Abstract: An in vitro dynamic system was employed in this study to simulate the infant digestion process of lactopontin (L-OPN). Throughout the process, the dynamic changes in gastric pH, hydrolysis degree, and peptide yield were tracked. The effect of lactopontin peptide (L-OPN-P) on the osteogenic activity of MC3T3-E1 cells was also evaluated. Results showed that gastric pH stabilized after 120 minutes of digestion. At the endpoint, the hydrolysis degree of L-OPN reached up to 40.38%, with a peptide yield of 56.77%. Mass spectrometry (MS) analysis identified 940 peptides derived from L-OPN, ranging from 3 to 67 amino acids in length and 361.148 5 to 7 269.676 Da in molecular mass. Among these, 725 peptides (77.1%) were phosphorylated. Cell assays demonstrated that L-OPN-P significantly promoted the proliferation of MC3T3-E1 osteoblasts, with enhanced activity observed at 24, 48 and 72 h. At a concentration of 50 μg/mL, L-OPN-P increased alkaline phosphatase (ALP) activity, ALP staining intensity, and calcium nodule formation by 3-fold, 87.27%, and 41.8%, respectively. These collective findings indicate that L-OPN-P effectively facilitated osteoblast differentiation and matrix mineralization. This study provided a theoretical basis for the application of L-OPN in infant formula and milk powder products.

Key words: lactopontin; dynamic in vitro simulated infant gastrointestinal digestion; osteogenic activity