Four novel sleep-promoting peptides screened and identified from bovine casein hydrolysates using a patch-clamp model in vitro and Caenorhabditis elegans in vivo

Bovine casein hydrolysates (CHs) have demonstrated sleep-promoting activities. However, only few peptides were identified from CHs with sleep-promoting effects. In this work, an in vitro model based on the electrophysiology of brain neurons was established for the evaluation of sleep-promoting effects. Based on this model, four novel peptides were systematically separated from CH. Compared with the control group, the action potential (AP) inhibitory rate of four peptides increased by 38.63%, 340.93%, 233.28%, and 900%, respectively, and the membrane potential (MP) change rate of four peptides increased by 319.78%, 503.09%, 381.22%, and 547.10%, respectively. These results suggested that four peptides have sleep-promoting activities. Furthermore, Caenorhabditis elegans (C. elegans) sleep behavior results indicated that all the four peptides could significantly increase the total sleep duration, the motionless sleep duration of C. elegans, implying that these four peptides can significantly improve sleep. The LC-MS/MS results showed that the primary structures of these novel peptides were HQGLPQEVLNENLLR (α_s1-CN, f8-22), YKVPQLEIVPNSAEER (α_s1-CN, f104-119), HPIKHQGLPQEVLNENLLR (α_s1-CN, f4-22), and VPQLEIVPNSAEER (α_s1-CN, f106-119). Overall, this study revealed that the four novel sleep-promoting peptides identified were strong candidates as potential functional ingredients in the development of sleep-promoting products.