Sainfoin (Onobrychis viciifolia L.) protein isolate as a new source of alternative plant-based protein: cytotoxicity, immunoreactivity, nutritional and functional properties

The objective of this research was to develop an alternative plant-based protein isolate using sainfoin (Onobrychis viciifolia L.) seeds. The extraction process was optimized using response surface methodology (RSM) based on the Box-Behnken Design, which examined the effects of key parameters: solvent/solid ratio (10–50 mL/g), pH (8–11), temperature (20–50 °C), and extraction time (30–120 min), aiming to maximize protein yield. The optimal extraction conditions identified were a solvent/solid ratio of 49.96 mL/g, pH of 10.99, temperature of 20 °C, and a duration of 38.55 min, achieving a protein yield of 56.36%. Additionally, the amino acid composition, cytotoxicity, immunoreactivity, and functional properties of the sainfoin seed protein isolate (SPI) were evaluated. SPI exhibited a high crude protein content of 91.44%, with arginine being the most abundant amino acid at 158.20 mg/g. The protein isolate comprised a remarkable value of 50.26% essential amino acids. Additionally, SPI demonstrated desirable functional properties, including solubility of 53.95% at neutral pH, water holding capacity of 2.36 g/g, and oil binding capacity of 4.68 g/g. Its emulsifying performance was notable, with emulsion activity and stability values of 66.67% and 77.50%, respectively. Moreover, in vitro cell culture studies demonstrated that sainfoin seed protein exhibited no adverse effects on cellular toxicity or immunoreactivity. This study highlights the potential of SPI as a novel, high-quality plant protein source with promising nutritional and functional properties and demonstrates its potential as a functional ingredient in the formulation of plant-based foods, meat analogs, and dietary supplements.