Timing matters: How delayed emulsifier addition shapes phospholipid-protein interfaces

Emulsions are multicomponent systems that often contain both phospholipids (PLs) and proteins such as β-lactoglobulin (β-LG). During industrial production, emulsification frequently occurs in multiple steps, meaning that surface-active components may enter the system at different time points. This can lead to dynamic interfacial restructuring driven by competitive displacement or protein-PL interactions. Industrial emulsions are further subjected to thermal stress before or after emulsification, which may induce PL phase transitions and modify interfacial organisation. Although the order of emulsifier addition and thermal treatments likely influence the final interfacial properties, this aspect has received little attention in interfacial research, and mixing order is often applied inconsistently across studies. This study investigates how temperature pretreatment and the order of emulsifier addition shape protein-PL interfaces. PLs with different headgroup structures (phosphatidylethanolamine, PE; phosphatidylcholine, PC) were used to assess the role of molecular structure in protein-PL interactions. Interfacial properties were analysed within and beyond the linear viscoelastic regime using dilatational and interfacial shear rheology. Introducing PLs before proteins hindered protein adsorption and network formation, resulting in PL-dominated deformation behaviour. When β-LG was added first, proteins unfolded and adsorbed effectively, yielding protein-dominated interfaces with embedded PLs. Smaller headgroups produced more compact interfaces, stronger protein-PL interactions, and increased interfacial stiffness. Thermal pretreatment further enhanced structural ordering through crystallisation-induced reorganisation. In conclusion, elevated temperatures enhance interfacial stability, and emulsifier mixing order markedly shapes interfacial structure, underscoring the need for consistent emulsification protocols. • Cooling led to crystalline interfaces with higher viscoelasticity • Without heating interfacial viscoelasticity is reduced due to weaker lateral interactions • Adding PLs first partly hinders protein adsorption and unfolding • When adding β-LG first, a protein network can form where the PL is embedded