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ORIGINAL ARTICLE
Year : 2015  |  Volume : 3  |  Issue : 3  |  Page : 64-69

Synergic antibacterial effect between Maillard reactive product (MRP) and hydrogen peroxide (H2O2) on Streptococcus mutans


Department of Oral Health Science, School of Dentistry, Hokkaido University, Sapporo, Japan

Date of Web Publication30-Oct-2015

Correspondence Address:
Dr. Morimichi Mizuno
Kita 13 Nishi 7, Sapporo
Japan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2321-4619.168731

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  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 H2O2in the presence and absence of MRP, was immersed into bacterial solution. The effect of MRP on H2O2degradation was examined by the measurement of H2O2content. Results: The resin composite containing MRP and H2O2showed stable antibacterial activity compared with resin containing H2O2only, and the effect of MRP was speculated to be the suppression of H2O2degradation, and the presence of H2O2correlated with the antibacterial activity of resin composite. These results indicated that the antibacterial activity of resin composite containing MRP and H2O2on S. mutans was dependent on the presence of H2O2, and MRP suppressed the degradation of H2O2after combination with H2O2. EDTA also suppressed the degradation of H2O2. Conclusions: An antibacterial effect of resin composite containing MRP and H2O2on S. mutans was observed. The effect of MRP on H2O2might be a metal chelating action. Application of resin composite containing MRP and H2O2to 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)


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

How to cite this URL:
Mizuno M, Inoue Ki. Synergic antibacterial effect between Maillard reactive product (MRP) and hydrogen peroxide (H2O2) on Streptococcus mutans. J Res Dent [serial online] 2015 [cited 2019 Sep 21];3:64-9. Available from: http://www.jresdent.org/text.asp?2015/3/3/64/168731


  Introduction Top


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]

This recognition is a basic concept to develop atraumatic 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 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]

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 Top


Synthesis of soluble MRP, and preparation of dental resin containing MRP and H 2O2

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 × 106 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 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 H2 SO4, 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 Top


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.

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.
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

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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 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.
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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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 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 H2O2in 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.
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

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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 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.
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

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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.

Conflicts of interest

There are no conflicts of interest.

 
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