Study And Application Of Lipid Profilings In Dairy Products By Chromatography Coupled Mass Spectrometry

Lipids are core molecules in all biological processes.Lipids are a primary form of energy storage and provide fat-soluble vitamins and essential fatty acids,that play a key role in the formation of biofilms and signal transmission.It is reported that lipids in human milk and other dairy products are the most complicated substance in nature,and there are several thousand of lipid species.Human milk is regarded as an excellent source of energy and nutrients for infants.As the next best alternative to human milk,commercial infant formula has always regarded human milk as the “gold standard” in production.Therefore,it has always been a hot research topic to rationally mix the lipids in infant formula to make it similar to human milk in lipid composition.Human milk contains 3-5%(w/v)of lipids.The extreme structural diversity and the large differences in physicochemical properties of lipids in human milk are challenging for lipids analysis.Limited information is available on the comprehensive lipid composition,therefore,an intensive study into human milk lipid profiling is important for infant formula research.In addition,the production process of infant formulas involves pasteurization,homogenization,spray drying,and other processes.There are few studies on whether the structure and composition of lipids change during the production processes.To improve the design of infant formula that similar to human milk and to better understand the role of lipids in infant development,the precise profiling of lipids(including classes,subclasses,and individual molecular species)is necessary.Based on supercritical fluid chromatography-mass spectrometry(SFC-MS)and hydrophilic interaction chromatography-mass spectrometry(HILIC-MS),lipids in human milk,mammalian milk,and infant formulas were comprehensively analyzed,and the changes of the lipids in the production chain of infant formulas were studied in this thesis.The results are as follows:(1)To distinguish the triacylglycerol(TAG)profile and fatty acids composition of human milk over lactation period from mammalian milk,supercritical fluid chromatography-quadrupole time-of-flight mass spectrometry(SFC-QTOF-MS)was applied for the determination of TAG of colostrum,transitional,mature,cow,buffalo,goat,yak,and camel milk.And the data was processed using PCA and HCA to compare the lipid similarity between human milk and other mammalian milk.The experimental results showed that there was a great difference in TAG profile between human milk and mammalian milk,and there are many different TAG species in different types of milk.Human milk TAG consisted of more unsaturated fatty acids(UFAs).Colostrum milk contained a relatively high proportion of TAG contained long-chain fatty acids(LCFAs),while in transitional and mature milk,more TAG with medium fatty acids(MCFAs)was observed.Camel milk was rich in TAG containing UFAs,LCFAs,and TAG with higher molecular weight(Mw?790).TAG containing short-chain fatty acids(SCFAs)and MCFAs were abundant in goat milk.Cow and yak milk contained relatively fewer TAG with higher molecular weight and were rich in TAG containing SFAs.Buffalo milk was rich in LCFAs and TAG with higher molecular weight(Mw?792).PCA and HCA results show different mammalian milk could be distinguished according to their TAG composition.In mammalian milk,camel milk was the most similar to human milk in terms of degree of saturation,length of fatty acids,and molecular weight distribution.The lowest similarity was found between goat and human milk because goat milk was rich in MCFAs and SCFAs.Therefore,according to the lipid characteristics of mammalian milk,it can formulate their lipids as human milk lipid substitutes for infant formulas.(2)A HILIC-QTOF-MS method was developed to separate phospholipids(PL).Under the optimized conditions,eight classes of PL were completely separated within 10 min and the individual composition of PL species was determined by high resolution mass spectrometry.The developed method was applied to analyze the PL profiles and fatty acids composition of human,cow,buffalo,and goat milk.The results show that the molecular species of phosphatidylethanolamine(PE),sphingomyelin(SM),and phosphatidylcholine(PC)in human and mammalian milk were the most abundant,and the content of these three PL classes was relatively higher.The PL composition of human milk was significantly different from that of mammalian milk.PE(18:1/18:1),SM(d18:1/22:0),and PC(18:1/18:1)were the most abundant species in human milk,PE(18:1/18:1),PC(16:0/18:1),and SM(d18:1/16:0)were the most abundant species in cow and buffalo milk.PE(18:1/18:1),PC(16:0/18:1),and SM(d18:1/16:1)were the most abundant species in goat milk.An indepth study into the composition and structure of PL in human and mammalian milk will further improve the design of infant formulas mimicking human milk.(3)The production of infant formulas usually includes several key processes such as heat treatment,homogenization,and spray-drying,etc.These processing techniques may have an impact on the structure and composition of lipids because of heating processes involved,especially for heat-sensitive PL.Thus,the HILIC-QTOF-MS method was used for qualitative and quantitative analysis of PL in raw,pasteurized,homogenized,and spray-dried milk samples to investigate the effects of processing on PL during the production chain of infant formulas.The results show that pasteurization caused minor changes on most PL species,whereas the spray-drying process led to a reduction in PC,PI,PS,and LPC.Multivariate data analysis(PCA and OPLS-DA)was used to further investigate the effect on processing technology.The most varied PL components in the production chain of infant formulas screened by chemometric techniques contain unsaturated fatty acids.The content of PL species such as LPE(16:0)and LPE(18:2)increased significantly after the spray-drying process.Therefore,more attention should be paid to supplement these PL species when optimizing the infant formulas,or other drying methods can be considered to reduce the loss of PL during the production chain to meet the nutritional needs of infants.(4)To achieve high-throughput,comprehensive,and accurate determination of milk lipids,SFC-ESI-QTOF-MS method for qualitative and quantitative analysis of both nonpolar and polar lipids was developed.Under the optimized chromatographic conditions,12 lipids classes(triacylglycerols,TAG;diacylglycerols,DAG;monoglyceride,MAG;fatty acids,FA;phosphatidylcholine,PC;phosphatidylethanolamine,PE;phosphatidylinositol,PI;phosphatidylserine,PS;phosphatidylglycerol,PG;sphingomyelin,SM;lyso-phosphatidylcholine,LPC;lysophosphatidylethanolamine,LPE)were effectively separated within 7 min.Chromatographic mechanisms and the fragmentation laws of lipids were discussed.The developed method was applied to identify and quantify lipid species in cow,bovine,goat,human milk,and infant formulas samples.250 lipid species from 12 different lipid classes in milk samples were detected in this study.The results obtained in this current study demonstrate that there were large differences in the concentration between different lipid classes,therefore,more attention should be paid to comprehensive lipid analysis in different types of milk samples to evaluate objectively the nutritional value of dairy products.