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Milk fat globules (MFGs) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid-protein particles termed "lactosomes" that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation, and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma high-density lipoproteins (HDL) (d > 1.063 g/mL) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk, day 8, were found to be similar in size as those from mature milk >28 days, averaging ∼ 25 nm in diameter. In total, 97 nonredundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1-laden reconstituted HDL that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition, and function between lactosomes and MFG, suggesting that these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid-protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant but to play unique, protective, and regulatory roles.

While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first ten days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.

Human milk contains large amounts of complex oligosaccharides that putatively modulate the intestinal microbiota of breast-fed infants by acting as decoy binding sites for pathogens and as prebiotics for enrichment of beneficial bacteria. Several bifidobacterial species have been shown to grow well on human milk oligosaccharides. However, little data exists on other bacterial species. In this work we examined 16 bacterial strains belonging to 10 different genera for growth on human milk oligosaccharides. For this propose, we used a chemically-defined medium, ZMB1, which allows vigorous growth of a number gut–related microorganisms in a fashion similar to complex media. Interestingly, Bifidobacterium longum subsp. infantis, Bacteroides fragilis and Bacteroides vulgatus strains were able to metabolize milk oligosaccharides with high efficiency, while Enterococcus, Streptococcus, Veillonella, Eubacterium, Clostridium, and Escherichia coli strains grew less well or not at all. Mass spectrometry-based glycoprofiling of the oligosaccharide consumption behavior revealed a specific preference for fucosylated oligosaccharides by Bifidobacterium longum subsp. infantis and Bacteroides vulgatus. This work expands the current knowledge of human milk oligosaccharides consumption by gut microbes, revealing bacteroides as avid consumer of this substrate. These results provide insight on how human milk oligosaccharides shape the infant intestinal microbiota.