| Breast milk was widely regarded as the ideal food for infants because of rich nutrients and biologically active factors.Protein was the fourth largest nutrient in human milk,most of which was highly glycosylated.Protein glycosylation was an important post-translational modification,and the most widespread one is N-glycosylation.Glycosylation modification increased the complexity of protein structure,and many proteins could perform their normal functions by relying on correct glycosylation modification.Thus,comparing the structural stability of native and deglycosylated protein will help to understand the importance of glycosylation modification.In addition,many researches had shown that free oligosaccharides in milk could entere the large intestine directly and be fermented by the intestinal microorganisms without gastrointestinal digestion,thereby regulating the composition of intestinal microorganisms of the gut.Besides that,certain microorganism in the gut could secrete specific glycosidases to release N-glycans from glycoproteins to maintain their own growth.However,there was few research about the impact of N-glycans from human whey protein on the composition of intestinal microorganisms.Therefore,studying the impact of N-glycans from human whey protein on the intestinal microecological environment and the composition of the intestinal flora will help to understand the importance of N-glycosylation on the biological functions of protein and provide an important theoretical basis for the development of infant formula milk and functional dairy products in the future.In this study,human whey protein was used as object.The N-glycans of glycoprotein was separated with protein firstly,and then compared the structural stability and digestibility of human whey protein and deglycosylated protein.On this basis,human whey protein,deglycosylated protein and N-glycans were used as substrates for the fermentation of intestinal microorganisms in vitro,then studied the utilization of N-glycans and analyzed the effects of these substrates on the intestinal microecological environment and the composition of intestinal microorganisms.The specific research contents are as follows:1.The effect of N-glycosylation on the structural stability and digestive properties of human whey proteinHuman whey protein was removed N-glycans firstly,and then compared the physicochemical properties of human whey protein and de-N-glycosylated protein,including secondary structure fluorescence intensity,Zeta-potential,particle size,solubility,and digestibility.The results showed that,after deglycosylation,the secondary structure of protein had no significant changes(P>0.05),the fluorescence intensity of protein increased,the Zeta-potential of protein significantly reduced(P<0.05),the particle size of protein significantly increased(P<0.05)and the solubility of protein decreased.Furthmore,the digestibility of human whey protein was significantly higher than that of deglycosylated protein(P<0.05),which means that the attaching of glycans could help protein resist gastrointestin digestion.In addition,the structure of N-glycans from human whey protein was not affected by gastrointestinal digestion.2.The utilization of N-glycans from human whey protein by mice gut microbiota and its effect on the gut microenvironmentHuman whey protein,deglycosylated protein and N-glycans were used as substrates for the fermentation of mice intestinal microorganisms in vitro.The glycosidase activity of mice fecal microorganisms and the utilization of N-glycans by the intestinal microorganisms were detected,and then we detected the changes of p H and SCFAs during the fermentation of different substrates.The activity of galactosidase,N-acetylglucosaminidase,fucosidase could be detected in mice fecal microorganisms.N-glycans and monosaccharides including galactose,mannose,glucose,fucose and Neu5 Ac could be utilized by fecal microorganisms during fermentation in vitro.In addition,during fermentation,the p H of control group,HMDGP group and HMGP group all decreased at the beginning and then increased,and the p H of HMGP group was lower than other two groups significantly.However,the p H of N-glycan group decreased slowly but continuously.The result of SCFAs changes showed that there was no significant difference between HMDGP group and control group(P>0.05),while HMGP group and N-glycan group were significantly increased compared with control group(P<0.05),especially the content of acetic acid and propionic acid.Therefore,N-glycans was important for the regulation of intestinal environment.3.The effect of N-glycosylation on the mice gut microbiota by the fermentation of human whey protein in vitroHuman whey protein,deglycosylated protein and N-glycans were used as substrates for the fermentation of mice intestinal microorganisms in vitro,and high-throughput sequencing was used to analyze the composition of the intestinal microorganisms.The results showed that these three substrates could significantly affect the composition of intestinal microbes.Compared with the control group,the diversity of intestinal microbes in the HMGP group and HMDGP group was significantly reduced(P<0.05),while the N-glycan group did not change significantly(P>0.05).At the phylum level,compared with control group,the abundance of Bacteroides group was significantly increased in the HMDGP,but decreased in the N-glycan group(P<0.05),the abundance of Firmicutes in the HMGP and HMDGP groups.And the abundance of Proteobacteria in the HMGP group and N-glycan group were significantly increased(P<0.05),and the abundance of Proteobacteria in the HMGP group and N-glycan group were significantly increased(P<0.05).At the genus level,compared with control group,the abundance of the Muribaculaceae＿norank in the HMDGP,HMGP and N-glycan groups were all significantly reduced(P<0.05),but the abundance of the Bacteroides were all significantly increased(P<0.05).Besides that,the abundance of Alloprevotella in the HMDGP and HMGP groups were significantly increased(P<0.05),the abundance of Escherichia-Shigella in N-glycan group was significantly decreased(P<0.05).but the abundance of Escherichia-Shigella in HMDGP group was increased significantly(P<0.05).Compared with HMDGP group,the abundance of Firmicutes,Muribaculaceae＿norank and Alloprevotella were significantly decreased(P<0.05).Therefore,HMGP,HMDGP and N-glycans could significantly affect the structure and composition of mice fecal microorganisms,and there were some differences between these three groups,which means that N-glycosylation has an impact on the development of intestinal microorganisms guided by human whey protein. |
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