Effect Of Baking Process On The Structure And Allergenicity Of Peanut Main Allergen Protein Ara H 2

Compared with other food allergies,peanut allergy has severe symptoms and high mortality.It is a huge threat to human safety and health,and also the main research object.There are 17 confirmed allergen proteins in peanuts.Ara h 2 is considered to be one of the most important allergen in peanuts because of its strong sensitization and high IgE recognition rate in the serum of allergic patients.Baking,as a common processing method,will change the advanced structure of peanut protein,the epitopes would be damaged?exposed or generated,thus affect its allergenicity.In this paper,homemade antibodies and immunoaffinity columns were used to purify Ara h 2 from raw and baked peanuts.The components of each solution obtained by the purification were identified by Western blotting together with mass spectrometry.And the circular dichroic spectrum and ultraviolet absorption spectrum were used to analyze the influence of baking processing on the advanced structure of Ara h 2.Different evaluation methods were employed to evaluate the IgE binding capacity of native and baked Ara h 2 and the ability to induce degranulation of human basophils KU812.The main research methods,results and conclusions were as follows:1.Firstly,Ion exchange chromatography was used to separate Ara h 2 from raw peanuts to obtain rabbit anti-peanut protein serum and rabbit anti-Ara h 2 serum,and the titer of rabbit serum were dected by ELISA.The homemade immunoaffinity column was prepared with rabbit anti-Ara h 2 Ig G purified by Protein G Column as ligand to purified Ara h 2 from raw and baked peanuts.The results showed that the purity of Ara h 2 purified by anion column was more than 90 %,the titer of rabbit anti-peanut protein serum was 1:60 000,the titer of rabbit anti-Ara h 2 serum was1:100 000.The homemade immunoaffinity column successfully purified Ara h 2 from raw peanuts,whose purity was 88.9 %;This column could also work on baked peanuts,from which could get Ara h 2,Ara h 6 and also a mount of high molecular weight protein,and the purity of Ara h 2 among them was only 13.4 %.2.The affinity purification products of raw and baked peanuts were identified by rabbit serum immunoblotting and mass spectrometry,combined with the Ara h 2structure model.The effect of baking processing on the structure of Ara h 2 was analyzed.The results showed that after affinity purification,Ara h 2(also with a small amount of Ara h 6)were obtained from raw peanuts,and the components of roasted peanuts purification products were more complex: except for Ara h 2 and Ara h 6monomer,a few of their cross-linked products with Ara h 1 and Ara h 3 were also present in the bands with molecular weights around 70 k Da and 43 k Da,proving the crosslinking reaction caused by baking process.The mass spectrometry results showed that there were 20 peptides detected in the native Ara h 2,and only 11 of them were detected in the baked Ara h 2,the number of Ara h 2 peptides detected after baking decreased,and the different peptides were mainly located in the end of the second helix,the front of the fifth helix and the random coil area.It showed that baking processing would affect the structure of Ara h 2,while exposing protein allergenic epitopes or generating new allergenic epitopes,which cause the difference between the peptides detected in native and baked Ara h 2.3.Using circular dichroism spectroscopy and ultraviolet spectroscopy to analyze the effect of baking on the structure of Ara h 2 monomer.The results showed that there was no significant difference in structure between ion-exchange chromatography purified native Ara h 2(IPN Ara h 2)and afficity chromatography purified native Ara h 2(APN Ara h 2),the ?-helix content of IPN Ara h 2 and APN Ara h 2 were 27.1 % and 25.7 %,respectively,the radom coil content were 27.9 %and 29.1 %,respectively,ultraviolet absorption peaks were similar,indicating that the purification method has no effect on the characterization of Ara h 2 structure.Baking would reduce the ?-helix content of Ara h 2 and increase the content of random coils,the ?-helix and radom coil content of affinity chromatography purified baked Ara h 2(APB Ara h 2)were 21.6 % and 34.3 %,also the decrease in absorption indicated that the protein under high-temperature baking tends to be disordered and the overall structure became more stretched.4.Baking process could lead to changes of protein structure,which in turn affects its allergenicity.Peanut allergy patients' serum immunoblotting and competitive inhibition ELISA were used to determine the IgE binding of the affinity separation products and the IgE binding capacity of Ara h 2 in raw and baked peanuts.the sensitization change of Ara h 2 monomer after baking were evaluated by detecting the level of different cytokines in the basophils KU812 degranulation model.The main coloring part of the affinity separation product of raw and baked peanuts in immunoblotting were at the position of Ara h 2 and Ara h 6,and the cross-linked products in baked peanuts was weakly colored,indicating the weak IgE banding capacity of cross-linked products.The IC50 values of native Ara h 2 monomer obtained by different purification methods were 0.156 ?g/m L and 0.158 ?g/m L,respectively,and the IC50 value of Ara h 2 monomer after baking was 0.098 ?g/m L,which indicated that the Ara h 2 obtained by different purification methods had the same sensitization potential,also the baking process would enhance the IgE binding capacity of Ara h 2.The result of KU812 cell model indicated that the determination results of different cytokines were different: the ?-HEX release rate and TNF-?release increased after baking processing,while histamine release decreased,indicating that Ara h 2 monomer after baking processing stimulating the degree of degranulation of human basophils increases,but the ability of recruiting granulocytes to pro-inflammatory factors decreases.