Glucansucrases As New Tools For Stevioside Modification

Stevioside(13-O-β-sophorosyl-19-O-β-D-glucosyl-steviol) is a non-cariogenic and lowcalorigenic diterpenoid glycoside. Stevioside presented at the highest amount among the steivol glucosyl constituents in the leaf of Stevia. It is content in the range between 4 and 20% of the dried leaves(wt/wt) and was 300 times sweeter than sucrose. It has slightly bitterness and bad aftertaste. The objectives of this study were; first to modification of stevioside by glucansucrases(alternansucrase and dextransucrase) with the purpose of obtaining new derivatives of modified stevioside with improved organoleptic properties, seconde to increase the production of modified stevioside products and third to reduce production time and characterization of modified stevioside products.Enzymatic modification with alternansucrase from Leuconostoc citreum SK24.002 was utilized in biotransformation of stevioside to remove fully or partially the bitter taste and an aftertaste of the stevioside. The effect of reaction conditions including, time course(1- 24 h), temperature(20- 40 oC), p H(4- 7), donor concentration(10- 100 mg/m L) and enzyme concentration(0.5- 2.5 U/m L) were investigated in order to maximize the transglucosylation yield. The highest transglucosylation yield of approximately 43.7% was achieved at 20 oC, p H 5.4 for 24 h using 10 mg/m L sucrose and 1 U/m L alternansucrase. LC/MS analysis confirmed that product composition of mono-di- and tri- glucosylated stevioside and their isomers.Enzymatic modification of stevioside(13-O-β-sophorosyl-19-O-β-D-glucosyl-steviol) by dextransucrase acceptor reaction to improve the organoleptic properties of stevioside(by removing bitter taste and an aftertaste of stevioside) was investigated, using sucrose as the sole donor molecule. The maximum stevioside conversion of 74.33% was achieved after 24 h of reaction. The results obtained from HPLC and LC/MS analysis revealed that dextransucrase has catalytic activitiy on the stevioside, which it can catalyses transglycosylation reaction, produced mainly mono- and di-glycosyl-stevioside. The maximum transglycosylation of stevioside to mono- and di-glycosyl-stevioside of 31.83% was obtained at stevioside: sucrose molar ratio(1:35) after 24 h.To optimize the modified stevioside products and minimize production time, effect of shaking velocity on modified stevioside(mono- and di-glycosyl-stevioside) production was investigated, with four different level of shaking velocity(75, 100, 125 and 150 rpm) up to 24 h at 25 °C, using sucrose as donor and stevioside as acceptor. The results revealed that monoglycosyl-stevioside yield significantly increased with increase in the reaction shaking velocity and reached maximum yield of 3.78 ±.02 mg/m L at 150 rpm shaking velocity after 6 h of reaction. The di-glycosyl-stevioside production was increased significantly with increasing shaking velocity of reaction, which was increased from 0.35±.0.01 mg/ml at 6 h with 75 rpm to 1.0667±.0152 mg/m L at 6 h with 150 rpm shaking velocity. Furthermore the increase in reaction time results in increasing di-glycosyl-stevioside production and was achieved 1.2833±.0153 mg/m L with 150 rpm shaking velocity after 24 h of reaction time. And an increase in the reaction shaking velocity up to 150 rpm reduced the final reaction time and increased the productivity of mono-glycosyl-stevioside product such that the high quantity of mono-glycosyl-stevioside product was produced in only 6 h rather than 24 h.The main products of mono-and di-glucosyl-stevioside were complete separated using macroporours resin AB-8 flowed by semi-preparative HPLC. Macroporous resin AB-8 revealed high selectivity and capacity toward mono- and di-glucosyl-stevioside. A corroding to extensive 1D and 2D NMR(1H and 13 C, COSY, HSQC, HMBC) and mass spectral data, the structures of mono- and di-glucosyl-stevioside were characterized, as 13-{[α-D-glucopyranosyl-(1→6)-β-Dglucopyranosyl-(1→2)-β-D-glucopyranosyl]oxy}kaur-16-en-19-oic acid β-D glucopyranosyl eater and 13-{[α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]oxy}kaur-16-en-19-oic acid β-D glucopyranosyl eater, respectively.The sensory properties of stevioside and modified stevioside in water solution was investigated, and stability of stevioside and mono- and di-glucosyl-stevioside(produced via Leuconostoc citreum SK24.002 alternansucrase acceptor reaction), and the possible formation of the steviol at elevated temperature and different p H levels were assessed, covering a typical p H range that simulated both relevant and extreme beverage storage conditions. The sensory data showed that modified stevioside and the stevioside were, respectively, 125 ± 21.0 times and 183.3 ± 16.7 times sweeter than 2 g/100 m L sucrose. And the threshold for bitter aftertaste of stevioside and modified stevioside were 18 ± 3 mg/100 m L and 27 ± 3 mg/100 m L respectively, which was significantly increased(p ≤ 0.01). The results indicated that the modified stevioside was more pleasant-tasting than stevioside. Acid solutions mixed with stevioside or mono- and di-glucosyl- stevioside after 24, 48, and 72 h of storage time at 50 and 80 °C were analysed. Under mild conditions(at a p H range of 2–6.5 over 72 h and 50°C) stevioside and mono- and diglucosyl-stevioside showed good stability. degradation of 35, 24 and 54% for stevioside, monoand di-glucosyl-sevioside, respectively were observed at p H 3 and 80°C after 72 h, and stevioside was less stable than mono-glucosyl-stevioside. Complete degradation of stevioside and mono- and di-glucosyl-stevioside was observed at p H 2 and 80°C after 72 h. Stevioside and mono- and di-glucosyl-stevioside and their degradation products were analysed by highperformance liquid chromatography with a diode array detector(DAD-HPLC) on an NH2 analytical column, and the identity of the degradation products was confirmed by liquid chromatography-electrospray ionization mass spectrometry(LC-ESI-MS) in negative mode.