Impact of Holder Pasteurization on Extracellular Vesicles and Immunoregulatory MicroRNAs in Human Breast Milk

Human breast milk contains bioactive molecules, including microRNAs (miRNAs) that regulate neonatal development and immunity. Pasteurization ensures microbiological safety in milk banks, but its effects on milk-derived miRNAs remain unclear. This study evaluated the impact of Holder pasteurization (62.5 °C, 30 min) on extracellular vesicle (EVs) morphology and immunoregulatory miRNA expression in exosome, fat, and serum fractions from six donor samples. EVs were characterized by transmission electron microscopy, dynamic light scattering, nanoparticle tracking analysis, and protein quantification. Thirteen miRNAs were analyzed by RT-qPCR. Pasteurization caused morphological alterations in exosomes, reducing particle size and increasing protein concentration, while total particle number remained stable. Expression of eight out of 13 exosomal miRNAs significantly decreased (<i>p</i> ≤ 0.05), whereas miRNA profiles in fat and serum fractions were largely preserved.