Determination of Stevioside and Rebaudioside A in <i>Stevia rebaudiana</i> Bertoni Leaves Using near Infrared Spectroscopy and Multivariate Data Analysis

Abstract

Rebaudioside A and stevioside are abundant steviol glycoside contained in Stevia rebaudiana leaves. These components are widely used as a natural sweetener. The objective of this study was to develop rapid determination method of stevioside, and rebaudioside A in S. rebaudiana leaves using near infrared trans-reflectance spectroscopy (NIRS) combined with multivariate analysis. The reference method used was HPLC. A prediction model was developed using partial least square (PLS) regression. Calibration parameters were calculated based on a calibration set of various stevioside, rebaudioside A from 23 samples. Performance of PLS model was assessed in term of optimum determination coefficient (R 2 ), and minimum root mean square error of cross-validation (RMSEV). Validation of PLS model was performed using cross-validation and leave one out calibration of PLS component. Rebaudioside A has well PLS model in wavenumber region of 4100-5100 cm -1 , and stevioside determination using difference wavenumber region of 47605016 cm -1 . PLS model for total (sum of stevioside and rebaudioside A content) was exploited in wavenumber region of 45684928 cm -1 . NIRS in combination with multivariate data analysis of PLSR can be applied as a rapid method for determining rebaudioside A and the total amount of steviol glycosides in S. rebaudiana leaves. ABSTRAKRebaudiosida A dan steviosida adalah senyawa steviol glikosida dominan yang terkandung dalam daun Stevia rebaudiana. Senyawa tersebut banyak digunakan sebagai pemanis alami. Tujuan penelitian ini adalah mengembangkan metode penetapan kadar setiosida dan rebaudiosida A dalam daun S. rebaudiana menggunakan spektroskopi Near Infrared (NIRS) trans-reflektan yang dikombinasi dengan analisis multivariat. Sebagai metode referens adalah HPLC. Model prediksi yang dikembangkan menggunakan regresi Partial Least Square (PLS). Parameter kalibrasi dihitung berdasarkan sejumlah 23 sampel kalibrasi set yang bervariasi kandungan steviosida dan rebaudiosida A-nya. Model PLS yang optimal ditentukan berdasarkan koefisien determinasi (R 2 ) dan root mean square error of cross-validation (RMSEV) yang minimum. Validasi model menggunakan model cross validation dan leave one out dari komponen PLS. Model PLS rebaudiosida A dikembangkan pada daerah bilangan gelombang 4100-5100 cm -1 dan steviosida pada daerah bilangan gelombang yang lain yaitu 47605016 cm -1 . Model PLS untuk jumlah steviol glikosida dominan dibangun pada daerah bilangan gelombang 45684928 cm -1 . Metode spektroskopi NIR yang dikombinasi dengan analisis multivariat dapat digunakan sebagai metode penetapan kadar steviosida dan rebaudiosida A serta jumlah totalnya dalam daun S. rebaudiana secara cepat.

Comparison of uridine diphosphate-glycosyltransferase UGT76G1 genes from some varieties of Stevia rebaudiana Bertoni

“…Different techniques are used to determine the glycoside content in plants ( e.g ., gas chromatography, HPLC, LC-MS, infrared spectroscopy). HPLC is a reliable method that has been used to determine the composition of Stevia rebaudiana 10–13 . Plant UDP-glycosyltransferases (UGTs) are a unique group of enzymes that transfer sugar residue from an activated donor to an acceptor molecule 14–19 .…”