Separation And Structural Analysis Of Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) play important roles in many biological processes including beneficial effect of intestinal bacteria, antiviral activity and postnatal brain development, etc. The structure-activity study of HMOs has been seriously affected by the challenge of synthetic approaches, therefore the separation of HMOs from natural sources is still a better choice. In this study, the human milk was used as raw materials, and the crude oligosaccharides were isolated from human milk in the yield of 53 g/L. After fat and protein removal, the total sugar and protein content of HMOs were determined to be 92.5% and 3.7%, respectively. Eighteen natural and eight sialylated HMOs were obtained by multidimensional chromatographies (MDC) including low pressure gel permeation chromatography (LPGPC), high performance anion exchange chromatography (HPAEC) and porous graphitic carbon high-performance liquid chromatography (PGC-HPLC). The structural properties of these oligosaccharides were characterized by electrospray collision-induced dissociation mass spectrometry (ESI-CID-MS/MS). Based on the MS analysis, each oligosaccharide was identified from trisaccharide to nonasaccharide, and structural isomers were observed due to the versatile linkages. Among the eighteen neutral HMOs, the number of isomers of tri-, tetra-, penta-, hexa-, hepta-, octa- and nanosaccharide was identified as two, two, three, two, two, four, two, respectively. Among the seven sialylated HMOs, the number of isomers of tri-, penta-, hexa-, hepta-, octasaccharide was classified as two, two, two, one, one, respectively. The core structures of HMOs were composed of disaccharide unit including Galβ1-3/4GlcNAc located at the nonreducing end and lactose unit (Galβ1-4Glc) located at the reducing end, and the linkages between disaccharide units were identified as β1-3 or β1-6 glycosidic bond. In addition, Gal, Glc and GlcNAc residues were fucosylated at C2, C3 and C3/C4, respectively. Meanwhile, sialylation often occurs at C6 of GlcNAc and C3/C6 of Gal.In summary, tweenty-six human milk oligosaccharides containing versatile isomers and complex structures were first obtained using a multidimensional chromatography (MDC) strategy, and their structural sequences were determined by ESI-CID-MS/MS technique. It was shown that a rapid and sensitive detection of structurally complex oligosaccharides had been successfully developed in this dessertation. We believe that the methodlogy developed in this study will be very helpful for the further investigation of structure-activity relationship of human milk oligosaccharides.