Separation Of Human Milk Oligosaccharides And Its Quantitative Analysis In Infant Formula

Human milk has long been considered the best source of nutrition for nearly all infants,which is the “gold standard for nourishing a baby's growth”.Human milk oligosaccharides(HMOs)are the third largest solid component after lactose and fat which have beneficial effects to infant's health.They play an irreplaceable role in the growth of infantile intestinal flora,the improvement of immune system function and the resistance to pathogen invasion.However,due to HMOs' complex structures,numerous isomers,and large difference in quantities,all of these have caused huge challenges to the study of the relationship between human milk oligosaccharides' structure and function.Therefore,it is vital to develop a suitable method for the separation and analysis of HMOs.Infant formulas are manufactured predominantly with mature bovine milk,which lacks most of the HMOs commonly found in human milk,and contains only very low levels of oligosaccharides,such as 3?-Sialyllactose(3?-SL)and 6?-Sialyllactose(6?-SL).With the development of biosynthesis technology of HMOs,in order to better simulate human milk to meet the needs of infant growth and development,the United States and the European Union have now been approved to add 2?-fucosyllactose(2?-FL)and Lacto-N-neotetraose(LNn T)to infant formulas.Therefore,a suitable method is needed to accurately and quantitatively determine the human milk oligosaccharides in infant formulas.Human milk oligosaccharides were separated and prepared from human milk by amultidimensional chromatography(MDC)strategy and their detailed structural analysis was performed.And then an online HILIC-HILIC method was developed for the determination of four HMOs in infant formulas.The main research contents and results are described as follows:1.Thirty-four human milk oligosaccharides including twenty-four neutral HMOs and ten sialylated ones were isolated and purified by a multidimensional chromatography strategy(MDC).Neutral HMOs were purified from a two-dimensional separation fraction with a polymerization degree of 6-8,including IFLNH-III/MFLNH-III/ MFLNH-I/ MFLNn H and other isomers.Sialylated HMOs were separated from the one-dimensional fraction,including 3?-SL/ 6?-SL,LST-a/ LST-b/ LST-c etc.Using electrospray ionization-quadrupole-time of flight mass spectrometry(ESIQ/TOF-MS),detailed structural analysis of the partial obtained HMOs compounds including many isomers was carried out.This work provides a scientific basis for the separation and purification of complex human milk oligosaccharide isomers,structural analysis and the discovery of novel HMOs.2.An online HILIC-HILIC method was developed to achieve fast and accurate determination of four kinds of HMOs including the high content of added 2?-FL and LNn T,and trace amounts of endogenous 3?-SL and 6?-SL in infant formulas.Following the improvement in extraction method and labeling with 2-aminobenzamide(2-AB),four kinds of oligosaccharides were divided into two parallel hydrophilic interaction chromatography(HILIC)to separate further and detected by ultraviolet and fluorescent detectors respectively.This analytical technique not only can be applied to determination of endogenous 3?-SL and 6?-SL in infant formulas of different brands,but also to the rapid and accurate quantification of all kinds of HMOs in novel infant formulas.