can be achieved with A‐FFFF/MALLS/UV in a single run. Therefore, this method appears to be able to detect significant modifications of MWD of wheat protein, whatever the factor inducing these alterations (i.e., genetic or environmental) and whatever the nature of these alterations (i.e., monomeric‐to‐polymeric ratio or MWD of polymeric protein). In the present study, we have indeed demonstrated that the MWD of wheat proteins can be altered from one cultivar to another in three main ways: by changing the relative amounts of monomeric and polymeric proteins, by changing the MWD of polymeric protein, and then by changing both the monomeric‐to‐polymeric ratio and the MWD of polymeric protein.     

Using Multivariate Techniques to Predict Wheat Flour Dough and Noodle Characteristics from Size‐Exclusion HPLC and RVA Data

Flour proteins of hard and soft winter wheats grown in Oregon were characterized by size‐exclusion HPLC (SE‐HPLC). Flour pasting characteristics were assessed by a Rapid Visco Analyser (RVA). Principle component scores (PCS) were calculated from both RVA data and from absorbance area and % absorbance values from SE‐HPLC. The PCS and cross‐products, ratios, and squares were used to derive wheat classification and quality prediction models. A classification model calculated from PCS of SE‐HPLC data could reliably separate these hard and soft wheats. The prediction models for mixing and noodle characteristics showed better performance when calculated from PCS values of both SEHPLC and RVA data than from SE‐HPLC data only. The R² values of prediction models for mixograph absorption, peak time, and tolerance were 0.827, 0.813, and 0.851, respectively. Prediction models for noodle hardness, cohesiveness, chewiness, and resilience immediately after cooking had R² values of 0.928, 0.928, 0.896, and 0.855, respectively. These results suggest that multivariate methods could be used to develop reliable prediction models for dough mixing and noodle characteristics using just SE‐HPLC and RVA data.     

Size‐Exclusion HPLC of Protein Using a Narrow‐Bore Column for Evaluation of Breadmaking Quality of Hard Spring Wheat Flours

The objective of this study was to investigate whether a narrow‐bore column (NBC) (300 × 4.5 mm, i.d.) improved analyses of unreduced proteins in flour by size‐exclusion HPLC (SE‐HPLC) and subsequent evaluation of breadmaking quality of hard spring wheat flours. Total protein extracts and SDS buffer extractable and unextractable proteins were analyzed by SE‐HPLC. NBC separated proteins in 10 min at a flow rate of 0.5 mL/min with similar resolution to a regular column (300 × 7.8 mm, i.d.) which took 30 min. SE‐HPLC absorbance area (AA) data obtained from an NBC showed comparable or superior repeatability and correlations with flour breadmaking characteristics when compared with those of a regular column. AA values of total protein that were calculated by adding AA values of SDS extractable and unextractable proteins showed greater repeatability and correlations with quality characteristics than those of actual total protein extracts. The improvements including employment of an NBC in SE‐HPLC provide enhancement of rapid quality evaluation and decreased consumption of hazardous organic solvents.     

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.     

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.     

Formation of Homopolymers and Heteropolymers Between Wheat Flour and Several Protein Sources by Transglutaminase‐Catalyzed Cross‐Linking

The effect of different protein sources (soy flour, lupin flour, egg albumin, gelatin powder, protein‐rich beer yeast flour) on wheat dough functionality was tested by determining gluten index, texture properties, and thermomechanical parameters. Transglutaminase (TG) was also added to improve the dough functionality by forming cross‐links. The presence of protein sources had a significant effect on the gluten index, with the exception of lupin flour. Gelatin and the presence of TG resulted in significant single effects on the texture properties of the wheat‐protein dough. All the protein sources significantly modified the mixing characteristics of the dough or the thermal behavior. Capillary electrophoresis studies of the water‐soluble, salt‐soluble, and glutenin proteins indicated that interactions were mainly within proteins, thus homologous polymers. Scanning electron microscopy studies of the doughs made from blends of wheat and protein sources doughs supported the formation of heterologous structures in the wheat‐lupin blends. The combination of TG and lupin would be a promising method to be used on the treatment of insect‐damaged or weak flours, to increase the gluten strength.     

Substitution of Concentrated Ethanol for Water in the Laboratory Washing Fractionation of Protein and Starch from Hydrated Wheat Flour

An unprecedented, ethanol-based, washing process was used at a laboratory scale to produce both concentrated protein and starch fractions from hydrated wheat flour. In this multistep process, flour was first hydrated and mixed to a batter and then chilled and rested. The cold batter was then mixed and washed in chilled and concentrated ethanol using a modified device that normally applies the water-based Martin process. Control of the separation was affected by each of these steps. For instance, the hydration of the flour, the time of mixing, the temperature of the wash, the ethanol concentration, and the time of washing were influential. The method produced a gluten concentrate similar in yield and protein content to that reported for a pilot-scale Martin process but without the need for added salt. Notably, ethanol washing resulted in nonsticky, partially disintegrated curds that dried easily, whereas water washing resulted in a sticky, glutinous, cohesive mass that dried slowly. The process has commercial potential to reduce water and energy use, reduce wastewater generation and environmental impact, and improve product recovery. The process also has the potential to reduce the capital complexity of the drying step and create convenient opportunities for protein subfractionation.     

Separation and Characterization of Barley Starch Polymers by a Flow Field‐Flow Fractionation Technique in Combination with Multiangle Light Scattering and Differential Refractive Index Detection

Flow field‐flow fractionation (flow FFF) with frit inlet and frit outlet mode (FIFO) was coupled online to multiangle light scattering (MALS) and refractive index (RI) detectors to investigate the molecular characteristics of normal and zero amylose barley starch polymers. Application of two different cross‐flows, 0.35 mL/min followed by 0.1 mL/min, and constant channel and frit flows of 0.1 and 1.0 mL/min, respectively, permitted a complete separation of amylose and amylopectin. The improved signals from the detectors due to application of the FIFO mode enabled the proper characterization of the small molecular weight species, as well as significantly enhanced the reproducibility of the measurements. The weight‐average molecular weight (M_w) and zaverage root‐mean‐square (RMS) radii of gyration (R_g) values for amylose and amylopectin in the normal starch samples were 2.3 × 10⁶ and 280 × 10⁶, and 107 and 260 nm, respectively. The M_w and R_g of amylopectin in the zero amylose starch samples were 360 × 10⁶ and 267 nm, respectively. The slopes (α) obtained by plotting log M_w versus log R_g for amylose and amylopectin were 0.6 and 0.3, respectively. These results are in good agreement with the theoretical prediction of the molecular conformation of amylose and amylopectin.     

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.     

Influence of Bran Particle Size on Bread‐Baking Quality of Whole Grain Wheat Flour and Starch Retrogradation

The influence of bran particle size on bread‐baking quality of whole grain wheat flour (WWF) and starch retrogradation was studied. Higher water absorption of dough prepared from WWF with added gluten to attain 18% protein was observed for WWFs of fine bran than those of coarse bran, whereas no significant difference in dough mixing time was detected for WWFs of varying bran particle size. The effects of bran particle size on loaf volume of WWF bread and crumb firmness during storage were more evident in hard white wheat than in hard red wheat. A greater degree of starch retrogradation in bread crumb stored for seven days at 4°C was observed in WWFs of fine bran than those of coarse bran. The gels prepared from starch–fine bran blends were harder than those prepared from starch–unground bran blends when stored for one and seven days at 4°C. Furthermore, a greater degree of starch retrogradation was observed in gelatinized starch containing fine bran than that containing unground bran after storage for seven days at 4°C. It is probable that finely ground bran takes away more water from gelatinized starch than coarsely ground bran, increasing the extent of starch retrogradation in bread and gels during storage.     

End Use Quality of Waxy Wheat Flour in Various Grain‐Based Foods

The practical applications of flour from waxy (amylose‐free) hexaploid wheat (Triticum aestivum L.) were assessed. The applications evaluated were bread, cakes, white salted noodles, and pasta for gyoza. An excessive addition of waxy hexaploid wheat flour to total wheat flour (>20%) resulted in poorer functional properties (sticky, lumpy, or less crispy textures) in almost every end use product. However, incorporation of <20% waxy hexaploid wheat flour, produced considerable improvement in shelf‐life characteristics. After one day of storage, the bread from flour including waxy hexaploid wheat flour maintained moistness, softness, and stickiness. This application of waxy hexaploid wheat flour as an antistaling ingredient was also confirmed in cake products. Tests were also conducted on alimentary pasta products. In alimentary pasta, waxy hexaploid wheat flour was most effective when utilized for frozen fried dumplings (gyoza). By using flour including 30 or 50% waxy hexaploid wheat flour, the problem of firmness was solved without other ingredients. In conclusion, flour from waxy hexaploid wheat may be useful in developing more increased staling‐ and freezing‐tolerant grain‐based foods. Starch properties could be responsible for these improved characteristics.     

Study of a Small‐Scale Laboratory Wet‐Milling Procedure for Wheat

The objectives of this research were to study the effects of slurry specific gravity, starch table slope, slurry pumping rate, and their interactions on starch recovery and purity; and to propose a small‐scale laboratory wet‐milling procedure for wheat. First‐order and second‐order response surface regression models were developed to study the effects and interactions of slurry specific gravity, starch table slope, and slurry pumping rate on starch and gluten separation for a 100‐g wheat wet‐milling procedure. The starch and starch protein content data fit the first‐order models (R² = 0.99 and 0.96) better than the second‐order models (R² = 0.98 and 0.93). Regression results from the first‐order models indicated that specific gravity, table slope, pumping rate, and their interactions all had a significant effect on starch yield and purity. However, these effects could be simplified as the effect of the resident time of starch and gluten slurry on the starch table and the specific gravity. Starch yield increased as resident time increased and specific gravity decreased. Protein content in starch decreased as the resident time decreased and the specific gravity increased. The separation condition with specific gravity of 3 Bé, table slope of 1.04 cm/m, and pumping rate of 50 mL/min was recommended. Under this condition, starch recovery was 85.6% and protein content of starch was 0.42%, which was similar to the 1.5‐kg laboratory methods in starch recovery. Total solids recovery was 98.1%, which is similar to that from 1.5‐kg laboratory methods. These results indicated that precision of the 100‐g wheat wet‐milling procedure was similar to that of the 1.5‐kg laboratory methods.     

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.     

Relationships of Quality Characteristics with Size‐Exclusion HPLC Chromatogram of Protein Extract in Soft White Winter Wheats

This study investigated relationships between molecular weight distributions of unreduced grain proteins and grain, flour, and end‐use quality characteristics of soft white winter wheats grown in Oregon. Absorbance area and area percentage values of protein fractions separated by size‐exclusion HPLC (SE‐HPLC) showed significant correlations with quality characteristics, indicating associations of molecular weight distributions of proteins with quality characteristics. Specifically, high molecular weight polymeric protein fractions appeared to have a detrimental effect on soft wheat quality. This was shown by significant positive correlations with single kernel hardness index, and mixograph water absorption and tolerance, and negative correlations with break flour yield, cookie diameter, and cake volume. Higher proportions of soluble monomeric protein fraction eluted after the main gliadin peak, were associated with soft wheat quality due to negative associations with single kernel hardness index and mixograph water absorption and tolerance, and positive associations with break flour yield, cookie diameter, and cake volume. Calibration models were developed by the application of multivariate analyses to the SE‐HPLC data. These models explained >90% of the variation in mixograph water absorption and cookie diameter and thickness.     

Farinograph Responses for Wheat Flour Dough Fortified with Wheat Gluten Produced by Cold‐Ethanol or Water Displacement of Starch

The objective of this research was to identify and define mixing characteristics of gluten‐fortified flours attributable to differences in the method for producing the gluten. In these studies, a wheat gluten concentrate (W‐gluten) was produced using a conventional process model. This model applied physical water displacement of starch (dispersion and screening steps), freeze‐drying, and milling. W‐gluten was the reference or “vital” gluten in this report. An experimental W‐concentrate was produced using a new process model. The new model applied coldethanol (CE) displacement of starch (dispersion and screening steps), freeze‐drying, and milling. Freeze‐drying was used to eliminate thermal denaturation and thereby focus on functional changes due only to the separation method. The dry gluten concentrates were blended with a weak, low‐protein (9.2%), soft wheat flour and developed with water in a microfarinograph. We found that both water and cold‐ethanol processed gluten successfully increased the stability (St) and improved mixing tolerance index (MTI) to create in the blended flour the appearance of a breadbaking flour. Notably, in the tested range of 9–15% protein, the St for CE‐gluten was always higher then the St for W‐gluten. Furthermore, the marginal increase in St (slope of the linear St vs. protein concentration) for the CE‐gluten was ≈57% greater than that for the W‐gluten. The slope of the MTI vs. protein data was lower for the CE‐gluten by 24%. Flour fortified with CE‐gluten exhibited higher water absorption (up to 1.8% units at 13.5% P) than flour fortified with W‐gluten.