Identification and Screening of Potential Bioactive Peptides with Sleep-Enhancing Effects in Bovine Milk Casein Hydrolysate

Casein tryptic hydrolysate (CTH) has been proven to possess stress-relieving and sleep-enhancing effects, but only one decapeptide YLGYLEQLLR (α-CZP) in CTH was reported to exhibit affinity for the benzodiazepine site of a GABAA receptor (GABAAR). This study aimed to compare the sleep-enhancing effects between CTH and α-CZP and to explore novel sleep-enhancing peptides. Our results showed that CTH significantly prolonged sleep duration in mice, which was almost 2-fold longer than that of α-CZP. The α-CZP in CTH was degraded more slowly than the synthetic α-CZP; meanwhile, CTH could release other potential sleep-enhancing peptides during gastrointestinal digestion. Additionally, two peptides YPVEPF and YFYPEL with strong sleep-enhancing activity were explored by virtual screening. Especially, YPVEPF could significantly prolong the sleep duration from 559.00 ± 272.24 to 2501.63 ± 1021.21 s and increase the sleep rate from 58.33 to 83.33% in mice. Moreover, YPVEPF and YFYPEL could bind with the Ser-205 and Phe-77 residues of GABAAR via hydrogen bonds and lipid contacts. They were largely released after digestion with 11.19 ± 0.15 and 1.78 ± 0.01 g/kg, respectively.