Study On The Preparation Of Hesperetin Monoglucoside By Separating An α-L-rhamnosidase From A Strain

α-L-rhamnosidase is an important hydrolase and can hydrolyze glycosidic bond of α-1,2、α-1,3、α-1,4 andα-1,6. Hesperidin is formed by a flaconoid ring and a α-1,6 glycosidic, with great physiological activities such as anti- inflammatory, anti-oxidant, anti-cancer, maintaining normal blood vessel of osmotic pressure and reducing blood fat. An α-L-rhamnosidase-producing strain was screened from rotten citrus, and using the α-L-rhamnosidase to hydrolyze hesperdin, getting the hesperetin monoglucoside(HMG) as a result. The main works of the paper are listed as follows:1.An α-L-rhamnosidase-producing strain was screened from rotten citrus, and using the α-L-rhamnosidase to hydrolyze hesperdin, getting the hesperetin monoglucoside(HMG) as a result. The identification of the strain species was studied and the strain was initially identified as A. niger Aspergillus sp. The fermentation conditions of α-L-rhamnosidase were optimized to find the best enzyme production nutrient as : potato 20%, glucose 2.0%, yeast extract 1.0%, K2HPO4 0.3%, hesperidin 0.75%; and the optimum reaction codition: culture the strain at 35℃, pH5.5, reacting for 72 h with the ratory speed 180r/min.2.The purification of α-L-rhamnosidase and its enzymatic properties were studied. The purification of α-L-rhamnosidase carried out by ammonium sulfate fraction and dialysis to remove the impurity protein and the salt. The molecular weight of α-L-rhamnosidase was identified to be about 60 KDa by SDS-PAGE after the purification, and the optimal temperature and p H of α-L-rhamnosidase were 55℃ and 6.0, and it was stable at the condition of 20-70℃, pH3.0-7.0, its Michaelis-Menten contant(K m) was 11.66 mmol/L.3.Separation and purification of hesperitin-7-glucoside with silica gel column and macroporous resin were studied. The eluent of silica gel column was methanol: acetic ether: chloroform =1:4:5, after purification by silica gel column, the purity of hesperitin-7-glucoside was 91.71%. The static adsorption, dynamic adsorption and the elution condition of macroporous resin were studied, and the result showed hesperidin and hesperitin-7-glucoside can be separated well by 30% ethanol solution with the florate of 1ml/min.