Di-glycosyl-stevioside production via Leuconostoc citreum sk24.002 alternansucrase enzymatic reaction and structural characterization

In this work, alternansucrase enzyme from Leuconostoc citreum SK24.002 was used to produce di-glycosyl-stevioside through acceptor reaction. This was detected by high-performance liquid chromatography (HPLC) and was isolated using AB-8 macroporous resin and semi-preparative HPLC. With the help of mass spectroscopy, 1D NMR (H 1 and C 13 ) and 2D NMR (COSY, HMQC, HMBC) the di-glycosyl-stevioside structure was identified to be 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. In order to increase the modified stevioside (di-glycosyl-stevioside) product and reduce production time, the impact of velocity of shaking on the di-glycosyl-stevioside production was also evaluated, using 75–150 rpm for 24 h at 25 °C. The results showed that the di-glycosyl-stevioside production increased significantly (p < 0.05) from 0.35 ± 0.0.01 mg/mL for 75 rpm to 1.0667 ± 0.0152 mg/mL for 150 rpm at 6 h during the glycosylation reaction.