Digestibilidad de alérgenos de leche y clara de huevo. Respuesta inmunológica y caracterización de péptidos y epítopos

Autor: Benedé, Sara
Přispěvatelé: Molina Hernández, Elena, López Expósito, Iván, López-Alonso Fandiño, Rosina, UAM. Departamento de Química Física Aplicada, Molina, Elena, López-Expósito, Iván, López-Fandiño, Rosina
Jazyk: angličtina
Rok vydání: 2013
Předmět:
Zdroj: Biblos-e Archivo. Repositorio Institucional de la UAM
instname
Digital.CSIC. Repositorio Institucional del CSIC
Popis: Memoria que presenta Sara Benedé Pérez para optar al grado de Doctora en Ciencia y Tecnología de los alimentos. Esta tesis doctoral se ha realizado en el Departamento de Bioactividad y Análisis de Alimentos del Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM).
[EN]: Milk and egg allergy is increasing in Europe and the United States, showing a very high incidence, especially in children. Major allergens of these products are glycoproteins, and to induce an allergic response, they should be able to cross the intestinal barrier maintaining sufficient structural integrity to interact with the immune system. Therefore, one of the main characteristics of food allergens is their resistance to gastrointestinal digestion and it is important to assess whether the fragments generated during this process retain IgE binding epitopes. Given the multiple factors involved in physiological digestion, the validity of the stability to the in vitro digestion as a criterion to assess the potential allergenicity of proteins is still controversial. In this thesis, digestions with oral, gastric and duodenal human and simulated fluids of three commercial allergenic food proteins, β-lactoglobulin (β-LG), β-casein (β-CN), and ovalbumin (OVA) have been performed. The results showed that beside the degradation profiles of the two models were different, the residual immunoreactivity, cleavage site of enzymes and the resultant peptides were very similar. The results of microarray and dot blot analysis showed that some of the peptides resulting from digestion maintain their allergenicity being the most immunoreactive areas β-LG (43-65), β-LG (86-99) and β-LG (124-140) in the case of β-LG, β-CN (57-68) and β-CN (82-93) for β-CN and OVA (370-385) for OVA. Furthermore we also studied the effect of carbohydrate residues attached to the ovomucoid (OM), immunodominant egg allergen, on its allergenicity and digestibility. Results indicated that after enzymatic deglycosylation, the structure of OM was unmodified and 80% of the patients used in the study showed reduced IgE binding capacity compared with OM glycosylated. The presence of IgE-reactive epitopes specific for glycosylated protein was also demonstrated. Deglycosylated OM was more susceptible to proteolysis during digestion but the resultant peptide pattern was very similar. Immunoreactivity of the digests measured by inhibition ELISA decreased to values between 1 and 4% at the end of digestion, but some of the identified peptides such as OM (80-89) and the regions OM (36-61), OM (100-122) and OM (133-180) showed maintain their IgE binding capacity reacting in 40-100% of patients tested. The last part of this thesis describes the identification of peptides generated during the in vitro gastrointestinal digestion of a major egg allergen, lysozyme (LYS). The results indicated that the gastric and duodenal digests retained their IgE binding and basophils activation capacities. This biological activity may be attributed to the presence of intact LYS, to the fragment LYS (24-129) produced by the action of chymotrypsin or to the immunoreactive peptides released during digestion and linked by disulphide bridges, containing the epitopes LYS (57-83) and LYS (108-122). The fact that major linear epitopes can be released during digestion of LYS led to study the case of an egg allergic child, which tolerate egg after a immunotherapy treatment, suffered a severe allergic reaction to a medication containing LYS and papain The hypothesis that the presence of papain, could increase the allergenic potential of the LYS was raised. The results indicated that LYS treated with papain produced fragments with a high IgE-binding capacity and they were identified as LYS (22-129), LYS (34-96) and LYS (62-128). Furthermore, these fragments could be linked by disulphide bridges forming large structures that could have an IgE-binding capacity increased.
Databáze: OpenAIRE
Externí odkaz: