| Fish is an important food source for human beings,not only delicious but also nutritious.Fresh fish contains 15%~20% full fat protein,1%~10% fat and rich unsaturated fatty acids.China is a large aquaculture country,which accounts for more than 70% of the world's aquaculture products,and the total area of freshwater fisheries is 17 million 600 thousand hectares,accounting for 1.8% of the total land area.According to the statistics of China's General Administration of customs,the total import and export volume of aquatic products in China was 8 million 279 thousand and 100 tons in 2016,and the total import and export volume was 30 billion112 million dollars.It can be seen that the development of fishery brings great benefits to the national economy.But fish and its products are one of the eight food allergen identified by FAO and WHO.Parvalbumin(PV)has been identified as fish allergen protein.It is a calcium binding acidic protein.Therefore,it has important theoretical and practical significance for the purification,hypersensitivity treatment and hypersensitivity development of parvalbumin.This study mainly includes the separation,purification and identification of small albumin,the preparation of small albumin polyclonal antibody,the treatment of low sensitivity and the development of low sensitive food.The specific research contents are as follows:1.Isolation and purification of small albumin Seven extracts were selected for preliminary extraction of fresh surimi.Heat treatment,ammonium sulfate fractionation,dialysis and dialysis were used to extract and purify small albumin.Through single factor experiment,the best p H,material liquid ratio and salting out ammonium sulfate concentration in the extraction process were explored.The best conditions for the final determination are as follows:The extract consisted of 10 m M Tris-HCl and 0.02 m M PMSF,p H was 8,the ratio of material to liquid was 1:20,the first salting out concentration was 60%.After calculation,the concentration of albumin was about0.3% of fish.The size of the small albumin was about 12 KD by electrophoresis of SDS-PAGE protein.Amino acid composition analysis showed that the amino acid components of parvalbumin were mainly Ala,Asp and Lys,and had strong homology between four populations.2.Preparation and low sensitivity treatment of parvalbumin monoclonal antibody.Two groups of mice were designed,each group was 4.The initial dose of PV in the first groups was 100 ug,and the second group was 200 ug.The follow-up immunization dose was the same.The titer of antiserum was measured by indirect ELISA.The highest titer of mice was 1:256000.Checkerboard method was used to determine the optimum working concentration of polyclonal antibody:The concentration of coated antigen was 2.5g/m L,and the polyclonal antibody was diluted 64000 times.After different time of high temperature and high pressure treatment to PV,the results of enzyme linked immunosorbent assay showed that the sensitivities of PV at 100 degrees centigrade remained almost unchanged with the prolongation of treatment time,and decreased by 55.65% at 121.In the Maillard reaction treatment,the glucose group's sensitization decreased,and the xylose group's sensitization decreased.When tea polyphenols were added to the tea leaves,PV was transferred to the sediment,and the sensitization was not decreased obviously.The browning degree showed that the browning degree increased with Maillard reaction.Through circular two chromatography,we know the change of structure of two stage:The ratio of alpha to helix decreased,the proportion of beta fold increased,the ratio of beta rotation decreased,and the rate of irregular curling decreased.It can be deduced that the two level spatial structure of parvalbumin has been damaged to varying degrees.3.Development of low sensitive products.Combine the product features and our diet style,select fish as low sensitive products.The composite low sensitive processing means for preparing low sensitive fish on the basis of a chapter on the research.Enzyme linked immunosorbent assay was used to detect the change of sensitization.The results showed that the sensitivities of the treated fish surimi combined with high temperature and high pressure were 81.88%,the glucose was treated with high temperature and high pressure,the sensitivities were reduced by34.64%,and the tea polyphenols were treated with compound high temperature and high pressure to the sensitivity of 20.11%.The sensitization model of BALB/c mice showed that the fish balls prepared by high temperature and high pressure treatment combined with Maillard reaction could reduce the allergic symptoms in mice,and showed that the fish balls prepared by high temperature and high pressure combined with Maillard reaction had lower sensitivities... |
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