| The incidence of crustacean aquatic allergies is increasing year by year.Scylla paramamosain is one of the important economic species of crustacean aquatic.The food allergenic diseases should not be ignored,which caused by S.paramamosain.Thermal processing can reduce the sensitization of single allergen effectively,and there is still has insufficient research on the influence of thermal processing on the allergenicity of food matrix.Hence,it is meaningful to explore the thermal processing influence the muscle allergen allergenicity of mud crab and the molecular mechanism,so as to study the processing methods of hypoallergenicity products and guarantee the food safety of mud crab food.In this study,the effects of three thermal processing,including cooking,steaming and high temperature pressure treatment,on the structure,digestive enzyme hydrolysis stability and immune binding activity of the crab muscle crude protein were evaluated.Continuous digestion of gastric and intestinal fluid was simulated for the crab muscle tissue by high temperature-pressure.Intestinal transport and absorption of digestive products were performed using the human intestinal epithelial cell Caco-2 transport model,and the basophil activation test was used to detect the allergenicity of transport products.The results showed that compared with the raw treatment,the content of ? helix increased significantly after heat processing,and the content of ? folding and ? corners decreased,which means that the stability of muscle crude protein decreased.After thermal processing,the enzymatic stability of muscle crude protein decreased,the digestibility increased,and the Immunoglobulin(Ig)E inhibition rate decreased,and the IgG-binding activity of heat-stable allergen reduced.Among the three thermal processing methods,high temperature-pressure treatment presented the lowest immune binding activity of muscle crude protein,IgE inhibition rate decreased by 26.66%±2.46% and IgG-binding activity decreased by 21.93%±0.83%.In addition,the fiber bundles of crab muscle tissue were loosened and porous after high temperature pressure treatment.The contents and types of proteins and allergens in muscle tissue were reduced effectively.The average particle size of muscle tissue suspension decreased,the maximum absorption peak shifted to the left,and the digestibility increased.It means that the spatial structures of food matrix,including allergens,were destroyed.The expression of CD63 and CD203 c of basophils were down-regulated after high temperature-pressure treatment,and the reduced ratio were 24.23%±0.07% and 4.54%±0.17%,respectively.The immune binding activity were investigated during the process of digestion and transport after high temperature-pressure of the five important allergens in mud crab muscle,including tropomyosin(TM),arginine kinase,myosin light chain,sarcoplasmic calcium-binding protein,and triosephosphate isomerase.The heat-resistant and digestion-stable epitopes of five important allergens were further analyzed.Meanwhile,the structure and allergenicity of heat-stable allergen TM were researched.The heat-resistant,digestion-stable and allergenic epitopes and the key amino acids in epitopes of TM were determined by immunological and bioinformatics analysis to clear the mechanism of the lower immune binding activity in high temperature-pressure treatment,which helps to clarify the molecular mechanism of reducing sensitization by high temperature-pressure treatment.The results showed that the disulfide bond and digestive enzyme cleavage site of allergens were easily destroyed by high temperature-pressure treatment.The IgG-binding activity of the five important allergens after digestion and transportation by high temperature-pressure treatment was reduced,compared with the raw treatment,however,the heat-resistant and digestion-stable allergen linear epitopes were retained,which reserved the allergenicity of crab.During the five important allergens,TM showed the better thermal and digestion-stable,and the most amount of heat-resistant and digestion-stable linear epitopes,which means that TM is the major and thermal stable allergen in S.paramamosain.Furthermore,the ?-helix of thermally stable allergen TM was opened,the hydrophobicity was enhanced,the spatial structure was changed,and the sensitization was significantly reduced after high temperature-pressure treatment.There were seven heat-resistant,digestion-stable and allergenic epitope peptides were identified in TM after digestion and transportation by high temperature-pressure treatment,which were AA19-29,39-48,99-109,110-123,153-162,170-188 and 211-221,respectively.In addition,seven key amino acids,R21,E103,E104,E115,A116,E122 and E156,showed a significant effect on the IgE-binding activity of TM epitopes.Based on the research,the Maillard reaction(dipping 5% L-arabinose for 2 hours)combined with high temperature-pressure were used to treat the mud crab in the study,which can modify the glycosylation site in the heat-resistant and digestion-stable epitopes,and further reduce the immune binding activity of mud crab.In summary,this study found that thermal processing increased the content of ? helix in the crude protein,the fiber bundles were loosened and porous,which was conducive to the contact between digestive enzymes and proteins,the content and species of allergens in food were reduced,and the digestibility was increased and allergenicity was decreased.High temperature-pressure treatment destroyed the spatial structure of food matrix,including allergens,and influenced the heat unresistant epitopes of allergens and reduced the allergenicity of mud crab.Based on the heat-resistant and digestion-stable epitopes,the glycosylation modification can further reduce the immunobinding activity of the crab.These results preliminary clarify the allergenicity and molecular mechanism of thermal processing affecting the allergenicity,lay a foundation for the research and development of hypoallergic food,and also provide a new reference for the deep assessment of the influence of thermal processing on food allergenicity. |
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