Yak bone is one of the major components of Tibetan medicine (traditional Chinese medicine) and its function is mainly related to an improvement of bone health. Yak bone collagens were considered one promising substrate to prepare osteogenic activity peptides. However, due to the complexed triple-helical structures, yak bone collagen is not easy and accessible for enzymatic cleavage of most proteases. Herein, the purpose of this study was to investigate the effects of five different pretreatment approaches (ultrasonic, formic acid, alkali, ultrasonic and formic acid, and ultrasonic and alkali) on the release of osteogenic activity peptides during the subsequent enzymatic hydrolysis. On the basis of our results, combined ultrasonic (ultrasonic power of 339 w, ultrasonic frequencies of 23.8 kHz, ultrasonic pulsed model of 6 s/5 s, and ultrasonic processing time of 36 min) and formic acid pretreatment of yak bone collagen was considered the optimum operating condition, which could significantly expedite the release of osteogenic activity peptides from the complex triple helix of yak bone collagen during the subsequent enzymatic hydrolysis, in terms of secondary structure, microstructures, the content of free sulfhydryl (SH) and/or disulfide bond (SS), degree of hydrolysis (DH), the content of soluble peptides (CSP), in vitro digestibility, and osteogenic bioactivity of yak bone collagen hydrolysates (P < 0.05). Based on the experimental data, yak bone collagen hydrolysates after ultrasonic combined acid pretreatment displayed the highest SH content of 43.4 μmol/g, DH value of 24.6%, highest digestibility of 61.4%, osteoblastic proliferation rate of 147.7%, and maximum soluble peptide content of 79.8 mg/100 mg, respectively. During these procedure, formic acid could assist the hydrolysis of highly and complexed cross-linked yak bone collagen and the ultrasonic could simultaneously break the covalent bonds between different yak bone collagen fibers. The combined effect could undoubtedly damage the complexed triple helical structures in yak bone collagens and ultimately expose more inner enzymatic cutting sites for the subsequent hydrolysis. Collectively, these findings of this work highlighted that ultrasound-assisted formic acid pretreatment method exhibited highest efficiency for pretreating yak bone collagen, and maybe one promising strategy to prepare osteogenic activity peptides from collagen-rich raw materials.
