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.

E40 glutenase detoxification capabilities of residual gluten immunogenic peptides in in vitro gastrointestinal digesta of food matrices made of soft and durum wheat.

Gluten degrading enzymes, which are commonly referred to as "glutenases," represent attractive candidates for the development of a pharmacological treatment of gluten related disorders, such as coeliac disease (CeD). Endoprotease-40 (E40), a novel glutenase secreted by the actinomycete Actinoallomurus A8 and recombinantly produced in S. lividans TK24, was shown to be active at pH 3 to 6 (optimum pH 5), resistant to pepsin and trypsin degradation, able to destroy immunotoxicity of both gliadin 33-mer peptide and whole proteins and to strongly reduce the response of specific T cells when added to gliadin in in vitro gastrointestinal digestion. This study aims to functionally assess the capabilities of Endoprotease-40 (E40) to detoxify residual gluten immunogenic peptides in gastrointestinal digesta of food matrices made of soft and durum wheat. The INFOGEST harmonized protocols were applied to the multicompartmental model of simulated human gastrointestinal digestion, for the quantitative assessment of residual gluten in liquid (beer) and solid (bread and pasta) foods, made of either soft or durum wheat. Proteomic and immunological techniques, and functional assays on intestinal T cell lines from celiac disease patients were used to identify gluten-derived immunogenic peptide sequences surviving in gastric and gastrointestinal digesta after the addition of E40 at increasing enzyme: wheat proteins ratios. During the gastric phase (2 h incubation time), the addition of E40 demonstrated an extensive (≥ 95%) dose-dependent detoxification of whole gluten in real food matrices. Overall, the residual gluten content was found at, or even below, the 20 ppm gluten-free threshold for soft and durum wheat-based food. Furthermore, unlike in untreated gastrointestinal digesta, none of the immunodominant α-gliadin peptides survived in E40-treated digesta. Traces of ω- and γ-gliadin derived immunogenic peptides were still detected in E40-treated digesta, but unable to stimulate celiac-intestinal T cells. In conclusion, E40 is a promising candidate for the oral enzymatic therapy of CeD, as a stand-alone enzyme being efficient along the complete gastrointestinal digestion of gluten.

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.

Beneficial effects of a T. monococcum wheat cultivar on diabetes incidence evaluated in non-obese diabetic mice and after in vitro simulated gastroduodenal digestion.

Wheat consumption can represent one of the nutritional factors involved in the onset of diabetes. We specifically investigated the potential diabetogenic effects of Hammurabi, a T. monococcum wheat cultivar, in non-obese diabetic (NOD) mice and analysed the levels of resistant starch in pasta manufactured with Hammurabi after in vitro gastroduodenal digestion. NOD mice were fed with Hammurabi, bread wheat or rice flour to evaluate diabetes incidence and insulitis score. An enzymatic method was applied to compare the content of resistant starch in Hammurabi pasta and durum wheat pasta (control). In NOD mice, the Hammurabi-based diet significantly delayed diabetes onset (p = 0.0042) and reduced insulitis score compared to rice or wheat-based diet. Furthermore, the resistant starch value following in vitro digestion of Hammurabi pasta was significantly higher (4.08%) than that of durum wheat pasta (2.28%). Taken together, these results highlighted the potential positive effects of the Hammurabi-based diet on diabetes incidence.

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.

Triticum monococcum amylase trypsin inhibitors possess a reduced potential to elicit innate immune response in celiac patients compared to Triticum aestivum.

SCOPE: Several studies reported a role of amylase/trypsin-inhibitors (ATIs) of common wheat species in promoting immune reactions. Here, we investigated in celiac disease (CD), the immunogenic properties of ATIs from diploid compared to common hexaploid wheats after an in vitro proteolytic hydrolysis. METHODS AND RESULTS: ATIs purified from two lines of diploid Triticum monococcum (TM), Monlis and Norberto-ID331, and from Triticum aestivum (TA), Sagittario, were digested with pepsin-chymotrypsin (PC) enzymes and analyzed using a proteomic approach, and subsequently their immune stimulatory properties were investigated on jejunal biopsies and T-cell lines from CD patients. No significant expression of IL-8 and TNF-α were detected on biopsies cultured with ATIs from TM in comparison with ATIs from TA. No significant IFN-γ production was observed in intestinal gliadin- raised T-cells in response to ATIs from both TM and TA wheats. Proteomic results revealed that both TM ATIs showed reduced stability to proteolytic enzymes compared to TA ones. CONCLUSION: TM ATIs are substantially different from those of TA, showing a reduced ability to trigger the innate immunity in CD and a higher susceptibility to enzymatic hydrolysis.

Bacteria do it better! Proteomics suggests the molecular basis for improved digestibility of sourdough products.

The aim of this study was to evaluate the dynamics of proteolysis during dough fermentation started with different lactic acid bacteria species, through the identification of intermediate and small-sized peptides generated during fermentation. Single-strain cultures of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Companilactobacillus alimentarius, and Leuconostoc pseudomesenteroides were assayed as sourdough starters. Assays were carried out at lab-scale for 48 h of fermentation, using both unstarted and yeast-leavened dough as controls. Physicochemical and microbiological analyses were combined with peptidomic and proteomic profiling, identifying several hundreds of peptides mainly released from the water-soluble wheat proteins, including ß-amylase, triticin, and serpins. Both α- and γ-gliadins were hydrolyzed, though only at the N-terminal domain, while the central protein region - encrypting celiac disease epitopes- remained unaffected. The bacterial-mediated consumption of sugars and the concomitant hydrolysis of starch degrading ß-amylase could underlie improved digestibility and several nutritionally beneficial effects of sourdough baked products.

In vitro gastroduodenal and jejunal brush border membrane digestion of raw and roasted tree nuts.

Heat treatments induce chemical/physical modifications, which may affect the stability to enzymatic digestion and consequently the allergenicity of food proteins to a varying extent, depending on the time/temperature regimen. Herein, we evaluated the stability to digestion of whole tree nut (walnuts, hazelnuts and almonds) allergens in a food digestion model reflecting the real one by, taking into consideration the allergen-containing processed (roasted) food. To this aim, whole raw and roasted tree nuts were subjected to in vitro digestion combining the harmonized oral-gastric-duodenal digestion models with brush border membrane enzymes (BBM) to simulate the jejunal degradation of peptides. The degradation of allergens was monitored by integrated proteomic/peptidomic and bio-informatic tools. Roasting increased digestibility of tree nuts, since very few peptides were detected in digested samples (<6.5 kDa fraction). After BBM digestion step, the degradation of peptides was enhanced in roasted walnuts and hazelnuts compared to the raw counterpart. Conversely, almond allergens showed a different behaviour, since the presence of resistant peptides was more evident for roasted almonds, probably because of the hydrolysis of high molecular weight aggregates generated during roasting. Our results provide new insight into the relationship between thermal processing and metabolic fate of tree nut allergens, highlighting the importance of investigating the digestion stability of whole allergenic food, rather than purified proteins.

Immunogenic Potential of Beer Types Brewed With Hordeum and Triticum spp. Malt Disclosed by Proteomics.

The protein/peptide composition of five beer kinds, including two experimental beer-like products brewed with einkorn (Triticum monococcum), a beer labeled as "gluten-free," a traditional all-barley malt and a wheat (T. aestivum) containing beer, was characterized with HPLC-ESI MS/MS-based proteomics. To enlarge the characterization of the components, the polypeptides were fractionated according to their molecular size (cut-off 6 kDa). All the beer types contained a variety of polypeptides arising from all the gliadin subfamilies (α-/ß-, γ-, and ω-gliadins) able to induce an immune response in celiac disease (CD) patients in addition to a panel of IgE-reactive food allergens. Wheat storage proteins were heavily hydrolyzed in the beer samples brewed with einkorn. The presence of gluten-like fragments, also including the 25-mer and 33-mer-like of α-gliadin, was confirmed in beer brewed with barley and wheat malt as well as in the gluten-free beer. Both CD-toxic and allergenic peptides of all beer samples were drastically degraded when subjected to a simulated gastroduodenal (GD) digestion. After in vitro digestion, the level of gluten-like peptides assayed with the G12 competitive ELISA, was below the threshold (20 ppm) for a food to be considered as "gluten-free." A few gliadin-derived epitopes occurred in the digests of beers crafted with wheat or Norberto-ID331 line of einkorn. In contrast, digests of all barley malt and gluten-free beers did not contain detectable gluten-like epitopes, but only minor fragments of hordeins and IgE-reactive food allergens. All beer samples evoked a weak immune response on gliadin-reactive celiac T cells isolated from intestinal biopsies of celiac patients. Compared to undigested polypeptides, the response was markedly reduced by GD digestion. Although the consumption of a moderate amount of beer brewed with barley or einkorn could deliver a relatively low amount of CD-toxic epitopes, the findings of this study emphasize the urgent need of a reliable and accurate quantification of gluten epitopes in all types of beer, also including the gluten-free one, to compute realistically the contribution of beer to the overall gluten intake, which can be responsible of intestinal tissue damages in celiacs.

Comparative Analysis of in vitro Digestibility and Immunogenicity of Gliadin Proteins From Durum and Einkorn Wheat.

Recent studies suggested that gliadin proteins from the ancient diploid einkorn wheat Triticum monococcum retained a reduced number of immunogenic peptides for celiac disease patients because of a high in vitro digestibility with respect to hexaploid common wheat. In this study, we compared the immunological properties of gliadins from two Triticum monococcum cultivars (Hammurabi and Norberto-ID331) with those of a Triticum durum cultivar (Adamello). Gliadins were digested by mimicking the in vitro gastrointestinal digestion process that includes the brush border membrane peptidases. Competitive ELISA, based on R5 monoclonal antibody, showed that gastrointestinal digestion reduced the immunogenicity of Triticum monococcum gliadins; conversely, the immunogenic potential of Triticum durum gliadins remained almost unchanged by the in vitro digestion. The immune stimulatory activity was also evaluated by detecting the IFN-γ production in gliadin-reactive T-cell lines obtained from the small intestinal mucosa of HLA-DQ2+ celiac disease patients. Interestingly, gastrointestinal digestion markedly reduced the capability of Triticum monococcum gliadins (p <0.05) of both cultivars to activate T cells, while it slightly affected the activity of Triticum durum. In conclusion, our results showed that Triticum durum was almost unaffected by the in vitro gastrointestinal digestion, while Triticum monococcum had a marked sensibility to digestion, thus determining a lower toxicity for celiac disease patients.

Comparative analysis of eliciting capacity of raw and roasted peanuts: the role of gastrointestinal digestion.

This study investigated the simultaneous impact of food matrix and processing on the food allergy eliciting capacity of peanuts in a physiologically relevant context. Whole raw and roasted peanuts were subjected to in vitro digestion combining the harmonized oral-gastric-duodenal digestion models with brush border membrane enzymes (BBM) to simulate the jejunal degradation of peptides. SDS-PAGE and HPLC analysis showed that roasting increased digestibility of peanuts and this trend was even more evident after BBM degradation. The eliciting properties of raw and roasted peanuts were assessed by Rat Basophil Leukemia assay in the presence of sera from peanut-allergic patients. As general features, the BBM digestion reduced allergenicity of roasted peanuts compared to the raw counterpart, suggesting that intestinal peptidases effectively contribute to further destroy specific domains of peanut allergens. These findings provide new and more realistic insights in the stability of peanut allergens within their natural matrix.

A Comprehensive Peptidomic Approach to Characterize the Protein Profile of Selected Durum Wheat Genotypes: Implication for Coeliac Disease and Wheat Allergy.

The wheat varietal selection undertaken by breeders in recent decades has been tailored mainly to improve technological and productivity-related traits; however, the latter has resulted in a considerable impoverishment of the genetic diversity of wheat-based products available on the market. This pitfall has encouraged researchers to revalue the natural diversity of cultivated and non-cultivated wheat genotypes in light of their different toxic/immunogenic potential for celiac disease and wheat-allergic patients. In the present investigation, an advanced proteomic approach was designed for the global characterization of the protein profile of selected tetraploid wheat genotypes (Triticum turgidum). The approach combined proteins/peptides sequence information retrieved by specific enzymatic digestions (single and dual proteolytic enzymes) with protein digestibility information disclosed by means of in-vitro simulated human gastroduodenal digestion experiments. In both cases, the peptide pools were characterized by discovery analysis with liquid chromatography high-resolution tandem mass spectrometry, and specific amino acid sequences were identified via commercial software. The peptide list was screened for in silico toxicity/immunogenicity risk assessment, with the aid of various open-source bioinformatics tools for epitopes matching. Given the global information provided by the designed proteomic approach, the in silico risk assessment not only tackled toxicity implication for celiac disease patients, but also scouted for immunogenic sequences relevant for wheat allergic patients, achieving a comprehensive characterization of the protein profile of the selected genotypes. These latter were assessed to encrypt a variable number of toxic/immunogenic epitopes for celiac disease and wheat allergy, and as such they could represent convenient bases for breeding practices and for the development of new detoxification strategies.

Hidden "Digestome": Current Analytical Approaches Provide Incomplete Peptide Inventories of Food Digests.

Analyzing an in vitro gastroduodenal digest of whey proteins by high-performance liquid chromatography (HPLC) coupled to high-resolution/high-sensitivity tandem mass spectrometry (MS/MS), we sought to evaluate if state-of-art peptidomics provide comprehensive peptide coverage of food "digestomes". A multitude of small-sized peptides derived from both α-lactalbumin and ß-lactoglobulin as well as disulfide cross-linked hetero-oligomers remained unassigned, even when the digests were compared before and after S-S reduction. The precipitation with 12% trichloroacetic acid demonstrated the occurrence of large-sized polypeptides that escaped the bioinformatic identification. The analysis of a HPLC-MS/MS run with different proteomic search engines generated dissimilar peptide subsets, thus emphasizing the demand of refined searching algorithms. Although the MS/MS fragmentation of monocharged ions with exclusion of non-peptide-interfering compounds enlarged the inventory of short peptides, the overall picture of the "digestome" was still incomplete. These findings raise relevant implications for the identification of possible food-derived bioactive peptides or allergenic determinants.

Comparative analysis of protein composition and digestibility of Ceratonia siliqua L. and Prosopis spp. seed germ flour.

By virtue of exclusive nutrient composition and nutritional properties, seed germ flours from both European carob (Ceratonia siliqua L.) and South American algarrobo (Prosopis alba and Prosopis nigra) or vinal (Prosopis ruscifolia) have potential as a high nutritional value and health-promoting ingredient for food formulations. In order to define their compositional and functional properties, we investigated the germ protein content of carob compared to the P. alba, P. nigra and P. ruscifolia counterparts, applying proteomics and complementary methods. The mono- and two-dimensional electrophoretic profiles of Prosopis spp. were very similar among one another, while C. siliqua exhibited significant differences. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy highlighted a dominant ß-sheet structural conformation for C. siliqua, suggesting that carob germ flour might more suited than Prosopis germ flour for baking and food technological applications. In contrast, Prosopis spp. contained a more adequate nutritional value than C. siliqua, in terms of essential amino acid complement. Both carob and algarrobo germ flour samples were highly digestible, as demonstrated by in vitro simulated gastrointestinal digestion, releasing high amounts of free amino acids and only minor proportions of low molecular weight peptides.

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).

Phytosterols in supplements containing Serenoa repens: an example of variability of active principles in commercial plant based products.

Phytosterols are one of the bioactive components responsible for the beneficial effects of Serenoa repens in Benign Prostate Hyperplasia. The aim of this study was to verify the actual variability of the phytosterols content in supplements containing serenoa, in order to provide useful elements to check the effectiveness of these preparations. The amount of campesterol, stigmasterol and ß-sitosterol were determined by gas-chromatography in commercial raw materials and supplements containing serenoa in association or not with other botanicals. The experimental data were used to calculate amounts of phytosterols for recommended daily dose. The overall results of this study show an extreme variability in the content and also in the amounts per daily dose of phytosterols of the examined supplements (both mono/multi components). These data confirm that the characterization of serenoa based supplements is insufficient to ensure comparable effects between different products.

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.

Identification of enzyme origin in dough improvers: DNA-based and proteomic approaches.

Enzymatic dough improvers (DIs) are increasingly used as baking co-adjuvants. Herein, an array of techniques, including Western blotting, PCR, electrophoresis-based and shotgun proteomics, was addressed to identify the enzymes in six commercial DI preparations. In particular, this work sought to exclude the possible undeclared use of amylolytic enzymes from porcine (or other animal origin) pancreas in DIs. PCR-amplified mitochondrial cytochrome b (mt cyt b) gene region and porcine pancreatic α-amylase were the targets of DNA-based and protein methods, respectively, both assuring a limit of detection lower than 0.5-0.1% (w/w). Aspergillum oryzae α-amylase and Hordeum vulgare (barley) ß-amylase were the most represented enzymes in all DI samples. Although one sample was PCR-positive, none among the DIs contained porcine pancreatic enzymes. Comparative gas chromatographic analysis of fatty acids suggested that the porcine contamination might arise from hard fats of porcine origin (lard), emphasizing the need of performing analyses at the protein level when the targets are enzymes or proteins.

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.

Differential representation of albumins and globulins during grain development in durum wheat and its possible functional consequences.

Durum wheat (Triticum turgidum ssp. durum (Desf.) Husn.) is an economically important crop used for the production of semolina, which is the basis of pasta and other food products. Its grains provide proteins and starch for human consumption. Grain development is a key process in wheat physiology; it is highly affected by a number of enzymes that control the metabolic processes governing accumulation of starch and storage proteins and ultimately grain weight. Most of these enzymes are present in the albumin/globulin grain fraction, which represents about a quarter of total seed proteins. With the aim to describe the dynamic profile of the albumin/globulin fraction during durum wheat grain development, we performed a proteomic analysis of this subproteome using a two-dimensional differential gel electrophoresis (2D-DIGE)-based approach and compared six developmental stages. A total of 285 differentially (237 over- and 48 under-) represented spots was identified by nanoLC-ESI-LIT-MS/MS, which were associated with 217 non-redundant Triticum sequence entries. Quantitative protein dynamics demonstrated that carbon metabolism, energy, protein destination/storage, disease/defense and cell growth/division functional categories were highly affected during grain development, concomitantly with progressive grain size increase and starch/protein reserve accumulation. Bioinformatic interaction prediction revealed a complex network of differentially represented proteins mainly centered at enzymes involved in carbon and protein metabolism. A description of 18 proteins associated with wheat flour human allergies was also obtained; these components showed augmented levels at the last developmental stages. By providing a comprehensive understanding of the molecular basis of durum wheat grain development, yield and quality formation, this study provides the foundation and reveals potential biomarkers for further investigations of durum wheat breeding and semolina quality. BIOLOGICAL SIGNIFICANCE: A 2D-DIGE-based comparative analysis of the albumin/globulin fraction from durum wheat caryopses at six developmental stages was performed to describe the dynamic subproteomic changes associated with grain development. Quantitative variations of 217 differentially proteins demonstrated that highly affected are the functional categories of carbon metabolism, energy, protein destination/storage, disease/defense and cell growth/division, which displayed a general over-representation, consistently with concomitant occurrence of grain size increase and starch/protein reserve accumulation. Bioinformatics revealed a complex protein network centered mainly at enzymes involved in carbon and protein metabolism. Differentially represented proteins and corresponding functional categories highly resembled those previously identified as variable in developing bread wheat grain. This suggests that the main differences in kernel hardness between durum and bread wheat probably do not depend on proteomic changes in corresponding albumins/globulins, but on other specific factors affecting the interaction between the starch granules and the endosperm protein matrix in the kernel.