Purification And Characterization Of Parvalbumin Isotypes And Effect Of Processing Methods On The Main Allergen Of Grass Carp

Fish is one of the major food allergen, and parvalbumin is the main allergen in fish. Parvalbumin is an acidic calcium-binding protein with a molecular weight of10-12KDa. Parvalbumin was reported inseveral kinds of sea fish species, such as cod, salmon and pilchard, and it was also reported in some freshwater fish in China, but there was no research about grass carp, one of the most frequently consumed freshwater fish in china. This study purified the main allergen in grass carp, and prepared the rabbit anti-parvalbumin antibody. This study also investigated the effect of processing methods on the antigenicity, allergenicity and digestibility of parvalbumin. The effect of Maillard reaction on the antigenicity of parvalbumin was studied. Parvalbumin isotypes were purified and characterized. And the pertential allergenicity of surimi products was evaluated in this study.1. Purification of parvalbumin from grass carp and establishment of ELISA methods. Heat treatment and Gel filtration were used to purify parvalbumin from grass carp muscle. The fractions with high IgG-binding were collected. Anti-grass carp parvalbumin antibody was produced in rabbit and then used in ELISA test. The allergen coated concentration was2μg/mL and the sera was diluted160000times in ELISA. The cross reactivity between the sera and carp, crucian, silver carp and bream were investigated, and the results revealed high cross reactivity between them. Different sera from fish allergic patients were collected and used as sera pool to test the IgE-binding capactity of parvalbumin.2. Effects of thermal processing on the antigenicity, allergenicity and digestibility of parvalbumin. Heat treatment caused higher antigenicity and allergenicity of white muscle when heated at80and100℃within90min. However, heat treatment at65℃was able to decrease the allergenicity of white muscle as the heating time increased. For the dark muscle, the antigenicity and allergenicity decreased with the increasing temperature. The effects of steam, boil, high peressure and bake on parvalbumin were different. High pressure (121℃,0.15MPa) was the most effective method to control the antigenicity of parvalbumin, and the parvalbumin in the grass carp muscle which treated by high pressure was more sensitive to digestion and the antigenicity and allergenicity of parvalbumin were decreased significantly during digestion. The water used in boil and the water obtained from steam and high pressure treated grass carp muscle contained parvalbumin, which can cause fish allergy.3.Effect of maillard reaction condition on the antigenicity and allergenicity of parvalbumin. A model for predicting antigenicity of PV and maltose Maillard reaction products was established. On the field of conditions studied, temperature had the most important effect on the antigenicity of PV. The predicted antigenicity of PV was reduced from26.54μg mL-1to0.276μg mL-1under the optimal Maillard reaction condition. Maillard reaction lead significantly suppressed IgE binding to PV and it was an effective method to control the major allergen of grass carp. After Maillard reaction, the essential amino acid content of PV decreased14.50%and the non-essential amino acid content of PV decreased23.21%.4. Purification andcharacterization of parvalbuminisotypes from grass carp. Three PV isotypes were purified and denoted as PⅥ, PⅦ and PⅧ. The molecular weights of PⅥ, PⅦ and PⅧ were determined to be11.968,11.430and11.512kDa, respectively. PⅥ showed74%sequence similarity with PV isotype4a from Daniorerio while PⅦ and PⅧ showed46%sequence similarities with PⅤ isotypes from Hypophthalmichthy smolitrix, respectively. PVII showed the highest IgG-binding and IgE-binding as well as the highest content among the three isotypes. Therefore, PⅦ was demonstrated to be the main allergen. However, PⅦ was liable to gastrointestinal enzymes compared with PⅥ, which was resistant to pepsin digestion. The IgE-binding capacity of pepsin digested PⅥ was significantly higher than other digested PV isotypes, and the undigested PVI in the peptic digest was responsible for its IgE-binding capacity. After thermal treatment, the structure, antigenicity and allergenicity of PⅦ and PⅧ were stable. But PVI was degraded when the heat time was1h, and the heat temperature was higher than70℃or when the heat temperature was99℃, and the heat time was longer than10min. And the degradation of PVI may be was the reason for the dereased IgG and IgE binding capacities.5. Analysis of the potential allergic of surimi products. The content and antigenicity of parvalbumin in grass carp white muscle were stable when stored at-20,-40,-60℃in14weeks. The content of parvalbumin decreased significantly during the surimi processing procedure. Rabbit anti-parvalbumin antibody and human sera pool were used to test parvalbumin in different concentration, and the results were used as a standard to measure the parvalbumin content in ten kinds of surimi products. The parvalbumin content of the surimi products were lower than0.125μg/mL except for2-1and7-3when tested by rabbit anti-parvalbumin antibody. The parvalbumin content of the surimi products were in the range of0.001-0.1μg/mL when tested by the human sera pool.