Studio dell'attività biologica e della stabilità alla digestione gastro-enterica degli inibitori e delle α-amilasi esogene di frumento

This doctorate thesis deals with the characterization of wheat exogenous α-amylase inhibitors (α-AIs) e with the evaluation of their role in food allergy to wheat. α-AIs are responsible for the onset of Baker’s asthma, and less frequently they have been associated with food allergy to wheat. In order to define a protein a food allergen, this must fulfil some prerequisites, among which the stability to gastro-enteric digestion. Since in scientific literature there are no data concerning the stability of α-AIs to digestion, the objective of this thesis was to study the biologic activity and the stability of one of the major wheat amylase inhibitor (the dimeric inhibitor 0.19) by means of in vitro and in vivo protocols. The first part of the thesis was addressed to the development of and anti-zymographic method in order to detect the inhibition activity of α-AIs directly in the electrophoresis gel. Thanks to this technique, it was possible to study for the first time the heterodimeric forms of these proteins. The stability to in vitro peptic digestion of 0.19 inhibitor showed that the occurrence of an emulsion contributes to the stability of the protein. This means that the inhibitor could reasonably reach the gut and sensitize/elicit an allergic reaction. In addition it may act as an antinutritional factor by inhibiting the human amylolytic enzymes. This hypothesis was tested by an in vivo study that led to the immunodetection of α-AIs in the intestine contents of rats fed a wheat cooked product. The IgE-blotting analysis of the same samples did not allow to observe any allergologically active α-AI. This result is in accordance with previous publications that indicated α-AIs as responsible for inhalant but not food allergic manifestations to wheat. However, it must not be excluded that the experimental condition might have altered the allergenicity of the α-AIs. In particular, the reduction of the disulfide bridges might have denatured some conformational epitopes, hindering the binding of IgEs. It is evident that the development of new experimental protocols for the analysis of the allergenicity of disulfide-rich proteins, i.e. prolamins, is a crucial issue.