Research On The Characterization Of The Allergens In The Roe And Milt From Large Yellow Croaker(Pseudosciaena Crocea)

Large yellow croaker, an important economic fish in China, its yield is increasing year byyear. With the formation of the special processing area of large yellow croaker in Fujian and theemphasis on increasing additional value of processing by-products from large yellow croaker, thedevelopment and utilization of the roe and milt from large yellow croaker attract more and moreattentions of researchers. But if the allergens in the roe and milt are not removed, it will causeallergy to eaters and bring food safety issues which is partly lead to restrict the development oflarge yellow processing industry. Therefore, it is very important to research the allergens in theroe and milt of large yellow croaker.In this study,20allergic paitents’ sera of the roe from large yellow croaker were used forELISA and Western-blotting analysis, confirming the16kDa protein was the major allergen inthe roe of large yellow croaker. After acetone precipitation, ammonium sulfate, and a series ofcolumn chromatography (DEAE-Sepharose, SP-Sepharose and Sephacryl-S-200HR), thisprotein was purified in the roe of large yellow croaker and it was identified as β′-component (β′-c)by peptide mass fingerprinting. The results of its physicochemical characterization analysisshowed that β′-c was a glycoprotein containing4.2%glucose and was resistant to acid/alkali andheating. In the simulated gastric and intestinal fluid digestion, although β′-c was easily degradedby pepsin, its digestion products still had immunoreactivity. By contrast, it was resistant totrypsin and α-chymotrypsin.The crude extracts of yolk proteins from other13species containing carp roe, crab egg andegg etc. had cross-reaction with rabbit polyclonal antibody against β′-c from the roe of largeyellow croaker. The results showed β′-c was a specific protein in fish roe. In addition, inhibitionELISA analysis was performed between positive mixed serum pool and crude extracts of yolkproteins from8fish roe, which was revealed that the cross-reactivity had some differences inspite of the allergen proteins had cross-reactivity between different fish roe.The yolk protein from large yellow croaker and β′-c were treated with Maillard reaction andother three physical processing methods (ultraviolet radiation, ultrasound-heat, and high pressuresteaming), then the crude extracts of yolk protein were extrated and were conducted in thesimulated gastric and intestinal fluid digestion with β′-c. It indicated that Maillard reaction couldeffectively decrease the IgG/IgE-binding ability of the roe allergen in large yellow croaker, andultrasound-heat and high pressure steaming could decrease the IgG/IgE-binding ability of their digestion products. On this basis, a method for making canned fish roe was invented by usingMaillard reaction and high pressure steaming treatment. The IgE-binding ability of the allergenin the canned fish roe was lower by this method.On the other hand,25allergic paitents’ sera of the milt from large yellow croaker were usedfor ELISA and Western-blotting analysis, confirming the12kDa protein was the major allergenin the milt in this study. After sulfuric acid, ethanol precipitation and Sephacryl-S-200HRchromatography,12kDa allergen protein and the components with similar molecular weightwere isolated, and they were protamine identified by circular dichroism spectrum. The results ofits physicochemical characterization analysis showed protamine was an alkaline glycoproteinwith2.5%glucose containing O-glycosidic bond and was resistant to acid/alkali and heating.RP-HPLC analyzed the components of3protamines from different fish species and foundthat there were4,3and6components of protamine in salmon, herring and large yellow croaker,respectively. After that6components of protamine in large yellow croaker were separatedthough Quick flow heparin affinity gel chromatography and Dot-blotting was used for assayingits IgE-binding ability with sera. It turned out that only the12kDa component5hadimmunoreaction with more than half sera.Different fish protamine and different components of protamine in large yellow croakerwere performed antibacterial experiment for5common bacteria (Escherichia coil, bacillussubtitles, staphylococcus aureus, Enterobacter aerogenes, Salmonella). The results showed that,protamine had good inhibition effect on positive bacteria compared with potassium sorbate.Furthermore, the6components of protamine in large yellow croaker had different inhibition onbacteria, among which component5had the best inhibition effect on gram positive bacteria andcomponent6had the best inhibition effect on gram negative bacteria.This article focused on the roe and milt of large yellow croaker, we finished separation,purification, identification and physicochemical characterization analysis of the allergens in them.Cross-reactivity between different species was researched for the safety of the allergen control.The influence of different processing methods on the immnoreactivity of allergens in the roe oflarge yellow croaker had been also studied. Then a method for making low allergic canned fishroe was invented. On the other hand, antibacterial activity of different components of protaminein large yellow croaker was compared and the antibacterial effect of6components to5commonbacteria was determined. All of these provided technical evidence for developing fish processingby-products which were low allergic even no allergic.