Detection Of Polar Lipids In Human Milk Based On UPSFC-Q-TOF-MS Technology And Its Application

Human milk is the most ideal food for infants,among which polar lipids are a large class of lipids located on the milk fat globule membrane,mainly from the endoplasmic reticulum membrane and lactating cell membrane,including phospholipids,cholesterol,gangliosides,etc.It is low in content(accounting for 2% of milk fat)but highly correlated with the gut,immune and cognitive development of infant.Due to the large variety of polar lipids in human milk,the large differences in properties and the wide distribution of polar ranges,it is difficult for polar lipids detection.And make it difficult to further study the function of polar lipids in human milk.Therefore,a method for the detection of polar lipids in human milk was established in this study by ultra-high efficiency supercritical fluid chromatography-mass spectrometry(UPSFC-Q-TOF-MS),and the influencing factors of the composition of polar lipids in human milk were studied.The main contents are as follows.Firstly,human milk polar lipids were separated and purified by solid phase extraction.The detection method of human milk polar lipids was established by UPSFC-Q-TOF-MS.The results showed that the amount of milk fat required for solid phase extraction was reduced from 200 mg in the classical method to 50 mg,and most triglycerides were removed,while high recovery of cholesterol,ceramides and phospholipids(95.05%～104.23%)was achieved.The established detection method can separate 18 lipid classes and 219 lipid molecular structures,and identify their fatty acid chain information.The separation time is shortened to10 min,and further quantitative analysis is performed by external standard method.Secondly,human milk polar lipids were determined by the above method,and their composition characteristics were analyzed.The results showed that a total of 179 polar lipids were identified in human milk,and sphingomyelin(SM)was the most abundant phospholipid in human milk with a content of 57%.Nervonic acid is the most abundant ultra long chain unsaturated fatty acid and only occurs in SM and ceramide(Cer)molecules.Phosphatidylethanolamine(PE),phosphatidylcholine(PC)and phosphatidylinositol(PI)are the main carriers of ARA in the polar lipids of human milk,and the ARA content accounts for52.30%,24.67% and 14.99% of the ARA content of polar lipid fatty acids in human milk,respectively.PE is the polar lipid of human milk.DHA is the most important carrier in sexual lipids,and the DHA content accounts for 67.79% of the DHA content of polar lipid fatty acids in human milk.Thirdly,the changes in human milk polar lipids with lactation period and region were investigated based on 126 human milk samples.The results showed that colostrum was significantly different from transitional milk and mature milk,while transitional milk was similar to mature milk.In terms of SM,PE and total phospholipid content,colostrum was significantly lower than mature milk,and transitional milk content was between those two with no significant difference.In terms of polar fatty acids,different fatty acids have different laws.On the DHA content of lysophosphatidylcholine(LPC)and lysophosphatidylethanolamine(LPE),transitional milk(3.662 mg/100 m L,59.797 mg/100 m L)and mature milk(3.456 mg/100 m L,60.701 mg/100 m L)were significantly higher than colostrum(2.391 mg/100 m L,35.299 mg/100 m L).At the mature milk stage,the differences between North China and South China were more significant than those between North China and Central China,and South China and Central China.The total contents of SM,PC,polar lipids and phospholipids containing nervonic acid in human milk from North China were significantly higher than those from South China.In addition,odd carbon fatty acids 15:1,17:0,21:0,23:0,and 25:0 varied across regions.Finally,the polar lipid composition of different milk samples was investigated.The results show that the content of SM in donkey milk is second only to PE in terms of the proportion of phospholipids,which makes it a potential raw material for infant formula.But in terms of phospholipid molecules,animal milk,infant formula and milk fat globule membrane ingredient are obviously different from those of human milk.The relative content of polyunsaturated fatty acids(11.79%)in polar lipids of human milk was much lower than that of animal milk,infant formula and milk fat globule membrane ingredients(23.12%～29.62%).In addition,this study detected abundant Cer in milk samples,and camel milk had the highest Cer content(60.885 mg/100 m L)in animal milk.Similar to human milk,the major fatty acids in SM are long or very long chain monounsaturated or saturated fatty acids,such as nervonic acid.And the total amount of Cer molecules containing nervonic acid in each dairy product Cer accounted for the percentage of the total Cer content ranging from22.290% to 38.237%.In summary,this subject used UPSFC-Q-TOF-MS technology combined with solid phase extraction to establish a method for the detection of polar lipids in human milk.In addition,the polar lipids of several animal milk,infant formula and milk fat globule membrane ingredients and that of human milk were determined,and their composition differences were analyzed.It provides technical support for in-depth research on polar lipids in human milk in the future,and also provides a reference for the design of infant formula based on human milk.