Enzyme-specific casein hydrolysates enhance calcium absorption and bone mineralization: Mechanistic insights from osteoblast activation and peptide profiling.

Calcium bioavailability and bone mineralization are critical for skeletal health; however, conventional calcium supplements often face limitations in absorption efficiency. This study investigates how enzyme-specific hydrolysis of CN generates bioactive peptides with distinct capacities to promote calcium absorption and bone formation. Papain-derived CN hydrolysate significantly outperformed calcium chloride in restoring bone health in osteoporotic mice, elevating serum osteocalcin levels by 1.8-fold and reducing tartrate-resistant acid phosphatase levels by 41% compared with inorganic calcium. Mechanistically, papain hydrolysates upregulated the expression of TRPV5 and TRPV6 calcium transporters in intestinal cells, thereby facilitating intestinal calcium uptake. Peptidomic profiling revealed enzyme-dependent cleavage patterns: papain preferentially targets glutamate- and lysine-rich sites (e.g., ES, EK, QS), yielding peptides such as QPKTKVIPYVRYL and RELEELNVPGEIVE, which synergistically enhance calcium chelation and osteogenic signaling. Notably, micro-computed tomography analysis confirmed that papain hydrolysates restored trabecular bone density and microarchitecture in murine femurs, outperforming inorganic calcium supplementation. These findings establish a structure-activity framework for designing enzyme-tailored CN peptides to address calcium deficiency disorders, offering a transformative strategy for the development of functional nutraceuticals.