Natural variation in avenin epitopes among oat varieties: Implications for celiac disease

Celiac disease (CD) is a chronic inflammatory disease affecting the small intestinal mucosa. The causative agents have been identified as gluten proteins from wheat, barley, and rye, and the only available treatment for CD patients is a lifelong gluten-free diet. Non-gluten containing cereals would be a valuable contribution to the gluten-free diet. In this respect, oats are a good choice. However, commercial lots of oat flakes and flour frequently are contaminated with wheat, barley, and rye, and two studies have reported that some peptides derived from the gluten-like avenin storage proteins of oat can trigger an immune response in some CD patients. In the present study we have initiated the investigation whether all oat varieties contain similar amounts of potentially harmful sequences by biochemical and immunological methods. We confirm that commercial oat preparations are contaminated with other cereals that contain gluten or gluten-like proteins. Moreover, our results demonstrate that contamination-free oat varieties differ in their capacity to stimulate an avenin-sensitive gamma-gliadin specific T cell line derived from a patient with CD, indicative for differences in the two known avenin epitopes among oat varieties, implying that selection and breeding of completely safe oat varieties for all CD patients may be a realistic possibility.     

Production of gluten-free wheat starch by peptidase treatment

The potential of peptidase-containing bran extracts from germinated cereals (wheat, emmer, barley) and a peptidase preparation from Aspergillus niger (AN-PEP) to degrade gluten in wheat starch below the threshold for gluten-free foods of 20 mg/kg was compared. The gluten-specific peptidase activity of the peptidases was determined by using gliadin as a protein-based substrate as well as the two celiac-active peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein). The peptidase activity of AN-PEP exceeded the activities of bran from germinated cereals by a factor up to 690,000. Three wheat starches with initial gluten contents of 110, 1679, and 2070 mg/kg, respectively, were incubated with bran extracts and AN-PEP, lyophilized, and residual gluten was quantitated by a competitive ELISA. Unlike peptidases from bran extracts, AN-PEP was capable of degrading gluten below 20 mg/kg in all starches. The absence of gluten in AN-PEP-treated starches was confirmed by liquid-chromatography-mass spectrometry. The properties of gluten-free starches were comparable to the native starches with the exception of a reduced viscosity after AN-PEP treatment. This problem could be overcome by using higher enzyme concentrations and shorter incubation times or by optimizing AN-PEP production for lower residual α-amylase activity.     

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.     

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.     

Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat

RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms. The elucidation of the mechanism of RNA silencing has stimulated its use as a reverse genetics tool, because RNA silencing strongly down-regulates the expression of the target gene in a sequence-specific manner. The major protein fraction of wheat grain is gluten which is largely responsible for the functional properties of dough. Gliadins contribute mainly to the extensibility and viscosity of gluten and dough, with the polymeric glutenins being responsible for elasticity. The aim of this work was therefore to silence the expression of specific γ-gliadins by RNA interference, to demonstrate the feasibility of systematically silencing specific groups of gluten proteins. The sequence of a γ-gliadin gene was used to construct the pghp8.1 plasmid. The hpRNA silencing fragment was designed on the basis of 169 base pairs (bp) in sense and antisense orientation with the sequence of the Ubi1 intron as spacer region between the repeats. Two lines of bread wheat were transformed by particle bombardment. Gliadins were extracted from 30 mg of flour, separated by acid-PAGE and determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Seven transgenic lines were obtained and all of them showed reduced levels of γ-gliadins. All seven transgenic plants were fully fertile and their grain morphology and seed weight were comparable to the control lines. MALDI-TOF MS showed that six peaks, present in the untransformed line, were missing in transgenic lines of the BW208 genotype whereas three peaks were missing in the BW2003 genotypes. The proportion of γ-gliadins was reduced, by about 55–80% in the BW208 lines and by about 33–43% in the BW2003 lines. The ELISA assay based on the R5 antibody showed reductions in total gliadins (μg/mg flour) in three of the BW208 lines and in one BW2003 line, but an increase in one BW208 line (C613).     

Effects of transglutaminase on the rheological and noodle-making characteristics of oat dough containing vital wheat gluten or egg albumin

Incorporating exogenous proteins into food production is a common practice for improving processing characteristics. In the present study, oat dough containing 15% (w/w, blends of protein-oat flour basis [POB]) vital wheat gluten (VWG) or 15% (w/w, POB) egg albumin (EA) was used to produce noodles with or without gluten (i.e., gluten-free). The rheological and noodle-making characteristics of oat dough containing exogenous proteins and the effects of added transglutaminase (TGase) were examined. The results indicate that the extent of TGase’s modification of the thermomechanical and dynamic rheological characteristics (G′ and G″) is dependent on the source of exogenous proteins in the oat dough. By adding 1.0% (w/w, POB) TGase, the cooking qualities of the resulting noodles (i.e., those containing VWG and EA) were significantly elevated with lower cooking loss; the elasticity of both types of noodles increased. The effects of TGase in different dough systems were analyzed by SDS-PAGE. In oat dough prepared with VWG, TGase was shown to catalyse the cross-linking of both oat protein and gluten protein; however, oat protein acted as the only substrate of TGase in the noodles that had been prepared with EA.     

Recent progress in analytical method development to ensure the safety of gluten-free foods for celiac disease patients

As laid down by the Codex Alimentarius, products bearing a gluten-free label must not contain gluten levels above 20 mg/kg to be safe for consumption by celiac disease patients. Analytical methods to detect gluten from wheat, rye and barley need to be sufficiently sensitive, specific, suitable for routine analyses and validated by collaborative studies. With continuous progress in the field of gluten analysis, the aim of this paper is to provide an up-to-date overview of legislation regarding gluten-free products worldwide, as well as immunochemical, proteomics-based, genomics-based and other methods designed to analyse gluten traces. While ELISA test kits and PCR are still most widely used in quality control, liquid chromatography tandem mass spectrometry (LC-MS/MS) is gaining more and more importance by providing unprecedented insights into gluten. Several other methods such as immunosensors, other sensors and microarrays are being developed. The pro's and con's of the different methods are discussed as well as the remaining challenges, including the need for improved extraction procedures, comprehensive reference materials and independent reference methods.     

Understanding the role of oat β-glucan in oat-based dough systems

B-glucan is one of the components that differentiate oats from other cereals and that contribute to the health-related value of oats. However, so far oats cannot easily be applied in bread-like products without loss of product quality. Here we have studied how the content and viscosity of oat β-glucan affect the technological properties of oat dough in both a gluten-free and a gluten-containing system. In both systems, increasing the β-glucan concentration resulted in an increase of dough stiffness and in a reduction of dough extensibility. β-glucan negatively impacted the elastic properties that additional wheat gluten conferred to oat dough. This effect was smaller for medium-viscosity β-glucan than for high-viscosity β-glucan. Interestingly, dough made from low β-glucan flour (<2%) had increased gas retention capacity. Overall, the impact of β-glucan on the properties of oat dough systems was governed by concentration and viscosity, with or without additional wheat gluten. Our findings indicate that β-glucan is a key component that determines the rheology of oat-based dough systems and, with that, the technological functionality of oat in dough systems.     

Milling,functional and thermo-mechanical properties of wheat,rye, triticale,barley and oat

The milling potential of hulled barley, hulled oat, triticale, rye and wheat was studied using a long tempering process and a laboratory four-roller mill. Regardless of the investigated cereal, the results indicated a significant influence (p < 0.05) of volume per surface area ratio on the milling yield and ash contents of the flour. The lowest milling yield was obtained in case of hulled oat. Solvent retention capacity profiles were determined for all investigated whole cereals and flours for predicting the contribution of different polymers to the functionality of samples. For all solvents higher values were obtained for the whole cereals compared to the corresponding flour. Thermo-mechanical properties of the whole cereals and refined flours were also investigated. If in case of wheat the gluten proteins play an essential role on dough behaviour during kneading at 30 °C, in case of triticale, rye, hulled barley and hulled oat, the fibers play a major role as well. Thermo-mechanical properties of starch registered a large variation between cereals and/or flours. The lowest torque value corresponding to starch gelatinization (C3) was registered in case of the hulled oat flour, 1.92 Nm, while the highest value in case of rye flour, 2.65 Nm.     

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.     

青稞B-hordein基因对小麦面粉加工特性的影响

B组醇溶蛋白是大麦的主要贮藏蛋白之一,其组成及含量与大麦的营养品质和加工品质密切相关。为探究大麦B组醇溶蛋白对小麦面粉加工特性的影响,以转青稞B-hordein基因的小麦转基因高代株系(T4、T5代)及其受体品种龙麦30为材料,分别对转B-hordein基因小麦高代株系及龙麦30进行分子检测、农艺性状测量、近红外分析和粉质分析。结果表明,转B-hordein基因小麦高代植株均为阳性植株;在农艺性状方面,转B-hordein基因小麦与龙麦30无显著差异;在品质方面,转B-hordein基因小麦的蛋白质含量、湿面筋含量的平均值均极显著高于龙麦30;粉质分析结果也表明转B-hordein基因小麦的粉质参数优于龙麦30。由此推测,B-hordein基因的成功转化能够改善小麦的面粉加工特性。     

Heterologous expression and protein engineering of wheat gluten proteins

A range of systems are available for the production of recombinant wheat gluten proteins, from simple and widely used systems based on Escherichia coli to more sophisticated eukaryotic systems in yeasts or cultured insect cells. The characteristics of these systems are summarised and their advantages and disadvantages for application to wheat gluten proteins discussed. We then review the applications of heterologous expression systems to the synthesis and characterisation of wheat gluten proteins, including the production of wild type and mutant proteins for structure–function studies. We also discuss the use of heterologous expression to establish model systems including perfect repeat peptides based on motifs present in gliadins and glutenin subunits and ‘analogue glutenin proteins’ based on C hordein of barley. It is concluded that the pET series of vectors and E. coli are suitable for most applications, providing high-level expression and being rapid and easy to use.     

Efficiency and Limitations of Immunochemical Assays for the Testing of Gluten-free Foods

This review considers the various immunochemical assays developed for gluten quantification and their feasibility for testing gluten-free food consumed by patients with coeliac disease. The first part concerns the major requirements of these assays: (a) detection of prolamins from the different cereals involved in gluten intolerance (wheat, rye, barley and oats) and avoidance of cross-reactions with prolamins from maize, rice, etc.; and (b) sensitive detection of native proteins as well as those denatured by cooking or other treatments. The second part evaluates several immunochemical assays in terms of their specificity, sensitivity and dependence on heat treatments.     

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.     

Malt hydrolysates for gluten-free applications: Autolytic and proline endopeptidase assisted removal of prolamins from wheat,barley and rye

Cereal based products intended for gluten sensitive individuals, particularly to celiac disease patients, tend to have poor organoleptic qualities and they contain low levels of healthy whole grain compounds. Adding whole grain ingredients, such as malt hydrolysates, could compensate these defects provided that the ingredients are adequately free from toxic prolamin epitopes. Here we demonstrate that the level of toxic prolamin epitopes in the malt autolysates (wheat, barley, rye) were substantially lower than in the native malts but too high to allow “very low in gluten” labelling. To further eliminate the residual levels of toxic prolamin epitopes, a proline-specific endoprotease from Aspergillus niger was added to the malt autolysates. In the resulting malt hydrolysates (of wheat and rye but not barley), the prolamins were indeed greatly reduced and were below the very low gluten limit of 100 mg/kg. Malt hydrolysates with adequately low gluten levels may potentially be used as novel ingredients within gluten-free foods.     

Wheat Bread Quality as Influenced by the Substitution of Waxy and Regular Barley Flours in Their Native and Extruded Forms

The substitution of wheat flour with barley flour (i.e. native or pretreated/extruded) reduced the loaf volume. Depending on the barley variety and flour pretreatments, the colour and firmness/texture of the bread loaves were altered. Amongst the barley breads prepared from native flours (at 15% barley flour substitution level), Phoenix had higher loaf volume and lower crumb firmness than Candle. However, amongst the barley breads prepared from extruded flours, CDC-Candle had higher loaf volume and lower crumb firmness than Phoenix. The lower loaf volume and firmer crumb texture of barley breads as compared with wheat bread may be attributed to gluten dilution. Also, the physicochemical properties of barley flour components, especially that of β-glucan, can affect bread volume and texture. β-glucan in barley flour, when added to wheat flour during bread making, could tightly bind to appreciable amounts of water in the dough, suppressing the availability of water for the development of the gluten network. An underdeveloped gluten network can lead to reduced loaf volume and increased bread firmness. Furthermore, in yeast leavened bread systems, in addition to CO₂, steam is an important leavening agent. Due to its high affinity for water, β-glucan could suppress the amount of steam generated, resulting in reduced loaf volume and greater firmness. In the present study, breads made with 15% HTHM CDC-Candle flour had highly acceptable properties (loaf volume, firmness and colour) and it indicated that the use of extruded barley flours would be an effective way to increase the dietary fibre content of barley breads.     

Effects of vacuum mixing and mixing time on the processing quality of noodle dough with high oat flour content

The effects of different mixing parameters (vacuum mixing and mixing time) on oat (70% oat flour) and wheat noodle dough were investigated on the basis of textural properties and gluten formation. The results showed that at a vacuum degree of −0.06 MPa and mixing time of 10 min, oat and wheat dough sheets exhibited the highest resistance to extension and glutenin macropolymer (GMP) content, and had the most compact and uniform gluten network. Compared with wheat noodle dough, oat dough had lower resistance to extension, lower tightly bound water content, and higher GMP content. Microstructural examination showed that oat noodle dough had a more aggregated distribution of gluten protein compared with wheat noodle dough under the optimum mixing parameters. Furthermore, the poor binding ability of vital wheat gluten with water molecules caused the indexes of oat noodle dough to be more strongly affected by the changes in mixing parameters than wheat noodle dough.     

Development of a standard test for dough-making properties of oat cultivars

Bread is consumed all over the world. However, so far, production of large volume bread is only possible with wheat. Alternatives, such as oats, are less suitable but this is partly due to the lack of knowledge about their functionality for other purposes than porridge, which is their most common use. Existing standard tests for the dough making characteristics of wheat flour are not suitable for oat flour, hampering research to optimize oats for bread-making purposes. We therefore set out to develop a test to evaluate oat in relation to mixing and dough making properties using wheat as a model. It was possible to reproduce the profile of various qualities of wheat flour using mixtures of oat flour and gluten in different proportions. Our standard test was based on a dough system composed of 87.2% oat flour and 12.8% gluten and it presented similar properties to a wheat flour with regard to resistance to extension. This dough system was sensitive and reliable (coefficient of variation lower than 10%) for detecting differences among oat cultivars, and it can be used to screen oat varieties and individual oat components in relation to relevant properties for bread-making purposes.     

Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat

RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms. The elucidation of the mechanism of RNA silencing has stimulated its use as a reverse genetics tool, because RNA silencing strongly down-regulates the expression of the target gene in a sequence-specific manner. The major protein fraction of wheat grain is gluten which is largely responsible for the functional properties of dough. Gliadins contribute mainly to the extensibility and viscosity of gluten and dough, with the polymeric glutenins being responsible for elasticity. The aim of this work was therefore to silence the expression of specific γ-gliadins by RNA interference, to demonstrate the feasibility of systematically silencing specific groups of gluten proteins. The sequence of a γ-gliadin gene was used to construct the pghp8.1 plasmid. The hpRNA silencing fragment was designed on the basis of 169 base pairs (bp) in sense and antisense orientation with the sequence of the Ubi1 intron as spacer region between the repeats. Two lines of bread wheat were transformed by particle bombardment. Gliadins were extracted from 30 mg of flour, separated by acid-PAGE and determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Seven transgenic lines were obtained and all of them showed reduced levels of γ-gliadins. All seven transgenic plants were fully fertile and their grain morphology and seed weight were comparable to the control lines. MALDI-TOF MS showed that six peaks, present in the untransformed line, were missing in transgenic lines of the BW208 genotype whereas three peaks were missing in the BW2003 genotypes. The proportion of γ-gliadins was reduced, by about 55–80% in the BW208 lines and by about 33–43% in the BW2003 lines. The ELISA assay based on the R5 antibody showed reductions in total gliadins (μg/mg flour) in three of the BW208 lines and in one BW2003 line, but an increase in one BW208 line (C613).     

Nutritional properties and ultra-structure of commercial gluten free flours from different botanical sources compared to wheat flours

Coeliac patients suffer from an immune mediated disease, triggered by the ingestion of a protein composite (gluten) found in wheat, rye and barley. Consequently, there is a need for products such as bread or pasta, made from alternative cereal grains or pseudocereals. A fair proportion of the gluten free products currently on the market are nutritionally inadequate. Hence, it was the aim of this study to investigate the nutrient composition of seven commonly used commercial gluten free flours (oat, rice, sorghum, maize, teff, buckwheat and quinoa) and compare them to wheat and wholemeal wheat flour. In addition to the levels of all major compounds, also mineral composition, fatty acid profile, phytate, polyphenols and folate content were determined. Furthermore, properties of carbohydrates were studied in greater detail, looking at total and damaged starch levels; total, soluble and insoluble dietary fibre content as well as amylose/amylopectin ratio. Proteins were further investigated by means of capillary electrophoreses. Additionally, the ultra-structure of these materials was explored using scanning electron microscopy. The results show that maize and rice flour are poor regarding their nutritional value (low protein, fibre, folate contents). In contrast, teff as well as the pseudocereals quinoa and buckwheat show a favourable fatty acid composition and are high in protein and folate. In particular, quinoa and teff are characterised by high fibre content and are high in calcium, magnesium and iron. Therefore these flours represent nutrient-dense raw materials for the production of gluten free foods.