Rapid, quantitative analysis of 3′- and 6′-sialyllactose in milk by flow-injection analysis-mass spectrometry: Screening of milks for naturally elevated sialyllactose concentration

Non-protein-bound oligosaccharides are important bioactive components of cow milk, with potential human-health benefits such as stimulation of the growth of beneficial gut bacteria and defense against pathogens. In bovine milk, the majority of oligosaccharides are sialylated; 3′-sialyllactose (3′-N-acetylneuraminyl-d-lactose; 3′-SL) is the predominant sialylated oligosaccharide, followed by 6′-sialyllactose (6′-N-acetylneuraminyl-d-lactose; 6′-SL). Both 3′-SL and 6′-SL have antimicrobial activity. As bovine milk products such as infant formula can be an important component of the human diet, and the concentrations of 3′-SL and 6′-SL are lower in bovine milk compared with human milk, we aimed to identify cows that naturally produce higher concentrations of sialyllactose in their milk. Milk from such cows could be used to produce foods with an increased sialyllactose content, potentially providing increased health benefits. We speculated that cows overexpressing 3′-SL and 6′-SL would exist at low frequency in the population and, to allow their efficient identification, we developed a novel assay for 3′-SL and 6′-SL utilizing flow-injection analysis-mass spectrometry, which could be used for high-throughput analysis of milk samples. We then determined 3′-SL and 6′-SL concentrations in milk samples from 15,507 cows from Friesian, Jersey, and Friesian-Jersey crossbred animals. We found 329 cows with concentrations of 3′-SL or 6′-SL >2-fold higher than the mean, 26 cows with concentrations of 3′-SL or 6′-SL >3-fold higher than the mean, and 1 cow with concentrations of 3′-SL >4-fold higher than the mean. Although these outliers were observed across the 3 groups of cows, breed had a strong effect on mean 3′-SL and 6′-SL concentrations.