Isolation and Purification of Colostrum Oligosaccharides

Oligosaccharides were isolated from the colostrum samples following a previously published procedure [3 (link)]. Briefly, thawed samples were mixed with 4 volumes of a 2:1 chloroform/methanol solution and centrifuged. The upper layer was mixed with two volumes of pure ethanol and proteins were left to precipitate in the freezer at − 30 °C for 1 h. After a second centrifugation, the supernatant was dried in a speed-vacuum (Eppendorf Vacufuge Plus centrifugal concentrator). The dried samples were reconstituted in Milli-Q purified water, diluted five times and further purified by solid phase extraction (Glygen FNSCAR; Columbia, MD, USA) using a previously established method using water as the solution for equilibration [13 (link)].