Allergenicity Assessment Of Peanut Protein After Thermal Processing

Peanut belongs to the eight major allergens.As common food and a kind of food ingredient,peanut can induce severe allergic reaction.Peanut is always eaten after cooking,however,the effect of processing on allergenicity of peanut remains uncertain.Which may owe to the various protein extract methods and sensitization assessment methods among related researches.Therefore,clarifying the influence of extraction and assessment methods on potential allergenicity assessment,and analyzing the effect of thermal processing on digestibility and potential allergenicity,will provide the basis knowladge for optimization of peanut products.The presented study,fresh peanut was taken for thermal processings,then,the defatted peanut powder was prepared using acetone.On the one hand,protein was extracted by different methods,the influences of extract methods,so the extractability,compositions and structure of protein effected by processing methods were analyzed.On the other hand,simulating digestion was used to analyze the effect of thermal processing on degestibility of protein in defatted peanut powders?DPP?.Finally,different sensitization assessment methods were employed to evaluate the potential sensitization of peanut in vitro,including the IgE binding capacity and the capacity of inducing basophil KU812 cell degranulation,using digested DPP productions.The main methods,results and conclusions were as follows:1.Fresh peanuts were thermally processed as follows:boiled peanut with shells?100°C for 15 min?,boiled peanuts without shells?100°C for 15 min?,roasted peanuts with shells?170°C for 35 min?,roasted peanuts without shells?170°C for 20 min?and fried peanuts without shells?152°C for 400 s?.After processing,peanut kernels were milled and then defatted by acetone to prepare the DPP.The total protein content was determined by kjeldahl.Results demonstrated that,the total protein content of fresh peanut was 51.84%.After processing,the contents were 52.29%?boiled with shells?,51.69%?boiled without shells?,51.78%?roasted with shells?,52.71%?roasted without shells?and 54.21%?fried?.The result indicated that thermal processing would not significantly influence the total protein content in peanut.2.Peanut protein was extracted from DPP by three methods.Method 1 was implemented using duplicate extraction by mixing the DPP with 50 mM Tris-HCl?1:10?w/v?,pH 7.2?,and extracted deplications.Method 2 was implemented by mixing DPP with 20 mM Tris-HCl?5%w/v,pH 8.2?.Method 3 was performed by mixing the DPP with chaotropic zwitterionic buffer?1:50?w/v??in an ultrasonic bath.Peanut protein content in each sample was determined by Bradford assay and the extractability was calculated,the components of protein were evaluated using SDS-PAGE assay.Results demonstrated that,protein extractability of processed peanut in method 1 and method2 were within the limits of 7.20%¹8.58%and 5.21%²3.24%respectively,which was significantly lower than that of raw peanut?45.35%,40.77%?.From SDS-PAGE assay,protein extracted by method 3 contained the most compositions.After thermal processing,the extractability of Ara h 1 monomer in Tris-HCl buffer?method 1 and 2?decreased obviously.Method 1 could extracted 29.55%of Ara h 1 monomer from raw peanut but no more than 1.57%from processed peanut.For method 3,although roasted treatment decreased extractability of Ara h 1 and Ara h 2 monomer,the high molecular weight protein aggregate could be obtained.3.Using circular dichroism?CD?spectroscopy and ultraviolet absorption?UV?spectroscopy,advanced structure of protein extraction was characterized,the digestibility of total protein in DPP was analyzed by Tricine-SDS-PAGE.Results showed that,protein extracted by different extraction methods had different advanced structures.the?-helix contents of peanut treated with roasted with shells,roasted without shells and fried in method 1 were 24.05%,15.70%,11.80%,which were higher than that in method 2?3.05%,8.75%,5.45%in order?.For method 3,protein structure could be destroied during extracted.Form raw peanut,the?-helix contents of peanut protein were 23.10%and 20.02%in method 1 and 2,but it was only 7.10%in method3.Besides extraction,thermal processing altered advanced structure of peanut protein,reduced the contents of?-helix or?-structure,unfold or unorder the protein structures.4.Competive inhibition ELISA assay was devoted to assess the IgE binding capacity of protein extractions and digestive productions of DPP,and the affinity constant between allergen and IgE was quantified using BLI method.Results illustrated that,the BLI assay had good consistency with the results from ELISA.Under Tris-HCl?method 1 and 2?extract methods,compared with raw peanut,boiled with shells and roased with shells could significantly decreased the IgE binding capacity of peanut protein,roasted without shells had no significant influence on the IgE binding capacity of protein extraction,fried treatment could enhance the IgE binding capacity.The IC₅₀values of proteins extracted by method 1 were:1.22?g/mL for raw,3.29?g/mL for boiled with shells,1.81?g/mL for boiled without shells,2.78?g/mL for roasted with shells,0.95?g/mL for roasted without shells and 0.73?g/mL for fried.For proteins extracted by method 2 were:1.14?g/mL for raw,3.77?g/mL for boiled with shells,2.74?g/mL for boiled without shells,4.93?g/mL for roasted with shells,1.25?g/mL for roasted without shells and 0.67?g/mL for fried.In proteins extracted by method 3,the IC₅₀ value of raw peanut was 11.02?g/mL,boiled peanut with or without shells both could not influence the IgE binding capacity of protein with the IC₅₀ values of13.12?g/mL and 10.76?g/mL,but roasted with or without shells and fried treatment showed dereased IC₅₀ values of 7.84?g/mL,6.64?g/mL and 3.29?g/mL,obviously increased the IgE binding capacity of protein.That is,the extract methods could influence the detection results of IgE binding capacity of protein.Moreover,after digested by gastric fluid or gastrointestinal fluid,the digestive productions of DPP of processed peanut showed decreased IgE binding capacity than raw one.After gastric digestion,the IC₅₀ values of protein samples were:3.13?g/mL for raw,5.67?g/mL for boiled with shells,4.70?g/mL for boiled without shells,20.42?g/mL for roasted with shells,6.33?g/mLfor roasted without shells,5.93?g/mL for fried.After gastric-intestinal digestion,the IC₅₀ values of protein samples were:0.48?g/mL for raw,3.92?g/mL for boiled with shells,5.05?g/mL for boiled without shells,22.90?g/mL for roasted with shells,3.43?g/mLfor roasted without shells and 3.98?g/mL for fried.5.KU812 cell model was created,by detecting the level of intracellular Ca²⁺,the release of?-hexosaminidase,histamine and IL-4 in supernatant after simulated by allergens,to reflect the capability of protein extraction and digestive productions of DPP to elicit cell degranulation.Results indicated that,roasting treatment could induce higher cell degranulation level when protein extracted by Tris-HCl buffer?method 1and method 2?.After sitimulating with protein form method 1,the intracellular Ca²⁺level of peanut protein from raw,roasted with shells and roasted without shells samples were 65.17%,80.26%and 74.41%,the release of histamine were 7.90 ng/mL,9.94ng/mL and 11.11 ng/mL.For method 2,the intracellular Ca²⁺levels of peanut protein from raw,roasted with shells and roasted without shells samples were 68.88%,73.23%and 70.79%,the release of histamine were 8.12 ng/mL,10.05 ng/mL and 9.56 ng/mL.And the inconsistent changes of cytokines were found in protein extracted by method3.Nevertheless,after gastrointestinal digesting,boiled treatment?with or without shells?could decrease the release of four cytokines,decreased the capability to trigger KU812cells degranulation,on the contrary,roased without shells could enhance the degree of KU812 cells degranulation with increased cytokines and intracellular Ca²⁺level.While treatment of roasted with shells and fried had no significant influence on the potential sensntization of peanut.