Simulative Fabrication of Milk Fortified with Sialyloligosaccharides and Its Prospective Applications

Sialyloligosaccharides, critical components of human milk oligosaccharides, are pivotal for infant immunity and development. Recent advances have identified sialidases with <i>trans</i>-sialylation activity as promising biocatalysts for cost-effective sialyloligosaccharide synthesis using economical substrates. Herein, we report a novel sialidase (<i>Ks</i>Siase1) from <i>Kribbella</i> sp. ALI-6-A with enhanced catalytic properties and broad substrate specificity, showing preferential hydrolysis: α2,3 > α2,6 > α2,8. The enzyme demonstrated robust <i>trans</i>-sialidase activity, achieving 3.40 g/L sialyloligosaccharide production using 0.5 U/mL <i>Ks</i>Siase1, 6% lactose, and 8% cGMP. Implementing a direct-fed enzymatic strategy in raw milk with 1 U/mL <i>Ks</i>Siase1 and 6% cGMP, we achieved in situ sialyloligosaccharide enrichment (2.49 g/L) while concomitantly reducing lactose. This biocatalytic system establishes a sustainable, scalable platform for functional food production, merging lactose valorization with sialyloligosaccharides biosynthesis through enzyme-substrate adaptability, addressing both industrial demands and nutritional needs in infant formula development.