The Research In The Fermentation And Application Of Hesteridinase And Naringinase

Hesterdinase and naringinase are the key enzymes for hydrolyzing hesperidinand naringin resperctively. After hydrolyzing by hesterdinase and naringinase,hesperetin-7-glucoside, naringenin-7-O-glucoside, hesperetin and naringenin will beobtained as Hydrolysate of great value in use.This study was about producing hesterdinase and naringinase at the same timefrom fermentation of Aspergillus niger WZ001. Optimized the conditions of cultureof thalli, it was found that when bran was seledted for carbon sources, with theamounts of 1％; the powder bean cake was seledted for nitrogen sources, with theamounts of 1～3％; and （NH₄）H₂PO₄ was seledted for phosphate. Enzyme activitywould be advanced to 1090.6U/mL（Hesteridinase） and 1089.3U/mL（Naringinase）,with the percentage of advance was 50％. Then research about fermentation dynamicsbased on above-mentioned terms. The result which was ture to fact reflected theconnection between the amount of thalli and produced enzyme in the curse offermentation, and it was adapted to guide manufacture of industry.The immobilized enzyme is not only can catalize, but also can be reclaimedeasily, and then can be used circularly to make the whole production consecutive androboticized. Optimized the means for immobilizing enzyme after chose a preferableone, then the immobilized hesterdinase and naringinase could be produced embed bysodium alginate, and studied the characteristics of them. The optimal temperature ofimmobilized hesterdinase and naringinase were 60℃and 50℃respectively, theoptimal pH of them were 3.5 and 5.5 respectively, and Mchaelis-Menten contant （Km） ofthem were 6.0 mmol/L and 2.67 mmol/L respectively. They were more stable than solubleenzyme in temperature and pH, and also had great stability when reacting group by group or in continuous reaction. In order to produce the mid production hesperetin-7-glucosideand naringenin-7-O-glucoside which has high worth of appliance, the filling reactor ofimmobilized enzyme was studied with hesterdinase. The results of the studies indicated: adversecurrent was excellent, velocity of flow should be 2～3mL/min; the reactor can be level using for 9days at least, and its productive strength kept in1.44～1.53μg/mL/min. The dynamics was bestudied at the same time which was adapted to guide manufacture of industry.The edible and floury enzyme preparation which was adaptive to conserving andconvenient for using was produced. The liquid enzyme was obtained by ultrafiltration.It made the activity of hesterdinase and naringinase get to 4725.9 U/mL and 4508.5U/mL respectively which were 5 to 6 times of original one. The white and farinaceousenzyme, which had symmetrical granule and preferable appearance, could beproduced as follows: Use absolute alcohol in 2.7 times of enzyme’s vol. for deposit,add in amylum 1％at the same time. Then collect deposition to drying at 45℃. Finallypulverize it. The activity of hesterdinase and naringinase could be get to 48150.4 U/gand 37596.5 U/g respectively, and the whole ratio of callback were 88.1％and 59.4％respectively. This technique could be industrialization.Separation and purification of modification of hesperidin and naringin withmacroporous resin were researched in this study. Adsorption and desorption ofmacropore resin for modification of hesperidin and naringin were studied. It wasfound that it was doable for absolute separation of hesperetin-7-O-glucoside andhesperetin, or naringenin-7-O-glucoside and naringenin through desorption for 20％ethanol-water solution used as eluting solvent with velocity of flow in 2mL/min.Finally the products could be obtained after decompressed distil, static placement,filtration and drying. The purity of hesperetin-7-glucoside andnaringenin-7-O-glucoside were 95％and 96.3％respectively, and the the ratio ofcallback were 83.8％and 71.2％respectively.