Liposomal encapsulation and characterization of casein-derived peptides obtained by enzymatic hydrolysis coupled with microfiltration: In vivo evaluation of anti-fatigue activity.

Bioactive peptides derived from casein have attracted increasing attention for their antioxidant properties. However, their application is limited by stability issues and insufficient understanding of their anti-fatigue mechanisms, highlighting the need for effective delivery strategies. In this study, we aimed to evaluate the antioxidant and anti-fatigue effects of < 1 kDa casein peptides (CP) and to determine whether liposomal encapsulation enhances their bioactivity. Casein micelles (MCCs) were enzymatically hydrolyzed and microfiltered to obtain < 1 kDa peptides (CP), followed by liposomal encapsulation. The CP were characterized by amino acid composition, peptide sequences, and antioxidant activity in vitro and in vivo. Results showed that YPFPGPI and FPKYPVEPF exhibited strong Kelch-like ECH-associated protein 1 (Keap1) and DPPH binding, respectively, with molecular dynamics (MD) simulations confirming stable bonding involving ARG415, ARG483, SER508, and SER555. CP showed strong DPPH radical scavenging and high hydrophobic amino acid (HAAs) content. In an exhaustive swimming test, CP administration significantly prolonged the swimming duration in mice (P < 0.05), with the liposome group showing a 25.95% increase over the control group. Additionally, CP enhanced glycolysis and muscle strength, indicating marked anti-fatigue effects. Compared with previous studies, this work integrates peptide identification with liposomal delivery to improve functional efficacy and provide mechanistic insight. These findings highlight the potential of CP in alleviating fatigue, suggesting promising applications in functional foods and nutraceuticals.