Gliadin Proteins Research Articles

Proteolytic breakdown of gliadin by Enterococcus faecalis isolated from Tunisian fermented dough

The aim of this work was to select strains with proteolytic activity on wheat gliadin, among lactic acid bacteria, previously isolated from Tunisian fermented wheat dough. Hydrolysis of gliadin, as sole nitrogen source, in an agar medium was visualized by a clear zone surrounding colonies. The increase in absorbance due to gliadin breakdown was measured spectrophotometrically using O-phthaldialdehyde (OPA) on Gliadin Glucose Broth medium. Fermented liquid dough inoculated with individual selected Enterococcus faecalis, showed a decrease of the gliadin concentration from 45 g/kg to 18 g/kg determined by sandwich ELISA test (R-7001). Only the enterococci strains show an hydrolysis of gliadin proteins. Strains showing proteolytic activity are gaining more and more importance in cereal based fermented foods and may help to reduce gliadin involved in coeliac disease.

Hypoallergenic Characteristics of Wheat Flour Produced by Stepwise Polishing

Hypoallergenic wheat flour was produced by polished-graded method using a rice polishing machine. Eight fractions (C1–C8) of polished-graded wheat flours were obtained step wise from the outer layer of whole wheat grains by 10% of the total weight, and the distribution of allergenic protein in each fraction was determined. The salt-soluble (albumin/globulin), salt-insoluble (glutenin) and alcohol-soluble (gliadin) proteins obtained from the polished-graded wheat flours were tested for the allergen assay with immunodetection using the sera of wheat allergenic patients. Immunoblotting results confirmed that the innermost fraction (C8) contained a smaller amount of allergenic proteins. Albumin/globulin groups in all fractions (C1–C8) showed different IgE-reactivity patterns, the 60–75 kDa proteins appeared in all of fraction flours. It was higher in C3–C5 fractions. Fractions C3 and C4 contained higher amount of specific wheat allergenic protein including 60–75, 35, 22, and < 20 kDa. IgE-antibody also bound to glutenin (25–37 kDa), especially in C3 and C4, but the binding proteins showed quite faint bands. Gliadin was found in all fractions of wheat flour. We propose that polishing is an appropriate method to obtain hypoallergenic wheat flours, and the fraction C8 may be possible to be consumed by people suffering from wheat allergy.

Turbidimetric Titration of a Haze-Active Polyphenol in Beer

At least two commercial instruments that perform automated turbidimetric titrations are available for determining haze-active (HA) constituents in beer. They have advantages over single-point measurements due to the interactions between beer HA polyphenols, HA proteins, and other beer constituents. Analysis of HA polyphenols is particularly difficult due to their low concentration in beer. Manual turbidimetric titrations with soluble polyvinylpyrrolidone (PVP) were carried out in model systems with a HA polyphenol (tannic acid [TA]), a non-HA polyphenol (epicatechin), a HA protein (gliadin), and a non-HA protein (lysozyme). Increasing concentrations of TA resulted in higher turbidity peaks at higher titrant volumes. Essentially no haze was seen with epicatechin or lysozyme. Gliadin solutions had an initial modest haze that decreased with increasing titrant. The effects of adding fixed amounts of epicatechin, lysozyme, or gliadin to TA were examined. In the presence of epicatechin or lysozyme, the titration endpoints were shifted higher. The endpoints with mixtures of gliadin and TA were similar to those found with TA alone. PVP titration also was applied to samples of unchillproofed beer treated with different amounts of silica or polyvinylpolypyrrolidone and showed responses of the expected magnitudes.

A method of extraction and separation of wheat gluten

After removing gliadin and other proteins with 7.5% 2-propanol and 0.3 mol/L NaI, the total glutenin subunits were extracted with two kinds of buffer, one containing 25% 2-propanol, 0.04 mol/L Tris-HCI (pH=8.0), 10% SDS and 2% DDT and the other containing 25% 2-propanol, 0.04 mol/L Tris-HCI (pH=8.0), 10% SDS and 1.4% VP. In SDS-PAGE electrophoresis (4% stacking gel and 13% resolving ge1) system HMW-GS and LMW-GS were clearly separated by the improved method with only a single step. The background of the SDS-PAGE gel was gliadin-free. It was more effective and applicable to separate and distinguish the complex LMW-GS. This method will be used in wheat protein separation, variety identification and improvement.

Characterization of antimicrobial properties on the growth of S. aureus of novel renewable blends of gliadins and chitosan of interest in food packaging and coating applications

The biocide properties of chitosan-based materials have been known for many years. However, typical antimicrobial formulations of chitosan, mostly chitosonium salts, are known to be very water sensitive materials which may impair their use in many application fields such as food packaging or food coating applications. This first work reports on the development and characterization of the antimicrobial properties of novel fully renewable blends of chitosan with more water-resistant gliadin proteins isolated from wheat gluten. Chitosan release to the nutrient broth from a wide range of blends was studied making use of the ninhydrin method. The results indicated that both pure chitosan and its blends with gliadins presented significant antimicrobial activity, which increased with increasing the amount of chitosan in the composite formulation as expected. The gliadins-chitosan blends showed good transparency and film-forming properties and better water resistance than pure chitosan. The release tests revealed that dissolution of the biocide glucosamine groups, i.e. the chitosan water soluble fractions, also increased with the amount of chitosan present in the formulation. The release of these groups was for the first time directly correlated with the antimicrobial properties exhibited by the blends. Thus, incorporation of chitosan into an insoluble biopolymer matrix was revealed as a very feasible strategy to generate novel chitosan-based antimicrobial materials with potential advantages, for instance active food packaging applications.

The Determination of the Basic Amino Acids of Proteins: the Work of Hubert Vickery

The Determination of the Basic Amino Acids of Proteins: the Work of Hubert Vickery

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

ABSTRACTContact 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.

6012 POSTER The existence of humoral immunity to gliadin and cow's milk proteins in patients with lymphoma

6012 POSTER The existence of humoral immunity to gliadin and cow's milk proteins in patients with lymphoma

Natural History of Antibodies to Deamidated Gliadin Peptides and Transglutaminase in Early Childhood Celiac Disease

Gliadin proteins play a key role in the pathogenesis of celiac disease; however, as a screen for celiac disease, anti-gliadin antibody testing has been replaced by the more sensitive and specific serological assays for transglutaminase autoantibodies (TGAA). A new generation of anti-gliadin antibody assays has been developed to detect synthetic, deamidated homologous gliadin peptides (DGP) with high sensitivity and specificity. Sera were collected prospectively from children with an increased risk for celiac disease as part of an ongoing study at Denver, and studied for the development of celiac autoimmunity. We investigated the high-performance DGP antibody assay in 50 TGAA-positive children both before the development of celiac autoimmunity and following the institution of a gluten-free diet to determine the relationship of DGP antibodies to TGAA. TGAA were measured by an in-house radioassay. DGP antibodies and TGAA parallel each other over the period of years children were studied. DGP antibodies resolved sooner than TGAA in subjects on a gluten-free diet. DGP antibodies appeared earlier than TGAA in 9 children. Measuring DGP antibodies may be more useful than TGAA in monitoring children on a gluten-free diet. DGP antibodies can precede the appearance of TGAA in some at-risk children.

The effect of aphid infection and cultivar on the protein content governing baking quality of wheat flour

AbstractBACKGROUND: Aphids cause both quantitative and qualitative yield losses in cereals. Aphid feeding resulted in a significant decrease in gliadin/glutenin ratio in soft‐kernel wheat. Effect of aphid feeding on hard‐kernel wheat was examined.RESULTS: Plants of two different hard‐wheat cultivars were artificially infected by two aphid species: Sitobion avenae and Diuraphis noxia. Three different levels of caging regulated aphid density. Aphid density was highest in the cages, followed by open cages and lowest under no‐cage conditions. Numbers of D. noxia were generally higher compared to those of S. avenae. There was no significant difference in head length, mean kernel number/head, mean kernel mass/head or mean individual kernel mass between the two cultivars. Glutenin, gliadin and total protein content was analysed using size‐exclusion HPLC. Quality parameters of wheat flour were significantly different between the two cultivars. Gliadin content, total protein content and gliadin/glutenin ratio were significantly higher in Mv Magdaléna compared to Mv Magvas regardless of the level of caging and infestation, while glutenin content of uninfected Mv Magvas was significantly higher than that of Mv Magdaléna in the open cages. Aphid feeding did not induce any significant reduction in the gliadin, total protein content and gliadin/glutenin ratio of the examined hard wheat cultivars.CONCLUSION: Individual kernel mass reduction is the major factor in yield loss caused by aphids. Aphid feeding did not change the protein structure of hard‐kernel wheat. Copyright © 2007 Society of Chemical Industry

Matrix‐assisted laser desorption/ionization time‐of‐flight based wheat gliadin protein peaks are useful molecular markers for wheat genetic study

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation has been used to analyze wheat seed gliadins as an alternative to other established methods, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), etc. The MALDI-TOF approach has shown to have many advantages such as high resolution, cost effectiveness and high throughput. MALDI-TOF-based gliadin profiles have been used for fast wheat cultivar identification. However, the genetic information represented by individual gliadin peaks has not been utilized. In this study a wheat doubled haploid population with a genetic linkage map of good coverage was used to assay individual gliadin peaks from MALDI-TOF profiles as molecular markers. Eight segregating peaks in the population were scored as polymorphic across the population. The 1 to 1 segregating ratios validated the scoring of the peaks and all peaks were mapped to the expected chromosomes or linkage groups on the available linkage map: 1 peak on chromosome 1A, 1 on 6A, 4 on 6B and 2 on 6D.

Elucidating the Mechanism of Laccase and Tyrosinase in Wheat Bread Making

Cross-linking enzymes generate covalent bonds in and between food biopolymers. These enzymes are interesting tools for tailoring dough and bread structures, as the characteristics of the biopolymers significantly determine the viscoelastic and fracture properties of dough and bread. In this study, the influence of oxidative cross-linking enzymes, tyrosinase from the filamentous fungus Trichoderma reesei and laccase from the white rot fungus Trametes hirsuta, on dough and bread were examined. Oxidation of low molecular weight phenolic model compounds of flour, cross-linking of gluten proteins, dough rheology, and bread making were characterized during or after the enzymatic treatments. In the dough and bread experiments, laccase and tyrosinase were also studied in combination with xylanase. Of the model compounds tyrosine, p-coumaric acid, caffeic acid, ferulic acid, and Gly-Leu-Tyr tripeptide, tyrosinase oxidized all except ferulic acid. Laccase was able to oxidize each of the studied compounds. The phenolic acids were notably better substrates for laccase than l-tyrosine. When the ability of the enzymes to cross-link isolated gliadin and glutenin proteins was studied by the SDS-PAGE analysis, tyrosinase was found to cross-link the gliadin proteins effectively, whereas polymerization of the gliadins by laccase was observed only when a high enzyme dosage and prolonged incubation were used. Examination of large deformation rheology of dough showed that both laccase and tyrosinase made doughs harder and less extensible, and the effects increased as a function of the enzyme dosage. In bread making, interestingly, the pore size of the breads baked with tyrosinase turned out to be remarkably larger and more irregular when compared to that of the other breads. Nevertheless, both of the oxidative enzymes were found to soften the bread crumb and increase the volume of breads, and the best results were achieved in combination with xylanase.

Characterization of two 1D-encoded ω-gliadin subunits closely related to dough strength and pan bread-making quality in common wheat ( Triticum aestivum L.)

Gliadin proteins of 113 common or bread wheat ( Triticum aestivum L.) cultivars and advanced lines from China and other countries, were analyzed by high performance capillary electrophoresis (HPCE) and reversed-phase high performance liquid chromatography (RP-HPLC). A major protein peak migrating at 3 min by HPCE and eluting at about 20 min by RP-HPLC was identified in the ω-gliadin region. It was present in cultivars with good pan bread-making quality, whereas most cultivars with poor bread-making quality lacked this protein peak. Quality testing and statistical analysis showed that this ω-gliadin peak was significantly related to dough strength, loaf volume and loaf score. It was separated into two apparent protein components by one-dimensional SDS-PAGE and two-dimensional electrophoresis (2-DE). According to their relative mobilities on the gels, the proteins were designated ω-15 and ω-16, and their accurate molecular masses (42590.5 Da for ω-15 and 41684.1 Da for ω-16) were determined by MALDI-TOF-MS. The ω-15 and ω-16 gliadins possessed the N-terminal amino acid sequences of ARELNPSNKELQQQQ and KELQSPQQQF, and therefore they belonged to 1D-encoded ω-2 type and ω-1 type gliadins, respectively. Both gliadin subunits were always present together among the 86 cultivars analyzed, suggesting that they were encoded by two closely linked genes at Gli-D1 locus. The accumulative characteristics of gliadins during grain development indicated possible additive quantitative effects of ω-15+16 on dough strength. The ω-15 and ω-16 gliadins could be used as valuable genetic markers for wheat quality improvement.

Impact of Redox Agents on the Extractability of Gluten Proteins during Bread Making

The gluten proteins gliadin and glutenin are important for dough and bread characteristics. In the present work, redox agents were used to impact gluten properties and to study gliadin-glutenin interactions in bread making. In control bread making, mixing increased the extractability of glutenin. The level of SDS-extractable glutenin decreased during fermentation and then further in the oven. The levels of extractable alpha- and gamma-gliadin also decreased during bread baking due to gliadin-glutenin polymerization. Neither oxidizing nor reducing agents had an impact on glutenin extractabilities after mixing. The redox additives did not affect omega-gliadin extractabilities during bread making due to their lack of cysteine residues. Potassium iodate (0.82-2.47 micromol/g of protein) and potassium bromate (1.07-3.17 micromol/g of protein) increased both alpha- and gamma-gliadin extractabilities during baking. Increasing concentrations of glutathione (1.15-3.45 micromol/g of protein) decreased levels of extractable alpha- and gamma-gliadins during baking. The work not only demonstrated that, during baking, glutenin and gliadin polymerize through heat-induced sulfhydryl-disulfide exchange reactions, but also demonstrated for the first time that oxidizing agents, besides their effect on dough rheology and hence bread volume, hinder gliadin-glutenin linking during baking, while glutathione increases the degree of covalent gliadin to glutenin linking.

Zinc nutrition influences the protein composition of flour in bread wheat ( Triticum aestivum L.)

Zinc (Zn) deficiency and heat stress during grain filling occur in a number of important wheat growing regions around the world. The changes in grain protein composition due to high temperature are well documented, but little is known about the effect of grain Zn and its interaction with heat stress. Six field experiments were conducted at sites differing in grain filling temperatures to examine these effects. Two varieties of bread wheat were grown at six rates of Zn, including foliar sprays of Zn. The relative amounts of gliadin and polymeric protein were measured by size exclusion HPLC. Applying Zn increased grain yield at three sites and altered protein quality at two of these. Foliar Zn applications doubled grain Zn concentration, reduced the proportion of gliadin and SDS-unextractable polymeric protein and increased the proportion of SDS-extractable polymeric protein. Heat stress during grain filling was associated with a high proportion of gliadin and low proportions of the polymeric protein in the grain. However, the proportions of gliadin and of SDS-extractable polymeric protein were less affected by high temperatures in grain with high Zn concentrations. The results demonstrate that Zn nutrition can alter protein composition and the effects of Zn may interact with grain filling temperatures.

Fluorescence Correlation Spectroscopy Assay for Gliadin in Food

Gliadin proteins are primarily responsible for celiac disease. As gliadin is a complex mixture of proteins difficult to solubilize and to extract from food, it is difficult to develop an assay capable of accurate quantization of gliadin in food for celiac patients. In this work, we present an advanced fluorescence assay for the detection of traces of gliadin in food. The described assay is based on measurement of the fluctuations of fluorescein-labeled gliadin peptides (GP) in a focused laser beam in the absence and in the presence of anti-GP antibodies. A competitive assay based on the utilization of unlabeled GP was developed. The obtained results indicate that the combination of high-avidity IgG antibodies together with the innovative fluorescence immunoassay strategy resulted in a gluten detection limit of 0.006 ppm, which it is much lower than the values reported in the literature.

Immune Cross-Reactivity in Celiac Disease: Anti-Gliadin Antibodies Bind to Neuronal Synapsin I

Celiac disease is an immune-mediated disorder triggered by ingestion of wheat gliadin and related proteins in genetically susceptible individuals. In addition to the characteristic enteropathy, celiac disease is associated with various extraintestinal manifestations, including neurologic complications such as neuropathy, ataxia, seizures, and neurobehavioral changes. The cause of the neurologic manifestations is unknown, but autoimmunity resulting from molecular mimicry between gliadin and nervous system proteins has been proposed to play a role. In this study, we sought to investigate the immune reactivity of the anti-gliadin Ab response toward neural proteins. We characterized the binding of affinity-purified anti-gliadin Abs from immunized animals to brain proteins by one- and two-dimensional gel electrophoresis, immunoblotting, and peptide mass mapping. The major immunoreactive protein was identified as synapsin I. Anti-gliadin Abs from patients with celiac disease also bound to the protein. Such cross-reactivity may provide clues into the pathogenic mechanism of the neurologic deficits that are associated with gluten sensitivity.

Gliadain, a gibberellin‐inducible cysteine proteinase occurring in germinating seeds of wheat,Triticum aestivumL., specifically digests gliadin and is regulated by intrinsic cystatins

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.

The Effect of Beer pH on Colloidal Stabilization with Adsorbents

Solutions of the haze-active (HA) protein gliadin were prepared in buffer at different pH's and treated with silica. Analysis using the bicinchoninic acid and HA protein methods produced similar patterns, with greater protein removal near pH 3.5 and 5.0. Portions of an unchillproofed beer were adjusted to different pH's, treated with silica, and subjected to HA and foam-active (FA) protein analyses. The HA protein results showed greater protein removal near pH 3.8 and least removal near pH 4.4. FA protein was hardly affected, although slightly more was removed near pH 4.4. Tannic acid (TA) and catechin were each combined with soluble polyvinylpyrrolidone in buffer solutions with different pH's, and turbidity was determined after incubation. TA formed maximum haze near pH 4, while catechin formed more haze above pH 5. Portions of unchillproofed beer were adjusted in pH and treated with polyvinylpolypyrrolidone (PVPP). The treatment pH had essentially no effect on total polyphenol removal. HA polyphenols were removed to a greater extent near pH 3.8 and 4.6, with lower effectiveness at intermediate pH's. The colloidal stabilization performance of both silica and PVPP was better at pH's near the extremes of the beer pH range. This may account for difficulties encountered in stabilizing some beers.

Genome-wide linkage analysis of 160 North American families with celiac disease

Celiac disease (CD) is a common autoimmune disease caused by exposure to the protein gliadin in wheat, and related prolamins in barley and rye. The prevalence of the disease in the US is 1:133. The aim of this study was to identify non-human leukocyte antigen (HLA) loci that predispose to CD. A genome-wide search of 405 microsatellite markers was performed on DNA samples from 160 families with a minimum of two cases of CD. Multipoint, parametric and non-parametric linkage (NPL) analyses were performed. Locations on chromosomes 1q, 3q, 6p, 6q, 7q, 9q and 10q showed linkage statistics (NPL scores or heterogeneity logarithm of the odds (HLOD) scores) of approximately 2.0 or larger. The greatest evidence for linkage outside of chromosome 6 was on 7q and 9q. An NPL score of 2.60 occurred at position 151.0 on 7q and a HLOD score of 2.47 occurred at position 144.8 on 9q under a recessive model. As expected, there was highly significant linkage to the HLA region on 6p, with NPL and HLOD scores exceeding 5.50. In conclusion, this genome-wide linkage analysis represents one of the largest such studies of CD. The most promising region is a putative locus on 7q, a region reported independently in previous genome-wide searches.