The Structural Analysis And Allergenic Assessment Of Allergen Components In Roasted Peanut

Peanuts,is one of the eight major allergic food globally,can induce severe anaphylactic reaction in peanut allergic patients.Peanut allergy has a low eliciting dose,is usually lifelong,and with a increasing prevalence.There are 17 allergens in peanut with potential allergenicity,including some major allergens such as Ara h 1,Ara h 2,Ara h 3 and Ara h 6.In daily consumption,roasted peanuts are commonly consumed on their own or used as an ingredient in processed foods,which may be related to the excellent flavor of roasted peanut.Roasted peanut was widely used,but the effect of roasting on allergenicity of peanut was disputable,the principal reason is that the relationship between the structure and allergenicity of roasted peanut allergen components,including the major allergen monomers and the formed aggregation during the roasting process,remains unclear.To analyze the improvement of roast on peanut flavor,further understand the changes in allergen components in roasted peanuts,and to provide a deeper analysis of the structural basis for changes in allergenicity,this study carried out the following works.Firstly,the nutrition,taste substances and aroma compounds were assessed,to explored the nutrition and flavor characteristics of roasted peanuts;Subsequently,relatively low-allergenic roasted peanut samples are selected by comparing the distribution of protein molecular weight and potential allergenicity of peanut protein under different roasting durations.Then,the structure and in vitro allergenicity of protein aggregation and allergen monomers(Ara h 1,Ara h 2,Ara h 3,Ara h 6)were analyzed by utilizing proteomics techniques and immunological methods.Furthermore,by establishing a peanut allergic mouse model,the allergenicity of roasted peanuts and their allergen components was further validated.Finally,to dig the structureallergenicity relationship of allergens in roasted peanuts.The main methods,results,and conclusions of this study were outlined below:1.Raw peanuts and boiled peanuts were used as control.The nutrition and flavor components of roast peanuts were assessed.Among three kinds of peanuts,the content of protein,fat,fatty acids,and sugar were compared,the changes of contents of free amino acids and taste were analyzed,the volatile flavor compounds were identified and the composition of aroma compounds were analyzed.Results showed that,processing may affect the contents of nutrition and free amino acids.Compared with boil,roast had little effect on the contents of nutrition and free amino acids,but made peanuts had more key taste and aroma substances,had a richer taste and a stronger aroma.The improved flavor made roast an irreplaceable processing method in peanut products production,which also made the study on the structural basis of allergenicity of roasted peanut more meaningful.2.Peanuts roasted at 170 ℃ for different durations were used as the research subjects,their protein patterns,allergen Ig E recognition pattern and potential allergenicity were analyzed.Firstly,the changes of distribution of protein products in peanut during roasting were analyzed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Ig E recognition pattern and Ig E binding capacity of peanut protein were analyzed by Western blot and ELISA individually.Also,the ability of peanut proteins to induce KU812 basophils degranulation was measured.Results showed that,during roasting,bands of allergen monomers become lighter,bands of protein exceeding 100 k Da(OH)obviously appeared,and Ig E from peanut allergic patients can recognize and combine the OH bands.Disulfide bonds played a crucial role in the formation and Ig E recognition of OH.Protein from roasted peanut had higher Ig E binding capacity,but decreased ability to induce KU812 cells degranulation.Especially for protein from peanut roasted for 12 min,it owned the most OH,the strongest Ig E binding capacity,and the lowest ability to induce KU812 cells degranulation.That make peanuts roasted for 12 min were excellent research subjects for studying the relationship between structure and allergenicity of allergens in potentially low-allergenic peanuts.3.Taken OH in peanut roasted for 12 min as the research objective,investigated the relationship between its structure and potential allergenicity.Tandem mass spectrometry-combination with liquid chromatography(LC-MS/MS)and proteomics server software were used to identify disulfide-linked peptides and chemical modification on side chains of amino acids within the OH.Structure simulation and analysis software were used to construct the structures of allergens in crosslinked form and analyze the influence of cross-linking on the structures of the allergens,to construct the modified peptides and analyze the influence of modification on the structures of the allergens.The Ig E binding capacity and the ability to induce KU812 cell degranulation of OH were assessed using ELISA and cell model.Results showed that,the disulfide bonds in OH involved in intermolecular disulfide bonds,intramolecular disulfide bonds,and loop-linked disulfide bonds,they had varying effects on the advanced structures of allergens,changed the structure and exposure of linear and conformational Ig E epitopes.Meanwhile,some modifications have been detected in the amino acid side chain of allergens in OH,which mainly affect the local electrostatic potential distribution of Ig E linear epitopes.Changes in the conformation,electrostatic potential distribution and exposure of the Ig E epitopes in allergens could affect its Ig E binding ability,and may lead to the decreased potential sensitization of OH.4.Regarding the four allergen monomers(Ara h 1,Ara h 2,Ara h 3 and Ara h 6)in peanut as the research objects,analyzed the relationship between their structure and allergenicity.Circular dichroism,ultraviolet spectrum,and fluorescence spectroscopy,were used to analyze the structural changes of allergen monomers in raw and roasted peanuts.Molecular dynamics simulation technique was used to analyze the conformational and local structural changes of the allergen monomers.LC-MS/MS was used to analyze the exposure of trypsin cutting sites and side chain modification of allergen monomers.WB and ELISA assays were used to assess the Ig E recognition abilities and Ig E binding capacities of individual allergen monomers,while the KU812 cell line was utilized to evaluate the abilities of allergens to induce cell degranulation.Results indicated that,compared to the corresponding allergens in raw peanuts,conformational changes of Ara h 1 and Ara h 6 were the largest.The conformational changes induced by roast may alter the structure and electrostatic potential distribution of Ig E epitopes,as well as lead to the exposure or cover of these Ig E epitopes.After roasting,the Ig E recognition ability of peanut allergenic monomers decreased,while the Ig E binding capacity increased.Compared with raw peanut,Ara h 1 and Ara h 6 in roasted peanuts exhibited significantly higher capacities to bind Ig E and to induce KU812 cell degranulation.However,Ara h 2 and Ara h 3 showed no significant changes.That is,the conformation of allergens plays a crucial role in their potential allergenicity.5.Taken raw peanut proteins,roasted peanut proteins,OH and low molecular components in roasted peanut(OL)in roasted peanuts as subjects.Utilizing a peanut allergic mouse model to assess the allergic manifestations and immune responses,levels of specific antibodies,and cytokine release,to compare the allergenicity of different allergen components.Results showed that,roasting peanut at 170 ℃ for 12 min reduced the abilities of peanut protein to induce Th2 cells differentiation and Th2 cytokines release,consequently reduced the ability of peanut protein to induce Th2 type immunoreaction,may decrease the potential allergenicity of peanut proteins.Comparing with roasted peanut protein,both OH and OL showed no significant difference on potential sensitization.The different structures of OH and OL may affect the response of T cells to allergen simulation,influence the release level of IFN-γ.In conclusion,roast is a better processing method for peanut processing,can reduce potential allergenicity of peanut by changing conformation of allergens and forming OH.The formation of OH was involved in the formation of new disulfide bonds,different types of disulfide bonds induced varied changes on structures of allergens,affected the structures of linear and conformational Ig E epitopes,and covered Ig E epitopes located on the contact surface,lead to the low potential allergenicity of OH.Among allergen monomers,different allergens in roasted peanut showed different changes of structures and potential allergenicity,Ara h 1 and Ara h 6showed significantly changed conformation and obviously enhanced abilities to bind Ig E and to elicit KU812 cell degranulation,while Ara h 2 and Ara h 3 showed no significant changes.Results in this study provided an idea for desensitization processing methods of peanut,provided a reference for the solution of peanut allergy,had important scientific and application value;as well as,put new evidence for the critical rule of conformation in the allergenicity of allergens,promoted researches on the relationship between structure and function of protein in the field of food processing.