Insight into covalent conjugates of β-lactoglobulin with rutin: Characterizing allergenicity, digestibility, and antioxidant properties in vitro

β-lactoglobulin (β-LG) is an essential nutrient in milk, but it is the primary allergen causing dairy allergy in humans. Currently, researchers are focusing on using flavonoids to covalently modify β-LG for improving its functionality. However, the impact and underlying mechanisms of rutin covalent modification on the functional properties and allergenicity of β-LG remain unclear. Here, we aim to investigate the changes in allergenicity, digestive characteristics, and antioxidant properties of β-LG after covalent modification using a combination of spectroscopy, enzyme-linked immunosorbent assay (ELISA), simulated digestion, and antioxidant assays. The results indicate that rutin forms covalent bonds with the free amino group, sulfhydryl group, and tryptophan of β-LG, leading to alterations in the secondary structure of β-LG. Furthermore, the modified β-LG exhibits improved antioxidant capacity and decreased allergenicity, along with reduced resistance to pancreatin digestion in vitro. This study provides novel insights and strategies to expand the functional application of β-LG.