Kochikyan V.T., Markosyan A.A., Abelyan L.A., Balayan A.M., and Abelyan V.A. Combined Enzymatic Modification of Stevioside and Rebaudioside A
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ISSN 0003-6838, Applied Biochemistry and Microbiology, 2006, Vol. 42, No. 1, pp. 31–37. © MAIK “Nauka /Interperiodica” (Russia), 2006.
Original Russian Text © V.T. Kochikyan, A.A. Markosyan, L.A. Abelyan, A.M. Balayan, V.A. Abelyan, 2006, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2006, Vol. 42,
No. 1, pp. 37–43.
31
Stevioside and rebaudioside A are low-calorie natu-
ral products isolated from
Stevia rebaudiana
Bertoni
leaves, which are widely used as sweeteners [1–6].
All diterpene glycosides isolated from
S. rebaudi-
ana
leaves have the same backbone (steviol) and differ
in the content of carbohydrate residues—mono-, di-,
and trisaccharides containing glucose and/or rhamnose
at positions C13 and C19 (Fig. 1) [7–11]. The major
diterpene glycosides are stevioside (5–10%), rebaudio-
sides A (2–4%), rebaudiosides C (1–2%), and dulco-
side A (0.4–0.7%) [12]. The sweetness of all these com-
pounds is greater than that of sucrose: rebaudioside A,
250–450; rebaudioside B, 300–350; rebaudioside C,
50–120; rebaudioside D, 250–450; rebaudioside E,
150–300; dulcoside A, 50-120; and steviobioside, 100–
125. On average, the sweetness of stevioside is 250–
300 times greater than the sweetness of sucrose [13].
Stevia glycosides possess valuable biological prop-
erties. Regular consumption of these compounds
decreases the content of sugar, radionuclides, and cho-
lesterol in blood; improves cell regeneration and blood
coagulation; suppresses neoplastic growth; strengthens
blood vessels [14–16]. They also exhibit choleretic,
anti-inflammatory, and diuretic properties and prevent
ulceration in the gastrointestinal tract [17].
Among these glycosides, stevioside and rebaudio-
side A have the greatest commercial potential. How-
ever, they possess residual bitterness and taste, which
affect their qualitative characteristics. Enzymatic mod-
ification of stevioside by pullanase, isomaltase [18],
β
-galactosidase [19], and dextrin saccharase [20] in the
presence of respective donors allows this problem to be
solved completely or in part.
Transglycosylation of rebaudioside A is associated
with certain difficulties. For this reason, there is no
industrial production of its modified form.
Our previous studies demonstrated the efficiency of
transglycosylation of stevioside catalyzed by cyclodex-
trin glucanotransferases (CGTases) from different
groups of microorganisms using cyclodextrins as
donors [21].
The goal of this study was to investigate the charac-
teristics of transglycosylation of stevioside and rebau-
dioside A by CGTases from different groups of micro-
organisms using starch as a donor.
MATERIALS AND METHODS
In this study, we used CGTases from thermophilic
and mesophilic strains exhibiting marked ability for
intermolecular transglycosylation, including
Bacillus
stearothermophilus
INMIA-B-5076,
B. stearothermo-
philus
INMIA-B-4019,
B. circulans
subsp.
thermophi-
lus
INMI-B-4024, as well as the mesophilic bacilli
B. macerans
INMIA-BIO-4m and
B. macerans
INMIA-BIO-12m. The strains were grown under sub-
surface conditions; CGTases were obtained as
described earlier [22, 23]. After separation of biomass
by centrifugation at 5000
g
for 20 min, the supernatant
was concentrated by ultrafiltration on an UPL-0.6
device equipped with an AP-0.2 column (Russia).
Determination of cyclizing activity of CGTases.
A
mixture of potato starch (2%, 2 ml, dissolved in appro-
priate buffer with pH optimum for each enzyme) and
unpurified enzyme (0.5 ml) was incubated at
50°ë
. At
certain intervals (0.5–1.0 min), aliquots (600
µ
l) of the
mixture were taken and mixed with phenolphthalein
(900
µ
l) [24] or bromcresol green [25] to identify
Combined Enzymatic Modification
of Stevioside and Rebaudioside A
V. T. Kochikyan*, A. A. Markosyan**, L. A. Abelyan*,
A. M. Balayan*, and V. A. Abelyan*
*Institute of Microbiology, National Academy of Science of Armenia, Abovyan, 375510 Armenia
e-mail: abelyan@sbc.com.my
**Biotechnological Corporation Stevian, Kuala Lumpur, 50450 Malaysia
Received November 11, 2004
Abstract
—Cyclodextrin glucanotransferases (CGTases, EC 2.4.1.19) produced by mesophilic, thermophilic,
alkaliphilic, and halophilic bacilli were used for transglycosylating stevioside and rebaudiosides A with the use
of starch as a donor. CGTases produced by
Bacillus stearothermophilus
B-5076
B. macerans
BIO-4m were the
most effective biocatalysts. This method can be used successfully for direct transglycosylation of stevia extract
without purification of its individual components.
DOI:
10.1134/S0003683806010030
32
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
Vol. 42
No. 1
2006
KOCHIKYAN
et al
.
β
-
and
γ
-cyclodextrin, respectively. The content of
α
-
cyclodextrin was determined by HPLC. The amount of
the enzyme that produced 1
µ
mol of CD in 1 min was
taken as one unit of activity.
Determination of transglycosylating activity of
CGTases
was performed as described in [26] with some
modifications. Briefly, the reaction mixture (1 ml) con-
taining the preparation of CGTase (4.0 U), soluble
starch (10 mg), sucrose (50 mmol), 10
µ
mol
CaCl
2
, and
0.1 M buffer (with optimum pH) was incubated at
50°C
for 15 min. The reaction was stopped by boiling for
10 min. After centrifugation (10000
g
, 10–15 min), the
content of maltosylfructose was determined by HPLC.
The amount of the enzyme that produced 1
µ
mol of
maltosylfructose in 1 min was taken as one unit of
activity.
Stevioside, rebaudioside A, and their derivatives
were determined by HPLC (Agilent Technologies 1100
Series, United States) using a Zorbax-
NH
2
column. A
mixture of acetonitrile and water (70 : 30 or 80 : 20, v/v)
was used as an eluent; the flow rate was 1 ml/min. The
compounds of interest were detected in UV light at
210 nm. The product was analyzed using the acetoni-
trile–water system gradient from 80 : 20 v/v (2 min) to
50 : 50 v/v (70 min) [9].
The content of protein was determined by the Lowry
method using BSA as a standard; the content of reduc-
ing saccharides, by the Somogyi–Nelson method.
In this study, we used a mixture of stevioside and
rebaudioside (1 : 1, w/w); the glycosides were obtained
from Shandong Huaxian Stevian Co., Ltd. (China).
Other reagents were from Wako Pure Chemical Indus-
tries, Ltd. (Japan).
RESULTS AND DISCUSSION
Transglycosylating activities of CGTases from vari-
ous groups of microorganisms were studied to obtain
glycosylated derivatives of stevioside AND rebaudio-
side A using starch as a donor. After purification and
Name R
1
R
2
1. Steviol H H
2. Steviobioside H
β
-Glc-
β
-Glc(2 1)
3. Stevioside
β
-Glc
β
-Glc-
β
-Glc(2 1)
4. Rebaudioside A
β
-Glc
β
-Glc-
β
-Glc(2 1)
β
-Glc(3 1)
5. Rebaudioside B H
β
-Glc-
β
-Glc(2 1)
β
-Glc(3 1)
6. Rebaudioside C
(dulcoside)
β
-Glc
β
-Glc-
α
-Rha(2 1)
β
-Glc(3 1)
7. Rebaudioside D
β
-Glc-
β
-Glc(2 1)
β
-Glc-
β
-Glc(2 1)
β
-Glc(3 1)
8. Rebaudioside E
β
-Glc-
β
-Glc(2 1)
β
-Glc-
β
-Glc(2 1)
9. Rebaudioside F
β
-Glc
β
-Glc-
β
-Xyl(2 1)
β
-Glc(3 1)
10. Dulcoside A
β
-Glc
β
-Glc-
α
-Rha(2 1)
CH
3
COO
CH
3
CH
2
OR
2
R
1
Fig. 1.
Structure of the major glycosides of
Stevia rebaudiana
leaves (Geuns, 2003).
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
Vol. 42
No. 1
2006
COMBINED ENZYMATIC MODIFICATION 33
identification of the compounds by different methods,
their gustatory properties were estimated.
Proportion and concentration of substrates.
To
determine the optimum concentration of the reaction
mixture, we prepared 10, 20, 30, 40, 50, and 60% solu-
tions of starch and the mixture of stevioside and rebau-
dioside at a ratio of 1 : 1 (w/w), pH 6.5–7.0. Starch was
thinned using bacterial
α
-amylase to a dextrose equiv-
alent of 15–20. The amount of CGTase was 50 U/g
starch. The reaction was performed for 24 h under con-
stant stirring at 55 and
50°ë
in the case of CGTase from
the thermophilic and mesophilic strains, respectively.
It was found that, as the initial concentration of com-
ponents in the reaction mixture increased, the reaction
rate and the amount of transformation products also
increased (Figs. 2a, 2b), with the amount of high-molec-
ular-weight products being increased as well (table).
Under these conditions, the reactions catalyzed by
CGTase from the thermophilic strains were preferable.
An increase in the starch-to-glycoside ratio corre-
lated with an increase in the yield of the transformation
products. For example, at a starch-to-glycoside ratio of
1 : 1 (w/w), the sweetness of the resultant product
(without additional purification) was 150–170 times
greater than the sweetness of sucrose; at a ratio of 10 : 1,
only 15–30 times greater.
Effect of pH and temperature.
To determine opti-
mum pH and temperature, starch and the mixture of
stevioside and rebaudioside A (50 g each) were dis-
solved in a buffer (150 ml) with the corresponding pH.
Then, the enzyme solution with activity of 10 U per
gram of glycoside was added to the solution, and the
mixture was incubated at 55–60
°
C for 24 h.
Similarly to transglycosylation with the use of
cyclodextrin as a donor [21], optimum pH for all
enzymes was in the range of 6.5–7.5 (Fig. 3a).
Optimum temperature for the enzymes from the
thermophilic strains, determined in the presence of
30
−
40% substrates, was in the range of 55–65
°
C;
mesophilic strains, 50–55
°
C (Fig. 3b).
Effect of enzyme content.
To determine the effect of
the enzyme content on the transglycosylation effi-
ciency, different amounts of enzymes were added to
40% solution of glycosides and starch (1 : 1, w/w;
pH 7.0) and incubated at
55°ë
for 24 h.
An increase in the amount of enzyme led to an
increase in the reaction rate and the yield of transforma-
tion products. Note that the amount of glycosylated
20
A
, %
100 (a)
40
60
80
1
2
3
20
010
A
, %
C
, %
60
20
(b)
30 40 50 60
30
40
50
1
2
3
10
010
C
, %
20 30 40 50 60
Fig. 2.
Effect of total concentration of substrates (
C
, %) on transferase activity (
A
, %) of CGTases from (a) the thermophilic strains
(
1
)
B. stearothermophilus
INMIA-B-5076, (
2
)
B. stearothermophilus
INMIA-B-4019, and (
3
)
B. circulans
INMI-B-4024 (a) and
(b) the mesophilic strains (
1
)
B. macerans
INMIA-BIO-4m, (
2
)
B. macerans
INMIA-BIO-2m, and (
3
)
B. macerans
INMIA-BIO-12m.
Transglycosylation of stevioside and rebaudioside A by CG-
Tases from thermophilic and mesophilic strains in the pres-
ence of starch as a donor
Strain
Transformation product yield, %
I II III IV V VI
Thermophilic
B-5076 51 19 15 11 4 53
B-4019 42 22 18 12 6 49
B-4029 48 21 13 10 8 23
Mesophilic
BIO-4m 47 21 15 12 4 37
BIO-2m 43 20 17 14 6 34
BIO-12m 45 23 14 12 6 28
Note: I–IV, Mono-, di-, tri-, and tetraglycosylated derivatives;
V, more high-molecular-weight derivatives; VI, total amount
34
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
Vol. 42 No. 1 2006
KOCHIKYAN et al.
derivatives was considerably greater in the case of
enzymes from the thermophilic strains (Fig. 4a).
The duration of reaction also had a marked effect on
the yield of glycosylated derivatives. At a starch-to-gly-
coside ratio of 1 : 1 (w/w), the reaction of transglycosy-
lation catalyzed by CGTase from B. stearothermophi-
lus B-5076 (10 U/g starch) was completed within 48 h
at 55–60°ë (Fig. 4b).
The results of HPLC analysis of the compounds pro-
duced by the most effective CGTases from the thermo-
philic (B-5076) and mesophilic (BIO-4m) strains during
incubation for 24 and 48 h are shown in Figs. 5a and 5b.
Notably, at the initial stages of transglycosylation
20
0
2
% of max
pH
100
3
80
60
40
1 95 6 7 84
1
2
3
4
5
6
(a)
20
0
% of max
°C
100
30
80
60
40
50 60 7040
1
2
3
4
5
6
(b)
Fig. 3. Dependence of transferase activity of CGTases (% of maximum) on (a) pH of reaction medium at 55°C (acetate buffer,
pH 3.0–3.5; phosphate–citrate buffer, pH 4.0–6.5; and sodium-phosphate buffer, pH 6.5–9.0) and (b) temperature at pH 7.0. Desig-
nations: 1, B. stearothermophilus B-5076; 2, B. stearothermophilus B-4019; 3, B. circulans B-4024; 4, B. macerans BIO-4m;
5, B. macerans BIO-2m; 6, B. macerans BIO-12m. The total concentration of stevioside/rebaudioside A and starch was 40% (1 : 1,
w/w); incubation duration, 24 h; CGTase concentration, 10 U/g glycosides.
05
A, % of max
CGTase, U/g starch
100
10
80
60
40
20
15 20
1
2
3
4
5
6
(a)
012
h
24 36 48
1
2
3
4
5
6
(b)
6 18 30 42
Fig. 4. Dependence of the total yield (A, % of maximum) of transformation products on (a) the amount of CGTase and (b) duration
of reaction (CGTase concentration, 10 U per gram starch). Designations: 1, B. stearothermophilus B-5076; 2, B. stearothermophilus
B-4019; 3, B. circulans B-4024; 4, B. macerans BIO-4m; 5, B. macerans BIO-2m; 6, B. macerans BIO-12m. The total concentration
of stevioside/rebaudioside A and starch was 40% (1 : 1, w/w; pH 7.0); incubation duration, 24 h; temperature, 55°C.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 1 2006
COMBINED ENZYMATIC MODIFICATION 35
1000
800
600
400
200
0
600
500
400
300
200
100
0
mAU
500
400
300
200
100
0
800
700
600
500
400
300
200
100
0
0 5 10 15 20 25 5 10 15 20 2503035
0 5 10 15 20 25 0 5 10 15 20 25 min
St
Reb A
G-St
G-Reb
2G-St
2G-Reb
3G-St
3G-Reb
St
Reb A
G-St
G-Reb
2G-St
2G-Reb
3G-St
3G-Reb
St
Reb A
G-St
G-Reb
2G-St
2G-Reb
3G-St
St
Reb A
G-St
G-Reb
2G-St
2G-Reb
3G-St
4G-St
III
III
(‡)
(b)
Fig. 5. Chromatogram of the products of transformation catalyzed by CGTase from (a) B. stearothermophilus B-5076 and
(b) B. macerans BIO-4m for (I) 24 and (II) 48 h. The total concentration of stevioside/rebaudioside A and starch was 40% (1 : 1,
w/w; pH 7.0); CGTase concentration, 10 U/g glycosides, temperature, 55°C.
1400
1200
1000
800
600
400
200
0
400
350
300
250
200
150
100
50
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
St
Reb A
G-St
G-Reb
2G-St
3G-St
St
Reb A
G-St
G-Rab
2G-St
2G-Rab
3G-St
4G-St
3G-Reb
(a) (b)
min
mAU
Fig. 6. Chromatogram of the products of transformation catalyzed by (a) α-amylase (Termamyl, Novozyme) and (b) CGTase from
B. stearothermophilus B-5076. The total concentration of stevioside/rebaudioside A and starch was 40% (1 : 1, w/w; pH 7.0); incu-
bation duration, 48 h; CGTase concentration, 10 U/g glycosides; α-amylase concentration, 0.3 U/g starch; temperature, 60°C.
36
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 1 2006
KOCHIKYAN et al.
(in the first 24 h), the reaction proceeded predomi-
nantly with the involvement of stevioside. Later, when
the concentration of stevioside decreased to 10–15%,
rebaudioside A entered the reaction process.
Note that it is reasonable to thin starch using the
same CGTase that catalyzes the major reaction (transg-
lycosylation), because the efficiency of transglycosyla-
tion considerably decreases when α-amylase is used as
a thinning agent (Fig. 6).
To perform transglycosylation of stevia glycosides,
CGTase (2 U per gram starch) was added to starch (100 g)
suspended in distilled or deionized water (300 ml;
pH 6.5–7.0), and the mixture was heated to 75–80°ë
under constant stirring until thinned starch suspension
with a dextrose equivalent of 0.15–0.3 was obtained.
The solution was cooled to 50–60°ë; mixed with 100 g
stevioside, rebaudioside A, or stevia extract containing
stevioside and rebaudioside A at a ratio of 1 : 1 (w/w);
and stirred until completely dissolved. Then, CGTase
(8 U per gram starch) was added, and the mixture was
incubated at 55–60°ë for 48 h under constant stirring.
The reaction mixture was treated with activated char-
coal and filtered; the filtrate was dried in a spray dryer
(Fig. 7).
To improve the gustatory characteristics of the reac-
tion product, before and after purification it latter was
additionally treated with α-amylase (5 U per gram total
glycosides) at 50°C for 22–24 h. As a result, the high-
molecular-weight derivatives of glycosides were trans-
formed predominantly into mono- and diglycosylated
forms, whose sweetness is greater than that of the high-
molecular-weight forms and taste is more delicate
(Figs. 8a, 8b).
Thus, comparative analysis showed that CGTases
produced by B. stearothermophilus B-5076 and
B. macerans BIO-4m may serve as effective biocata-
lysts in enzymatic transglycosylation of stevia glyco-
sides with the use of starch as a donor. These CGTases
exhibit high activity with respect not only to stevioside
but also rebaudioside A, whose degree of modification
can be varied by changing the technological parameters
of the reaction.
Fig. 7. Scheme of stages of transglycosylation of stevioside
and rebaudioside A with the use of starch as a donor.
350
300
250
200
150
100
50
0
mAU
0 102030405060min
St
Reb A
G-St
G-Reb
2G-St
2G-Reb
3G-Reb
3G-St
4G-St
3G-Reb
St
Reb A
G-St
G-Reb
2G-St
2G-Reb
3G-St
4G-St
3G-Reb
250
200
150
100
50
0
mAU
0 102030405060min
(a) (b)
Fig. 8. Chromatogram of the products of transformation catalyzed by B. stearothermophilus B-5076 for 48 h, which were identified
by the gradient method (a) before and (b) after treatment with β-amylase.
Starch, 100 g
Water, 300 ml
Starch suspension, 25%, pH 6.5–7.0
CGTase, 2 U/g starch
75–85°ë; 30–40 min
Thinned starch
Cooling, 55–60°ë
Stevioside, rebaudioside A,
or their mixture, 100 g
Reaction mixture
CGTase, 8 U/g starch
Transglycosylation, 55–60°ë; 48 h
Activated charcoal, 1% of dry weight
Discoloration, 60–70°ë; 20–30 min
Filtration
Spray dryer
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 42 No. 1 2006
COMBINED ENZYMATIC MODIFICATION 37
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