Effects of gut bacteria and diet on unusual trisaccharides in mouse milk

Milk free oligosaccharides play critical roles in infant health, supporting beneficial gut microbiota and protecting against pathogens. While most milk free oligosaccharides follow well-characterized biosynthetic pathways initiated by lactose, recent studies have revealed several unusual trisaccharides that deviate from these canonical motifs. To investigate the origin of such atypical milk free trisaccharides, we analyzed the free milk trisaccharide profiles of conventional (microbiota-colonized) and germ-free mice using porous graphitized carbon high-performance liquid chromatography coupled with tandem mass spectrometry. One unique trisaccharide (compound Gal-β1→4-Glc-β1↔1β-Gal) was present exclusively in conventional mice, suggesting microbial contribution to its biosynthesis. In contrast, several other unusual milk free trisaccharides including Gal-β1→4-Glc-β1→4-Glc, Gal-β1→4-[Glc-α1→2]-Glc and Gal-β1→4-[Gal-β1→2]-Glc were detected in both conventional and germ-free mice, indicating an endogenous production. Comparison of the oligosaccharide Gal-β1→4-Glc-β1→4-Glc isolated from milk of mice fed on the normal diet and those maintained on a cellulose-free diet for four weeks revealed no difference in intensity. It suggests that its formation is independent of dietary cellulose and likely arises from endogenous biosynthetic processes.