Study On Enzymatic Transglycosylation Of Stevioside Using Hydrolyzed Cornstarch Catalyzed By Cyclodextrin Glucanotransferase Toruzyme 3.0L

Stevioside is a safe and low-calorie natural sweetener. Enzymatic modification of stevioside could improve its aftertaste. In this paper, aiming at improving the aftertaste with introducing one or two glucose residues to stevioside, the transglycosylation of stevioside catalyzed by a commercial cyclodextrin glucanotransferase(CGTase) Toruzyme 3.0L was investigated. Enzymatic modification of stevioside conducted by double-enzyme method which is catalyzed by CGTase in the mixture of stevioside and cornstarch hydrolyzed byα-amylase and single-enzyme method which only used CGTase in the absence ofα-amylase. The convention and low power microwave assisted transglycosylation of stevioside using CGTase Toruzyme 3.0L were investigated, respectively. In addition, the immobilization of CGTase Toruzyme 3.0L was studied using AB-8 resin and then the immobilized CGTase Toruzyme 3.0L applied to catalyze the mixture of stevioside and cornstarch. The main contents and results are as follows:1. Recrystallization of industrial stevioside was investigated, the highest stevioside content reached 87.32% and the recrystallization rate reached 67% by using methanol as recrystallized solvent. The CGTase from Thermoanaerobacter (Toruzyme 3.0L) which presented the highest activity was chosen for the experiment. By using CGTase Toruzyme 3.0L, 14 stevioside analogues were found including mono-, bi-, tri-, tetra-, penta-glucosylated stevioside and their isomers, while mono- and bi-glucosylated stevioside are the main product. The bitter aftertaste of the product significantly improved. The highest conversion of stevioside of 77.11% was obtained with a mass ratio of 1:1 (stevioside:cornstarch), the final concentration of the cornstarch was 1%, and catalyzed by 100 U CGTase /g stevioside at 60 oC for 4 h.The immobilization of CGTase Toruzyme 3.0L was studied by using AB-8 resin. The immobilization of CGTase Toruzyme 3.0L conducted at the ratio of enzyme to resin of 45.6 U mL-1 g-1 with an enzyme loading of 9.12 U/mL and shaked at 200 rpm, 15 oC for 5 h, giving the immobilization and specific activity 88.8% and 81%, respectively. St conversion reached 68.23% by using immobilized CGTase Toruzyme 3.0L which was more stable than free CGTase and the immobilization of CGTase Toruzyme 3.0L could reuse for 3 times.2. Transglycosylation of stevioside only using CGTase in the absence ofα-amylase produced mono-, bi-, tri-, tetra-, penta-glucosylated stevioside and their isomers. The highest conversion of stevioside of 71.38% was obtained with a mass ratio of 1:1 (stevioside to cornstarch), the final concentration of the cornstarch was 1%, and catalyzed by 100U CGTase /g stevioside in water at 55 oC for 1 h. Mono- and bi-glucosylated stevioside are the main product. The comparison of double-enzyme method and single-enzyme method concluded that the initial rate and St conversion in double-enzyme method were higher than that in single-enzyme method and the reaction last long in double-enzyme method.3. Transglycosylation of stevioside was speeded up to 20 times with CGTase Toruzyme 3.0L first employing low power microwave in water system which produced mono-, bi-, tri-, tetra-, penta-glucosylated stevioside and their isomers. The highest conversion of stevioside of 64.48% was obtained in double-enzyme method with a mass ratio of 0.5:1 (stevioside:cornstarch), the final concentration of the stevioside was 1%, and catalyzed by 100U CGTase/g stevioside at 60 oC for 4 h and that of 56.82% in single-enzyme method under same reaction conditions mentioned above.