Study On Mass Spectrometric Analysis Of Goat Milk Oligosaccharides And The Changes Of Oligosaccharides In Heating Process

Milk contains complex nutrients,and the types of oligosaccharides in milk are numerous.The content of oligosaccharides in breast milk is higher(10-15 g/L)than any other milk.However,only the low molecular weight oligosaccharides in human milk can be synthesized by chemical methods due to differences of size,charge and structure.Therefore,alternative resources for human milk oligosaccharides have attracted the attention of researchers.The content of oligosaccharides in animal milk was 0.02-1.5 g/L,and the content of oligosaccharides in goat milk was higher about0.25-0.30 g/L.Goat milk oligosaccharides were an alternative resources for human milk oligosaccharides.In this paper,ultra-high performance liquid chromatography coupled with mass spectrometry was used to evaluate oligosaccharides in goat milk.The changes in the types and contents of oligosaccharides in Guan-zhong and Sa-neng goat milk were studied.Effects on the types and contents of goat milk oligosaccharides by different sterilization temperatures were evaluated.The effect of oligosaccharides in the separation of pilot membranes was observed.The research results are as following:(1)Establishment of detection method of ultra-high performance liquid chromatography coupled with mass spectrometry for goat milk oligosaccharides.The protein removal method was optimized in goat milk,Sep-PakC18,graphitized carbon column,ultrafiltration,dialysis and Sephadex-G10 gel column chromatography were used to purify oligosaccharides and remove lactose from crude extracts.The removal rate of lactose and retention rate of oligosaccharides were compared.Furthermore,the liquid phase conditions and mass spectrometry conditions of UPLC-Q-Exactive focus mass spectrometry were optimized by optimal extraction and enrichment methods.The final optimization conditions were 0-20 min,95%-78%A;20-35 min,78%-73%A;35-38 min,73-62%A;38-45 min,62%-50%A.The mass spectrometry conditions were set as followings:The electrospray ion(ESI)source was operated under positive polarity using 3.8 kV capillary voltage and negative polarity using 3.8 kV capillary voltage at 320?.The spherical gas pressure was 25 arb,the auxiliary gas pressure was 8 arb,and the heating temperature was 350?.Mass of m/z 300 to 2000oligosaccharides was selected in positive and negative polarity.Finally,the results showed that the ratio of skim milk to absolute ethanol was 1:2,the reaction temperature was-20?,and the removal rate of protein was 87.01%under the condition of 2 h.The goat milk purified by Sephadex-G10 gel column chromatography showed the highest removal rate of lactose and retention rate of oligosaccharides.The numbers of oligosaccharides of 62 were significantly higher in the samples purified by gel chromatography than the crude extract samples of 29.(2)The types and abundances of oligosaccharides in Guan-zhong and Sa-neng goat milk were analyzed.The results showed that the types and abundances of oligosaccharides were different between individual Guan-zhong goat milk and Sa-neng goat milk.52 kinds of oligosaccharides were identified in Guan-zhong goat milk.The abundance of oligosaccharides was 1.96×10~9.54 kinds of oligosaccharides were identified in Sa-neng goat milk.The abundance of oligosaccharides was1.00×10~9.The number of oligosaccharides in Sa-neng milk was greater than that of Guan-zhong goat milk,while the abundance of oligosaccharides in Guan-zhong goat milk was greater than that of Sa-neng goat milk.(3)Effects on the types and abundance of goat milk oligosaccharides by different sterilization temperatures were evaluated.The results showed that 60 types of oligosaccharides were identified in goat milk,and 42 types of oligosaccharides were identified in bovine milk.In the goat milk,18 types of oligosaccharides were identified in goat milk more than bovine milk.The oligosaccharide content gradually reduced with increased of sterilization temperature.The abundance of oligosaccharide of goat milk and bovine milk was 2.04×10~9 and 1.58×10~9 in the uncontrol group,respectively.However,the abundance of oligosaccharide of goat milk and bovine milk was 9.58×10~8 and 6.64×10~8 in the 137?/5s sterilization temperature,respectively.The loss rate of oligosaccharides reached to 53.11%and 57.99%,respectively.(4)Separation of goat milk oligosaccharides by pilot membrane.When the dilution factor was 6,the removal rate of lactose was increased with the increase of the concentration factor,whereas the retention rate of oligosaccharide standards remains remained constant.When the concentration factor reached 4,the removal rate of lactose reached to 44.18%,and the retention rate oligosaccharide standards was about50%.0.096 g of iGb3,3'-SL and 6'-SL oligosaccharides were obtained in 1 L of goat milk,which supported a theoretical foundation and data support for industrial production.