Intolerances and food allergies: assessment of the stability of allergenic proteins to gastrointestinal digestion

The objectives of the present PhD project are summarized as follow: A1) evaluation of the digestion stability of major nut allergens (peanuts, hazelnuts, walnuts and almonds) as whole food, using an in vitro static model that simulates the gastrointestinal digestion process, including oral, gastric, duodenal and intestinal (brush border membrane enzymes) phases; A2) determine the stability of nut allergens following gastrointestinal digestion of whole food using proteomic techniques (SDS-PAGE, RP-HPLC, LC- HR-MS/MS); A3) assess the allergenic properties of nuts following gastrointestinal digestion of whole food using immunological methodology (ELISA, western-blot, dot-blot, RBL assay); A4) investigate how in vitro gastro-intestinal digestion affects the immune toxic properties of gliadin from diploid (Triticum monococcum) compared to hexaploid (Triticum aestivum) wheat by an immunological and proteomic approach

Differential Protein Expression in Berry Skin from Red Grapes with Varying Hybrid Character

Protein expression from the berry skin of four red grape biotypes with varying hybrid character was compared at a proteome-wide level to identify the metabolic pathways underlying divergent patterns of secondary metabolites. A bottom-up shotgun proteomics approach with label-free quantification and MaxQuant-assisted computational analysis was applied. Red grapes were from (i) purebred Vitis vinifera (Aglianico cv.); (ii) V. vinifera (local Sciascinoso cv.) grafted onto an American rootstock; (iii) interspecific hybrid (V. vinifera × V. labrusca, Isabel), and (iv) uncharacterized grape genotype with hybrid lineage, producing relatively abundant anthocyanidin 3,5-O-diglucosides. Proteomics supported the differences between hybrids and purebred V. vinifera grapes, consistently with distinct phenotypic metabolite assets. Methanol O-anthraniloyltransferase, which catalyses the synthesis of methyl anthranilate, primarily responsible for the "foxy" odour, was exclusive of the Isabel hybrid grape. Most of the proteins with different expression profiles converged into coordinated biosynthetic networks of primary metabolism, while many possible enzymes of secondary metabolism pathways, including 5-glucosyltransferases expected for hybrid grapes, remained unassigned due to incomplete protein annotation for the Vitis genus. Minor differences of protein expression distinguished V. vinifera scion grafted onto American rootstocks from purebred V. vinifera skin grapes, supporting a slight influence of the rootstock on the grape metabolism

Analytical and functional approaches to assess the immunogenicity of gluten proteins

Gluten proteins are the causative agents of celiac disease (CD), a lifelong and worldwide spread food intolerance, characterized by an autoimmune enteropathy. Gluten is a complex mixture of high homologous water-insoluble proteins, characterized by a high content of glutamine and proline amino acids that confers a marked resistance to degradation by gastrointestinal proteases. As a consequence of that, large peptides are released in the gut lumen with the potential to activate inflammatory T cells, in CD predisposed individuals. To date, several strategies aimed to detoxify gluten proteins or to develop immunomodulatory drugs to recover immune tolerance to gluten are under investigation. This review overviews the state of art of both analytical and functional methods currently used to assess the immunogenicity potential of gluten proteins from different cereal sources, including native raw seed flours and complex food products, as well as drug-treated samples. The analytical design to assess the content and profile of gluten immunogenic peptides, described herein, is based on the oral-gastro-intestinal digestion (INFOGEST model) followed by extensive characterization of residual gluten peptides by proteomic and immunochemical analyses. These approaches include liquid chromatography–high-resolution mass spectrometry (LC-MS/MS) and R5/G12 competitive ELISA. Functional studies to assess the immune stimulatory capabilities of digested gluten peptides are based on gut mucosa T cells or peripheral blood cells obtained from CD volunteers after a short oral gluten challenge

Peanut digestome: Identification of digestion resistant IgE binding peptides

Stability to proteolytic degradation in the digestive tract is considered a general feature shared by most food allergens. Current digestibility models exclusively utilize purified allergen proteins, neglecting the relevant effects of matrix that occur for foodstuff systems. In the present study, we investigated digestion stability of the major peanut allergens directly in the natural matrix using an in vitro static model that simulates the gastrointestinal digestion including the oral, gastric, duodenal and intestinal (brush border membrane enzymes) phases. Immunogenicity was evaluated by Western Blot using N=8 pooled sera of peanut allergic pediatric subjects. Immunoreactive, large-sized and fragments of Ara h 2, Ara h 6 and Ara h 3 survived hydrolysis as assessed by SDS-PAGE. Smaller resistant peptides mainly arising from Ara h 3 and also Ara h 1 were detected and further identified by LC-high resolution-MS/MS. RP-HPLC purification followed by dot-blot analysis and MS/MS-based identification demonstrated that stable IgE-binding peptides derived from Ara h 3. These results provide a more realistic picture of the potentially allergenic determinants of peanuts that survived the human digestion, taking into account the role of the food matrix, which may significantly affect gastrointestinal breakdown of peanut allergens

Effect of thermal/pressure processing and simulated human digestion on the immunoreactivity of extractable peanut allergens

Peanut allergy is one of the most widespread types of food allergies especially affecting developed countries. To reduce the risk of triggering allergic reactions, several technological strategies have been devised to modify or remove allergens from foods. Herein we investigated the combination of high temperature and pressure on the modulation of peanuts immunoreactivity after simulated gastro-duodenal digestion. Extractable proteins of raw and autoclaved peanuts were separated on SDS-PAGE and immunogenicity was assessed by ELISA and Western Blot analyses. Proteins surviving the heat treatment and reacting towards allergic patients' sera were analysed and attributed to Ara h 3 and Ara h 1 proteins by untargeted LC-high resolution-MS/MS. A progressive reduction in the intensity of the major allergen proteins was also highlighted in the protein fraction extracted from autoclaved peanuts, with a total disappearance of the high molecular allergens when samples were preliminary exposed to 2 h hydration although the lower molecular weight fraction was not investigated in the present work. Furthermore, raw and processed peanuts underwent simulated digestion experiments and the IgE binding was assessed by using allergic patients' sera. The persistence of an immunoreactive band was displayed around 20 kDa. In conclusion, the synergistic effects of heat and pressure played a pivotal role in the disappearance of the major peanut allergens also contributing to the significant alteration of the final immunoreactivity. In addition, the surviving of allergenic determinants in peanuts after gastrointestinal breakdown provides more insights on the fate of allergenic proteins after autoclaving treatments

Un polymorphisme mendélien sous-jacent aux variations quantitatives de la caséine αs1 caprine

National audienceUsing SDS-polyacrylamide gel electrophoresis and rocket immunoelectrophoresis, 3 new alleles, designated αs1-CnB-,αs1-CnF and αs1-Cno, were identified at the goat αs1-Cn locus, in addition to alleles αs1-CnA, αs1-CnB and αs1-CnC previously reported by BOULANGER al. (1984). Alleles αs1-CnA, αs1-CnB and αs1-CnC are associated with a high content of αs1-casein (approximate mean contribution of each allele being 3.6 g/I) compared to αs1-CnF with a low content (0.6 g/I) and αs1-CnB- with an intermediate content (1.6 g/1) ; αs1-Cno appears to be a true null allele. In a sample of 213 Alpine females from 49 flocks in West Central France, the frequencies of the 6 alleles were : αs1-CnA = 0.14 ; αs1-CnB = 0.05 ; αs1-CnC = 0.01 ; αs1-CnB- = 0.34 ; αs1-CnF = 0.41 ; and αs1-Cno = 0.05. In a sample of 159 Saanen females from 52 flocks of the same region, the frequencies were : αs1-CnA = 0.07 ; αs1-CnB = 0.06 ; αs1-CnC = 0 ; αs1-CnB- = 0.41 ; αs1-CnF = 0.43 ; αs1-Cno = 0.03. Additional data confirm that loci αs1-Cn and αs2-Cn are closely linked. Preliminary investigations indicated a significant superiority in casein content of milks from goats possessing the allele αs1-CnA, as compared to that of milks from goats of genotypes αs1-CnF / αs1-CnF and αs1-CnB- /αs1-CnF and, in a large herd (N = 251), a strong correlation was observed between the αs1-casein content and the rennet-casein content of milk (r = 0.68 ; b = 0.64).A l’aide d’électrophorèses en gel de polyacrylamide SDS et d’immuno-électrophorèses « rocket », 3 allèles, appelés αs1-CnB-, αs1-CnF et αs1-Cno ont été identifiées au locus αs1-Cn de la chèvre, en plus des allèles αs1-CnA, αs1-CnB et αs1-CnC déjà détectés par BOULANGER et al. (1984). Les allèles αs1-CnA, αs1-CnB et αs1-CnC sont associés à un taux élevé de caséine αs1 (contribution approximative de chaque allèle : 3,6 g/I), l’allèle αs1-CnF a un taux faible (0,6 g/I) et l’allèle αs1-CnB a un taux intermédiaire (1,6 g/1). Dans un échantillon de 213 femelles Alpine provenant de 49 troupeaux du centre-ouest de la France, les fréquences des 6 allèles actuellement identifiés étaient les suivantes : αs1-CnA = 0,14 ; αs1-CnB = 0,05 ; αs1-CnC = 0,01; αs1-CnB- = 0,34 ; αs1-CnF = 0,41 et αs1-Cno = 0,05. Dans un échantillon de 159 femelles Saanen provenant de 52 troupeaux de la même région, les fréquences étaient : αs1-CnA = 0,07 ; αs1-CnB = 0,06 ; αs1-CnC = 0; αs1-CnB- = 0,41 ; αs1-CnF = 0,43 ; αs1-Cno = 0,03. Des données supplémentaires confirment que les loci αs1-Cn et αs2-Cn sont étroitement liés. Des investigations préliminaires révèlent que le taux de caséine des laits des chèvres possédant l’allèle αs1-CnA est significativement supérieur à celui des laits des chèvres de génotype αs1-CnF / αs1-CnF ou αs1-CnB- /αs1-CnF; parailleurs, dans un grand troupeau (N = 251), une forte corrélation a été observée entre le taux de caséine αs1 et le taux de matières azotées coagulables (r = 0,68 ; b = 0,64)

Comprehensive analysis of the peanut allergome combining 2-DE gel-based and gel-free proteomics

In this work, we explored the “deep” seed peanut proteome by using both two dimensional electrophoresis (2- DE)-based analysis run under reducing and non-reducing condition (protein-centric) and LC-MS/MS gel-free proteomic (peptide-centric). The former approach allowed to identify high molecular weight disulfide-linked Ara h 1 and Ara h 3 heteroligomers and Ara h 1 homoligomers linked through covalent bonds other than disulfides. The occurrence of these protein complexes revealed natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut. The second approach, also referred to as shot-gun proteomics, allowed the identification of 149 gene products, including low-abundance proteins escaped the 2-DE detection. Interestingly, we identified 60 proteins never catalogued previously. The complementary exploitation of two proteomic approaches enabled the access to new relevant information about the complexity of the peanut proteome, with special emphasis to the complement of allergens (allergome)

Tritordeum: Promising Cultivars to Improve Health

Tritordeum is an amphiploides species resulting from the hybridization between durum wheat (T. durum) and wild barley (H. chilense). This new cereal is considered a natural crop as it is obtained by traditional breeding techniques. Given its appreciable organoleptic characteristics, agronomic features, presence of interesting components, and good technological properties, Tritordeum is of promising interest for the development of health-oriented foods. In this study, we evaluated two registered Tritordeum cultivars, Bulel and Aucan. T. durum (Provenzal) was employed as the positive control. The extracted proteins were digested by gastric/pancreatic proteases, and their biological effects on Caco-2 differentiated on transwell inserts were determined. Changes in cell viability, monolayer permeability, organization of F-actin microfilaments, and ER stress triggered by protein-digested samples (DPs) were inspected. Our results showed that exposure to Provenzal-DPs promptly disrupted the tight junction barrier. Conversely, Aucan-DPs did not enhance monolayer permeability, whereas Bulel-DPs exerted only slight effects. Provental-DPs-induced toxicity was also confirmed by changes in cell viability and by the deep reorganization of the enterocyte cytoskeleton. In contrast, Aucan-DPs and Bulel-DPs did not affect monolayer viability and cytoskeleton structure. Overall, our findings suggest that both Tritordeum cultivars could be potential candidates for mitigating the toxicity of wheat flour

Protective effects of ID331 Triticum monococcum gliadin on in vitro models of the intestinal epithelium

A growing interest in developing new strategies for preventing coeliac disease has motivated efforts to identify cereals with null or reduced toxicity. In the current study, we investigate the biological effects of ID331 Triticum monococcum gliadin-derived peptides in human Caco-2 intestinal epithelial cells. Triticum aestivum gliadin derived peptides were employed as a positive control. The effects on epithelial permeability, zonulin release, viability, and cytoskeleton reorganization were investigated. Our findings confirmed that ID331 gliadin did not enhance permeability and did not induce zonulin release, cytotoxicity or cytoskeleton reorganization of Caco-2 cell monolayers. We also demonstrated that ID331 omega-gliadin and its derived peptide omega(105-123) exerted a protective action, mitigating the injury of Triticum aestivum gliadin on cell viability and cytoskeleton reorganization. These results may represent a new opportunity for the future development of innovative strategies to reduce gluten toxicity in the diet of patients with gluten intolerance. (C) 2016 Elsevier Ltd. All rights reserved

Proteomic characterization of pistachio nut allergen proteins

Pistachio nut (Pistacia vera) is an important tree nut cultivated widely worldwide and is commonly appreciated based on its organoleptic characteristics and nutritional healthfulness. Similar to other tree nuts, the consumption of pistachio nuts is associated with moderate or severe allergic reactions in sensitized/allergic individuals. In the present study, we investigated the proteome composition of pistachio seeds using gel-based (mono- and two-dimensional electrophoresis) and gel-free (shotgun and label free quantitative) proteomics analyses. The gel-based approach allowed the identification of proteins belonging to the cupin protein family (11S and 7S globulins) and suggested the occurrence of multiple isoforms. The shotgun proteomic analysis extended the identification to allergens 2S albumin and heat shock protein, whereas label free quantitative was employed to investigate the proteomes of four different pistachio varieties. Protein pattern characterization through sophisticated techniques, such as those developed by proteomics science, allowed the identification of distinct protein markers on different pistachio cultivars.Fil: Di Stasio, Luigia. Consiglio Nazionale delle Ricerche; ItaliaFil: Sciammaro, Leonardo Pablo. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: De Caro, Salvatore. Consiglio Nazionale delle Ricerche; ItaliaFil: Salinas, Maria Victoria. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Puppo, Maria Cecilia. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Mamone, Gianfranco. Consiglio Nazionale delle Ricerche; Itali