mayonnaise

Mayonnaise, a protein-stabilized oil-in-water emulsion, undergoes irreversible destabilization during freeze–thaw (FT) cycling, yet the relative contributions of aqueous-phase ice formation, protein denaturation, and fat crystallization remain debated. Here, we investigated FT destabilization using cryo-microscopy, differential scanning calorimetry (DSC), particle size analysis, interfacial protein assays, and a non-freezing control experiment. Cryo-microscopy revealed heterogeneous ice distribution, with an ice area fraction of 83% at the surface compared with 65% internally, indicating mechanical crowding of oil droplets during freezing. DSC analysis showed cooling-rate-dependent ice crystallization accompanied by severe freeze-concentration, with the aqueous-phase pH decreasing from 3.92 to 3.82 and NaCl concentration increasing ~1.7-fold at 40% ice formation. Despite repeated FT cycles, egg-yolk LDL retained 82–94% of its native emulsifying activity, while surface hydrophobicity decreased by only 25–32%, suggesting that protein functionality loss alone cannot explain the observed 45–70% oil separation under slow cooling conditions. Critically, replacing the aqueous phase with a non-freezing medium (90% ethylene glycol, stable at −50 °C) completely prevented destabilization (oil separation 0%; ΔD₅₀ < 5% versus ~520% in water-based controls). These findings indicate that aqueous-phase ice crystallization is the dominant driver of FT instability via two coupled pathways: (1) mechanical compression of oil droplets by growing ice crystals, and (2) freeze-concentration that generates chemically hostile unfrozen microenvironments (acetic acid >5%, NaCl >11%) that inactivate interfacial LDL. This water-centric mechanism provides quantitative targets for designing freeze-tolerant emulsion systems.