Molecular characterisation of 36 oat varieties and in vitro assessment of their suitability for coeliacs’ diet

Coeliac disease (CD) is a chronic intolerance to gluten, contained mainly in wheat, rye and barley. The only therapy at present is the lifelong exclusion of gluten from the diet.Whether oats can be considered safe for CD patients has long been debated, and oats have been included among gluten-free ingredients only recently (EU Regulation 41/2009), provided the gluten content does not exceed 20 ppm.The aim of this study was to evaluate the suitability of 36 different oat cultivars for CD patients using biochemical and immunochemical approaches. The cross-reactivity between avenins and gliadins was evaluated by both SDS-PAGE/Immunoblotting and ELISA.The protein pattern of each oat cultivar showed both qualitative and quantitative differences that correlated with different binding affinity for specific anti-gliadin antibodies in immunoblotting. In most oat samples, the content of cross-reactive proteins measured by ELISA was below 20 ppm, but in a few varieties was above 80 ppm.Although the taxonomic and biochemical characteristics of oats allow to conclude that their use could be safe for CD patients, it is essential to select those cultivars having the lowest level of gluten-like proteins.     

Reducing the incidence of allergy and intolerance to cereals

This paper reviews studies on allergy, intolerance and sensitivity to cereals, especially to wheat, barley, rye, maize, rice and oats with regard to reducing their incidences. Prevalence of allergy to cereals is generally low; prevalence of other cereal-related diseases are highest for wheat and lowest for oats. Compared to the other cereals, wheat (and its components wheat starch and vital gluten) are most abundantly applied in a broad range of food products world-wide, which justifies the major focus of this review on wheat. Current knowledge on diagnosis of the cereal-related diseases and on detection and characterization of the relevant proteins is discussed in the context of the development of prevention strategies. Aiming at their design and implementation, such strategies require building of knowledge frameworks at the primary, secondary and tertiary prevention levels. In this regard, selection and breeding of low-allergenic/low-intoleragenic crop varieties, application of processing and technological approaches, and the introduction of alternative safe cereal crops is discussed. Sustainable reduction of immune-related diseases in general (including cereal allergies and intolerances) is discussed with regard to eating habits and lifestyle factors, human genetic and physiological characteristics, and the role of the intestinal micro-flora.     

Using the Gluten Peak Tester as a tool to measure physical properties of gluten

The functional properties of wheat are largely dictated by composition and interactions of the gluten proteins. All flours contain gliadin and glutenin, but produce baked products of varying quality, which provides evidence that gluten proteins from different wheats possess different properties. A common method to study differences in gluten properties, which is utilized in this study, is fractionation/reconstitution experiments to understand how various gliadin to glutenin ratios and how fractions from different wheat sources affect gluten aggregation properties. Gliadin and glutenin from a vital wheat gluten were fractionated with 70% ethanol and reconstituted at various gliadin to glutenin ratios. Gliadin and glutenin from a Canadian eastern soft, eastern hard and western hard wheat (14% moisture) were fractionated and substituted between flours at the native gliadin to glutenin ratio. Gluten combinations were evaluated with a Gluten Peak Tester at constant temperature and mixing. Varying gliadin to glutenin ratio showed that 50:50 is optimal for fast gluten aggregation while amount of glutenin dictates strength. Substitution experiments showed that replacing good quality gluten fractions with those from a lower quality wheat decreases gluten quality, and vice versa. Data also showed that cultivar specific differences in gliadin and glutenin are more important in dictating gluten strength (torque), while gliadin to glutenin ratio dictates aggregation time (PMT) independent of the source of fractions. The study demonstrated the ability of the improved method to evaluate gluten aggregation by controlling for all variables except the one being tested. The data also revealed information about gluten aggregation properties never before seen.     

Improving wheat to remove coeliac epitopes but retain functionality

Coeliac disease is an intolerance triggered by the ingestion of wheat gluten proteins. It is of increasing concern to consumers and health professionals as its incidence appears to be increasing. The amino acid sequences in gluten proteins that are responsible for triggering responses in sensitive individuals have been identified showing that they vary in distribution among and between different groups of gluten proteins. Conventional breeding may therefore be used to select for gluten protein fractions with lower contents of coeliac epitopes. Molecular breeding approaches can also be used to specifically down-regulate coeliac-toxic proteins or mutate coeliac epitopes within individual proteins. A combination of these approaches may therefore be used to develop a “coeliac-safe” wheat. However, this remains a formidable challenge due to the complex multigenic control of gluten protein composition. Furthermore, any modified wheats must retain acceptable properties for making bread and other processed foods. Not surprisingly, such coeliac-safe wheats have not yet been developed despite over a decade of research.     

Does wheat make us fat and sick?

After earlier debates on the role of fat, high fructose corn syrup, and added sugar in the aetiology of obesity, it has recently been suggested that wheat consumption is involved. Suggestions have been made that wheat consumption has adverse effects on health by mechanisms related to addiction and overeating. We discuss these arguments and conclude that they cannot be substantiated. Moreover, we conclude that assigning the cause of obesity to one specific type of food or food component, rather than overconsumption and inactive lifestyle in general, is not correct. In fact, foods containing whole-wheat, which have been prepared in customary ways (such as baked or extruded), and eaten in recommended amounts, have been associated with significant reductions in risks for type 2 diabetes, heart disease, and a more favourable long term weight management. Nevertheless, individuals that have a genetic predisposition for developing celiac disease, or who are sensitive or allergic to wheat proteins, will benefit from avoiding wheat and other cereals that contain proteins related to gluten, including primitive wheat species (einkorn, emmer, spelt) and varieties, rye and barley. It is therefore important for these individuals that the food industry should develop a much wider spectrum of foods, based on crops that do not contain proteins related to gluten, such as teff, amaranth, oat, quinoa, and chia. Based on the available evidence, we conclude that whole-wheat consumption cannot be linked to increased prevalence of obesity in the general population.     

Deamidation of gluten proteins and peptides decreases the antibody affinity in gluten analysis assays

Wheat gluten is a widely used ingredient in the food industry due to its unique properties and relatively low price. Modification of wheat gluten makes it a versatile ingredient and, thus, increases its applicability in foods. Therefore, gluten proteins can be found in unexpected sources, and this makes the gluten-free diet challenging to follow. Deamidation is one way to modify protein structure. It increases solubility and surface activity of gluten improving its functionality, but consequently, also influencing the accuracy of quantification by immunoassays. In this study, the effect of deamidation on the antibody recognition with gluten analysis methods based on monoclonal R5, omega-gliadin or G12 antibodies was studied. Random deamidation decreased the intensities to 13–54% of the intensity obtained for the intact peptides. Deamidation representing the transglutaminase deamidation decreased the intensities to 4–8%. Deamidation of gluten proteins abolished the recognition by omega-gliadin and G12 antibodies and decreased the recognition of R5 by 600 times when analyzed by the sandwich method and 125 times by the competitive method. In conclusion, with all of the investigated gluten-specific antibodies, deamidation decreased the affinity of antibodies to gluten peptides and proteins, which needs to be considered when assays and regulations are developed for gluten-free products.     

Immunological analysis of serum for buckwheat fed celiac patients

Buckwheat (Fagopyrum esculentum, Moench), a herbaceous plant of the Polygonaceae family, does not have any affinity for the Gramineae family. It is commercially rated as a cereal, which has led to some misunderstanding concerning its use in gluten induced enteropathy or celiac disease. The effect of buckwheat flour ingestion by celiac patients was evaluated through the indirect immunofluorescence technique. Samples of serum were collected 30 days after the flour ingestion. Cryostat performed cuts of wheat and buckwheat grains were accomplished in order to compare the reactivity of their proteins and serum of celiac and normal patients. The assay revealed that the buckwheat flour presents no toxicity for the celiac patient and no anti-protein antibodies formation in the grain was observed for serum dilutions of 1:2 up to 1:32 with 0.15 M NaCl solution in 0.01 M phosphate buffer, pH 7.2 throughout the 30 day assay period. The results of the immunological assays showed that the buckwheat flour does not present toxic prolamines to celiac disease patients.     

Effect of legume flours on baking characteristics of gluten-free bread

The objective of this work was to study the characteristics of four gluten-free bread formulations and the possibility of substituting soya protein with other legume proteins. Four bread recipes were prepared with chickpea flour, pea isolate, carob germ flour or soya flour. Carob germ flour batter structure was thicker compared with the other batters, probably due to the different protein behaviour and the residual gums present in carob germ flour. However, carob germ flour bread obtained the lowest specific volume values (2.51 cm³/g), while chickpea bread obtained the highest (3.26 cm³/g). Chickpea bread also showed the softest crumb. Confocal scanning-laser microscopy results showed a more compact microstructure in carob germ flour bread compared with soya and chickpea formulations. Chickpea bread exhibited the best physico-chemical characteristics and, in general, good sensory behaviour, indicating that it could be a promising alternative to soya protein.     

Composition of peptide mixtures derived from simulated gastrointestinal digestion of prolamins from different wheat varieties

Gliadin content of wheat is highly variable, both qualitatively and quantitatively, in function of the plant genetic and of the growing conditions. The gliadins are among the major triggers of celiac disease: peptides derived from the gastrointestinal digestion of these proteins and absorbed from the lamina propria cause the immunological reactions that damage the villous structure in affected subjects. In the present work, the peptide mixtures generated by simulated gastrointestinal digestion of the prolamin fraction extracted from different wheat varieties (Triticum turgidum subsp. durum and Triticum aestivum) were characterized by LC/MS and LC-MS/MS techniques. Peptides related to the amount of α-gliadin, as well as toxic and immunogenic peptides for celiac patients have been identified and quantified using an isotopically labeled internal standard. The quantification demonstrated strong differences among the varieties tested. Some samples, belonging to the same varieties and/or cultivation area, showed a lower α-gliadin content, and a smaller amount of toxic and immunogenic peptides.     

Avenin diversity analysis of the genus Avena (oat). Relevance for people with celiac disease

Oat is widely consumed by people with celiac disease (CD). Its safety has been disputed because two peptides from oat avenins can be recognized as T cell epitopes by some CD patients. Differential signals of gluten-specific monoclonal antibodies and in-vitro T cells to oat varieties have suggested the existence of differences in immunogenicity. We aimed to clarify the nature of such responses by cloning avenin genes from 13 Avena species. A single oat plant contained up to 10 avenin genes. Avenin proteins clustered in four groups of which two contained the two avenin CD epitopes. All Avena species examined harbored avenins of these two groups, and as a consequence all contained avenins with the two avenin-specific epitopes, which makes it very unlikely to find oat cultivars that are devoid of these sequences. The established gluten epitopes from wheat, rye and barley were not present in oat avenins; some variants with two and three amino acid substitutions occurred, but they were predicted not to resist proteolysis in the gastro-intestinal tract. Perfect recognition sites of antibodies R5 and G12 were also not present in avenins. Thus, their signals to oat should not be interpreted as differences in immunogenicity for CD patients.     

Optimisation of droplet digital PCR for determining copy number variation of α-gliadin genes in mutant and gene-edited polyploid bread wheat

Targeted and random mutagenesis of gene families require accurate quantification. Droplet digital PCR (ddPCR) enables high-throughput screening of copy number variation (CNV). We tested the accuracy of ddPCR for CNV analysis in the large α-gliadin gene family, using degenerate primers. First, duplex ddPCR assays measured α-gliadins in diploid (15–17 copies) and tetraploid (70–76 copies) wheat accessions and a corresponding number in resulting Synthetic Hexaploid Wheat, demonstrating linear amplification up to 86–95 genes. Second, we amplified 61 α-gliadin genes in Chinese Spring. Most α-gliadins of the homoeologous chromosomes 6A and 6D were correctly amplified, as corroborated using deletion and nullisomic-tetrasomic lines, but one group of genes from 6B were not amplified with these primers. Third, in Paragon we amplified 61 α-gliadin genes while selected γ-irradiated mutant lines revealed reductions of 25–50%. Finally, using two duplex ddPCR assays, we showed that CRISPR/Cas9-targeting of α-gliadins in Fielder produced indels (1–50 bp) in up to 10 α-gliadin genes plus large deletions (>300 bp) in 20 of 87 amplified α-gliadin genes. ddPCR is suitable for high-throughput screening of CNV and gene-editing-induced mutations in large gene families, in polyploids. In wheat, ddPCR enables screening of gliadins in breeding programs towards hypoimmunogenic gluten for coeliac patients.     

Reactivity of gluten detecting monoclonal antibodies to a gliadin reference material

People affected by coeliac disease need to adhere to a life-long gluten-free diet to avoid symptoms. ELISA-tests are seen as the mainstay for the detection of gluten in gluten-free food because of their sensitivity. They can, however, yield different gluten amounts depending on the antibody and reference material used. We compared the reactivity of three prominent mouse anti-gliadin-antibodies to a reference gliadin isolated from 28 common bred European wheat varieties. The reference material proteins were labelled with fluorescent dye Cy3. They were then separated by 2DE and transferred by Western blot onto low fluorescent PVDF-membranes, followed by incubation with the three primary anti-gliadin antibodies one by one. Detection of the reacting proteins used anti-mouse antibody which was labelled with fluorescent dye Cy5. The use of this technique made it possible to co-detect the 2DE-image of the reference material proteins (Cy3) and proteins reacting with the respective antibody (Cy5). The three investigated antibodies had dissimilar reactivities with different proteins of the reference gliadin. Antibodies R5 and PN3 reacted mainly with gliadin fractions, antibody 401.21 mainly with high molecular weight glutenins. The results confirm the individual specificity of these antibodies and demonstrate the importance of validating immunochemical methods for gluten detection.     

Characterization of a gluten reference material: Wheat-contaminated oats

Oat could be a good addition to the Gluten Free diet, but the purity of the oat supply is under scrutiny. As celiac disease becomes more prevalent, better detection tools for gluten in oats are necessary. We aimed to produce reference materials (RMs) of Canada Western Red Spring wheat (CWRS)-contaminated oat flour. Pure, uncontaminated oats flour (cultivars Navan and Gehl) was supplied by Cream Hill Estates. CWRS samples were provided by the Canadian Grain Commission from the 2009 Harvest Sample program. RMs containing 0, 20 and 100 ppm CWRS gluten-contaminated oats were created using a V-shell blender and tested by sandwich-type ELISA for gluten. Marked variations in ELISA results for the RMs were found among different test kits due to differences in capture antibodies and kit construction. The Veratox test was accurate at the 0, 20 and 100 ppm levels but detected only 30% (Veratox) and 50% (Veratox R5) of gluten at the 1000 ppm level; the Ridascreen test was accurate at all levels; the Biokits test detected roughly 10% of the gluten dosage; the Gluten-Check test detected some 30% of the gluten dosage. The RMs created could serve as standards for gluten detection in oat containing foods.