Supplimentary Material

Manuscript No. BL-10408

Supplimentary material

Isolation and purification of stevioside

Air dried leaf powder (1 kg) of Stevia rebaudiana was extracted with MeOH/H2O (80:20, v/v) for 12 h at room temperature (25 0C). The hydromethanolic percolation was repeated three times (1000 ml × 3). The combined percolations were evaporated to dryness (250 g) and fractionated with hexane, chloroform, ethylacetate and butanol. The sub-fractions were dried with anhydrous Na2SO4 filtered and concentrated under reduced pressure at 50±5 0C yielding hexane (30 g), chloroform (10 g), ethylacetate (10.5 g) and butanol (150.2 g) fractions respectively.

Butanol fraction (150.2 g) was subjected to column chromatography over silica gel (60-120 mesh) using gradient elution of CHCI3 /MeOH with increasing proportion of 5%, 10%, 20% and 30% methanol(v/v) in chloroform to give four fractions (i-iv). Fraction (iv) was rechromatographed over silica gel using gradient elution of 30 % methanol (v/v) in chloroform yielding pure stevioside (8 g), m.p 196-198 0C.

Purification of transglycosylated products

Stevioside (1.24 mmol) and β-cycodextrin (1.76 mmol) were dissolved in sodium phosphate buffer (5ml) pH 7 at 50 0C. To this β-CGTase (2 U/g) was added and the reaction mixture was held in a microwave oven (2450 MHz, 300 W) at 80 W and 50 0C for 1 min for carrying out the enzymatic reaction. The reaction was stopped by raising the temperature to 100 0C for 10 min. The reaction product was centrifuged at 5-10 0C to precipitate residual β-cyclodextrin followed by filtration and then passed through diaion HP-20 column (1.2x25cm, stevioside to gel ratio, 10:1, w/v). The column was washed with distilled water (100 ml), before loading the reaction mixture. The reaction mixture was eluted with water, 10, 80 and 96 % alcohol in water (100ml each), and the solutions were evaporated until dry at 50-55 0C under vacuum. The aqueous and 10% ethanol fractions contained only glucose, linear maltosaccharides and residual β-CD. The products were eluted with 80% and 96% ethanol in water (100 ml each) and further purified by column chromatography using silica gel (mesh 60-120) in a linear gradient system CHCl3/MeOH/H2O (30:10:1-15:7:1-10:5:1, by vol) yielding two main glycosylated products I and II.

Separation of I and II by prep-HPLC

The two purified products were separated on waters 2487 Prep-HPLC using agilent-zorbax-NH2 column (9.4x250 mm) particle size 5 μM, PDA (photodiode array) detector and peak detection was at 205 nm. The products were collected with mobile phase, MeCN/H2O (80:20, v/v) having flow rate of 10 ml/min.

Anakytical HPLC

Analytical HPLC of reaction products I and II was performed for quantification on automated waters system equipped with waters 717 plus autosampler, waters 996 photodiode array detector, WatersTM 600 pump, waters online degasser AF, waters 600 controller fitted with LichrocartR 100 NH2 (5 μm) column. The samples were eluted with two solvents: solvent A, 80% acetonitrile (HPLC grade, J T Baker, USA) and solvent B 20 % water (HPLC grade, J.T. Baker, USA). Ten microlitre of samples were injected to the column and eluted at a flow rate of 0.8 ml/min. with isocratic system of MeCN/H2O (80:20, v/v). Column temperature was fixed at 25 0C throughout the analysis, stabilized at initial conditions and washed with acetonitrile. Two peaks corresponding to product I and II were detected at 205 nm using PDA (photodiode array) detector. The two reaction products were also analyzed using gradient system of MeCN/H2O (80:20, v/v) for 2min to (60:40, v/v) at 40 min. Quantification of the transglycosylated products was achieved using standard calibration curve yielding 66 and 24 % (w/w) of I and II respectively. HPLC chromatograms of reaction mixture, stevioside and product I and II are given in Supplementary Figure1(A–D).