Abstract
The aim of this study was to isolate and identify osteogenic bioactive peptides from yak bones collagen, while simultaneously investigating their underlying mechanisms for promoting osteoblast proliferation. Response surface methodology (RSM) was employed to investigate the effect of hydrolysis variables on the production of peptides with osteoblast proliferation-promoting activity (OPPA). The concentration of soluble peptides reached 0.5169 mg mL-1, which was well matched with the value (0.5189 mg mL-1) predicted by the model, with the following optimized conditions: hydrolysis time, 3.6 h; pH, 6.12; hydrolysis temperature, 54 °C; E/S (enzyme to substrate) of 5637 U g-1. Hydrolysates were then separated using an ultrafiltration membrane system, and the peptides (<3 kDa) possessed excellent OPPA with a dose-response relationship. A total of 59 novel peptides were identified by HPLC-MS/MS with Mascot analysis. GPSGPAGKDGRIGQPG (GP-16) and GDRGETGPAGPAGPIGPV (GD-18) were selected for docking to investigate the underlying mechanisms of interaction. The molecular docking study revealed that osteoblast proliferation stimulation activity of GP-16 and GD-18 was mainly attributed to the formation of very strong hydrogen bonds with the epidermal growth factor receptor (EGFR). These results indicate that such peptides are promising in the discovery of potential candidates for the future industrial production of functional peptides, which could be used in the mediated treatment of osteoporosis.