Homogenization and thermal processing reduce the concentration of extracellular vesicles in bovine milk

Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Industrial processing treatments may affect the amount or bioactivity of EVs intrinsic to bovine milk. We investigated how the content and concentration of EVs were affected by homogenization and thermal processing of raw bovine milk. Raw milk was processed by homogenization, low-temperature (LT) heat, or pasteurization [high-temperature short-time (HTST) and ultra-high-temperature (UHT)] in a pilot processing facility. EVs were isolated from the raw and processed bovine milk using differential ultracentrifugation and quantified using a nanoparticle tracking analyzer. Bovine milk EVs were assessed for total miRNA and protein concentrations standardized to particle count using a fluorometric assay. There were 1.01 × 10¹⁰ (±3.30 × 10⁹) EV particles per ml of bovine milk. All industrial processing treatments caused >60% decrease in EV concentration compared to the raw bovine milk. Homogenization and heat treatments independently and additively reduced the content of EVs in bovine milk. The averages of total miRNA/particle and total protein/particle concentrations were elevated threefold by low-temperature heat-processing treatment relative to HTST and UHT pasteurizations. The average diameter of EVs was reduced by 11%-16% by low temperature compared to raw milk (127 ± 13 nm). Homogenization and pasteurization indiscriminately reduce the EV concentration of bovine milk. Smaller EVs with higher protein content resist degradation when processing bovine milk at sub-pasteurization temperature. This new foundational knowledge may contribute to food product development on the preservation of EVs in processed dairy products, including bovine milk-based infant formulas that some newborns are dependent on for adequate growth and development.