Abstract: In our study, the formation mechanism of the ternary complex of β-lactoglobulin (β-LG) with folic acid (FA) as a hydrophilic molecule and retinol (VA) as a hydrophobic molecule was explored by fluorescence spectroscopy. The changes in the antioxidant activity and allergenicity of β-LG after complexation were examined by 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging, 1,1-diphenyl-2-picrylhdrazyl (DPPH) radical scavenging and ferric ion reducing antioxidant power (FRAP) assays as well as indirect enzyme-linked immunosorbent assay (ELISA), respectively. Fluorescence spectroscopy indicated that FA and VA had a strong ability to quench the fluorescence of β-LG mainly in a static mode．FA and VA could form stable ternary complexes with β-LG through hydrophobic interaction, which was reflected by the binding constant (Ka) > 104 L/mol, the number of binding sites of β-LG for FA and VA (about 1) and the thermodynamic parameters. The presence of VA affected the affinity of FA for β-LG, while the presence of FA did not affect the affinity of VA for β-LG. The results of ABTS, DPPH and FRAP assays showed that the antioxidant activity of β-LG-FA and β-LG-VA was not significantly different compared with that of β-LG, but lower than that of β-LG-FA-VA. Different binding sequences had no significant effect on the antioxidant activity of the ternary complex. Indirect competitive ELISA showed that β-LG-FA-VA gave a lower inhibition of 42.23% against β-LG antigenicity, while β-LG-VA-FA exhibited a higher inhibition of 70.32%. These results indicate that different binding sequences had different effects on the antigenicity of β-LG.

Key words: β-lactoglobulin; folic acid; retinol; ternary complexes; antioxidant; allergenicity