Study On The Reduction Of Allergenicity Of Major Allergen In Penaeus Chinensis By Enzyme Cross-Linking

Shrimp is very popular with consumers all over the world because of its delicious flavor and rich nutrients.However,in recent years,shrimp allergy has aroused great attention of the government and become a hot food safety issue.In this study,we investigated the effect of enzyme crosslinking on the allergenicity of Penaeus chinensis tropomyosin.We explored the effect of enzyme cross-linking on the structure of tropomyosin,and evaluated the allergenicity after cross-linking by in vivo and vitro experiments.The main contents are listed as follows.1.Tropomyosin was extracted and purified from Penaeus chinensis,and the cross-linking reaction was performed by using transglutaminase and tyrosinase.SDS-PAGE was used to evaluate the cross-linking reaction with different enzyme amount and reaction time.Serum samples from 20 allergic patients were used to determine the allergenicity change.The optimal conditions for transglutaminase was 37 ?,1 U/mg and 24 h.The optimum conditions for tyrosinase was 37 ?,800 U/mL and 24 h.Scanning electron microscopy(SEM)was used to observe the state of the cross-linked proteins,and the result showed that the cross-linked proteins were closely interwoven.In vitro digestion experiments showed that while TM was totally digested in 60 minutes,the cross-linking products can tolerate for longer time.In order to further verify the allergenicity reduction after enzyme cross-linking,human mast cell degranulation assay was carried out.Mast cells were stimulated with 10 ?g/mL protein,and the degranulation was assessed by the release of histamine,?-hexosaminase,IL-8 and TNF-?.The results showed that the degranulation-induction ability of tropomyosin was decreased after cross-linking.2.Circular dichroism was used to analyze the secondary structure change of tropomyosin after cross-linking.The contents of ?-helix,?-turn and random coil were 49.7%,24.7% and 25.6% when the reaction time was 0 h,respectively.With the prolongation of cross-linking reaction time,the content of ?-helix decreased,the content of random coil and ?-turn increased,indicating that the secondary structure was changed.After deconvolution and second derivative treatment,the protein acyl I band spectra were obtained,the percentages of secondary structure were calculated.The ?-helix content reached the lowest and the ?-sheet content reached the highest when the reaction time was 24 h,indicating that ?-helix was changed into ?-sheet.In addition,the fluorescence spectroscopy showed that the surface hydrophobicity of tropomyosin was increased after cross-linking,and reached the strongest at 24 h,indicating that the tertiary structure of the protein was also changed.In the ultraviolet spectrum analysis,the maximum ultraviolet absorbance at 280 nm was enhanced and a blue shift occurred after cross-linking,indicating that the protein polarity and structure were changed.In addition,the relationship between protein structure change and allergenicity reduction after transglutaminase catalyzed cross-linking was analyzed by Pearson bivariate correlation analysis.The results showed that the allergenicity was significantly correlated with the secondary structure of tropomyosin.3.The effect of enzymatic cross-linking on tropomyosin allergenicity was comprehensively evaluated by an in vivo mice model.Compared with the intact tropomyosin(TM group),cross-linked tropomyosin induced much weaker allergic symptoms.The levels of serum histamine and IgE in the cross-linking groups were lower than those in TM group.Cytokine(IL-10?IL-4)productions of the cross-linking groups were also lower than that of TM group significantly,indicating that the pro-inflammatory ability of the protein was decreased.The proportion of dendritic cells in TM group was significantly higher than that in the other three groups,and the proportions of regulatory T cells in the cross-linking groups were significantly higher than that in TM group,suggesting that cross-linked protein may inhibit food allergy by inducing immune tolerance.In conclusion,the mouse model demonstrated that the allergenicity of tropomyosin was decreased after of enzyme cross-linking.