补充materials.docx

In previous studies, we also determined the calcium transport volume in the intestinal epithelial cell model (Figure S1) (Jin et al., 2024). The results indicated that the hydrolyzed products from different enzymes exhibited significantly enhanced calcium absorption (P < 0.05). Furthermore, among the hydrolyzed casein products, those treated with trypsin, bromelain, and neutral protease demonstrated the most effective calcium absorption promotion. The final step of bone formation is mineralization, where the deposition of calcium phosphate in the extracellular matrix of bone cells is regarded as a hallmark of bone regeneration and formation (Gaharwar et al., 2013). Alizarin red, which possesses a hydroxyl anthraquinone structure, binds with calcium ions in calcium salts to form a complex that is marked by its red color. Consequently, numerous studies have assessed the effect of bone cell mineralization by analyzing the proportion of the red-stained areas in alizarin red staining experiments (Bensimon-Brito et al., 2016). We also determined the mineralization results of different enzymatic hydrolysates on MC3T3-E1 cells by alizarin red staining (Figure S2). The results indicate that different enzymatic hydrolysates of casein promote bone cell mineralization compared to the control group, with the highest proportion of red-stained areas observed in the case of trypsin and bromelain. This suggests that the hydrolysates of casein produced by trypsin and bromelain are the most effective in promoting osteoblast mineralization, with a significantly stronger effect than that of other enzymatic hydrolysates of casein (P<0.05).