| Soy is rich in protein, and it is a very valuable food-borne plant proteins.It contains healthful such substances as soyasaponin, isoflavone,unsaturated fatty acid, ete. are medicinally healthful. Soybean has played an important role on human nutrition, health and the development of animal husbandry. However, soybeans contain a number of antinutritional factors(ANFs), such as trypsin inhibitors, producing adverse effects on the animal growth and human health. It is a kind of Health products, and it has a potential applications on Anti-tumor, trypsin inhibitor have some effects on diabetes treatment and regulation of insulin. Therefore, the study about purification of soybean trypsin inhibitor has a certain theoretical and practical significance.It is usual to subject the raw soybean to some types of heat treatments. But the prolonged high temperature treatment required to destroy all ANFs is 1ikely to damage the availability of certain essential amino acids. Because some ANFs are chemically proteins, it seems that proteolysis is a promising approach to inactivate them. The topics include extraction technique of soybean trypsin inhibitor(STI), to determine theconditions of Ultrafiltration, optimization of extraction technique of soybean trypsin inhibitor by response surface methodology, purification of STI, electrophoresis detection, selection of protease, Single-factor test to determine the hydrolysis reaction conditions of soybean trypsin inhibitor and Response surface analysis to determine optimal conditions of alkaline protease enzyme on soybean trypsin inhibitor. The main conclusions are as follows:1) The extraction rate of purification of STI was investigated indicators. Effects of extraction pH value, thermal denaturation temperature and ammonium sulfate saturation on extraction rate of soybean trypsin inhibitor(SBTI) were investigated respectively in single-factor tests. The crude conditions have determined by single-factor test: thermal denaturation temperature60～80℃, pH value4.5～5.5, aturation of ammonium sulfate50 %～60 %. The crude conditions have determined by response surface methodology. The optimum extraction pH value, thermal denaturation temperature and saturation of ammonium sulfate are 5.1, 72.8℃and 54.5 %, respectively. Under the above conditions, the extraction rate of STI reaches 89 %.2) Ultrafiltration conditions: In the present conditions, 5 kDa membrane was choosing for experimental. Under these conditions, determine the operating pressure is 1.5 bar, the operating temperature is 30℃, and cleaning ultrafiltration membrane every 40 min.3) Purification of STI. DEAE-52 ion exchange chromatography and Sephadex G-75 gel filtration were used to purification. BAPNA method was used to detect whether there is activity on each peak. molecular weight has been determined according to the standard curve. Compared to the active peak and the peak time of standard products KTI and BBI can be determined.4) The determine of protease. The passive rate of hydrolysis soybean trypsin inhibitor as a reference, Considering the enzyme specificity, mechanism of action, safety, economy, effectiveness, and ultimately alkaline protease enzyme was chosen to the hydrolysis STI.5) Determination of optimum Hydrolysis conditions. As passivity rate of Alkaline protease enzyme soybean trypsin inhibitor for the indexes, The reaction temperature, reaction time, enzyme dosage and pH value as the factors that affect the passivity rate, The Hydrolysis conditions have determined by single-factor test: Reaction temperature 30 ~ 70℃, reaction time 40 ~ 120min, the amount of enzyme 20000 ~ 40000 units/mL, pH value of 7 ~ 11. The optimum Hydrolysis conditions Reaction temperature 53.13℃, pH9.11, the amount of enzyme 41754.75 u/mL reaction time 90.65min were determined by response surface methodology. The passivity rate of 3 time s average that Alkaline protease Hydrolysis soybean trypsin inhibitor can reach to 94.47 percent. |
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