Anti-cariogenic Characteristics of Rubusoside

Article abstract of DOI:10.1007/s12257-018-0408-0

Streptococcus mutans plays an important role in the development of dental caries in humans by synthesizing adhesive insoluble glucans from sucrose by mutansucrase activity. To explore the anti-cariogenic characteristics of rubusoside (Ru), a natural sweetener component in Rubus suavissimus S. Lee (Rosaceae), we investigated the inhibitory effect of Ru against the activity of mutansucrase and the growth of Streptococcus mutans. Ru (50 mM) showed 97% inhibitory activity against 0.1 U/mL mutansucrase of S. mutans with 500 mM sucrose. It showed competitive inhibition with a K_i value of 1.1 ± 0.2 mM and IC₅₀ of 2.3 mM. Its inhibition activity was due to hydrophobic and hydrogen bonding interactions based on molecular docking analysis. Ru inhibited the growth of S. mutans as a bacteriostatic agent, with MIC and MBC values of 6 mM and 8 mM, respectively. In addition, Ru showed synergistic anti-bacterial activity when it was combined with curcumin. Therefore, Ru is a natural anti-cariogenic agent with anti-mutansucrase activity and antimicrobial activity against S. mutans.

Full text of DOI:10.1007/s12257-018-0408-0

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Biotechnology and Bioprocess Engineering 24: 282-287 (2019)
DOI 10.1007/s12257-018-0408-0
pISSN 1226-8372
eISSN 1976-3816
RESEARCH PAPER
Anti-cariogenic Characteristics of Rubusoside
Jeesoo Kim, Thi Thanh Hanh Nguyen, Juhui Jin, Iis Septiana, Gyu-Min Son, Gang-Hee Lee, You-Jin Jung,
Dilshad Qureshi, Il Kyoon Mok, Kunal Pal, Soo-Yeon Yang, Seong-Bo Kim, and Doman Kim
Received: 17 October 2018 / Revised: 28 November 2018 / Accepted: 18 December 2018
© The Korean Society for Biotechnology and Bioengineering and Springer 2019
Abstract Streptococcus mutans plays an important role in Keywords: Anti-cariogenicity, mutansucrase, natural
the development of dental caries in humans by synthesizing sweetener, rubusoside, Streptococcus mutans
adhesive insoluble glucans from sucrose by mutansucrase
activity. To explore the anti-cariogenic characteristics of
rubusoside (Ru), a natural sweetener component in Rubus
suavissimus S. Lee (Rosaceae), we investigated the
1. Introduction
inhibitory effect of Ru against the activity of mutansucrase Dental caries affects 80% of the world’s population. It is
and the growth of Streptococcus mutans. Ru (50 mM) showed costly to health care. Dental caries is initiated by interaction
97% inhibitory activity against 0.1 U/mL mutansucrase of between acidogenic bacteria and sucrose in susceptible
S. mutans with 500 mM sucrose. It showed competitive hosts. Streptococcus mutans is an acidogenic and acid-
inhibition with a K value of 1.1 0.2 mM and IC of tolerant bacterial species. It has been recognized as the
i
50
2.3 mM. Its inhibition activity was due to hydrophobic and primary pathogen of dental caries because of its ability to
hydrogen bonding interactions based on molecular docking produce glucansucrase synthesizing water insoluble glucan
analysis. Ru inhibited the growth of S. mutans as a called mutan [1]. Mutan can mediate the adherence of
bacteriostatic agent, with MIC and MBC values of 6 mM S. mutans and other oral bacterial species to tooth surfaces,
and 8 mM, respectively. In addition, Ru showed synergistic contributing to the formation of dental plaque biofilms
anti-bacterial activity when it was combined with curcumin. [2,3]. Glucansucrases are a member of glycoside hydrolase
Therefore, Ru is a natural anti-cariogenic agent with anti- (GH) family 70 that catalyze the formation of glucan with
mutansucrase activity and antimicrobial activity against various types of glucosidic linkages such as α(1-3), α(1-4),
S. mutans.
or α(1-6) bonds, from sucrose via transglucosylation
reactions. Glucansucrases are classified at mutansucrase,
dextransucrase, alternansucrase, and reuternansucrase,
which catalyzed α(1-3), α(1-6), α(1-3 and 1-6), and α(1-4
and 1-6) bond formation, respectively [4]. S. mutans
produces three extracellular glycosyltransferases (GTFs),
Jeesoo Kim , Thi Thanh Hanh Nguyen , Juhui Jin, Iis Septiana, Gyu-Min
Son, Gang-Hee Lee, You-Jin Jung, Il Kyoon Mok, Soo-Yeon Yang,
Doman Kim
Graduate School of International Agricultural Technology and Institutes GTF-I, GTF-SI and GTF-S, which are encoded by gtfB,
of Green Bioscience & Technology, Seoul National University,
gtfC, and gtfD, respectively. GTF-I and GTF-SI are
Pyeongchang 25354, Korea
mutansucrases that synthesize mainly insoluble glucan
Tel: +82-33-339-5720; Fax: +82-33-339-5716
E-mail: kimdm@snu.ac.kr
with α(1-3) glucosidic linkages [5,6]. In addition, other
crucial virulence factors of S. mutans include a membrane-
Dilshad Qureshi, Kunal Pal
Department of Biotechnology and Medical Engineering, National Institute
bound F1Fo-ATPase system, agmatine deiminase system,
of Technology, Rourkela, India
enolase, and lactate dehydrogenase [7]. Diet, especially
high sucrose intake, elevates the risk for dental caries. The
Seong-Bo Kim
CJ CheilJedang, Life Ingredient & Material Research Institute, Suwon
cariogenic nature of sucrose has led food and beverage
16495, Korea
industries to develop alternative sweeteners without having
The first two authors contributed equally to this work.
carcinogenicity.

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Anti-cariogenic Characteristics of Rubusoside
283
Rubusoside (13-O-β-glucosyl-19-O-β-D-glucosyl-steviol; [9]. The partially purified enzyme was kept at –20°C for
Ru) is one of the steviol glycosides present in the leaves of further study. One unit (U) of mutansucrase activity was
Rubus suavissimus S. Lee (Rosaceae). It is a well-known defined as the amount of enzyme required to release 1 μM
natural nontoxic sweetening agent. It has been used in fructose from sucrose per min at 37°C and pH 6.8. Fructose
various food and beverage products [8]. In addition, Ru has concentration in the reaction mixture was measured with a
been used to increase the solubility of several pharma- D-fructose kit (Megazyme, Ireland). The kinetic parameter
ceutically and medically important compounds with poor of mutansucrase activity (0.1 U/mL) was measured using
solubility in water [9]. It can decrease the growth and 0-200 mM sucrose as the substrate for 35 min. Reaction
glass-surface adherence of S. mutans [10] when Ru and responses were linear within this time frame. The Michalis-
sucrose are incubated together and then the growth, Menten constant (K ) was calculated from Lineweaver-
m
adherence, and acid production of S. mutans are measured. Burk plot [11].
The mechanism involved in the inhibition of Ru against
mutansucrase or its reduction of the growth of S. mutans is 2.3. Inhibition activity of rubusoside against mutansucrase
currently unknown. Therefore, the objective of this study Ru was dissolved in distilled water to prepare a 250 mM
was to investigate the mechanism of action of Ru against stock solution. The reaction mixture was composed of
mutansucrase and S. mutans growth. Kinetic characterization 0.1 U/mL mutansucrase, 500 mM sucrose, and 50 mM Ru
of Ru against mutansucrase activity and molecular docking in 20 mM sodium phosphate buffer (pH 6.8). After
analysis were carried out. The growth inhibition type of Ru incubating at 37°C for 12 h, water-insoluble glucan was
against S. mutans was also studied.
harvested by centrifugation at 12,000 × g for 15 min. The
pellet was dissolved in 1 M NaOH. The amount of water-
insoluble glucan was measured via thin-layer chromatography
(TLC) [12]. One μl of sample was spotted onto a precoated
TLC plate (Merck, Darmstadt, Germany) and developed
with two ascents of acetonitrile:water (85:15, v/v). Carbo-
2. Materials and Methods
2.1. Preparation of rubusoside
Ru was prepared using stevioside as a substrate with hydrates were visualized by dipping the TLC plate into
immobilized β-galactosidase expressed in Escherichia coli 0.5% (w/v) N–(1-naphtyl) ethylenediamine dihydrochloride
BL21 (DE3) pLysS and was purified as described in our (Sigma Aldrich, USA) and 5% (w/v) sulfuric acid (Duksan
previous study [9].
Chemicals, South Korea) in methanol (Duksan Chemicals,
South Korea) followed by heating at 120°C for 5 min. The
amount of carbohydrates was analyzed by conversion to
2.2. Preparation of mutansucrase from S. mutans
S. mutans KCTC 3065 was obtained from Korean Collection the integrated density value using the AlphaEaseFC 4.0
for Type Cultures (KCTC, Korea). Mutansucrase was Image Program (Alpha Inotech, CA, USA) [12].
prepared by culturing S. mutans in brain heart infusion
The 50% inhibitory concentration (IC ) was defined as
50
media (BHI; BD Difco, USA) supplemented with 2% (w/v) the concentration of Ru necessary to reduce mutansucrase
glucose at 37oC with shaking (at 150 rpm) until all glucose activity by 50% relative to a reaction mixture containing
was consumed. After fermentation, cells were separated mutansucrase without inhibitor. IC and K value of Ru
50
i
from the broth by centrifugation at 8,000 × g for 15 min. against mutansucrase were calculated based on the amount
The supernatant was concentrated using a Centriprep of fructose released into the reaction mixture using a
Centrifugal Filter Unit with Ultracel-YM10 (Merck, fructose kit. Inhibition activity was calculated using the
Germany) and loaded onto a DEAE-Sepharose ion-exchange following formula:
chromatography column (1×1×60 cm) equilibrated with
20 mM sodium phosphate buffer (pH 6.8). Mutansucrase Inhibition activity (%) = 100 – [(S – S ) / (C – C )] × 100,
0 0
was eluted with a salt gradient of 0-1,000 mM NaCl in
20 mM sodium phosphate buffer (pH 6.8). Fractions where C was the absorbance of control (enzyme, buffer,
containing mutansucrase were pooled and dialyzed with and substrate) after 35 min of reaction, C was the
0
20 mM sodium phosphate buffer (pH 6.8) at 4°C. These absorbance of control at the zero-time point, S was the
dialyzed fractions were concentrated using a Centriprep absorbance of the sample (enzyme, Ru, buffer, or substrate)
Centrifugal Filter Unit with Ultracel-YM10 and analyzed after 35 min of reaction, and S was the absorbance of
0
by sodium dodecyl sulfate polyacrylamide gel electrophoresis sample at the zero-time point. Inhibitor kinetic studies of
(SDS-PAGE). Protein concentration was measured by the Ru were done like those used in a kinetic study for
Bradford method using crystalline bovine serum as standard mutansucrase, except that multiple concentrations (0-

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284
Biotechnology and Bioprocess Engineering 24: 282-287 (2019)
10 mM) of Ru and various concentrations (35-175 mM 99.9% of cells. All experiments were carried out in triplicate.
sucrose) of substrate were used. The type of inhibition was
identified using a Lineweaver-Burk and Dixon plot (1/v) as
a function of inhibition concentration [I]. The kinetic
parameter (K) was calculated using the SigmaPlot program.
3. Results and Discussion
i
3.1. Characterization of mutansucrase from S. mutans
2.4. Antibacterial effect assay by disc diffusion method Mutansucrase from Streptococcus mutans was purified using
S. mutans was cultured in brain-heart infusion (BHI) at DEAE-Sepharose column (Fig. S1). The specific activity
37oC until OD reached approximately 1.0 [13,14]. The of mutansucrase after purification was 14.4 U/mg protein
600
antibacterial effect of Ru solution (250 mM) against when using sucrose as a substrate. The Michaelis-Menten
S. mutans was measured using epigallocatechin gallate constant (K ) of mutansucrase using sucrose as a substrate
m
(EGCG) (250 mM) as a control [15]. Both the Ru solution was 34.5 4.6 mM (Fig. S2) based on the amount of
and EGCG (250 mM) were filtered with a Minisart® fructose released in the reaction mixture. The K value of
m
syringe filter (0.2 μm, Satorius, Germany). A disc-diffusion mutansucrase in this study was higher than that of
antimicrobial test was carried out by spreading 100 µL of mutansucrase from S. mutans Ingbritt c (K = 9.4 mM) [18]
m
suspension containing 108 CFU/mL onto a BHI agar plate and that of S. mutans 6715 (K = 2.4 mM)[19]. The K
m
m
[15]. Then a 6-mm diameter disc containing 20 µL of values of S. mutans Ingbritt c and 6715 were determined by
250 mM Ru, 250 mM EGCG, or dimethyl sulfoxide (DMSO) the phenol sulfuric acid assay, which measured measuring
was processed in triplicate. These discs were impregnated the total glucan formation in the reaction mixture containing
into the inoculated agar plate. These plates were then sucrose and dextran T10 [19].
incubated at 37°C for 12 h.
3.2. Inhibition activity of rubusoside against mutansucrase
2.5. Molecular docking of Ru against mutansucrase
Ru was tested for its ability to inhibit mutansucrase. At
The three-dimensional structure of mutansucrase was 50 mM, Ru showed 97% inhibition against mutansucrase
retrieved from the Protein Data Bank (http://www.rcsb.org, (Fig. 1A) based on insoluble glucan formation. The IC
50
accession code 3AIC). Alpha acrabose (ACR) is located in value of Ru against mutansucrase was 2.3 mM. Based on
the active site of 3AIC [6]. All water molecules and co- both Lineweaver-Burk and Dixon plots, Ru exhibited
crystal ligand ACR were removed. Structure information competitive inhibition toward mutansucrase, because the
containing only amino-acid residues of mutansucrase Lineweaver-Burk plot of 1/v versus 1/[S] resulted in a
enzyme was used for molecular docking. All docking family of straight lines with the same y-axis intercept
experiments were carried out according to the previous (Fig. 1B). The K value of Ru was found to be 1.1 0.2 mM
i
published method [16].
(Fig. 1C). To our best knowledge, this is the first report
about the inhibitory effect of Ru against mutansucrase.
2.6. Minimum inhibition concentration (MIC) and
minimum bactericidal concentration (MBC) against 3.3. Molecular docking of Ru against mutansucrase
S. mutans
To have a better comprehension of the molecular recognition
Curcumin was purchased from Sigma. Water-soluble process between mutansucrase and Ru, docking experiments
curcumin (Ru-CUR) was prepared as described previously were done using the crystal structure of mutansucrase
[9]. A broth microdilution method was used to find the (3AIC) [6] and Autodock 4.2 for docking simulations. The
minimum inhibitory concentration (MIC) and minimum free binding energy of Ru was -8.27 kcal/mol (Fig. 2A). Ru
bacterial concentration (MBC) [17]. To measure MIC, Ru inhibited mutansucrase from S. mutans potentially through
or Ru-CUR was placed into each well of a 96-well-plate. hydrophobic and hydrogen bond (H-bond) interactions
Each well was then inoculated with 5 μL of S. mutans between amino-acid residues in the active site pocket of
culture (1.5 × 107 CFU/mL) and incubated at 37oC for mutansucrase and Ru (Fig. 2B). Carbon atoms of Ru
24 h. Thereafter, turbidity of each well was measured with interacted hydrophobically with Leu 433, Leu434, Ala478,
a SpectraMax M3 (Molecular devices, USA) at a wavelength Asp480, Glu515, Trp517, and Tyr916. Ru formed three H-
of 600 nm. The lowest concentration that inhibited bacterial bonds with residues in the catalytic binding pocket of
growth was considered to be the MIC. MBC was measured mutansucrase. The O7 atom of the 2-hydroxyl group of the
by subculturing the medium from MIC on a BHI agar glucosyl group at C19 of Ru accepted an H-bond with the
plate. These plates were incubated at 37oC for 24 h. MBC amino group of Lys977 at a distance of 2.61 Å. The O
was defined as the consequent concentration that killed atom of the side chain carboxyl group of Asp909 formed

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Anti-cariogenic Characteristics of Rubusoside
285
Fig. 1. Effect of Ru on reducing insoluble glucan formation using mutansucrase from S. mutans (A) and the plots of Lineweaver-Burk
(B) and Dixon (C) analyzing Ru inhibition of mutansucrase activity. Kinetic constants K and K were calculated using linear regression
m
i
analysis. In (A), Mut, mutansucrase reaction without Ru and Mut+Ru, mutansucrase reaction with 50 mM Ru. The while color material
indicates the formation of insoluble glucan. The mutansucrase reaction mixture comprised of 0.1 U/mL mutansucrase, 500 mM sucrose,
o
and 50 mM Ru in 20 mM sodium phosphate buffer (pH 6.8) and reacted at 37 C for 12 h. The amount of water-insoluble glucan was
determined using thin-layer chromatography (TLC). The amount of carbohydrates was analyzed by the AlphaEaseFC 4.0 Image
Program which measures the integrated density value. In (B), the Ru concentration was 0 mM (
●), 1 mM (○), 2.5 mM (►), and 10 mM
(∆). In (C), the sucrose substrate concentration was 35 mM (●), 70 mM (○), 10 mM (▲), and 175 mM.
Fig. 2. Computation docking and hydrophobic and hydrogen bond interactions of Ru with amino-acid residues in the active site of
mutansucrase. (A) Comparison of the binding mode of Ru (yellow) in the active site of mutansucrase. (B) Hydrophobic and H-bond
interactions between Ru and amino-acid residues in the active site of mutansucrase. H-bond interactions are represented by green dashed
lines (Red, oxygen; cornflower blue, nitrogen; black, carbon).
two H-bonds with O11 and O12 of the glucosyl group at against S. mutans growth were assessed by an inhibition
C13 of Ru at a distance of 2.91 Å and 3.04 Å, respectively. zone assay and zone diameter measurement (Fig. S3). Ru
inhibited the growth of S. mutans. The inhibition zone at
3.4. Antimicrobial susceptibility test of rubusoside and 5 mM/disc was 11.3 1 mm. EGCG was used as a positive
rubusoside-curcumin complex against S. mutans growth control. Its inhibition zone at 5mM/ disc was 19 1 mm.
The antimicrobial activities of Ru against S. mutans have MIC and MBC values of Ru against S. mutans were 6 mM
been reported previously by Chu et al. (2016) after and 8 mM, respectively (Fig. 3A). MBC was higher than
incubating Ru with S. mutans in the presence of sucrose. MIC, indicating that Ru could inhibit S. mutans as a
Our study confirmed the antibacterial activity of Ru by bacteriostatic agent [20]. Ru is known to be less acidogenic
MIC and MBC studies. The antimicrobial activities of Ru than is glucose, maltose, fructose, or sucrose, which can be