| Peanut is a high-fat, high-protein and high nutritional value foods, as one of the most important allergy in food, peanut has caused worldwide concern for its serious symptons, higher incidence and lots of deaths. Ara h2is the most sensitive allergen in peanut which can be recognized by at least90%of allergic patients serum IgE, Therefore research on reducing the Ara h2allergencity has important significance.Our work started from separation and purication Ara h2from peanut and got enzyme extraction of polyphenol oxidase from agaricus bisporus. Then comfirmed the optimal conditions of the polyphenol oxidase catalyzed cross-linking Ara h2, and studied the simulated gastrointestinal digestion and allergencity of Ara h2and cross-linked protein, then analysised microstructure change of these products. The main contents are as follows:1. Used Ara h6which has59%homology to Ara h2as the template, homology modeled Ara h2.01and Ara h2.02, both of them have only one model. After software analysised the allergen epitopes and cross-linking sites, we found Ara h2.01may have three crosslinking sites and Ara h2.02may have four crosslinking sites; most of these crosslinking sites are between the three major allergens epitope aa60-69, aa70-75in Ara h2, for these reasions, it may has significant allergenic effects on the Ara h2allergencity.2. Optimized the anion exchange chromatography column one-step separation and purification allergen Ara h2in peanut, including salt gradient elution concentration, buffer pH and extraction of crude protein, the purity is more than90%after coomassie blue staining, and the purification yield is23.31%, this method has good reproducibility. Prepared polyphenol oxidase enzyme solution, the enzyme activity is6,000U/mL and can be used for follow cross-linking experiments.3. Comfirmed the optimal cross-linking processing conditions by polyphenol oxidase catalyzed. When the protein concentration of Ara h2was1mg/mL, the optimum system was polyphenol oxidase final activity1600U/mL, the enzymatic reaction conditions:50℃, pH7.0, reaction time80minutes. Multi-factor experimental results show that there is a significant difference between enzyme activity and temperature. There are also no significant difference changes in different ionic strength. Clarifed different reducing agent on the polyphenol oxidase-catalyzed cross-linking Ara h2and found β-mercaptoethanol completely inhibited the enzymatic crosslinking reactions occur, the natural Ara h2still retains two clear bands. Further study showed that when β-mercaptoethanol concentration is less than0.1%, the bands of the natural Ara h2has been blurred, the Ara h2spatial structure has changed.4. Evaluated the digestion of peanut Ara h2and the crosslinked product, and we evaluated microstructure change of Ara h2, the cross-linked product, simulated gastrointestinal digestion product by means of microscopy at the same time, the study show that the Ara h2resist to gastric digestion and easily digested in the intestinal solution, while Ara h2crosslinked product relative can be gastric digestion and the intestinal digestion rate slow down due to the formation of high dimer. The microstructure results show that the natural Ara h2is easy to gather into spherical, the average particle diameter of Ara h2is287nm, and the formed crosslinked polymer average particle diameter is1705nm. After simulated gastrointestinal digestion, the electron microscopy results and the particle size detected results coincide, the particle size of Ara h2was less than287nm due to the pepsin or the acidic environment influence, and this particle size is not uniform. The cross-linked protein after gastric digestion, we observed the particle diameter about300nm residual which was Ara h2protein, and cross-linked protein after simulated intestinal digestion,also Ara h2of approximately400nm and polymer of900nm which has not been fully digestion were observed, in short, by means of microscopy further explained the digestion change.5. Used indirect competitive ELISA assessed the antigenicity of Ara h2, the crosslinked product and indirect ELISA assessed the allergenicity of Ara h2, Ara h2crosslinked product, the results showed that, compared with Ara h2, Ara h2crosslinked product has lower antigenicity in low concentrations (<0.32μg/mL) and the Ara h2is higher when in high concentration (>0.32μg/mL), the crosslinked protein is higher than Ara h2, but there is no significant difference between them. The allergencity of Ara h2significantly reduced after cross-linking by IgE binding capacity experiment. For the simulated gastric digestion of Ara h2and cross-linked product, the strongest IgE-binding ability is nature Ara h2, followed crosslinked protein, the weakest is gastric digestion of Ara h2; also after simulated intestinal digestion of Ara h2and the crosslinked protein, the strongest IgE binding capacity belong to nature Ara h2, followed Ara h2after simulated intestinal digestion, the weakest is cross-linked protein. Final study showed that0.5%of β-mercaptoethanol is the critical point to control the cross-linking reaction which has the lowest allergenicity... |
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