Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana.
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.
Department of Biochemistry, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.
Somsiri
S
Udompaisarn
Department of Tropical Nutrition & Food Science, Faculty of Tropical Medicine, Mahidol University , Rachawithi Road, Bangkok 10400, Thailand.
Dumrongkiet
D
Arthan
Department of Biochemistry, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.
Jamorn
http://orcid.org/0000-0002-2883-0693
J
Somana
D001426:Bacterial Proteins / Q000737:chemistry*; D000070077:Bacteroides thetaiotaomicron / Q000201:enzymology*; D055162:Biocatalysis; D003124:Colorimetry / Q000379:methods; D045786:Diterpenes, Kaurane / Q000032:analysis; D057075:Enzyme Assays / Q000379:methods*; D005949:Glucose Oxidase / Q000737:chemistry; D005960:Glucosides / Q000032:analysis; D010936:Plant Extracts / Q000032:analysis*; D036625:Stevia / Q000737:chemistry*; D013549:Sweetening Agents / Q000032:analysis*; D001617:beta-Glucosidase / Q000737:chemistry*
D001426:Bacterial Proteins; D045786:Diterpenes, Kaurane; D005960:Glucosides; D010936:Plant Extracts; D013549:Sweetening Agents; C012043:stevioside; D005949:Glucose Oxidase; D001617:beta-Glucosidase
Stevia rebaudiana; enzymatic method; method validation; stevioside; stevioside determination; β-glucosidase