Intestinal Tract Controlled Release Of Fish Allergen And The Mechanism Of Inducing Immune Tolerance

As the popular food, fish is eutrophy and delicious, but fish allergen makes manyconsumers shrink back at the sight of fish, so allergen have a strong impact on theamplification of cusumers. At present there are two methods for food allergy,removing the allergen or remedying the symptom, but these two methods just curethe symptom and consumers are still hypersensitive. So this paper focuses on thepreparation of a safe and efferctive microspheres encapsulating fish allergen, andalso discusses the mechanism of oral tolerance.In this study, fish allergen was extracted and encapsulated in sodium alginate toinduce two kinds of beads, after FTIR、DSC and release in PBS, we chose one kindof microsphere and used in allergic organism. The main results are as follows:1. This study focused on extraction and purification of fish main allergen,Parvalbumin. We used salting out and gel chromatography to purify Parvalbuminfrom Turbot and get12kDa protein. The yield of Parvalbumin was0.286%,westernblot showed that the protein had immunocompetence.2. After preparation, compared the encapsulation efficiency and release rate invitro, and then determined the materials of mice oral toterance experiments. Themethod of instilment got optimum microspheres using3%sodium alginate solutioninstilled in5%calcium chloride solution with pH1.0. FTIR, DSC and SEM showedmicrospheres had rough surface and a mass of holes in the meantime Parvalbumin hadsuccessfully encapusulated into microspheres. Release experiements in vitro showedthat release rate could reach to61%and release kinetics fit Higuchi model andRitger-Peppas model, the mechanism could be fit with skeleton erosion in additionwith protein diffusion mechanism. Meanwhile, we used emulsion method to preparealginate-Parvalbumin microshpheres and the encapsulate efficience was83.79%,grain diameter was decreased and release rate could reach73%, release kinetics fitwith zero equation and the mechanism was linear diffusion. Compared with instilment,emulsion method could get smaller, efficient and released longer microspheres, it was appropriate for oral tolerance raw material.3. We confirmed the effectiveness of oral tolerance induced by allergenencapsulated by microspheres and investigated the mechanism of oral tolerance.Compared to allergy model group, mice orally intake microspheres encapsulatingallergen reduced specific IgE antibodies by25.85%, total IgE antibodies by36.61%and serum IL-4by27.75%. The allergic organism induced oral tolerance. Speakingdosage, the low allergen dosage was more effective. Meanwhile speaking proteincondition, allergen protein encapsulated by microspheres could stimulate oraltolerance as well as protein solution, and through slow release the safety of toleranceprogress was higher. We analysed the mechanism and found low dose fish allergenpromoted IgG antibodies to reduce the production of specific IgE antibodies as wellas promoted immune system Th2cell mass transforming to Th1cell mass and recoverthe balance of cell mass while high dose allergen just promoted the cell masstransformation.In sum, this study established two kinds of general manufacture method and thenseparately researched the structure characteristic, probed into the release curve invitro and confirmed the efficiency of leading to oral tolerance. In addition, themechanism of microspheres encapsulated with allergen induced oral tolerance wasclarified and this study provided technological base of oral tolerance food.