The document describes the enzymatic synthesis of glucosyl rebaudioside A from rebaudioside A using recombinant dextransucrase from Leuconostoc lactis. Glucosyl rebaudioside A was produced with 86% yield and purified. It showed improved solubility and stability in acidic and thermal conditions compared to rebaudioside A, indicating its potential as a highly pure and stable sweetener. The structure of glucosyl rebaudioside A was characterized through NMR and LC-MS analysis.

1. Food
Engineering,
Materials
Science,
&
Nanotechnology
Enzymatic Synthesis of Glucosyl Rebaudioside A
and its Characterization as a Sweetener
So-Hyeon Lee, Jin-A Ko, Hae-Soo Kim, Min-Ho Jo, Joong-Su Kim, Doman Kim, Jeong-Yong Cho, Young-Jung Wee,
and Young-Min Kim
Abstract: Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001
in Escherichia coli BL21 (DE3), forming single O-α-D-glucosyl-(1
→6
) rebaudioside A with yield of 86%. O-α-D-
glucosyl-(1
→6
) rebaudioside A was purified using HPLC and Diaion HP-20 and its properties were characterized
for possible use as a food ingredient. Almost 98% of O-α-D-glucosyl-(1
→6
) rebaudioside A was dissolved after
15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O-α-
D-glucosyl-(1
→6
) rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus,
O-α-D-glucosyl-(1
→6
) rebaudioside A could be used as a highly pure and improved sweetener with high stability in
commercial drinks.
Keywords: glucosylation, glucansucrase, Leuconostoc lactis, Rebaudioside A, sweetener
PracticalApplication: The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation.
Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks.
This method can be applied to obtain high value-added bioactive compounds by enzymatic modification.
Introduction
Steviol glycosides are plant-derived zero-calorie sweeteners
from Stevia rebaudiana BERTONI (Philippe, De Mey, Anderson
Ajikumar, 2014). Leaf extract of S. rebaudiana BERTONI con-
tains more than 30 steviol glycosides (Chaturvedula et al., 2011;
Wolwer-Rieck, Tomberg Wawrzun, 2010). Stevia leaf extract
also contains various ent-kaurene-type diterpene glycosides; re-
baudioside C–E, dulcoside A, rubusoside, stevioside, and rebau-
dioside A as major components. Steviolbioside and rebaudioside B
are obtained by partial degradation during the extraction and pu-
rification process (Makapugay, Nanayakkara Kinghorn, 1984).
Commercial production of stevioside began in the 1970s. Since
then, stevioside has been mainly used as a cheap sweetener in
seasonings, salted foods, and pickles (Ko et al., 2012). However,
its bitter aftertaste restricts its use for human consumption. It also
limits its application in other food and pharmaceutical products.
Many researchers have tried to modify glucosylation at steviol C-
13 hydroxyl group and/or C-19 carboxylic acid of stevioside to
enhance its sweetness (Chaturvedula Prakash, 2011a, 2011b).
Among major components in Stevia, rebaudioside A is a target
compound, because it has an additional glucose attached to the
JFDS-2019-0492 Submitted 4/5/2019, Accepted 8/24/2019. Authors Lee, Kim,
Jo, Cho, and Kim are with Dept. of Food Science Technology, Chonnam National
Univ., Gwangju 61186, Republic of Korea. Author Ko is with Radiation Breeding
Research Center, Advanced Radiation Technology Inst., Korea Atomic Energy Resea-
rch Inst., Jeongeup, Republic of Korea. Author Kim is with Bio-industrial Pro-
cess Research Center, Korea Research Inst. of Bioscience and Biotechnology, Jeongeup
56212, Republic of Korea. Author Kim is with Research Inst. of Food Industrial-
ization, Inst. of Green Bio Science Technology, Seoul National Univ., Pyeongchang
25354, Korea Author Wee is with Dept. of Food Science and Technology, Yeungnam
Univ., Gyeongsan Gyeongbuk, 38541, Republic of Korea. Direct inquiries to author
Cho, Wee, and Young-Min Kim (E-mail: jyongcho17@jnu.ac.kr; yjwee@ynu.ac.kr;
u9897854@jnu.ac.kr).
Authors So-Hyeon Lee and Jin-A Ko contributed equally to this work.
C-13 hydroxyl position of stevioside, making it sweeter and more
pleasant-tasting than stevioside (Brandle, Starratt Gijzen, 1998).
Some researchers have tried to synthesize rebaudioside A from
stevioside using microbial enzymes such as extracellular enzymes
from Actinomycete Kusakabe Watanabe, Morita, Terahara Mu-
rakami, 1992), alternansucrase from Leuconostoc citreum SK24.002
(Musa, Miao, Zhang Jiang, 2014), dextransucrase from L. cit-
reum (Ko et al., 2012), dextrin-dextranse from Acetobacter capsula-
tus (Yamamoto, Yoshikawa Okada, 1994), β-fructofuranosidase
from Arthrobacter sp. (Ishikawa Kitahata, Ohtani, Ikuhara Tanaka,
1990), cyclomaltodextrin glucanotransferase from Bacillus mega-
terium (CGTase; Darise et al., 1984; Jaitak et al., 2009; Li, Li,
Xiao Xia, 2013), and β-glucosidase from Streptomyces W19-1
(Kusama, Kusakabe, Nakamura, Eda Murakami, 1986). More-
over, it has shown that plant-derived glucosyltransferases such as
UGT76G1 (Wang et al., 2016) can insert and produce rebau-
dioside A from stevioside in Saccharomyces cerevisiae (Olsson et al.,
2016; Li et al., 2016) and Escherichia coli system (Wang et al., 2016).
Production of rebaudioside I, rebaudioside D, and glucosyl rebau-
dioside A from rebaudioside A by enzymatic biotransformation
has also been reported (Gerwig, Te Poele, Dijkhuizen Kamer-
ling, 2017; Te Poele et al., 2018; Chen et al., 2018). Lactobacillus
reuteri 180 glucansucrase can transferred several glucosyl residues
from one moiety to nine moieties into rebaudioside A from sucrose
(Gerwig et al., 2017). Although there have been many biotransfor-
mation trials for stevioside or rebaudioside A, cyclomaltodextrin
glucanotransferase is the most well-known enzyme for stevioside
derivatives and the sole one used in industrial production of enzy-
matically modified stevioside. To elucidate the structure-sweetness
relationship, the stevioside or rebaudioside A derivatives produced
by cyclomaltodextrin glucanotransferase, glucansucrase from L. cit-
reum (Ko et al., 2012), β-fructofuranosidase, or glucansucrase from
Lactobacillus returi by transglucosylation (Te Poele et al., 2018).
However, the structure-sweetness relationship of various steviol
derivatives remains to be elucidated.
C
2019 Institute of Food Technologists R
doi: 10.1111/1750-3841.14821 Vol. 0, Iss. 0, 2019 r Journal of Food Science 1
Further reproduction without permission is prohibited

2. Food
Engineering,
Materials
Science,
Nanotechnology
Production of glucosyl rebaudioside A . . .
Dextransucrase can transfer the α-d-glucosyl moiety from su-
crose onto a hydroxyl group of carbohydrates as well as noncarbo-
hydrates. It has been investigated for the modification of various
bioactive compounds, including salicin (Seo et al., 2004), acarbose
(Yoon Robyt, 2002), gallic acid (Nam et al., 2017), and cate-
chin (Moon et al., 2006). These glycoconjugates are useful for the
manufacture of pharmaceuticals, fine chemicals, and food ingre-
dients under mild reaction conditions in white biotechnology. In
a previous study, we have found that Leuconostoc-derived dextran-
sucrase can transferred a glucosyl moiety to stevioside with high
regio-selectivity, high conversion yield, and improved sweetness
(Ko et al., 2012). We have also optimized the production process
of glucosyl rebaudioside A using a combination of factorial design
and response surface methodology (RSM).
Here, we report enzymatic modification and structural deter-
mination of glucosyl rebaudioside A. We also evaluated its thermal
stability and water solubility after long-term storage. In addition,
we also investigated the stability of glucosyl rebaudioside A in
commercial drinks by analyzing its hydrolysis products. Moreover,
we described parameters required for the maximum production
of glucosyl rebaudioside A by RSM.
Materials and Methods
Compound and enzyme
Rebaudioside A with a purity of ࣙ96% was purchased from
Sigma-Aldrich (St. Louis, MO, USA). It was used as a standard ma-
terial. The recombinant dextransucrase of Leuconostoc lactis EG001
was produced in E. coli BL21 (DE3) and purified by Ni-NTA
affinity chromatography as described previously (Kim et al. 2010).
Enzyme assay
A reaction mixture containing 0.4 M sucrose, 30 mM sodium
acetate buffer (pH 5.2), 13 mM MgCl2, and 0.42 U/mL dex-
transucrase was incubated at 30 °C for 30 min. Free fructose
released from sucrose was measured using fructose assay kit (Ab-
nova, Taoyuan, Taiwan). One unit of enzyme activity was defined
as the enzyme amount required to generate 1 µmol of fructose
per minute at given reaction conditions.
Synthesis and carbohydrate analysis
To glucosylate rebaudioside A, a reaction mixture (1 L) con-
sisting of 80 mM rebaudioside A, 0.4 M sucrose, 30 mM sodium
acetate buffer (pH 5.2), 13 mM MgCl2, and 2.1 U/mL dextransu-
crase was incubated at 30 °C for 6 hr. After enzymatic conversion,
the reaction mixture was placed in a water bath at 90 °C for
10 min to halt enzymatic activity. At designated time intervals,
10-µL aliquots were withdrawn to analyze the reaction products
via high performance liquid chromatography (HPLC). For
quantitative analysis, chromatographic separation was achieved
using a Shimadzu Prominence modular HPLC system (Shimadzu,
Tokyo, Japan) consisting of an LC-20AD liquid chromatograph
with a DGU-20A3R degassing unit. A SIL-20A auto sampler,
a CBM-20A communications bus module, a SPD-20A UV/vis
detector set at 210 nm, a CTO-20A column oven set at 30 °C.
Separation was performed on a ZORBAX 300SB-C18 (5 µm,
4.6 × 150 mm; Agilent Technologies, Palo Alto, CA, USA). Elu-
tion was done by a linear gradient of 100% H2O and 100% acetoni-
trile: starting with 100% H2O and increasing to 100% acetonitrile
for 60 min. The flow rate was 1.0 mL/min and the compounds
were monitored at 210 nm. The calibration curves (N = 6 point)
were constructed using transglucosyl rebaudioside A product
(1 to 50 µM) isolated in this study. Accuracy and reproducibility
was evaluated using the standard spike method. External standards
of the transglucosyl rebaudioside A product were added to enzy-
matic reaction solution and other samples at three concentrations
(N = 3, six replicates). The external calibration curve of the
transglucosyl rebaudioside A product at a concentration range of 1
to 50 µM produced a good linear correlation (Y = 1196770X +
74183, R2
0.999) and its precision was 5.0%. The content of
the transglucosyl rebaudioside A in each sample was determined
in triplicate experiments.
Isolation and structural elucidation of glucosyl
rebaudioside A
Reaction digests were centrifuged at 12,000 rpm for 10 min at
4 °C. Then the supernatant was collected, added to two volumes
of chilled ethanol, and incubated at −80 °C for 50 min to elimi-
nate glucan. After separation by centrifugation (at 12,000 rpm for
10 min at 4 °C) again, the mixture containing target materials was
concentrated under a vacuum using a rotary evaporator (Eyela,
Tokyo, Japan) at 40 °C. The concentrated mixture was then sub-
jected to a two-step separation procedure. After the resultant digest
(4 g/150 mL) was loaded onto a Diaion HP-20 column (2.2 ×
16 cm) washed with water to remove by-products, and then eluted
with ethanol to afford target materials. The target materials were
purified and isolated from ethanol fraction by a semipreparative
ODS-HPLC equipped with YMC-Pack Pro C18 column (5 µm,
20 × 250 mm; YMC Co., Ltd., Kyoto, Japan). Elution was per-
formed by a linear gradient of 100% H2O and 50% acetonitrile:
starting with 100% H2O and increasing to 50% acetonitrile for
60 min. The flow rate was 7.0 mL/min and the compounds were
monitored at 210 nm. The target materials were collected by the
repeated purification of ODS-HPLC and concentrated by vac-
uum rotary evaporation. The production yield (%) of glucosyl
rebaudioside A was calculated as the concentration (mM) of the
purified product to initial rebaudioside A.
LC-MS analysis of isolated compounds was then performed
with a Surveyor HPLC in line with an Agilent 6410B (Ag-
ilent technology, Wilmington, PA, USA). The isolated com-
pounds were separated under the following HPLC conditions:
column, XDB C18 (5 µm, 2.0 × 150 mm) (Agilent); flow rate,
0.23 mL/min. The sample was eluted using a gradient system of
10% water containing 0.1% formic acid to 100% acetonitrile con-
taining 1% formic acid for 20 min. LC-MS mass spectrometer
equipped with an ESI source. To confirm the position of sugar
bound in rebaudioside A, glucosyl rebaudioside A (8 mg) was hy-
drolyzed by the addition of 2 N sodium hydroxide (2 mL) and
heating at 90 °C for 2 hr. After neutralization by 1 N HCl solu-
tion, the reaction mixture was partitioned with water-saturated n-
butanol (3 mL, two times) and the organic layer was concentrated
in vacuo to afford glucosyl rebaudioside A hydrolysate, which was
analyzed by LC-MS analysis. For the structural elucidation of the
glucosylaton products, approximately 20 mg of the isolated glu-
cosyl rebaudioside A was dissolved in 600 µL of pyridine-d5 and
placed into 4.2-mm nuclear magnetic resonance (NMR) tubes.
The NMR spectra were acquired on an INOVA 500 spectrome-
ter (Varian, Palo Alto, CA, USA) operating at 500 MHz for 1
H
and 13
C. Connection between the steviol and glucoses were as-
signed using the 2D-NMR experiments, including nuclear over-
hauser effect spectroscopy (NOESY), homonuclear correlation
spectroscopy (COSY), heteronuclear single quantum coherence
(HSQC), heteronuclear multiple bond correlation (HMBC), and
total correlation spectroscopy (TOCSY).
2 Journal of Food Science r Vol. 0, Iss. 0, 2019

3. Food
Engineering,
Materials
Science,
Nanotechnology
Production of glucosyl rebaudioside A . . .
Retention time (min)
0 10 20 30 40
a
Rebaudioside A
Glucosyl
rebaudioside A
b
c
d
A
Alkaline hydrolys
[M-H]-
B
[M
sis
M+Na]+
Figure 1–Time course of glucosyl rebaudioside A synthesis from rebaudioside A and sucrose with dextransucrase (A) and LC-MS spectrum and structural
elucidation of glucosyl rebaudioside A (B).
a–d: Reaction time of 0, 2, 4, and 6 hr. The reaction mixture consisting of 80 mM rebaudioside A, 0.4 M sucrose, and 2.1 U/mL dextransucrase in
30 mM sodium acetate buffer (pH 5.2) was incubated at 30 °C for 6 hr.
Table 1–1H- and 13C-NMR data of O-α-D-glucosyl-(1→6) rebaudioside A.
Position δH (int., mult., J in Hz) δC Position
δH (int., mult., J in
Hz) δC Position
δH (int., mult., J
in Hz) δC
1a 1.78 (1H, d, 12.0) 41.1 1 6.05 (1H, d, 8.0) 95.8 1 5.57 (1H, d, 7.5) 105.2
1b 0.75 (1H, td, 12.0, 3.5)
2a 2.19 (1H, m) 19.8 2 4.10 74.0 2 4.20 76.7
2b 1.43 (1H, d, 13.5)
3a 2.38 (1H, d, 13.0) 38.6 3 4.11 79.3 3 4.29 78.9
3b 0.99 (1H, td, 12.5, 4.5)
4 - 44.4 4 4.14 71.9 4 4.30 72.3
5 1.03 (1H, br. d, 12.0) 57.6 5 3.98 77.4 5 3.94 78.6
6a 2.43 (1H, m) 22.6 6a 4.49 68.2 6a 4.44 63.0
6b 1.92 (1H, br. d, 12.0) 6b 4.23 6b 4.09
7 1.28 (2H, m) 42.0 1 5.37 (1H, d, 3.5) 100.8 1 5.34 (1H, d, 7.5) 105.1
8 - 43.0 2 4.13 (1H, dd, 8.0, 3.5) 74.3 2 4.07 75.6
9 0.88 (1H, br. d, 6.0) 54.3 3 4.58 (1H, t, 8.0) 75.7 3 4.24 79.0
10 - 40.2 4 4.27 (1H, t, 8.0) 72.2 4 4.19 71.9
11 1.69 (2H, m) 21.0 5 4.26 (1H, m) 79.0 5 4.10 (1H, m) 74.4
12a 2.27 (1H, m) 37.3 6a 4.59 62.7 6a 4.47 63.2
12b 2.01 (1H, m) 6b 4.32 6b 4.39
13 - 86.8 1 5.08 (1H, d, 8.0) 98.6
14a 2.65 (1H, d, 11.0) 44.7 2 4.38 (1H, dd, 8.0, 8.0) 82.0
14b 1.82 (1H, d, 11.0)
15 2.05 (1H, s) 48.0 3 4.21 (1H, dd, 8.0, 8.0) 88.2
16 - 154.6 4 3.88 (1H, dd, 8.0, 8.0) 71.0
17a 5.65 (1H, d, 15.5) 104.9 5 3.78 (1H, m) 77.7
17b 5.00 (1H, d, 15.5)
18 1.32 (3H, s) 15.9 6a 4.46 62.9
6b 4.39
19 - 177.4
20 1.29 (3H, s) 28.6
Long-term storage test at room temperature
Aqueous solutions of 0.04 to 2% (w/v) rebaudioside A and O-α-
D-glucosyl-(1
→6
) rebaudioside A were prepared in volumetric
flasks. These solutions were transferred to 1.5 mL microtubes.
Each tube was stored at 25 °C for up to 1 month. After 0, 7, 17,
and 30 days, aliquots were analyzed by the same HPLC method
described in section of synthesis and carbohydrate analysis.
Stability of glucosyl rebaudioside A at different pH
conditions
Aqueous solutions of rebaudioside A and O-α-D-glucosyl-
(1
→6
) rebaudioside A (0.8 mg/mL) were mixed with each
Robinson-Britton buffer at different pH conditions ranging from
pH 1.4 to pH 10. As a result, the final concentration of glucosyl
rebaudioside A was 0.4 mg/mL. Samples were heated in a dry
Vol. 0, Iss. 0, 2019 r Journal of Food Science 3

4. Food
Engineering,
Materials
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Nanotechnology
Production of glucosyl rebaudioside A . . .
Im
1
H
N
mportant HMBC correla
H-1
H COSY and /or TC
OESY correlation
ations
COSY correlations
Figure 2–Structure of O-α-D-glucosyl-(1→6) rebaudioside A.
oven at 80 °C for 2 hr. For quantitative analysis, the remaining
undecomposed glucosyl rebaudioside A was measured by the same
HPLC method described in section of synthesis and carbohydrate
analysis.
Heat stability of O-α-D-glucosyl-(1
→6
) rebaudioside A
To confirm that the material was stable at high temperatures, re-
baudioside A and O-α-D-glucosyl-(1
→6
) rebaudioside A were
prepared to a concentration of 0.4 mg/mL and incubated at 40,
60, and 80 °C for up to 2 hr. The degree of stability was confirmed
by HPLC analysis.
Stability of O-α-D-glucosyl-(1
→6
) rebaudioside A in
commercial drinks
Degradation of steviol glycosides was analyzed in two com-
mercial drinks, Coke (Coca Cola Co., Atlanta, GA, USA) and
orange juice (Coca Cola Co.), following published procedures
(Wolwer-Rieck et al. 2010). Briefly, degassed samples were added
to volumetric flasks containing 0.4 mg/mL of rebaudioside A and
O-α-D-glucosyl-(1
→6
) rebaudioside A. Each sealed tube was
stored for up to 48 hr in such a way that air was used as heat
transfer medium at 24 °C, 40 °C, 60 °C, and 80 °C. After 0,
24, and 48 hr, aliquots were removed and analyzed by the same
HPLC method described in section of synthesis and carbohydrate
analysis.
Optimization procedure and experimental design
A three-level Box-Behnken design with three-factors was ap-
plied to produce glucosyl rebaudioside. An optimization procedure
using Design Expert 7.00 software, including six replicates at the
central point, was utilized in the fitting of a second-order response
surface. Dextransucrase (8.4 U/mL) (x1), sucrose (x2), and rebau-
dioside A concentrations (x3) were modified to prepare each of 18
cultivation conditions summarized in Table 3. Optimization was
conducted using a desirability function to determine the effects
of x1, x2, and x3 on glucosyl rebaudioside A production. A total
of 18 experiments composed of 16 factorial points, eight axial
points, and six center points were conducted to determine the 14
coefficients of the model as follows (1):
y = β0 + β1x1 + β2x2 + β3x3 + β12x1x2 + β13x1x3 + β23x2x3
+ β11x1
2
+ β22x2
2
+ β33x3
2
(1)
where y was the predicted response; β0 was the intercept; β1, β2,
and β3 were linear coefficients; β11, β22, and β33 were squared
coefficients; and β12, β13, and β23 were interaction coefficients.
Once an appropriate model was obtained, it was used to determine
the optimum conditions for the process.
Results and Discussion
Enzymatic glucosylation of rebaudioside A
For glucosylation of rebudioside A, sucrose was added to the
reaction mixture (1 L) consisting of 80 mM rebaudioside A,
30 mM sodium acetate buffer (pH 5.2), 13 mM MgCl2, and
2.1 U/mL dextransucrase at 2-hr intervals. The 2-hr interval was
chosen because sucrose would be completely consumed within
2-hr. After stopping the reaction to remove glucans, each reaction
mixture containing rebaudioside A was analyzed by HPLC. HPLC
analysis of the mixture showed that rebaudioside A (retention time
= 23.93 min) was converted into putative transglucosyl product
(retention time = 20.93 min) identified as glucosyl rebaudioside
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5. Food
Engineering,
Materials
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Production of glucosyl rebaudioside A . . .
Figure 3–Stability of rebaudioside A (A) and glucosyl rebaudioside A (B) in long-term storage, and pH stability (C) of rebaudioside A (•) and O-α-D-
glucosyl-(1→6) rebaudioside A (), and thermal stability of rebaudioside A (D) and O-α-D-glucosyl-(1→6) rebaudioside A (E).
Each sample was dissolved at a concentration of 0.4 mg/mL (•), 1 mg/mL (◦), 5 mg/mL (), 10 mg/mL (), and 20 mg/mL () in an aqueous solution
and incubated at room temperature for 0, 7, 17, and 30 days. Stabilities of these compounds were evaluated at pH 1.4 to pH 10 after incubating at
80 °C for 2 hr. The mount of soluble compound was analyzed by HPLC, and calculated based on the original amount added. Vertical bars represent
standard deviations.
A, showing a high conversion rate of 86.5% (Figure 1A). The
synthesized rebaudioside A were separated from the transglucosyl
product mixture by Diaion HP-20 column chromatography and
semipreparative ODS-HPLC.
Structural elucidation of glucosyl rebaudioside A
The molecular weight (M.W.) of the synthesized rebaudioside A
was determined to be 1,128, as established by its quasi-molecular
ion peak of m/z 1151.50 [M+Na]+
in LC-MS (positive) spectrum
(Figure 1B). The synthesized rebaudioside A was suggested to be
mono-glucosyl rebaudioside A conjugated with one molecule of
glucose to rebaudioside A (M.W. 966.4). The M.W. of the synthe-
sized rebaudioside A hydrolysate (steviol 13-O-triglucoside) after
alkaline hydrolysis was 804, as established by its quashi molecular
ion peak at m/z 803.1 [M-H]−
in LC-MS (negative) spectrum
(Figure 1B), indicating that diglycose esterified to the C-19 of
rebaudioside A was hydrolyzed. Therefore, it was suggested that
one sugar moiety was conjugated to glucose bound to the C-19 of
rebaudioside A (Figure 1B). When the 1
H and 13
C-NMR spectra
of the transglucosyl product were compared to those of rebaudio-
side A, one sugar moiety corresponding to seven proton signals
(δ 4.13, 4.26, 4.27, 4.32, 4.58, 4.59, and 5.37) and six carbon
signals (δ 62.7, 72.2, 74.3, 75.7, 79.0, and 100.8) was additionally
observed (Table 1; Supplementary NMR data S1 and S2). In par-
ticular, the coupling constant (J) of the anomeric proton (δ 5.37)
in the 1
H-NMR spectra was 3.5 Hz and coupling constant (J) of
other protons confirmed by 1
H-1
H COSY, 1D-ROESY, and 1D-
TOCSY experiments (Figure 2 and Supplementary NMR data)
were 8.0 Hz, indicating that the sugar moiety bound to rebaudio-
side A was α-D-glucose. In addition, to confirm the position of
the sugar moiety bound to rebaudioside A, we analyzed the 2D-
NMR of 1
H-1
H COSY, TOCSY, HSQC, and HMBC (Supple-
mentary NMR data). Specifically, the 1
H-1
H COSY and TOCSY
analyses were able to determine proton and carbon signals corre-
sponding to five sugar moieties. The compound was confirmed
as rebaudioside A by the correlations of δ 5.57 (H-1
) → δ 82.0
(C-2
), δ 5.57 (H-1
) → δ 82.0 (C-2
), δ 5.34 (H-1
) → δ
88.2 (C-3
), and δ 6.05 (H-1
) → δ 177.4 (C-19) observed in the
HMBC experiment (Figure 2 and Supplementary NMR data S9).
In particular, the correlation of δ 5.37 (H-1
) → δ 68.2 (C-6
) was
observed, indicating that C-1
of α-D-glucose was etherified with
C-6
of rebaudioside A. This result was additionally confirmed by
the 1D-NOESY experiment that irradiation of H-1
at δ 5.37
enhanced signals of H-6
at δ 4.49 and 4.23 (Figure 2 and Sup-
plementary NMR data S8). Therefore, the transglucosyl product
was determined to be O-α-D-glucosyl-(1
→6
) rebaudioside A.
Stability of O-α-D-glucosyl-(1
→6
) rebaudioside A in an
aqueous solution
From the above mentioned results, dextransucrase hydrolyzes
sucrose to form a glucosyl enzyme intermediate complex and
acts as the effective acceptor molecule in glucosylation at the
19-hydroxyl group of rebaudioside A. The main glucosyl prod-
uct, glucosyl rebaudioside A, was purified and used for the
food additive stability experiments. To observe storage stability in
aqueous solutions, rebaudioside A and O-α-D-glucosyl-(1
→6
)
Vol. 0, Iss. 0, 2019 r Journal of Food Science 5

6. Food
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Production of glucosyl rebaudioside A . . .
Table 2–Stability of rebaudioside A and O-α-D-glucosyl-(1→6)
rebaudioside A in drinks.
Stability in commercial
drinks (%)
Drink
Temperature
(°C)
Time
(hr)
Rebaudioside
A
O-α-D-glucosyl-
(1→6)
rebaudioside A
4 24 100 100
48 100 100
24 24 100 100
48 100 100
Coke (pH
2.49)
40 24 99.8 99.9
48 99.1 99.2
60 24 88.3 91.0
48 78.5 82.2
80 24 25.5 32.6
48 7.2 11.6
4 24 100 100
48 100 100
24 24 100 100
48 100 100
Orange juice
(pH 3.52)
40 24 99.3 99.6
48 98.8 98.3
60 24 91.5 97.0
48 73.5 96.8
80 24 77.7 84.9
48 66.1 74.0
rebaudioside A were incubated at 25 °C for 30 days. As a result,
rebaudioside A showed low stability in 1% to 2% (w/v) aqueous
solution. After adding to 1% and 2% aqueous solutions, rebau-
dioside A showed 46.35% or 74.72% of the initial compounds,
respectively. It precipitated as a white colored product (Figure 3A
and 3B). In contrast, in 2% (w/v) aqueous solution, O-α-D-
glucosyl-(1
→6
) rebaudioside A only showed a tiny amount of
precipitation (0.04% of compound initial compound). These prop-
erties of O-α-D-glucosyl-(1
→6
) rebaudioside A were due to
glucosylation of the glucosyl molecule bound to the 19-carboxyl
group of rebaudioside A. The synthesized O-α-D-glucosyl-
(1
→6
) rebaudioside A could be used as a high-intensity sweet-
ener since it improved water solubility of rebaudioside A that
would be easily precipitated in aqueous solution. Moreover, due
to glucosylation of 19-carboxyl groups of rebaudioside A, O-α-
D-glucosyl-(1
→6
) rebaudioside A may affect water solubility in
aqueous solutions as previously reported for stevioside (Ko et al.,
2012).
pH stability and thermal stability of
O-α-D-glucosyl-(1
→6
) rebaudioside A
Quantitative analysis of the stability at different pH conditions
and high temperature revealed that rebaudioside A was more than
82% stable while glucosyl rebaudioside A was slightly more stable
(86.4%) at different pH conditions (2.0 to 10). At pH 1.4, rebu-
dioside A had a stability of 52.3% of initially added compound,
while O-α-D-glucosyl-(1
→6
) rebaudioside A had a stability of
53.3% of initially added compound, indicating a low stability at
low pH (Figure 3C). This suggests that ester and ether bonded
glucose in steviol were chemically degraded at low pH. Regard-
ing thermal stability, rebaudioside A and glucosyl rebaudioside A
were remarkably stable at temperature of 40 °C to 120 °C, indi-
cating that both compounds were highly stable (about 100%) even
at extreme temperature (Figure 3A and 3B).
Stability of O-α-D-glucosyl-(1
→6
) rebaudioside A in
commercial drinks
Rebaudioside A in aqueous solution was chemically degraded
at high temperature (80 °C) and acidic pH conditions. Based
on this information, we observed the stability of rebaudioside A
and O-α-D-glucosyl-(1
→6
) rebaudioside A in two commercial
drinks (Coke and orange juice) at temperatures ranging from 4 °C
to 80 °C. With increasing temperature, the decomposition of
both materials also increased. Most of thermal-induced steviol
glycosides began degradation at 80 °C. At this temperature after
24 hr, the concentration of the remaining rebaudioside A decreased
by 74.5% in Coke (pH 2.49, Table 2). The highest degradation
(92.8%) was seen in Coke after 2 days at 80 °C. In the orange juice
(approximately, pH 3.52), rebaudioside A and O-α-D-glucosyl-
(1
→6
) rebaudioside A were degraded to 33.9% and 22.3% of
their initial amounts, respectively. O-α-D-Glucosyl-(1
→6
) re-
baudioside A was more stable in both commercial drinks than re-
baudioside A for 24 hr at the designated temperature conditions.
After 48 hr, final degradation rate was 88.4% in Coke and 26% in
orange juice. However, O-α-D-glucosyl-(1
→6
) rebaudioside A
Table 3–Box-Behnken design of three independent variables with actual and predicted responses.
Coded level Actual level Product (mM)
Run x1 x2 x3 Dextransucrase (U/mL) Sucrose (mM) Rebaudioside A (mM) Experimental Predicted
1 −1 −1 0 0.2 50 65 4.97 2.64
2 1 −1 0 2.5 50 65 6.70 5.57
3 −1 1 0 0.2 1,200 65 4.31 5.44
4 1 1 0 2.5 1,200 65 25.23 27.57
5 −1 0 −1 0.2 625 10 3.04 1.62
6 1 0 −1 2.5 625 10 4.93 2.31
7 −1 0 1 0.2 625 120 5.69 8.31
8 1 0 1 2.5 625 120 31.26 32.68
9 0 −1 −1 1.35 50 10 3.55 7.30
10 0 1 −1 1.35 1,200 10 6.70 6.99
11 0 −1 1 1.35 50 120 13.40 13.11
12 0 1 1 1.35 1,200 120 41.99 38.24
13 0 0 0 1.35 625 65 30.81 30.33
14 0 0 0 1.35 625 65 25.73 30.33
15 0 0 0 1.35 625 65 33.34 30.33
16 0 0 0 1.35 625 65 33.38 30.33
17 0 0 0 1.35 625 65 26.12 30.33
18 0 0 0 1.35 625 65 32.57 30.33
6 Journal of Food Science r Vol. 0, Iss. 0, 2019

7. Food
Engineering,
Materials
Science,
Nanotechnology
Production of glucosyl rebaudioside A . . .
Figure 4–Response surface and contour plots of O-α-D-glucosyl-(1→6) rebaudioside A production.
showed higher stability than rebaudioside A in both drinks. After
acidic saponification of glucosyl esters of steviol glycoside and its
glucosylated derivatives, stevioside, rebaudioside A, or O-α-D-
glucosyl-(1
→6
) rebaudioside A, the resulting glucosyl moiety is
usually modified with an alkali and cannot be isolated in an intact
form (Catharino Santos, 2012).
Optimal synthesis of O-α-D-glucosyl-(1
→6
)
rebaudioside A
RSM was used to investigate the interaction of variables im-
portant to the production the O-α-D-glucosyl-(1
→6
) rebau-
dioside A. Through preliminary observations, three important
factors were found to be able to optimize O-α-D-glucosyl-
(1
→6
) rebaudioside A production: rebaudioside A concentra-
tion (10 to 120 mM), sucrose concentration (50 to 1,200 mM),
and enzyme activity (0.2 to 2.5 U/mL). The Box-Behnken de-
sign for O-α-D-glucosyl-(1
→6
) rebaudioside A production is
shown in Table 3. Based on central points of corresponding con-
tour plots (Figure 4), these three important factors for O-α-D-
glucosyl-(1
→6
) rebaudioside A production (that is, rebaudio-
side A, sucrose concentration, and dextransucrase activity) had
optimal values of 2.24 U/mL, 1170.75 mM, and 119.4 mM, re-
spectively. O-α-D-Glucosyl-(1
→6
) rebaudioside A production
was measured to be 43.67 mM under the obtained conditions.
Its production can be expressed with the following regression
equation:
y = 30.33 + 6.26x1 + 6.20x2 + 9.27x3 + 4.80x1x2 + 5.92x1x3
+ 6.36x2x3 − 12.60x2
1 − 7.42x2
2 − 6.49x2
3
where x1, x2, and x3 were values of dextransucrase activity
(U/mL), sucrose concentration (mM), and rebaudioside A con-
centration (mM), respectively. The regression equation yielded a
high R2
value (0.9614) using analysis of variance (ANOVA). This
was an estimate of the fraction of three variations in the data
obtained based on the model. This model was capable of explain-
ing 96% of the variation in the response. The predicted response
for O-α-D-glucosyl-(1
→6
) rebaudioside A production was
43.67 mM, and the real observed experimental production was
39.94 ± 0.48 mM, demonstrating almost identical results between
the predicted and real observed O-α-D-glucosyl-(1
→6
) rebau-
dioside A production.
Conclusions
The glucosylation of rebaudioside A by dextransucrase has
many advantages. First, dextransucrase showed the highest
conversion yield (approximately 86%) of rebaudioside A compared
to another enzymatic synthesis (55% yield, Gerwig et al., 2017).
Second, compared to other applied glucosyl transferases (Gerwig
et al., 2017), dextransucrase yielded a single product with differ-
ent regio-selective glucosyl derivative at the 19-carboxyl group
of rebaudioside A. Third, the transglucosylation product showed
a lower precipitation than rebaudioside A in aqueous solutions
after 15 days of incubation at room temperature. Finally, O-α-
D-glucosyl-(1
→6
) rebaudioside A showed higher stability in
two commercial drinks, Coke and orange juice, than rebaudio-
side A. Thus, O-α-D-glucosyl-(1
→6
) rebaudioside A, a new
steviol glucoside synthesized in this study, could be used as a novel
sweetener. Further studies on the synthesis of various rebaudio-
side A derivatives by glucosyltransferase are ongoing to reveal the
relationship between structure and sweetness.
Acknowledgments
This research was supported by a grant (NRF-
2019R1F1A1058385) of the Basic Science Research Program
through the National Research Foundation of Korea (NRF).
It was also supported by “Cooperative Research Program for
Agriculture Science and Technology Development (Project No.
PJ01256503)” funded by Rural Development Administration,
Republic of Korea.
Author Contributions
YM, JY, and YJ conceived and designed this study. SY, JA, HS,
and MH performed experiments and analyzed data. YM, YJ, and
JY wrote the paper. JA and JS reviewed and edited the manuscript.
All authors read and approved the manuscript.
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Supporting Information
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
8 Journal of Food Science r Vol. 0, Iss. 0, 2019