The present study aims to investigate the structure of covalent conjugates of bovine β-
lactoglobulin (BLG) and
flavonoids (
luteolin,
myricetin, and
hyperoside), and their effect on the allergenicity and human intestinal microbiota. Covalent modification of
amino acids in BLG by
flavonoids was confirmed by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
o-phthaldialdehyde assay. The secondary and conformational structures of BLG were changed by the covalent modification, which were determined by the circular dichroism, Fourier transform infrared spectroscopy, fluorescence spectroscopy, and UV spectroscopy. The
enzyme-linked
immunosorbent assay (ELISA) and cell experiments indicated that BLG covalent conjugates could reduce
IgE/
IgG binding capacities and suppress the
allergy reactivity of RBL-2H3 cells, suggesting that the covalent modification modulated the balance of T cells. Meanwhile, covalent modification of BLG with these
flavonoids can alter the diversity of human intestinal microbiota and the community abundance at phylum, family, and genus levels. The results revealed that covalent modification of BLG with
flavonoids alters human intestinal microbiota, might result in the reduction of allergenicity, which could provide information for confirming the relationship between
food allergy and the intestinal microbial ecosystem.