Techno-functional properties of clover grass protein and the effect of different process technologies

Abstract This study investigated the effects of different downstream processes for protein isolation on the bulk properties and composition of clover grass protein prototypes (CGPs). A clarified clover grass juice, obtained using membrane filtration (MF), underwent precipitation by acid (AP), heat (HP), or acid+heat (AHP), or underwent ultra- and diafiltration to produce a concentrate (DC) as well as subsequent tryptic hydrolysis of DC (DCH). HP had the highest protein content (p<0.05) and was whiter than other CGPs, although it showed lower aqueous solubility. In contrast, DC showed excellent solubility across a broad pH range. CGPs efficiently decreased oil-water interfacial tension (16-13 mN/m) and displayed viscoelastic and solid-like interfacial behavior. CGPs-stabilized emulsions displayed low physical stability with larger droplets despite high absolute ζ-potentials. CGPs were rich in RuBisCO (37-47%) but had varying levels of other proteins. Despite significant protein-level differences, overall protein composition of CGPs was comparable, highlighting that protein state governs bulk functionality more than subtle compositional changes. Graphical abstract Created with BioRender.com Highlights The effect of different processes on functional properties of CGPs was explored. Heat treatment increased protein purity and whiteness at the expense of solubility. CGPs efficiently reduced O/W interfacial tension but produced unstable emulsions. CGPs were found rich in RuBisCO (34-47%) using quantitative proteomics. Protein state had larger influence on functionality than protein-level composition.