Regulating the heat stability of protein-phospholipid stabilised oil-water emulsions by changing the phospholipid headgroup or fatty acyl chain

Stabilising oil-water emulsions remains a central challenge across food, pharmaceutical and cosmetic applications. β-lactoglobulin (β-LG) and phospholipids (PLs) can act synergistically at oil-water interfaces: PLs adsorb rapidly, while β-LG forms a viscoelastic protein network that enhances long-term stability. However, competitive adsorption between proteins and PLs can disrupt interfacial structure. In addition, for commercial production, emulsions are often exposed to heat treatment during or after manufacture, for instance due to food safety requirements. Yet, the combined effects of PL structure and heat treatment on interfacial organisation and emulsion stability remain poorly understood. Here we show that PL saturation and processing temperature jointly determine interfacial organisation, protein-PL interactions and emulsion stability. Using β-LG-PL emulsions, we combined ζ-potential measurements, small-angle X-ray scattering (SAXS), micro-differential scanning calorimetry (μDSC), X-ray diffraction and confocal laser scanning microscopy (CLSM) to link interfacial composition with functional stability. Below the β-LG denaturation temperature (≤75 °C), saturated PLs promoted partial unfolding of β-LG at the interface without displacement, producing mixed protein-PL networks with enhanced viscoelasticity and stability. Unsaturated PLs displaced β-LG, yielding less elastic interfaces and promoting protein aggregation in the bulk. At ≥75 °C, increased hydrophobicity intensified protein-protein interactions irrespective of PL type. Our findings reveal that saturated PLs shift the β-LG denaturation temperature upward by restricting molecular mobility, without preventing quaternary-level protein-protein interactions. Thermal denaturation, regardless of PL type, promoted interfacial multilayer formation at 90 °C. These results provide a mechanistic framework for tailoring emulsion stability via lipid saturation and processing temperature.