Study On The Effects Of N-glycosylation In Human And Bovine Milk Proteomes' Anti-pathgenic Activity

Oligosaccharides including free oligosaccharides and oligosaccharides attached to proteins are the third abundant big molecule component in milk.Breast-fed infants are much healthier than formula-fed infants despite great efforts have been given in improving the nutritional value of infant formula by adjusting the levels of major milk components,such as protein,fatty acids,vitamins and trace elements,etc.This phenomenon indicates that formula optimization needs a new direction which could be milk oligosaccharides.Extensive studies have been reported on the function of free oligosaccharides,but lack of oligosaccharides attached to proteins which are at equal importance.As is known to all,glycosylation act an extremely important role in biological function of protein molecule in the organism.What can glycosylation do on the funcation of milk glycoprotein as an exogenous protein?Our laboratory already did thorough research on structure and funcation of human and bovine milk N-glycans early,and found that both human and bovine milk N-glycans have growth suppression and anti-adhesion activities,but human N-glycans is better than bovine N-glycans.This result is direct at released N-glycans.However,N-glycans were attached to protein when been taking in by infants.When sugars enter babys' body in the form of glycoproteins,its function and mechanism keeps unknown.Therefore,it has great significance to do further research on milk protein glycosylation.The entire human and bovine N-glycomes and(deglycosylated)proteomes were investigated as the objects of study.First,a technology method for deglycosylation on milk glycoprotein was established and optimized.Based on this,the anti-pathogen activity of the milk N-glycomes and(deglycosylated)proteomes including growth inhibition and anti-adhesion of pathogenic bacteria to host cells was conducted.The details of the results are as follows:1.Establishment and optimization of the technology method for deglycosylationIn this study,the following technologies were tested and optimized:lipid removal,remove free sugars,milk protein deglycosylation,the separation of N-glycans and deglycosylated protein and the further purification of N-glycans.The method for deglycosylation of glycoprotein which includes multi-step and multi-technology is established.After comparing,the optimal combination of techniques comprise free oligosaccharides removing by dialysis with 8000?14000 Da dialysis tube at low temperature,N-glycans release with PNGase F and do three consecutive reactions with total 16×3 h,separation of deglycosylated protein and N-glycans with 10 kDa ultrafiltration tube at low temperature,N-glycans further purification with sephadex G-25 chromatographed column.2.Effect of N-glycosylation on antibacterial activity of milk protein——Growth suppression effectIn order to investigate antibacterial effect of the milk N-glycome and(deglycosylated)proteomes,we selected Escherichia coli.0157,Salmonella typhimurium,Listeria monocytogenes and Staphylococcus aureus as the target bacteria in this study.And milk N-glycomes and(deglycosylated)proteomes were used as the target substreates.The growth suppression activity of target substreates was studied by investigating OD 600 value.The results showed that With N-glycosylation or not,the suppression of human and bovine milk protein sample have no unified rule,and the differences between human and bovine milk protein is not significant.In short,N-glycosylation makes no significant influence on growth suppression activity of both human and bovine milk proteomes.Milk N-glycomes have obvious growth suppression activity.No matter human source or bovine milk source.No matter glycoprotein or deglycosylated protein,bacteriostatic activity of protein samples is significantly lower than N-glycomes.Bovine and human milk proteins' inhibition activity has no significant difference.And after digestion,the results have no difference with undigested group.3.Effect of N-glycosylation on antibacterial activity of milk protein-Anti-adhesion effectSalmonella typhimurium was used as the target bacteria and human colonic cancer cells(Caco2 cell)as adhesion cell model in this study.Anti-adhesion of pathogenic bacteria to intestinal cells was investigated on milk N-glycomes and(deglycosylated)proteomes with cell culture and plate counting method.The results showed that N-glycosylation can enhance anti-adhesion activity of human and bovine proteomes.The milk N-glycomes have obvious anti-adhesion activity and human milk source sample has equal anti-adhesion activity with the bovine milk.The milk proteomes have anti-adhesion activity and human milk source samples show equal anti-adhesion activity with the bovine milk.After digestion,human milk source samples show better anti-adhesion activity than the bovine milk.