Raffinose Inhibits Streptococcus mutans Biofilm Formation by Targeting Glucosyltransferase

Abstract

Streptococcus mutans is a representative biofilm-forming bacterium that causes dental caries through glucosyltransferase (GTF) activity. Glucans are synthesized from sucrose by GTFs and provide binding sites for S. mutans to adhere tightly to the tooth enamel. Therefore, if a novel compound that interferes with GTF function is developed, biofilm formation control in S. mutans would be possible. We discovered that raffinose, an oligosaccharide from natural products, strongly inhibited biofilm formation, GTF-related gene expression, and glucan production. Furthermore, biofilm inhibition on saliva-coated hydroxyapatite discs through the reduction of bacterial adhesion indicated the applicability of raffinose in oral health. These effects of raffinose appear to be due to its ability to modulate GTF activity in S. mutans. Hence, raffinose may be considered an antibiofilm agent for use as a substance for oral supplies and dental materials to prevent dental caries.
IMPORTANCE Dental caries is the most prevalent infectious disease and is expensive to manage. Dental biofilms can be eliminated via mechanical treatment or inhibited using antibiotics. However, bacteria that are not entirely removed or are resistant to antibiotics can still form biofilms. In this study, we found that raffinose inhibited biofilm formation by S. mutans, a causative agent of dental caries, possibly through binding to GtfC. Our findings support the notion that biofilm inhibition by raffinose can be exerted by interference with GTF function, compensating for the shortcomings of existing commercialized antibiofilm methods. Furthermore, raffinose is an ingredient derived from natural products and can be safely utilized in humans; it has no smell and tastes sweet. Therefore, raffinose, which can control S. mutans biofilm formation, has been suggested as a substance for oral supplies and dental materials to prevent dental caries.

Full text

Raffinose Inhibits Streptococcus mutans Biofilm Formation by
Targeting Glucosyltransferase
So-Young Ham,
a
Han-Shin Kim,
b
Eunji Cha,
a
Taehyeung Lim,
c
Youngjoo Byun,
c
,
d
Hee-Deung Park
a
,
e
a
School of Civil, Environmental, and Architectural Engineering, Korea University, Seoul, South Korea
b
Division of Biotechnology, College of Environmental and Bioresource Sciences, Jeonbuk National University, Iksan, Jeonbuk, South Korea
c
College of Pharmacy, Korea University, Sejong, South Korea
d
Biomedical Research Center, Korea University Guro Hospital, Seoul, South Korea
e
KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, South Korea
ABSTRACT Streptococcus mutans is a representative biofilm-forming bacterium that
causes dental caries through glucosyltransferase (GTF) activity. Glucans are synthe-
sized from sucrose by GTFs and provide binding sites for S. mutans to adhere tightly
to the tooth enamel. Therefore, if a novel compound that interferes with GTF func-
tion is developed, biofilm formation control in S. mutans would be possible. We dis-
covered that raffinose, an oligosaccharide from natural products, strongly inhibited
biofilm formation, GTF-related gene expression, and glucan production. Furthermore,
biofilm inhibition on saliva-coated hydroxyapatite discs through the reduction of
bacterial adhesion indicated the applicability of raffinose in oral health. These effects
of raffinose appear to be due to its ability to modulate GTF activity in S. mutans.
Hence, raffinose may be considered an antibiofilm agent for use as a substance for
oral supplies and dental materials to prevent dental caries.
IMPORTANCE Dental caries is the most prevalent infectious disease and is expensive
to manage. Dental biofilms can be eliminated via mechanical treatment or inhibited
using antibiotics. However, bacteria that are not entirely removed or are resistant to
antibiotics can still form biofilms. In this study, we found that raffinose inhibited bio-
film formation by S. mutans, a causative agent of dental caries, possibly through
binding to GtfC. Our findings support the notion that biofilm inhibition by raffinose
can be exerted by interference with GTF function, compensating for the shortcom-
ings of existing commercialized antibiofilm methods. Furthermore, raffinose is an in-
gredient derived from natural products and can be safely utilized in humans; it has
no smell and tastes sweet. Therefore, raffinose, which can control S. mutans biofilm
formation, has been suggested as a substance for oral supplies and dental materials
to prevent dental caries.
KEYWORDS raffinose, Streptococcus mutans, biofilm, glucosyltransferase
In oral microbial communities, beneficial bacteria are advantageous for oral health,
whereas harmful bacteria, which cause various oral diseases, also exist (1). Among
oral diseases, dental caries is the most prevalent infectious disease and is expensive to
manage (2). Left untreated, cavities can lead to inflammation of the tissue around the
teeth, infection or abscess formation, and even loss of teeth (3). Bacteria that cause
dental caries are not usually sufficiently concentrated to cause problems. However,
when sugar concentrations increase, acidogenic bacteria in oral microbial communities
metabolize sugars to organic acids and synthesize polysaccharides (4). Acidic condi-
tions skew the bacterial community toward dominance by aciduric bacteria, and ele-
vated polysaccharide levels promote biochemical and structural changes in the biofilm
matrix (dental plaque) (5, 6).
Editor Justin R. Kaspar, Ohio State University
Copyright © 2022 Ham et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Hee-Deung Park,
heedeung@korea.ac.kr.
The authors declare no conflict of interest.
Received 28 October 2021
Accepted 13 April 2022
Published 16 May 2022
May/June 2022 Volume 10 Issue 3 10.1128/spectrum.02076-21 1
RESEARCH ARTICLE

Streptococcus mutans is more viable under acidic conditions than other Streptococcus
species, colonizing the human oral cavity environment (7). Streptococcus mutans is a well-
known causative bacterium of dental caries and utilizes sugars (sucrose, starch, glucose,
and fructose) as sources of carbon (8). Streptococcus mutans secretes glucosyltransferase
(GTF) and fructosyltransferase (FTF) (9). These enzymes synthesize glucan and fructan,
respectively, using sucrose as a substrate, which damage the tooth enamel due to acid pro-
ductionandformationofbiofilms (10).
Streptococcus mutans produces three genetically distinct GTFs (GtfB, GtfC, and GtfD).
Each GTF has a unique role but is eventually involved in the formation of dental plaques
(11). GtfB and GtfC are associated with initial microbial adherence and the structural stabil-
ity of the extracellular matrix (12). GtfB synthesizes insoluble glucans rich in
a
-1,3-glycosidic
linkages and is absorbed onto the bacterial cell surface to promote tight cell clustering for
microcolony formation. GtfC, which is adsorbed to the enamel within the pellicle, produces
a mixture of insoluble and soluble glucans with
a
-1,6-linkages and serves as an attachment
site for bacteria. The role of GtfD in plaque formation remains unclear; however, it synthe-
sizes soluble glucans, which serve as primers for GtfB activity (6, 13, 14).
Overall, GTFs synthesize soluble and insoluble glucans from sucrose, permitting the
bacteria to adhere to glucan and subsequently colonize the dental surfaces (12). GTF
enzymes are involved in the formation of glucan, a glucose multimer, accounting for
10 to 20% of dental biofilm dry weight (7). Glucan plays an important role in glucan-
cell-tooth surface binding by increasing cell aggregation (11). Streptococcus mutans
can easily adhere to tooth surfaces and contribute to the formation of the biofilm and
its structural integrity (15).
To prevent oral diseases such as dental caries, dental biofilms are often eliminated
via nonspecific mechanical removal treatments, such as brushing and flossing, or inhib-
ited by inactivating the growth of cariogenic bacteria through the use of antibiotics
(16). Fluoride is the most effective anticaries agent (17); low concentrations of fluoride
inactivate a variety of enzymes in intact cells, whereas high concentrations of fluoride
enhance the proton permeability of cell membranes as a transmembrane proton car-
rier. Based on these mechanisms, fluoride affects the production and tolerance of acid
and the antimicrobial abilities of S. mutans (18). However, continuous and high concen-
trations of fluoride treatment cause the emergence of resistant bacteria and fluorosis.
Oral bacteria adapted to fluoride treatment exhibit stable resistance to high fluoride
levels (19). Furthermore, excess fluoride treatment hypomineralizes tooth enamel by
increasing the porosity of the surface and subsurface of tooth enamel (20). This alters
the appearance of the tooth, from fine white lines to pitting or staining of enamel.
Therefore, other biofilm-inhibiting agents need to be developed to compensate for
these drawbacks.
Recently, some researchers have focused on developing biofilm inhibitors that can
interfere with the function of GTFs in S. mutans. Many natural compounds (e.g., hops,
green tea, traditional medicinal plants, and food extracts) have been used as antibio-
film agents targeting GTFs (21–23). Koo et al. found that propolis collected by Apis mel-
lifera bees is a potent inhibitor of GTF enzymes, with high inhibitory activities against
GtfB and GtfC (24). They also discovered that apigenin decreases the accumulation of
S. mutans biofilms by affecting the formation of insoluble and soluble glucans in the
polysaccharide matrix (25).
We previously reported that raffinose can reduce Pseudomonas aeruginosa biofilm
formation by decreasing cellular cyclic diguanylate levels (26). Raffinose is widely pres-
ent in various vegetables (e.g., ginger, garlic, kohlrabi, onion, parsnip, and scallion) and
fruits (e.g., apricot and melon) (27). It is a trisaccharide consisting of galactose, glucose,
and fructose. Galactose in raffinose can bind to the LecA protein of P. aeruginosa,
which indicates that galactose and raffinose compete for the same binding site of the
LecA protein. Similarly, S. mutans contributes to biofilm formation by binding sucrose-
derived glucan to GTFs (11). Therefore, the development of novel compounds that
Inhibition of S. mutans Biofilm Formation by Raffinose Microbiology Spectrum
May/June 2022 Volume 10 Issue 3 10.1128/spectrum.02076-21 2

interfere with the binding of glucan to GTFs should be considered to inhibit S. mutans
biofilm formation.
The objective of this study was to develop a novel S. mutans biofilm inhibitor; we
hypothesized that raffinose affects S. mutans biofilm formation by reducing the activity
of GTFs. We investigated S. mutans biofilm formation and the molecular mechanisms
with raffinose treatment to prove the hypothesis. Biofilm formation was tested under
both static and flow conditions, and the antibiofilm mechanism was deduced by esti-
mating sucrose consumption and GtfC binding affinity. GTF-related gene expression
levels and glucan production were measured using reverse transcription-quantitative
PCR (RT-qPCR) and colorimetric methods, respectively. Furthermore, the applicability
of raffinose to dental caries was evaluated using bacterial adhesion tests and scanning
electron microscopy (SEM) analysis of biofilms formed on artificial-saliva-coated hy-
droxyapatite (HA) discs.
RESULTS
Effects of raffinose on S. mutans biofilm formation. Raffinose reduced biofilm for-
mation by most Streptococcus species (KCOM 1136 to KCOM 1228) isolated from human
mouths (Fig. 1). Biofilm formation by S. mutans and S. sobrinus was inhibited, on average,
by 12 to 25% and 11 to 17%, respectively, compared to untreated biofilm (i.e., control
biofilm), when treated with 100 or 1,000
m
Mraffinose. In particular, biofilm formation by
S. mutans KCOM 1136, a representative biofilm-forming bacterium in this study, was
decreased most significantly following raffinose treatment, which resulted in biofilm forma-
tion being reduced by .50%, compared to control biofilm, after 1,000
m
Mraffinose
treatment.
The inhibition of KCOM 1136 biofilm formation by raffinose treatment was analyzed
under static and flow conditions. Under static conditions, biofilm formation was dra-
matically decreased by 44% at a high concentration of raffinose (1,000
m
M) (Fig. 2A).
Under flow conditions, although the morphology of the biofilm seemed to be similar,
with a bumpy shape in control and raffinose-treated biofilms, the average volume and
thickness of the raffinose-treated biofilm were decreased by 54 to 64%, compared to
the control biofilm (Fig. 2B).
Raffinose is a trisaccharide consisting of galactose and sucrose (see Fig. S1A in the sup-
plemental material). However, contradictory patterns in S. mutans biofilm formation were
observed for sucrose and galactose treatments. Galactose exhibited biofilm-inhibiting activ-
ity similar to that of raffinose; in contrast, biofilm formation was enhanced in the presence
of sucrose (see Fig. S1B). Moreover, sucrose increased S. mutans growth at a concentration
of 1,000
m
M (see Fig. S2), while there were no differences in the growth of S. mutans with
raffinose treatment (0 to 1,000
m
M).
Mechanism of S. mutans biofilm inhibition by raffinose. Sucrose consumption of
S. mutans following raffinose treatment was monitored for 24 h to investigate the
FIG 1 Biofilm formation of Streptococcus species following raffinose treatment for 24 h. Biofilms of S.
mutans and S. sobrinus were formed following raffinose treatment (0 to 1,000
m
M) under static
conditions. Error bars indicate the standard deviations of five measurements. **,P,0.005; *,
P,0.05, versus the control. Raf, raffinose.
Inhibition of S. mutans Biofilm Formation by Raffinose Microbiology Spectrum
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relationship between raffinose and sucrose. The method used for assessment of sucrose
consumption was suitable for detecting sucrose concentration but did not respond to
high concentrations of raffinose, as indicated in the standard curves for sucrose and raffi-
nose (see Fig. S3). Streptococcus mutans consumed 88% of the sucrose over 24 h
(Fig. 3A). However, as the concentrations of raffinose with sucrose in S. mutans increased,
sucrose consumption decreased, compared to that in the control (i.e., no raffinose treat-
ment). When 10, 100, and 1,000
m
Mraffinose was used together with 100
m
Msucrose,
79%, 70%, and 56% of the sucrose, respectively, was consumed over 24 h.
Furthermore, competitive biofilm formation was analyzed by adding various con-
centrations of raffinose and sucrose (0 to 1,000
m
M) simultaneously to the culture me-
dium of S. mutans. Biofilm formation decreased by 50% following treatment with
1,000
m
M raffinose, whereas it decreased by 26% with simultaneous treatment with
FIG 2 Streptococcus mutans biofilm formation following raffinose treatment under static and flow conditions. (A) CV-
stained biofilm following raffinose treatment (0 to 1,000
m
M). Quantification was performed by measuring the OD under
static conditions (OD
545
/OD
595
). Error bars indicate the standard deviations of five measurements. **,P,0.005, versus the
control. Raf, raffinose. (B) Volume and thickness of DAPI-stained biofilm based on CLSM images; 1,000
m
M raffinose was
added to the S. mutans biofilm for 48 h under flow conditions. Raf, Raffinose.
Inhibition of S. mutans Biofilm Formation by Raffinose Microbiology Spectrum
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FIG 3 Possible mechanisms underlying the inhibition of Streptococcus mutans biofilm formation following raffinose
treatment. (A) Sucrose consumption in S. mutans biofilm cells treated with raffinose. Streptococcus mutans biofilm was
formed following sucrose (100
m
M) and raffinose (0 to 1,000
m
M) treatments under static conditions for 24 h. Error bars
indicate the standard deviations of three measurements. **,P,0.005, versus the control. Raf, raffinose; Suc, sucrose. (B)
Competitive biofilm formation tests between raffinose and sucrose. Streptococcus mutans biofilm was formed following
treatment with raffinose and sucrose at concentrations of 0 to 1,000
m
M. Error bars indicate the standard deviations of
five measurements. **,P,0.005; *,P,0.05, versus the control. (C) Best-docked poses of raffinose in S. mutans
glucansucrase (GtfC [PDB code 3AIC]).
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1,000
m
M sucrose, compared to that in the control (i.e., no raffinose treatment) (Fig. 3B,
left). This finding indicated that the inhibition of biofilm formation following raffinose
treatment decreased as the sucrose concentration increased. Similarly, as the raffinose
concentration increased, biofilm formation following sucrose treatment decreased
(Fig. 3B, right). These results suggest that raffinose and sucrose have a competitive
relationship regarding sucrose consumption and biofilm formation in S. mutans.
The possibility of raffinose acting as a glucansucrase inhibitor in S. mutans was investi-
gated using molecular docking studies. Figure 3C shows the best-docked poses of raffinose
intheactivesiteoftheS. mutans glucansucrase.Thedockedposeofraffinose was fitted
inside the active site of glucansucrase and formed hydrogen bonds with Asp593, a key
amino acid residue considered to be the most critical point for acceptor sugar orientation,
which influences the transglycosylation specificity of S. mutans glucansucrase (28).
However, the docked pose of sucrose was located only in subunit 1, which consists of Arg
475, Asp477, Glu515, and Asp909, without interaction with Asp593, as shown in Fig. S4A in
the supplemental material. The glucosyl and fructosyl moieties, which are common in both
raffinose and sucrose, made close contact with the amino acid residues of the subunit 1
site. For D-galactose, the best-docked pose was also in close contact only with the amino
acid residues of subunit 1, including Asp475, Asp477, Asp909, and Gln 960 (see Fig. S4B). In
particular, the OH groups at the C-3 and C-4 positions of the galactose moiety in raffinose
interacted with Asp593 as well as with the amino acid residues of the subunit 1 site, which
might enhance its binding affinity for glucansucrase. Overall, the docking studies sug-
gested that raffinose, sucrose, and galactose might be in contact with the subunit 1 site of
glucansucrase, with total scores of 9.90, 7.88, and 6.42, respectively.
Effects of raffinose on GTFs in S. mutans.GTF-related gene expression levels and
glucan production were analyzed to assess the effects of raffinose on GTFs in S.
mutans. As shown in Fig. 4A, all GTF-related genes were significantly downregulated in
S. mutans biofilm cells treated with 1,000
m
M raffinose. The expression of gtfB,gtfC,
and gtfD was repressed by 69%, 74%, and 38%, respectively, compared to that in the
control (S. mutans biofilm cells without raffinose treatment). Galactose also evenly
decreased GTF-related gene expression by 40 to 43%, whereas sucrose increased the
expression levels of GTF-related genes, especially gtfC and gtfD, by 1.9- to 5.5-fold,
compared to those in the control (see Fig. S5A and B). Furthermore, when raffinose
and sucrose were used together, most GTF-related gene levels were not significantly
different from those in the control (see Fig. S5C). The expression of the 16S rRNA refer-
ence gene was not significantly affected by any of the treatments.
FIG 4 GTF-related changes in Streptococcus mutans following raffinose treatment. (A) GTF gene expression levels in S. mutans biofilm cells. Streptococcus
mutans biofilm was formed following raffinose treatment (1,000
m
M) under static conditions for 24 h. Relative fold changes were evaluated by RT-qPCR
analysis. Error bars indicate the standard deviations of five measurements. **,P,0.005; *,P,0.05, versus the control. Raf, raffinose. (B) Relative glucan
production of S. mutans following raffinose treatment. Extracted insoluble glucan was reacted with raffinose (0 to 1,000
m
M). Glucan production was
evaluated using the colorimetric method. Error bars indicate the standard deviations of three measurements. **,P,0.005, versus the control. Raf, raffinose.
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To evaluate the production of water-insoluble glucans, GTFs were extracted, using
solid ammonium sulfate, from S. mutans cultured in brain heart infusion (BHI) medium
containing sucrose. The precipitated GTFs were then reacted with raffinose (0 to
1,000
m
M). Using sucrose as a substrate, the production of glucans was estimated by
measuring the intensity of glucan color using a spectrophotometer. There were no sig-
nificant changes in glucan production in S. mutans at concentrations below 10
m
M raf-
finose (Fig. 4B). However, glucan production was reduced by 18 to 39% after raffinose
treatment at 100 to 1,000
m
M, in a concentration-dependent manner. These results
implied that raffinose affected the downregulation of GTF-related gene expression and
decreased glucan production in S. mutans (Fig. 5).
Application of raffinose on artificial-saliva-coated HA discs. Artificial-saliva-coated
HA discs were used to simulate conditions of the oral environment in humans.
Adhesion of S. mutans was evaluated by staining and counting biofilm cells on HA
discs. As shown in Fig. 6A, S. mutans biofilm cells on HA discs treated with 1,000
m
M
raffinose were not stained well, compared to the control, which indicated that S.
mutans cells had difficulties in adhering to HA discs after raffinose treatment. Similar
results were observed with the colony-counting method, in which the number of colo-
nies on HA discs was diminished following raffinose treatment (Fig. 6B).
The effects of raffinose on S. mutans biofilm formation on HA discs were also dem-
onstrated via SEM analysis. There was no difference between HA discs coated with or
without artificial saliva in the SEM images (Fig. 7A and B). Figure 7C and D show SEM
images of S. mutans biofilms formed on HA discs. Biofilm formation after 1,000
m
M raf-
finose treatment was reduced significantly, compared to the control (no raffinose treat-
ment). Galactose also adversely affected biofilm formation, whereas sucrose increased
it (see Fig. S6). This finding indicates that raffinose can effectively control S. mutans
FIG 5 GTF-mediated biofilm inhibition in Streptococcus mutans following raffinose treatment. GTFs
secreted by S. mutans are adsorbed on the pellicle and bacterial surfaces. The adsorbed GTFs bind to
sucrose-derived glucan. Glucan provides binding sites on the surfaces for S. mutans, mediating
adherence to the tooth enamel and tight bacterial clustering and eventually promoting biofilm
formation. However, if the S. mutans biofilm is treated with raffinose, then the raffinose is expected
to prevent sucrose-derived glucan from binding to GTFs. Therefore, the activity of glucan production
in S. mutans is reduced, which may retard biofilm formation.
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biofilm formation under conditions similar to those of the human oral environment.
However, these results do not reliably indicate the applicability of raffinose in the treat-
ment of dental caries. To prevent or treat dental caries with raffinose, further research
should be conducted, including clinical demonstration and development of efficient
methods for raffinose application.
DISCUSSION
In this study, we discovered that raffinose reduced biofilm formation by bacteria that
cause dental caries in humans. The common cariogenic bacteria, S. mutans and S. sobrinus,
adhere to the enamel salivary pellicle and other plaque bacteria to form biofilms (29).
Raffinose inhibited S. mutans biofilm formation more effectively than S. sobrinus biofilm for-
mation (Fig. 1). S. mutans is predominantly found in dental biofilm development (74 to
94% of the diverse carious bacterial population), whereas S. sobrinus is less prevalent and is
FIG 6 Adhesion of Streptococcus mutans to artificial-saliva-coated HA discs following raffinose treatment
(1,000
m
M). (A) Bacterial adhesion evaluation using CV staining. Streptococcus mutans biofilm was formed on
HA discs for 24 h under static conditions, and OD
545
was assessed. Error bars indicate the standard deviations
of three measurements. *,P,0.05, versus the control. Raf, raffinose. (B) Bacterial adhesion evaluation using
the cell-counting method. The number of colonies of separated biofilm cells that formed on the HA discs was
calculated using the standard plate culture method. Error bars indicate the standard deviations of three
measurements. **,P,0.005, versus the control.
FIG 7 SEM images of Streptococcus mutans biofilm cells following raffinose treatment (1,000
m
M) on
artificial-saliva-coated HA discs. (A) HA discs. (B) Artificial-saliva-coated HA discs. (C) Streptococcus
mutans biofilm cells on artificial-saliva-coated HA discs. (D) Raffinose-treated S. mutans biofilm cells
on artificial-saliva-coated HA discs.
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usually detected along with S. mutans (30). S. mutans mainly synthesizes polysaccharides
from sucrose as a substrate, contributing to an increase in bacterial cell surface hydropho-
bicity and biofilm formation (31). Streptococcus mutans biofilm formation increased in
response to sucrose, as shown in Fig. S1B in the supplemental material.
Sucrose accelerates S. mutans biofilm formation by targeting GTFs (32). Our RT-qPCR
results showed that raffinose mainly downregulated the expression of gtfB and gtfC in
S. mutans (Fig.4A).However,gtfD expression slightly decreased after raffinose treatment.
GtfB and GtfC are highly homologous and share 75% amino acid sequence identity in an
operon-like arrangement. In contrast, GtfD, which is not linked to the gtfBC locus, shares
,50% identity with GtfB and GtfC (25). The different patterns for gtfB-C and gtfD may be
related to various factors (carbohydrate availability and source, environmental pH, and
growth rate/phase) that influence the transcription of gtf genes (25).
Streptococcus mutans synthesizes sticky, water-insoluble/soluble extracellular glu-
cans from sucrose as the sole substrate via the production of GTFs (33). Sticky glucans
enhance the pathogenic potential of dental plaque by providing binding sites for
S. mutans colonization and promoting accumulation on tooth surfaces. A large propor-
tion of glucans contribute to the establishment of the extracellular polysaccharide
matrix and provide bulk and structural integrity to dental biofilms (24, 25). In particular,
insoluble glucans are directly related to bacterial attachment during biofilm formation
by S. mutans in dental caries. In contrast, soluble glucans indirectly contribute to bio-
film formation as a nutrient source for biofilm cells (31). The production of insoluble
glucans decreased in proportion to the concentration of raffinose (Fig. 4B).
Most studies regarding biofilm inhibitors for dental caries have focused largely on
enzymatic activity in solution, without considering the importance of enzymes
adsorbed on the pellicle of the tooth surface. Current commercially available antibio-
film agents show high levels of resistance to surface-adsorbed GTF enzymes; thus, it
should be considered that inhibitors of GTF function effectively under conditions simi-
lar to those of the oral environment (34). In this study, we used saliva-coated HA, the
main constituent of tooth enamel, because GTFs are detected in whole saliva at high
levels in individuals with dental caries (11). Furthermore, saliva-coated HA can be
tightly bound with GTFs, although GTFs have a low binding affinity for HA and lose
their activity over time. As shown in Fig. 7, S. mutans biofilm formation on saliva-coated
HA discs was decreased following raffinose treatment.
Although all GTFs interact with saliva-coated HA discs, GtfC has a higher binding af-
finity, compared with those of GtfB and GtfD (12). GtfC is significantly adsorbed onto
the pellicle when exposed to sucrose, thereby enhancing the adherence of bacterial
cells to dental surfaces (35). Therefore, inhibition of GTF activities, especially those of
GtfC and mainly those adsorbed to the salivary pellicle, is prioritized to prevent the for-
mation of pathogenic dental plaque (24). GtfC secreted by S. mutans is commonly
incorporated into the pellicle, and surface-adsorbed GtfC utilizes sucrose to produce
glucans (11). Glucans provide binding sites on the surface of S. mutans to easily adhere
to tooth enamel, facilitating biofilm formation. In our study, raffinose treatment
degraded glucan production, and S. mutans had difficulty adhering to saliva-coated HA
discs, eventually resulting in inhibition of biofilm formation on HA discs (Fig. 5).
This phenomenon might be related to the interruption of GTF by raffinose. Streptococcus
mutans consumes sucrose to synthesize glucan, thereby increasing biofilm formation (11).
When raffinose was combined with sucrose in S. mutans, sucrose consumption and biofilm
formation decreased (Fig. 3A), suggesting that raffinose may control GTF activity in S.
mutans. In particular, this may be related to the possibility of raffinose binding to GtfC
(Fig. 3C). Galactose in raffinose has additional interactions with Asp593, a key amino acid res-
idue, and amino acid residues of the subunit 1 site, which might enhance its binding affinity
for glucansucrase. Furthermore, galactose showed inhibitory activities similar to those of raf-
finose in biofilm formation and GTF gene expression in S. mutans, whereas sucrose showed
the opposite effects (Fig. S1B and Fig. S5). Ryu et al. also discovered that galactose signifi-
cantly inhibits S. mutans biofilm formation by decreasing the expression of three GTF genes
Inhibition of S. mutans Biofilm Formation by Raffinose Microbiology Spectrum
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(36). These results suggest that galactose may play an important role in the various activities
of raffinose in S. mutans. However, these results do not provide direct evidence that raffi-
nose binds to GtfC. Further studies are required to elucidate the detailed mechanism by
which raffinose affects GTF activity in S. mutans.
Nagasawa et al. reported that raffinose induces S. mutans biofilm formation with
low concentrations of sucrose, which is contrary to our findings (31). Although there
may be many reasons for the difference in biofilm formation between the two studies,
we cautiously suggest that the difference is correlated with the concentration of raffi-
nose. Streptococcus mutans biofilm formation decreased in proportion to the raffinose
concentration (0 to 1,000
m
M) in our study. However, the inhibition of S. mutans bio-
film formation by raffinose treatment decreased as the sucrose concentration
increased (see Fig. S7A). This can be explained by the fact that raffinose is insufficient
to inhibit biofilm formation at high levels of sucrose but this does not decrease the
activity of raffinose. Conversely, raffinose treatment above 0.03% increases S. mutans
biofilm formation (31). A raffinose concentration of 0.03% is equal to 620
m
M, which is
relatively higher than the raffinose concentrations used in our study. This difference
was expected to be due to the remaining raffinose used to inhibit S. mutans biofilm
formation. Raffinose can serve as a substrate for FTF in Streptococcus species but is not
used by GTF (37–39). Nagasawa et al. also found that raffinose induces S. mutans bio-
film formation through fructan synthesis (31). Furthermore, raffinose treatment gener-
ally shows biofilm formation patterns; however, at a concentration of less than 0.03%
raffinose, there are slight biofilm inhibition patterns. Similarly, in this study, S. mutans
biofilm formation increased following treatment with millimolar concentrations of raffi-
nose (see Fig. S7B). Therefore, determination of the raffinose concentrations suitable
for environmental conditions should be considered when applying raffinose to regu-
late S. mutans biofilm formation.
Raffinose, an oligosaccharide found in natural products, greatly reduced S. mutans bio-
film formation under static and flow conditions. Furthermore, GTF-related gene expression
levels and glucan production were decreased following 1,000
m
Mraffinose treatment.
Moreover, the reduction in bacterial adhesion following raffinose treatment delayed S.
mutans biofilm formation on saliva-coated HA discs. The activities of raffinose can be
explained by the possibility of binding to GtfC by raffinose.Hence,raffinose has the poten-
tial to be utilized as a natural substance to prevent S. mutans biofilmformationintheoral
environment.
MATERIALS AND METHODS
Bacteria and chemicals. S. mutans (strain KCOM 1136; Korean Collection for Oral Microbiology
[KCOM]) used in this study was clinically isolated and identified in the oral microbiota of Korean subjects.
Streptococcus mutans cultured overnight was prepared by incubation with BHI medium (Difco, Detroit,
MI, USA) at 37°C in a shaking incubator (200 rpm).
D-(1)-Raffinose pentahydrate, sucrose, and D-(1)-galactose were selected as chemicals that affect S.
mutans biofilm formation. These chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and
dissolved in dimethyl sulfoxide (DMSO) (Carl Roth, Karlsruhe, Germany).
Static biofilm formation test. Streptococcus mutans cultured overnight was adjusted to an optical
density at 595 nm (OD
595
) of 1.0 using a UV-visible spectrophotometer (UV mini-1240; Shimadzu, Kyoto,
Japan) and then diluted 1:20 (vol/vol) in BHI medium containing 10
m
M sucrose. The diluted bacterial
culture was grown in the presence of 0 to 1,000
m
M sugars for 24 h in 96-well polystyrene microtiter
plates (Sigma-Aldrich) and borosilicate bottles. The plates and bottles were incubated at 37°C without
agitation, to allow biofilm formation. After 24 h of incubation, the OD
595
of the suspended cultures in
the plates and bottles was measured using an iMark microplate reader (Bio-Rad, Richmond, CA, USA) to
analyze the growth of the bacterial cells. The suspended culture was then discarded, and biofilm cells
attached to the plates and bottles were washed with phosphate-buffered saline (PBS) (137 mM NaCl,
2.7 mM KCl, 10 mM Na
2
HPO
4
, and 2 mM KH
2
PO
4
[pH 7.2]) to remove the remaining suspended culture
medium. Biofilm cells were stained with 0.1% crystal violet (CV) for 30 min. After the staining period, CV
was washed using sterile deionized water to remove the remaining dye and dissolved in 100% ethyl
alcohol. The remaining CV was measured at 545 nm using a microplate reader. The extent of CV staining
indicated the amount of biofilm formed. Biofilm formation was normalized by dividing OD
545
by OD
595
.
Flow biofilm formation test. To form biofilms under flow conditions, glass slides were inserted into a
drip-flow reactor (DFR-110; BioSurface Technologies Corp., Bozeman, MT, USA). BHI medium containing a
dilution of S. mutans cultured overnight (OD
595
of 1.0) with or without raffinose (1,000
m
M) was fed into the
reactor using a peristaltic pump (Masterflex C/L tubing pump; Cole-Parmer, Vernon Hills, IL, USA) at a flow
Inhibition of S. mutans Biofilm Formation by Raffinose Microbiology Spectrum
May/June 2022 Volume 10 Issue 3 10.1128/spectrum.02076-21 10

rate of 0.3 mL/min. The reactor was operated at 37°C for 48 h. At the end of the reactor operation, the slides
were carefully removed from the reactor. Biofilms formed on the slides were washed with PBS twice and
stained with 49,6-diamidino-2-phenylindole (DAPI) (Carl Roth) for confocal laser scanning microscopy (CLSM)
(LSM 700; Carl Zeiss, Jena, Germany) analysis.
Biofilms were measured in z-stack mode under blue fluorescent light (excitation wavelength, 350 nm;
emission wavelength, 470 nm) with a 20lens objective (W N-Achroplan 20/0.5 W [DIC] M27). Biofilm mor-
phology was analyzed using ZEN 2011 software (Carl Zeiss), and the volume and thickness of biofilms were
measured using the Comstat2 tool of ImageJ software (National Institutes of Health, Bethesda, MD, USA) (40).
Sucrose consumption test. A sucrose consumption test was conducted by modifying the method
presented in a previous study to suit the experimental conditions (41). A dilution of S. mutans cultured
overnight (OD
595
of 0.05) in BHI medium treated with sucrose (100
m
M) and raffinose (10 to 1,000
m
M)
was aliquoted in borosilicate bottles at 37°C for 24 h. To calculate sucrose consumption, bacterial sus-
pensions were sampled every 6 h and passed through a 0.22-
m
m membrane filter. Sucrose consumption
by the suspension was examined using a sucrose assay kit (Sigma-Aldrich). The filtered suspension
(100
m
L) was incubated at 25°C for 10 min with 100
m
L of sucrose assay reagent. After the addition of
2,000
m
L of glucose assay reagent, the reaction mixture was incubated again at 25°C for 15 min. Sucrose
consumption was evaluated by measuring the OD
340
using a spectrophotometer.
In silico docking studies. All compounds were rendered as two-dimensional (2D) and three-dimensional
(3D) structures using ChemDraw Ultra v.12.0.2.1076 and Chem3D Pro v.12.0.2, respectively. Ligand preparation
and optimization were performed using the Sanitize preparation protocol in SYBYL-X v.2.1.1 (Tripos Inc., St.
Louis, MO, USA). The GtfC protein structure (Protein Data Bank [PDB] code 3AIC) in PDB format was down-
loaded from the Research Collaboratory for Structural Bioinformatics (RCSB) PDB. The SYBYL-X v.2.1.1 program
was employed for protein preparation, including fixation of conflicting side chains of amino acid residues.
Water molecules were removed from the protein crystal structure, and chains other than chain A were also
removed. Hydrogen atoms were added under the application of AMBER7 FF09 for the force field setting. The
minimization process was performed using the POWELL method. The initial optimization option was set to
zero. Docking studies involving the ligands were performed using the Surflex-Dock GeomX module in SYBYL-
X v.2.1.1. Docking was guided by the Surflex-Dock protocol, and the docking site was defined by the Ligand
method with the complexed ligand
a
-acarbose with a threshold value of 0.50. Other parameters were applied
with default settings in all runs.
RT-qPCR. A dilution of S. mutans cultured overnight (OD
595
of 0.05) in BHI medium containing 10
m
Msu-
crose and treated with or without raffinose (1,000
m
M) was incubated in borosilicate bottles at 37°C for 24 h.
Biofilm samples were collected by scraping bacterial cells attached to the bottles with PBS. Total RNA was
extracted from the biofilm samples using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). RT-
qPCR was performed with SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) on a Bio-Rad CFX 96 real-time PCR sys-
tem (Bio-Rad). The primer sets for GTF-related genes were designed using Primer 3 v.0.4.0 (http://frodo.wi.mit
.edu) (see Table S1 in the supplemental material). Thermocycling conditions for the designed primer sets
were as follows: initial denaturation at 95°C for 10 s, followed by 40 cycles of denaturation at 95°C for 10 s,
annealing at 60°C for 10 s, and extension at 63°C for 34 s. Fluorescent signal intensities were measured at the
end of the final extension step. Relative gene expression was normalized to that of 16S rRNA as a reference
gene (42) and analyzed using the 2
2DDCT
relative expression method (43).
Glucan production test. A glucan production test was conducted by modifying the methods used
in previous studies (44, 45). A dilution of S. mutans cultured overnight (OD
595
of 0.01) in BHI medium
containing 10
m
M sucrose was incubated at 37°C for 24 h with shaking (200 rpm). The supernatant of
incubated S. mutans obtained by centrifugation at 4°C for 30 min was precipitated with solid ammonium
sulfate (Sigma-Aldrich). After agitation at 4°C for 1 h and centrifugation at 10,000 gat 4°C for 30 min,
the precipitate was diluted with 10 mM potassium phosphate buffer (pH 6.0). The extracted crude GTFs
were stored at 280°C.
To measure the effects of raffinose treatment on water-insoluble glucan production, mixtures of GTFs
treated with raffinose (0 to 1,000
m
M) and 20
m
L of 0.0625 M potassium phosphate buffer (pH 6.5) were
reacted with 12.5
m
g/L sucrose and 0.25
m
g/L sodium azide (Sigma-Aldrich) and then incubated at 37°C for
24 h. Precipitated water-insoluble glucans were dispersed using a sonicator (VCX 750; SONICS, Newtown, CT,
USA) for 4 cycles of 5 s of operation and 5 s of pause, at a frequency of 3.5 Hz. The glucan production was
estimated by measuring the OD
550
using a UV-visible spectrophotometer.
Bacterial adhesion test. Dense ceramic HA discs (Clarkson Chromatography Products, Williamsport,
PA, USA) were coated with artificial saliva. Following an incubation period at 25°C for 12 h, the HA discs
were rotated at 5 rpm for 1 h with artificial saliva. Artificial saliva was prepared according to the ISO/
TR10271 standard, i.e., 0.04% sodium chloride (NaCl), 0.08% calcium chloride dihydrate (CaCl
2
2H
2
O),
0.04% potassium chloride (KCl), 0.0005% sodium sulfide dihydrate (Na
2
S2H
2
O), 0.08% sodium dihydro-
gen phosphate (NaH
2
PO
4
2H
2
O), and 0.1% urea (CH
4
N
2
O) (30). The coated discs were then washed for
30 min with buffered KCl containing 5 mg/mL bovine serum albumin.
A dilution of S. mutans cultured overnight (OD
595
of 0.01) in BHI medium containing 10
m
M sucrose
was adhered to artificial-saliva-coated HA discs by incubating the bacterial cells with or without raffinose
treatment (0 to 1,000
m
M) at 37°C for 1.5 h. Adherent bacterial cells on HA discs were washed twice with
PBS and then analyzed using CV staining and cell-counting methods. CV staining was performed as
described above for the static biofilm formation test. For cell counting, HA discs were sonicated for
10 min at an amplitude of 30%, using a sonicator, to detach bacterial cells from the discs. The detached
bacterial cells were dispersed, diluted, spread on BHI agar plates, and then incubated at 37°C for 24 h.
CFU were calculated by counting the number of bacterial colonies formed on agar plates.
Inhibition of S. mutans Biofilm Formation by Raffinose Microbiology Spectrum
May/June 2022 Volume 10 Issue 3 10.1128/spectrum.02076-21 11

SEM analysis. A dilution of S. mutans cultured overnight (OD
595
of 0.05) in BHI medium containing
10
m
M sucrose was incubated with glass slides at 37°C for 24 h. Biofilm cells on the slides were fixed
with 4% glutaraldehyde solution (Sigma-Aldrich) at 4°C for 1 h. Then, the fixed biofilm cells were sequen-
tially dehydrated in 50%, 80%, and 100% ethyl alcohol for 20 min each. Dehydrated biofilm cells were
dried in a vacuum desiccator for 12 h and coated with platinum for 90 s using an E-1030 ion-sputter
coater (Hitachi, Tokyo, Japan). The coated sample was observed using a model S-4300 scanning electron
microscope (Hitachi) operated at a magnification of 5,000 and a voltage of 15.0 kV.
Statistical analysis. Pvalues were estimated via Student's ttest using SigmaPlot (Systat Software,
Inc., San Jose, CA, USA).
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.7 MB.
ACKNOWLEDGMENTS
This work was supported by the National Research Foundation of Korea (grant
2020R1A6A1A03045059 to H-D.P. and grant 2019R1A6A1A03031807 to Y.B).
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