Dynamic Simulated Gastrointestinal Digestion of Lactopontin and Effects of Its Products on the Proliferation, Differentiation and Mineralization of Osteoblasts

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 361.1485 to 7269.676 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.