Effect of Aelia spp. and Eurygaster spp. Damage on Wheat Proteins

The effect of Aelia spp. and Eurygaster spp. wheat bugs on the protein fractions of different wheat cultivars has been studied by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and free‐zone capillary electrophoresis (FZCE). Those methods were used to quantify and characterize the extent of protein modification. A decrease in the amount of alcohol‐insoluble polymeric proteins along with an increase in the alcohol‐soluble polymeric proteins and gliadins were observed in damaged wheat. The high molecular weight (HMW) and low molecular weight (LMW) glutenin fractions were barely detected in the incubated damaged wheat from some cultivars, which indicated hydrolysis of those proteins by the bug proteinases. In damaged wheats, both incubated and unincubated, gliadin electrophoregrams revealed the presence of some new peaks with mobilities similar to the ω gliadins. The overall results suggest that the bug proteinases are potent enzymes that appear to be nonspecific because they hydrolyze all gluten proteins.     

Biochemical Characterization and Quantification of the Storage Protein (Secalin) Types in Rye Flour

Kernels of the rye cultivars Danko and Halo were milled into white flour and compared with flour of the wheat cultivar Rektor. Flour proteins were extracted stepwise with a salt solution (albumins‐globulins), 60% ethanol (prolamins), and 50% 2‐propanol under reducing conditions (glutelins). The quantification by reversed‐phase HPLC indicated that the extractable proteins of both rye flours consisted of ≈26% albumins‐globulins, 65% prolamins, and 9% glutelins. Compared with wheat flour, rye flours comprised significantly higher proportions of nonstorage proteins (albumins‐globulins) and lower proportions of polymerized storage proteins (glutelins). SDS‐PAGE revealed that the prolamin fractions of rye contained all four storage protein types (HMW, γ‐75k, ω, and γ‐40k secalins), whereas the glutelin fractions contained only HMW and γ‐75k secalins. The quantification of secalin types by RP‐HPLC showed a close relationship between the two cultivars.The γ‐75k secalins contributed nearly half (≈46%) of the total storage proteins, followed by γ‐40k secalins (24%) and ω secalins (17%); HMW secalins (≈7%) were minor components, and 6% of eluted proteins were not identified. The amino acid composition of γ‐40k secalins corresponded to those of γ‐gliadins of wheat, whereas γ‐75k secalins were characterized by higher contents of glutamine and proline. Matrix‐assisted laser desorption/ionization and time of flight mass spectrometry (MALDI‐TOF MS) indicated molecular masses of about 52,000 (γ‐75k) and 32,000 (γ‐40k), respectively. N‐terminal amino acid sequences were homologous with those of wheat γ‐ gliadins except for position 5 (asparagine in γ‐75k and glutamine in γ‐40k secalins) and position 12 (cysteine in γ‐75k secalins). The N‐terminal amino acid sequences of HMW and ω‐secalins were homologous with those of the corresponding protein types of wheat. Gel‐permeation HPLC of prolamin fractions revealed that rye flours contained a significantly higher proportion of ethanol‐soluble oligomeric proteins than wheat flour.     

Effect of Microbial Transglutaminase on Dough Proteins of Hard and Soft (Triticum aestivium) and Durum (Triticum durum) Wheat Cultivars

Microbial transglutaminase (MTGase), a protein‐glutamine γ‐glutamyl transferase (E.C. 2.3.2.13), catalyzes acyl transfer reactions by introducing a covalent cross‐link between l ‐lysine and l ‐glutamine residues. The use of this enzyme has been proposed as an improver to increase dough strength. The objective of this study was to assess and compare the effect of MTGase on different fractions of dough proteins found in hard, soft, and durum wheat. Three different concentrations of the MTGase (0, 5, and 10U/g of gluten) were tested. Moisture, protein, and dry gluten contents were determined for each concentration in addition to rheological measurements done with the farinograph. Following each treatment, the dough proteins were extracted and analyzed by SE‐HPLC and RP‐HPLC. Soluble polymeric protein, gliadins, albumins, and globulins were quantified in addition to the gliadin subclasses and glutenin subunit types. The combustion procedure was used to determine the amount of insoluble polymeric protein. Differences were observed in susceptibility to MTGase catalysis among the dough proteins of the cultivars studied: the cultivar Cortazar (soft wheat) was the most susceptible. The proteins of this cultivar had a characteristically higher amount of ω and α+β gliadins when compared with the other cultivars. As reported earlier, solubility of high molecular weight glutenin subunits and ω‐gliadins was reduced because of the MTGase treatment. However, all gliadin subclasses, including the γ and α+β gliadins, also participated in cross‐linking. The proteins of the cultivar Altar (durum wheat) were the least susceptible to the effects of MTGase. Albumins and globulins did not show any reduction in solubility, implying that they did not participate in cross‐linking.     

Sequential extraction and quantitative recovery of gliadins, glutenins, and other proteins from small samples of wheat flour

Methods to sequentially extract and fractionate wheat flour proteins were evaluated to reliably quantify gliadins, glutenins, and albumins/globulins in single flour samples. Compositions of the resulting protein fractions were analyzed by RP-HPLC combined with SDS-PAGE. Unknown proteins were identified by mass spectrometry or N-terminal sequencing. The best separation and recovery of discrete albumin/globulin, gliadin, and glutenin fractions from the same flour sample was achieved by extraction with 0.3 M NaI in 7.5% 1-propanol followed by 2% SDS, 25 mM DTT in 25 mM TRIS, pH 8.0, and precipitation of the solubilized proteins with ammonium acetate/methanol followed by acetone. Average flour composition for the variety Butte86 was 10% albumin/globulin, 40% gliadin, and 48% glutenin. This method should be useful for determining flour composition in diverse samples and evaluating relationships between proteins and end-use functionality.     

Detection of smoked paprika "Pimentón de La Vera" adulteration by free zone capillary electrophoresis (FZCE)

The purpose of this work was to develop a procedure based on protein analysis by free zone capillary electrophoresis (FZCE) that can be used in the determination of smoked paprika "Pimentón de La Vera" adulteration with paprika elaborated from varieties of pepper foreign to the "La Vera" region, in central western Spain. Two autochthonous varieties of pepper, Jaranda and Bola, and the variety Papri Queen, foreign to the "La Vera" region, were used in the study. Several aqueous solutions for solubilization of the methanol-soluble proteins were tested, and the FZCE conditions of capillary dimensions, FZCE buffer concentrations, and detection wavelengths were optimized. On the basis of the results, 30% (v/v) acetonitrile was adopted as the suspending solution for routine analysis, and the optimal FZCE parameters were 75 microm inner diameter and 57 cm total length capillaries, 8.75 mM phosphate/20.6 mM tetraborate as run buffer, and 256 nm as detection wavelength. This method was found to give excellent repeatability of the corrected migration time (CMT) with coefficients of variation (RSD %; n = 5) of <1% for most of the proteinaceous compounds analyzed and showed greater effectiveness in discriminating paprika varieties than the SDS-PAGE technique. Four peaks found in the FZCE electropherograms were investigated as a basis for detecting and estimating the adulteration of smoked paprika with paprika elaborated from the Papri Queen variety. The adulteration detection limits varied from 5 to 40% of the Papri Queen variety within a satisfactory working range of mixture (5-80%) sufficiently large to cover the adulteration levels of interest. The use of peak 6 as a marker for determining adulteration gave the best results, with an adulteration detection limit of 5-10% (w/w).     

Capillary Electrophoresis of Gliadins as a Tool in the Discrimination and Characterization of Hulled Wheats (Triticum dicoccon Schrank and T. spelta L.)

Twenty‐two lines of emmer (T. dicoccon Schrank) and 10 of spelt (T. spelta L.) were analyzed using capillary electrophoresis for their gliadins. These proteins were separated on an uncoated fused‐silica capillary (30 cm long, 22 cm to detector, 50 μm i.d.) using the isoelectric buffer 40 mM aspartic acid, 4M urea, 0.5% (w/v) HEC, and 20% (v/v) acetonitrile. Samples were run for 20 min at 22kV and 42°C. By using these conditions, gliadins were separated into 21–30 components (peaks and shoulders). The major peaks eluted between 4.5 and 8.5 min. Electrophoregrams of tested lines showed qualitative and quantitative differences, including number of peaks, presence or absence of some major peaks, and areas of peaks. Lines belonging to the same species can be discriminated mainly on the basis of β‐ and ω‐gliadin patterns. The γ‐and ω‐gliadins seem to be more useful in the differentiation of emmer from spelt. The comparison of electrophoregrams relative to hulled and unhulled species evidenced the high similarity between species with the same genome composition (durum wheat‐emmer, and common wheat‐spelt).     

Refrigerated Storage of Yellow Alkaline Durum Noodles: Impact on Color and Texture

Durum wheat straight‐grade flour samples, representing the cultivars Commander and Strongfield, a composite cargo mixture of Canada Western Amber Durum cultivars and a Japanese commercial durum flour were used to make yellow alkaline noodles. A Canada Western Red Spring common wheat composite straight‐grade flour was included in the study for comparative purposes. Alkaline noodles were prepared using 1% w/w kansui reagent (sodium and potassium carbonates, 9:1) and stored for 1, 2, 3 and 7 days at 4°C to duplicate a normal convenience store operation. The raw noodle color of the durum alkaline noodles exhibited significantly better noodle brightness, L*, and yellowness, b*, as compared to noodles prepared from common wheat at all storage periods. The number of discolored specks in the durum flour based noodles was significantly lower as well as significantly lighter than those of common wheat at all time intervals. Noodles prepared from Commander, Strongfield, or the cargo composite flours displayed significantly lower water uptake during cooking than both the commercial durum flour and the common wheat noodles. The commercial durum flour noodles displayed the thinnest cooked noodles, while the common wheat flour noodles were the thickest. Evaluation of cooked noodle texture, immediately after production and subsequent storage of the raw noodles at 4°C for 1, 2 and 3 days before cooking showed a general increase in maximum cutting stress (MCS) with storage. Noodles prepared from Commander flour consistently display MCS values exceeding those of CWRS as well as the highest resistance to compression (RTC) and recovery (REC) measurements. The visual improvements in noodle brightness, enhanced yellowness, reduced speck numbers and darkness in combination with equivalent to improved cooked noodle texture attributes compared with common wheat flour suggests that durum flours are an ideal material for fresh, refrigerated yellow alkaline noodles.     

Proteolysis in model sourdough fermentations

Model wheat doughs started with six different lactic acid bacteria (LAB), with or without a commercial baker's yeast culture, were used to study proteolysis in sourdough fermentations. Cell counts, pH, and free amino acid concentration were measured. Sequential extraction of dough samples was performed to separate wheat proteins. The salt-soluble protein fraction (albumins and globulins) was analyzed by RP-HPLC and SDS-PAGE, whereas propanol-soluble (gliadins) and insoluble (glutenins) protein fractions were analyzed by SDS-PAGE only. Multivariate statistical methods were used for the analysis of results. The presence of yeasts and LAB affected RP-HPLC and SDS-PAGE patterns of the salt-soluble fraction in a complex way. The only changes in the gluten proteins that could be related to the presence of LAB were the appearance of new protein fragments (20 and 27 kDa) from gliadins and the degradation of high molecular weight glutenin subunits.     

Impact of Genotype and Environment on the Quality of Amber Durum Wheat Alkaline Noodles

Canada Western Amber Durum wheat cultivars (4), Canada Western Red Spring (1), and Canada Western Hard White Spring (1) wheat were grown at three sites in 2007 to evaluate the effect of genotype (G) and environment (E) on the quality of yellow alkaline noodles (YAN). YAN were evaluated for color, appearance, and cooked texture. Brightness (L*) and yellowness (b*) of YAN made from durum cultivars were significantly higher than common wheat. Durum flour yellow pigment content was approximately fourfold greater than common wheat while noodle speckiness was approximately half of CWRS at 2 hr with environment accounting for >75% of the variance for each parameter. Resistance to compression (RTC) and recovery (REC) of cooked durum alkaline noodles were equivalent or superior to common wheat noodles even when lower grade durum wheat flour was used. In conclusion, cooked durum noodle texture parameters were all significantly influenced by genotype and environment, with environment accounting for 66–71% of their variance.     

Immunochemical and Electrophoretic Analysis of the Modification of Wheat Proteins in Extruded Flour Products

Antibodies specific for wheat proteins were used to identify protein fractions modified during extrusion of Hard Red Spring wheat flour (14% protein) under four different combinations of extrusion conditions (18 and 24% feed moisture and 145 and 175°C die temperature). Antibody binding was assessed on immunoblots of proteins extracted from flour and extrudates separated by SDS‐PAGE. Antibodies to high molecular weight glutenin subunits (HMW‐GS) and to B‐group low molecular weight glutenin subunits (LMW‐GS) recognized intact subunits from both flour and extrudates. Antibodies to C‐group LMW‐GS had diminished binding to extruded proteins. Glutenin‐specific antibodies also recognized protein in the extrudates migrating as a smear at molecular weights higher than intact subunits, indicating cross‐linked proteins. Antibodies recognized albumins or globulins in flour but not in extrudates, evidence that these fractions undergo significant modification during extrusion. Acid‐PAGE and antibody reaction of gliadins extracted in 1M urea and in 70% ethanol revealed total loss of cysteine‐containing α, β, γ‐gliadins but no obvious effects on sulfur‐poor ω‐gliadins, suggesting gliadin modification involves replacing intramolecular disulfides with intermolecular disulfide cross‐links. Identifying protein fractions modified during different extrusion conditions may provide new options for tailoring extrusion to achieve specific textural characteristics.     

Characterization of protein fractions from Bt-transgenic and non-transgenic maize varieties using perfusion and monolithic RP-HPLC. Maize differentiation by multivariate analysis

Protein fractions from transgenic Bt and non-transgenic maize varieties, extracted by the Osborne solvent fraction procedure, were characterized for the first time by perfusion and monolithic RP-HPLC in very short analysis times. Albumins and globulins from different transgenic Bt maizes as well as from their non-transgenic isogenic varieties were eluted in four peaks using perfusion RP-HPLC, whereas prolamins and glutelins were separated in seven peaks. Monolithic RP-HPLC enabled the separation of maize proteins in a large number of peaks showing 6 and 10 main peaks for albumins and globulins, respectively. Prolamins migrated at retention times higher than 5 min as seven peaks, whereas glutelins were separated in three main peaks appearing at retention times higher than 6.0 min. Moreover, chromatograms of the whole protein extracts showed 8 and 11 components for perfusion and monolithic RP-HPLC, respectively. A comparison of the chromatograms of the whole protein extracts relative to transgenic and non-transgenic varieties evidenced quantitative differences on the percentages of area, mainly for peaks 2 and 3 by perfusion RP-HPLC and for peaks 3 and 7 by monolithic RP-HPLC. A discriminant analysis based on these proteic profiles was carried out to classify and predict transgenic Bt maize lines, achieving 100% correct classification using perfusion RP-HPLC.     

Detection of soft wheat in semolina and durum wheat bread by analysis of DNA microsatellites

The aim of this work was to evaluate the analysis of DNA microsatellites for the detection of soft wheat (Triticum aestivum L.) in semolina and durum wheat bread (prepared from Triticum turgidum L. var. durum). The results enabled selection of an efficient D-genome-specific repetitive DNA sequence to detect common wheat in semolina and breads by qualitative PCR with a threshold of 3 and 5%, respectively, lowered to 2.5% by real-time PCR. This is of major importance for checking during production of some typical products recently awarded the European Protected Designation of Origin (PDO) mark such as Altamura bread, which should not contain soft wheat flour. The feasibility of quantification of common wheat adulteration in semolina using real-time PCR was also demonstrated.     

Accessibility of Amino Groups in Gluten Proteins Studied by a Combination of Chemical Labeling and Immunochemical Detection

The accessibility of primary amino groups to an external probe in durum wheat gluten proteins in the flour itself, and after the extraction and separation of gluten proteins, was studied by labeling the free amino groups with the hemisuccinate of 2‐(2,4‐dichlorophenyl)‐3‐(1H‐1,2,4‐triazol‐1‐yl) propanol (FF18) and evidencing the label immunochemically. Within the flour, the amino groups were less available to the probe than after extraction, and gliadins were less accessible than glutenins, differences that decrease after solubilization and separation of the proteins. Data are discussed in relation to the structural organization of proteins within gluten.     

Wheat Flour Proteins as Affected by Transglutaminase and Glucose Oxidase

Enzymes are good tool to modify wheat proteins by creating new bonds between the protein chains. In this study, the effect of the addition of glucose oxidase (GO) and transglutaminase (TG) on the wheat flour proteins is presented. The modification of wheat proteins was determined by analyzing the changes in gluten quality, alveograph parameters, and protein modifications. The amount of wet gluten increased with the addition of GO and TG, but the gluten quality was not improved in any case. Regarding the alveograph parameters, the effect of GO was readily evident obtaining wheat dough with higher tenacity and lower extensibility than the control, while TG led to doughs with lower tenacity and that were also less extensible. The protein modifications were characterized by free‐zone capillary electrophoresis (FZCE). FZCE data indicated that TG polymerizes mainly glutenins and, of those, the high molecular weight glutenin subunits were the most affected.     

Comparing Small‐Scale Testing Methods for Predicting Wheat Gluten Strength Across Environments

Gluten strength is the main factor determining the rheological and processing properties of wheat. Rapid, small‐scale tests that can indirectly predict gluten strength are extremely important for wheat‐breeding selection, particularly when using pedigree methodology. The efficiency and reliability of three small‐scale tests (SDS sedimentation volume [SDSS], swelling index of glutenin [SIG], and lactic acid retention capacity [LARC]) across three environments (E1, no stress; E2, drought stress; and E3, heat stress) were evaluated by using 15 common wheat and nine durum wheat cultivars. In the case of common wheat, SIG highlighted its advantage for predicting gluten strength, even under stress environments, compared with LARC and SDSS, whereas SDSS showed the best relationship with bread loaf volume. For durum wheat, SIG showed the best predicting value in E1 and E3; however, under drought stress, SDSS, SIG, and LARC all lost their good ability for predicting gluten strength in durum wheat, which needs further investigation. Also, the comparison between two mixograph parameters (mixograph peak time and mixograph peak integral) for predicting gluten strength and the suitability of testing SIG and LARC with whole meal (or semolina) instead of refined flour were also investigated.     

Production of Flours with Reduced Epitope Content Using Milling Technology

Epitopes on the α‐gliadins are known to give rise to immune responses that may lead to the development of celiac disease in genetically predisposed individuals. The reduction of epitope levels in wheat‐based products would likely benefit this group of consumers and also consumers with non‐celiac gluten sensitivity. Conventional breeding of wheats with lowered epitope levels will take time, but in this study we show for the first time that milling technology can be used to produce flour mill streams that are depleted in α‐20 gliadin epitopes. Fifteen mill streams from two New Zealand wheat cultivars, Sapphire (a biscuit wheat) and Monad (a bread wheat), were tested with reversed‐phase HPLC and an α‐20 gliadin epitope ELISA kit. The level of α‐20 epitope measured in Sapphire gliadins was significantly less than that found in Monad gliadins, even taking into account differences in total protein content. For both cultivars, compared with the straight‐run flour, the break flours had similar or significantly higher proportions of α‐20 epitope per unit of protein, whereas most of the reduction streams had significantly lower proportions of α‐20 epitope per unit of protein. Theoretically, combining selected (mainly reduction) flour streams may produce flour with ∼75% of the epitope content of the straight‐run flour.     

Wheat Proteins Extracted from Flour and Batter with Aqueous Ethanol at Subambient Temperatures

Contact of wheat flour with aqueous ethanol may enrich protein by starch displacement or deplete protein by extraction depending on 1) extraction conditions and 2) the form of the substrate. Extraction at subambient temperatures has not been described for specific gliadins for either dry flour with the protein in native configurations or for wet, developed batter or dough. This limits the ability to interpret technologies such as the cold-ethanol method. Here, we describe specific albumin and gliadin composition of flour extracts by capillary zone electrophoresis CZE in 0–100% (v/v) ethanol from –12 to 22°C. Extraction was reduced for albumin and gliadin protein as the temperature was reduced and the concentration range for extraction narrowed. Extraction dropped precipitously between 0 and –7°C for both albumins and gliadins. Electrophoretically defined gliadins extracted in constant proportion at 22°C and 30–80%(v/v) ethanol, but at lower temperature, the α-gliadins were enriched and β-gliadins depleted in the 30–55% (v/v) range. For extracts from wheat flour batter, depletion of α and β and enrichment of γ relative to the dry flour contact suggested that the electrophoretically slow migrating γ- and ω-proteins are less well incorporated to the dough matrix than electrophoretically fast migrating α and β types.     

Separation of Wheat Proteins by Two-Dimensional Reversed-Phase High-Performance Liquid Chromatography Plus Free Zone Capillary Electrophoresis

Gliadins and glutenins from four hard red winter wheat cultivars were separated by a novel two-dimensional (2D) technique. Protein extracts were separated by reversed-phase high performance liquid chromatography as the first dimension with each 30-sec interval collected separately. Those fractions were then separated by free-zone capillary electrophoresis (FZCE) for the second dimension. Data was combined into 2D surface contour plots similar to traditional gel electrophoresis 2D maps. For HPLC, C₈ and C₁₈ columns were used in the first dimension to separate gliadins and glutenins, respectively. Uncoated fused silica capillaries (27 cm × 25 μm, i.d.) were used for the 2D FZCE separations. Differences in the 2D maps of both gliadin and glutenin fractions were found between pairs of both closely related and sister lines that varied in quality.     

Proteomic analysis by two-dimensional gel electrophoresis and starch characterization of Triticum turgidum L. var. durum cultivars for pasta making

Criteria for durum wheat quality are continuously evolving in response to market pressure and consumer's preference. Specific attributes of durum wheat for different end products require more rapid and objective means to grade and classify wheat parcels based on processing potential. A total of 10 durum wheat cultivars were compared for compositional, protein, and starch characteristics. Mean values for the gross composition differed for total protein, gluten, and starch. Two-dimensional electrophoresis (2DE) analysis showed the proteome diversity among the cultivars. As shown by the principal component analysis (PCA) applied to 2DE data of gliadin and glutenin fractions, cultivars differed mainly from the number of proteins and levels of protein expression. As determined by the rapid viscoanalyzer (RVA), swelling power, starch damage, amylose content, and starch pasting properties of 10 cultivars differed significantly. 2DE fingerprinting and amylose content seemed to distinguish specific cultivars being useful tools for selecting suitable durum wheat cultivars for pasta making.     

Quantification of Common Wheat Adulteration of Durum Wheat Pasta Using Real‐Time Quantitative Polymerase Chain Reaction (PCR)

Common wheat adulteration of durum wheat pasta was quantified using real‐time duplex polymerase chain reaction (PCR). The total DNA content of pasta was determined by amplifying part of a wheat gene encoding a lipid transfer protein, and common wheat DNA was quantified by amplifying part of the puroindoline‐b gene. Under the conditions defined by this study, for pasta with a theoretical adulteration of 3%, the experimentally determined mean value was 2.6–3.4%, depending on drying temperature. Pure durum wheat pastas were distinguished from adulterated pastas without ambiguity. This study demonstrates the feasibility of using real‐time duplex PCR to quantify common wheat adulteration of pasta dried at high temperature, quantification that was impossible with the French official peroxidase‐marker method.