Study On The Extraction, Purification And Refining Of Isoflavones From Soybean

Soybean isoflavones are the secondary metabolites formed in the growing process of soybean, which are seen as bad ingredients to try to eliminate for a long time because of their bitter flavour. In recent years, Epidemiological studies have shown that isoflavones play a vital role in preventing and curing various diseases. During the gestating process of malignant tumour, Genistein can inhibit vascular hyperplasying and cut off the nourishment that is supplied to the tumour efficiently, and the deterioration of tumour was staved and held back. Isoflavone glycosides were transformed into compounds with close similarity in structure to estrogens by intestinal flora, so consumption of soybean isoflavones-containing foods can weaken and suspend menopausal symptoms which are caused by decrease of estrogen for the postmenopausal women. Soybean isoflavones were shown to have inhibitory effects on bone resorption and hypothyroid calcium secretion, which might reduce the risk of osteoporosis. Moreover, It was also shown the positive effects on cardiovascular system, immune system and antioxidant properties. Soybean isoflavones have attracted increasing considerable attentions because of their beneficial functions, especially preventing cancer. And this might advance the exploitation and utilization of isoflavones products widely.Presently, the methods of processing soybean isoflavones usually utilize refluxing with ethanol, and often in combination with extraction or adsorption. Although the products of thus made could be of high purity, the economical, environmental and safety problems would appear because of using large quantities of organic solvents. Aiming at these problems, numerous experiments were done in order to find a low costs, little pollution and"Green"working line of soybean isoflavones, and these are just the objectives of this paper.Under the principles of utilizing local materials reasonably and effectively, Soybean cake, a by-product which is abundant in northeast and can be obtained during processing of soybean oil was selected. The high-performance liquid chromatography and UV spectrophotometry were used to determine the content of different forms and total isoflavones separately. Isoflavones were separated by the modern ultrafiltrate membrane separation technique, and refined by macroporous resin adsorptive technique.The parameters of membrane processing and the properties of adsorption and desorption for resin were studied, which are dependent on the loss amounts and purity of isoflavones. The aims of this study were (i) to find a way to solve the problems in present production, (ii) to obtain several products (soybean isoflavones, soybean protein and soybean ogligosaccharides etc.) synchronously on one working line. The idiographic works are as follows:1. The optimum detection conditions of HPLC were obtained after optimization of the influencing factors such as the ratio of mobile phase, the acidity and the flow rate, etc. And the working condition was validated by the accuracy test, stability test and repeated test. Then the quantifications of isoflavones (Daidzein, Genistein, Daidzin, Genistin) in samples and standard mixtures were detected.2. After a large number of experimentations, the influencing factors of extracting isoflavones and the relationships between every factor and the extracting rate were found, and the suitable value-scale of every factor was selected simultaneously. These data which come from the single factor tests were optimized and recombined by the orthogonal tests. Finally, the optimum extracting conditions of isoflavones were owned: The 50% ethanol was used as extraction solvent. The water bath temperature was set at 70℃, and the defatted soybean flour which was soaked at the flour-to-solvent ratio of 1:50 (g/ml) were incubated for 7 hours, and it was extracted for only one time. The results was given by the repeated tests that the relative standard errors was 0.94%, and it was shown that the test result was accurate.3. The isolated and purified conditions of soybean isoflavones were studied and the best techniques were confirmed as follows: the sample, whose concentration was 0.25~0.374 mg/ml, obtained by decompression concentration was ultrafiltered by a membrane with a molecular weight cut-off (MWCO) of 10 k Da. The ultrafiltrate was passed the macroporous adsorption resin ADS-21. The impurity such as sugars and salts could be eluted by distilled water from the macroporous adsorption resin column and the isoflavones could be eluted by 80% ethanol. In succession the eluate was concentrated and freeze-dried, and the high purity soybean isoflavones were obtained.4. The losses of soybean isoflavones were studied during the ultrafiltration under different working conditions. It was shown that both increasing the MWCO and elevating the trans-membrane temperatures would decrease the losses of isoflavones, and the loss amount was cut by 4/5 and 2/3 respectively. The purity of ultrafiltered flour was compared with the non-ultrafitered, we noticed that the former is higher by 26.1%, and what it meant that ultrafiltration have an effective effect on eliminating the impurities such as soybean protein. 5. Macrophorous resin ADS-21 was selected by comparing the properties of adsorption and desorption of 3 types of macrophorous resins for soybean isoflavones. The adsorption and desorption kinetics of resin ADS-21 were also studied. It had been found the static adsorption and desorption curves, dynamic elution curve of distilled water, dynamic desorption curve of ethanol and adsorption dynamic equation at room temperature.6. By comparing the different isolated and purified methods, the results were given that the use of solvents and the losses of isoflavones were cut by a half, while the purity of final product was higher.Because of the reduction of solvents, costs and pollutions, the isolated and purified method of soybean isoflavones that studied in this paper has displayed the validity and advantage to solve the problems during production. But it was also needed to study further about other products in order to carry out the project mentioned"obtaining several products synchronously on one working line"earlier.