Impacts of hesperidin on whey protein functionality: Interacting mechanism, antioxidant capacity, and emulsion stabilizing effects

Abstract

The objective of this work was to explore the possibility of improving the antioxidant capacity and application of whey protein (WP) through non-covalent interactions with hesperidin (HES), a citrus polyphenol with nutraceutical activity. The interaction mechanism was elucidated using several spectroscopic methods and molecular docking analysis. The antioxidant capacity of the WP-HES complexes was analyzed and compared to that of the proteins alone. Moreover, the resistance of oil-in-water emulsions formulated using the WP-HES complexes as antioxidant emulsifiers to changes in environmental conditions (pH, ion strength, and oxidant) was evaluated. Our results showed that HES was incorporated into a single hydrophobic cavity in the WP molecule, where it was mainly held by hydrophobic attractive forces. As a result, the microenvironments of the non-polar tyrosine and tryptophan residues in the protein molecules were altered after complexation. Moreover, the α-helix and β-sheet regions in the protein decreased after complexation, while the β-turn and random regions increased. The antioxidant capacity of the WP-HES complexes was greater than that of the proteins alone. Non-radiative energy transfer from WP to HES was detected during complex formation. Compared to WP alone, the WP-HES complexes produced emulsions with smaller mean droplet diameters, exhibited higher pH and salt stability, and had better oxidative stability. The magnitude of these effects increased as the HES concentration was increased. This research would supply valuable information on the nature of the interactions between WP and HES. Moreover, it may lead to the creation of dual-function antioxidant emulsifiers for application in emulsified food products.

Keywords: emulsions; hesperidin; interaction; molecular docking technology; whey protein.

FIGURE 1

Fluorescence emission profiles of WP at various concentrations of HES (0–26.7 μM) at 298K (A), 304K (B), and 310K (C). The Double-log plots (D) and Stern-Volmer plots (E) of the interaction between WP and HES at different temperatures.

FIGURE 2

The overlapping area of the UV-VIS absorption spectrum of HES and the WP fluorescence spectrum (A), the red line and black line represent UV-VIS absorption spectrum and fluorescence spectrum, respectively; FTIR spectrograms of WP and WP-HES (B).

FIGURE 3

The synchronous fluorescence spectrum of WP in the absence and presence of HES at Δλ = 15 nm (A) and Δλ = 60 nm (B).

FIGURE 4

3D diagrams of α-LA-HES (A) and β-LG-HES (B); the hydrophobicity surface of α-LA (C) and β-LG (D) interacted with HES, the red color and blue color represent hydrophobicity and hydrophilicity, respectively; 2D schematic interaction diagram α-LA-HES (E) and β-LG-HES (F), the color of amino acid residue is drawn by interaction.

FIGURE 5

Diphenyl-2-picrylhydrazyl free radical scavenging activity (A) and ferric-reducing antioxidant power (B) of WP-HES complexes.

FIGURE 6

The visual appearance of WP-based emulsion (A), WP-HES (1 mM) based emulsion (B), and WP-HES (2 mM) based emulsion (C) under different pH values (numbers on the tubes).

FIGURE 7

Effects of pH on the particle size (A) and zeta potential (B) of WP or WP-HES complex stabilized emulsions.

FIGURE 8

The microstructure of WP based emulsion or WP-HES based emulsions at different pH values.

FIGURE 9

The particle size (A–C) and zeta-potential (D–F) changes of WP based emulsion or WP-HES based emulsions under different pH values and salt concentrations.

FIGURE 10

The microstructure of emulsions with different salt ion concentrations under acidic condition.

FIGURE 11

Change in PV (A) and TBARS (B) values for WP based emulsion or WP-HES based emulsions during storage at 55°C for 15 days.