Immunoreactivity of β-lactoglobulin and identification of the peptides generated after simulated orogastrointestinal digestion

Abstract

[Background]: The aim of the study was to evaluate the allergenicity of one of the main allergens from cow milk, β-lactoglobulin (β-Lg) after being digested through a simulated orogastrointestinal digestion and to identify those peptides generated during the digestion process. [Methods]: The digestion was performed in three steps by using simulated oral, gastric and duodenal fluids. Digestibility of β-Lg was assessed by SDS-PAGE and RP-HPLC. IgE binding of native β-Lg and hydrolysates was evaluated by indirect ELISA, using the sera from six milk-allergic patients. The peptides produced during the orogastointestinal digestion, were identified by liquid chromatography tandem mass spectrometry analysis. [Results]: Results showed that β-Lg was progressively degraded during the digestion. Intact β-Lg was observed after the gastric phase and in the first stages of the duodenal digestion. However, no residual β-Lg was observed at the end of the duodenal phase. Immunoassays showed that during the in vitro gastric and duodenal digestion immunoreactivity decreased progressively with an EC50 value increased 150 times at the end of the digestion. Among the products of digestion, 146 peptides were identified. No peptides were found in the oral phase. Forty five peptides were detected in the gastric phase, 71 in the duodenal, and 30 were common in both phases. Between those identified peptides, four of them with the sequences LIVTQTMK, GLDIQK, IDALNENK, and VLVLDTDYK had been previously described as epitopes of β-Lg. [Conclusions]: β-Lg is progressively degraded during the digestion process. Similarly, β-Lg allergenicity is reduced through the simulated digestion with a severe reduction at the end of the duodenal stage. From the digestion products, 147 peptides have been identified. Studies are underway to evaluate the ability to cross the intestinal barrier and to bind to human-IgE of the most relevant identified peptides.