Patterns and Pathways of Proteolysis of Gluten Proteins in the Gastrointestinal Tract

UoM administered thesis: Phd

  • Authors:
  • Frances Smith

Abstract

Introduction: Wheat is one of the most cultivated cereal grains in the world and is used for the manufacture of a wide range of food products; however its consumption has been linked to several health issues. Food products containing wheat flour commonly elicit a high glycaemic response (GR) through rapid breakdown of starch and absorption of the resulting glucose. Regular over-consumption of such foods has been linked to obesity and development of type 2 diabetes. Dietary fibre may alter GR after meal consumption indirectly through modification of chyme viscosity. Wheat can also elicit immune-mediated adverse reactions, such as immunoglobulin E(IgE)-mediated wheat allergy and coeliac disease (CD), which are most often associated with gluten proteins consisting of gliadins and glutenins. Resistance to digestion may impact the allergenicity of such protein components. Digestion of gluten and its epitopes important for CD have been enhanced in vitro and in vivo using a prolyl endopeptidase from Aspergillus niger (AnPEP) however the impact on IgE-mediated allergy has yet to be considered. Additional information is needed about the digestion of wheat. Specifically the impact of food matrix, digestion conditions and effect of AnPEP require further investigation. Methods: First, the effect of food matrix on proteolysis was tested by in vitro batch oral-gastric digestion of a purified total gliadin fraction (TGF), flour and bread. As the most physiologically relevant material, bread was also processed through the duodenal/intestinal phase in varying conditions to assess the impact of enzyme inclusions on macronutrient breakdown. Second, results from the batch digestions were compared to bread digestion in dynamic models, where the effect of natural variations in soluble fibre was also tested. Increasing levels of AnPEP were used in two in vitro batch oral-gastric models. Protein breakdown in digestions was assessed using a combination of 1D PAGE, immunoblots with a variety of wheat-specific antibodies, kinetic analysis and inhibition ELISA. Immunoassays were performed with sera from 23 wheat-allergic patients and some digestions were analysed in terms of starch digestion. Finally, LC-MS/MS was used to obtain specific sequence information and relative intensity of peptides from in vitro batch model digestions. Thus, digestion of selected allergens and key epitopes was monitored. Results and Discussion: Wheat proteins were very resistant to in vitro batch gastric digestion in bread compared to the TGF, with flour proteins somewhat intermediate. Thus, studies digesting purified proteins are not always indicative of protein digestion in a processed food matrix. Digestion of bread protein was enhanced by starch digestion and vice versa. This has implications for patients with deficiency in pancreatic amylase, which is often observed in childhood, so may play a role in food allergy development by influencing polypeptides reaching the gut mucosa. Digestion model conditions also had a large impact on wheat protein digestibility with differences observed between batch and dynamic models, and the two batch models used. This may reflect biological variations observed in vivo. Unexpectedly, the wheat cultivar with higher soluble fibre digested slightly more quickly which may be due to alterations in other macronutrients present. In most cases patient sera were poly-sensitized to a number of wheat proteins and IgE-binding was mostly unaffected by baking. Gastric digestion reduced IgE-reactivity of bread but large polypeptides of high relative intensity remained. Addition of AnPEP further reduced IgE-reactivity of digestion samples by digesting gluten proteins into smaller peptides of lower relative intensity. This reduced the presence of epitopes important for IgE-mediated allergy and CD. Therefore, AnPEP may have an application for treatment of accidental wheat consumption for patients with IgE-mediated wheat allergy.

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Original languageEnglish
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Award date1 Aug 2017