Effect And Mechanism Of Different Cooking Methods On Allergenicity Of Egg Proteins

Eggs are a crucial food source in Chinese daily life.More than 33%of the world’s egg production comes from our country.Meanwhile,eggs are the second most common food allergen.Egg allergy can lead to allergic clinical reactions,even life-threatening in severe cases,which seriously affects patients’ quality of life.Cooking is one of the main egg processing methods.Different cooking methods influence egg allergen allergenicity differently.In this study,the allergenicity changes of soluble protein in whole hen’s eggs after different cooking processes were analyzed.The mechanism of action was further explored.The B-cell linear epitopes of the egg yolk allergen Gal d 6 were predicted and identified.The main research results and conclusions are as follows:(1)Four cooking methods,boiling(100℃,10 min),steaming(100℃,10 min),baking(160℃,15 min)and frying(170℃,5 min)were used to process the eggs.After extracting the soluble protein from the hen’s eggs,molecular weight changes of the protein were detected.It was observed that the bands of the major egg allergens Gal d 1-6 were all lightened.This suggests that the soluble protein composition of hen’s eggs was significantly changed by the four cooking methods.In vitro simulated digestion experiments were used to analyze egg protein digestion resistance changes.It was found that the soluble protein digestion resistance of hen’s whole egg decreased after cooking,most of which could be decomposed by simulated gastroenteric digestion in vitro.This suggests that different cooking treatments could reduce the presence of soluble proteins that make hen’s eggs.Using BALB/c mouse egg allergy model,RBL-2H3 mast cell model and human serum experiments,it was found that after boiling,steaming,baking and frying,the ability of chicken whole egg soluble protein to induce egg allergy in BALB/c mice was decreased,the level of inducing RBL-2H3 mast cell degranulation was decreased,and the binding ability to serum IgE in patients with egg allergy was decreased.In addition,the egg yolk allergen Gal d 6 still exists after cooking and processing.It has strong binding ability with serum of patients with egg allergy,and its related properties need further study.(2)A variety of spectral analysis methods were used to analyze soluble protein structural changes in hen’s eggs.It was found that after cooking,the secondary structure of soluble protein in hen’s eggs changed remarkably:the proportion of α-helix and β-Turn decreased(p<0.05),while the proportion of β-sheet increased(p<0.05).The tertiary structure also changed significantly,and protein surface hydrophobicity decreased.Further,a BALB/c mouse egg allergy model was used to study the immunological mechanism of egg proteininduced food allergy induced by different cooking methods.The Th2 cytokines GATA-3 and T-bet in mouse spleen and Jagged-1 and Jagged-2 in spleen and jejunum were detected.It was found that the ability of hen’s egg soluble protein to induce Th2 type immune response in BALB/c mice was decreased after four cooking methods of egg,including boiling,steaming,baking and flying,which may be caused by the change in egg protein structure caused by cooking treatment.(3)The recombinant protein Gal d 6 was successfully expressed by constructing a pET30a-Gal d 6 expression vector.It was found that the recombinant Gal d 6 protein was expressed as an inclusion body with a molecular weight of about 35 kDa.The recombinant Gal d 6 protein was further purified by affinity chromatography.The B-cell linear epitopes of Gal d 6 protein were predicted by the bioinformatics method,and the predicted epitopes contained 8 peptide segments.The peptide sequences were 28SFTCSFN34,58VDHQSL63,94SVLSDSGVSE103,156NDREKHN162,165LMPNHKLAHS174,187ETCSGGCKLQR197,227DCTPIEKTS235,respectively.263ASEDV267.By simulating digestion in vitro and combining LC-MS/MS experiments,we finally determined the linear epitopes of Gal d 6 as 28SFTCSFN34,58VDHQSL63,156NDREKHN162 and 165LMPNHKLAHS174.This study is of substantial significance to elucidate the influence of different cooking methods on the allergenicity of whole hen’s eggs.At the same time,the linear B-cell epitopes of Gal d 6,a representative egg yolk allergen,were predicted and identified,providing a basis for the study of the related mechanism of egg allergens.