Chiara Nitride

Shotgun proteome analysis of beer and the immunogenic potential of beer polypeptides

The majority of beer proteins originate from barley (Hordeum vulgare) which is used for brewing. Barley is known to contain celiacogenic gliadin-like prolamins (hordeins) along with other immunogenic proteins which endure malt proteases and the harsh conditions of brewing. In addition, a multitude of peptides that may retain or even amplify the immune-stimulating potential is released in beer because of proteolysis. The comprehensive annotation of the beer proteome is challenged both by the high concentration range of the protein entities and by a severe degree of processing-induced modifications. Overcoming the pitfalls of the classical two-dimensional electrophoresis approach coupled to mass spectrometry (MS), the gel-free shotgun proteomic analysis expanded the current inventory of a popular Italian beer to 33 gene products, including traces of intact B- and D-hordeins and 10 proteins from Saccharomyces spp. The high performance liquid chromatography-electrospray MS/MS peptidomic analysis of the low-molecular weight beer components disclosed a panel of hordein-derived peptides that encrypt gluten-like sequence motifs, potentially harmful to celiacs. The presence of antigliadin IgA-immunoresponsive prolamins was assayed by Western and dot blot using sera of N=4 celiac patients. Gliadin-reactive T-cell lines isolated from the intestine of N=5 celiacs activated an IFN-γ response when challenged with deamidated beer polypeptides.

Tracking the fate of pasta (T. Durum semolina) immunogenic proteins by in vitro simulated digestion.

The aim of the present study was to identify and characterize the celiacogenic/immunogenic proteins and peptides released during digestion of pasta (Triticum durum semolina). Cooked pasta was digested using a harmonized in vitro static model of oral-gastro-duodenal digestion. The course of pasta protein digestion was monitored by SDS-PAGE, and gluten proteins were specifically analyzed by Western blot using sera of celiac patients. Among the allergens, nonspecific lipid-transfer protein was highly resistant to gastro-duodenal hydrolysis, while other digestion-stable allergens such as α-amylase/trypsin inhibitors were not detected being totally released in the pasta cooking water. To simulate the final stage of intestinal degradation, the gastro-duodenal digesta were incubated with porcine jejunal brush-border membrane hydrolases. Sixty-one peptides surviving the brush-border membrane peptidases were identified by liquid chromatography-mass spectrometry, including several gluten-derived sequences encrypting different motifs responsible for the induction of celiac disease. These results provide new insights into the persistence of wheat-derived peptides during digestion of cooked pasta samples.

New insights on the features of the vinyl phenol reductase from the wine-spoilage yeast Dekkera/Brettanomyces bruxellensis

Vinyl phenol reductase activity was assayed in extracts from 19 strains of Dekkera bruxellensis isolated from wine. In all strains, vinyl phenol reductase activity was insensitive to the presence/absence of 4-vinyl guaiacol, confirming that expression is not related to the presence of the substrate. D. bruxellensis CBS 4481 showed the highest vinyl phenol reductase activity toward 4-vinyl guaiacol. Vinyl phenol reductase from D. bruxellensis CBS 4481 was purified to mass spectrometric homogeneity, and sequenced by trypsinolysis and mass spectrometry. The sequence of the purified protein showed convincing homology with a Cu/Zn superoxide dismutase in the D. bruxellensis AWRI 1499 genome, and indeed it was found to possess both vinyl phenol reductase and superoxide dismutase activities. A bioinformatics analysis of the sequence of vinyl phenol reductase/superoxide dismutase from D. bruxellensis CBS 4481 reveals the presence in this protein of cofactor-binding structural features, that are absent in sequences of superoxide dismutases from related microorganisms, that do not display vinyl phenol reductase activity.

Stability and bioactivity of a Bowman–Birk inhibitor in orange juice during processing and storage

The present research aimed to evaluate the feasibility of a soybean Bowman-Birk inhibitor (BBI) as a natural functional food ingredient. The influence of food processing, storage and digestion on the structure and bioactivity of BBI added to a food matrix was evaluated. The cytotoxic effect of the digested samples was also tested with the aim to provide information regarding the safe use of BBI as a functional food ingredient. Samples of freshly squeezed orange juice (OJ) and two orange juice model systems (OJ-ms) supplemented and non-supplemented with BBI were prepared and processed mimicking pasteurization and sterilization industrial conditions. Moreover, pasteurized samples were stored at 4 °C for two months. The samples were digested in vitro under oral-gastrointestinal physiological conditions. Results seem to indicate that the activity of BBI supplemented to the food matrix sufficiently survives thermal processing, storage and digestion processes. Changes in BBI bioactivity may be ascribed among others to the Maillard reaction. Formation of early Maillard reaction products also called Amadori products has been detected in the food sample. No cytotoxic effect was observed for the samples under study. In conclusion, our findings support that BBI has significant potential as a natural functional food ingredient in pasteurized orange juice stored at 4 °C.

The Role of Proteomics in the Discovery of Marker Proteins of Food Adulteration

Food products are subjected to adulteration, with consequent nutritional or economic loss for consumers and with damage to the commercial reputation of producers and trade labels. Substances used for adulteration range from synthetic chemicals to poor-quality plant or animal materials added to food preparations. Currently, a variety of analytical methods can be exploited to determine the presence of undeclared or unexpected ingredients in food products. Although conventional analytical tools have good potential for detecting the synthetic adulterants of food and agricultural foodstuffs, these methods often fail to identify the addition of low-quality materials to higher-value products, as in the case of typical and Protected Denomination of Origin foods. In the last years, the application of the “omic” technologies in food science has assumed a leading role in the definition of the entire and detailed (bio)chemical composition of a food and its modification along the artisanal or industrial production chain to evaluate its technological properties. These developments are driven by the need to meet the instances of food industries, regulatory agencies, and consumers in order to guarantee food quality and authenticity. Although, in principle, any class of food constituents can be a marker of the food characteristics, more than other fractions, proteins retain the record of the treatment and processes a food undergoes from raw materials to end products. This chapter focuses on the application of the newly born proteomic technologies to the discovery and characterization of reliable molecular markers of food adulteration for the assessment of food quality, typicality, and authenticity.

Proteomic Analysis of Beer

Proteins determine several key sensory and technological properties of beer. Most of the protein components of beer are barley pathogenesis–related proteins, which endure the harsh brewing treatments. Due to their intrinsic resistance, they can also survive gastrointestinal digestion in humans, evoking immune responses in predisposed individuals. For these reasons, beer proteins have been studied for several decades. However, only the recent development of the mass spectrometry–based proteomic techniques is disclosing the “deep” proteome of beer. Several dozens of barley- and yeast-derived proteins constitute the current “core” proteome of beer, while many others are specific to beer brands or styles. Proteolytic peptides of beer include a multitude of sequences derived from prolamins that are potentially harmful for celiacs. In this chapter, we survey the compositional and functional aspects of the beer proteome/peptidome emphasizing the most recent findings obtained using the newest proteomic approaches.

Proteomics of Hazelnut ( Corylus avellana )

Abstract Hazelnut (Corylus avellana) is widely used in food production in raw, roasted, salted, and paste form. Proteins are key components conferring favorable sensory, technological, and functional properties to hazelnut. For these reasons, the protein fraction of hazelnut has been the subject of extensive research over the last years. In addition, the complete annotation of the hazelnut proteome at the molecular level is of great interest, in particular in clinical research because hazelnut proteins can elicit even severe allergic reactions in sensitive subjects. This chapter provides an overview of the current knowledge on the hazelnut proteome.

PO23 PROTEOMIC AND IMMUNOASSAY CHARACTERIZATION OF A NEW FOOD ALLERGEN FROM HAZELNUT (CORYLUS AVELLANA)

Methods Otherwise healthy children with oral food challenge confirmed hazelnut allergy were prospectively evaluated. Crude protein extracts were obtained from 5 hazelnut varieties, including autochthon, Northern Italy and Oregon (USA) cultivars, with phosphate saline buffer, pH 7.2. The immunoreactive protein components were identified by SDS-PAGE electrophoresis and Western immunoblotting, using patients sera as source of specific IgE. The IgE-binding protein bands were characterized by advanced proteomic strategies and tandem mass spectrometry (MS)-based de novo peptide sequencing.