Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 65, Issue 15, Pages 3223-3229Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.6b05793
Keywords
stevioside determination; enzymatic method; beta-glucosidase; Stevia rebaudiana; method validation; stevioside
Funding
- Agricultural Research Development Agency (Public Organization, Thailand) Grant [CRP5605011990]
- Dean's Research Fund, Faculty of Tropical Medicine, Mahidol University
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An enzymatic method for specific determination of stevioside content was established. Recombinant beta-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at beta-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 degrees C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r(2) = 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.
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