Gliadin Research Articles

Wheat gliadin/xanthan gum intermolecular complexes: Interaction mechanism and structural characterization

In this study, the interactions between wheat gliadin (GL) and xanthan gum (XG) were investigated to design new systems with potential applications as a gluten-free substitute product. Combining spectral with morphological and molecular docking methods allowed the establishment of the complexation mechanism between globular hydrophobic GL and the hydrophilic XG with an extended and partially disordered backbone. GL maintains intact its hydrophobic core even at high GL/XG ratios and organizes into small aggregate–type assemblies. The stable and uniform complexes have a low GL content, based on intermolecular hydrogen bonds and hydrophobic interactions. The GL/XG combining ratio influences the size, structure and interaction mechanism of the microparticles. The preferred sites of interaction and the binding affinities were determined by molecular docking on GL libraries and XG models. This research may provide significant knowledge for the development of low-GL wheat food products using a dietary fiber polysaccharide as a functional compound.

Sonochemical Effects on the Preparation, Structure and Function of Gliadin-(−)-Epigallo-Catechin 3-Gallate Conjugates

It is essential to understand the mechanism of action of ultrasound synergistic free radical oxidation to promote covalent reactions between proteins and polyphenols. (-)-epigallo-catechin 3-gallate (EGCG) with rich bioactivity could be used to increase the functional properties of cereal protein-gliadin (GL). This study systematically explored the role of ultrasound treatment (US) on the binding mechanisms of GL and EGCG. Electrophoresis and high-performance liquid chromatography (HPLC) confirmed the greater molecular mass of the covalent complexes in the ultrasound environment. Quantitative analysis by the phenol content revealed that the ultrasound environment increased the EGCG content in the covalent complex by 15.08 mg/g of protein. The changes in the spatial structure of the proteins were indicated by Fourier infrared and ultraviolet spectroscopy. Additionally, scanning electron microscopy (SEM) and atomic force microscopy (AFM) found that US disrupted the aggregation of GL and the clustered structure of the covalent complexes. The results demonstrated that the water solubility of ultrasonic conjugates was significantly increased by 8.8-64.19%, the digestion rate was more efficient, and the radical scavenging capacity was twice that of GL. This research contributes to the theoretical basis for broadening the application of polyphenols in modifying protein.

Epitope-imprinted polydopamine and reduced graphene oxide-based sensing interface for label-free detection of gliadin

Detection of gluten and its components in foods is paramount in ensuring safety of gluten-sensitive individuals. However, the conventional immunodetection methods including gold standard enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, and advanced proteomic methods suffer from high cost, complicated instrumentation and protocol, and slow assay turnaround time. Herein, we developed an inexpensive electrochemical sensor for gliadin (GLI) detection using electrochemically reduced graphene oxide for sensitivity enhancement and PQQPFPQQ epitope-imprinted polydopamine as selective recognition interface. The sensing performance was improved by optimizing key process factors including template/monomer ratio, pre-polymerization period, electropolymerization and electrochemical reduction cycles, elution time, and rebinding pH and time. The sensor shows a linear relationship between current response and GLI concentration in the 1–15 ppm range with high affinity to the epitope template (imprinting factor IF = 8.68) and target GLI (IF = 3.14), low limits of detection (0. 70 ppm) and quantification (2.33 ppm). The sensor also exhibits excellent selectivity against potential interferents including ovalbumin (selectivity factor SF = 0.15), folic acid (SF = 0.25), and casein (SF = 0.39) in buffered matrix. The obtained sensor-to-sensor reproducibility, repeatability, and stability ensure its sensitive determination of native GLI in rice flour matrix with coefficient of variance in the range of 2.52–4.27 %, and recovery rates of 99.1–101.9 %. The results fare better than those analyzed by traditional ELISA and similar methods. The developed sensor could be used as a reliable platform for rapid detection of allergic gluten components in processed food products.

Preparation and characterization of magnetic molecularly imprinted polymer for specific adsorption of wheat gliadin

In this study, a magnetic molecularly imprinted polymers (MIPs) was successfully synthesized by surface imprinting technology for the specific recognition of wheat gliadin (GLI). MIPs showed an obvious hierarchical characteristic by investigations of micromorphology, the magnetic Fe3O4 core were wrapped with SiO2 layer (approximately 40 nm) and imprinted layer (approximately 10 nm). The results of vibrating sample magnetometer (VSM) proved the superparamagnetism of MIPs, which was contributed to the rapid magnetic separation. Furthermore, MIPs displayed excellent selective adsorption ability for GLI through adsorption kinetic model, adsorption isotherm model and adsorption selectivity experiment. The saturated adsorption capacity of MIPs to GLI was 73.55 mg/g under the optimized conditions and the imprinting factor was 3.10. The high performance liquid chromatography (HPLC) manifested that the MIPs were successfully applied to separate template GLI from gluten, reflecting the excellent adsorption specificity of MIPs. Therefore, this research provided a promising method for rapidly separation and specific adsorption of GLI.

Fabrication of cumin seed oil loaded gliadin-ethyl cellulose nanofibers reinforced with adipic acid for food packaging application

In this study, the water-insoluble cumin seed oil (CSO) loaded gliadin (GN)-nanofibers (NFs) mat was prepared in the presence of ethyl cellulose (EC) and adipic acid (AD) using electrospinning method. The NFs were prepared with mixing 30 % w/v of GN and 15 %w/v of EC at the ratio of 80:20 v/v and 5%w/v of AD were selected as optimum formulation based on electrospinning efficiency and SEM images. In the presence of EC, the spinnability of GN fibers improved. Moreover, in the presence of AD the fibers diameters decreased to 50 % (250 ± 14 nm), and uniformity enhanced in all formulations. AD acted as hydrogen bond cross-linker between mixed biopolymers that led to increase in the viscosity of the polymer solutions. The selected formulation was loaded with the CSO based on 10 %w/v of polymers. Thermal stability of the GN fibers was improved in the presence of EC and AD based on DSC results. The FTIR results confirmed the hydrogen bond crosslinking between polymers and AD. Based on antioxidant activities results, the prepared NFs successfully retarded the release of CSO. CSO loaded NFs showed the strong antibacterial and antifungal activity against Escherichia coli, Staphylococcus aureus, and Aspergillus flavus. NFs had no significant toxicity on the viability of normal human umbilical vein endothelial cell (HUVEC). The results of the current study indicated that the prepared NFs mat may have a great potential for using in active food packaging due to its sustained control release properties for loaded antimicrobial and antioxidant active agent.

Gold nanoparticles phytoreduced with Cornus mas extract mitigate some of gliadin effects on Caco-2 cells.

Celiac disease (CD) is an autoimmune condition that occurs in genetically predisposed people where the ingestion of gluten produces damage in the small intestine. The treatment accepted until now is a strict gluten free diet. This implies the need for novel or adjuvant treatments, in addition to the standard of care. The present study aimed to assess the effect of gold nanoparticles phytosynthesized with Cornus mas extract (AuCM) compared to Cornus mas extract (CM) and luteolin (LT) on Caco-2 cells, exposed or not to gliadin. Ultraviolet-visible spectroscopy and transmission electron microscopy were used for the characterization of AuCM. Measured cellular outcomes included oxidative stress markers (malondialdehyde level, catalase and superoxide dismutase activities), inflammatory response and cellular signaling and transcription factors involved in apoptosis (NFκB, pNFκB, NOS2, TNF-α, TRAIL, Bax, Bcl-2, p53). The internalization of gold nanoparticles in cells was evidenced by transmission electron microscopy (TEM). The gliadin administration induced oxidative stress, improved the activity of antioxidants enzymes, increased NOS2 and NFκB expressions and reduced pNFκB/NFκB ratio. In addition, gliadin enhanced TRAIL and Bcl-2 levels and reduced p53 expression in Caco-2 cells. The pretreatment with AuCM, CM extract and LT diminished oxidative stress and reduced NOS2 activity. AuCM and CM treatment amplified the expression of p53 and pNFκB/NFκB ratio and diminished Bcl-2, NFκB and pNFκB, especially AuCM. The results obtained confirmed that AuCM mitigate some of gliadin effects on Caco-2 cells through modulation of oxidative stress and inflammation.

Deamidated Gliadin Peptide Antibodies in Celiac Disease: A Diagnostic Driver or just along for the Ride?

Anti deamidated gliadin peptides antibodies are considered as celiac disease associated diagnostic antibodies. They are in clinical use for almost the last two decades. In the first decade they were preferentially used in early childhood, in face of IgA deficiency and occasionally recommended as the prime serological marker, outperforming the anti-tissue transglutaminase autoantibody. Notably, they were recommended in combination with the tissue transglutaminase as enhancer of the diagnostic performances. No more, the circle turned over. In the second decade (2012-2019), most of the studies limited and criticized their past published advantages. They suggested that deamidated gliadin peptides antibodies do not have any advantage over anti-tissue transglutaminase autoantibodies in terms of early childhood, IgA deficiency, diagnostic performances and when both antibodies are combined. It seems that the deamidated gliadin peptide are losing their place in the celiac disease algorithmic diagnostic flow chart.

DNA damage in human skin fibroblasts from patients with dermatitis herpetiformis.

Dermatitis herpetiformis (DH) and celiac disease (CD) are considered to be autoimmune diseases that share a specific trigger (gluten) and a common genetic background (HLA-DQ2/DQ8). However, the pathogenesis of DH is not yet fully understood and no data are available regarding a possible role of fibroblasts in this disease. The aim of this study was to assess baseline DNA damage in fibroblasts in DH-diagnosed patients vs. fibroblasts of controls without DH or CD. Primary fibroblast cultures were derived from dermal biopsies from DH patients and controls (without DH or CD). In vitro genotoxic damage was investigated using the comet assay and ɣH2AX test after different treatments (with 33mer peptide and digested gliadin [DG]) in order to investigate a correlation between oxidative stress (evaluated by reactive oxygen species formation) and glutathione content. Our results demonstrate a difference in baseline DNA damage between cutaneous fibroblasts of controls and DH patients, moreover, DNA damage significantly increased after exposure to gluten (DG and 33mer peptide) in fibroblasts from DH patients. DNA damage in fibroblasts from patients under dapsone treatment was similar to that of the control group. Our data indicate that oxidative stress and DNA damage may be characteristics of fibroblasts from DH patients who are not treated with dapsone, particularly after exposure to gliadin peptides.

Neurological Complications of Coeliac Disease

Coeliac disease (CD) is a chronic autoimmune enteropathy of the small intestine that occurs in genetically predisposed individuals. It is characterized by atrophy of intestinal villi accompanied by an increase in the number of intraepithelial lymphocytes and crypt hypertrophy as well as the presence of specific endomysial antibodies (EMA), IgA tissue transglutaminase antibodies (IgA-tTG), and IgA and IgG deaminated gliadin peptide antibodies (DGP IgA and IgG). CD is associated with a wide spectrum of clinical signs and symptoms secondary to malabsorption (vitamin deficiency, anaemia, osteoporosis) or unrelated to the gastrointestinal tract. Neurological disorders are a common problem in patients with CD and are not always accompanied by gastrointestinal symptoms. The most common neurological manifestations of CD are cerebellar ataxia, epilepsy, and peripheral neuropathy.

3D-printed scaffolds of mesoporous bioglass/gliadin/polycaprolactone ternary composite for enhancement of compressive strength, degradability, cell responses and new bone tissue ingrowth.

BackgroundDue to the increasing number of patients with bone defects, bone nonunion and osteo-myelitis, tumor and congenital diseases, bone repair has become an urgent problem to be solved.MethodsIn this study, the 3D-printed scaffolds of ternary composites containing mesoporous bioglass fibers of magnesium calcium silicate (mMCS), gliadin (GA) and polycaprolactone (PCL) were fabricated using a 3D Bioprinter.ResultsThe compressive strength and in vitro degradability of the mMCS/GA/PCL composites (MGPC) scaffolds were improved with the increase of mMCS content. In addition, the attachment and proliferation of MC3T3-E1 cells on the scaffolds were significantly promoted with the increase of mMCS content. Moreover, the cells with normal phenotype attached and spread well on the scaffolds surfaces, indicating good cytocompatibility. The scaffolds were implanted into the femur defects of rabbits, and the results demonstrated that the scaffold containing mMCS stimulated new bone formation and ingrowth into the scaffolds through scaffolds degradation in vivo. Moreover, the expression of type I collagen into scaffolds was enhanced with the increase of mMCS content.ConclusionThe 3D-printed MGPC scaffold with controllable architecture, good biocompatibility, high compressive strength, proper degradability and excellent in vivo osteogenesis has great potential for bone regeneration.

Nanoporous diopside modulates biocompatibility, degradability and osteogenesis of bioactive scaffolds of gliadin-based composites for new bone formation.

IntroductionIt is predicted that with increased life expectancy in the whole world, there will be a greater demand for synthetic biomedical materials to repair or regenerate lost, injured or diseased tissues. Natural polymers, as biomedical materials, have been widely applied in the field of regenerative medicine.Materials and methodsBy incorporation of nanoporous diopside bioglass (nDPB) into glia-din (GL) matrix, macro–nanoporous scaffolds of nDPB/GL composites (DGC) were fabricated by method of solution compressing and particles leaching.ResultsThe results revealed that the DGC scaffolds possessed well-interconnected macropores of 200–500 μm and nanopores of 4 nm, and the porosity and degradability of DGC scaffolds remarkably increased with the increase in nDPB content. In addition, in vitro cell experiments revealed that the adhesion and growth of MC3T3-E1 cells on DGC scaffolds were significantly promoted, which depended on nDPB content. Moreover, the results of histological evaluations confirmed that the osteogenic properties and degradability of DGC scaffolds in vivo significantly improved, which were nDPB content dependent. Furthermore, the results of immunohistochemical analysis demonstrated that, with the increase in nDPB content, the type I collagen expression in DGC scaffolds in vivo obviously enhanced, indicating excellent osteogenesis.Discussion and conclusionThe results demonstrated that the DGC scaffolds containing 30 wt% nDPB (30nDGC) exhibited good biocompatibility and new bone formation ability, which might have a great potential for applications in bone regeneration.

Deamidated Gliadin Peptide Antibodies; is the Time to Use as a Routine Test for Celiac Disease

Antibodies to deamidated gliadin peptides (DGP-AGA) have newly been presented into the extensive variety of serologic examinations for celiac disease (CD). In compare with the modern AGA, DGP-AGA indicated a higher sensitivity and specificity for CD and simply identified by ELISA kits. Nevertheless, even though the very favorable outcomes, DGP-AGA are not yet regularly used in the serologic checkup for CD. An antibody approach could initiate development in the diagnostic precision of serology for CD screening.

The Significance of Key Amino Acid Sequences in the Digestibility and Toxicity of Gliadin Peptides in Celiac Disease

The importance of alternative or adjunct treatments to the gluten-free diet in celiac disease is now being recognized. This paper discusses the scientific principles behind the use of caricain for enzyme therapy. Objective: To review the structures of the toxic peptides in A-gliadin that relate to those found by other workers insofar as having key sequences of amino acids or motifs which relate to toxicity, especially in regard to difficulty of digestion or immunogenicity. Methods: Structures of synthetic A-gliadin peptides shown to be toxic in the fetal chick assay were examined before and after digestion with duodenal mucosa from patients in long remission. Synthetic peptides corresponding to the undigested residues were also assayed and the key amino acid sequences compared in order to determine if they could be related to direct toxicity and immunogenicity of the peptides. Results: The results showed that the smallest toxic peptides from celiac mucosal digestion were octa-peptides and that they were obtained in greater yield than similar products from normal digestion. One of those peptides corresponded to residues 12-19 of A-gliadin and contained the key motifs PSQQ and QQQP of De Ritis et al. , whilst the other corresponded to residues 72-79 and contained the key motif PYPQ (extending to PYPQPQ), observed by other workers, especially those who have been investigating immunological activity over the past two decades. Conclusions: The presence of key motifs in undigested residues from celiac mucosal digestion and the greater prevalence of these residues compared with residues from normal digestion justifies our work on enzyme therapy. These studies have also indicated that our use of caricain as an enzyme capable of digesting peptides with two different types of toxicity has a sound scientific basis.

Multiwalled Carbon Nanotubes Based Immunosensor for Diagnosis ofCeliac Disease

Celiac Disease (CD) is an autoimmune disorder, represented by the ingestion of gliadin protein usually found in wheat, barley and rye. CD is closely associated with genes that code HLA-II antigens, mainly of DQ2 and DQ8 classes. Small intestinal biopsy is considered the gold standard for diagnosis of celiac disease. In addition, the most sensitive tests for the diagnosis of celiac disease include the increase in level of IgA isotype of anti-gliadin antibodies, connective tissue antibodies and tissue transglutaminase antibodies. In the present study, an amperometric immunosensor was fabricated for CD diagnosis using multiwalled carbon nanotubes screen printed electrode. Antigen (gliadin) was covalently linked to carbon nanotubes through 1-Ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDC)- N-hydroxysuccinimide (NHS) cross linking chemistry. Different concentrations of anti-gliadin antibodies were added on working electrode surface and change in current was measured by Cyclic Voltammetry using redox indicator Potassium ferricyanide [K3Fe(CN)6]. Electrochemical studies showed that the immunosensor was able to detect the antibody concentration as low as 0.13ng/μl. Sensitivity of the immunosensor was found to be 119.2 (μA/cm2)/ng. The present sensor can be used for diagnosis of celiac patients who are sensitive to gluten ingestion.

Comparison of Spectral and Molecular Analyses for Classification of Long Term Stored Wheat Samples.

This study aimed to determine whether NIR spectroscopy and protein band analysis can differentiate the grain samples of 15 wheat genotypes stored for different periods: Group Ⅰ (91 weeks), Group Ⅱ (143 weeks), Group Ⅲ (194 weeks), and Group Ⅳ (246 weeks). Samples were harvested from previously-conducted field trials, and stored at +4 ℃. A-PAGE and SDS-PAGE methods were utilized to separate gliadin and glutenin fractions, respectively. A qualitative calibration model based on the Support Vector Machine (SVM) method was generated and validated using NIR spectra taken from samples. Results indicated storage length did not have an effect on molecular band fractions. Use of this method would not be considered an effective tool for discrimination of samples stored for different lengths of time. Spectral techniques may have potential in sorting samples based on their storage time. The SVM calibration model generated here had an acceptable true classification rate (over 80%) for separating all groups, while only Groups Ⅱ and Ⅳ were precisely separated (100% true classification rate) in the validation step.

Preparation and Characterization of Chitosan Oligo-Saccharide-Hydrolyzed Gliadin Copolymer

Preparation and Characterization of Chitosan Oligo-Saccharide-Hydrolyzed Gliadin Copolymer

Correlations between gluten aggregation properties defined by the GlutoPeak test and content of quality-related protein fractions of winter wheat flour

The gluten aggregation properties of 19 winter wheat cultivars were investigated using a rapid small-scale (7 min, 8.5 g flour) technique (GlutoPeak test, GPT). Correlations between GPT indices and gliadin (GLIA), glutenin (GLUT), SDS-soluble protein (SDSS), and glutenin macropolymer (GMP) concentrations were established. Two groups of samples were distinguished based on the shape of their GPT curves. Flours of group 1 gave curves with a slower buildup in torque (measured as aggregation time) and a higher area under the curve at peak (maximum torque), compared to those of group 2. Group 1 was characterized by lower GLIA and higher GLUT and GMP contents than group 2. GLIA (r = 0.70, p ≤ 0.001) and SDSS (r = 0.59, p ≤ 0.01) concentrations were significantly correlated to peak torque. GLUT and GMP contents were correlated to the aggregation time (GLUT: r = 0.72, p ≤ 0.001; GMP: r = 0.77, p ≤ 0.001) and the area under the curve at peak (GLU: r = 0.68, p ≤ 0.01; GMP: r = 0.78, p ≤ 0.001), confirming the importance of GMP for gluten strength. GPT could be an alternative to the labor-intensive quantitation of quality related protein fractions of wheat flour.

Spectrophotometric and fluorimetric quantitation of quality-related protein fractions of wheat flour

Spectrophotometric and fluorimetric methods for quantitating quality-related parameters of wheat flour such as the gliadin (GLIA), glutenin (GLUT), and glutenin macropolymer (GMP) contents were adapted. The bicinchoninic acid (BCA) assay, the Bradford assay, the fluorescamine assay, the Naphthol Blue Black (NBB) assay, and the acid orange 12 assay were studied. Calibration functions were generated by using protein references isolated from a mixture of flours of 10 wheat cultivars. Among the assays studied, only the Bradford, the fluorescamine and the NBB assays provided useful results. The Bradford assay succeeded in quantitating the GLIA and GLUT fractions, but it was unsuitable for the GMP fraction because of the presence of SDS. All relevant protein fractions could be quantitated with the fluorescamine and the NBB assays. While the former showed higher coefficients of variation, the NBB assay was more reliable. NBB and HPLC data as well as NBB and micro-scale baking test data were strongly correlated. The NBB assay is a good alternative to chromatographic methods, which require time and sophisticated equipment. It can be adapted as easy-to-use, high throughput method that enables screening of a large number of flour samples for quality parameters in a short time per sample.

Relationship between storage protein banding pattern and growth type of some bread wheat varieties.

Seed storage proteins are one of the most important food resources that play important role on bread making quality, in this respect distinguishing the genetic diversity was crucial in breeding aims. In order to study genetic diversity in 20 wheat commercial varieties with different growth type, we analysed the banding patterns of gliadin proteins by SDS-PAGE technique. Our results showed 11 different bands that were polymorphic in 20 studding varieties. Cluster analysis classified these genotypes into 3 groups. These differentiated groups had different growth types; also cluster analysis based on Jaccard similarity coefficient showed that some lines had higher similarities than the others. Principle coordinate analysis showed that 80% of total variance was determined by the first 3 components and cophenetic correlation coefficient equal to 0.75 was resulted and this confirmed the significant relation between gliadins and growth type. Existence of relation between growth type and gliadin proteins could help breeders in variety discrimination and selection perspectives.

Proteínas del gluten y reología de trigos harineros mexicanos influeciados por factores ambientales y genotípicos

El objetivo de este estudio fue conocer el efecto de factores ambientales y genotípicos sobre los parámetros de calidad industrial y sobre la cantidad y relación de proteínas monoméricas y poliméricas del gluten en 24 líneas recombinantes de trigos harineros de temporal. El cultivo se desarrolló en cinco condiciones ambientales generadas por manejo agronómico, ciclo otoño-invierno 2006/2007, en Roque, Guanajuato, México. Se evaluaron el tiempo de amasado (TMA), fuerza (ALVW), extensibilidad (ALVPL) de la masa, fracción rica en gliadina (50PS) y en glutenina (50PI), y su relación (50PS/50PI). Las mejores combinaciones de gluteninas de alto y bajo peso molecular para TMA y ALVW fueron los genotipos con 1, 17+18, 5+10/Glu-A3c, Glu-B3g, Glu-D3b; 1, 17+18, 5+10/Glu-A3c, Glu-B3h, Glu-D3b, y 2*, 17+18, 5+10/Glu-A3c, Glu-B3g, Glu-D3b; para ALVPL, 2*, 17+18, 2+12/Glu-A3e, Glu-B3h, Glu-D3b; para 50PS, 2*, 17+18, 2+12/Glu-A3e, Glu-B3h, Glu-D3b; y 1, 17+18, 5+10/Glu-A3e, Glu-B3h, Glu-D3b. La relación 50PS/50PI fue mayor en genotipos con 2*, 17+18, 2+12/Glu-A3e, Glu-B3g, Glu-D3b. El TMA es mayor cuando aumenta la temperatura y la mejor ALVPL se obtiene en el ambiente bajo condiciones normales. La fracción 50PS y la relación 50PS/50PI son mayores cuando se realiza la fertilización con azufre, y se obtiene incremento de 50PI con riego limitado y aumento de temperaturas durante el llenado de grano.