Gluten Fractions Research Articles

Occurrence of serum antibodies against wheat alpha-amylase inhibitor 0.19 in celiac disease.

The alcohol-soluble fraction of wheat gluten (gliadins) induces in genetically susceptible individuals immunologically mediated celiac disease (CLD). However, gliadins and related cereal proteins are not unique foodstuff targets of CLD patients´ immune system. Non-gluten wheat alpha-amylase inhibitor 0.19 (AAI 0.19) has been found to be capable of activating human monocyte-derived dendritic cells and inducing pro-inflammatory status in intestinal mucosa of patients with celiac disease (CLD). The possible contribution of this reactivity in incomplete remission of CLD patients on a gluten-free diet (GFD) is matter of contention. In an attempt to characterize the antigenicity of AAI 0.19 in patients with active CLD, patients on a GFD and healthy controls we developed ELISA employing wheat recombinant AAI 0.19. Using this test we revealed a significant (P<0.001) elevation of IgA anti-AAI 0.19 antibodies (Ab) in patients with active CLD (12 out of 30 patients were seropositive) but also in CLD patients on a GFD (15/46), in contrast to healthy controls (2/59). Anti-AAI 0.19 IgG Ab levels were increased (P<0.001) only in patients with active CLD (14/30) in contrast to the controls. Interestingly, the levels of anti-AAI 0.19 IgG Ab were decreased in CLD patients on a GFD (P<0.001, 1/46) compared to the controls (1/59). Notably, 20 out of 30 patients with active CLD were positive either for IgA or for IgG anti-AAI 0.19 Ab. Thus, the majority of CLD patients developed a robust IgA and IgG Ab response against AAI 0.19. These findings may contribute to the broadening of the knowledge about CLD pathogenesis.

Grass-Allergic Children Frequently Show Asymptomatic Low-Level IgE Co-Sensitization and Cross-Reactivity to Wheat

Background: Specific immunoglobulin E (IgE) sensitization to wheat is more common than a doctor’s confirmed wheat allergy and is also frequently observed in grass pollen-allergic patients (pollinosis patients). Thus, the objective of this study was to investigate the level and feature of serological IgE cross-reactivity between grass pollen and wheat in a cohort of pollinosis subjects with no diagnosis of wheat allergy. Methods: Seventy-two children, aged 5–17 years, with a doctor’s diagnosis of pollinosis, IgE towards grass pollen, and currently eating wheat were recruited. Serum samples were analyzed for IgE against wheat, timothy grass/wheat-specific allergen components, Pru p 3, and cross-reactive carbohydrate determinants (CCD) and specific IgE-binding inhibition experiments were performed. Results: Sixty percent of the grass pollen subjects were sensitized to wheat with a median of 0.5 kU_A/L. Wheat-sensitized subjects were more often sensitized to the two allergens, Phl p 12 and CCD, known to be cross-reactive between grass and wheat. Sensitizations to seven wheat-specific allergens derived from the gluten fraction were, with the exception of one individual, only found in wheat-sensitized subjects. These subjects also more often reported current and past history of allergy to staple foods (milk, egg, wheat, soy, and fish). Conclusion: Wheat sensitization caused by cross-reactivity but also by sensitization to wheat-specific allergens was common in the grass-allergic children and also associated with allergy to staple foods other than wheat. The results indicate the presence of a subgroup of pollinosis patients with simultaneous sensitization to wheat and food allergy not only caused by cross-reactions.

Changes in enzymatic activities and functionality of whole wheat flour due to steaming of wheat kernels

The effects of steaming wheat kernels on lipolytic degradation of resulting whole flour was studied by quantifying enzyme activities and lipid degradation products during storage. Lipase, lipoxygenase, polyphenol oxidase, and peroxidase activities were decreased by up to 81%, 63%, 22%, and 34%, respectively, as the time of steaming increased up to 90 s. Steaming had no effect on starch and gluten properties. Upon storage free fatty acids decreased with respect to time of steaming. Time of steaming did not affect lipid oxidation in flour; however, total carbonyls produced in dough made from stored flour were decreased with the increase in steaming duration. Thus, steaming wheat kernels prior to milling reduced lipase activity and consequently hydrolytic rancidity during storage without affecting starch and gluten fractions. Steam treatment did not affect oxidative rancidity in flour during storage, but did reduce oxidation once the flour was made into a dough.

Effect of Environment and Genotype on the Protein Quality Attributes and Baking Characteristic of Newly Developed Wheat Inbred Lines

The present work examined the effect of genotype and environment on protein content and fractions, gluten and starch fraction, SSL (sodium stearoyl-2-lactylate) and DMG (distilled mono glyceride) binding ability of starch and specific loaf volume (SLV) of six wheat genotypes grown in three different environment. Genotype and environment significantly affected all quality attributes under investigation. However, protein content and fractions showed differences in relative effects of genotype and environment. Most of the protein quality characteristics were influenced more by genotype than environment. Size distribution of gluten subunits was significantly affected by genotype and environment. It was observed that as the flour protein content increased, the magnitudes of monomeric proteins appeared to rise, but glutenin decreased. Flour protein content was expressively associated with gliadin and dough making characteristics. Environment influenced b eedings.

Exercise Lowers Threshold and Increases Severity, but Wheat-Dependent, Exercise-Induced Anaphylaxis Can Be Elicited at Rest

Wheat-dependent, exercise-induced anaphylaxis (WDEIA) is a severe form of allergy in which exercise is being considered as mandatory. The diagnosis is often complex and the clinical reproducibility low. The aims of this study were to establish a standardized challenge method for the diagnosis of WDEIA and to investigate whether exercise is an essential trigger factor or alternatively an augmentation factor able to lower threshold and increase severity. We investigated 71 patients (age, 18.6-73.7 years) with a case history of WDEIA. Skin prick test (SPT) and measurement of specific IgE (sIgE) were followed by an oral food challenge with gluten at rest and in combination with treadmill exercise. A clinical reaction was elicited in 47 of 71 (66%), and in 26 of these (37%) the reaction could be elicited at rest. The median dose required at rest was 48 g (8-80 g) and in combination with exercise 24 g (4-80 g). Severity was significantly higher with exercise (2.3) than at rest (1.1) using Sampson severity score. In the challenge, SPT was positive to wheat in 93.6% (44 of 47) and to gluten in 95.7% (45 of 47). sIgE to wheat, gliadin, and omega-5 gliadin was present in 78.7% (37 of 47), 76.5% (36 of 47), and 91.4% (43 of 47) of the patients. Receiver operating characteristic-curve analysis for sIgE to omega-5 gliadin, a component of the gluten fraction and the major allergen in WDEIA, showed best sensitivity (91%) and specificity (92%) when gluten was combined with exercise. A challenge test with gluten at rest and combined exercise is a safe confirmatory test for WDEIA. A reaction can be elicited at rest (without exercise), but exercise is able to lower the threshold and increase the severity.

How leaf rust disease and its control with fungicides affect dough properties, gluten quality and loaf volume under different N rates in wheat

The unique viscoelastic properties of gluten make bread wheat (Triticum aestivum L.) a staple food. The specific balance between gluten fractions, together with grain protein content (GPC), defines nutritional and end-use properties. The leaf rust (Puccinia triticina Eriks.) and its control using succinate dehydrogenase inhibitors (SDHIs) fungicides together with nitrogen (N) fertilization could alter the GPC and its composition during the grain-filling period, modifying dough properties. The aim of this study was to investigate the effects of leaf rust, its control using fluxapyroxad (SDHI) and the interaction with N fertilization on breadmaking quality of wheat. Two field experiments were conducted during 2014 and 2015 in a split-split plot design with three fungicide treatments as main plots and three N fertilization rates as subplots using a susceptible cultivar. Leaf rust reduced GPC and modified its composition. The disease shortened the grain-filling period and reduced gluten content. Gluten tenacity (AlvP) was more affected than gluten extensibility (AlvL) resulting in doughs with lower AlvP/L ratio, minor gluten strength and inferior loaf volume. The treatment containing fluxapyroxad showed better levels of disease control and it also evidenced additional beneficial effects in most of the evaluated parameters compared to the treatment without this active ingredient.

Specificity of combinations of qualitative and quantitative characteristics of glucovine in genotypes of allocytoplasmatic spruce wheat with allel of wild type Wx-B1a

As a result of screening of the allelic composition of genes associated with baking properties, a significant genotypic variety of forms of allocytoplasmic spring wheat (ATSGG) from the ATI PFUR collection was established. In addition to the altered forms, 15 genotypes were isolated as a result of recombinations and introgression, in the genome of which the presence of a allel of wild type Wx-B1a (primer 4F / 4R) was detected. Analysis of the content and quality of gluten in these forms of ACPG made it possible to differentiate these genotypes according to their functional characteristics, which are related to baking properties. The amplitude of the differences in the genotypes of the ACPG in terms of the mass fraction of gluten is from 21.7% to 37.8%, the quality of gluten according to the IDK parameters in the majority of the studied genotypes of the I-st group. The genotypes of the category of strong wheat are of particular value: No. 24 (cytoplasm T. timopheevii), in which the mass fraction of gluten is 37.8% (class of super-strong wheat), and also genotypes of the 1st class, in which the gluten content is not less than 32%, and the quality of gluten is not lower than the I-st group (IDK - 43-77 units.). These are genotypes No. 25 (the cytoplasm of T. timopheevii) and No. 29 (the cytoplasm of T.aestivum L., as a result of backcrossing). To the category of strong wheat of the 2nd class (mass fraction of gluten is not lower than 28%, and the quality of gluten is the I-st group), four genotypes are classified. The category of valuable wheat of the 3rd class includes two genotypes, in which the mass fraction of gluten is not less than 25%. However, the quality of gluten in these genotypes is not II-th group, but higher - it corresponds to the I-st group. Genotypes with a specific combination of the mass fraction of gluten characteristic of strong and valuable wheat, with the qualitative characteristics of gluten of the I-th group, expand the range of their intended use in the production of bakery products.

The approach to Celiac Disease in children

Celiac Disease (CD) is a permanent, irreversible but treatable multifactorial disease triggered by the ingestion of gluten (a plant storage protein contained in wheat, barley and rye) in genetically predisposed individuals and resulting in an autoimmune small intestinal inflammation with systemic implications. While the gastrointestinal manifestations secondary to an inflammatory enteropathy with variable degrees of severity are what defined it for many decades, CD is in fact characterized also by a wide range of extra-intestinal complaints and elevated titers of celiac-specific autoantibodies. 1.1. Epidemiology and pathogenesis The prevalence of CD is increasing at a remarkable pace during the past few decades [1], [2], [3]. Once thought to be a rare condition, affecting no more than 1/10,000 people, thanks to the availability and widespread use of specific and sensitive serological markers, CD is now recognized worldwide as a common disorder, with a prevalence varying between 0.3 and 3 per 100. Only a limited portion of the expected celiac patients are however actually identified, with proportions varying between different countries: in the USA, even though overall CD prevalence is estimated to be around 1%, only about 15% of this population (including children and adults) has been diagnosed and can therefore be treated [4]. This phenomenon of under-diagnosis is likely due to a combination of inadequate awareness and a high prevalence of asymptomatic or oligo-symptomatic patients. Like most multifactorial disorders, CD is the result of a complex interaction between genes, immune status of the host, and environmental triggers. Gluten is a heterogeneous molecule. The gluten fractions that are toxic to celiac patients are a mixture of alcohol-soluble proteins called gliadins. Gliadins are rich in glutamine and proline residues, which even the healthy human intestine cannot fully digest. As a result, intact gliadin peptides are left in the lumen, and some cross the intestinal barrier. These fragments come into contact with the intracellular enzyme tissue transglutaminase (tTG), which deamidates them, leading to a change in shape and increased negative charge. This creates peptides that can easily be captured by the HLA-DQ2 and/or DQ8 molecules expressed on the surface of the lamina propria-associated antigen-presenting cells (APCs) and are presented to CD4+ T cells triggering an inflammatory reaction [5]. The end result of this autoimmune-triggered inflammatory reaction is a varied degree of small intestinal mucosal damage, typically more severe proximally than distally. The role of additional environmental factors is still the object of an intense research. Recently, our group showed evidence for a plausible role of an otherwise innocuous viral infection (Reovirus) in creating a pro-inflammatory milieu conducive to the development of overt CD [6].

LAOS behavior of the two main gluten fractions: Gliadin and glutenin

Crude gliadin and glutenin fractions were studied using Large Amplitude Oscillatory measurements. LAOS measurements were carried out at three different frequencies (20, 10, 1 rad/sec) between the strain values of 0.01–200%. The beginning of non-linearity for glutenin occurred at ∼2.5%, while an initial region of strain hardening was observed for gliadin (2.5–10%) at 1 rad/sec frequency and up to 15% at the higher frequencies applied. Lissajous curves showed in the elastic analysis of both fractions glutenin was more elastically dominated since Lissajous curves were narrower, while for gliadin the ellipses were much broader suggesting more fluid-like behavior and each ellipse depended on the magnitude of frequency. Decreasing frequency increased the viscous behavior of both glutenin and gliadin in the non-linear region, but the change in gliadin was much more pronounced. Gliadin molecules only display intramolecular disulfide bonds creating a great deal of mobility whereas for glutenin molecules, which contain both intermolecular and intramolecular disulfide bonds, the strong network structure formed by this molecular arrangement results in very pronounced strain stiffening.

An Effective Gluten Extraction Method Exploiting Pure Choline Chloride-Based Deep Eutectic Solvents (ChCl-DESs)

Gluten analysis is still affected by the lack of an efficient and universal extraction solvent for gliadin, i.e., the prolamine fraction of gluten which is the analytical target used to quantify gluten in food. In this investigation, the possibility of adopting pure choline chloride deep eutectic solvents (ChCl-DESs) as gliadin extraction media was tested. As prototypes of ChCl-DESs, ethaline and reline were chosen in view of their good dipolar and hydrogen bond donor (HBD) properties, as well as of their moderate enough viscosity. Their ability to act as effective solvents for gliadin and their inability to affect the subsequent gliadin detection by a frequently adopted and commercially available ELISA kit were first assessed. Ethaline and reline extraction performance was then tested by evaluating gliadin recoveries achieved on gluten-free food real samples before and after their spiking with controlled gliadin amounts. Higher recoveries were found by using these DESs in comparison with those gained by the usually adopted 60% (v/v) ethanol-water solvent. In fact, gluten recoveries ranging from 78 to 113%, with a RSD better than 13%, were achieved by using ethaline. Less good recoveries (from 67 to 132% with a RSD of 20%) were achieved with reline, even though they were however better than those found with 60% v/v ethanol-water solvent. These results make the proposed media profitable solvents for gliadin extraction and its subsequent detection by a commonly used immunoassay kit, without any change of the usual detection procedure.

Antibody reactivity in patients with IgE-mediated wheat allergy to various subunits and fractions of gluten and non-gluten proteins from ω-gliadin-free wheat genotypes.

[/b] The observed differentiation of immunodetection spectra allows to model highly specific IgE-binding profiles of allergenic wheat proteins attributed to individual patients with symptoms of gluten intolerance. Highly immunoreactive subunits and fractions among GP and NGP were identified. The observed immunoreactivity of 49 kDa NGP is worth to emphasize, as it has never been reported as wheat allergenic protein before.

Osteomalacia with Looser zones caused by celiac disease

Celiac disease is considered a malabsorption syndrome and is characterized by chronicsmall intestinal disease caused by hypersensitivity to the gliadin fraction of gluten. Celiac disease comes with diarrhea, occasional steatorrhea, weight loss, and other complications which might be caused by anemia. Reports of osteomalacia as the only symptom of celiac disease are very rare; however, osteomalacia can be a detected sign of celiac disease. Herein is described a case of osteomalaciawith a Looser zone in a 39-year-old woman who had low bone mineral density caused by severe osteomalacia associated with chronic celiac disease. In patients with pain in the spine and proximal muscle, the risk of osteomalacia should be considered in any kind of diagnosis.

A new label-free impedimetric aptasensor for gluten detection

Celiac disease is a serious autoimmune disorder caused by the ingestion of gluten. Gliadin is the gluten fraction responsible for the triggering of disease. The only cure for the celiac patients, known until now, is a diet with gluten-free foods, classified by the European regulation doesn't exceed 20 ppm in gluten. With the aim to guarantee the food safety for celiac patients in this study the developing and optimization of a fast and reliable label free impedimetric aptasensor for gliadin detection is reported. The aptamer (Gli1) at 0.5 μmol and poly (amidoamine) dendrimer of fourth generation (PAMAM G4) at 2 mg/ml were chosen during the developing steps of the sensing platform because the best ones for the detection of low gluten concentrations with the highest sensitivity. The aptasensor showed linearity in the range of 5–50 μg/l and 50–1000 μg/l in gliadin, a limit of detection of 5 μg/l corresponding to 5 ppm of gluten, a reproducibility lower than 5% and a storage stability at 4 °C of two months. Finally the aptasensor was used to measure gluten, in gluten and gluten-free food products, showing a good agreement with the results obtained with official R5 ELISA method.

New bread formulation with improved rheological properties and longer shelf-life by the combined use of transglutaminase and sourdough

The combined use of the protein reticulating enzyme transglutaminase (TGase) and a selected microbial consortium of Lactobacillus sanfranciscensis and Candida milleri for improving the rheological properties, aroma, and shelf-life of a bakery product was evaluated. A microbial TGase, showing the highest activity over a wide temperature range on different protein substrates, was selected among different types. Results showed that this TGase was able to produce isodipeptide bonds, especially in the gluten fraction, leading to the formation of protein aggregates, which improved the structure of a sourdough bakery product. The microbial TGase in combination with sourdough exhibited a positive synergistic effect allowing the production of flavor-enriched bread, with rheological properties similar to those of standard bread.

Chromatographic and mass spectrometry analysis of wheat flour prolamins, the causative compounds of celiac disease.

Immunogenic gluten peptides trigger Celiac Disease (CD), an adaptive immune response in genetically predisposed individuals. Given the structural similarity between all gluten proteins their individual CD influence is not clear. Hence, the extraction, separation and characterization of wheat gluten proteins have become relevant to measure their individual potential immunoreactivity. Wheat proteins were extracted from commercial wheat flour and further isolated by preparative HPLC. The resulting richest gliadin sub-fractions were characterized by nano-LC-MS/MS following a shotgun proteomic approach in order to identify the prolamins in the original commercial wheat flour. It was found that the gliadin extract was additionally composed of glutenins and avenin-like proteins. Accurate prolamin identification has emerged as a need to delve deep into the influence of each fraction on the onset of celiac disease. After protein characterization, the immunoreactivity towards the main epitope related to CD was verified by ELISA and western blotting for several different gluten fractions.

INSOLUBILITY AND ALTERATION OF ALLERGENIC ACTIVITY OF WHEAT PROTEINS IN PROCESSED FOODS.

Food processing causes decomposition, denaturation or polymerization of protein, which may alter an allergic reaction. This study aimed to investigate the insolubility and alteration of wheat allergens in processed foods and the reactivity to patient sera. We extracted proteins from wheat flour, udon and bread using different extracts and conducted SDS-polyacrylamide gel electrophoresis. IgE-immunoblotting was also conducted using sera from children with wheat allergy. Soluble protein was extracted from wheat flour, and gluten fractions were also extracted by adding SDS. However, no proteins were able to be extracted from udon or bread witout severing the disulfide bonds under reducing condition. Only trace amounts of protein were detected in the water after boiling udon noodles. The reactivity of IgE antibody to the extracted protein did not differ among the different processed food types. Wheat allergens became strongly insolubilized after gluten formation and heating. However, the reactivity of IgE antibody to each allergen was not affected by food processing. Further studies are needed for the effects on clinical symptoms.

Model development for prediction of the allergic response to the wheat proteins ω-5 gliadin and HMW-glutenin

As one of the eight foods that account for 90 % of food allergies, wheat must be excluded from the diet in patients suffering from wheat allergies. From studies of wheat-dependent exercise-induced anaphylaxis (WDEIA), which has been used as a model to develop hypoallergenic wheat, we now know that the gluten fraction of wheat protein, particularly ω-5 gliadin and high-molecular-weight (HMW)-glutenin, is responsible for the allergic response. However, studies of allergic responses with WDEIA have been performed with a single wheat cultivar. Thus, in an effort to provide more information for the development of hypoallergenic wheat, we compared various cultivars with different countries of origin and characteristics. For the first step, we compared the allergen contents (ω-5 gliadin and HMW-glutenin) in each cultivar and the allergic response caused by each cultivar. Domestic wheat cultivars had lower contents of ω-5 gliadin and HMW-glutenin than those of imported wheat cultivars. Additionally, some cultivars caused varying allergic responses due to their allergen components. From regression analysis of allergen contents and allergic responses in vivo, we suggest a prediction model to estimate the extent of allergic response based on the ω-5 gliadin and HMW-glutenin contents. Further studies are needed to analyze the biological interactions between allergens from various cultivars and allergic response factors.

English

The study was aimed to evaluate the effect of thermo-molding on gluten and gluten fractions composition and its extractability with 1.0 ml 0.05 M sodium phosphate buffer (pH 6.8) with 2.0% sodium dodecyl sulfate (SDS) at 130 and 150&deg;C for 5 and 25 min. The gluten, gliadin and glutenin composition as affected by thermo-molding was analysed with reverse phase high performance liquid chromatography (RP-HPLC) and amino acid was analysed with acid hydrolysis using high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD). The extractability and composition were significantly affected. Gluten extracts were found in equivalent proportions; gliadin (51%) and glutenins (49%). Gluten, gliadin and glutenin subunits (GS) extracts were significantly affected and a reduction with increased molding condition explains the occurrence of polymerization reactions due to SH-disulfide interchange reactions. The glutenin fraction contains 15% gliadin while the gliadin fraction contains 2.5% glutenin after extraction with 60% ethanol. The extractability of the gliadin types and GS decreased with increasing molding time and temperature. Key words: Gluten, gliadin, glutenin, extractability, composition, thermo-molding, amino acids.

Improved Quantitation of Gluten in Wheat Starch for Celiac Disease Patients by Gel-Permeation High-Performance Liquid Chromatography with Fluorescence Detection (GP-HPLC-FLD).

Purified wheat starch (WSt) is commonly used in gluten-free products for celiac disease (CD) patients. It is mostly well-tolerated, but doubts about its safety for CD patients persist. One reason may be that most ELISA kits primarily recognize the alcohol-soluble gliadin fraction of gluten, but insufficiently target the alcohol-insoluble glutenin fraction. To address this problem, a new sensitive method based on the sequential extraction of gliadins, glutenins, and gluten from WSt followed by gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD) was developed. It revealed that considerable amounts of glutenins were present in most WSt. The gluten contents quantitated by GP-HPLC-FLD as sum of gliadins and glutenins were higher than those by R5 ELISA (gluten as gliadin content multiplied by a factor of 2) in 19 out of 26 WSt. Despite its limited selectivity, GP-HPLC-FLD may be applied as confirmatory method to ELISA to quantitate gluten in WSt.

Impact of the preparation procedure on gliadin, glutenin and gluten contents of wheat starches determined by RP-HPLC and ELISA

Celiac disease patients rely on safe gluten-free products, and the use of purified wheat starch (WSt) as part of the diet is usually well tolerated. However, uncertainties about residual gluten amounts in WSt remain, because ELISAs target the alcohol-soluble prolamin (gliadin) fraction of gluten, but hardly detect the alcohol-insoluble glutelin (glutenin) fraction. Therefore, gliadin, glutenin and gluten contents of WSt prepared from doughs on a laboratory scale were monitored by RP-HPLC. WSt washed with water from optimally or overmixed doughs had the lowest gluten contents. Gliadins were removed more extensively than glutenins during consecutive washing steps, so that gluten contents analyzed by ELISA were lower than those by RP-HPLC in 17 out of 24 WSt samples. The inability to detect glutelins by ELISA may thus lead to an underestimation of gluten contents in WSt. These findings highlight the need for improved analytical methods capable of detecting both prolamins and glutelins in processed food samples.