Multi-Enzyme Hydrolysis and Ultrafiltration-Based Optimization, Identification, and Characterization of Buttermilk...

Dairy peptides and plant polyphenols attract significant attention for their bioactive properties; however, the synergistic antioxidant mechanism of buttermilk peptides and myricetin remains unclear. In this study, buttermilk proteins from Lacprodan MFGM-10, a milk fat globule membrane (MFGM)-enriched buttermilk powder, were subjected to multi-enzyme hydrolysis using papain (Pap), alkaline protease (AP), and neutral protease (NP) at an enzyme concentration of 6000 U/g in different combinations. The results showed that the synergistic application of enzymes significantly increased the degree of hydrolysis (DH), with the AP group reaching a peak DH of approximately 42% at 150 min. Two candidate peptides, WGSPP and SWPWQ, were selected from the screened buttermilk peptide fractions. Through Caco-2 transmembrane transport validation and molecular docking, it is confirmed that these peptides can non-competitively bind to acetylcholinesterase (AChE) via molecular docking, with binding energies of -12.7 and -11.1 kcal/mol, respectively. Additionally, myricetin exhibits a strong binding energy, with a binding energy of -9.5 kcal/mol. In an L6 myoblast injury model induced by rotenone, combined treatment significantly enhances AChE inhibitory rate, reduces reactive oxygen species (ROS) and malondialdehyde (MDA) levels, restores superoxide dismutase (SOD) activity and the NAD⁺/NADH ratio, and improves mitochondrial membrane potential and ATP levels, without demonstrating cytotoxicity. Buttermilk-derived peptides and myricetin exert synergistic antioxidant effects by improving redox homeostasis and mitochondrial function, thereby alleviating oxidative stress-induced injury in rotenone-induced L6 cells. These findings provide preliminary evidence for the potential role of these compounds in oxidative stress-associated skeletal muscle health; however, direct sarcopenia-related endpoints were not evaluated in the present study.