| As we all know,breast milk is very crucial for the growth and development of newborns,especially oligosaccharides,which has important biological functions as the third largest solid component in milk other than lactose and fat.The structure of milk free oligosaccharides is complex and diverse,especially human milk oligosaccharides.Compared with human milk and bovine milk,there are fewer researchs on the structure and function of oligosaccharides in goat milk,especially the research on the structure and biological functions of human milk,bovine milk and goat milk oligosaccharides is less.In addition,milk oligosaccharide is a non-digestible oligosaccharide,which can reach the intestine to produce organic acids through fermentation,inhibit the growth of pathogenic bacteria and promote the proliferation of beneficial bacteria,it also has the function of maintaining the stability of the intestinal flora.Studies have shown that milk free oligosaccharides can promote the intestinal microbial level of abnormal mice to normal levels,but the regulation of milk free oligosaccharides is mainly concentrated on the effect on the abnormal flora,so it is necessary to expand the effect of milk free oligosaccharides on the structure of normal flora.In addition,clinical studies have shown that formula-fed infants are far more likely to contract disease than breast-fed infants,but improvements to formula have been focused on the addition and balance of proteins,vitamins,and trace elements,relatively little research on free oligosaccharides.Therefore,a comprehensive analysis helps to better understand the special biofunction of milk free oligosaccharides from different species,as well as provide an important scientific basis for finding out the differences between breast milk and infant formula,and then improving the efficacy of formula.In this paper,the structure of human,bovine and goat milk oligosaccharides were analyzed and quantified.On this basis,the inhibitory effects of human,bovine and goat milk free oligosaccharides to five common foodborne pathogens were compared.Effects to mice intestinal microflora abundance of three different types of milk free oligosaccharides were studied at the same time.The details of results are shown as follows:1.Structural analysis and quantitation of milk oligosaccharides 1)Structural analysis of human,bovine and goat milk free oligosaccharidesIn this chapter,UHPLC and MAIDI-TOF MS were used to analyze the milk free oligosaccharide structures.The milk oligosaccharides were extracted by chloroform-methanol method.After analysis by UHPLC,single peaks were collected for MALDI-TOF MS detection.According to the m/z value and GU value,the accurate structure was obtained by GlycoWorkbench,and the accuracy of the structure can be verified by MALDI-TOF MS/MS results.Structural analysis results showed that 34 oligosaccharide structures were detected in human milk,contains 5 sialylated oligosaccharides and 10 fucosylated oligosaccharides,of which 11 structures were only detected in human milk,not in bovine and goat milk.28 structures were detected in bovine milk,of which 12 sialylated structures and 4 fucosylated structures;26 structures were detected in goat milk,and which contains 8 sialylated structures,6 fucosylated structures.2)Relative quantification of milk oligosaccharidesIn his chapter,we use maltopentaose as a standard for the quantification of the milk oligosaccharide.The peak areas of maltopentose with different concentrations were obtained according to UHPLC,and a standard curve of peak area and concentration was drawn,then the peak area of milk oligosaccharides were substituted into the linear regression equation to obtain the relative content.The results showed that the amount of human milk oligosaccharides extracted from the same volume of milk samples was 10-15 times higher than that of bovine milk and goat milk oligosaccharides without considering the loss of oligosaccharides during extraction.2.Antibacterial activity of milk free oligosaccharidesIn this chapter,five common foodborne pathogens such as Escherichia coli O157,Salmonella,Listeria monocytogenes,Shigella,and Staphylococcus aureus as the target bacterias.The antimicrobial effect of milk oligosaccharides was study by inhibiting the growth of pathogens and antiadhesion effect of bacterias to the Caco-2 cells.According to the growth curves and inhibition zoon,the results showed that both human,bovine and goat milk free oligosaccharides had no inhibitory effect on the growth of pathogenic bacteria.The results of anti-adhesion test showed that the three milk oligosaccharides all had an inhibitory effect on the adhesion of five pathogenic bacteria to cells when compared with the control group,and human milk oligosaccharide had the highest anti-adhesion rate to S.aureus.3.Effects of milk free oligosaccharides to the intestinal microorganisms in miceIn this chapter,mice were used as models to investigate the effects of human,bovine and goat milk oligosaccharides to the intestinal microbial abundance of mice.Five-week-old C57/6J mice were given a certain amount of milk free oligosaccharide by gavage.After 21 days,the feces of the mice were collected for high-throughput sequencing.Sequencing results showed that the composition and abundance of intestinal microorganisms in mice ingesting milk free oligosaccharides had changed significantly.Bacteroidetes and Firmicutes were the dominant bacteria in the HMOs and GMOs group,which was consistent with the control group,while the amount of Proteobacteria in the BMOs group increased significantly and becomes the dominant bacteria.In addition,although the dominant flora in HMOs and GMOs groups unchanged,the abundance of Firmicutes increased significantly in HMOs and GMOs groups,and Proteobacteria in the GMOs group decreased slightly.Conclusion:This article has comprehensively elucidated the structure difference,the antibacterial activity and effects to intestinal microorganisms of human,bovine milk,and goat milk oligosaccharides at the glycomics level.The results showed that there are significant differences in the oligosaccharides structures and content,as well as the effect of milk oligosaccharides to intestinal microorganisms.the human,bovine and goat milk oligosaccharides all exhibited the antiadhesion activity,but can not inhibite the growth of pathogenic bacterias,and human milk oligosaccharide has better anti-adhesion activity than bovine and goat milk oligosaccharide.The results of this study indicate that differences in the structure of milk oligosaccharides are likely to be the main reason for the difference in health status between formula milk and breast-fed infants,and which can provide an important basis for improving formula milk or finding alternatives. |
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