Research On The Allergens Of Octopus

Food allergy as a widespread allergic disease is a hypersensitive reaction mediated by IgE,and is mainly caused by allergens in the food. Sensitied individuals could develop diarrhea,asthma, vomiting, shock and even life-threatening anaphylaxis. Eight major sources of foodallergens were proposed by the Food and Agriculture Organization (FAO) of the United Nationsand World Health Organization (WHO), and two of them were belonged aquatic foods whichtook a very great proportion of the allergic foods. Octopus was very popular by consumer for itsfresh meat, delicious taste, and high nutritional value. With the increase in consumption ofoctopus, the reports of allergy induced by octopus were growing in quantity, but the reaserch onthe allergens of octopus was less. It was reported that the major allergen of octopus wastropomysin (TM). So far, octopus TM had been purified and analyzed, but there had been noreports that how to develop hypoallergenic octopus products and whether there were other novleallergens in the octopus.In this study, octopus TM was purified, identified and analyzed, and anti octopus TMpolyclonal antibody was obtained. And then the purified TM was treated by ultrasound combinedwith heat, UV irradiation, high temperature combined with high pressure, and maillard reaction,respectively. Digestive stability of treated TMs was analyzed by simulated gastrointestinaldigestion assay, and immunological activity was identified by polyclonal antibody and patients’serum. Finally, the muscle of octopus was treated by heat and high temperature combined withhigh pressure, respectively, and the digestive stability of whose crude extracts was studied. Theresults showed that the molecular mass of purified octopus TM was35kDa, and immunologicalcross-reaction could be appeared between the octopus TM and crab TM. Octopus TM wasrelatively stable to heat, acid or alkali, and was a glycoprotein contained1.3%sugar. Thedigestive stability and the immunological activity of TMs had not obvious changes after treatedby ultrasound combined with heat and UV irradiation, while both of the digestive stability andthe immunological activity of TM after high temperature combined with high pressure treatmentwere reduced signally. With the reaction time increasing, the immunological activity of maillardreaction products between purified TM and glucose was reduced more and more, but thedigestive stability of them was increased. In addition, the digestive stability and immunologicalactivity of crude extracts of muscle after heat or high temperature combined with high pressurewere also reduce, and the effect of the latter was more conspicuous. In this study, arginine kinase (AK) was also isolated from octopus muscle and identified bymass spectrometry, and the anti octopus AK polyclonal antibody was prepared. Then theimmunological, molecular biological and physicochemical characterizations of octopus AK wereanalyzed. Finerally, octopus AK was compared with crustacean AK, and the differences andsimilarity of them were analyzed. The results showed there were3kinds of protein fractions withthe molecular mass of50kDa,38kDa and26kDa in the myogen could react with allergicpatients’ sera, and the major protein fraction of them was38kDa. The38kDa protein waspurified in further, and was confirmed to be octopus AK by mass spectrometry. The IgE bindingactivity of octopus AK was appraised by inhibition ELISA, and was proved to be a novleallergen of octopus in first time, and then the higher titre rabbit anti octopus AK polyclonalantibody was also obtained. Octopus AK was a glycoprotein contained1.1%sugar, but wasunstable to heat, acid or alkali, and was digested by pepsin, trypsin and chymotrypsin easily.Through molecular cloning, the full-length gene sequence of octopus AK was obtained, whichencompassed1209bp, and was predicted to encode a protein with348amino acid residues. Theamino acid sequence of octopus AK was less8amino acid residues than that of crustaceans AK.The homology of octopus AK and crustacean AK was about54%, but the similarity betweentheir three-dimensional structures was high, and the parts of predicted epitope of them wereshared. Octopus AK could appeare obviously immunological reaction with anti crustacean AKpolyclonal antibody, and crab AK could also inhibit the reaction between the octopus AK andIgE in the positive sera of allergic patients.In conclusion, TM and AK of octopus were analyzed in detail in this study. Firstly, purifiedTM was treated in different processing ways, in order to screen the methods which could reducethe digestive stability and immunological activity of TM. Sencondly, octopus AK was proved tobe a novle allergen of octopus in the first time, and whose characterizations were analyzedsystematicly. All these results were expected to supply the technical reference for developing thehypoallergenic pruducts of octopus, and were also significant in allergy diagnosis and thetreatment of octopus-allergic disorders.