Study On Characterization Of β-glucosidase From Lotus Seeds And Its Effect On Lotus Leaves Beverage's Aroma

β-glucosidase(EC 3.2.1.21), a kind of hydrolytic enzyme, is one of the key enzymes which plays an important role in the alcoholic aroma formation of the plant . it can hydrolyzeβ-D-glucosidase bond to the non-redox molecule chain's end accompany with releasing theβ-D-glucose and relative ligands. Recently, many literatures about the research on P-glucosidase has been reported, but little aboutβ-glucosidase extracting from aquatic plant. This experiment,primarily, explored the process of extraction, analytic measure of its activity, seperation and purification ofβ-glucosidase, and research its enzymatic property. The beverage, made from lotus leaves and treated withβ-glucosidase, was detected by the HS-SPME and GC-MS ,compared with the non-adding group, to study the impact ofβ-glucosidase's on aroma enhancement effect.Main results are as follow:1 Optimizing activity analysis and extraction method ofβ-glucosidase from lotus seeds.The enzymatic reaction should be performed at 56℃,pH 5.0 in 60min and the testing wavelength ought to be 400nm. Measured by this method,the activity of fresh lotus seeds was 1.78U/g, the the activity changed to 1.26U/g after lotus seeds be dried.The different kinds of buffer and pH has crucial impact on the activity of enzyme during the process of extraction. This experiment found out that the optimum measurement to extract theβ-glucosidase from lotus seeds during the process of extraction, was pH 6.0 citric acid - Na₂HPO₄ buffer and proper ratio of sample to PVPP was 5:1.2 Seperation and purificationβ-glucosidase from lotus seeds.The range of ammonium sulfate precipitation ofβ-glucosidase in the system was 50%-70% saturated solubility, 87.6% activity ofβ-glucosidase concentrated in this rang. After the sample was separated by ion-exchange chromatography of DAEA-52 cellulose, theβ-glucosidase was purified by 18.28 fold, and specific activity reached to 286.6×10^-3U/mg. Through Sephadex G-100 gel filtration chromatography,β-glucosidase, was father purified 32.57 fold with a specific activity of 510.83×10^-3U/mg. But after a series of process of purification, the total activity of enzyme decreased sharply, and the yield of enzyme was only 1.29%.3 The property ofβ-glucosidase from lotus seeds.The optimum temperature and pH of activity ofβ-glucosidase was 56℃, 5.0 respectively. The thermo-stability experiment indicated that the enzyme remianed comparitive high activity for 150 mins in 30℃-50℃. The preservation-stability experiement showed thatβ-glucosidase have very well preservation-stability at 4℃, even after half a month it possessed high activity about 91%. And pH stability experiment showed that, pH5.0-7.0, theβ-glucosidase had best acivity. When it in the alkalescence environment, the stability and activity of enzyme decreased sharply. Km was 2.281mmol/L, and Vmax was 2.308 U/L. SDS-PAGE electrophoresis indicated thatβ-glucosidase from lotus seeds contained two monomers, the molecule weight was 39.2KDa and 30.1KDa.In the lower concentration environment of Ca²⁺,Mg²⁺,Al³⁺,Zn²⁺, the ions had little impact on the activity of enzyme. When the concentration of Mg²⁺ reached to as high as 4.0 mmol/L, it can promote the activity of enzyme conspicuously. But when the concentration Al³⁺ and Zn²⁺ rose over 6.0 mmol/L, they could inhibit the activity of enzyme conspicuously. Low concentration of Vc and cysteine-HCl could promote enzyme activity slightly. Sodium chloride and sodium sulfite could inhibit the activity ofβ-glucosidase. More higher concentration, more inbihition effect could be observed.4 The aroma enhancement function ofβ-glucosidase on lotus leaves beverageThe optimum extracting condition,using HS-SPME, from lotus leaves beverage, the extracting time was 60min; extracting temperature was 50℃Headspace volume was 40ml. The GC-MS results demonstrated that the main flavor in the lotus leaves beverage was aldehyde, ketone, alcohol, their proportion were 19.69%,16.24%,6.25% in the whole odor material. The proportion of flavor in the total odor components ,in the lotus leaves beverage, were enhanced after the treatment ofβ-glucosidase to 12%. And the relatively content of aldehyde, ketone, alcohol in the whole aroma, change to 17.18%,16.54%,8.82% respectively. Meanwhile,β-glucosidase had major impact on the content of Ester, its relatively content had increased from 3.21% to 7.64%.The flavor substances of lotus leaves beverage ,added withβ-glucosidase, had critically been enhanced with merit of more flavorful and tender aroma.