Precipitation and recovery of soy protein extracted in natural deep eutectic solvents: Emphasizing solvent reusability for sustainable processing

Soy protein is one of the most important plant-based protein source used in food formulations due to its favorable nutritional profile and functional properties. However, the increasing interest in sustainable processing has led to the search for environmentally friendly extraction systems that can reduce reliance on conventional solvents. In this context, natural deep eutectic solvents (NADES) have attracted attention as effective media for plant protein extraction; however, protein recovery and solvent reuse after extraction still present significant practical challenges. In this study, soy proteins extracted from defatted soybean meal using choline chloride: glycerol NADES were recovered through isoelectric precipitation, antisolvent precipitation using water, ethanol, or water: ethanol (1:1) at extract: antisolvent ratios (1:2 to 1:6), and a sequential combined strategy involving pI adjustment followed by water: ethanol addition. Both the type of antisolvent and the dilution ratio had a marked influence on precipitation behavior. Water did not produce visible precipitation at low dilution ratios, whereas ethanol-containing systems more effectively facilitated protein aggregation. Among all approaches, the combined pI + water: ethanol (1:4) method gave the most favorable results, yielding a precipitation yield of 6.03%, a protein recovery of 77.47%, and the highest protein purity (88.60%). ATR-FTIR analysis showed that the main protein bands (Amide I-III) were preserved after recovery, while SEM images revealed irregular aggregated particles characteristic of proteins. In terms of solvent reuse, NADES recovery remained high over three consecutive cycles (96.4 % to 89.6 %), whereas extraction efficiency retention gradually declined. This reduction was accompanied by an increase in viscosity from 63.2 to 88.8 mPa·s, although no major structural changes were observed in the FTIR spectra of the recycled solvent. These findings indicate that combining charge neutralization with polarity modulation is an effective strategy for recovering high-purity soy protein and support the short-term reuse of NADES in a closed-loop extraction system.