Final Activity Report Summary - CasAll (Why casein is a major food allergen ?)
This project had two main objectives, namely the development of an infant in vitro digestion model along with the application of dairy samples of various complexities to the infant gut and the identification of the sequences that were resistant to in vitro digestion. In this first part, we developed a new infant gut model that was applied to the digestion of three model proteins. Hydrolysis of b-CN and Ova was reduced in this infant model compared to the adult one. Surprisingly, b-lg was more hydrolysed in the infant model than in the adult one because of decreased protection of the molecule by the lower concentration of phosphatidylcholine which was used in this model.
15 different proteins or dairy products obtained from the same original raw milk were submitted to the infant gut model. Digested samples were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed phase high performance liquid chromatography (RP-HPLC) and enzyme-linked immunosorbent assay (ELISA) and four samples, namely raw, pasteurised, sterilised whole milks and full-fat yoghurt, were further investigated to determine the areas that were resistant to digestion. The regions b-CN(f76-93), k-CN(f98-169) and as2-CN(f36-75) were found resistant to in vitro digestion as well as as1-CN(f149-199) to a lesser extent.
LC-MS-MS of the digested raw, pasteurised and sterilised milk and yoghurt showed that some area of the proteins generated peptides was found in all analysed samples whereas other area generated peptides were only found in the heavily processed materials. When the protected peptides were matched to the hydrophobicity profiles of as1-, as2-, b- and k-casein at pH 3.0 i.e. the ones used for the gastric phase of in vitro digestion, it was shown that all areas that were resistant to digestion were those that were highly hydrophobic at pH 3.0.
As a general conclusion, a physiologically-relevant infant in vitro digestion model was developed and applied to a variety of dairy samples. It showed that some regions of the casein molecule were resistant to digestion and could therefore elicit an allergic response. This resistance could be explained by the low pH in the stomach that made these areas highly hydrophobic and hardly cleavable by the proteases.
15 different proteins or dairy products obtained from the same original raw milk were submitted to the infant gut model. Digested samples were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed phase high performance liquid chromatography (RP-HPLC) and enzyme-linked immunosorbent assay (ELISA) and four samples, namely raw, pasteurised, sterilised whole milks and full-fat yoghurt, were further investigated to determine the areas that were resistant to digestion. The regions b-CN(f76-93), k-CN(f98-169) and as2-CN(f36-75) were found resistant to in vitro digestion as well as as1-CN(f149-199) to a lesser extent.
LC-MS-MS of the digested raw, pasteurised and sterilised milk and yoghurt showed that some area of the proteins generated peptides was found in all analysed samples whereas other area generated peptides were only found in the heavily processed materials. When the protected peptides were matched to the hydrophobicity profiles of as1-, as2-, b- and k-casein at pH 3.0 i.e. the ones used for the gastric phase of in vitro digestion, it was shown that all areas that were resistant to digestion were those that were highly hydrophobic at pH 3.0.
As a general conclusion, a physiologically-relevant infant in vitro digestion model was developed and applied to a variety of dairy samples. It showed that some regions of the casein molecule were resistant to digestion and could therefore elicit an allergic response. This resistance could be explained by the low pH in the stomach that made these areas highly hydrophobic and hardly cleavable by the proteases.