Identification and exploration of sleep-enhancing peptides from goat milk casein by peptidomics and virtual screening

Dietary proteins display sleep-enhancing properties, primarily due to the active peptides they release. However, the identification of specific sleep-enhancing peptide sequences remains limited. This study aimed to rapidly identify sleep-enhancing peptides in goat milk protein. In silico computational simulation predicted that papain, chymotrypsin, alcalase, and bromelain generate peptides with the highest activity. Among the four goat casein hydrolysates, papain hydrolysates significantly improved sleep in mice and effectively regulated the levels of neurotransmitters, including gamma-aminobutyric acid (GABA, 5.61 ± 0.76 μmol/mL), 5-hydroxytryptamine (5-HT, 108.90 ± 12.15 ng/mL), and orexin (54.42 ± 9.16 pg/L). Furthermore, 118 peptides were identified using peptidomics. Molecular docking analysis revealed that peptides containing aromatic amino acids and N-terminal proline exhibited strong binding affinity with the GABA_A receptor (GABA_AR) and orexin 2 receptor (OX2R) at the N and C terminals. The peptides FAWPQY and PVVVPPF demonstrated the highest binding affinities, which were - 10.2 and - 9.5 for GABA_AR, and - 10.5 and - 10.5 for OX2R, respectively. Notably, FAWPQY was particularly effective in extending sleep duration (37.08 ± 3.88 min) and decreasing sleep latency (5.06 ± 1.03 min) in mice. These results indicated that combining peptidomics, molecular docking, and animal testing is an effective strategy for screening sleep-enhancing peptides.