Optimisation of novel method for the extraction of steviosides from Stevia rebaudiana leaves

Stevioside, a diterpene glycoside, is well known for its intense sweetness and is used as a non-caloric sweetener. Its potential widespread use requires an easy and effective extraction method. Enzymatic extraction of stevioside from Stevia rebaudiana leaves with cellulase, pectinase and hemicellulase, using various parameters, such as concentration of enzyme, incubation time and temperature, was optimised. Hemicellulase was observed to give the highest stevioside yield (369.23 ± 0.11 μg) in 1 h in comparison to cellulase (359 ± 0.30 μg) and pectinases (333 ± 0.55 μg). Extraction from leaves under optimised conditions showed a remarkable increase in the yield (35 times) compared with a control experiment. The extraction conditions were further optimised using response surface methodology (RSM). A central composite design (CCD) was used for experimental design and analysis of the results to obtain optimal extraction conditions. Based on RSM analysis, temperature of 51-54 °C, time of 36-45 min and the cocktail of pectinase, cellulase and hemicellulase, set at 2% each, gave the best results. Under the optimised conditions, the experimental values were in close agreement with the prediction model and resulted in a three times yield enhancement of stevioside. The isolated stevioside was characterised through ¹H-NMR spectroscopy, by comparison with a stevioside standard.