Effects Of Quercetin On The Allergenicity Of Walnut Protein Under Oxidation Conditions

Walnut protein is a common food allergen and severe allergic reactions are observed.Walnut allergies has become a major global public health problem in recent years.This paper focuses on the Juglans sigillata which is the main species of walnut,Yunnan.The walnut allergenic protein（WAP）and rabbit antiserum were first prepared to be established an indirect competitive ELISA（ic-ELISA）detection method.Quercetin-WAP complexes were further prepared,while their and WAP were oxidized with hydroxyl radical oxidation system.The allergenicity,construct and digestive stability of the samples were studied to explore the effect of quercetin and hydroxyl radical oxidation on the allergenicity of walnut protein,and may provide a new idea for desensitization of walnut protein in the future.The key findings of the paper were as follows:（1）WAP was extracted and prepared from Juglans sigillata,which contained five walnut allergens by LC-MS/MS,Jug r 1,Jug r 2,Jug r 3,Jug r 4 and Jug r 6.The antiserum with the highest titer of 1:40 000 000 was obtained after immunized the New Zealand White rabbits with WAP.Further established the ic-ELISA detection method with the most suitable detect range of 0.125～256μg/m L.（2）Quercetin and hydroxyl radical oxidation system were established to be processed in batches.Quercetin and hydroxyl radical oxidation could be reduced the allergenicity of WAP with the surface microstructure,particle size,endogenous fluorescence and thermal stability were changed.WAP was treated with quercetin（137.66μmol/g）and hydroxyl radical oxidation（10 mmol/L H₂O₂）,the allergenicity of WAP was reduced by 95.18%and 77.77%,respectively.However,the allergenicity of WAP which was treated with quercetin then oxidized by hydroxyl radicals was also reduced by 78.53%.The results showed the most significant desensitization on WAP using quercetin（P<0.05）,but hydroxyl radical oxidation could affect the desensitization of quercetin on WAP.（3）In vitro simulated digestion model test found that the digestion stability of WAP treated with quercetin,hydroxyl radical oxidation,and quercetin-oxidation were all significantly reduced（P<0.05）,and the WAP treated with quercetin-oxidation has higher digestive stability.The results indicated that both quercetin and hydroxyl radical oxidation could reduce the digestion stability of WAP,but the interaction between them could affect their effect on reducing the digestion stability of WAP respectively.（4）141 allergenic peptides were detected on WAP by shotgun mass spectrometry,and the cluster analysis of the correlation showed that oxidation and in vitro simulated digestion had a greater impact on WAP,while quercetin had a lesser impact.GO and KEGG enrichment analysis showed that differential proteins were concentrated in functions such as protein folding mainly,and participated in signaling pathways such as protein processing in the endoplasmic reticulum.PRM showed that hydroxyl radical oxidation and in vitro simulated digestion could be reduced the WAP allergen content,while quercetin treatment had a little effected on WAP.The results showed that the desensitizing effect of hydroxyl radical oxidation on WAP was directly dominated by irreversible modification,while the desensitizing effect of quercetin was indirectly dominated by reversible complex masking of allergens.In conclusion,both quercetin and hydroxyl radical oxidation could be effectively reduced the allergenicity of WAP,and the quercetin was the best,but there is an interaction between the quercetin and hydroxyl radical oxidation which was affected the desensitization of WAP.However,the effect of protein oxidation during storage and processing on quercetin desensitization should be considered,while studying the desensitization of walnut protein by quercetin.This paper provided new ideas for quercetin in food protein desensitization.