Study On Enzymatic Extract And Biotransformation Of Isoflavones From Soybean Residue

Soybean isoflavone is a kind of phytoestrogen with important physiological effects. It has vital economical significance to extract isoflavones from soybean residue, The dissertation has studied the isoflavones extraction and structure modification from soybean residue by enzymes from different microbes. The results were as follows:A simple and new method to analyze isoflavones in soy products by measuring both aglycones and glucosides was developed. The conditions were as follows: the column temperature was 40℃, wavelength was 254 nm , injection volume was 20 uL and flow rate was 1.0mL/min. The mobile phases were 10% Acetonitrile(with 0.1% tricholroacetic acid) and 90% Acetonitrile(with 0.1% tricholroacetic acid).The gradient was as follows: 0 min,100% A;30min,30% A. It was reported for the first time about the relationship between total isoflaovne content by UV and by HPLC: as to isoflavone glucosides, the total isoflaovne content determined by HPLC was 60% of which determined by UV; as to isoflavone aglycones, the total isoflaovne content determined by HPLC was 75% of which determined by UV. Thin layer chromatography(TLC) was set up to determine whether the isoflavone glucosides was changed to their aglycones. The conditions were as follows:V(chloroform): V(methanol): V(water) =(5:1:0.5). The retardation factors(Rf) were 2.31(daidzin),2.46(genistin),8.72(daidzein) and 8.84(genistein) respectively.When using the cellulase from Bacillus sp ZU-4, the isoflavone yield was as 1.4 times as that of untreatment and the isoflavone composition was unchanged since ; When using the cellulase from aspergillus niger ZU-7,the isoflavone yield was not enhanced but isoflavone glucosides were changed to aglycones. By cellulase pretreatment from Trichoderma reesei ZU-5, the isoflavone yield was enhanced by 0.6 times and at the same time, the isoflavone composition was changed to aglycones with higher physiological activity. The optimal cellulase dosage from Trichoderma reesei ZU-5 was 15FPIU/g soybean residue and the treating time was 36 hours. The optimal xylanase dosage was 200IU/g soybean residue and the treating time was 36hours. The isoflavone yield of ethnol extract obtained by xylanase pretreatment was 0.67 times higher than that of untreatment. The synergetic reaction by cellulase and xylanase could enhance the extract yield of isoflavone . When the cellulase and xylanase dosage per gram soybean residue were 5FPIU and 100IU respectively, the isoflavone yield was as 1.72 times as that of untreatment.3 -glucosidase had been obtained from Aspergillus niger ZU-7 by solid-state fermentation. β -glucosidase was immobilized efficiently and easily by entrapping the Aspergillus niger spores which was rich in β -glucosidase into calcium alginate. In repeated batch processes, the yield of isoflavone aglycones kept higher than 90% during 7 batches. In a continuous enzyme treatment, the yield of isoflavone aglycones reached 95% at 0.12 h^-1 dilution rate. The research results are meaningful in the structure modification of isoflavone glucosides.The ethanol stirring method was selected to extract isoflavone aglycones. The optimum extraction conditons were as follows :solvent was 80 % ethanol; temperature was 50℃;treating time was 4h;sample and extractant ratio was 1:5.Furthermore, the total yield of isoflavone was enhanced from 0.31% to 0.37% by counter-current extraction technology.The resin SD200 with strong polarity exhibited good performance in seperating isoflavone glucosides. The optimum separating conditions were as follows : isoflavone glucosides concentration was 0.5 mg/mL, resin volume was 7BV, pH was 4 , absorption rate was 1.0 mg/mL, eluant was 80% ethnol and desorption rate was 2mL/min. The purity of isoflavone glucosides could reach 53% by the treatment of SD200.The resin HZ801 with weak polarity exhibited good performance in seperating isoflavone aglycones. The optimum separating conditions were as follows: isoflavone aglycones concentration was 0.5 mg/mL, resin volume was 6BV, pH was 3-4 , absorption rate was1.0 mg/mL, eluant was 95% ethnol and desorption rate was 2.5mL/min. The purity of isoflavone aglycones could reach 53% by the treatment of HZ801.The dissertation has gained important research results on isoflavone enzymeextraction from soybean residue, isoflavone glucosides directional modification by immobilized β -glucosidase and separation and extraction on isoflavone glucosides and isoflavone aglycones. The results is important on thorough understanding of cellulose operation mechanism, widening the application of enzyme preparations from microbes and promoting the industrialization of extracting high value product from plant materials.