Effects Of The Maillard Reaction On Epitopes And Allergenicity Of Tropomyosin From Chlamys Nobilis

Chlamys nobilis is an important economic shellfish in the coastal areas of southern China.In this thesis,Chlamys nobilis was used as the experimental object to purify its main allergen tropomyosin（TM）.The allergenicity,physicochemical properties and IgE epitopes of TM were systematically analyzed,and the effects of Maillard reaction on IgE epitopes and allergenicity of TM were explored,which provided a theoretical basis for the accurate development of hypoallergenic food.In this research,a single protein of 36 kDa was isolated and purified from the adductor muscle of Chlamys nobilis.It was proved to be the main allergen TM by liquid chromatography tandem mass spectrometry and serological experiments.TM was proved to be a glycoprotein with an isoelectric point of 4.5 by two-dimensional electrophoresis and PAS staining.The results of thermal stability and p H stability tests show that TM is heat-resistant,acid-resistant and alkali-resistant.The results of simulated gastrointestinal juice digestion showed that pepsin and trypsin had different degradation modes of TM.TM was resistant to pepsin and easy to be digested by trypsin,but the digested products still had immune binding activity.Using IgE as the target protein in the serum of patients with shellfish allergy,the phage display peptide library and one bead and one compound peptide library were used to screen the epitopes of TM,and seven epitopes of TM were obtained by bioinformatics software analysis.The peptides were synthesized by Fmoc method and verified by inhibitory ELISA method.Finally,seven IgE linear epitopes of TM(K₇-L₂₁,Q₁₉-E₃₃,N₅₅-E₆₆,K₇₆-G₈₇,A₁₂₀-S₁₃₄,S₁₃₄-L₁₄₈,K₁₈₉-A₂₀₂)were identified.In order to reduce the allergenicity of TM,the Maillard reaction conditions were optimized for heating at 100℃for 30 min（p H 8.5）with D-Xyl.It was found that the transport ability of Maillard reaction product（MR-TM）decreased by 25.3±0.2%after being transported by human cloned colon adenocarcinoma cell model for 4 h.The results of Dot blot analysis showed that the IgG/IgE-binding activity of MR-TM decreased by 58.9±0.3%and 79.2±0.1%,respectively.The results of basophil activation assay showed that MR-TM significantly down-regulated the expression of CD63 and CD203c on the surface of basophils（p<0.05）.Circular dichroism and surface hydrophobicity analysis showed that Theα-helix content of Maillard reaction product（MR-TM）decreased and the surface hydrophobicity increased.Furthermore,the modified sites of Maillard reaction were detected by shotgun proteomics,and 11 specific amino acids(K₁₂,R₁₅,K₂₈,K₇₆,R₁₂₅,R₁₂₇,K₁₂₈,R₁₃₃,R₁₄₀,K₁₄₆,K₁₈₉)were found on 6 IgE epitopes of TM.As a result,the sensitization of TM was significantly reduced（p<0.05）.In conclution,the main allergen TM of Chlamys nobilis was purified and identified,and its seven IgE linear epitopes were identified,and a Maillard reaction system to reduce the allergenicity of TM was established.It was clarified that Maillard reaction modification of specific amino acids of TM IgE epitopes was the main reason for the significant decrease of allergenicity of Chlamys nobilis.