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222]

Original Article

Synergic antibacterial effect between

Maillard reactive product (MRP)
and hydrogen peroxide (H 2O 2) on
Streptococcus mutans
Morimichi Mizuno, Ki‑ichiro Inoue
Department of Oral Health Science, School of Dentistry, Hokkaido University, Sapporo, Japan

Address for correspondence: Dr. Morimichi Mizuno, Kita 13 Nishi 7, Sapporo, Japan. E‑mail: mmizuno@den.hokudai.ac.jp

ABSTRACT Objectives: To evaluate the antibacterial activity of resin composite containing Maillard reactive product
(MRP) and hydrogen peroxide (H2O2) on Streptococcus mutans (S. mutans), and to investigate the
antibacterial mechanism involved. Methods: The growth of S. mutans was investigated after dental
resin containing H2O2 in the presence and absence of MRP, was immersed into bacterial solution. The
effect of MRP on H2O2 degradation was examined by the measurement of H2O2 content. Results: The
resin composite containing MRP and H2O2 showed stable antibacterial activity compared with resin
containing H2O2 only, and the effect of MRP was speculated to be the suppression of H2O2 degradation,
and the presence of H2O2 correlated with the antibacterial activity of resin composite. These results
indicated that the antibacterial activity of resin composite containing MRP and H2O2 on S. mutans was
dependent on the presence of H2O2, and MRP suppressed the degradation of H2O2 after combination
with H2O2. EDTA also suppressed the degradation of H2O2. Conclusions: An antibacterial effect of resin
composite containing MRP and H2O2 on S. mutans was observed. The effect of MRP on H2O2 might be
a metal chelating action. Application of resin composite containing MRP and H2O2 to a caries dentine
could be an alternative therapy to or serve as an additional minimally invasive antibacterial treatment.

Keywords: Hydrogen peroxide (H2O2), maillard reactive product (MRP), Streptococcus

mutans (S. mutans)

INTRODUCTION This recognition is a basic concept to develop atraumatic

Today, the concept of minimally invasive dentistry that
aims to manage dental caries by providing optimal
preventive care and minimally invasive operative
interventions has been widely accepted.[1]
restorative treatment (ART) system. ART involves the
removal of carious tooth tissues with hand instruments
followed by the restoration of cavity with an adhesive
dental material (currently a high‑viscosity glass ionomer
cement) that simultaneously seals the remaining pits

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DOI: How to cite this article: Mizuno M, Inoue Ki. Synergic antibacterial effect
10.4103/2321-4619.168731 between Maillard reactive product (MRP) and hydrogen peroxide (H2O2)
on Streptococcus mutans. J Res Dent 2015;3:64-9.

64 • © 2015 Journal of Restorative Dentistry | Published by Wolters Kluwer - Medknow

[Downloaded free from http://www.jresdent.org on Wednesday, November 04, 2015, IP: 114.125.170.222]
Mizuno and Inoue: Synergic effect of MRP plus H2O2 enhanced the antibacterial activity
and fissures. By this treatment, the blockage of bacterial
invasion into the dentin is expected.
Glass ionomer cement contains fluoride for facilitating
remineralization but has less antibacterial activity,
which is one of reasons for the unreliability of the ART
system.[2]
One way of removal of the bacteria is the application
of antibiotics complex into caries or cavities and this
procedure was recognized to show good clinical
outcomes.[3]
However, antibiotics show limited bacteria spectrum
due to the difference of inhibitory action for each
bacterial metabolic pathway. Apart from this, long‑term
application of antibiotics induces the tolerance of bacteria
against antibiotics.
Chlorhexidine is a typical antibacterial agent[4]   and was
used to study the efficacy of antibacterial activity provided
on glass ionomer cement.[5]
Hydrogen peroxide  (H2O2) is an antibacterial reagent,
which kills most species of anaerobes by strong oxidizing
action.[6] The antibacterial action is due to hydroxyl
radical, which is produced by the interaction of H2O2
with iron ion present in the bacteria.[7] By the reaction
of H2O2 with iron ion, water and oxygen are produced.
Therefore, H2O2 might be a suitable antibacterial reagent
for the removal of bacteria  from the carious dentin.
H2O2 has been widely utilized as a liquid and in the
vaporized form.  However, the application and fixing of
H2O2 solution at the carious dentin is difficult owing to
the high fluidity of the H2O2 solution.
In this study, we developed carbohydrate polymers
containing H2O2 and produced antibacterial dental resin
involving H2O2.
Maillard reactive product  (MRP) is an aminocarbonyl
reaction product caused by maillard reaction between
amino compounds (amino acid, peptide, and protein)
and the reducing carbohydrate.[8] It performs several
activities such as antioxidative,[7,9] antihypertensive,[8,10]
metal chelating,[9,11] and antibacterial activities.[12,13]
From the chemical structural analysis it was found that
MRP acts as an anionic material,[14] and is able to form
stable complexes with metal cation.[15] These findings
imply that MRPs could possess anionic charge and
chelate some cations such as Fe, Zn, and Cu,[16] which
are essential for the growth and survival of some
bacteria.[17]
Journal of Restorative Dentistry / Vol - 3 / Issue - 3 / Sep-Dec 2015 • 65
On the basis of these researches, we examined the
potential of synergic effect between MRP and H2O2 and
investigated the antibacterial effect on Streptococcus
mutans (S. mutans) in order to investigate the mechanism
involved.
MATERIALS AND METHODS
Synthesis of soluble MRP, and preparation
of dental resin containing MRP and H2O2
The soluble MRP was prepared by modification of the
method described by Morales and Babber.[18] Glucose of
50 mg and the same amount of histidine were dissolved
in 1.5 mL or 3 mL of physiological saline (PBS) and was
heated at 120°C for 30 min by autoclave and then cooled
until it reached room temperature.
MRP solution 150 μL (5 mg and 10 mg of MRP/150 μL)
and the same volume of 1% H2O2 solution (1.5 mg/150
μL of H2O2), which is of a similar concentration to that
used as an oral disinfection,[19] were mixed equivalently
and then adsorbed to 50 mg of carbohydrate polymer
compound of amylase (60%), amylopectin (30%), and
cellulose (10%).
It was held at room temperature under anaerobic
condition to avoid an oxidation of polymer compound
during the dry process. After we confirmed the absence
of water in the polymers by the moisture meter (MT‑900,  
Kett co., Tokyo, Japan), the polymers were made into fine
powder, the particle size of which was below 100 μ, by the
powder mixer (Dr. Fritsch - Sondermaschine co., Fellbach,
Germany). The fine powder was kept at ‑30°C until use.
Two hundred mg of light cure type resin (Kuraray
medical Inc; Kurashiki, Japan) was mixed with 50 mg
of polymer and formed to disc (3‑mm diameter, 1‑mm
thickness). The resin contained 5 mg and 10 mg of MRP,
and 1.5 mg of H2O2.
Measurement of the antibacterial activity of
MRP plus H2O2
In this study, we used 1 × 10 6 colony forming
unit  (CFU)/mL of S. mutans (ATCC25175) cultured in
brain‑heart infusion (BHI) media (Eiken, Tokyo, Japan)
under an anaerobic condition at 35°C.
The bacterial samples (1 mL) were incubated with dental
resin disc for 2 h at room temperature in anaerobic
condition. Then, cell suspensions were centrifuged at
1,000 rpm for 15 min. The antibacterial activity was
evaluated by the bacterial growth, which was determined
by measuring the optical density at OD660 of each sample
using a spectophotometer  (Shimazu, Kyoto, Kyoto
Prefecture, Japan) as described by Kim et al.[20] All the
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Mizuno and Inoue: Synergic effect of MRP plus H2O2 enhanced the antibacterial activity
experiments were performed in duplicate and were
repeated thrice (n = 6).
Measurement of H2O2 content in dental
resin in the presence and absence of MRP
The contents of H2O2 in dental resins were measured by
the modified method described by Gay et al.[21] The dental
resins were immersed in 1 mL of distilled water and held
for 2 h at room temperature followed by centrifugation at
10,000 g for 3 min at 20°C. Then, 10 μL of the supernatant
was mixed with 1 mL of the reagents to give final
concentrations of 25 mM of H2SO4, 100 μM of xylenol
orange (XO), and 150 μM of ferrous ammonium sulfate
in a volume of 1 mL. After the mixtures were held at
room temperature for 30 min in a dark environment, the
density of the mixture was measured at the absorbance of
560 nm with XO as blank. The measurement was carried
out triplicate in the independent experiments.
Statistical analysis
The effect of MRP on bacterial growth and content of
H2O2 were assessed by comparing the samples with the
presence and absence of MRP using the t‑test. All the
applied tests were two‑tailed and a P value below 0.05
was considered statistically significant.
RESULT AND DISCUSSION
An antibacterial activity of MRP and H2O2
The bacteria in tooth might interfere with remineralization
of the dentin by the acidic metabolites produced by them.
Therefore, removal of the bacteria   from the carious
dentin may facilitate remineralization.
Figure 1: The antibacterial activity of dental resin involving MRP or
H2O2. Bacterial growth was expressed as the percentage OD660 of the
nonincubated bacteria medium with dental resins (100%). Values were
expressed as mean ± standard deviation (SD) from six samples and
P < 0.05 indicated statistically significance
66 • Journal of Restorative Dentistry / Vol - 3 / Issue - 3 / Sep-Dec 2015
Deyhle et al. demonstrated that there was no difference
of collagen network between normal dentin and  carious
dentin in an early infected stage,[22] and Zhang et al.
reported that the phosphorylation combined with the
calcium hydroxide pretreatment definitely induced
remineralization on the surface of the demineralized
dentin.[23] They assumed that the increase of negative
zeta potential of collagen molecules induces remarkable
remineralization of the demineralized dentin not
containing bacteria.[23]
These findings indicate that the demineralized dentin is
able to remineralize when networks of collagen fibers
keep normal quality and arrangement and that removal
of the bacteria from the demineralized carious dentin
prefers to induce remineralization of the dentin.
First of all, we investigated the antibacterial activity of
MRP used in this study. Rufián‑Henares et al. reported
that 2 ~ 8 mg/mL of MRP is sufficient to inhibit bacterial
growth.[13] Then, we formed dental resins involving
5 mg and 10 mg of MRP followed by incubation at 37°C
under 100% humidity for 7 days. Then, the resins were
immersed in bacterial medium (1 mL) for 2 h at room
temperature in anaerobic condition (MRP concentration
in bacteria medium was 5 mg and 10 mg/mL).
As shown in Figure 1, nonincubated dental resins
containing 1.5  mg of H2O2 inhibited bacterial growth;
however, the inhibitory effect was lost after incubation for
1 day at 37°C in 100% humidity. On the other hand, the
dental resins containing 5 mg and 10 mg of MRP did not
suppress bacterial growth during the experimental period.
These findings indicate that 5 mg/mL and 10 mg/mL of
MRP did not show antibacterial activity. Our finding
was different from the report of Rufián et al.[13] and
Figure 2: The antibacterial activity of resins including MRP (5 mg and
10 mg) and H2O2. Bacterial growth was expressed as the percentage
OD660 of the nonincubated bacteria medium with dental resins (100%).
Values were expressed as mean ± SD from six samples and P < 0.05
indicated statistically significance
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Mizuno and Inoue: Synergic effect of MRP plus H2O2 enhanced the antibacterial activity
the discrepancy might be due to the difference of the
experimental condition. Rufián et al. used several bacteria
instead of S. mutans and the difference of bacteria species
might propose the different sensitivity toward MRP.
Second, they used MRP derived from coffee, which
contains several kinds of high molecular weight MRP.
On the other hand, the MRP we used in this study had
low molecular weight and the difference in the quality
of MRP might reflect the difference in the experiment
results.
Next, we investigated the antibacterial activity of MRP
and H2O2.
The dental resins containing MRP  (5  mg and 10  mg)
plus 1.5 mg of H2O2 were incubated at 37°C under 100%
humidity for 7 days and were then immersed into 1 mL of
bacterial medium (1.5 mg/mL of H2O2, 5 mg and 10 mg/mL
of MRP in bacterial medium). As shown in Figure 2, the
antibacterial activity of resin including MRP and H2O2
was similar to the resins containing H2O2 without MRP
before the incubation. After incubation for 1 day, the
antibacterial activity of resins including H2O2 plus 5 mg
of MRP decreased to one‑third fold compared with the
nonincubated resins; however, resins containing H2O2 plus
10 mg of MRP maintained initial antibacterial activity. At
day 2, resins involving H2O2 plus 10 mg of MRP decreased
the antibacterial activity to one‑fourth fold compared with
the initial activity and resins containing 5 mg of MRP lost
the antibacterial activity. At day 3, the antibacterial activity
was not recognized in all the resins.
These findings reveal that MRP did not enhance the
antibacterial activity of H2O2; however, the antibacterial
activity was expressed for a longer duration by the
presence of MRP dose dependently. Statistical analysis
Figure 3: The content of H2O2 in dental resins involving MRP (5 mg and
10 mg). H2O2 content was measured in 1 mL of   distilled water in which
the resins were immersed. Values were expressed as mean ± SD from
six samples and P < 0.05 indicated statistically significance
Journal of Restorative Dentistry / Vol - 3 / Issue - 3 / Sep-Dec 2015 • 67
showed that the effect of MRP plus H2O2 was definite.
Therefore, it might be concluded that the antibacterial
mechanism of MRP against H2O2 was the conservation
of antibacterial activity of H2O2.
Contents of H2O2 in the dental resins
We speculated that MRP might suppress the degradation
of H2O2. To confirm this hypothesis, we measured H2O2
content in the dental resins after incubation at 37°C under
100% humidity for 7 days.
As shown in Figure 3, resins containing H2O2 or H2O2
plus MRP (5 mg and 10 mg) showed 55.5 mM, 55.1 mM,
and 55.3 mM of H2O2 before incubation, respectively, and
approximately 80% of bacterial growth was inhibited as
shown in Figure  2. Our results that H2O2 released from
the resins suppressed bacterial growth were not in conflict
with the finding of Feuerstein that 10 mM of H2O2 showed
65% of growth inhibition of S. mutans.[24] After the resins
were incubated for 1 day, the content of H2O2 in the resins
without MRP decreased to 5.6 ± 4.29 mM. On the other
hand, resins containing 5 mg and 10 mg of MRP showed
16.7 ± 7.22 mM and 44. 4 ± 9.11 mM, respectively. At day 2,
H2O2 content in resins containing 5 mg and 10 mg of MRP
was 5.6 ± 3.39 mM and 15.6 ± 7.78 mM and H2O2 was not
detected in resins without MRP. At day 3, 5.6 ± 2.23 mM
of H2O2 was detected in resins containing 10 mg of MRP;
however, H2O2 was not detected in resins containing 5 mg
of MRP. Statistical analysis indicated that MRP inhibited the
degradation of H2O2 but did not increase the H2O2 content.
Our results also showed that the presence of H2O2 in
resins is a main reason for the inhibition of bacterial
growth (coefficient of determination R2 = 0.995).
Therefore, the mechanism of antibacterial effect of MRP
against H2O2 might be suppression of H2O2 degradation
by MRP.
The possible suppressive mechanism of MRP against
H2O2 degradation is (1) interaction between MRP and
H2O2, which may form a stable complex and (2) the metal
chelating action of MRP that inhibits the production
of the hydroxyl radical by the suppression of H2O2
degradation.
In these possibilities, the formation of a complex between
MRP and H2O2 is hard to assume because of no reports
concerning the complex formation so far.
Repine et al.[25] reported that 1.5 μg of iron enhanced
1,000‑fold of bacteria killing activity of H2O2 compared
with 0.1 μg of iron against Staphylococcus aureus and
this finding means that hydroxyl radical production is
accelerated by the above amount of ion. It was reported
that iron ions present in bacteria act as catalytic agent
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Mizuno and Inoue: Synergic effect of MRP plus H2O2 enhanced the antibacterial activity
Figure 4: The content of H2O2 in dental resins involving 10 mM of
EDTA. H2O2 content was measured in 1 mL of   distilled water in which
the resins were immersed. Values were expressed as mean ± SD from
four samples and P < 0.05 indicated statistically significance
for the enhanced production of hydroxyl radical from
H2O2[26] and MRP is reported to show a metal‑chelating
action.[13]
Then, we investigated the effect of the chelating agent
on the degradation of H2O2. As shown in Figure 4, H2O2
content in resins involving ethylenediaminetetraacetic
acid (EDTA) was fourfold higher than the control (resins
containing H2O2 only) after incubation for 1 day and
6.3  ±  7.78 mM of H2O2 was detected at day 2. On the
other hand, H2O2 in the control resin was not detected at
day 2. These results indicate that EDTA suppressed the
degradation of H2O2 and support our speculation that the
metal chelating activity of MRP might be an antibacterial
effect of the dental resins containing H2O2.
Glass ionomer cement is widely recognized to be a
preferable dental material for ART therapy. We first
tried to produce H2O2 containing glass ionomer cement.
However, we found that H2O2 containing glass ionomer
cement was extremely fragile after incubation for 3 days
in 100% humidity (data not shown). Then, we judged
that glass ionomer cement was not suitable in this
study and that resin composite was preferable for our
experiment.
Dental resins are continuously exposed to bacteria in the
oral cavity and have a high risk of inducing secondary
caries and periodontitis. Application of dental resin
containing MRP plus H2O2 to   a carious dentin could
be   an alternative therapy or serve as an additional
minimally invasive antibacterial treatment.
Financial support and sponsorship
Nil.
68 • Journal of Restorative Dentistry / Vol - 3 / Issue - 3 / Sep-Dec 2015
Conflicts of interest
There are no conflicts of interest.
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