Investigation of the Structure and Allergic Potential of Whey Protein by Both Heating Sterilization and Simulation with Molecular Dynamics

Abstract

As the main allergens in milk, whey proteins are heat-sensitive proteins and are widespread in dairy products and items in which milk proteins are involved as food additives. The present work sought to investigate the effect of heating sterilization on the allergenicity of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG), the main composite and allergen in whey protein isolate (WPI), by combining molecular dynamics with experimental techniques for detecting the spatial structure and IgE binding capacity. The structure of WPI was basically destroyed at heat sterilization conditions of 95 °C for 5 min and 65 °C for 30 min by SDS-PAGE analysis and spectroscopic analysis. In addition, α-lactalbumin (α-LA) may be more sensitive to temperature, resulting in exposure to allergic epitopes and increasing the allergic potential, while the binding capacity of β-lactoglobulin (β-LG) to IgE was reduced under 65 °C for 30 min. By the radius of gyration (Rg) and root-mean-square deviation (RMSD) plots calculated in molecular dynamics simulations, α-LA was less structurally stable at 368 K, while β-LG remained stable at higher temperatures, indicating that α-LA was more thermally sensitive. In addition, we observed that the regions significantly affected by temperatures were associated with the capacity of allergic epitopes (α-LA 80-101 and β-LG 82-93, 105-121) to bind IgE through root-mean-standard fluctuation (RMSF) plots, which may influence the two major allergens. We inferred that these regions are susceptible to structural changes after sterilization, thus affecting the allergenicity of allergens.

Keywords: heat sterilization; molecular dynamics simulation; structure and allergy; whey protein isolate.

Figure 1

SDS-PAGE analysis of WPI with different sterilizations.

Figure 2

Structural changes in WPI after different sterilizations by spectroscopic analysis. The analysis of spatial structure by (A) UV spectrum and (B) fluorescence spectra and secondary structure by (C) CD spectra.

Figure 3

Effects of different sterilizations on the microstructure of WPI by SEM. The magnification is ×30,000 (A), ×2000 (B), and ×5000 (C), respectively.

Figure 4

Effects of different sterilizations on the binding capacity of major allergens and specific IgE in WPI. Different uppercase letters indicate significant differences in α-LA after different treatments (p < 0.05). Different lowercase letters indicate significant differences in β-LG after different treatments (p < 0.05).

Figure 5

MD simulates the differences of α-LA in different temperature environments, and (A) Rg plot and (B) RMSD plot.

Figure 6

MD simulates the stability of allergic epitopes of α-LA in different temperature environments: (A) RMSF plot of protein, and (B) conformational differences in region 80–101 in different environments. The arrow indicates the amino acid sequence and protein structure of this position.

Figure 7

MD simulates the differences of β-LG in different temperature environments, and (A) Rg plot and (B) RMSD plot.

Figure 8

MD simulates the stability of allergic epitopes of β-LG in different temperature environments: (A) RMSF plot of protein, and (B) conformational differences in regions 82–93 and 105–121 in different environments. The arrow indicates the amino acid sequence and protein structure of this position.