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Mar 26, 2017 - stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination...
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Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana Somsiri Udompaisarn,† Dumrongkiet Arthan,‡ and Jamorn Somana*,† †

Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Department of Tropical Nutrition & Food Science, Faculty of Tropical Medicine, Mahidol University, Rachawithi Road, Bangkok 10400, Thailand



S Supporting Information *

ABSTRACT: 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: stevioside determination, enzymatic method, β-glucosidase, Stevia rebaudiana, method validation, stevioside



(HPLC),14 near-infrared spectroscopy (NIR),15,16 and mass spectrometry techniques.17−19 Among these, HPLC is the most well accepted method for simultaneous screening and quantitative determination of steviol glycosides in samples. However, there are several limitations to the use of HPLC for steviol glycosides analysis,20 and these include the time required for testing of each sample and the need for exclusive use of equipment and facilities resulting in a high cost for analysis. Mizukami et al. reported an enzymatic procedure using crude hesperidinase from Aspergillus niger for the quantitative analysis of stevioside in methanol extract of Stevia.21 Each stevioside molecule contains three molecules of glucose: one linked at C19 and two others linked at C-13 in the form of a sophorose, β1,2-D-glucopyranosyl moiety. Therefore, glucose liberation during enzymatic hydrolysis of stevioside can be utilized for quantitation of stevioside content. However, crude hesperidinase is not specific for hydrolysis of stevioside and can also degrade rebaudioside A. The amount of glucose produced from rebaudioside A is 3% of that obtained from stevioside, when both compounds are present in Stevia samples at equal concentrations. While the contribution of glucose from rebaudioside A could be factored into the calculation for stevioside content, estimation is difficult due to varying levels of rebaudioside A in Stevia samples. For accurate enzymatic determination of steviol glycosides, a novel enzyme that is highly specific for stevioside is required. The present study aims to develop a validated selective enzymatic method for stevioside quantitation.

INTRODUCTION Stevioside, 1, and rebaudioside A, 2, (Figure 1) are natural sweet compounds found at high concentrations in the leaves of Stevia rebaudiana Bert. They have been accepted as safe for human consumption; in addition to their use as sweeteners, these compounds exhibit pharmacological and therapeutic properties.1−6 The Stevia plant is native to the southeastern regions of South America and has a long history of use by local inhabitants. Moreover, Stevia can be cultivated in other parts of the world where climate conditions are suitable, such as tropical and subtropical regions in Asia, including Thailand. At present, the demand for stevioside for use as a natural sugar substitute is increasing.7 Furthermore, there is growing interest for the utilization of stevioside as a substrate for production of rubusoside, 3 (Figure 1), a natural solubilizing agent.8−11 Because of the difficulty of rubusoside extraction from its natural source, Rubus plant, alternative sources are in demand. In contrast to rubusoside, stevioside is abundant in Stevia plant and can be easily extracted.12 Therefore, use of stevioside as a raw material for rubusoside preparation is feasible. To respond to the increasing need for stevioside to feed the Stevia market as well as the rubusoside production industry, there is a continuing need to identify high steviosideproducing clones of the Stevia plant. A fast, safe, inexpensive, and easy method for monitoring stevioside content from a large number of samples is essential to screen and identify high stevioside producing clones. In addition, this methodology would have application in quality control procedures to determine the amount of stevioside in raw materials, samples during extraction, as well as the final Stevia sweetener products. Generally, common methods for measurement of steviol glycosides content in Stevia sample rely on standard chemical analyses such as high-performance thin layer chromatography (HPTLC), 13 high-performance liquid chromatography © 2017 American Chemical Society

Received: Revised: Accepted: Published: 3223

December March 23, March 26, March 26,

28, 2016 2017 2017 2017 DOI: 10.1021/acs.jafc.6b05793 J. Agric. Food Chem. 2017, 65, 3223−3229

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of (1) stevioside, (2) rebaudioside A, and (3) rubusoside. kanamycin. Colonies were subsequently examined by colony PCR with T7 promoter primer and reverse primer of the gene. The plasmid of positive PCR clones was extracted using Presto mini plasmid (Geneaid Biotech Ltd., New Taipei, Taiwan) and was sent for sequencing at Macrogen Inc. (Seoul, South Korea). The native nucleotide sequence of BT_3567 was submitted as GenBank accession number KU845310. The cloned BT_3567 gene was in frame with a C-terminal 6xHis tag in pET-28a. Protein Expression and Purification. The recombinant plasmid designated as pBT_3567His was transformed to E. coli Rosetta2 (DE3) expression strain. Transformants of pBT_3567His were inoculated into 10 mL of LB broth containing 34 μg/mL of chloramphenicol and 50 μg/mL of kanamycin and incubated at 37 °C for 16−18 h. Ten milliliters of the preculture was then inoculated into 800 mL of LB broth and allowed to grow until the OD600 nm reached 0.6−0.8. Cells were induced with 250 μM isopropyl β-1thiogalactopyranoside and incubated for an additional 16−18 h at 25 °C with shaking. Cells were harvested and resuspended in 10 mL of lysis buffer (50 mM of sodium acetate buffer (pH 6.0), 0.2 mg/mL of lysozyme, 0.1 μg/mL of RNase A, 1 U/mL of DNase I, 50 mM of MgCl2, and 1 mM of phenylmethylsulfonyl fluoride) and incubated at 37 °C for 10 min. Cell debris was removed by centrifugation, and the crude protein extract was loaded onto an affinity purification column containing with 2 mL of Ni-NTA agarose resin (1 × 2 cm2), equilibrated with 50 mM of sodium acetate buffer pH 6.0 containing 300 mM of NaCl (buffer A). A stepwise gradient of 75, 100, and 150 mM imidazole in buffer A was used to elute rBT_3567. The eluted fractions containing enzymatic activity were concentrated, bufferexchanged using Amicon Ultra Centrifugal Filters (Merck Millipore, Billerica, MA), and analyzed by 10% SDS-PAGE with Coomassie blue R-250 staining. Isolation and Identification of the Stevioside-Hydrolytic Product. Partially purified stevioside (10 mg/mL) was incubated with the purified rBT_3567 in 50 mM sodium acetate buffer (pH 6.0) at 37 °C for 24 h. The reaction was applied to TLC silica gel 60 F254 plates with a mobile phase consisting of ethyl acetate/isopropanol/acetone/ water (53:30:2:15, v/v). TLC plate was removed from the chamber after mobile phase migrated up to about 90% of TLC plate height, and then the TLC plate was dried in chemical fume hood. The TLC plate was partially cut and sprayed with 5% sulfuric acid followed by heating at 100 °C until the brown spots of standards and the product were observed. The region of the TLC plate containing the hydrolyzed product was scraped, and material was eluted using methanol extraction. The product was , and bright-yellow crystals were obtained. Approximately 5 mg of the compound was used for structure elucidation by mass spectrometry and NMR analysis. Mass analysis utilized electrospray ionization-time of fight (ESITOF) spectroscopy with a MicrOTOF (Bruker, Bremen, Germany) in the positive mode. Parameters for mass acquisitions are as follows: scan 50 m/z to 3000 m/z, capillary exit 180 V, hexapole RF 400 V, skimmer 1 70 V, hexapole 1 25 V, corrector fill 50 V, pulsar pull 337 V, pulsar push 337 V, reflector 1300 V, flight tube 9000 V, and detector

Previously, a group of human intestinal microorganisms, including Bacteroidaceae, bifidobacteria, clostridia, coliforms, enterococci, and lactobacilli, were examined for steviol glycosides degradation activity.22 The results demonstrated that Bacteroidaceae exhibited the ability to degrade both rebaudioside A and stevioside. However, in this study, protein extract from Bacteroides thetaiotaomicron HB-13 was found to exhibit only stevioside hydrolytic activity, but rebaudioside A degradation was not observed. The enzyme from B. thetaiotaomicron HB-13 involved in stevioside hydrolysis has not been previously described. To identify the enzyme responsible, several β-glucosidase genes from B. thetaiotaomicron HB-13 were cloned, expressed, and tested for stevioside hydrolysis. The β-glucosidase encoded by BT_3567 gene was identified as responsible for hydrolysis of stevioside. Therefore, we expressed and purified recombinant BT_3567 (rBT_3567) and analyzed its enzymatic properties as part of the development of an enzymatic method for stevioside determination. Finally, the method utilizing rBT_3567 was validated for affordability, specificity, accuracy, and robustness in the determination of stevioside in crude Stevia extracts and other stevioside containing products.



MATERIALS AND METHODS

Chemicals. Purified stevioside and rebaudioside A and others Stevia materials were kindly provided by Sugavia Co., Ltd. (Nakhonratchasima, Thailand). The column used was 3 μm, Acclaim 120 C18, 100 mm × 4.6 mm (Thermo Fisher Scientific, Waltham, MA). Precoated silica gel 60 F254 plate was purchased from Merck KGaA Co., Ltd. (Darmstadt, Germany). Ni-NTA His Bind resin for protein purification was obtained from Merck Millipore Headquarters (Billerica, MA). Synthesized primers were ordered from Pacific Science Co., Ltd. (Bangkok, Thailand). Reagents, buffers, and enzymes for molecular biology applications were from New England Biolabs Inc. (Ipswich, MA). Bacteroides thetaiotaomicron HB-13 culture stock was kindly provided by Dr. Kwanrawee Sirikanchana (Chulabhorn Research Institute, Bangkok, Thailand). Cloning and Vector Construction. The full-length gene encoding BT_3567 was amplified from gDNA of B. thetaiotaomicron HB-13 by polymerase chain reaction (PCR). Primers were designed based on the sequence of BT_3567 β-glucosidase gene from the reference strain, B. thetaiotaomicron VPI-5482 (GenBank accession number NC_004663.1). The BT_3567 forward primer was 5′TATACCATGGCGATGATAAATAAGAAAATATTTTTTTC-3′ (NcoI site underlined) and the reverse primer was 5′-CGAATTCTTTAGCGTGAATCGTG-3′ (EcoRI site underlined). These primers introduced NcoI and EcoRI restriction sites for cloning the gene product into the pET-28a expression vector and removed the stop codon. After PCR, amplification products were digested and ligated into pET-28a. The ligation reaction was transformed into Escherichia coli DH5α strain, and clones were screened on media containing 3224

DOI: 10.1021/acs.jafc.6b05793 J. Agric. Food Chem. 2017, 65, 3223−3229

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Journal of Agricultural and Food Chemistry TOF 2295 V. Data were acquired using Bruker Daltonics Data Analysis software version 3.3. For NMR analysis, the 1H and 13C NMR spectra were recorded with a Bruker Ascend 400 MHz NMR spectrometer (Bruker, Bremen, Germany) at 25 °C. Hydrolytic Activity Assays. The activity of rBT_3567 was measured using 2 mM p-nitrophenyl β-D-glucopyranoside (pNPG) or 2 mM of stevioside as substrates. For determination of p-nitrophenol (pNP) levels, 100 μL of enzyme solution was incubated with 700 μL of substrate solution in 50 mM sodium acetate buffer (pH 6.0) under the indicated conditions. The reaction was stopped by addition of 200 μL of 0.5 M Na2CO3. The pNP product was measured at 405 nm with a UV-2501 spectrophotometry (Shimadzu, Duisuburg, Germany). Standard pNP was prepared using serial dilution with final concentration ranging from 5−40 μmol/L. A standard curve generated with pNP was used to quantitate the products of enzyme hydrolysis. For stevioside analysis, 100 μL of enzyme solution was incubated with 700 μL of substrate solution in 50 mM sodium acetate buffer (pH 6.0) with described conditions. The reaction was stopped by heating at 80 °C for 10 min. Liberated glucose was subsequently determined using a coupling reaction of glucose oxidase in the presence of peroxidase reaction reagent (1 mg/mL glucose oxidase, 0.1 mg/mL of peroxidase, 0.6 mg/mL of ο-dianisidine, in 50 mM of sodium acetate buffer, pH 6.0). The reaction was incubated at 37 °C for 20 min, and the resulting colored peroxidase product, oxidized ο-dianisidine color, was enhanced with 200 μL of 80% (v/v) sulfuric acid. The stable oxidized ο-dianisidine was examined by measuring absorbance at 540 nm. Standard glucose was prepared using serial dilution with final concentration ranging from 2−32 μmol/L. A standard curve of glucose was used to quantitate the product. The amount of enzyme required to release 1 μmol per minute of either pNP or glucose was defined as one unit (U). Effects of pH and Temperature on Enzyme Activity. To determine the optimum pH, 100 μL of enzyme solution was incubated with 700 μL of substrate solution at 45 °C for 10 min in 50 mM of Britton-Robinson buffer (pH 3−12). The buffer consists of a mixture of 0.05 M of H3BO3, 0.05 M of H3PO4 and 0.05 M of CH3COOH that has been titrated to the desired pH with 0.25 M of NaOH. In addition, the enzyme was preincubated at room temperature for 2 h in 50 mM of Britton-Robinson buffer (pH 3−12), and the remaining enzyme activity was measured to determine the pH stability. The optimum temperature was determined by preheating the substrate solution for 2 min before incubating the enzyme at 25−65 °C for 10 min in 50 mM of sodium acetate buffer (pH 6.0). The thermal stability was ascertained by preincubating the enzyme at 25−65 °C for 0−160 min in the same buffer, and the remaining enzyme activity was measured. Effect of Different Metal Ions on Enzymatic Activity. The enzyme in 50 mM sodium acetate buffer (pH 6.0) was incubated with 1 mM BaCl2, CaCl2, CoCl2 CuCl2, KCl, FeCl3, MgCl2, MnCl2, NaCl, ZnCl2, HgCl2, or EDTA for 30 min at room temperature. Then 2 mM of pNPG was added to the reaction. After incubation at 45 °C for 10 min, the reaction was stopped, and the hydrolysis product, pNP, was then measured. Substrate Specificity Assays. The substrate specificity of the rBT_3567 was evaluated by incubating the enzyme with 2 mM of stevioside, rebaudioside A, pNP-β-D-glucopyranoside, pNP-α-Dglucopyranoside, pNP-β-D-galactopyranoside, pNP-N-acetyl-β-D-glucosaminide, amygdalin, arbutin, octyl-β-D-glucopyranoside, cellobiose, lactose, sucrose, trehalose, and isomaltose in 50 mM sodium acetate buffer (pH 6.0) at 45 °C for 10 min. The amounts of hydrolytic product, pNP, or glucose were determined as previously indicated. The rates of hydrolysis on different substrates were determined as relative values of the initial rate of hydrolysis obtained with stevioside. Kinetic Analysis of Recombinant BT_3567. The initial velocities of rBT_3567 were determined using various concentrations of 0.1−5 mM pNPG as substrate and were measured at 45 °C and pH 6.0. The kinetic study of rBT_3567 with SV was performed using SV concentrations ranging from 0.1−6 mM at 45 °C and pH 6.0. Plotting and data analysis were performed using Prism 6.0 software (GraphPad

Software, San Diego, CA). Michaelis−Menten kinetic parameters for rBT_3567 were determined by fitting data to nonlinear regression analysis. Crude Stevia Extract Preparation. Dry leaf samples from several clones of the Stevia plant were provided by Sugavia Co. Ltd. (Nakhonratchasima, Thailand). To prepare crude Stevia extract, dried leaves were first crushed into small pieces and ground to a powder. The leaf powder, 5 mg, was mixed with 1 mL of deionized water and then heated at 70 °C for 10−20 min. Crude extract was removed from insoluble material by centrifugation 13 000g for 1 min. The supernatant was collected, filtered using 0.22 μM syringe filter, and kept at −20 °C until needed for experiments. Method Optimization in Crude Stevia Extract. Cosubstrate concentrations and pH were optimized for the analysis of glucose in crude extracts. Three different cosubstrates: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-dianisidine, and 3,3′diaminobenzidine (DAB) were tested using buffers containing glucose oxidase and peroxidase reagent solution (GPO) at pH 3.0, 4.0, and 6.0. The enzymatic reagent, glucose oxidase, and peroxidase (690 μL) were prepared by mixing of 10 μL of glucose oxidase (100 mg/mL), 10 μL of peroxidase (10 mg/mL) with 10 μL of different cosubstrates: ABTS (10 mg/mL), o-dianisidine (6 mg/mL), or DAB (10 mg/mL) in 660 μL of 50 mM sodium acetate buffer at pH 3.0, 4.0, and 6.0. Crude extract samples were prepared using 100 μL of crude Stevia extract (5 mg/mL) mixed with or without 10 μL of standard glucose (3 mM). Crude extract without glucose added was used as a sample blank. Glucose standard samples at concentrations between 5 and 160 μmol/ L were prepared in 50 mM sodium acetate buffer (pH 6.0) with a final volume of 110 μL. Sodium acetate buffer (pH 6.0) without glucose addition was used as a sample blank. Crude extract and glucose standard samples were incubated with 690 μL of the GPO reagent at 37 °C for 1 h. The reactions were stopped by addition of 200 μL of 80% (v/v) sulfuric acid and mixed gently and thoroughly. The colored compounds from each reaction using ABTS, o-dianisidine, and DAB as a cosubstrate were measured using a UV−vis spectrophotometer at absorbances of 405, 540, and 450 nm, respectively. To test the effect of crude Stevia concentrations on the glucose assay, crude extract (5 mg/mL) was diluted in 50 mM sodium acetate buffer (pH 6.0) to make concentrations ranging from 0.5−2.5 mg/mL. One-hundred microliters of crude Stevia extract (at each concentration) was mixed with 10 μL of glucose standard (3 mM) and then incubated with 690 μL of GPO reagent (pH 3.0) containing DAB (GPO−DAB) at 37 °C for 1 h. The reaction was stopped by addition of 80% (v/v) sulfuric acid, as described above, and the product of oxidized DAB was measured at 450 nm. Time Course Analysis. To monitor stevioside hydrolysis over time, 400 μL of crude extract (5 mg/mL) was incubated with or without 40 μL of rBT_3567 solution (0.05 U) in 50 mM of sodium acetate buffer (pH 6.0) at 37 °C for 5, 10, and 15 min. The reactions were then stopped by heating at 70 °C for 5 min. Insoluble materials were removed by centrifugation at 13 000g for 10 min. The amount of stevioside in samples incubated with rBT_3567 was monitored using an HPLC analysis. The HPLC quantitation was performed using a Waters HPLC system equipped with a model e2695 separation module system, 2487 dual wavelength detector (Waters Corporation, Milford, MA) and Acclaim 120 C18 column (Thermo Fisher Scientific, 4.6 mm × 100 mm, 3 μm). The temperature of the column oven was 60 °C. The mobile phase was an isocratic mixture of 68% water and 32% acetonitrile. Ten microliters of crude Stevia samples or standard compounds (purified stevioside, rebaudioside A and rubusoside) was injected with a flow rate of 0.75 mL/min. The UV absorption was measured at 210 nm. The time for glucose analysis was evaluated using 110 μL of crude Stevia extract incubated with or without rBT_3567 for 15 min from the previous experiment. Experimental procedures for the determination of glucose were performed to test the effect of crude Stevia extract concentration on glucose analysis as described earlier. Determination of Stevioside in Crude Extract and Products of Stevia. In this experiment, analysis of stevioside content in crude Stevia extract and Stevia products, including steviol glycosides extract 3225

DOI: 10.1021/acs.jafc.6b05793 J. Agric. Food Chem. 2017, 65, 3223−3229

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Journal of Agricultural and Food Chemistry powder and syrup form, was performed. Crude Stevia extract was prepared as described in the Crude Stevia Extract Preparation section. Stevia products were dissolved and diluted 50 mM sodium acetate buffer (pH 6.0) to make a final concentration, which corresponded to the standard curve of each individual experiment. Standard stevioside was prepared using serial dilutions with final concentrations ranging from 0.25−4 mg/mL. HPLC analysis was performed using 10 μL of crude Stevia samples, Stevia product samples, or stevioside standards. A standard curve for stevioside was generated to quantitate the amount of stevioside in Stevia samples. HPLC analysis was performed as described previously. Enzymatic stevioside analysis was performed using 100 μL of crude Stevia extract and Stevia products samples incubated with or without 10 μL of rBT_3567 solution (0.05 U) at 37 °C for 15 min. Samples were heated to inactivate the enzymes and then clarified by centrifugation at 13 000g for 10 min. The supernatant was transferred to a new tube followed by the addition of 690 μL of GPO−DAB. After incubation at 37 °C for 45 min, the reaction was stopped with 80% sulfuric acid and color change was monitored using spectrophotometry. Samples without rBT_3567 were used as individual sample blanks. In this assay, five standard glucose solutions with concentrations between 5 and 160 μmol/L were prepared as described previously to generate a standard curve for measurement of liberated glucose in Stevia samples.

Mass spectrometric analysis of the purified hydrolysis product produced a signal at m/z corresponding to [M+Na]+, giving a molecular weight of 665.31. According to analysis of NMR spectra and comparison with previous reports,11,23 the product here was identified as rubusoside. In summary, rBT_3567 specifically cleaved the β-1,2-Dglucopyranosyl linkage of the sophorose moiety at C-13 of stevioside to yield one mole of rubusoside and glucose. Biochemical Characterization of rBT_3567 and Kinetic Analysis. The effect of pH and temperature on rBT_3567 hydrolysis activity is shown. Optimum pH and temperature for the hydrolytic activity of pNPG and stevioside by rBT_3567 were 6.0 and 45 °C, respectively. The enzyme maintained more than 80% of its maximal activity in a pH range from 6.0−8.0. The enzyme is thermally stable below a temperature of 25 °C and was completely inactivated after heating at 65 °C for 5 min. Strong inhibitors of rBT_3567 are HgCl2 and CuCl2, which completely inhibited the activity of rBT_3567. In addition, ZnCl2 also inhibited the hydrolysis activity of the enzyme with residual activity measured as 43% remaining. Others metal ions: Na+, Mn2+, Mg2+, K+, Fe3+, Co2+, Ca2+, Ba2+ did not significantly reduced activity of rBT_3567. The substrate specificity of rBT_3567 was determined using different substrates. Hydrolysis activity of rBT_3567 was apparent with stevioside and pNPG, but little degradation was observed with pNP-α-D-glucopyranoside, pNP-β-D-galactopyranoside, octyl-β-D-glucopyranoside, amygdalin, lactose, and rebaudioside A. In contrast, no hydrolysis activity on pNP-N-acetyl-β-D-glucosaminide, arbutin, cellobiose, sucrose, trehalose isomaltose was detected. The kinetic parameters of rBT_3567 were also examined. Optimization of Glucose Analysis in Crude Stevia Extracts. Generally, crude plant extracts consist of a mixture of complex compounds, which could interfere with the enzymatic determination of glucose. Therefore, the reaction conditions were first optimized to limit the effect of components in the extract. Initially, the effect of cosubstrate and pH on the assay was determined. Many types of cosubstrates have been reported to be used in the assay of glucose.24−30 In this experiment, cosubstrates ABTS, o-dianisidine, and DAB were prepared in three buffers at pH 3.0, 4.0, and 6.0, containing the glucose oxidase and peroxidase reagent (GPO). The percentage of glucose recovery was calculated to monitor the accuracy of glucose analysis. As shown in Table 1, glucose analysis from crude Stevia extract, performed with GPO (pH 3.0) and DAB, gives the best glucose recovery, reaching nearly 100%, indicating the accuracy of the downstream assay. Therefore, further analysis of glucose in both crude Stevia extracts and Stevia products was performed in GPO at pH 3.0 containing DAB (GPO−DAB). The optimal extract concentration for glucose determination was next evaluated. The analysis of glucose in Stevia extract was performed by varying the final concentration from 0.5 to 2.5 mg/mL. The result showed that a final concentration at 0.5 mg/mL of crude Stevia extract gave the highest percentage recovery, at nearly 100%, in the glucose determination assay. After the concentration of extract was raised above 0.5 mg/mL, the glucose recovery percentage diminished indicating an increase in interference from components of the Stevia plant extract. Hence, the concentration of crude Stevia extract for use in the enzymatic glucose assay should be restricted to 0.5 mg/ mL. In contrast, the hydrolysis reaction can be performed with a final concentration of crude Stevia extract of up to 50 mg/mL



RESULTS AND DISCUSSION Cloning, Expression, and Purification of rBT_3567. Recombinant BT_3567 with a polyhistidine tag was successfully cloned into the pET28a expression vector. The recombinant protein was subsequently expressed in using the Rosetta 2 (DE3) E. coli strain. Using a Ni2+ Sepharose column, rBT_3567 was successfully purified to homogeneity. The purified rBT_3567 gave a single band (Lane 4) with an apparent molecular weight (Mr) of 86 kDa (Figure 2) as analyzed by 10% SDS-PAGE.

Figure 2. SDS-PAGE analysis of rBT_3567 expression. Lane 1, molecular weight standard; lane 2, crude extract of noninduced cells; lane 3, crude extract of the cells induced by IPTG; lane 4, purified rBT_3567.

Isolation and Identification of Stevioside-Hydrolysis Products. To prepare hydrolysis products, the enzyme− substrate reaction was performed by incubation of rBT_3567 with partially purified stevioside. At the end of hydrolysis reaction, stevioside was monitored using TLC and HPLC assay to ensure that it was completely hydrolyzed, as shown in Figure 3. Products were subsequently isolated from the reaction mixture using a preparative TLC procedure, for further analysis. 3226

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Figure 3. Result of enzymatic stevioside hydrolysis using (A) TLC and (B) HPLC analysis.

well as other components from the Stevia extract, did not have significant inhibitory effect on enzymatic stevioside determination. Overall, these finding establish the selectivity, accuracy, and robustness of the assay. Precision and Accuracy. Finally, within day and between days precision of the assay was examined and judged by the coefficient of variation (CV) and accuracy range. As shown in Table 2, the CV result of within-day precision was 1.86−3.21% with stevioside content ranging from 40−70 μmol/L. The accuracy ranges for stevioside at concentrations of 40, 50, and 70 μmol/L were 98−105%, 97−105%, and 98− 104%, respectively. The CV result of between days precision was 1.80−3.28% with stevioside content ranging from 47−77 μmol/L. The accuracy ranges for stevioside at concentrations of 47, 58, and 77 μmol/L were 96−100%, 98−105%, and 95− 103%, respectively. The CV percentage and accuracy range for within day and between days assays are acceptable criteria for method validation according to ICH guidelines.31 Therefore, this method is acceptable as an alternative procedure for determination of stevioside. Determination of Stevioside in Various Stevia Samples. Beyond crude Stevia extracts, in which SV was accurately and precisely determined, the amount of SV in others Stevia samples was also tested to verify the usefulness of the enzymatic assay. Finished Stevia products obtained from Sugavia Co., Ltd. (Nakhonratchasima, Thailand) such as steviol glycosides powder (n = 4) and syrup (n = 4) were examined for stevioside content by both enzymatic assays and the reference method, HPLC. The similarity for stevioside content detected from these methods can be evaluated by calculating the relative error percentage (%RE). The results of relative error percentage were satisfactory established, as shown in Table 3. They ranged from 2.33−4.97 and 0.15−3.62% for the assay of stevioside from steviol glycosides powder and syrup samples, respectively. Hence, the enzymatic assay, developed in this study, can also be applied to the determination of stevioside in finished Stevia products. However, additives in others products have the potential to impede the enzymatic analysis and should be evaluated for interference before using the assay. In conclusion, the enzymatic stevioside determination developed in this study is a simple, eco-friendly technique that can be utilized in a general laboratory setting without the

Table 1. Effect of Cosubstrate and pH on Glucose Assay of Crude Stevia Extract Sample cosubstrates ABTS

o-dianisidine

DAB

a

pH 3 4 6 3 4 6 3 4 6

% recoverya (mean ± SD) 1.99 2.45 2.57 19.79 23.71 7.34 100.58 85.87 71.42

± ± ± ± ± ± ± ± ±

0.085 0.087 0.004 1.11 0.26 0.19 1.39 0.97 1.44

% Recovery = (measured-glucose value/added-glucose value) × 100.

without any detectable inhibitory effects from the Stevia plant matrix on rBT_3567 activity (data not shown). Time Course of Analysis. To estimate time course for the stevioside hydrolysis step, crude extracts containing 150 μmol/ L of stevioside were incubated with rBT_3567 (0.05 U), and the amount of stevioside remaining in the extract was monitored using HPLC analysis. Stevioside in crude extract sample was completely hydrolyzed after 15 min, as shown in Figure 4. To determine the time required for the completion of the glucose assay, the colorimetric measurement of oxidized DAB was performed. A glucose standard sample (20 μmol/L) and crude Stevia extract samples, with and without stevioside hydrolysis, were incubated with GPO−DAB. After 40 min of incubation, the reaction had reached completion, as judged by no additional increase in the absorbance at 450 nm. Therefore, the time needed for stevioside determination, including stevioside hydrolysis and glucose assay processes in crude extract, was estimated to be 1 h. Validation of Enzymatic Stevioside Determination. Intermethod Comparison Data. To validate the enzymatic determination of stevioside, the selectivity and precision of the method were evaluated with 16 different Stevia plant clones. This analysis compared results using enzymatic stevioside determination and those from HPLC analysis. The result showed a good correlation between the two methods and statistical parameters were: y = 0.9733x + 1.4712 (r2 = 0.9629). This result demonstrates that additional steviol glycosides, as 3227

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Figure 4. Time course analysis. (A) HPLC chromatogram of standard compounds; rebaudioside A and stevioside. (B) HPLC chromatogram of standard rubusoside. (C) HPLC profile of crude Stevia extract samples (containing 150 μmol/L of stevioside). (D−F) HPLC profile of crude Stevia extract after incubation with rBT_3567 (0.05U) for 5, 10, and 15 min, respectively.

need for sophisticated instruments. Additionally, this method should be applicable for use in high-throughput analysis. Use of multiwell plates in conjunction with a microplate reader would allow for time saving and simple analysis of stevioside content in a large number of samples. The estimated cost of analysis in 96-well plate in our facility situation for materials and facility service is about 0.22 USD per sample compared with 0.71 USD per sample for HPLC. The reduction cost of analysis at the same condition is more than three-times by this method compare to HPLC and might even be cheaper if the enzyme is commercialized. Enzymatic stevioside determination is also suitable for screening for high stevioside-produced plant clones as well as quality control of Stevia products. The future development of an assay for enzymatic rebaudioside A measurement in crude extract would also beneficial and aid in the analysis of the two major steviol glycosides in S. rebaudiana.

Table 2. Analysis of within Day and between Days Precision within day precision (n = 12) stevioside (μmol/L) original (mean ± SD) 29.37 ± 1.00

38.99 ± 1.71

detected (mean ± SD)

added

% CVa

% accuracy rangeb

10 40.21 ± 1.10 2.74 20 50.25 ± 1.61 3.21 40 70.43 ± 1.31 1.86 between days precision (n = 12) 47.98 ± 0.86 58.67 ± 1.34 77.97 ± 2.56

10 20 40

98−105 97−105 98−104

1.80 2.29 3.28

96−100 98−105 95−103

% Coefficient variation = (SD/mean) × 100. b% Accuracy range = [measured value/(original value + added value)] × 100.

a

Table 3. Comparative Result of Determination of Stevioside Content (%) in Finished Stevia Powder and Syrup Products detected stevioside (%)a HPLC method samples powder (n = 4)

syrup (n = 4)

mean ± SD

% CV

± ± ± ± ± ± ± ±

0.88 3.17 2.55 1.07 2.53 0.46 0.60 2.47

25.53 22.26 21.88 33.30 29.11 25.42 27.86 29.41

0.22 0.70 0.56 0.35 0.72 0.11 0.16 0.74



enzymatic method mean ± SD

% CV

% REb

± ± ± ± ± ± ± ±

2.28 0.98 2.15 1.29 2.27 2.02 2.64 1.85

3.48 2.33 4.97 4.04 2.79 0.15 1.68 3.62

26.42 22.80 20.79 34.65 29.93 25.46 28.33 30.48

0.60 0.22 0.44 0.44 0.67 0.51 0.74 0.56

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05793. Effect of pH and temperature on rBT-3567 enzymatic activity; time course for glucose assay; correlation plot and statistical parameters; comparative 13C NMR spectral data of hydrolytic product from stevioside; effects of metal ions on activity of rBT_3567; substrates specificity assay; kinetic parameters of rBT_3567; effect of crude extract concentration on glucose assay on crude Stevia extract sample (PDF)

a

Detected stevioside (%) was presented in term of weight per weight (w/w). bRelative error = |(enzymatic method value − HPLC method value)/HPLC method value| × 100. 3228

DOI: 10.1021/acs.jafc.6b05793 J. Agric. Food Chem. 2017, 65, 3223−3229

Article

Journal of Agricultural and Food Chemistry



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +66 2 201 5468. Fax: +66 2 354 7174. ORCID

Jamorn Somana: 0000-0002-2883-0693 Funding

This work was partially supported by Agricultural Research Development Agency (Public Organization, Thailand) Grant No. CRP5605011990 and the Dean’s Research Fund, Faculty of Tropical Medicine 2011, Mahidol University. Notes

The authors declare no competing financial interest.



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DOI: 10.1021/acs.jafc.6b05793 J. Agric. Food Chem. 2017, 65, 3223−3229