Industrial Scale Production and Characterization of a Whey Fraction Enriched in Extracellular Vesicle Material

Human milk serves the sole nutritional role for the developing infant. During lactation, nano-sized extracellular vesicles (EVs) in milk containing a multitude of biologically active components are transferred from mother to offspring. Infant formula (IF) based on cow milk-derived ingredients has been reported to contain reduced levels of EVs as compared to human milk. There is therefore an unmet need to produce large-scale volumes of milk EVs to improve IF composition.

Here, we report a scalable industrial production protocol for a bovine whey-derived ingredient that is highly enriched in EV material using a large-scale sequential ceramic membrane filtration setup. Furthermore, we demonstrate a robust and generally applicable analytical approach to determine the relative contributions of EVs and milk fat globule membrane (MFGM) using molar ratios of the membrane-bound proteins butyrophilin (BTN) and CD9 as surrogate markers for MFGM and EVs, respectively. Taken together, our findings provide a basis for comparing bovine milk-containing foods and aid in developing specialized ingredients that can minimize the compositional difference between infant formula and human milk.