Infant formula-derived casein phosphopeptide ameliorates sleep deprivation-induced memory impairment via the modulation of the NMDAR-cAMP pathway in zebrafish

Infant formula (IF) serves as a key nutritional intervention in early life, where sleep disturbances are prevalent and may contribute to memory impairment. Although dietary bioactive peptides have been reported to regulate melatonin (MT) and potentially improve sleep, their effects on memory remain unclear. In this study, we investigated the efficacy of 3 different IF (I, II, III) in a para-chlorophenylalanine (PCPA)-induced zebrafish model of sleep deprivation. All formulas improved sleep architecture and modulated key neurochemical indicators related to sleep and memory, including MT, γ-aminobutyric acid (GABA) and N-methyl-d-aspartate receptor (NMDAR). Formula III exhibited the most significant effects, increasing MT from 90.64 pg/mL to 191.10 pg/mL, GABA from 9.60 μmol/L to 20.71 μmol/L, and NMDAR activity from 1,441.67 μg/mL to 2,654.00 U/L. It also markedly prolonged rest time, from 2,087.01 s to 3,568.77 s. Further analysis revealed that casein phosphopeptide (CPP) content increased across the formula stages and was highest in formula III. By itself, CPP dose-dependently enhanced the listed biomarkers, outperforming the intact formulas. From the CPP fraction, a novel tripeptide, histidine-proline-phenylalanine (HPF), was identified via amino acid sequencing and MS as a high-affinity binder to NMDAR. Molecular docking and dynamics simulations demonstrated that HPF interacts with key residues (D547, E572, M544) of NMDAR through hydrogen bonds and hydrophobic interactions, potentiating NMDA binding and receptor activation. These results uncover a mechanism through which HPF modulates the NMDAR-cAMP (cyclic adenosine monophosphate) signaling pathway, thereby improving sleep quality and memory under sleep-deprived conditions. Our study highlights the potential of peptide-based functional ingredients derived from IF in developing nutritional strategies for sleep and cognitive health.