Crosslinking-degree-dependent TGase–EGCG synergy stabilizes WPI-based walnut oil HIPPEs for use as a butter replacer

This study explores the synergistic effects of the degree of transglutaminase (TGase) crosslinking and epigallocatechin-3-gallate (EGCG) complexation on whey protein isolate (WPI)-stabilized walnut oil high internal phase Pickering emulsions (HIPPEs) for butter replacement in processed cheese. By adjusting TGase levels with or without ultrasound, crosslinking degrees of 12.7–27.8% were achieved. Moderate crosslinking (22.8%) optimally unfolded WPI, improving molecular flexibility, surface hydrophobicity, and the three-phase contact angle, whereas excessive crosslinking (27.8%) caused aggregation and impaired interfacial performance. EGCG further enhanced the molecular flexibility and surface charge via non-covalent interactions. The optimal HIPPE (H-WPI-EO) exhibited superior emulsifying stability, freeze-thaw resistance, and oxidative stability (61.6% thiobarbituric acid reactive substances (TBARS) reduction versus unmodified emulsion). Crosslinking-degree-dependent covalent (TGase) and non-covalent (EGCG) modifications synergistically improved the interfacial properties of WPI, positively correlating with molecular flexibility. In processed cheese, the 58.8% (w/w) butter-replaced product with H-WPI-EO showed hardness and cohesiveness comparable to the full-fat control, improved moisture retention, and equivalent oxidative stability, which is a key advantage for polyunsaturated fatty acid rich oils such as walnut oil. This study provides a degree-dependent design principle for protein–polyphenol interfaces and a practical route for stable, nutritionally enhanced cheese with polyunsaturated oils.