Milk oligosaccharides (OS) are a key factor that influences the infant gut microbial composition, and their importance in promoting healthy infant development and disease prevention is becoming increasingly apparent. Investigating the structures, properties, and sources of these compounds requires a host of complementary analytical techniques. Relative compound quantification by mass spectral analysis of isobarically labeled samples is a relatively new technique that has been used mainly in the proteomics field. Glycomics applications have so far focused on analysis of protein-linked glycans, while analysis of free milk OS has previously been conducted only on analytical standards. In this paper, we extend the use of isobaric glycan tags to the analysis of bovine milk OS by presenting a method for separation of labeled OS on a porous graphitized carbon liquid chromatographic column with subsequent analysis by quadrupole time-of-flight tandem mass spectrometry. Abundances for 15 OS extracted from mature bovine milk were measured, with replicate injections providing coefficients of variation below 15% for most OS. Isobaric labeling improved ionization efficiency for low-abundance, high-molecular weight fucosylated OS, which are known to exist in bovine milk but have been only sporadically reported in the literature. We compared the abundances of four fucosylated OS in milk from Holstein and Jersey cattle and found that three of the compounds were more abundant in Jersey milk, which is in general agreement with a previous study. This novel method represents an advancement in our ability to characterize milk OS and provides the advantages associated with isobaric labeling, including reduced instrumental analysis time and increased analyte ionization efficiency. This improved ability to measure differences in bioactive OS abundances in large datasets will facilitate exploration of OS from all food sources for the purpose of developing health-guiding products for infants, immune-compromised elderly, and the population at large.

Milk oligosaccharides are of high interest due to their bioactive properties. This study is the first to characterise milk oligosaccharides from native North European cattle breeds, as represented by 80 milk samples collected from eight native breeds originated from Norway (Norwegian Doela cattle and Norwegian Telemark cattle), Sweden (Swedish Mountain cattle), Denmark (Danish Red anno 1970), Iceland (Icelandic cattle), Lithuania (native Lithuanian Black and White) and Finland (Western Finncattle and Eastern Finncattle). Using high-performance liquid-chromatography chip/quadrupole time-of-flight mass-spectrometry, 18 unique monosaccharide compositions and a multitude of isomers were identified. No N-glycolylneuraminic acid was identified among these breeds. Western Finncattle milk was most abundant in neutral, acidic and fucosylated oligosaccharides. Further, Eastern Finncattle milk was significantly higher in acidic oligosaccharides and Icelandic cattle milk significantly higher in fucosylated oligosaccharides, compared to the mean. This study highlights specific native breeds of particular interest for future exploitation of milk oligosaccharides and breeding strategies.

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O-linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including β-galactosidase, N-acetyl-β-d-glucosaminidase, certain oligosaccharide isomers as well as O-linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p-value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.