Rapid Quantification And Allergenicity Study Of Major Seafood Allergens

Crustaceans and fish are consumers' favorite aquatic products,however,crustacean and fish allergy are serious food safety problems worldwide.Tropomyosin(TM)and ariginine kinase(AK)are the two major allergens of crustaceans,and parvalbumin and collagen I(CO-I)are the two main allergens of fish.Analytical approaches to food allergens often relied on ELISA and PCR,but there were some limitations on these methods,including complicated and tedious operation,long dection time and false positive results.Although the allergenicity of CO-I has been confirmed in 2001,few researches on reducing antigenicity and identifying epitope of CO-I were performed until now.In the present study,a simple,fast and accurate method was established to simultaneously detect the two major allergens of crustaceans.On the other hand,the effects of high hydrostatic pressure(HHP)and enzymatic hydrolysis on the antigenicity of fish allergen CO-I were investigated.Moreover,the linear epitope of the rainbow trout allergen CO-I was predicted and validated.The main results are as follows:(1)We established ion-exchange chromatography coupled with dynamic coating capillary electrophoresis(IEC-DCCE)method to simultaneously detect TM and AK of crustaceans.IEC was first used to concentrate TM and AK crude extract.Then we optimized the capillary electrophoresis(CE)separation conditions.And the best conditions of CE are as follows:the separation voltage was 18 kV,running buffer was 30 mM borate-borax,pH 9.0 with 0.3%Tween-20.Under that condition,the migration time,separation efficiency and electrophoretic resolution greatly improved,and TM and AK could be accurately detected within 5.5 min.The standard curves of TM and AK were y=0.1x-0.12 and y=0.1x+0.07,R2 were 0.9935 and 0.9968,linear range were both 5-100?g/mL,LOD were 1.1 ?g/mL and 1.2 ?g/mL,LOQ were 3.7 ?g/mL and 4.0 ?g/mL,RSD were both lower than 10%,recovery were 94.0%-109.5%and 91.5%-106.1%,respectively.All those results indicated the accuracy and realiability of the method.(2)The antigenicity of Oncorhynchus mykiss allergen CO-I was reduced by HHP and enzymatic hydrolysis technique.The antigenicity of collagen I can be reduced up to 42.66%by HHP with the condition of 200 MPa,25 ?,and 10 min duration.On the other hand,by comparing the effect of protease papain,a-chymotrypsin and trypsin,we obtained the optimal enzymatic hydrolysis conditions for collagen I was 37 ?,pH 8.0,E:S for 1:10 by trypsin,which decreased 97.69%of the antigenicity of CO-I.(3)The hydrophobicity,flexibility,accessibility,antigenicity and secondary structure of the ?1 and 2 subunits of Oncorhynchus mykiss CO-I were analyzed by DNAStar software.By combing with Bepipred,ABCpred and BCPRED severs,10 peptide epitopes of al or a2 subunit were predicted.Meanwhile,sequences similarities of the al and a2 chains of different fish were compared with MegAlign software.We finded that the similarities of various type ? collagen al and ?2 sequences was more than 79%.Peptides were synthesized using standard Fmoc solid phase peptide synthesis and validated by LAD2 cells degranulation assay.Finally,peptides P2,P5,P6,P7,P11-20 were considered to be the linear epitopes of Oncorhynchus mykiss CO-I.Rapid quantification,reduction,and allergenicity study of major seafood allergens including TM,AK and CO-I would be benefical to customers' health and life and help to establish the basis for the hypoallergenic food.