Enzymatic production of a suite of human milk oligosaccharides directly in milk

Human milk oligosaccharides (HMOs) denote specific glycans in human breast milk. They function as prebiotics, immune modulating, and antimicrobial agents in the gut of breastfed infants, and certain HMOs even promote the cognitive development of the baby. HMOs are virtually absent in cow's milk and hence in infant formula, which provides a huge incentive for identifying ways in which HMOs can be produced to improve infant for-mulas. Here, we show that different sialylated and fucosylated HMOs can be generated in cow's milk via different simultaneous enzymatic transglycosylation reactions catalyzed by an engineered sialidase (EC 3.2.1.18, from Trypanosoma rangeli) and an 1,2-alpha-L-fucosidase (EC 3.2.1.63, from Tannerella forsinthia) acting on the lactose in the milk and on casein glycomacropeptide, two types of commercially available HMOs, i.e. 2 '-fucosyllactose and lacto-N-neotetraose, added to the milk. We also outline the details of the individual reactions in aqueous systems, demonstrate that the enzymatic reactions can be accomplished at 5 degrees C, and validate the products formed by LC -MS and NMR analysis. Enzymatic production of HMOs directly in milk provides opportunities for enriching milk and infant formulas and extends the use of enzymatic transglycosylation reactions to synthesis of HMOs in milk and eventually in other beverages.

Automated summary

Human milk oligosaccharides (HMOs) are specific glycans in human breast milk that have various beneficial functions for breastfed infants. This study demonstrates the enzymatic production of different types of HMOs in cow's milk through simultaneous enzymatic transglycosylation reactions. The results show that these reactions can be accomplished at low temperature and the products are validated by analytical techniques. This method provides opportunities for enriching milk and infant formulas with HMOs.