Metabolomic Analysis\nReveals the Linkage between Sleep-Enhancing\nEffects and Metabolite Biomarkers and Pathways of Different Casein\nHydrolysates in Chronic Unpredictable Mild Stressed Mice

Casein hydrolysates have been proven\nto exert varying\nsleep-enhancing\nand anxiolytic effects due to their distinct release of potential\npeptides. However, their underlying sleep-enhancing mechanisms at\nthe metabolic level remain unclear. This study aims to investigate\nthe potential sleep-enhancing mechanism of casein hydrolysates through\nan integrated approach of untargeted and targeted metabolomics in\nCUMS-induced anxiety mice for the first time. The results showed seven\npotential biomarkers were identified and screened using orthogonal\npartial least-squares discriminant analysis, random forest model,\nand pathway analysis, including ornithine, l-proline, l-prolinamide, inhibitory neurotransmitters gamma-aminobutyric\nacid, 5-HIAA, fumaric acid, and 4-oxoglutaramate. Moreover, casein\nhydrolysates exerted sleep-enhancing effects through multiple metabolic\npathways, mainly including the GABAergic system, tryptophan metabolism,\nand cAMP response signaling pathway, which was validated by targeted\nmetabolomics and vital protein expressions. It was interesting that\ncasein hydrolysates with diverse representative peptide compositions\nexhibited varying activity, which could be attributed to distinct\nalterations in metabolites via different pathways.