Free Oligosaccharides in human milk (HMO) have multiple biological functions, for example, the growth of beneficial bacteria in the gut and the development of brain. It has been known that the concentration of free oligosaccharides in bovine milk and infant formula is relatively low compared with HMO. In thisstudy, we have profiled and quantified free oligosaccharides extracted from various milks and dairy products. HMO and free oligosaccharides extracted from domestic milk (bovine, porcine), commercial milk (milk, low-fat milk), four infant formulas made from goat and bovine milk were used for this study. For quantitative analysis, initially same volume 5ml of milk was taken. Extraction of free oligosaccharides was performed by following steps including the removal of lipids by Folch method and protein precipitation by ethanol. Extracted free oligosaccharides were further purified by solid phase extraction using a porous graphitized carbon cartridge.
Purified and enriched free oligosaccharides were redissolved in water prior to mass spectrometry analysis. Overall glycan profiling was obtained by MALDI-TOF/TOF MS. We found that human milk has more free oligosaccharides than the others. In addition, the ion at m/z 730.237 corresponding to [Hex]3[HexNAc]1 is the base peak while the ion at m/z 568.179 corresponding to [Hex]2[HexNAc]1 is the base peak in bovine milk. Interestingly, we found NeucGc in bovine milk as a major glycan, which is not produced in human anymore. Quantitative analysis of targeted glycans was performed by UPLC-Triple quadrupole. This study can be applied as an analytical platform to extract, profile, and quantify free oligosaccharides in milk and dairy products.