Extraction And Passivation Of BBI Anti-nutritional Factors In Soybean And Effects Of BBI On HepG₂ Cells

Trypsin inhibitor (TI) is a natural bioactive substances, widely consist in animals,plants and microorganism. Excessive intake of TI will cause some adverse physiological reactions, such as pancreas enlargement, allergic reactions, and so on. Recent studies have shown the effect of anti-tumor and AIDS by TI is significant. This study include optimization of extraction process of BBI, passivation of BBI by immobilized enzyme technology, and the affect for morphology and proliferation of HepG2 cell by BBI.1.Extraction, isolation and identification of BBI: Firstly, on the base of single factor test, three significant factors were filter by Plackett-Burman (P-B), which is concentration of alcohol, solid-liquid ratio and extraction temperature. Secondly, the center points of factor level were selected by Steepest Ascent. Then, the extract process was optimized by central composite design. The result shows that the optimum condition is medium powder, 51% ethanol, solid-liquid ratio is 7:1, extraction temperature is 69℃, extraction time is 60min, pH 8.0, twice. Under this condition, the content of BBI could reach 8.47mg/g theoretically. Finally, the extraction rate of BBI which were purified by ultra filtration is 6.1mg/g, and activities of extracted BBI were identified.2. The study of BBI properties and passivity technology of BBI by subtilisin: The fixed carrier embedded subdivision is sodium alginate. Firstly, three Significant factors were filter by Plackett-Burman(P-B)experiment, which is fixed time, sodium alginate concentration, calcium chloride concentration. Secondly, the center points of factor level were selected by Steepest Ascent. Then, the extract process was optimized by central composite design. The result shows that the optimum condition is that immobilized time is 269min, 3.55% sodium alginate concentration is 3.55%, calcium chloride concentration is 2.53%, Activity is 4000BAEE, fixed temperature is 35℃, glutaraldehyde concentration is 1.0%, pH is 7. Under this condition, the relative activity of immobilized BBI can reach 88.48%. The study of immobilized enzyme activities showed that the ability of immobilized temperature of immobilized enzyme is higher 20℃than free enzyme's, the adaptability to acid-base environment of BBI was enhanced, the maximum initial reaction rate dropped by 25BAEE, the half-life time is about 4.2 days.3.The effects of HepG2 cell morphology and proliferation by BBI: The changes in the HepG2 cell morphology was observed after they were treated by BBI by using the method of Haematoxylin and Eosin(H&E)staining and directly by light microscopy. The result show that The cells were characterized by vacuole-shape in the cytoplasm, granular in chromatin and part of cell membranolysis after treated 72 hs under the concentration of 3.0 g/L by BBI. HepG2 was treated by different concentrations BBI for 1-4days .The experiment suggested that a linear relationship of effect and affected time and a linear relationship of effect and concentration. Survival rate gradually decreased with prolonged affected time and increased concentrations.