Wheat Protein Fractions Research Articles

Delicate Analysis of Interacting Proteins and Their Assemblies by Flow Field-Flow Fractionation Techniques.

We study the efficiency of several asymmetrical flow field-flow fractionation (AF4) techniques to investigate self-associating wheat gluten proteins. We compare the use of a denaturing buffer including sodium dodecyl sulfate (SDS) and a mild chaotropic solvent, water/ethanol, as the eluent, on a model gluten sample. Through a thorough analysis of the data obtained from coupled light scattering detectors and with the identification of molecular composition of the eluted protein, we evidence coelution events in several conditions. We show that the focus step used in conventional AF4 with the SDS buffer leads to the formation of aggregates that coelute with monomeric proteins. By contrast, a frit-inlet device enables the fractionation of individual wheat proteins in the SDS buffer. Interestingly conventional AF4, using water/ethanol as eluent, is an effective method for fractionating gluten proteins and their complex dynamic assemblies, which involve weak forces and are composed of both monomeric and polymeric proteins.

Genomic prediction reveals unexplored variation in grain protein and lysine content across a vast winter wheat genebank collection.

Globally, wheat (Triticum aestivum L.) is a major source of proteins in human nutrition despite its unbalanced amino acid composition. The low lysine content in the protein fraction of wheat can lead to protein-energy-malnutrition prominently in developing countries. A promising strategy to overcome this problem is to breed varieties which combine high protein content with high lysine content. Nevertheless, this requires the incorporation of yet undefined donor genotypes into pre-breeding programs. Genebank collections are suspected to harbor the needed genetic diversity. In the 1970s, a large-scale screening of protein traits was conducted for the wheat genebank collection in Gatersleben; however, this data has been poorly mined so far. In the present study, a large historical dataset on protein content and lysine content of 4,971 accessions was curated, strictly corrected for outliers as well as for unreplicated data and consolidated as the corresponding adjusted entry means. Four genomic prediction approaches were compared based on the ability to accurately predict the traits of interest. High-quality phenotypic data of 558 accessions was leveraged by engaging the best performing prediction model, namely EG-BLUP. Finally, this publication incorporates predicted phenotypes of 7,651 accessions of the winter wheat collection. Five accessions were proposed as donor genotypes due to the combination of outstanding high protein content as well as lysine content. Further investigation of the passport data suggested an association of the adjusted lysine content with the elevation of the collecting site. This publicly available information can facilitate future pre-breeding activities.

Fourier transform-based protein fraction analysis of whole-seed wheat

Abstract The principle of the Fourier transform is explored in this paper to compare protein structure similarity, transforming protein structure into a distance sequence, and performing spectral analysis on a fast Fourier transform. Secondly, protein fraction classification and similarity analysis of whole-seed wheat protein fractions were performed using the fast Fourier transform. Fourier transform infrared spectra were analyzed using two parameters: diatomic vibrations and molecular leaps. Finally, the whole seed wheat protein fraction content test analysis was analyzed experimentally. The results showed that the spectral range of whole seed wheat protein fractions was selected from 10539 cm −1 to 6080 cm −1, the fraction of fractions was determined as 9, the parameter R 2 val was 0.9614. This paper provides practical reference material for researching whole-seed wheat protein fields.

Proteomic Characterization of Wheat Protein Fractions Taken at Different Baking Conditions.

Food processing conditions affect the structure, solubility, and therefore accurate detection of gluten proteins. We investigated the influence of dough, bread, and pretzel making on the composition of different wheat protein fractions obtained by Osborne fractionation. The albumin/globulin, gliadin, and glutenin fractions from flour, dough, crispbread, bread, and pretzel were analyzed using RP-HPLC, SDS-PAGE, and untargeted nanoLC-MS/MS. This approach enabled an in-depth profiling of the fractionated proteomes and related compositional changes to processing conditions (mixing, heat, and alkali treatment). Overall, heat treatment demonstrated the most pronounced effect. Label-free quantitation revealed significant changes in the relative abundances of 82 proteins within the fractions of bread crumb and crust in comparison to flour. Certain gluten proteins showed shifts or reductions in particular fractions, indicating their incorporation into the gluten network through SS and non-SS cross-links. Other gluten proteins were enriched, suggesting their limited involvement in the gluten network formation.

Functional and Bioactive Properties of Wheat Protein Fractions: Impact of Digestive Enzymes on Antioxidant, α-Amylase, and Angiotensin-Converting Enzyme Inhibition Potential.

This research aimed to determine the biofunctional properties of wheat flour (WF) protein fractions and modifications to the antioxidant, anti-α-amylase and anti-angiotensin-I converting enzyme (ACE) activities induced by the action of digestive endopeptidases in vitro. A molecular characterization of the most abundant protein fractions, i.e., albumins, glutelins-1, glutelins-2 and prolamins, showed that low- and high-MW polypeptides rich in cysteine, glutamic acid and leucine were present in albumins and glutelins, whereas low-MW subunits with a high proportion of polar amino acids prevailed in prolamins. Prolamins exhibited the second-highest water holding capacity (54%) after WF (84%), while albumins provided superior foam stability (76%). Prolamins, glutenins-1 and globulins demonstrated the highest antioxidant activity (up to 95%, 68% and 59%, respectively) both before and after hydrolysis with pepsin (P-H) or trypsin-chymotrypsin (TC-H). Prolamins, globulins and WF strongly inhibited α-amylase (>90%) before and after TC-H, and before P-H (55-71%). Moreover, P-H significantly increased α-amylase inhibition by albumins from 53 to 74%. The fractions with strong ACE inhibitory activity (70-89%) included prolamins and globulins after TC-H or P-H, as well as globulins before TC-H and WF before P-H. This novel evidence indicates that WF protein fractions and their peptide-enriched P and TC hydrolysates are excellent sources of multifunctional bioactives with antioxidant, antihyperglycemic and antihypertensive potential.

Clipping Effect on the Grain Nitrogen and Protein Fractions of Ancient and Old Wheats Grown in a Mediterranean Environment

This study is the first to assess the effects of clipping, cultivar, season, and their interactions on the protein composition of six old and ancient wheat cultivars (n = 6). For this, nitrogen content, the proportion of wheat protein fractions, and the molecular weight distribution of the extractable and unextractable glutenin polymers were investigated as a function of cultivar and clipping in two consecutive seasons. The relationships between genotypic variation in grain nitrogen and protein fraction content under clipping and non-clipping conditions were also assessed. Clipping delayed and shortened the grain filling period of all of the cultivars. The protein composition of some cultivars behaved differently to clipping due to differences in the environmental conditions of S1 (exceptional dry season) and S2 (rainy season). In S1, clipping decreased the ratio of gliadins over glutenins (GLI/GLU) (<1) of Cappelli and Giovanni Paolo, while in S2, clipping improved the GLI/GLU of Giovanni Paolo, Monlis, and Norberto. The unextractable polymeric proteins were not affected by clipping. Khorasan was shown to be indifferent to clipping in S1 and S2. These results suggest that it is possible to have ancient/old wheats suitable for a dual-purpose system, in different climatic conditions, while maintaining good grain quality traits. The increased market demand for ancient and old wheats presents an economic opportunity for farmers who adopt the dual-purpose technique to cultivate these resilient crops again and increase their profit margins and revenues.

Physicochemical and Rheological Characteristics of Commercial and Monovarietal Wheat Flours from Peru.

In Peru, wheat (Triticum aestivum L.) is one of the main resources in the food industry; however, due to its low harvested area, it is the second most imported cereal. The quality of wheat flour was studied to verify that it has desirable characteristics for the preparation of bakery products. The quality of commercial and monovarietal wheat flours was assessed by measuring their physicochemical and rheological parameters, as well as the gluten content and wheat protein fractions. Eight commercial wheat flours and four monovarietal wheat flours (Barba negra, Candeal, Espelta, and Duro) from Peru were evaluated. Commercial wheat flours presented significantly higher levels of protein and gluten index compared to monovarietal wheat flours (p < 0.05). Between both groups, no significant differences were observed in the content of wet and dry gluten. Interestingly, monovarietal wheat flours presented a higher percentage of gliadins and albumins/globulins, as well as lower levels of glutenin, compared to commercial wheat flours (p < 0.05). According to the logistic regression models, the baking strength (W) was the most important parameter to evaluate the quality of commercial and monovarietal wheat flours. Our results show that monovarietal wheat flours show a lower quality compared to commercial wheat flours.

Effect of Sitophilus zeamais (Coleoptera: Curculionidae) Infestation on the Protein Physicochemical and Structural Properties of Wheat Grain.

Boring pests such as Sitophilus zeamais (S. zeamais) are major threats in grain storage. However, how these pests affect the proteins of stored grains remains largely unknown. Here we aimed to investigate the effect of S. zeamais infestation on wheat protein during postharvest storage. In this study, wheat grain infested by S. zeamais was sampled at egg (4 d), larval (20 d), pupal (35 d), and adult stages (45 d), respectively. The protein's physicochemical and structural properties and the edible quality of whole wheat noodle were analyzed. The results showed that S. zeamais infestation significantly decreased the quality of wheat protein by altering its constitution and structure properties. Especially, compared with the control, the content of wet and dry gluten, gluten index, sodium dodecyl sulfate sedimentation volume, sulfhydryl groups, and disulfide bonds in insect-infested wheat decreased by 19.40, 5.42, 18.40, 8.12, 29.13, and 14.30%, respectively, during the storage period of one life cycle of S. zeamais. Additionally, the proportions of wheat protein fractions (albumin [1.16-fold], globulin [0.96-fold], gliadin [1.16-fold], and glutenin [0.95-fold]) and secondary structures (α-helix [0.91-fold], β-fold [0.96-fold], β-turn [1.06-fold], and random coil [1.05-fold]) of protein changed significantly, and the gluten network structure was broken in S. zeamais-infested wheat. Furthermore, the color of whole wheat noodle became darker, cooking loss rate increased, and textural properties (hardness, adhesiveness, springiness, cohesiveness, chewiness, and resilience) decreased as well. The results in the present study provided new insights for analyzing the quality deterioration mechanism and further quality improvement of boring pests-infested wheat grain.

A sensitive sandwich enzyme-linked immunosorbent assay (sELISA) targeted multiple wheat protein fractions for the detection of several cereal grains in processed foods.

Wheat allergy has become a global public health and food safety concern; however, there is no accessible cure for wheat allergy. The complete exclusion of wheat-containing foods and environmental exposure is the most efficient allergy management to avoid the adverse reactions, which can be severe and occasionally life threatening. Therefore, the assay for accurate detection of wheat residues is demanded urgently for appropriate labeling guidelines and consumer safety. Thus, a sandwich enzyme-linked immunosorbent assay (sELISA) targeting multiple wheat protein fractions was fabricated in the present study. The results showed that the limit of detection (LOD) and limit of quantitation (LOQ) of the constructed sELISA were 0.25 and 0.5µg/g with high specificity for wheat. No cross-reactivity was observed in 32 foods or food ingredients tested, except barley and rye. The developed sELISA can also discriminate against many commercial foods containing declared or undeclared wheat residues except for Chinese yellow wine. Furthermore, high heat also can obtain a higher level of proteins extracted with corresponding enhanced detectability up to 100°C from heated samples and 160 °C in baked samples. PRACTICAL APPLICATION: Wheat is the most common food ingredient and wildly applied in various processed foods. However, wheat can cause severe and life-threatening symptoms in some allergic patients and must be labeled and tested accurately to protect those with a wheat allergy. Developing a new test assay can serve as a powerful tool for food manufacturers and regulatory agencies to accurately quantify wheat residues in processed foods and ensure their absence due to unintended contamination.

Determination of soluble wheat protein fractions using the Bradford assay

AbstractBackground and objectivesDetermination of different grain protein fractions in wheat cultivars is an important task in analyzing bread baking quality. In many laboratories, the Bradford assay is used to determine protein concentrations in solutions. In any protein assay (including Bradford), the ideal protein to use as a standard is the purified protein being assayed. In the absence of such an absolute reference, protein another protein must be selected as a relative standard such as bovine serum albumin (BSA) which is widely used. The aim of this work was to find conversion factors for BSA to determine correct albumin–globulin, gliadin, and glutenin concentrations, because these purified wheat grain protein fractions are mostly not available to be used for calibration purposes.FindingsIn case of BSA calibration, gluten concentration was underestimated (50%–54%) compared to calibration with the respective purified wheat proteins (65%–70%) in extracts of wheat grain samples. This result is explained with the different amino acid composition of BSA and wheat protein fractions leading to a more intense signal with BSA in the Bradford assay. Calibration of the Bradford assay using BSA as well as purified wheat protein fractions allowed to calculate the conversion factors of 2.11 for BSA/albumin–globulin, 4.25 for BSA/gliadin, and 3.42 for BSA/glutenin. Application of these conversion factors proved to accurately adjust protein concentrations of wheat fractions originating from ten cultivars, determined with BSA calibration of the Bradford assay.ConclusionsBSA calibration of the Bradford assay in combination with the conversion factors can be used to determine protein concentration of wheat grain fractions.Significance and noveltyFindings of this study make a contribution toward the correction of a common method, to provide a basis for better comparability of research results from different laboratories.

Development of a concept for the production of wheat snacks with the elimination of gluten by the biocatalysis

The increase in the number of cases of allergic reactions and celiac disease is an important problem. The solution to this problem is the search and development of relevant and effective ways to eliminate gluten. Specific amino acid sequences glutamine and proline determine the resistance to protease hydrolysis of the structural domains of gluten fractions. The analysis of the literature data showed that an alternative to the gluten-free diet is the use of biotechnological methods for modifying ingredients containing gluten. Such methods include the use of leavens on the base of lactic acid bacteria or enzyme preparations containing peptidases specific to gluten biocatalysis. In addition, the pretreatment of raw materials by extrusion cooking contributes to an increase in the degree of gluten hydrolysis. The effect of the thermoplastic extrusion and various enzyme systems containing proteases, amylolytic, cellulolytic and hemicellulolytic enzymes on the changes in the molecular weights of wheat protein fractions was studied. It was found that extrusion as a factor of protein modification significantly affects the proteolysis of wheat proteins using enzyme systems of different substrate specificity. The most effective hydrolysis was shown by the use of a complex enzyme preparation Amyloprotoorizin. including The effect was also noted after bioconversion of non-extruded wheat. An algorithm for the technology of wheat snacks based on the processes of extrusion and biocatalysis of proteins with specific proteases for the elimination of gluten is devepoped. The practical implementation of the technology will make it possible to obtain ready-to-eat snacks, which will be investigated for the preservation or elimination of antigenic properties during clinical trials.

Extraction of total wheat (Triticum aestivum) protein fractions and cross-reactivity of wheat allergens with other cereals

A one-step mild extraction of total wheat protein fractions was developed in this study, and the allergic cross-reactivity among dietary cereals were assessed by SDS-PAGE, western blotting, indirect ELISA, and inhibition ELISA using sera from 12 wheat allergic patients. The fractions of albumin, globulin, gliadin and glutenins in wheat flour can be obtained by a one-step extraction with Na2CO3-NaHCO3 (20 mM, pH 9.6, 0.5 M NaCl, 40% ethanol, 1 mM PMSF) in comparison to sequential extractions. Results showed high cross-reactivity in wheat, barley and rye due to close resemblance and high sequence identity (>50%), whereas nearly negligible cross-reactivity among rice, buckwheat, and quinoa was observed. Our research findings suggest that people with wheat allergy should rely primarily on the use of rice, quinoa and non-grain buckwheat, which is an effective substitute for wheat, while those with hypersensitivity should avoid the use of barley and rye in their diet.

The mesoscopic structure in wheat flour dough development

The Farinograph time-to-peak is an important wheat flour quality parameter. It is well-established that insoluble glutenins correlate with the strength of the gluten network and dough mixing time. To learn more about the physical changes at the mesoscopic level, dough samples were prepared in the Farinograph for study with diffusion wave spectroscopy. It was confirmed that a space-filling network was formed by wheat gluten proteins (mainly glutenin). At peak development (9.0 min) it was shown that the starch granules were confined in the gluten network. After the time-to-peak, dough resistance weakened, showing an increase of the starch granule movement. Kneading disrupts insoluble glutenin particles, the disrupted glutenin becomes part of the Sodium Dodecyl Sulfate (SDS)-extractable proteins. Both soluble and insoluble wheat protein extracts have been characterized by light scattering techniques. The results derived from light scattering of the wheat protein fractions: particle radii, apparent molar mass and geometrical shapes, suggests that the disrupted glutenin aggregate shape and glutenin size heterogeneity could be more important for gluten network bulk consistency, connectivity and resistance at dough peak, than the apparent molar mass of the solubilized glutenins, reaching a maximum after dough peak.

Mucosal Gamma/Delta T Cells Increased by i.n. Administration of Gliadin to Four-Week-Old NOD Mice Prevent Diabetes in the NOD-SCID Model of Adoptive Cotransfer of Diabetes

Beta-cell autoantigens, e.g., insulin, proinsulin, or GAD65, have been investigated both in animal models and human trials for induction of beta-cell specific tolerance to prevent the development of type 1 diabetes (T1D). On the other hand, environmental factors that significantly contribute to the recent outbreak of T1D were less studied, probably due to their complexity. The dietary wheat protein fraction has been associated with high penetrance of the disease, whereas gluten-free diets prevented T1D in animal models. We have previously reported that i.n. administration of gliadin prevented diabetes in NOD mice. Herewith we investigated mucosal gamma/delta T cells that were induced by i.n. administration of gliadin for their regulatory capacity to prevent diabetes in the NOD-SCID model of diabetes co-transfer. I.n. administration of gliadin to 4-week-old NOD mice increased proportion of gamma/delta T cells in pancreatic and mesenteric lymph nodes. Adoptive co-transfer of 2x106 gamma/delta T cells with 1x107 diabetogenic splenocytes led to statistically significant (p&amp;lt;0.01) diabetes prevention. The gamma/delta T cells were characterized for their memory-naïve and mucosal homing properties, or NK-like features by assessing expression of CD8alpha, CD45RB, CD62L, CCR7, CD103 and NKG2D by flowcytometry. In conclusion, similar to i.n. administration of insulin, i.n. vaccination with gliadin, an environmental antigen with possible ethiological influence in T1D, led to increased proportion of mucosal gamma/delta T cells. These gamma/delta T cells possess regulatory capacity to prevent diabetes transfer in the NOD-SCID model and may represent a novel and safer strategy for prevention or an early intervention in T1D. Disclosure D. Funda: None. J. Golias: None. M. Haupt-Jorgensen: None. K. Buschard: None.

The effects of protein-phenolic interactions in wheat protein fractions on allergenicity, antioxidant activity and the inhibitory activity of angiotensin I-converting enzyme (ACE)

Abstract Protein fractions were isolated from wheat flour including albumin, globulin, glutelin-1, glutelin-2 and prolamin. The free and bound phenolics were extracted from each protein fraction to determine the impact of their removal on allergenicity, antioxidant activity and the inhibitory activities of the angiotensin converting enzyme. The glutelin-1 fraction showed the highest protein content and yield of 71.02% and 21.02%, respectively. The highest antioxidant activity was noted in the glutelin-1 fraction (48.17%). The angiotensin converting enzyme inhibitory activities among the protein fractions ranged from 6.87% to 84.52%. The greatest reduction of allergenicity (39.66%) was demonstrated in the prolamin fraction. We conclude that the removal of phenolics appears to decrease significantly the allergenicity of wheat protein fractions while maintaining significant bioactivities related to anti-hypertensive, antioxidant, and anti-diabetic properties.

The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process.

Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

Transglutaminase from newly isolated Streptomyces sp. CBMAI 1617: Production optimization, characterization and evaluation in wheat protein and dough systems

The popularity of transglutaminase (TG) by the food industry and the variation in functionality of this enzyme from different origins, prompted us to isolate and evaluate a high-yielding TG strain. Through the statistical approaches, Plackett-Burman and response surface methodology, a low cost fermentation media was obtained to produce 6.074±0.019UmL−1 of TG from a novel source; Streptomyces sp. CBMAI 1617 (SB6). Its potential exploitation was compared to commonly used TG, from Streptomyces mobaraensis. Biochemical and FT-IR studies indicated differences between SB6 and commercial TG (Biobond™ TG-M). Additions of TG to wheat protein and flour based doughs revealed that the dough stretching depended on the wheat protein fraction, TG amount and its origin. A higher degree of cross-linking of glutenins and of inclusion of gliadin in the polymers was seen for SB6 as compared to commercial TG. Thus, our results support the potential of SB6 to tailor wheat protein properties within various food applications.

Characterization of phenolic compounds extracted from wheat protein fractions using high-performance liquid chromatography/liquid chromatography mass spectrometry in relation to anti-allergenic, anti-oxidant, anti-hypertension, and anti-diabetic properties

ABSTRACTThe purpose of this study was to identify novel natural bioactive phenolic compounds with anti-oxidant, anti-allergic, anti-hypertensive, and anti-diabetic properties in wheat protein fractions. Free and bound phenolic compounds were isolated from the albumin, glutelin-1, glutelin-2, prolamin, and globulin wheat protein fractions. The biological properties of the extracted phenolics were analyzed in vitro using 1,1-diphenyl-2-picryl-hydrazyl assays, enzyme-linked immunosorbent assays, angiotensin-1 converting enzyme assays, and α-amylase assays. The free and bound phenolic compounds were identified using liquid chromatography electrospray ionization tandem mass spectrometry methods. The aromatic rings in globulin were the highest in both before and after the removal of phenolic compounds (1.13 and 1.05 mg/g). The highest values of angiotensin-1 converting enzyme inhibitory and α-amylase inhibition (%) were obtained in glutelin-1 (73.17 and 96.41%, respectively) before removal phenolic compounds. The biological activity was affected by the presence or absence of phenolic compounds. Allergenicity was minimized in the presence of phenolic compounds. Correlation coefficients between wheat protein fractions and biological properties are described.

Minor Wheat Protein Fractions Analysis by Using Capillary Zone Electrophoresis

The wheat proteins soluble in chloroform-methanol mixtures are associated with several kinds of food allergies. A separation method based on capillary zone electrophoresis (CZE) with UV detection was developed for the analysis of these mixtures. An acidic phosphoric acid/β-alanine (pH 2.5) buffer containing HPMC, urea and acetonitrile was used for the separation. The capillary electrophoresis (CE) was able to complete the analysis in six minutes. The electrophoregrams of extracts of both durum and common wheat commercial cultivars were compared. The registered cultivar (cv.) Kamut® was included as a representative of rustic cereal species. A different number of peaks were detected in the profile relative to the tetraploid and exaploid analyzed cultivars. Three main peaks were observed for all tetraploid cultivars, while four peaks were detected for the common wheat cultivars. The peak corresponding to the α-amylase inhibitor type III was identified in the common wheat electrophoregram. The possibility of quantitative determination of this inhibitor has been investigated.

Celiac Disease and the Risk of Infertility

Celiac Disease is a chronic inflammatory disease, caused by an abnormal immune response triggered by intestinal exposure to dietary gluten, the protein fraction of wheat, rye and barley, in genetically susceptible individuals. Among atypical presentations of Celiac Disease, reproductive disorders and adverse pregnancy outcomes have been reported. Infertility is defined as the impossibility of conceiving after 12 months of unprotected intercourse. It can be related to both male and female factors, with the latter subdivided into ovarian, tubal, endometrial or unexplained causes. Celiac Disease has been related to infertility, but the evidence is inconclusive. In this review, we discuss the effect of Celiac Disease in both male and female fertility, as well as the possible mechanisms that may be involved.