Two Novel β-Casein Iron-Chelating Peptides: Isolation, Identification, Prediction of Iron Binding Modes, and Enhancement Intestinal Iron Absorption Activity

ABSTRACT

In this study, β-casein was used to prepare iron-chelating peptides through the enzymatic hydrolysis. β-casein hydrolysates were purified and identified, and combined with molecular docking to screen high-activity iron-chelating peptides. Additionally, their iron binding modes and enhancement of intestinal iron absorption activities were investigated. The results indicated that 232 peptides were identified from high-activity M4 fractions, and two novel peptides, SpEESITRINKKIE (1706.77617 Da) and SpSpSpEESITRINKKIE (1800.87390 Da), exhibited the strongest iron chelation potential. These peptides bound to Fe²⁺ through metal-acceptor and charge-–charge interactions, achieving Fe²⁺ chelation rates of 87.18 ± 0.89% and 91.15 ± 0.31%, respectively. In Caco-2 cell monolayers, the iron transport quantities of SpEESITRINKKIE and SpSpSpEESITRINKKIE within 30 min significantly (p < 0.05) increased by 79 ± 1% and 129 ± 2%, respectively, compared to the control group. These peptides demonstrated excellent enhancement of intestinal iron absorption activities. This study provides a theoretical basis for developing dietary iron supplements utilizing β-casein iron-chelating peptides.