Proteins Separation And Allergenicity Assessment Of Dried Peanut Treated By Three Thermal Processings

Peanut is listed among eight major food allergens by FAO/WHO.Peanuts allergy can cause a severe allergic reaction,even endanger lives,and the allergic reaction generally is life-long,attracting widespread attention.Peanut is always consumed or used in food industry after drying and thermal processing,hence,it is important to analyze the structure and potential allergenicity change of dry peanut after thermal processing.In this work,sun-dried peanuts were treated by different thermal processing(boiling,roasting and frying),followed by defined the changes of structure and allergenicity of thermal processed peanut.The protein samples were extracted from raw and thermal processed peanut by three methods;the protein extraction rate,allergens components and structural changes were analyzed;then the digestibility of raw and thermal processed peanut was compared after simulated digestion;finally,the IgE binding ability of peanut protein extracts and simulated digestion products of defatted powders were quantified using ELISAand basophilic granulocyte in vitro degranulation model.The main methods,results and conclusions are summaried as following:1.The sun-dried peanut kernel were processed(boiling: 15 min at 100 ℃;roasting: 10 min at 170℃;frying: 400 s at 152℃),followed by ground and defatted.Then the content of whole protein was determined by Kjeldahl.Protein was extracted using three methods(method 1: stirred with 50 mmol/L Tris-HCl at 4 °C;method 2: stirred with 20 mmol/L Tris-HCl at 4 °C;method 3: extracted by ultrasonic bath with chaotropic zwitterionic buffer at 60 °C),and the content of peanut protein in each sample was determined by Bradford assay to calculate the protein extraction ratio.SDS-PAGE was performed to detect the protein patterns of these samples.Results shown that the extraction ratio of method 3 was higher than the other two.Its extraction ratio on roasted peanut was 25.85%,and its extraction ratio of other three samples were more than 50%.The extraction ratio of roasted peanut was significantly lower than the other samples.The extraction ratio of three methods on roasted peanut were 3.72%,4.08% and 25.85% respectively.The result of SDS-PAGE showed that patterns of protein extracted by method 1 were the most diverse.Thus,compared with the other two method,method 1 could extracted morevarious allergen protein from thermal processed peanut.2.Far-UV CD spectra and the UV absorption spectra were performed to analyze the structure of peanut protein.The digestibility of defatted thermal processed peanut powder were investigated using simulates stomach and intestine digestion in vitro.Then the protein patterns were compared by Tricine-SDS-PAGE.The results showed that the structure of boiled peanut protein contained more compact tertiary structure,while the secondary structure changed little.The secondary structure of roasted peanut protein changed much,and its UV absorption peak was lower than others,which means that roast effected protein advanced structure severely.The structure of fried peanut protein extracted by method 1 and method 3 changed broadly,while the structure of protein extracted by method 2changed slightly.Thus,the extraction method can affect the advanced structure of peanut protein.The result of Tricine-SDS-PAGE showed that fried peanut protein left the least after 80 min of pepsin digestion,and roasted peanut protein left left least protein after 40 min of trypsin digestion.Which means roasted peanuts were more easily digested by trypsin,and fried peanuts are more easily digested by pepsin.Thermal processing can enhance the digestibility of peanut protein.3.The protein structure and digestibility of peanuts were changed by thermal processing,which could influence its allergenicity.The IgE-binding capacity of peanut protein was determined by competitive inhibition ELISA.The result showed that compared to raw peanut protein extracted by method 1,the IgE-binding capacity of fried peanut protein was higher while boiled and roasted peanut protein were lower.The IC50 values of protein samples extracted by method 1 were 1.9 μg/mL(fried),2.1 μg/m L(raw),3 μg/mL(boiled)and 4.5 μg/mL(roasted).Comparing with peanut protein extracted by method 2,the IgE-binding capacity of fried peanut protein was the highest while the other three samples were similar,their IC50 values were 3.5 μg/mL(fried),7.0 μg/mL(raw),6 μg/mL(boiled),7.2 μg/mL(roasted).For protein samples extracted by method 3,the IgE-binding capacity of four samples was similar.The IC50 values were 0.9 μg/mL(roasted),1.0 μg/mL(fried),1.2 μg/mL(boiled),1.3 μg/mL(raw).Compared with gastro digestive products of raw peanut defatted powder,the IgE-binding capacity of fried peanut was similar with it,but that of boiled and roasted peanut reduced slightly.Compared with gastro-intestinal digestive products of raw peanut defatted powder,the IgE binding capacity of fried peanut was increased,and that of boiled and roasted peanut were decreased.Thisresult was similar to the result of protein extracted by method 1,which may relate to the various allergens were extracted.The assessment result of potential allergenicity also influenced by the processed method to peanut(include extraction method and simulates digestion),and the result of simulate digestive products of peanut defatted powder should be more reliable.4.The basophilic granulocyte degranulation model was used to assessment the potential allergenicity in vitro.It was shown that,the assessment result of different factors were different for extracted proteins.For instance,compared to raw peanut protein extracted by method 2,the level of KU812 cells degranulation stimulated by boiled peanut was reduced,while the level of histamine was enhanced which means the ability of recruit granulocyte pro-inflammatory factor enhanced.The potential allergenicity assessment result of simulate digestive products of peanut defatted powder were consistent.The result was that compared to raw peanut,the level ofβ-HEX and intracellular calcium released by KU812 cells stimulated by fried peanut was reduced,while it stimulated by roasted peanut was enhanced.Which means the level of KU812 cells degranulation stimulated by fried peanut was reduced,while it was enhanced when stimulated by roasted peanut.Therefore,the allergenicity assessment was influenced by allergenicity assessment methods,digested product combined with cell model could be good choice for a reliable result.