Synergic Effect of Enzyme‐Assisted Extraction and Natural Deep Eutectic Solvents for Bioactives Recovery in Orange Peel Waste

Citrus wastes are a source of valuable compounds that have potential application in food industries. Despite the several methods employed for bioactive recovery, most of them are detrimental for both human health and the environment. Recently, the use of "green" extraction methods with natural deep eutectic solvents (NaDES) represents a promising ecofriendly approach. Moreover, the employment of enzymes could facilitate the breakdown of food matrix-bioactive interactions and enhance biomolecules extraction. The present study aimed to investigate the potential synergistic application of combined enzyme-assisted extraction (EAE) and NaDES formulation on polyphenols and proteins/peptides recovery from orange peel, in comparison with conventional extraction methods. Hesperidin appeared to be the most abundant polyphenol, and its extraction seemed higher with malic acid-containing NaDES, regardless of the EAE used. In terms of antioxidant capacity, the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay had the highest capacity with both aqueous and ethanolic extraction, whereas the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method revealed the highest values with l-proline:malic acid (LP:MA) pretreated with cellulase, 3% pectinase, and 0.75% protease. Moreover, the level of protein hydrolysis and peptide content was generally higher in malic acid:glucose-containing NaDES. The extract obtained from the most promising condition in term of bioactives extracted and antioxidant activities, identified as 3% pectinase followed by LP:MA, shows a cytoprotective effect at 10 µL/mL against the vehicle LP:MA by using in vitro an intestinal cell model. In conclusion, EAE and NaDES represent a combined effective and sustainable method of recovering bioactives from citrus waste, as opposed to the conventional extraction methods.