Triglycerides in food products such as mayonnaise are a vital source of energy and essential for a complete and healthy diet. Their molecular structures consist of a glycerol backbone esterified with fatty acids on the two outer and the middle positions. During the digestion of triglycerides by pancreatic lipase in the small intestine, the ester bonds on the outer positions are hydrolyzed,
leading to amphiphilic monoglycerides and free fatty acids as products. Depending on their chain length and degree of saturation, these products can self-assemble into a variety of structures in excess water. In this study, we report the discovery of highly ordered nanostructures inside of the mayonnaise emulsion droplets during in vitro digestion of mayonnaise under simulated in vivo conditions using timeresolved synchrotron small-angle X-ray scattering. The formation of these structures is most likely linked to their function as a carrier and controlled release system for food nutrients, especially poorly water-soluble components, in the aqueous milieu of the digestive tract. This detailed understanding of nanostructure formation during the digestion of triglyceride-containing food products such as mayonnaise may have fundamental implications for the development of foods with improved nutritional and functional properties.