| Food allergen is a common immunity disease, which mainly caused by the protein of food. The symptom of food allergy contained urticaria, asthma, and even life-threatening anaphylaxis. With the development of society and globalization, food allergy becomes more and more obvious, the rate of food allergy become higher. Food allergy is now a global attention food safety problem. Among the foods which cause anaphylaxis, seafood is an important type. Researches nowadays have confirmed parvalbumin to be the major allergen of fish, tropomyosin to be the major allergen of crab, and arginine kinase to be the secondary allergen of crab. At recent years, collagen was reported to be a new allergen.Tilapia (Tilapia zillii) and Carp(Cyprinus carpio carpio), which distributed widely in Fujian, China, with rich nutrition tastes good and welcome to consumers, are used in our researches. Our research focuses on the purification, antibody preparation, property comparison, specific IgE activity comparison, and allergen detection of tilapia and Carp. In our study collagen and the subunitα1,α2 was confirmed to be potential allergens.Collagen was isolated from skin of two fish by alkali extraction, acid extraction and NaCl precipitation. SDS-PAGE shows both collagen were consisted ofα1 chain (120 kDa),α2 chain (115kDa),βchain (250 kDa), and small amount ofγchains. The result of 2D-PAGE of tilapia confirmed the purity quotient of collagen, and showed the pI of collagen was approximate 9.0. Subunitα1 was isolated by carboxymethyl (CM)-cellulose column chromatography with 0-1 M NaCl linear gradient elution. Subunitα2 was isolated by carboxymethyl (CM)-cellulose column chromatography with stepwise elution using 0.13 M NaCl and 0.17 M NaCl, respectively.Through heating at different temperature, treating with buffer at different pH, simulated gastric fluid (SGF) digestion, the stability of collagen and parvalbumin was compared. The result indicated that the collagen was less stable under heat and high temperature high pressure condition, but more resistant to SGF digestion, pH stability of both did not present distinct difference. Heating did not accelerate the SGF digestion of collagen. Both pepsin and trypsin can decompose the collagen.The specific IgE activity of collagen and the subunits was confirmed by ELISA, western-blotting and dot-blotting. Result of ELISA indicated that there were specific IgE activity to collagen and the subunits. But the specific IgE activity among collagen, subunitα1, subunitα2, was different. Dot-blotting of patient sera presented antibody-antigen spot to both tilapia and Carp collagen. Western-blotting of patient sera and tilapia collagen presented IgE-binding bands, includingα1,α2 andβ. But no bands had been detected while the subunitα1,α2 reacted with patient sera individually.Collagen detection of tilapia and Carp muscle crude protein was achieved by IgG antibody prepared in rabbit. Two bands at about 100 kDa and 200 kDa was detected by western-blotting, another band at approximate 62 kDa detected in crude protein of tilapia muscle, was considered to the degradation of collagen. The detection of crude protein under heat treatment also showed the degradation bands. The method might be used in detection of collagen fragments after different industrial processing.
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