Abstract: To enhance the application value of lactoferrin (LF) in the health field by exploiting nutrient interactions, this study explored the covalent binding potential between LF and epigallocatechin gallate (EGCG). The aim is to augment the biological efficacy of LF while concurrently reducing its production costs. The effects of different durations of covalent reaction on the structure, thermal stability, and osteogenic activity of LF-EGCG complexes were investigated using circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, endogenous fluorescence spectroscopy, Raman spectroscopy, and differential scanning calorimetry (DSC). The results revealed that a 0.5 h covalent reaction between EGCG and LF led to significant structural changes in LF, while reducing the surface hydrophobicity to 1 304.00 (85.03% lower than that of LF). Furthermore, the resulting complex at a concentration of 100 μg/mL significantly promoted osteoblast proliferation (which increased the proliferation rate to 1.43 after 24 hours) and enhanced alkaline phosphatase activity (after treatment for 7 days, the enzyme activity increased to 15.27 U/g, 56.61% higher compared with the control group). This study provides new ideas for the functional modification of LF and lays the foundation for its application in the field of bone health.

Key words: lactoferrin; epigallocatechin gallate; covalent reaction; structural characterization; osteogenic activity