Ultrasound-tailored structural remodeling of oleosome-derived proteins enables interfacial engineering and stability of probiotic-loaded high internal phase emulsions

Oleosomes are intracellular lipid-storage organelles found in oilseeds and represent a promising yet underexplored source of proteinaceous emulsifiers for structuring food-grade colloidal systems. In this study, extrinsic proteins (OP) from soybean oleosomes were extracted under controlled alkaline-ultrasonic treatment (0–400 W) to elucidate how ultrasound-mediated protein remodeling governs interfacial adsorption kinetics, high-internal-phase emulsion (HIPE) formation, rheological behavior, and probiotic protection. Alkaline-ultrasonic treatment from 0 to 300 W significantly increased the protein recovery from 40.15% to 58.84%, solubility from 74.10% to 87.61%, and zeta potential from −8.81 to −13.31 mV, while reduced the particle size from 651 to 389 nm. Spectroscopic analyses revealed controlled protein remodeling characterized by increased exposure of hydrophobic and aromatic residues, reduced α-helix content from 22.95% to 15.03%, and increased β-sheet structures from 34.16% to 42.14%. Ultrasonic treatment (300 W) effectively improved the protein diffusion rate (0.106 to 0.182 mNm −1 s −0.5 ) and penetration rate (3.971 × 10 −4 to 4.682 × 10 −4 s −1 ) at the oil–water interface, indicating the formation of strengthened interfacial protein films. HIPEs stabilized by OP-300 revealed the smallest droplet size, intensified droplet–droplet interactions, superior viscoelastic properties, and excellent storage stability. LAOS analysis further confirmed that HIPEs prepared with OP-200 and OP-300 maintained structural integrity under large deformation amplitudes ≤100%, compared to the untreated sample ≤10%, indicating the formation of stronger and more cohesive gel networks. Moreover, encapsulated Lactobacillus reuteri showed improved survival rate during simulated digestion, increasing viable cell counts from 4.12 × 10 7 to 7.21 × 10 7 CFU/mL. These findings established that ultrasound tailors OP structure–function relationships, enabling their use as sustainable emulsifiers for robust HIPEs with enhanced probiotic delivery.