Determination Of Protein N-Glycome Profile In Milk And Its Application In The Identification Of Cow Milk Adulteration In Goat Milk

Goat milk is popular among consumers because of its high nutritional value,high digestion and absorption rate and low allergy rate.In recent years,the adulteration of high-value goat milk with low-value cow milk for profit has been widespread.The incorporation of cow milk into goat milk not only disturbs the order of food market,but also threatens the health of people with cow milk intolerance and the elderly and infants with fragile gastrointestinal tract.The common methods to identify adulteration of milk at home and abroad,such as immunoassay,electrophoresis,gene assay,spectroscopy,chromatography and mass spectrum methods,all have their own shortcomings.N-glycans are one kind of oligosaccharides linked to glycoprotein in milk.The composition and structure of N-glycans are regulated by glycosidase and glycosyltransferase in organisms,so they are highly specific among species.Therefore,N-glycome can be used as biomarker to develop a new glycomics based method for the identification of adulteration of dairy products.The advantages of this method are good stability of markers,high species-specificity,independent of whether the sample is degreased or not,and no cross-reaction.In this paper,the N-glycans of goat milk and cow milk were taken as the research object.Firstly,the N-glycans in goat milk and cow milk were extracted,separated and analyzed structures,compared with each other.Secondly,based on the UPLC differences of N-glycans between goat milk and cow milk,the method was established to identify goat milk adulteration using N-glycans as a marker.The details of research contents and results are as follows:1.Extraction,separation,structural analysis and difference comparison of N-glycans from goat and cow MilkIn this chapter,TCA method was used to extract milk glycoprotein,and PNGase F exoglycosidase was used to release N-glycans in goat milk and cow milk.All of possible N-glycans structures was inferred by UPLC separation and GU value,and further analyzed by MALDI-TOF-MS detection of mass/charge ratio.The results of structures analysis show that there are significant differences between the two milk species in UPLC results.A total of 37 kinds of N-glycans structures,including 5 kinds of high mannose,3 kinds of hybrid,and 29 kinds of complex were obtained from goat milk by detection.There are 14 kinds of sialylated N-glycans and 12 kinds of fucosylated N-glycans in goat milk.Comparing with cow milk,7 unique structures were detected in goat milk,which were:F1M3,A2G1,F1M5A1G1S1(Ac),M5A2G1S1(Ac),F1A3G3S1(Ac),F1A4G4S1(Ac),A4G4(β)G1(α)S1(Ac).2.Identification of milk adulteration based on UPLC analysis of goat milk and cow milk N-glycansIn this chapter,goat milk and cow milk of different varieties were used as research objects to verify the differences of N-glycans among different species,but the differences between different varieties of the same species are small.Then,the peaks with RPA greater than 2%in UPLC results of all goat milk and cow milk were collected,and the data set was established in the order of GU value from small to large.Firstly,the visual identification model of goat milk and cow milk was established through PCA,and the model contribution rate reached 80.30%.Secondly,PCA and HCA were used to qualitatively identify the adulteration proportions of cow milk in goat milk.Both PCA and HCA methods could significantly distinguish between pure goat milk and adulterated goat milk.Also,5%of cow milk adulterated in goat milk could be detected,and the contribution rate of PCA model reached 76.66%.By contrast,PCA is more clear and visual,while HCA can reflect the degree of species kinship between different samples.Based on this result,the PLS prediction model of milk adulteration in goat milk was further established.The established model R_c~2=0.902,R_v~2=0.931,RMSECV and RMSEP were 0.676%and 0.457%respectively.The prediction ability was excellent and could be used in laboratory detection.The results of this study provide a new method for the detection of milk adulteration,which can provide a new idea for detection of milk safety.