Purification, Pilot-scale Production Design Of Bovine Allergen β-lactoglobulin And Cytological Evaluation Of Allergenicity In Vitro

In recent years, food allergy has raised the attention of public and become a major public health issue. Food allergy is caused by intaking the allergens, and the body produce abnormal immune response, leading to the body’s physiological function disorder, tissue damage and a series of clinical symptoms.Milk allergy is the most common among eight kinds of food allergens and beta lactoglobulin is regarded as one of major allergen. Beta lactoglobulin is synthesized by ruminant mammary, but has not found in human milk. It accounts for 55% of whey protein. In our research, the high purity of bovine allergen beta lactoglobulin was obtained by preparative reverse phase high performance liquid chromatography. The structural changes of allergens were characterized by spectroscopic methods. Meanwhile, the changes of sensitivity was evaluated by KU812 human peripheral blood eosinophils. the approaches, results and conclusions were depicted as follows.1. A simple method of purifing β-lactoglobulin for laboratory preparation was gained by RP-HPLC(C18). The whole experiment only required 25 min, and the purity of β-lactoglobulin was about 98.2% by SDS-PAGE, and the recovery-ratio was 32.25%。2. β-lactoglobulin was treated with irradiation(5 k Gy) in coordination with ultrasonic(40 KHz, 300 W, 30 min), they were including irradiated β-lactoglobulin, utrasound after irradiation treated β-lactoglobulin and irradiation after utrasound treated β-lactoglobulin. After processing, the protein aggregated in different extent by SDS-PAGE and the structure has changed as well. Structure of processed protein gradually turned into more ordered form, leading to more compact of the internal molecular, the embedding of hydrophobic, the decreased of fluorescence intensity and the destruction of their conformational epitopes. Moreover, the binding capacity of Ig G to beta lactoglobulin has decreased, which may caused by the destroy of antigenic epitopes, inducing the reduction of the protein allergenicity.3. KU812 cells( l.0×106/m L) were carried out for allergenicity evaluation. Bioactive mediators, such as beta-HEX, histamine, TNF-α, IL-6 and intracellular Ca2+ concentration, were detected from activated KU812 cells. The results showed that the higher contents of bioactive mediators were induced by the unproscessed compared with the other three processed groups.The calcium influx and KU812 cells degranulation were the most signicificant in unprocessed group. The contents of Lyn、Syk、NF-κB and MAPK family produced by actived KU812 cells was tested by Western blot. The phosphorylated protein contents in upstream and downstream of signaling pathway were lower in the processed groups compared with the unprocessed BLG. In further,the lower content of phosphorylated proteins in signaling pathway was caused by inhibiting the activity of the headwaters kinases Lyn and Syk, thus restraining the production of relevant proteins in downstream. Moreover, it was confirmed that the KU812 cells degranulation was indeed regulated by Ig E/FcεRI signal pathway. All the results has proved that irradiation coupled with ultrasonic treatment can reduce the allergenicity of BLG.