Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana

J Agric Food Chem. 2017 Apr 19;65(15):3223-3229. doi: 10.1021/acs.jafc.6b05793. Epub 2017 Apr 6.


An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r2 = 0.9629, n = 16). The percentages of coefficient variation (CV) of within day (n = 12) and between days (n = 12) assays were lower than 5%, and accuracy ranges were 95-105%. This analysis demonstrates that the enzymatic method developed in this study is specific, easy to perform, accurate, and yields reproducible results.

Keywords: Stevia rebaudiana; enzymatic method; method validation; stevioside; stevioside determination; β-glucosidase.

Publication types

  • Evaluation Study

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacteroides thetaiotaomicron / enzymology*
  • Biocatalysis
  • Colorimetry / methods
  • Diterpenes, Kaurane / analysis
  • Enzyme Assays / methods*
  • Glucose Oxidase / chemistry
  • Glucosides / analysis
  • Plant Extracts / analysis*
  • Stevia / chemistry*
  • Sweetening Agents / analysis*
  • beta-Glucosidase / chemistry*


  • Bacterial Proteins
  • Diterpenes, Kaurane
  • Glucosides
  • Plant Extracts
  • Sweetening Agents
  • stevioside
  • Glucose Oxidase
  • beta-Glucosidase