Digestomika alergena kikirikija i karakterizacija fragmenata otpornih na proteolizu

Abstract

INFOGEST metoda predstavlja standardizovani protokol za in vitro simulaciju digestije kompletne hrane, zasnovanom na fiziološki relevantnim uslovima. Predmet rada ove disertacije je ispitivanje digestibilnosti alergena kikirikija iz celog zrna primenom INFOGEST metode, kao i karakterizacija njihovih fragmenata otpornih na proteolizu. Za odstranjivanje lipida primenjena je metoda taloženja proteina, koja se pokazala kao superiornija u odnosu ekstrakciju lipida organskim rastvaračem, usled manjeg kvalitativnog i kvantitativnog gubitka proteina. U ovoj tezi je pokazano da termički tretmani kikirikija, pored matriksa hrane, dodatno otežavaju oslobađanje proteina iz zrna, što čini glavne alergene kikirikija Ara h 1, Ara h 2 Ara h 3 i Ara h 6 nedostupnijim za pepsinsku hidrolizu. Oslobađanje proteina kikirikija, kao i digestibilnost, u gastričnoj fazi se pokazala znatno izraženijom, u odnosu na intestinalnu fazu, s tim da je digestija kod pečenog kikirikija otežana u odnosu na sirovi. Nakon oralno-gastrične digestije celog zrna sirovog kikirikija, glavni alergeni kikirikija u velikoj meri ostaju intaktni, a njihovi peptidi otporni na digestiju zadržavaju alergeni kapacitet. Pokazano je da većina Ara h 2 i Ara h 6 alergena ostaje rezistentna na digestiju. Ara h 1 i Ara h 3 kaskadno podležu pepsinolizi, do fragmenata koji i dalje zadržavaju IgE vezujući potencijal. Mali peptidi koji potiču od Ara h 2 alergena, su se pokazali kao najpotentniji inhibitori vezivanja IgE iz seruma pacijenata alergičnih na kikiriki, u odnosu na male Ara h 1 i Ara h 3 peptide. U ovoj disertaciji je pokazana izuzetno važna uloga efekata matriksa hrane, kao i njene termičke obrade, na digestiju proteina hrane, koji mogu povećati stabilnost alergena hrane tokom digestije, i time omogućiti zadržavanje potencijala aktivacije alergijske reakcije nakon oralno-gastrične faze digestije.

INFOGEST method is standardized protocol for in vitro simulation of complete food digestion, based on physiologicaly relevant conditions. The objective of this dissertation was to investigate digestibility of peanut allergens from whole peanut kernel by INFOGEST method, as well as to characterize their fragments resistant to proteolysis. For delipidation, protein precipitation approach was applied, showing to be superior in comparison to delipidation by organic solevent, due to lower qualitative and quantitative protein loss. In this thesis it was shown that peanut thermal processing, in addition to effect of food matix, further complicates the extractability and digestibility of proteins from the grain, making peanut allergens Ara h 1, 2, 3 and 6, less accessible for pepsin hydrolysis. Extractability and digestibility of peanut proteins in the gastric phase have shown to be significantly more pronounced, in comparison to intestinal phase, and roasted peanut digestion was impaired compared to the raw. It was shown that after oral and gastric digestion of whole raw peanut grains peanut allergens largely remain intact, and their digestion resistant peptides retain allergenic capacity. The most Ara h 2 and Ara h 6 allergens have been shown to remain resistant to digestion. Ara h 1 and Ara h 3 undergo pepsinolysis with cascade pattern to consequently smaller peptide fragments with retained IgE binding capacity. Small peptides from Ara h 2 allergens were the most potent inhibitors of IgE binding from sera of peanut allergic patients, compared to small peptides from Ara h 1 and Ara h 3. This thesis points to the great importance of the effects of food matrix, as well as food thermal processing, on protein digestibility, which can create additional stability of food allergens during digestion, and thus enable retaining of their potential for the sensitization or triggering of allergic reactions.