Effect Of Heat-treatment, Irradiation And High-pressure Microfluidization On The Structure And Antigenicity Of Peanut Allergen Ara H 6

Peanut is one of the useful plant proteins source for human diet. It is also recognized as one of "big eight" allergens. Due to the increasing incidence of peanut allergy, the investigations on how to prevent peanut allergy is highlighted. Ara h 6 is one of the major peanut allergens, and it could be recognized by 63% peanut-allergic individual serum IgE. In order to determine the relationship between structure and antigenicity of processed peanut allergens, the effects of processing on the structure and antigenicity of peanut allergen Ara h 6 were investigated in this work.The study mainly focused on the purification of Ara h 6, preparation of high specifical anti-Ara h 6 polyclonal antibody, and the effect on the structure and antigenicity of Ara h 6 induced by heat-treatment, irradiation and high-pressure microfluidization. The methods, results and conclusion were shown as follows.1. Ara h 6 was purified by anion-exchange chromatography from natural peanut kernal and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS). It was shown that the purity of Ara h 6 was above 95% and the recovery rate was more than 18.3%. A simple and efficient approach to purify Ara h 6 is achieved, resulting providing reliable experimental materials for further research on peanut allergy.2. Specific rabbit antibody was raised against Ara h 6 from Newland rabbits immunized on the basis of a routine way. The antigenicity of Ara h 6 was evaluated by indirect enzyme-linked immunosorbent assay (ELISA) and Western blotting. The results showed that the titre of the antibody was 1:100,000 by ELISA with high specificity, and It can meet the needs of antigenicity evaluation.3. To investigate the effect of heat-treatment on the structure and antigenicity of Ara h 6, the purified Ara h 6 and crude peanut protein extracts were heated at 0,55, 70,85,100 and 115℃for 30min, or heated at 100℃for 15,30,45 and 60min, respectively. Alternation in structure of Ara h 6 were characterized by SDS-PAGE, circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy and fluorescence spectroscopy, and antigenicity was evaluated by indirect ELISA with anti-Ara h 6 polyclonal antibody. Heating treatment induced significant alternation in the secondary and tertiary structure of Ara h 6, and heat-treatment at 115℃induced proteins to aggregate. Indirect ELISA showed both the antigenicity of purified Ara h 6 and crude peanut protein extracts reduced following the increasing temperature or increasing time. These results implied that the structure of Ara h 6 is crucial to its antigenic potential. The conformational changes of Ara h 6 lead to the decreasing of antigenicity.4. To investigate the effect of irradiation and high-pressure microfluidization on the structure and antigenicity of Ara h 6, the purified Ara h 6 and crude peanut protein extracts were irradiated at 0,1,3,5, 10kGy, or treated at 0,60,120,180MPa, respectively. Alternation in structure of Ara h 6 were characterized by SDS-PAGE, CD spectroscopy, UV absorption spectroscopy and fluorescence spectroscopy, and antigenicity was evaluated by indirect ELISA with anti-Ara h 6 polyclonal antibody. Irradiation and high-pressure microfluidization treatment induced significant alternation in the secondary and tertiary structure of Ara h 6, and protein aggregation appeared after irradiation. Indirect ELISA showed both the antigenicity of purified Ara h 6 and crude peanut protein extracts was reduced following the increasing irradiation dose or increasing pressure. It suggests that the reducing in antigenicity of Ara h 6 is due to the loss of structure induced by irradiation or high-pressure microfluidization treatment. The structure of Ara h 6 plays an important role in its antigenicity.