A PROTEOMIC-BASED STUDY TO ASSESS THE EFFECT OF EXTRUSION IN THE ALLERGENIC PROFILE OF WHEAT FLOURS OF DIFFERENT TECHNOLOGICAL QUALITIES

Although wheat is widely produced and consumed worldwide, predisposed people can present sensitivities or inflammatory reactions to the wheat proteins. These reactions are caused by the stimulation of the immune system of individuals presenting celiac disease, non-celiac gluten sensitivity, wheat allergy, dermatitis herpetiformis, amongst other disorders. This study aimed to understand how wheat flours of contrasting technological end-use qualities submitted to thermoplastic and cold extrusion behave concerning the expression of allergenic proteins. Wheat flours (Triticum aestivum) were extracted from two wheat genotypes of different end-uses (ORS Agile, classified as bread/improver wheat; ORS Vintecinco, classified as biscuit wheat). Then, extrudates were produced using single-screw extruder, applying two different screw rotation speeds (150 and 300 rpm). Pasta was also produced by the cold-extrusion process with further cooking. Proteins were sequentially extracted based on the Osborne solubility classification: salt-soluble (AG), ethanol-soluble (Gli), SDS-soluble with reduction (Glu) and then quantified by RP-HPLC. Extracts were reduced (DTT), alkylated (IAM), and digested (thermolysin, 1:20). Peptides were diluted and analyzed by nanoLC-MS/MS in a Q Exactive Plus Orbitrap by using data‐dependent acquisition (DDA) in positive ion mode. MS data was processed in MaxQuant using a Triticum database from UniprotKB using the following parameters: maximum of two missed cleavages, methionine oxidation and N-terminal protein acetylation as variable modifications, cysteine carbamidomethylation as fixed modification, maximum of 5 modifications per peptide, minimal length of 7 amino acids and 1% peptide and protein FDR. Filters (at least 2/3 replicates, score ≥ 1, no reverses, no potential contaminants) were applied to processed data to assign protein identification. Uni, multivariate and hierarchical clustering analysis were performed, and the identified proteins were searched against a compiled of wheat allergenic proteins built from official allergenic databases available online. After processing proteomic data, a total of 339 proteins were evaluated, from which 250 were present in ORS Agile and 162 in ORS Vintecinco. PCA allowed the separation of samples in three distinct clusters (1: flour; 2: 300 rpm extrudates; 3: 150 rpm extrudates, raw and cooked pasta) with the same pattern for both samples. Flours and expanded extrudates (300 rpm) presented a more distinct profile from the samples in third cluster. In the latter cluster, these samples presented a related protein profile among themselves. Dendrograms and its corresponding heatmaps corroborate, showing flours as the most hierarchically distant from products. Preliminary results concerning the expression of allergenic proteins showed that from the set of identified proteins, only 3% were stablished as wheat allergens. Proteins representative of all fractions (AG, Gli, Glu) were present in this list. From those, five were shared by samples from ORS Agile and ORS Vintecinco. ORS Agile flour had the highest expression intensity from the samples, while ORS Vintecinco expanded extrudate has the lowest. Although interesting and pointing to some effect of processing, further studies are necessary to determine the role of technological quality and processing in causing changes to the allergenic potential of wheat products.