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

Abstract

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 BoxBehnken 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.