Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana
Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana
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DOI：
10.1021/acs.jafc.6b05793
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发表时间：
2017-04-19
影响因子：
6.1
通讯作者：
Somana, Jamorn
中科院分区：
文献类型：
--
作者：
Udompaisarn, Somsiri;Arthan, Dumrongkiet;Somana, Jamorn
建立了一种特异性测定甜菊苷含量的酶法。多形拟杆菌HB-13重组β-葡萄糖苷酶BT_3567（rBT_3567）对甜菊苷的β-1，2-糖苷键具有选择性水解作用，产生甜茶苷和葡萄糖。该酶与葡萄糖氧化酶和过氧化物酶的偶联允许通过使用基于比色的方法定量甜菊样品中的甜菊苷含量。在37 ℃条件下，甜菊苷测定的一系列反应可在1 h内完成。使用酶法测定的甜菊苷测定与从HPLC定量获得的结果强烈相关（r（2）= 0.9629，n = 16）。日内（n = 12）和日间（n = 12）测定的变异系数（CV）均小于5%，准确度范围为95- 105%。该分析表明，本研究中开发的酶法具有专属性、易于执行、准确度高且结果重现性好。
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.