Unlocking Pea Protein's Emulsifying Potential: Achieving Submicron Droplets through a Dual-Modification Strategy of Enzymatic Hydrolysis and Heat-Assisted pH Shifting

Pea protein offers valuable nutritional and environmental advantages, yet its application in food emulsions is hindered by poor solubility and emulsifying capacity—a consequence of its inherently aggregated and rigid structure. To address this limitation, the present study developed a dual modification strategy combining controlled enzymatic hydrolysis (DH 3%) with heat assisted pH shifting. PPI was first hydrolyzed with either trypsin or pepsin, then subjected to pH 3 or pH 9 at 70°C. Trypsin hydrolysis proved markedly more effective than pepsin treatment, generating fragments with enhanced surface hydrophobicity, reduced aggregate size (~280 nm), and improved solubility, dispersibility, and emulsifying performance—benefits attributable to the stringent cleavage specificity of trypsin for lysine and arginine residues. Subsequent alkaline pH shifting (pH 9) with heating further exploited electrostatic repulsion to dissociate the aggregates to ~160 nm, yielding additional functional gains. In contrast, acidic pH shifting (pH 3) under the same heating conditions promoted extensive protein reaggregation and conferred no functional benefit. The optimally modified PPI—obtained by combining trypsin hydrolysis with alkaline pH shifting—enabled formulation of submicron sized oil in water emulsions (D_4,3=0.607±0.001 μm). These emulsions withstood 30 days of storage (D_4,3=0.837±0.003 μm) and heating at 95°C for 60 min (D_4,3=1.18±0.05 μm) with only modest droplet size increases, demonstrating notable stability. This work establishes that the synergy between enzymatic digestion and alkaline pH shifting effectively dissociates protein aggregates while avoiding the generation of excessively small peptides that would compromise emulsion stability. The dual strategy thus transforms pea protein into an efficient molecular emulsifier, offering a promising route for developing advanced plant based ingredients.