Refractive index of milk fat globules and extracellular vesicles in human milk

Human milk contains milk fat globules (MFGs) and extracellular vesicles (EVs). Knowing their refractive index is essential for accurate data interpretation obtained by optical methods, such as laser diffraction analysis.

As the refractive index of MFGs and EVs in human milk is undescribed, we aimed to determine the refractive index of these particles and compare their refractive index values to those in bovine milk, which has been more extensively studied.

Using flow cytometry, particles ranging from 200 to 650 nm were analyzed in 21 human milk samples and pooled bovine milk samples from four dairy farms.

Results show that the refractive index distribution of human MFGs ( $1.51\pm 0.01$ ) has a significantly higher mode compared with the distribution of bovine MFGs ( $1.49\pm 0.02$ ). In addition, EVs in human and bovine milk were best approximated by a bimodal refractive index distribution, suggesting the presence of two distinct EV populations. These populations had refractive indices of $1.38\pm 0.01$ and $1.41\pm 0.03$ for human milk, which differed from the bovine values of $1.38\pm 0.01$ and $1.42\pm 0.03$ .

This work provides insight into the refractive index of particles in human milk, which is essential for understanding milk-light interactions. Importantly, applying bovine MFG refractive index values in laser diffraction analysis for human MFGs can lead to significant errors in the estimation of particle size distributions.