Simplified Dilute Acetic Acid Based Extraction Procedure for Fractionation and Analysis of Wheat Flour Protein by Size Exclusion HPLC and Flow Field‐Flow Fractionation

A simple, highly efficient and reproducible two‐step extraction procedure using dilute acetic acid without (AN) and then with sonication (AS) has been developed for the fractionation of wheat flour protein. Approximately 97% of total protein was extracted from a Canadian hard red spring wheat flour; an additional 1.2% protein could be recovered by further extraction with 1% DDT and 50% 1‐propanol (AR). Size‐exclusion HPLC (SE‐HPLC) and flow field‐flow fractionation (flow FFF) showed that the AN extract, which accounted for most of the total extractable protein (AN + AS + AR), consisted primarily of monomeric protein. The AS extract was composed primarily of polymeric proteins. Flow FFF showed that AN polymeric protein, including that eluting at the SE‐HPLC void volume, showed smaller Stokes diameters than AS polymeric protein. Flow FFF profiles of AS SE‐HPLC subfractions showed that the void volume subfraction contained monomeric and small polymeric protein in addition to large polymeric protein, indicating formation of larger complexes through interaction between some or all of the components. AN and AS extracts, as well as SE‐HPLC and flow FFF fractions thereof, showed a fairly wide range of values among 12 Canadian hard red and white spring wheat cultivars. The proportion of total protein in the AS extract and in the larger sized polymeric protein fractions from SE‐HPLC and flow FFF were highly positively correlated to farinograph mixing time.     

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.     

Relationship Between Physicochemical Properties of Wheat Flour,Wheat Protein Composition,and Textural Properties of Cooked Chinese White Salted Noodles

Physicochemical properties and protein composition of 39 selected wheat flour samples were evaluated and correlated with the textural properties of Chinese hard‐bite white salted noodles. Flour samples were analyzed for their protein and wet gluten contents, sedimentation volume, starch pasting properties, and dough mixing properties by farinograph and extensigraph. Molecular weight distribution of wheat flour proteins was determined with size‐exclusion (SE) HPLC, SDS‐PAGE, and acid‐PAGE. Textural properties of Chinese hard‐bite white salted noodles were determined through texture profile analysis (TPA). Hardness, springiness, gumminess, and chewiness of cooked noodles were found to be related to the dough mixing properties. Both protein content and protein composition were found to be related to TPA parameters of noodles. The amount of total flour protein was positively correlated to hardness, gumminess, and chewiness of noodles. The absolute amounts of different peak proteins obtained from SE‐HPLC data showed positive correlations with the hardness, gumminess, chewiness, and springiness of noodles. The proportions of these peak proteins were, however, not significantly related to texture parameters. The proportions of low‐molecular‐weight glutenins/gliadins and albumins/globulins, as observed from SDS‐PAGE, were correlated positively and negatively, respectively, to the hardness, gumminess, and chewiness of cooked noodles. Among the alcohol‐soluble proteins (from acid‐PAGE data), β‐gliadins showed strong correlations with the texture properties of cooked noodles. For the selected flour samples, the total protein content of flour had a stronger relationship with the noodle texture properties than did the relative proportion of different protein subgroups. Prediction equations were developed for TPA parameters of cooked noodles with SE‐HPLC and rapid visco analysis data of the 30 flour samples, and it was found that about 75% of the variability in noodle hardness, gumminess, and chewiness values could be explained by protein composition and flour pasting properties combined together. About 50% of the variations in cohesiveness and springiness were accounted for by these prediction equations.     

Sorghum Protein Extraction by Sonication and Its Relationship to Ethanol Fermentation

The objectives of this research were to develop a rapid method for extracting proteins from mashed and nonmashed sorghum meal using sonication (ultrasound), and to determine the relationships between the levels of extractable proteins and ethanol fermentation properties. Nine grain sorghum hybrids with a broad range of ethanol fermentation efficiencies were used. Proteins were extracted in an alkaline borate buffer using sonication and characterized and quantified by size‐exclusion HPLC. A 30‐sec sonication treatment extracted a lower level of proteins from nonmashed sorghum meal than extracting the proteins for 24 hr with buffer only (no sonication). However, more protein was extracted by sonication from the mashed samples than from the buffer‐only 24‐hr extraction. In addition, sonication extracted more polymeric proteins from both the mashed and nonmashed samples compared with the buffer‐only extraction method. Confocal laser‐scanning microscopy images showed that the web‐like protein microstructures were disrupted during sonication. The results showed that there were strong relationships between extractable proteins and fermentation parameters. Ethanol yield increased and conversion efficiency improved significantly as the amount of extractable proteins from sonication of mashed samples increased. The absolute amount of polymeric proteins extracted through sonication were also highly related to ethanol fermentation. Thus, the SE‐HPLC area of proteins extracted from mashed sorghum using sonication could be used as an indicator for predicting fermentation quality of sorghum.     

Effects of Micronization on Protein and Rheological Properties of Spring Wheat

This research investigated the effects of micronization, at different moisture levels, on the chemical and rheological properties of wheat. A set of tests designed to analyze protein fraction characteristics and rheological behaviors were conducted on samples from four wheat cultivars (AC Karma, AC Barrie, Glenlea, and Kanata). After being subjected to infrared radiation at three moisture levels (as‐is, 16%, and 22%), the seeds were milled to produce straight‐grade flour. The protein fractionation test revealed significant decreases (P ≤ 0.01) in both monomeric proteins (from 54% of total protein in the control to 37% in the tempered micronized sample) and soluble glutenins (9.4–2.5%). There was a strong negative correlation (r = ‐0.98) between the percentages of monomeric proteins and insoluble glutenins. Total extractable proteins of micronized samples tempered to 22% moisture decreased 43.5% when compared with nonmicronized control samples using size‐exclusion HPLC (SE‐HPLC). Micronization had a significant effect on gluten properties, as seen from a decrease in water absorption (P ≤ 0.01) and dough development time (P ≤ 0.01). Results showed that micronization at 100 ± 5°C had detrimental effects on wheat flour gluten functionality, including a decrease in protein solubility and impairment of rheological properties. These phenomena could be due to the formation of both hydrophobic and disulfide bonds in wheat during micronization.     

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.     

Quantitation of LMW‐GS to HMW‐GS Ratio in Wheat Flours

A comparison was made of methods for measuring the LMW/HMW glutenin subunit (GS) ratio for glutenin. A set of near‐isogenic wheat lines with the number of HMW‐GS varying from 0 to 5 was utilized to provide a wide range of LMW/HMW‐GS. Glutenin preparations were obtained from ground whole meal after solubilization of monomeric proteins by dimethyl sulfoxide (DMSO) or 50% propanol or by fraction collection from a preparative SE‐HPLC column. Analyses were made on the reduced glutenin from each of the three preparations by RP‐HPLC, SE‐HPLC, and SDS‐PAGE. Both solvents, DMSO and 50% propanol, extracted appreciable amounts of polymeric protein, thus casting some doubts on the accuracy of the determinations. This problem was largely avoided when the polymeric fraction was collected from the eluate of a total glutenin extract run on a preparative SE‐HPLC column. Less glutenin was removed by the two solvents for lines with a greater number of HMW‐GS or with strength‐associated HMW‐GS 5+10 coded by the 1D chromosome. Collection of the polymeric protein in SE‐HPLC, followed by separation of the glutenin subunits in RP‐HPLC, was the best method for quantitating the LMW/HMW‐GS ratio. SE‐HPLC gave a clear separation of the two groups of subunits as well as HMW albumins. RP‐HPLC has the potential advantage of being able to quantitate individual subunits.     

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.     

Interaction Between Sorghum Protein Extraction and Precipitation Conditions on Yield,Purity, and Composition of Purified Protein Fractions

Sorghum proteins have the potential to be used as a bio‐industrial renewable resource for applications such as biodegradable films and packaging. This project was designed to evaluate the effect of interactions between sorghum protein extraction and precipitation conditions on the yield, purity, and composition of sorghum protein fractions. Proteins were extracted with 70% ethanol under nonreducing conditions, with ultrasound, or under reducing conditions using either sodium metabisulfite or glutathione as the reducing agent. Several conditions were used to isolate the extracted proteins through precipitation, including lowering ethanol concentrations alone or in combination with lowering to pH 2.5, or by adding 1M NaCl to the extract. Combinations of these conditions were also tested. All precipitation conditions effectively precipitated proteins and lowering the pH and adding 1M NaCl to the extracts enhanced precipitation in some cases. However, the conditions that precipitated the maxium amount of protein or highest purity of protein varied according to how the proteins were initially extracted. Precipitated proteins were characterized by RP‐HPLC, SEC, HPCE, and SDS‐PAGE to compare the protein fractions composition. Nonreduced and sonicated samples had a much wider M_w distribution than reduced extracts. Thus, extraction and precipitation conditions influenced the isolated proteins yield, purity, and composition. Because the extraction and purification processes influenced the composition, purity, and biochemical properties, it may be possible to prepare protein fractions with unique functionalities for specific end‐uses.     

Storage of Wheat Grains at Elevated Temperatures Increases Solubilization of Glutenin Subunits

Grains of two wheat (Triticum aestivum L.) cultivars, Sunco and Sunsoft, were stored at 4°C and 30°C for 270 days to examine changes in proteins during storage. When whole meal flour extracted from the grains was analyzed using an unfractionated protein extraction procedure, no significant changes were found in protein content or SDS‐PAGE profile for either cultivar in samples stored at 30°C compared with those stored at 4°C. Fractionation of the flour samples from stored grain into soluble and insoluble proteins revealed increases in soluble protein content for both cultivars stored at 30°C compared with 4°C. The soluble protein content, expressed as a percentage of the total protein, increased by 1.5% (P = 0.032) for Sunco and by 8.0 % (P = 0.158) for Sunsoft during storage at 30°C compared with those samples stored at 4°C. Analysis by SDS‐PAGE and subsequent protein identification revealed that the most evident change that occurred during storage at 30°C was an increase in the content of high molecular weight glutenin subunits (HMW‐GS) in the soluble fraction. The potential effect of changes in solubility of HMW‐GS on functional properties is discussed.     

Characteristics and Protein Subunit Composition of Flour Mill Streams from Different Commercial Wheat Classes and Their Relationship to White Salted Noodle Quality

Proximate characteristics and protein compositions of selected commercial flour streams of three Australian and two U.S. wheats were investigated to evaluate their effects on the quality of white salted noodles. Wheat proteins of flour mill streams were fractionated into salt‐soluble proteins, sodium dodecyl sulfate (SDS)‐soluble proteins, and SDS‐insoluble proteins with a sequential extraction procedure. SDS‐soluble proteins treated by sonication were subsequently separated by nonreducing SDS polyacrylamide gel electrophoresis (SDS‐PAGE). There was a substantial amount of variation in distributions of protein content and protein composition between break and reduction mill streams. SDS‐insoluble proteins related strongly to differences in protein quantity and quality of flour mill streams. The soluble protein extracted by SDS buffer included smaller glutenin aggregates (SDS‐soluble glutenin) and monomeric proteins, mainly gliadin (α‐, β‐, γ‐, and ω‐types) and albumin and globulin. SDS‐soluble proteins of different flour mill streams had similar protein subunit composition but different proportions of the protein subunit groups. Noodle brightness (L) decreased and redness (a) increased with increased SDS‐insoluble protein and decreased monomeric gliadin. Noodle cooking loss and cooking weight gain decreased with increased glutenin aggregate (SDS‐soluble glutenin and SDS‐insoluble glutenin) and decreased monomeric gliadin. Noodle hardness, springiness, cohesiveness, gumminess, chewiness, tensile strength, breaking length, and area under the tensile strength versus breaking length curve increased with increased glutenin aggregate. Monomeric gliadin contributed differently to texture qualities of cooked noodles from glutenin aggregate. Monomeric albumin and globulin were not related to noodle color attributes (except redness), noodle cooking quality, and texture qualities of cooked noodles. The results suggested that variation in protein composition of flour mill streams was strongly associated with noodle qualities.     

Textural Comparisons of Gluten‐Free and Wheat‐Based Doughs,Batters, and Breads

Studies were conducted with two newly developed gluten‐free bread recipes. One was based on corn starch (relative amount 54), brown rice (25), soya (12.5), and buckwheat flour (8.5), while the other contained brown rice flour (50), skim milk powder (37.5), whole egg (30), potato (25), and corn starch (12.5), and soya flour (12.5). The hydrocolloids used were xanthan gum (1.25) and xanthan (0.9) plus konjac gum (1.5), respectively. Wheat bread and gluten‐free bread made from commercial flour mix were included for comparison. Baking tests showed that wheat and the bread made from the commercial flour mix yielded significantly higher loaf volumes (P < 0.01). All the gluten‐free breads were brittle after two days of storage, detectable by the occurrence of fracture, and the decrease in springiness (P < 0.01), cohesiveness (P < 0.01), and resilience (P < 0.01) derived from texture profile analysis. However, these changes were generally less pronounced for the dairy‐based gluten‐free bread, indicating a better keeping quality. Confocal laser‐scanning microscopy showed that the dairy‐based gluten‐free bread crumb contained network‐like structures resembling the gluten network in wheat bread crumb. It was concluded that the formation of a continuous protein phase is critical for an improved keeping quality of gluten‐free bread.     

Network Formation in Gluten‐Free Bread with Application of Transglutaminase

One of the main problems associated with gluten‐free bread is obtaining a good structure. Transglutaminase (TGase), an enzyme that catalyzes acyl‐transfer reactions through which proteins can be cross‐linked could be a way to improve the structure of gluten‐free breads. The objective of this study was to evaluate the impact of TGase at different levels (0, 0.1, 1, and 10 U of TGase/g of protein) on the quality of gluten‐free bread. The recipe consisted of white rice flour (relative amount: 35), potato starch (30), corn flour (22.5), xanthan gum (1), and various protein sources (skim milk powder [SMP] [12.5], soya flour, and egg powder). The influence of the various proteins in combination with the different addition levels of TGase on bread quality (% bake loss, specific volume, color, texture, image characteristics, and total moisture) was determined. Confocal laser‐scanning microscopy (CLSM) was used to evaluate the influence of TGase on the microstructure of the bread. Baking tests showed that TGase had an effect on the specific volume of the bread. For instance, the SMP bread with 10 U of enzyme contained the most compact structure, which was reflected in the crumb texture profile analysis results (highest values) (P < 0.05), digital image analysis (highest level of cells/cm²) (P < 0.05), and CLSM micrographs (network formation). Finally, it can be concluded that it is possible to form a protein network in gluten‐free bread with the addition of TGase. However the efficiency of the enzyme is dependent on both the protein source and the level of enzyme concentration.     

Application of High‐Intensity Ultrasound and Surfactants in Rice Starch Isolation

High‐intensity ultrasound was evaluated as an alternative method to isolate rice starch without the use of chemicals as in the traditional alkaline steeping method. Surfactants, including sodium dodecyl sulfate (SDS), sodium stearoyl lactylate (SSL), and Tween 80, at 0.1, 0.3, or 0.5% combined with high‐intensity ultrasound were also investigated for rice starch isolation. A rice flour slurry (33%) was subjected to sonication for 15, 30, or 60 min at an amplitude of 25, 50, or 75% and at 40 or 50°C. The starch yield was not significantly affected by the treatment temperature and ranged from 46.7 to 76.2% (starch dry basis) after the sonication treatment; the protein and damaged starch contents of the isolated starches were 0.9–1.7% and 3.1–3.5% (dry basis), respectively. The combination of 0.5% SDS and high‐intensity ultrasound improved the starch yield to 84.9% with low residual protein, however, little improvement was observed with SSL or Tween 80. The pasting properties of isolated starch as measured by a Rapid Visco‐Analyser were affected by the treatment temperature and by the amount of residual protein and damaged starch. The thermal properties of the isolated starch were not changed by sonication and the amylose content remained unchanged. The surface of the isolated starch was not damaged by sonication as shown by scanning electron microscopy. High‐intensity ultrasound, alone or combined with SDS, showed a great potential for rice starch isolation in a short period of time without generating alkaline effluent.     

Proteins Extracted by Water or Aqueous Ethanol During Refining of Developed Wheat Dough to Vital Wheat Gluten and Crude Starch as Determined by Capillary‐Zone Electrophoresis (CZE)

Fluids applied to large‐sale, technical separation of wheat starch and protein also extract soluble proteins. The degree and rate of extraction and the specific components extracted depend on the flour, the flour hydration and development, the starch‐displacing fluid composition, the temperature, and the mechanical processing method. This study sought to identify major extracted protein groups using high‐performance capillary zone electrophoresis (CZE) applied directly to fluids obtained during laboratory‐scale technical separations. A dough‐ball or compression separation method was applied using a Glutomatic system and a batter or dispersion method was applied using a a McDuffie mixer and Pharmasep vibratory separator. Process fluids were water at 22°C to model commercial practice and 70 vol% ethanol in water at ‐13°C to model the cold ethanol process being developed here. Data were referenced to use of 70 vol% ethanol in water at 22°C in the Glutomatic compression method. The dough processed by each method was developed by mixing to a separable state. When flooded with excess water, this dough immediately released starch and water‐soluble or albumin proteins. When flooded with excess cold aqueous ethanol, neither the albumin nor gliadin proteins appeared in significant amounts until the bulk of the starch had been displaced, regardless of the mechanical method. Even with extraction and manipulation well beyond that necessary for starch displacement, the net amount of gliadin proteins dissolved was only ≈10% of that available from wet developed dough using 70 vol% ethanol at 22°C. There was more gliadin protein in the fluids at earlier stages of processing when the batter dispersion method was applied using cold ethanol. The most common soluble proteins revealed in the electrophoresis patterns for the batter compression method using cold aqueous ethanol were initially albumins and later γ‐gliadins. Albumins not appearing as soluble in cold 70 vol% ethanol were found in the insoluble crude starch, suggesting their precipitation in the dough fluids during the change from free water to cold aqueous ethanol. These results establish that some protein is dissolved during starch displacement by cold aqueous ethanol, but that the amounts may be limited by control of the mechanical working of the dough in the presence of the displacing fluids.     

Simple Determination of Gluten Protein Types in Wheat Flour by Turbidimetry

A simple method based on turbidimetry has been developed for the quantitative determination of total gliadins, glutenin subunits, and high and low molecular weight (HMW and LMW) subunits of glutenin. The standard procedure includes the subsequent extraction of wheat flour (100 mg) with a salt solution, with 50% 2‐propanol (gliadins), and with 50% propanol under reducing conditions and increased temperature (glutenin subunits). Aliquots of the gliadin and the glutenin extracts are mixed with 2‐propanol to a final concentration of 83%, and the turbidity of the precipitates is measured photometrically at 450 nm and 20°C after 40 min. Another aliquot of the glutenin extract is mixed with acetone to a final concentration of 40% acetone, and precipitated HMW subunits are determined turbidimetrically after 30 min. The sample is then filtered, and an aliquot of the filtrate is mixed with 2‐propanol to a final concentration of 77% to determine the precipitated LMW subunits. Control analyses with reversed‐phase HPLC on C₈ silica gel indicate that the precipitation of the different protein types is quantitative and specific, and studies of 16 different wheat flours demonstrate the strong correlation between quantification by HPLC and turbidimetry. The turbidimetric measurements are reproducible, linear over a wide absorbance range (0.2–1.7), and sufficiently sensitive to analyze 40 μg of protein or 20 mg of flour. The absolute amounts of protein types in flour can be determined by means of calibration curves with protein standards (gliadins, HMW, and LMW subunits). Altogether, the developed method is simple, accurate, sensitive, and specific for the different protein types. The total procedure takes ≈6 hr for the analysis of six flour samples in parallel or ≈4 hr for three samples in overlapping extraction steps. The chemicals used are inexpensive, scarcely toxic, and easy to dispose.     

Defining the Overall Quality of Cowpea‐Enriched Rice‐Based Breakfast Cereals

The development of innovative legume‐enriched rice products is a promising way to exploit rice varieties with a low sensory grade. In this work, a multidisciplinary approach was applied to the characterization of extruded breakfast cereals prepared from African‐grown Oryza glaberrima (cv. Viwonor) or Oryza sativa (cv. Jasmine 85) enriched with 30% cowpea flour, obtained from sprouted or nonsprouted cowpea. Regardless of the rice species, addition of sprouted cowpea flour conferred a peculiar volatiles profile, rich in sour, bitter, and astringent taste. Protein structural indices provided molecular insights about the macroscopic differences among samples. Extruded products from O. glaberrima were characterized by lower expansion rates with respect to those obtained from O. sativa , regardless of the type of cowpea flour. Sprouting time had a positive influence on the hardness of extruded glaberrima‐based products, facilitating formation of a more compact matrix, but it did not influence sativa‐ based products. Therefore, the breakdown of protein during sprouting appeared fundamental for the incorporation of legume proteins in more compact matrices, such as the one from sativa rice. In the glaberrima‐based products, addition of sprouted cowpea resulted in further loosening of the structure, and this was more evident at increased sprouting times.     

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.     

Improved Method for Isolating and Quantitating α‐Amino Nitrogen as Nonprotein,True Protein,Salt‐Soluble Proteins,Zeins, and True Glutelins in Maize Endosperm

The conventional Landry‐Moureaux method for selective extraction of maize proteins was modified by reducing the contact time of meal with extractants and by removing 55% 2‐propanol as extractant. The new procedure, coupled with a method for quantitating protein at microgram level, was used for assessing the nitrogen distribution of four soluble protein fractions present in 100‐mg samples of endosperm originating from six maize inbreds and opaque‐2 versions. Proteins extracted with 55% 2‐propanol plus reductant were made up of α‐, β‐, γ‐, and δ‐zeins. Proteins extracted subsequently with salt plus reductant were minor and poor in lysine (1 mol%).They were associated with zeins. Comparison of present data with those available in the literature showed a close similarity for a given genotype between the percentage of total α‐amino nitrogen extracted by 2‐propanol plus reductant than by salt plus reductant under conditions of the modified procedure and that of total Kjeldhal nitrogen extracted by 2‐propanol with and without reductant, and by salt plus reductant, using the conventional procedure. A simplified protocol was described and tested for isolating and quantitating α‐amino nitrogen as nonprotein, true protein, salt‐soluble proteins, zeins, and true glutelins in any sample of maize endosperm.