| HMOs is one of the active ingredients in human milk and have a variety of biological functions,such as promoting brain development,immune modulation and intestinal flora maintenance.However,all kinds of oligosaccharide including it’s isomers are mixted in nature,which hinders the study of specific oligosaccharides in it’s structure-activity relationship.Therefore,it is necessary to establish a method for isolating and preparing individual oligosaccharids with intact structure.In this study,maltodextrin was used as standard oligosaccharides.a method with higher resolution and ability to prepare a certain amount of oligosaccharide monomer with its original structure was established.As an application,neutral and acidic human milk oligosaccharides fraction from DEAE-52 and PGC were furher separated into individual oligosaccharides.In addition,the detailed structure of neutral individual oligosaccharides was investigated by permethylation and MS~n fragmentation.The main results are described as follows:1.A method for separating and regenerating reducing oligosaccharides was proposed and established.For the natural free reducing oligosaccharides mixtures,they was derivatived with hydrazine reagents to introduce a chromogenic group and then used for high performance liquid chromatography(HPLC)separation.The sample solution of each fraction collected after separation is treated with aqueous solution of 5%acetic acid at 70℃for 45min to remove the tag and regenerate the parental-free reducing glycans.By this method,the reducing maltose monomers can be regenerated from it’s derivatives labeled with seven hydrazine reagents respectively,and the derivatization efficiency of the seven reagents and the recovery yield of the corresponding reducing oligosaccharides are compared,As a result,BSH is the most suitable derivatization reagent for the preparation of reducing oligosaccharide monomers,followed by Girard’s P.The method was successfully used to prepare 14 reducing maltose monomers and 11 N-glycan monomers of albumin without the formation of by-products.2.A method for isolation and purification of human milk oligosaccharides was established.Crude human milk oligosaccharides was separated into the neutral and acidic oligosaccharides fraction by the DEAE-52 anion exchange column firstly.As for the neutral oligosaccharides fraction,which was seperated by the graphite carbon chromatography column to desert the lactose;As for the acidic oligosaccharides fraction,which was purified by the dialysis bag to remove the salt.In addition,the method was combined with the Girard’s P target derivatization method,selective sialic acid derivatization method(distinguishingα2,3-linked sialic acid withα2,6-linked sialic acid)to analysis of the seperated oligosaccharides fraction based on MALDI-TOF-MS detection technique.The results showed that there were 63 oligosaccharides in mature milk,including 36 neutral oligosaccharides and 27 acidic oligosaccharides,of which 28 fucosylated neutral oligosaccharides,8 oligosaccharides withα2,3-linked monosialic acid,and 2 withα2,3-linked diasialic acid;10 oligosaccharides withα2,6-linked monosialic acid,and 2 disialic acid;5 disialylated oligosaccharides modified byα2,3-andα2,6-linked sialic acid.3.The neutral oligosaccharide and the acidic oligosaccharide from DEAE-52 and PGC are further separated by the method of separating and regenerating reducing oligosaccharides to prepare reducing human milk oligosaccharide monomer.After HILIC-HPLC separation,10neutral oligosaccharide components and 9 acid oligosaccharide components were obtained.And the 10 neutral oligosaccharide components were futher separated by PGC-HPLC,a total of 17 neutral oligosaccharide monomer components were obtained and following analysed by permethylation MS~n,and 19 structures of oligosaccharide molecules were identified as2’-FL,3’-FL,GL,DFL,LNT,LNnT,LNFPⅡ,LNFPⅢ,LNFPV,LNnDFHI,LNnDFII,LNDFI,LNDFII,LNH,MFLNnH,MFLNH,DFpLNnH,DFpLNH and DFLNnH respectively. |
PDF Full Text Request