Effects Of Simulated Gastric Fluid Digestion On The Epitopes And Allergenicity Of Major Allergen Tropomyosin In Shrimp

With the development of the food processing industry,food allergy has become a serious public health problem,and the degree of response can range from slight symptoms to severe systemic reactions.Aquatic crustaceans,represented by shrimp,are one of the eight categories of allergenic food identified by the FAO,allergens in shrimp are tropomyosin(TM),arginine kinase(AK),sarcoplasmic calcium binding protein(SCP)and myosin light chain(MLC),among which TM is the main allergen in shrimp.The main cause of allergic reactions is some proteins in food.The allergens in the human body can still cause allergic reactions after digestion.Therefore,studying the effects of human digestive system on the immunological activity of allergen and epitopes is the key to studying the allergenicity changes of allergen in the human digestive process and analyzing the mechanism of food allergic reactions.In this paper,the main allergen TM in Litopenaeus Vanname was enriched and purified,and TM was digested in vitro simulated gastric fluid(SGF).The immunological activity of TM in the digestion process was analyzed by SDS-PAGE,Western-blot and indirect ELISA.The structure changes of TM epitopes in SGF were analyzed by mass spectrometry combined with known TM epitopes.In order to confirm the correlation between TM epitopes and sensitization,a sensitizing animal model was established in Balb/c mice.The structure-activity relationship of TM epitopes was discussed by analyzing the changes of specific IgG antibody,IgE antibody and histamine in serum of immunized mice.The main research contents and results are as follows:(1)TM was enriched from Litopenaeus Vanname and digested in vitro SGF which was prepared according to the US Pharmacopoeia.Digested products were analyzed by SDS-PAGE,Western-blot and indirect ELISA.SDS-PAGE and Western-blot results showed that TM bands were gradually decreased with the increasing of digestion time,but the immunological activity remained after 2 h digestion in vitro SGF.Indirect ELISA results appeared that after 2 h digestion,the immunological activity and allergenicity of TM lost 89.1% and 80.8%,respectively.The above results indicate that TM lost most of its immunological activity by enzymatic hydrolysis in vitro SGF.(2)SDS-PAGE and Western-blot were performed on the products of TM in vitro SGF.Protein bands with different degrees of degradation were selected according to the analysis results,and the structural changes of TM epitopes were analyzed by LTQ Orbitrap Velos Pro mass spectrometer.Mass spectrometry results showed that most epitopes were destroyed during in vitro SGF,but the immunological activity of TM remained after 2 h digestion,probably because there were 3 undestroyed epitopes(peptides 111-125,137-141,and 153-161)and 4 antigenic epitopes(peptides 50-66,144-151,145-164,and 150-163)in digested fragments.These anti-digestive epitopes may be a key factor in shrimp allergy.(3)In order to confirm the correlation between TM epitope and sensitization,6 anti-digestive epitopes and 2 non-digestible epitopes were synthesized artificially.A Balb/c mouse model of sensitization was established by intragastric administration,with TM as the positive control group and normal saline as the negative control group.Changes of specific IgG antibody and IgE antibody in serum of immunized mice were analyzed by indirect ELISA method,and histamine content in mice was analyzed by HIS ELISA kit.The results showed that the specific IgE antibody and histamine content of the epitope 111-125(TKLAEASQAADESER),epitope 137-141(DEERM)and epitope 50-66(MQQLENDLDQVQESLLK)were high in mice,indicating the high sensitization and further confirming the structural changes of TM epitopes in vitro SGF.In summary,the major allergen TM in Litopenaeus Vanname produces some small molecule degradation products during in vitro SGF,and some epitopes were destroyed,but the immunological activity of TM remained after 2 h digestion.It was probably due to the presence of 3 undestroyed epitopes and 4 antigenic epitopes that are highly resistant to digestion.The sensitized animal model verified that the epitope 111-125(TKLAEASQAADESER),epitope 137-141(DEERM),and epitope 50-66(MQQLENDLDQVQESLLK),these three epitopes were likely to be the key to shrimp allergy.