Abstract: In this study, calcium-chelating casein hydrolytic peptide (CHP-Ca) was prepared by the chelation reaction between CHP, which was prepared from enzymatic hydrolysis of micelle casein, and calcium chloride at a mass ratio of 2:1. Five enzymes (flavourzyme, alcalase, trypsin, papain and neutral protease) were screened based on hydrolysis degree (DH) and calcium-chelating capacity. A mixture of flavourzyme and trypsin was found to be the best enzyme for the enzymatic preparation of CHP. Furthermore, response surface methodology (RSM) was used to optimize the enzyme hydrolysis conditions. The results showed that the optimal conditions were enzyme-to-substrate ratio 6 000 U/g, flavourzyme-to-trypsin ratio 1:1, hydrolysis time 90 min, pH 6.9, and temperature 42 ℃. The calcium chelation capacity of the peptide prepared under these conditions was (90.46 ± 0.72) μg/mg. Finally, the structural properties and formation mechanism of CHP-Ca were investigated by X-ray diffraction (XRD), thermogravimetry (TG), zeta potential, particle size analysis and infrared (IR) spectroscopy. The results showed that CHP-Ca bound mainly by the interaction of carboxyl, amino and phosphate groups, and the zeta potential and particle size decreased after chelation, and the structure of CHP-Ca became more compact.

Key words: micellar casein hydrolytic peptides; peptide-calcium chelate preparation; multi-enzymatic hydrolysis; structural characterization; chelation mechanism