Detection of ovalbumin amyloid-like fibrils at the oil-water interface in oil-in-water emulsions by spinning disk confocal microscopy

Protein amyloid-like fibrils (ALFs) and amyloid fibrils (AFs) can display positive techno-functional properties in oil-in-water emulsions. AFs are widely studied in a medical context, but similar structures occur in certain food products. Whereas the confirmation of their nature as AFs follows from the outcome of specific transmission electron microscopy, Congo Red birefringence, X-ray diffraction, and Thioflavin (ThT) fluorescence measurements, that as ALFs is less strict. It has been suggested that upon intensive emulsification of both unheated and heated ovalbumin (OVA) dispersions as continuous phase, fibrillar structures are formed. However, the presence of OVA ALFs at the oil-water interface was not unambiguously proven. Here, the presence of ThT-positive fibrillar proteins at the oil-water interface was investigated. A clock scan image analysis approach was developed and optimized for this study. Whereas emulsions stabilized by either unheated or heated OVA did not clearly show an increased ThT fluorescence at the interface, ThT fluorescence was clearly detected when re dispersing the cream phase obtained by ultracentrifugation. The increased ratio of ThT fluorescence at the oil water interface to the background signal at the oil-water interface of the three times washed emulsions allowed concluding that ALFs accumulate at the interface. Furthermore, comparison with Fast Green For Coloring Food (FCF)-labelled emulsions suggested that ALFs more than other protein structures preferentially adsorbed at the interface. Confocal microscopy confirmed that ALFs accumulate at the oil-water interface and as such provided evidence for the hypothesis that hydrophobic interactions between proteins and oil droplets induced the formation of ALF structures during emulsification. The present observations contribute strongly to the interpretation of the interfacial properties of ALFs in O/W emulsions.