Protein profiling and modulation of digestive proteolytic enzymes by NaDES extracts of citrus by-product

Aim: Food processing generates high waste causing significant disposal expenses. Many of these biomaterials, including citrus wastes, are a source of valuable compounds that could be used in food industries. Recently, "green" novel extraction techniques have been used to optimize the extraction of bioactive compounds and nutrients. NaDES are composed of two or more components that interact each other’s and form a eutectic mixture. The advantages of NaDES are biodegradability, low costs, and low toxicity. Anyway, considering the extraction recovery of nutrients and bioactive compounds, the NaDES formulation should be deeply evaluated. Aim of this study was to characterize the protein profile of extracts from citrus peel obtained using 4 NaDES formulation (choline chloride:fructose, choline chloride:xylose, choline chloride:glycerol, choline chloride:glycerol:citric acid) and 1 hydroalcoholic solution. Moreover, since the orange peel is a considerable source of polyphenols which possess an enzymatic modulating activity, the effect of extracts on the proteolytic activity of pepsin, trypsin, and chymotrypsin was evaluated. Method: Proteins were collected mixing extracts with acetone. After phases separation, acetone parts were mixed with PBS and loaded on 12% SDS-PAGE for protein profile separation. Enzymatic activity was determined measuring spectrophotometrically the release of aromatic aminoacids after protein cleavage using hemoglobin as substrate. Results: Results indicated as protein recovery and profile is depending by NaDES formulation, where choline chloride:glycerol:citric acid seems to be the most promising formulation with several bands associated to proteins with a Mr comprised between 66 and 45 KDa. Similarly, NaDES modulated the proteolytic activity of digestive enzymes, indicating as these innovative green solvents may exert a pivotal role in digestive tract. Conclusion: The data obtained indicate how NaDES may be a viable alternative to organic solvents in the extraction of nutrients from food by-products for their direct use in food preparations. Further studies are needed to evaluate their biological effects by considering NaDES as active ingredients thus bypassing the difficulties of solute recovery. This work was partially supported by Italian Piano di Sostegno della Ricerca 2021-Azione A (Linea 2) and by the Ministry of Science and Innovation (Spain) -State Research Agency (PID-2019-111331RB-I00/ AEI/10.13039/501100011033).