Enzymatic Transglycosylation And Hydrolysis Of Stevioside

Steviol glycosides are widely used as low-calorie intensive sweeteners obtained from Steviarebaudiana. They are the most ideal sucrose substitutes in nature and possess many bioactivities. Stevioside（St） is one of the main components in the extract; however, its bitterness aftertaste affects its application infood and beverage. The aim of this dissertation is to elimite the bitterness aftertaste of St and develop highvalue-added St derivatives. In this experiment, several glycosyltransferases and glycosyl hydrolases havebeen utilized to catalyze the conversion of St, and novel catalysis activities were found. Based on studies onthe catalysis behavior and process kinetics, transglycosylation products were obtained with improvedsweetness and no more bitterness aftertaste. Subsquntly, some rare St derivatives existed in nature wereprepared through regioselective hydrolysis of the three glycosidic linkages in St, respectivly. In addition,microwave irradiated transglycosylation and hydrolysis were investigated.The cyclodextrin glucosyltransferase （CGTase） showed the best transglycosylation activity among ofall assayed enzymes. In the transglycosylation catalyzed by α-CGTase from Paenibacillus maceransJFB05-01, more than6transglycosylation products were produced, and the mono-glucosyl stevioside（St-Glu1） presented the best taste. The optimized transglycosylation conditions were: the mass ratio ofstarch hydrolysate to St was1.5:1,150mg/mL St in DI water with15U/g St of the enzyme, the Stconversion and St-Glu1yield reached59.2%and32.4%in3h at60oC, respectively. The hydrolysis oftransglycosylated products with glucoamylase increased St-Glu1content1.6folds, and left the tri-glucosylstevioside and higher grafted St in a content less than1.1%.Enzymatic hydrolysis of St was studied using some glycosyl hydrolases. A natural sweetenerrubusosidewas prepared from the specific hydrolysis of β-1,2glucosidic linkage of St with β-galactosidasefrom Aspergillus sp. This β-galactosidase showed lower transglycosylation activity accompanying with thehydrolysis of St, even in the presence of additional donors. The β-galactosidase also possesses substratespecificity in hydrolysis. Analogues of St, such as rebaudioside A, rebaudioside C, etc. can’t be hydrolyzedusing the β-galactosidase. However, steviolmonoside was produced from hydrolysis of steviolbioside withthis enzyme. Optimal hydrolysis conditions were:60oC in DI water with a substrate concentration of80mg/mL, St conversion and rubusoside yield reached98.3%and91.4%in6h, respectively. The activity ofenzyme can be improved by Ca²⁺, Co²⁺, Mn²⁺and Mg²⁺. The synthetic rubusoside tastes better than thecommercial rubusoside extracted from Rubus Suavissimus. rubusoside has a higher solubility than St, andcan improve the solubility of St. In a solution of3mg/mL rubusoside, the St solubility increased6.25folds.Subsequently, the ester linkage at C19of St was hydrolyzed selectively using the β-galactosidase fromKluyveromyces lactis with steviolbioside as the only hydrolysis product. St conversion reached99.5%at12h under37.5oC,25mg St/mL and16000NLU/g St. Besides of aforementioned transglycoslyationactivity, the β-galactosidase from Sulfolobus sp. can cleavage all three glycosides of St to release steviolThe conversion of St reached99.0%using20mg/mL of St and625U/g St of enzyme at pH4.6,75oC, in6h. The in-situ purification in the reaction could be realized because of the big difference between thesolubilities of products and materials.Microwave irradiation assisted transglycosylation and hydrolysis of St were investigated. Onlyα-CGTase from Paenibacillus macerans JFB05-01showed higher activity under irradiation comparing tothat with conventional heating, the catalytic efficiency under irradiation increased21.7folds under thesame conditions. The optimized transglycosylation conditions were:20W,55oC,1000U/g St of enzyme,the mass ratio of starch hydrolysate to St was1:1,200mg/mL of St, St conversion reached71.6%with23.7%of St-Glu1yield in1min. Microwave irradiation was more efficient than conventional heating for shortentime, but which does not favor to yield St-Glu1.Besides of sweetness and solubility, antiradical activity of steviol glycosides, steviol, isosteviol,rubusoside, steviolbioside, stevioside, rebaudioside A and rebaudioside C was compared. The amount ofglucose of steviol glycosides and antiradical activity was related.