Which casein micelle removal method is suitable for studies of human milk extracellular vesicles? A systematic comparison of four different treatments for casein depletion before extracellular vesicle isolation from human milk

Aim: This study aimed to systematically compare four casein micelle removal methods on the particle and protein characteristics of the isolated human milk EVs. Methods: The defatted milk was treated with 1% sodium citrate, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% acetic acid, or 1% chymosin/calcium chloride for 30 min at 4 °C to remove casein micelles. EV isolation was performed using qEV size exclusion chromatography. Milk turbidity at the optical density 350 nm and dot immunoblot with casein antibody were applied to monitor the qEV fractions. Particle analyses were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The enrichment of human milk EV markers, i.e., tetraspanins, Alix, lactadherin, butyrophilin, and xanthine dehydrogenase, and casein depletion capabilities were evaluated by proteomics and immunoblotting. Results: Compared to the untreated condition, sodium citrate and EDTA decreased milk turbidity by disrupting casein micelles, while acetic acid and chymosin removed them by inducing precipitation/coagulation. All treatments shifted casein immunoreactivity in the qEV fractions from large micelles (the exclusion volume) to small molecular sizes (gel-infiltrated fractions). Acidification affected human milk EV morphology, while EDTA, acetic acid, and chymosin methods slightly altered EV particle numbers. Different casein micelle removal methods confer different degrees of human milk EV marker enrichment and casein depletion. The method performances could be ranked as follows: chymosin > EDTA > acetic acid > sodium citrate. Conclusion: Our findings suggest that chymosin and EDTA should be considered as the method of choice for casein micelle removal in future studies involving human milk EV isolation and characterization.