Natural variability in bovine milk oligosaccharides from Danish Jersey and Holstein-Friesian breeds

Abstract

Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds, Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contains levels of fucosylated oligosaccharides significantly higher than that of Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid), suggesting it may therefore offer advantages in term of a wider array of bioactivities.

Figure 1

(A) Representative base BPC of both Holstein-Friesian and Jersey milk oligosaccharides (Cow id: Holstein-Friesian: 4529, Jersey: 0502). (B) Neutral and acidic oligosaccharide abundances in 14 samples measured in duplicates (values: mean ± SD). Samples are colored according to neutral OS (black) and acidic OS (white).

Figure 2

Extracted ion chromatograms and calculated AUC in 14 samples measured in duplicates (mean ± SD) (A) HexNAc-lactose. (B) the two isomers of sialyllactose (3-SL and 6-SL). (C) sialyl-hexosyl-lactose.

Figure 3

(A) PCA score scatter plot of PC1 and PC2 showing targeted analysis of 29 oligosaccharides EIC being integrated by Molecular Feature using MassHunter software (Agilent). (B) Corresponding loading plot. Oligosaccharide IDs refer to Table 2 and 3.

Figure 4

Extracted ion chromatogram and calculated AUC in 14 samples measured in duplicates (mean ± SD). (A) 3Hex, 6HexNAc. (B) 5Hex, 4HexNAc, 1Fucose. (C) 4Hex, 5HexNAc, 1Fucose.

Figure 5

(A) PCA score scatter plot of PC1 and PC2 showing untargeted analysis of 20 different milk samples purified for oligosaccharides. Base peak chromatograms were extracted and 761 continuous variables were used. (B) Corresponding loading line plot.