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The reasons behind the increasing prevalence of celiac disease (CD) worldwide are still not fully understood. This study adopted a multilevel approach (in vitro, ex vivo, in vivo) to assess the potential of gluten from different wheat varieties in triggering CD. Peptides triggering CD were identified and quantified in mixtures generated from simulated gastrointestinal digestion of wheat varieties ( n = 82). Multivariate statistics enabled the discrimination of varieties generating low impact on CD (e.g., Saragolla) and high impact (e.g., Cappelli). Enrolled subjects ( n = 46) were: 19 healthy subjects included in the control group; 27 celiac patients enrolled for the in vivo phase. Celiacs were divided into a gluten-free diet group (CD-GFD), and a GFD with Saragolla-based pasta group (CD-Sar). The diet was followed for 3 months. Data were compared between CD-Sar and CD-GFD before and after the experimental diet, demonstrating a limited ability of Saragolla to trigger immunity, although not comparable to a GFD. Ex vivo studies showed that Saragolla and Cappelli activated immune responses, although with great variability among patients. The diverse potential of durum wheat varieties in triggering CD immune response was demonstrated. Saragolla is not indicated for celiacs, yet it has a limited potential to trigger adverse immune response.

Germination is already a well-accepted process by consumers with many products made from sprouted seeds or containing limited amounts of flour form sprouted grains. The present work aimed assessing the usefulness of germination in reducing gluten peptides associated with celiac disease, at the same time evaluating some technological features of the obtained germinated wheat. In the first part of the work, celiac disease (CD)-triggering peptides were tracked as a function of germination kinetics (from day 1 to day 6). Using simulated gastrointestinal digestion and liquid chromatography coupled to mass spectrometry, ten celiac disease triggering peptides were identified: seven peptides presumably involved in the adaptive immune response (TI) and three peptides mainly involved in the innate immune response (TT). All the identified peptides belonged to gliadins. TI track pattern showed three phases: the first two days displayed a significant degradation, a stability phase was observed from day 3 to day 5, and finally a drastic reduction occurred on the 6th day. For TT peptides, important degradation was exclusively observed at the 6th day. In the second part, some techno-functional features of germinated whole wheat flour were assessed to estimate its potential as an alternative to conventional flour. Functionality comparison of the non-germinated versus germinated flours revealed that germination significantly influenced solvents retention capacities as well as swelling and solubility. Thus, with a reduced amount of celiac disease triggering peptides, but also with different technological behavior compared to traditional wheat flour., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPβ transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In this work, the aim is to study the effectiveness of germination on wheat protein degradation, with a specific focus on proteins involved in adverse reactions to wheat. The effects of 8 days of germination at 25 °C on the chemical composition and the protein profile were determined. Germination did not have a significant effect on starch, protein, lipid, and ash contents. General protein profile, as indicated by SDS-PAGE analysis, revealed that germination induced a relevant degradation in protein fraction. After in vitro gastrointestinal digestion, gluten peptides involved in celiac disease (CD) were identified and quantified using UPLC/ESI-MS technique. Also, CM3 protein, involved in baker's asthma and intestinal inflammation, was quantified by measuring a marker peptide. Statistical analysis underlined that germination and genotype had significant impact on the amount of both components. Regarding gluten peptides related to CD, germination enabled an average reduction of 47% in peptides eliciting adaptive immune response and 46% in peptides eliciting innate immune response. CM3 protein showed also a high average reduction (56%). Thus, this study suggests that germination might be a good bioalternative to provide a low "impact" raw ingredient for special wheat-based foodstuffs.

The aim of this study was to compare immunogenic and toxic gluten peptides related to celiac disease (CD). 100 accessions of genotypes selected during the 20th century in Tunisia were in vitro digested and then analyzed by UPLC/ESI-MS technique using an isotopically labeled internal standard. The first MANOVA confirmed a high variability in the content of immunogenic and toxic peptides reflecting high genetic diversity in the germplasm released during the past century in Tunisia, consistently with PCA and clustering analysis results. Our finding showed also important variability in CD epitopes due to growing season's climate scenarios. Moreover, the second MANOVA revealed significant differences between abandoned and modern cultivars' CD-related peptide amounts. Although we could not conclude that there was an augment of allergens in newly selected durum wheat lines compared to abandoned ones, we demonstrated that modern genotype peptides were less sensitive to climate variation, which is a useful indicator for wheat breeders.

Coeliac disease is an autoimmune enteropathy that develops in genetically predisposed subjects after the ingestion of gluten or related proteins. Coeliac disease has an increasing incidence in the last years in western countries and it has been suggested that wheat breeding might have contributed to select more toxic forms of gluten. In this work, we analysed gluten peptides generated by in vitro digestion of different old and modern Triticum varieties, using LC-MS. We concluded that old varieties analysed produced a higher quantity of peptides containing immunogenic and toxic sequences than modern ones. Thus old wheat lines are not to be considered "safer" for subjects that are genetically predisposed to celiac disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

During wheat digestion, gluten-derived proteolytic resistant peptides are generated, some of them involved in celiac disease. In vitro digestion models able to mimic the peptides generated in the human gastrointestinal tract are extremely useful to assess the pathogenicity of wheat-derived products. In this paper, samples belonging to three different durum wheat varieties were taken at six different steps of the pasta production chain and two different digestion models present in the literature were assessed on the different samples: a more complex one using artificial fluids simulating the exact composition of digestive juices, and a simplified method based on a peptic-tryptic/chymotryptic treatment of wheat ethanolic extract. An extensive characterization of the peptides generated using two in vitro digestion models was performed through LC-MS/MS techniques and the two methods were compared in order to evaluate qualitative and quantitative differences and their possible implications for varietal screening. Strong differences in the type of peptides produced with the two methods were detected, indicating that the simplified method can still be used for a varietal screening but is not representative of the peptides really generated after physiological human digestion. Results indicate a clear necessity of physiologically accurate models for simulating human gastrointestinal digestion of wheat products.

Old wheat genotypes are perceived by consumers as healthier than modern ones. The release of gluten peptides with in vitro digestion and the content of potentially prebiotic carbohydrates (i.e. resistant fraction of starch and cell-wall associated dietary fiber) were evaluated in tetraploid wheats, namely 9 old and 3 modern Triticum turgidum ssp. genotypes. Simulated digestion of wholemeal flours yielded 152 major peptides, 59 of which were attributed a sequence. Principal component analysis revealed that peptide profiles were variable in old genotypes, unlike in modern ones. Digestion of old genotypes generally yielded peptides in greater concentration. In particular, 5 peptides of γ-gliadin, known to trigger the adaptive immune reaction, and two peptides of α-gliadin, known to be toxic to celiac patients, were particularly abundant in some old varieties. Resistant starch (RS) was negligible in modern genotypes (<0.6%), but it was remarkably abundant in some old varieties, reaching the highest value in Dauno III (8.5%, P < 0.05). Dauno III also presented the highest amount of soluble fiber (4.2%, P < 0.05). Pasta was made with an old and a modern genotype (Dauno III and PR22D89, respectively) with opposite RS content. Pasta making and cooking affected starch digestibility, overtaking differences between genotypes and yielding the same amount of RS for both the varieties (approx. 1.7%). The data herein presented suggest that the wholemeal flours of old tetraploid wheat genotypes could not boast particular claims associated to a lower exposure to gluten peptides and, if cooked, to a prebiotic potential. Unlabelled Image • Gluten peptides, fiber, and starch were analyzed in old and modern wheat genotypes. • Peptides associated to celiac disease were released abundantly in some old genotypes. • Resistant starch is abundant in the wholemeal of some old wheat genotypes. • Pasta cooking overtook differences in resistant starch content between genotypes. • Old wheats cannot claim prebiotic potential or low gluten compared with the new ones. [ABSTRACT FROM AUTHOR]

Abstract: Gluten proteins are the basis of the rheological properties of wheat derived products, such as bread and pasta. Their particular amino acidic composition (high proline and glutamine content) is responsible for the poor gluten digestibility. Some of the high molecular weight peptides that are generated in the gastrointestinal tract are involved in an autoimmune entheropathy called celiac disease. In this work we compared the amount of peptides containing sequences involved in adaptive and immune responses, which were produced after simulated gastrointestinal digestion of prolamins extracted from different durum wheat varieties and in-bred lines. Peptides containing sequences involved in celiac disease were quantified using an isotopically labeled peptide as internal standard. The results demonstrated a very high variability in the amount of pathogenic peptides produced by different lines, showing a strong contribution of the genetic component. At the same time, the variability in total protein and gluten content was lower; the weak correlation between pathogenic peptides and the amount of gluten proteins gives rise to the possibility of a varietal selection aimed to maintain good rheological properties, but simultaneously reducing the exposure to peptides eliciting an immunological response in celiac predisposed subjects. These varieties might be useful for celiac disease prevention. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

• Major effect of environment on accumulation of gliadins and CD-triggering peptides. • Genetic factor contributes to toxic peptides accumulation. • GDD and rainfall affected gliadins and peptides involved in celiac disease. • Low evapotranspiration decreases level of gliadins and peptides involved in CD. • Selection for TP, IP, α-GliA2-6, γ-Gli-5 could be uncoupled from GPC and GY. The impact of environment, genetic selection and their interactions on grain yield of durum wheat genotypes has been extensively studied; however, limited information is available for their influence on gluten quality associated with effects on the amount and composition of glutenins, gliadins and celiac disease (CD)-triggering peptides. In this study, a set of six commonly cultivated durum wheat genotypes were assessed in a multi-environment trial of eight site-year combinations in different Italian regions during two consecutive harvest years (2016 and 2017). While high-molecular-weight glutenin subunits (HMW-GS) were more stable between years, differences in total gluten proteins were mainly due to low-molecular-weight glutenin subunits (LMW-GS) and gliadins accumulation. After mass separation and quantification, two gliadin proteins – γ-Gli-5 and α-GliA2-6 (41.1 and 33.8 kDa, respectively) – were further studied together with toxic (TP) and immunogenic (IP) celiac disease-triggering peptides obtained via simulated gastrointestinal digestion. While TP accumulation was strongly influenced by the genotypes, IP showed marked variation in the different sites with significant genotype-by-year and genotype-by-site interaction. Specific agrometeorological variables (i.e. growing degree days and aridity index) in different growing phases showed a strong negative correlation with α-GliA2-6 and CD-associated peptides. Statistical analysis revealed that the level of gliadins and TP/IP peptides were uncorrelated with grain protein content and yield. The selection of plant materials with good technological properties but with a low content of CD-triggering peptides should combine with ad hoc environment (e.g. site) selection and management practices reducing crop evapotranspiration in the vegetative phase. [ABSTRACT FROM AUTHOR]