Studies On Enzymatic Modification Of Bioflavonoid In Citrus

In this paper, it was summarized that using of citrus by-products and studies of bioflavonoid in home and abroad.At the same time ,new progresses in present about structure-modification of hespeidin and it's derivate was introduced.According to screening model, Aspergillus D7 (1) was selected from over 20 different soils and strains reserved by our lab. The optimized formula of fermentation medium with orthogonal experiment comprised: bran 8.0%, soybean powder 2.0%, KH2PO4 0.1% and 5ml 0.1% hesperidin per 30ml culturing base. The highest output of hesperidase could be obtained when the optimized fermentation medium with 30ml in 250ml flask, kept fermenting for 4 days, at the starting pH6 and the optimum temperature 50℃, after incubated 2% seed. After the treatment of UV mutation, Aspergillus D7 (1)-1 was obtained with hesperidinase activity more 19% than strain D₇ (1).After separation and purification of cultured broth by means of salting-out, hydrophobic interaction chromatograph (Hiprep 16/10) and cationg-exchange (Hitrap), it showed that hesperidinase was made of rhamnosidase and glucosidase.In the hesperidinase; the activity of rhamnosidase was 8.5 times higher than that of glucosidase. The studies showed that it's optimal pH for activity was 4.0 ,stability range was 3.6-6.0,the optimum temperature was at 55 ℃ ,the enzyme was stable below 55℃ and unstable above 60℃ The activity of raw hesperidinase was remained in 4 days at 4℃ . Enzymatic parameters (Km=0.54mmol/ml, Vm=0.8ummol/l.min ) had been obtained by Lincweaver-Burk with hesperidin as substrate.Merchanic of hesperidinase reaction was analysised: the bond between rhamnoside and glucose was cut down firstly and glucopyranoside was hydrolyzed rapidly by hesperidinase, so the content of glucopyranoside was very low during the reaction.The low solubility of hesperidin in water was solved by adding DMF (N,N-dimethylformamide),DMF have a little effect on the activity of hesperidinase, hesperidinase has remained 90% activity when DMF took up 40% in reaction system, the optimal reaction condition:5% hesperidn DMF/buffer ,1/4 enzyme of total volume, pH4.5,reaction temperature 60℃,DMF: buffer =5:15,reaction time 30 minute. Through immobilizing hesperidinase and 2% yeast together with 4% PVC, 2%alginate, 0.15%tannin and 10% hesperidinase, the feedback restrain of glucose and the separation of rhamnoside and glucose was settled down. The experimental results show that the highest outputs of monoglucosyhesperetin in the column reaction can be attained at 60 ℃ ,velocity of flow 5ml/min, reacting time 60min. After 10 times recycle, immobilized hesperidase had remained about 85% of activity.it's halflife is 1200 minutes.Comparing many isolating methods of different products, the final method was determined as follow: Through extraction by aether and ethyl acetate in turns, raw hesperetin and monoglucosyhesperetin was gained. After column chromatography with D₁₀₁ macroporous resin as adsorbent, eluted by grads aqueous-alcohol ,the products in which the purity of monoglucosyhesperetin was 98.02% was obtained.Antioxidation, scavenging hydroxy radical and superoxide radical and inhabiting a-glucosidase enzyme of hesperidin and its hydrolysate was studied. The results showed as follow: 1) hesperidin and it's hydrolysate have strong antioxidation to plant oil, hesperetin excelled VE and mixture of BHA and BHT.But early stage was better than later stage to the effect. 2) Hesperidin and it's hydrolyte showed good ability to scavenge hydroxyl radical and superoxide radical. About the effect to scavenge hydroxyl radical and superoxide radical, hesperetin was better than monoglucosyhesperetin and hesperidin. 3) hesperidin and it's hydrolysate both have strong inhibitive to a-glucosidase. The inhibitive activity of monoglucosyhesperetin was 82% of glucobay in water and 84.5% in DMF solvent.The enzymologic property of hesperidin and it's hydrolysate had strong affinity to a-glucosidase.