Influence of hydrophobic interfaces and shear on ovalbumin amyloid-like fibril formation in oil-in-water emulsions

Abstract

Animal proteins are widely used because of their good techno-functional (e.g. emulsification) properties. However, the large ecological impact of animal-derived proteins is often debated and therefore alternatives are evaluated in which animal proteins with increased functionality are employed. Amyloid fibrils are a distinct type of protein fibrils characterized by an intermolecular cross β-sheet structure, mostly studied because of concerns regarding their role in certain human diseases. However, they have also been detected in processed food proteins such as heated ovalbumin (OVA) and other egg proteins. Previous work also mentioned superior emulsification properties of amyloid-like fibrils. Here, the effect of different shear conditions during the emulsification of oil in water emulsions stabilized by heated OVA was investigated. Emulsification with intensive shear treatment (using both an ultra-turrax and a microfluidizer) resulted in submicron sized droplets, whilst ultra-turrax only gave rise to rapidly creaming emulsions (>10 μm). Thioflavin T (ThT) fluorescence, used as an indicator of cross β-sheets, did significantly increase for submicron emulsions, whereas supermicron emulsions showed a similar fluorescence as the aqueous phase used for emulsification. The increase in ThT fluorescence in submicron emulsions was in line with a higher amount of larger fibrillar structures, which showed high ThT fluorescence, measured using size exclusion-HPLC and transmission electron microscopy images. Similar observations were also made for unheated OVA, suggesting that high-shear emulsification induced amyloid-like fibril formation. In contrast, shearing of aqueous solutions without the creation of interfaces did not show increased fibril formation. Therefore, it is claimed that the large specific surface area induced amyloid-like fibril formation or maturation as a result of a better interaction between hydrophobic amyloid-like fibril fragments. In this work, we were able to characterize the protein fibrillar structures present in emulsions by considering commonly used techniques for the study of fibril formation. This creates opportunities towards the implementation of these protein mixtures in food applications.