Screening Of Hypoallergenic Peanut And Effect Of Electron Beam Irradiation On Immunoreactivity Of Peanut Allergens

Food allergy, which was also known as food hypersensitivity (FH), is a kind of excessive immune response, and it will lead to ill or even death of human being. With the rapid development of the contemporary food processing and international trade, the incidence of FH increased year by year at home and abroad. The only effective method of the treatment about food hypersensitivity is the strict control of food allergens; therefore, seeking a method to reduce or eliminate food allergenicity has become one of the key point about food safety research. Peanut, as the main oil crop in China, is also one of the eight major food allergies. Rich germplasm resources and various types of Chinese peanuts provided materials for the screening of hypoallergenic or non-allergic peanut. My researches focus on lowering peanut proteins allergenicity by using processing method of electron beam irradiation, and evaluation of allergenicity of Chinese planting peanuts varieties.Preparation of peanut allergen was established first. After defatted by hexane, the protein extracts was made by the method of direct extraction using phosphate buffer solution, and then ultrasonic wave treatment was used to break cells wall to improve the rate of extracts, the allergic solution was dialysis after centrifugation, and then the protein samples was freeze-drying and cryopreservation. The serum of patients suffered from peanut hypersensitivity was used and three major allergen Ara h1, Ara h2, Ara h3are initially identified for Chinese.Altogether33kinds of Chinese peanut’s varieties and23kinds of Japanese peanut’s varieties were collected for the evaluation of the peanut allergenic difference. The kinds of peanut allergy and their content were evaluated using of SDS-PAGE electrophoresis and Gel Imaging System, and the level of immunoreactivity among varieties was investigated by indirect ELISA test. Results showed that, the lack of Ara h3(35,35kDa) was found in peanut varieties of Suizhong and Shuyangdazhanyang, lack of Ara h3(24kDa) was found in peanut varieties of Xinjinfuda, Rudongwanerqing, Xingchengdahuasheng, Japanese varieties16and19. The relative content of Ara h1was the highest among the three kinds of allergens in all the species, content range25～47.6%for Chinese varieties, and20.2～39.6%for Japanese varieties. The relative content was followed by of Ara h2(20kDa)and Ara h3(14kDa). The immunoreactivity of Chinese peanut allergen is higher than Japanese peanut by indirect ELISA test, combined with the serum of patients. The lowest immunoreactivity varieties is Tielingsilihong, the highest is Yingkousilihong in Chinese varieties, and the JP-10is the highest, the JP-6is the lowest Japanese varieties. Because the immunoreactivity of peanut depends on complex factors, it is difficult to explain the immunoreactivity simply by the lack of allergen bands or the level of content.The effects of electron beam (EB) irradiation on lowering immunoreactivity and changing the biochemical properties of peanut allergens were evaluated in this study. The defatted peanut powder and extracted protein solution were treated by EB irradiation at the doses of2,4,6,8,10,15and20kGy. SDS-PAGE electrophoresis was used to observe the change of protein molecular weight. Changes in allergenicity of irradiated peanut major allergen (Ara h1, Ara h2, Ara h3) were analyzed by immunoblotting and indirect enzyme-linked immunosorbent assay (CI-ELISA). The changes of concentration, turbidity and hydrophobicity of the irradiated protein solution were determined by UV and fluorescence spectrophotometer. Results showed that allergen solution is more sensitive to EB irradiation than allergen in solid state. When the irradiated dose was lower than lOkGy, the immunoreactivity of allergen solution became little stronger; but the immunoreactivity was decreased when irradiation dose was higher than lOkGy. The IC50of20kGy irradiated samples was1ltimes of control. The concentration and turbidity of the allergen solution increased with the increase of irradiation dose. Hydrophobicity first increased with increasing of irradiation dose and then decreased when the irradiation dose was higher than15kGy. The effects of EB irradiation on immunoreactivity and biochemical properties of peanut allergen in solid state is not significantly.In conclusion, the method of the allergen extraction was established, the three major allergens were initially identified, lack of one allergenic protein was found in tested peanut’s varieties, and20kGy irradiation could be used to lower immunoreactivity of peanut allergen solution.