Protein concentrates from pilot-scale biorefining of clover grass: Interfacial properties and their effects on the physical and oxidative stability of fish oil-in-water emulsion

Clover grass blends are promising sources of nutritional and techno-functional proteins, but currently mainly utilized for animal feeding. The application as a physical and oxidative stabilizer in food emulsions remains underexplored. In this study, the stabilizing effects of clover grass proteins (CGPs), produced through a pilot-scale, two-stage membrane filtration process yielding a native GPC concentrate (DC), as well as enzymatic hydrolysate hereof (DCH), were compared with commercial plant proteins (soy and pea) and animal sources (sodium caseinate). Both DC and DCH produced emulsions (0.4% (w/w) protein and 5% fish oil) with smaller size droplets and larger electrostatic repulsion between droplets compared to the other proteins tested. Moreover, DC and DCH exhibited higher protection against the generation of both primary and secondary oxidation products. Furthermore, emulsions stabilized with CGPs were well-protected from off-flavor compounds. Mass spectrometry-based proteomics analysis revealed that DC included a high RuBisCO content (38%) and the membrane process successfully depleted pigment-binding proteins affiliated with grassy color and sensory attributes. Moreover, DC was enriched (compared to the initial green juice) in known antioxidant proteins, constituting 10% of the total protein. In the hydrolysate (DCH), 30% of the total MS1 peptide signal originated from peptides predicted as probable free radical scavengers. These findings demonstrate that refined, native CGP, as well as its hydrolysate, improved both physical and oxidative stability of emulsions compared to plant and animal-based reference proteins due to a high endogenous antioxidant properties of the protein.