Enrichment of Zinc-Chelating Peptides from Casein Using IMAC and Their Zinc Binding Modes and Promoting Intestinal Zinc Absorption Activity.

In this study, casein was used as the raw material to prepare casein hydrolysates, and the fraction with high activity (F-IMAC) was enriched by immobilized metal affinity chromatography. Then, F-IMAC and its zinc chelates (F-IMAC-Zn, 99.1 mg/g) were characterized. Analysis of amino acid composition indicated that the contents of Asp, Thr, Ser, Glu, Cys, Ile, His, Lys, and Arg in F-IMAC-Zn increased. Ultraviolet-Visible spectroscopy, Fourier transform infrared spectroscopy, and scanning electron microscopy revealed structural differences between F-IMAC and F-IMAC-Zn. The carboxyl, carbonyl, and amide bonds serve as zinc binding sites. The results of HPLC-MS/MS and molecular docking indicated that YPVEPF (750.3588 Da), EMPFPK (747.3625 Da), and EAMAPKHKEMPFPK (1639.8214 Da) exhibited the strongest zinc chelating potential among the 50 peptides identified from F-IMAC, and the three mainly combined with Zn²⁺ through Metal-Acceptor bonds and Charge-Charge bonds. In the zinc transport experiment, the zinc transport amounts of YPVEPF, EMPFPK, and EAMAPKHKEMPFPK increased by 99.80, 67.40, and 123.37%, respectively, compared with the control group (the ZnSO₄), which indicated that all three exhibited strong activities in promoting intestinal zinc absorption. This study provides theoretical bases and new ideas for the development of dietary zinc supplements using casein zinc-chelating peptides.