Breakdown of ß-Casein after in vitro digestion model. Identification and immunoreactivity of the products generated at different phases

Abstract

[Background]: The aim of the study was to evaluate the allergenicity of one of the main allergens from cow milk, β-casein (β-CN) after being digested through a simulated gastrointestinal digestion and to identify those peptides generated during the digestion process. [Methods]: The digestion was performed in three steps by using simulated gastric and duodenal fluids. Digestibility of β-CN was assessed by SDS-PAGE and RP-HPLC. IgE binding of native β-CN and hydrolysates was evaluated by indirect ELISA, using the sera from six milk-allergic patients. The peptides produced during the gastrointestinal digestion, were identified by liquid chromatography tandem mass spectrometry analysis. [Results]: Results showed that β-CN was quickly degraded at the early stages of the digestion, being broken down in 4 large fragments with molecular weight close to 23, 18, 14 and 12 kDa and other peptides with less than 10 kDa. No residual β-CN was observed after the gastric phase. Immunoreactivity of β-CN increased at the end of the gastric digestion showing values between 150 and 200% of IgE binding compared to the undigested protein and then fell drastically at the end of digestion until values close to zero. Among the products of digestion, 98 peptides were identified. Thirty nine peptides were detected in the gastric phase, 68 in the duodenal. Between those identified peptides, three of them with the sequences RELEELNVPGEIVE, YQEPVLGPVR and VYPFPGPIPN had been previously described as epitopes of β-CN. [Conclusion]: β-CN is completely degraded during the digestion process. Similarly, allergenicity is severely reduced at the end of the duodenal stage, but the increase of the IgE binding capacity at the end of the gastric phase suggests that hidden epitopes may have been exposed as result of the hydrolysis. From the digestion products, 98 peptides have been identified. Studies are underway to evaluate the human-IgE binding capacity of the identified peptides by peptide microarray technique