Influence of Transglutaminase and the Reducing Agent Glutathione in the Gastric Digestion and Immunogenicity of Beta-Lactoglobulin

Background: Among the milk whey proteins, β-lactoglobulin (β-Lg) is the main allergen. In native state, β-Lg is resistant to pepsin, which is considered an indicator of its allergenic potential. Protein antigenicity can be reduced by treatment with transglutaminase (TG), an enzyme which catalyses inter or intra crosslink between Lys and Gln residues. Reducing agents have been used to increase the access of TG to the Lys and Gln residues. This study aimed at investigating the antigenicity and digestibility by pepsin of β-Lg modified by TG in the presence of the reducing agent glutathione (GSH). Methods and Results: The polymerization of β-Lg (Davisco, MN, USA) by microbial TG (WM, Ajinomoto Ltda, Brazil) was studied using a central-composite experimental design with 2 independent variables, GSH concentration [GSH] (0 - 0.2 mmol L^-1) and  enzyme:substrate ratio (E/S) (0 - 44.1 U g^-1). The dependent variable was the response of specific IgE obtained from the serum of BALB/c mice sensitized with native β-Lg (IgE anti-β-Lg) against the modified protein. Polymerization was carried out at 50°C/180 min and stopped by heating at 80 °C/5 min. Digestion was simulated using pepsin (E/S 1:20 w/w, pH 2, 37°C, 1h), and SDS-PAGE and the ELISA assay used to characterize and evaluate the antigenicity of the samples, respectively. The linear and quadratic factors of [GSH] and E/S, and their interaction, showed no significant effect on the response of lgE. No treatment resulted in a decrease in the response of IgE anti-β-Lg, but a significant (p<0.05) increase was observed in the response of IgE against treatments 1, 7 and center point. Intact β-Lg was observed in the electrophoretic profile of the digested samples, indicating resistance to pepsin. After digestion, the samples not presented difference with respect to the digested native β-Lg sample (213.06 ± 9.59 ng mL^-1). Conclusion: Polymerization of the β-Lg with TG in the presence of the GSH did not alter its digestibility by pepsin or decrease the antigenicity. Under some of the conditions studied the antigenicity of the β-Lg increased, indicating that the treatment could have created or exposed epitopes.