Phospholipid Remodeling and Tri‐Layer Membrane Reconstruction Mediate Cognitive Effects of Humanized Milk Fat Globules in Neonatal Rats

Abstract Human milk fat globules (MFG) are critical for cognitive development during early life. Here, the first study is presented to simulate the phospholipid composition of human milk fat globules and reconstruct their tri‐layer membrane to evaluate their effects on cognitive function. Humanized milk fat globule membrane phospholipids (MFGM‐PL+SM) significantly promoted the proliferation and neuronal differentiation of hippocampal neural progenitor cells (NPC). Transcriptomic and proteomic analyses reveal that MFGM‐PL+SM upregulated cholinergic signaling and neurotrophic factor pathways in differentiated NPC. Layer‐by‐layer deposition produced tri‐layer milk fat globules (T‐MFG) with enhanced stability and digestibility over single‐layer milk fat globules (S‐MFG). Formula supplementation with humanized MFG modulated serum metabolite profiles in rat pups, improving neurodevelopment and increasing hippocampal sphingomyelin levels. Moreover, key neurodevelopmental proteins in formula‐fed pups approached levels observed in breastfed controls. These findings highlight the potential of humanized MFG to support infant cognitive development and provide insights for designing enriched infant formulas.