Successive extraction using natural deep eutectic solvents and pressurized liquids for a greener and holistic recovery of proteins from pomegranate seeds

This work describes a novel and sustainable strategy for the recovery of proteins by successive extractions using natural deep eutectic solvents (NADES) and pressurized liquid extraction (PLE). This strategy was applied to the valorisation of pomegranate seeds. Nine different NADES were screened and that constituted by choline chloride and acetic acid was chosen due to its best performance. A Response Surface Methodology was employed to optimize other conditions in this extraction step: time, temperature, amount of sample, and HIFU amplitude. Protein recovery, under optimal conditions, was 13.3 g of proteins/100 g of milled and dried defatted seeds. Proteins were next characterized by their separation using RP-HPLC, SDS-PAGE, isoelectrofocusing electrophoresis, and by the evaluation of their digestibility and antioxidant properties. Comparison of these results with those from extracts obtained by other techniques supported the interest of combining the extraction using acid NADES with PLE, under alkaline conditions. The successive extraction by both methodologies enabled to double the total recovery of proteins. The analysis of samples by UHPLC-MS/MS, after a simulated gastrointestinal digestion, and de novo identification revealed the presence of 19 peptides in the NADES hydrolysate, while the successive extraction by PLE enabled to observe 15 additional peptides. Additional 83 peptides were found by database searching against Punica granatum and by homology with other organisms. Differences between peptides and the proteins in both hydrolysates confirmed the different protein selectivity of both strategies and the potential of NADES for extracting larger proteins and PLE for the extraction of smaller ones. Some phenolic compounds, amino acids, and fatty acids were also co-extracted with proteins in both extractions.