Pilot-scale biorefining of clover grasses for production of food protein ingredients: Interfacial properties, molecular composition, and effects on the physical and oxidative stability of fish oil-in-water emulsions

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 CGP concentrate (DC), as well as enzymatic hydrolysate hereof (DCH), were compared with commercial plant proteins (soy and pea) and animal sources (sodium caseinate). DC and DCH were investigated by mass spectrometry (MS)-based quantitative proteomics and subsequent bioinformatic analysis to link molecular composition to observed bulk functionality. DC presented a high RuBisCO content (38% mol/mol) and the membrane process successfully depleted pigment-binding proteins affiliated with grassy attributes. Moreover, DC was enriched in known antioxidant proteins, constituting 10% of the total protein. In DCH, 30% of the total peptide intensity (MS1) originated from peptides predicted as probable free radical scavengers. 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. Both DC and DCH exhibited higher protection against the generation of primary and secondary oxidation products. Furthermore, emulsions stabilized with CGPs were less prone to form compounds associated with off-flavors. These findings demonstrate that refined, native CGP, as well as its hydrolysate, can produce emulsions with good physical and particularly oxidative stability compared to the included plant and animal-based reference proteins. This property is likely attributed to the enrichment of proteins with strong endogenous antioxidant properties in DC and high content of peptides with predicted radical scavenging properties in DCH.