Comparative Evaluation of Isolation Methods on the Biophysical Properties of Milk Derived Exosomes from Bovine Subspecies

Milk-derived exosomes (MDEs) are stable, nanosized extracellular vesicles secreted by mammary epithelial cells, encapsulating bioactive proteins, lipids and nucleic acids within a phospholipid bilayer. Their stability and resistance to enzymatic degradation make them promising candidates for targeted delivery systems and nutraceutical applications. This study standardized a robust, reproducible protocol for isolating high-purity MDEs from Bos indicus (Sahiwal), Bos taurus (Holstein Friesian) and crossbred (Karan Fries) cows and evaluated subspecies-specific variations in their properties.

Differential ultracentrifugation and precipitation kit based methods using specific polymers were compared for efficiency, yield and preservation of exosomal integrity. Comprehensive characterization comprising transmission electron microscopy, dynamic light scattering, nanoparticle tracking analysis and western blotting for exosomal markers.

Our results demonstrate that differential ultracentrifugation is the superior method for yielding morphologically intact, homogeneous MDEs from large milk volumes, irrespective of the milk source. This precision was crucial, as it allowed us to discern subtle but significant subspecies-specific differences. MDEs from Bos indicus exhibited a markedly smaller particle size than those from Bos taurus and crossbreds. This distinction is critical because exosome size directly impacts their functional performance and utility. Bos indicus MDEs potentially offer enhanced cellular uptake and bioactivity, while larger MDEs from taurine and crossbred cattle may be advantageous for delivering bulkier molecular cargos or modulating immune responses. This work not only validates an optimized isolation technique for large-scale recovery of high-quality MDEs but also highlights bovine subspecies nanostructural traits with implications for précised applications in dairy biotechnology and healthcare. Identification of smaller, potentially more bioavailable MDEs from Bos indicus highlights the importance of native cattle resources for high value therapeutic applications, promoting value addition and sustainable utilization of native cattle genetic resources.