The Studies On Detection Methord Of Fish Allergens Based On Proteomics

Fish is rich in proteins,which contains many known and unknown allergens.These allergen proteins are often resistant to digestive enzymes.Studying the stability of fish proteins in simulated gastric juice in vitro has important reference value for predicting which proteins are allergens.The Paralichthys olivaceus protein and Larimichthys crocea protein samples were digested in simulated gastric juice in vitro,and the fish protein was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE)and mesoporous materials.Combining proteomics and protein sequence homology analysis methods,the proteins in enzymatic hydrolysate of Paralichthys olivaceus and Larimichthys crocea were studied for the screening of potential allergens in food.The main results are as follows:(1)The extracted proteins of Paralichthys olivaceus and Larimichthys crocea were digested in vitro simulated gastric juice.Pepsin-resistant proteins in enzymatic hydrolysis were separated by SDS-PAGE,and proteomics and LC-MS/MS techniques were used for protein analysis.Five kinds of pepsin-resistant proteins were identified in Paralichthys olivaceus and seven in Larimichthys crocea.Homology analysis of the protein sequence with known allergen proteins showed that the sequence similarity of tropomyosin alpha-1 in Paralichthys olivaceus and tropomyosin alpha-1 and tropomyosin alpha-4 in Larimichthys croceawere high with the known allergen Ore m 4.The identities of the three groups were 96.5%,81% and 78% and the positiveswere 97.9%,82% and 89%.Because of the high similarity,the three kinds of proteins were considered as potential allergens.The sequence of creatine kinase 1 in Paralichthys olivaceus and creatine kinase M in Larimichthys crocea were compared with Pen M 2.The identities were 42% and 45%,and the positives was 63%.The identities and positives between glyceraldehyde-3-phosphate dehydrogenase in Paralichthys olivaceus and the known allergen Tri a 34 were 72% and 83%.Glyceraldehyde-3-phosphate dehydrogenase was also potential allergen.(2)Two kinds of mesoporous materials SBA-15 and MCM-41 were prepared and the externalsurfaces of two mesoporous materials were modified with alkyl-diolgroups.The mesoporous materials were characterized by FTIR and pore size distribution test.Alkyl-diol groups were successfully grafted onto the external surfaces of mesoporous materials.The effects of pH and concentration of enzymatic hydrolysate on the adsorption performance of mesoporous materials were investigated.The adsorption capacity of four kinds of mesoporous materials increases with the concentration of enzymatic hydrolysate.When the pH is in the range of 5.5~7,the adsorption properties of four mesoporous materials are high.(3)Ultrafiltration method and mesoporous material adsorption method were used for the separation of pepsin-resistant proteins in the enzymatic hydrolysate of simulated gastric juice digestion of Paralichthys olivaceus and Larimichthys crocea,and LC-MS/MS techniques were used for protein analysis.Both methods can effectively enrich and separate the potential allergens in the enzymatic hydrolysate.15 kinds of proteins were identified in the ultrafiltration retentate of Paralichthys olivaceus which contains five potential allergens.38 kinds of proteins were identified in the ultrafiltration retentate of Larimichthys crocea which contains five potential allergens.Two proteins(including 1 potential allergen)were identified in Paralichthys olivaceus and 6 proteins(including 2 allergens)in Larimichthys crocea by alkyl-diol-SBA-15 adsorption.Although the number of protein identified by the mesoporous material adsorption is less than that by the ultrafiltration method,the selectivity of mesoporous material for the potential allergen protein was high.