Purification Of Soybean Glycinin And β-Conglycinin And Development Of Quantitative Detection Method

Soybean glycinin andβ-conglycinin, which accounted for 40% and 30% respectively in soybean protein, were one of the most important soybean anti-nutritional factors. Thus, the utilization of the soybean products in animal feed was restricted.The study isolated and purified the soybean glycinin andβ-conglycinin from the low-temperature soybean meal. The polyclonal antibodys of glycinin andβ-conglycinin were prepared by purified protein. A single radial immunodiffusion (SRI) based on the polyclonal antibodys were established.Soybean glycinin andβ-conglycinin were isolated by adjusting the pH of Tris-HCl buffer and the concentration of ammonium sulfate solution. Then, they were further purified through affinity chromatography with ConA-Sepharose 4B column. Purified soybean glycinin andβ-conglycinin were judged to be over 90% and 80% pure respectively according to SDS-PAGE analysis with the software of Quantity one.Young adult rabbits were injected with purified soybean glycinin andβ-conglycinin in the soles of their feet and popliteal lymph nodes, the back skin and ear vein. When the antiserum titer which was detected by the double immuno-diffusion was over 1:32, the whole-blood was collected. Serums were purified by ammonium sulfate salting out. Antibodies were frozen drying after adding 1% Gly and 1% fucose. There were not cross-reaction between anti-glycinin serum and anti-β-conglycinin serum with double immunodiffusion analysis. Antibody also could not react with peanut meal, cottonseed meal, rapeseed meal, corn protein powder and fish meal respectively. They show good specificity.The study optimized rapid extraction of sample antigen protein through the single element experiment. The SRI based on the purified soybean glycinin andβ-conglycinin was established. Calibration curve showed well linear relation between 50 and 200μg/ml. Detection limit was 1 mg/g. The recovery tests and validation studies of the single radial immunodiffusion showed that the coefficient of variation for the same group was no more than 5% and for the different groups was no more than 10%. The error of average recoveries was between 98% and 113.6%. This methed showed a good repeatability and accuracy. It was suitable to detect soybean products glycinin andβ-conglycinin with the SRI.