Research On Extraction By Supercritical Fluid Technology And Transformation By Immobilized β-Glucosidase Of Isoflavone Glucosides From Defatted Soybean Residues

Soybean isoflavones, which are the secondary metabolites, have important biological effects. To date 12 soybean isoflavones have been reported, including three aglycones (genistein, daidzein and glycetein) and their nine glycosides (glucosides, acetyl-β-glucosides and malonyl-β-glucosides). In total soybean isoflavones the content of glycosides reaches to 97% to 98 %, while the content of aglycones content only 2% to 3%. It has been revealed that the biological effects of soybean isoflavones are not due to the glycosides instead their aglycones, such as genistein and daidzein. The glycosides can play the functional role in humans' health when converted in theirs aglycones. In the present dissertation, extraction by supercritical fluid technology and transformation by immobilizedβ-glucosidase of isoflavone glucosides from the defatted soybean residues were studied. The results were as follows:Effects of the supercritical fluid extraction conditions, including flow rate of CO₂, static and dynamic extraction time, entrainer dosage, extraction temperature and pressure, on the extraction rate of isoflavones from the defatted soy bean residues were investigated by both single factor and orthogonal experiments. The results showed that the flow rate of CO₂, extraction temperature and pressure in the separation tank had a little effect on the extraction yield. The maximum extraction yield of 794.5μg/g was obtained under the optimum extracting conditions (flow rate of CO2, 8 L/h; static extraction time, 120 min; dynamic extraction time, 60 min; ethanol dosage, 300 mL/100g soy bean residues; extraction temperature, 55℃; extraction pressure, 30 MPa). The kinetic model of extraction was developed based on adsorption and desorption theory and the model parameter of k_Lαwas determined by BP neural network. Training error and prediction error were 1.8 % and 3.7 %, respectively. The error value of k_Lαbetween simulated and experimental data was about 9%. The kinetic model with determined parameters could be used to describe the genistin distribution in the extraction bed with higher accuracy.Activities ofβ-glucosidase from 32 plant species with high content of isoflavones and 32 strains from different microbes were compared. Theβ-glucosidase activity (633.76 U/mg protein) toward genistin was found to be highest from Aspergillus niger 5-12.β-glucosidase from Aspergillus niger 5-12, after isolated and purified through ammonium sulphate precipitation, DEAE-Cellocuse -52 ion exchange and Sephadex G-100 gel filtration chromatography, has a molecular mass of about 151.7 kDa and contains two subunits of 75.8 kDa. CD (circular dichroism) revealed that theβ-glucosidase consisted of 7.1% a-helixes, 49.0%β-sheets, 21.7% turns and 22.2% random coils. Theβ-glucosidase had better heat stability, pH stability and storage stability.The submerged fermentation technology forβ-glucosidase production in 5 L fermentor by Aspergillus niger 5-12 was optimized through orthogonal tests and quadratic rotational combination designs. The optimum technology was rotary rate of 400 r/min and temperature of 30℃with inoculum size of 10%. The optimum medium compositions were wheat bran of 28.4 g/L, (NH₄)₂SO₄ of 1.2 g/L and KH₂PO₄ of 2.0 g/L. Under the optimum conditions,β-glucosidase acitivity reached 1493.90 U/mL after 3 days of fermentation. Based on Monod equation and Luedeking-Piret equation, the kinetic models describing cell growth andβ-glucosidase production were established. During the process of fermentation, Aspergillus niger 5-12 had a trend to form mycelia aggregates. Based on this growth property, immobilized fermentation system of Aspergillus niger 5-12 with non-carrier was established to produceβ-gluoosidase.With sodium alginate as a carrier and glutaraldehyde as the crosslinking agent, an improved immobilization method ofβ-glucosidase for production of soybean genistein from genistin was developed. When compared to the free enzyme, the optimum temperature, pH value and K_m of the immobilizedβ-glucosidase were shifted from 50℃to 40℃, 4.5 to 4.0 and 2.99μg/mL to 2.02μg/mL, respectively. The immobilizedβ-glucosidase was employed to genistein production, 71.1% of the activity and 76.5% of conversion were kept after consecutive use of 8 timesβ-glucosidase was immobilized with magnetic nanoparticles of aluminum nitride for 3.5 h under the temperature of 20℃, pH value of 5.0 and rotary rate of 100 r/min. The results of SEM, IR and DSC analysis revealed the structure of immobilized enzyme. The Michaelis constant of K_m for genistin of the immobilizedβ-glucosidase, with good storage stability and operation stability, was 6.41 mmol/L under the optimal temperature of 55℃and the optimal pH value of 5.0. Although the affinity and conversion ofβ-glucosidase immobilized with magnetic nanoparticles of aluminum nitride were slightly lower than those ofβ-glucosidase immobilized with sodium alginate, the magnetic aluminum nitride nanoparticle as a good support for enzyme immobilization could provide a fast and convenient approach to separate enzyme and products under the magnetic field.