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 lg) in 1 h in comparison
to cellulase (359 ± 0.30 lg) and pectinases (333 ± 0.55 lg). 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.