| Peanut allergy is one of the most severe food allergies.Only trace amounts of peanut allergens can cause a life-threatening allergic reaction,which generally does not vanish with age.As a common method of processing peanut products,thermal processing changes the conformational structure of protein,while exposing peanut allergenic epitopes or generating new allergenic epitopes,which have a certain impact on the potential allergenicity of peanut allergen proteins.To reduce the risk of triggering allergic reaction,many studies have investigated different thermal treatments to reduce the allergenicity of peanut allergens,However,the specific sites of the structural change and the mechanism of influencing epitopes caused by thermal processing is rarely reported.In this study,raw peanut,as the research object,was treated by different thermal processings,followed by proteins separation,then analyzed the structure and potential allergenicity of the major peanut allergens.Firstly,the raw and thermally processed peanuts were prepared into powder,total protein was extracted from defatted peanut powder,and the major peanut allergens,such as Ara h 1,Ara h 2 and Ara h 3 were initially separated.Secondly,the peptides of the corresponding allergens combined with the conformational structure model was analyzed respectively,reflect the changes in the conformational structure of these three major peanut allergens after different thermal processings.Finally,combining with the epitopes of three major peanut allergens,the effects on the potential allergenicity of peanut major allergens Ara h 1,Ara h 2 and Ara h 3 were analyzed.The main methods,results and conclusions were summaried as follows:1.Raw peanuts were thermally processed as follows:boiled peanut with shell or without shell(boiling:100? for 15 min),roasted peanut with shell(roasting:170?for 35 min),roasted peanut without shell(roasting:170? for 20 min)and fried peanut without shell(frying:152? for 400 s).After peanuts were shelled and grinded uniformly,they were defatted with acetone to obtain defatted peanut powder.Total protein was extracted from the defatted peanut powder by chaotropic salt solution,and the major peanut allergens Ara h 1,Ara h 2 and Ara h 3 were separated by SDS-PAGE.The interest protein bands of Ara h 1 monomer were excised and subjected to enzymatic digestion.The peptides' information of Ara h 1 was obtained by mass spectrometric detection.Then combined with the conformational structure model of Ara h 1,the changes of the conformational structure of Ara h 1 protein after different thermal processings were analyzed.Meanwhile,the changes of potential allergenicity of Ara h 1 after different thermal processings were analyzed combined with the epitopes of Ara h 1.There was no significant change in Ara h 1 protein bands after boiling.The Ara h 1 monomer decreased significantly after roasting and frying,and the peanut polymer bands also appeared above 180 kDa.A total of 79 peptides of Ara h 1 were detected in raw peanut and different thermal treated peanut samples.After different thermal processing,the number of Ara h 1 peptides detected in samples decreased,and the roasted peanut with shell was the least.Among them,72 peptides were detected in raw peanut,57 peptides for boiled peanut with shell,65 peptides for boiled peanut without shell,32 peptides for roasted peanut with shell,56 peptides for roasted peanut without shell and 42 peptides for fried peanut.The amino acid coverage of Ara h 1 was 79.2%for raw peanut,69.88%for boiled peanut with shell,71.38%for boiled peanut without shell,43.93%for roasted peanut with shell,70.55%for roasted peanut without shell,and 55.41%for fried peanut.This indicated that thermal processing destroyed the protein structure of Ara h 1 and affected the exposure of trypsin digestion sites.The peptides of Ara h 1 detected in raw peanut involved 20 linear epitopes.17 linear epitopes were detected in both boiled peanut with and without shell,and the amount of linear epitopes detected in roasted peanut without shell,fried peanut,and roasted peanut with shell were decreased orderly,which were 19,16 and 13 respectively.After mass spectrometric pre-processing,the amount of linear epitopes destroyed by enzymatic digestion were:17(raw peanut),15(boiled peanut with shell),15(boiled peanut without shell),10(roasted peanut with shell),14(roasted peanut without shell),13(fried peanut).Different thermal processings destroyed the structure of Ara h 1.After roasting with shell or frying,Ara h 1 had the highest degree of structure compactness.Compared with these two processings,the structure of Ara h 1 was less compactness after boiling.The structure of Ara h 1 tended to be compactified after thermal processings,part of the trypsin cleavage sites were covered,thus reducing the damage to enzymatic hydrolysis to the linear epitope of Ara h 1.2.The Ara h 2 protein bands of different thermal processed peanuts were excised,and then subjected to in-gel digestion and mass spectrometric detection.Ara h 2 protein bands were shallowed at different levels after different thermal processings.Roasted and fried peanut both showed diffuse Ara h 2 protein bands,especially the roasted peanut with shell.A total of 27 peptides of Ara h 2 were detected in all peanut samples.The amount of Ara h 2 peptides detected in peanut increased after thermal processings,the fried peanut reached the highest.15 peptides were detected in raw peanut,16 for boiled peanut with shell,19 for boiled peanut without shell,18 for both roasted peanut with and without shell,20 for fried peanut.The amino acid coverage of Ara h 2 was 61.59%in both raw peanut and boiled peanut with shell,75.5%for boiled peanut without shell,72.85%for roasted peanut with shell and 74.83%for roasted peanut without shell;fried peanuts reached the highest amino acid coverage with 80.13%.The amount of Ara h 2 peptides and amino acid coverage were all increased after thermal processings except for boiled peanut with shell.It indicated that the conformational structure of Ara h 2 might be damaged to varying degrees,exposing more enzymatic sites.8 linear epitopes were detected in both raw and boiled peanut with shell.The boiled peanut without shell,roasted and fried peanut all involved 10 linear epitopes.After mass spectrometric pre-processing,the amount of linear epitopes destroyed by enzymatic digestion were:5(raw peanut),5(boiled peanut with shell),6(boiled peanut without shell),6(roasted peanut with shell),7(roasted peanut without shell),7(fried peanut).Different thermal processings could break the disulfide bonds,resulting in the expansion of Ara h 2 protein structure,among which roasting with shell had the most destructive effect on the structure of Ara h 2,while boiling with shell had the least destructive effect.The expansion of Ara h 2 exposed more trypsin cleavage sites,in which boiling with shell had few effects on the linear epitope of Ara h 2,while boiling without shell,roasting and frying all enhanced the damage to the linear epitope of Ara h 2.3.The Ara h 3 protein bands of different thermal processed peanuts were excised,and then subjected to in-gel digestion and mass spectrometric detection.Ara h 3 protein bands were shallowed at different levels after different thermal processings.Roasted peanut with shell showed narrow and diffuse protein bands.A total of 74 peptides of Ara h 3 were detected in all peanut samples.The amount of Ara h 3 peptides detected in peanut increased after thermal processings,the boiled peanut with shell reached the highest.Only 48 peptides were detected in raw peanut,64 for boiled peanut with shell.The boiled peanut without shell,roasted peanut with shell and fried peanut were all detected 55 peptides,and 51 peptides for roasted peanut without shell.The amino acid coverage of Ara h 3 was 65.49%in raw peanut,70.98%for boiled peanut(with shell or without shell)and roasted peanut without shell,73.33%for roasted peanut with shell,and 71.18%for fried peanut.Only two linear epitopes detected in all peanut samples.Under enzymatic digestion,there was no difference of destructions among the linear epitopes in all peanut samples.Boiling had the lowest damage to the structure of Ara h 3,while the roasting and frying both had a greater damage to the structure of Ara h 3,especially in roasted peanut with shell.The structural changes of Ara h 3 caused by thermal processing affected the exposure of the trypsin cleavage site,but did not change the damage of the linear epitope by enzymatic hydrolysis before and after thermal processing. |
PDF Full Text Request