Super-resolution microscopy reveals heterogeneity in the coverage of oil-in-water food emulsions

Oil-in-water food emulsions such as mayonnaise and dressings are stabilized by proteins and low-molecular weight surfactants binding to the oil/water interface. One common source of emulsifying proteins is egg yolk containing the iron-binding protein phosvitin. Here, we applied super-resolution microscopy to quantify the distribution of phosvitin on the droplet interfaces of binary SDS/phosvitin model emulsions prepared by high-pressure homogenization (HPH). We targeted phosvitin either via fluorescently labeled, primary antibodies or with affimers, which are short polypeptides. Re-scan confocal microscopy (RCM) revealed a bimodal droplet size distribution in which small droplets were primarily covered by SDS and large droplets by phosvitin. This inter-droplet heterogeneity was in line with expected kinetics of emulsifier coverage of droplet interfaces during HPH. Stochastic optical reconstruction microscopy (STORM) indicated that changing the concentration of phosvitin did not affect the intra-droplet distribution at the droplet interface. STORM further provided a direct visualization of the redistribution of phosvitin upon prolonged low shear treatment, resulting in diffusion-assisted exchange of SDS and phosvitin between droplet interfaces and the continuous aqueous phase. Our RCM- and STORM-based approaches allow a direct and quantitative view on the intricate balance between kinetic and thermodynamic forces governing inter- and intra-droplet interfacial distributions of proteins. • Localization of phosvitin at interfaces in phosvitin/SDS stabilized model emulsions. • Quantification of intra-and inter-droplet interfacial distribution with phosvitin. • Demonstration of protein redistribution at droplet interface upon low shear treatment.