Abstract: In this study, pepsin, trypsin, and chymotrypsin were used in combination to prepare eel bone collagen peptide (EBCP) with high calcium-binding ability. The optimal preparation conditions of EBCP-calcium chelate that provided maximum calcium chelation rate of over 80% were determined as follows: 40 ℃, pH 7.5, 40 min, and mass ratio of peptide to calcium 5:1. Fluorescence, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopic (SEM) observation indicated that the carboxyl and amino nitrogen atoms of EBCP could chelate calcium to form EBCP-Ca. EBCP-Ca was stable toward temperature, pH and simulated gastrointestinal digestion. In addition, this chelate exhibited low toxicity to mouse Caco-2 cells. In addition, the Caco-2 cell monolayer model was used to evaluate the calcium uptake-promoting activity of EBCP-Ca in vitro. The effects of EBCP-Ca concentration and culture time on the calcium content and alkaline phosphatase (AKP) activity in Caco-2 cells were investigated. It was demonstrated that EBCP-Ca enhanced the AKP activity, thereby promoting the absorption of calcium in the human gastrointestinal tract. This research provides a scientific basis for the development of new calcium supplements and the high-value utilization of eel bones.

Key words: eel bone; calcium-binding peptide; stability; cell absorption