Extracellular Vesicles from Olive Wastewater and Pomace By‐products: Isolation, Characterization of Their Lipid and Phenolic Profiles, and Evaluation of Their Radical Scavenging Activity

Extracellular vesicles (EVs) are nanoscopic structures that are involved in intercellular communication. Recent works have highlighted the existence of these assemblies in several plants and shown that they are able to vectorize hydrophilic and lipophilic molecules. In this study, we have isolated EVs from the two main olive oil by-products (wastewaters [WWs] and pomace) by differential centrifugation/ultracentrifugation and have characterized their main physicochemical properties (size, charge, multimolecular structure, lipid and phenolic contents) and radical scavenging activity. Lipid content in EV fractions was 3.4 (0.2) % (% dry material) for WWEVs and 7.7 (0.3) % and 5.9 (0.9) % for EVs, respectively, from plurivarietal or monovarietal pomaces. Polar lipids represented around 49% of total lipids, and their profiles were globally similar in all EVs. Phosphatidylcholine and phosphatidic acid were the more abundant molecules. Their phenolic contents ranged from 2.1 to 4.6 mg hydroxytyrosol (HT) eq g(-1) of raw material, with HT, oleuropein, and verbascoside being among the most abundant. Transmission electron cryomicroscopy showed the presence of spherical vesicles delimited by a single bilayer of amphiphilic lipids. Finally, the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of EVs was high and depended on their original by-product type. Practical Application: Recent works have highlighted the existence of extracellular vesicles in several plants and shown that they are able to vectorize hydrophilic and lipophilic molecules. Herein, we have isolated and provided a chemical characterization of such vesicles from olive wastewater and pomace. Results showed that these vesicles are rich in the phenolic compounds that are generally found in olives and that the potential radical scavenging activity of extracellular vesicles from olive could be valorized as new antioxidants for the food or cosmetic sectors.