Composition comprising stannous fluoride and labeling material for inhibiting oral pathogens

Abstract

The present invention relates to a composition comprising stannous fluoride and a labeling material for selectively inhibiting pathogens and a method for detecting pathogens at the same time. According to the present invention, in an oral environment in which resident strains producing hydrogen peroxide and pathogens forming circular architectures coexist, tin using hydrogen peroxide as a substrate is used as a catalyst to produce reactive oxygen, thereby enabling pathogens to be specifically inhibited and pathogens in circular architecture form to be detected.

Description

Composition for inhibiting oral pathogens comprising tin fluoride and a labeling substance

[0001] The present invention relates to a composition for selectively inhibiting pathogens comprising tin fluoride and a labeling substance, and a method for labeling pathogens simultaneously.

[0002]

[0003] The term "microbiome" is a compound of "microbiota" (microbial community) and "biome" (ecosystem), encompassing a concept that includes diverse microbial communities as well as the ecosystems in which they live. Human microbiome research, built upon next-generation sequencing (NGS) technology in the early to mid-2000s, is being applied to the diagnosis of various diseases based on microbiome changes and is even leading to the development of novel microbiome drugs for the treatment of specific diseases. Microbiome research has evolved from the initial analysis of the microbiomes of the human body and the environment to the exploration of correlations between health and disease. In particular, microbiome research regarding the correlation between oral health and systemic diseases is important, as maintaining oral health can influence the maintenance and improvement of systemic diseases.

[0004] The oral microbiome exists in the form of particles suspended in saliva and biofilms attached to teeth or soft tissues; the formation of these biofilms can be attributed to the microbiome's environmental adaptation and evolution. Dental caries occurs when various microorganisms in the oral cavity coexist—either cooperatively or sometimes competitively—and when oral hygiene is poor or sugary foods are frequently consumed, leading to the formation of biofilms on the tooth surface (enamel), which is mineralized hard tissue. Microorganisms within the biofilm produce acid during the metabolic process using sugar; when acidic conditions are established between the biofilm formed by acid-resistant microorganisms and the tooth surface (enamel), demineralization occurs due to acid dissolution, which serves as the initial lesion of dental caries.

[0005] According to the analysis of the causal relationship between microbiome structure and disease, frequent exposure to sugar, the resulting dominance of pathogens, and the formation of polysaccharides lead to the formation of biofilms with structures resembling circular architecture. Since the structural characteristics of these circular biofilms distinguish them from other structures, there is a need for technology to prevent their formation through precision dental approaches that target this feature. As not all biofilms formed on tooth surfaces cause disease, selectively targeting pathological biofilms to inhibit or control their formation could serve as a method for preventing or treating diseases.

[0006] The virulence factor of caries-causing biofilms is exopolysaccharide (EPS), an extracellular polysaccharide that acts as a building material for forming circular structures. EPS provides the scaffold essential for biofilm formation and offers cohesive physical properties that connect microorganisms and adhesive properties that are stable against the external environment. Consequently, biofilms maintain a physically stable and acidified environment through the production of microbial metabolites, particularly lactic acid, in the continuous presence of sugars. While the precise diagnosis and control of such biofilms are crucial strategies for the future of dentistry, research and product development regarding strategies to selectively inhibit highly virulence-prone biofilms are currently lacking among the various approaches available.

[0007]

[0008] Accordingly, the inventors have completed the present invention, in which a tin substrate with hydrogen peroxide is used as a catalyst in an oral environment where a commensal strain that produces the antibiotic hydrogen peroxide and pathogens that form a circular structure coexist, thereby producing active oxygen and specifically inhibiting pathogens, and simultaneously enabling the detection of pathogens in the form of a circular structure.

[0009] This patent is based on research conducted with support from the Ministry of Science and ICT (MSIT) and the National Research Foundation of Korea (NRF) (No. 2710067740), and research conducted with support from the Bio-Medical Technology Development Project (No. 2710074767) (jointly led by the lead institution) supported by the Ministry of Science and ICT (MSIT) and the National Research Foundation of Korea (NRF).

[0010]

[0011] The object of the present invention is to provide a composition for inhibiting oral pathogens comprising tin fluoride (SnF2).

[0012] Another objective of the present invention is to provide a composition for labeling oral pathogens comprising tin fluoride (SnF2) and a labeling substance.

[0013] Another objective of the present invention is to provide a composition for the simultaneous inhibition and targeting of oral pathogens comprising tin fluoride (SnF2), commensal strains, hydrogen peroxide (H2O2), and a labeling substance.

[0014] Another objective of the present invention is to provide a method for specifically labeling oral pathogens using a composition comprising tin fluoride (SnF2), a commensal strain, hydrogen peroxide (H2O2), and a labeling substance.

[0015]

[0016] To achieve the above objective, the present invention provides a composition for inhibiting oral pathogens comprising tin fluoride (SnF2).

[0017] In the present invention, the composition may be characterized in that tin fluoride produces active oxygen (ROS) through a catalytic reaction using hydrogen peroxide (H2O2) present in the oral cavity as a substrate.

[0018] In another embodiment, the present invention provides a composition for labeling oral pathogens comprising active oxygen generated from a reaction between tin fluoride and hydrogen peroxide and a labeling substance (fluorescent substance or chromogenic reagent).

[0019] In the present invention, the composition is characterized by specifically reacting with commensal strains that produce hydrogen peroxide in the oral cavity, and may be characterized by producing hydrogen peroxide under conditions of 0.1 to 2% (w / v) of sucrose or 0.2 to 2% (w / v) of glucose, that is, under conditions of low to high sugar content.

[0020] In the present invention, the above-mentioned commensal strain may be characterized as being a Streptococcus of the mitis group, and specifically, Streptococcus oralis, which is one of the Streptococcus of the mitis group, and specifically, Streptococcus oralis subsp. oralis KCTC 13048 (=ATCC 35037), Streptococcus oralis KCTC 5662, Streptococcus oralis subsp. tigurinus J22 (BioProject ID / acession no. PRJNA1094609).

[0021] In the present invention, the composition may be characterized in that the catalytic action of tin fluoride on a hydrogen peroxide substrate in the oral cavity activates the generation of active oxygen under acidic conditions of pH 4 to 5, and at this time, a labeling substance reacts with the active oxygen to develop color.

[0022] In the present invention, the composition for inhibiting oral pathogens may be characterized by having antibacterial activity against Streptococcus mutans.

[0023] In addition, the above-mentioned labeling substances may include natural pigments that react with ROS, for example, purple sweet potato extract with anthocyanin (cyanidin acyl glucoside) as the main component and cochineal extract with carminic acid as the main component; colorimetric labeling substances, for example, TMB (3,3',5,5'-Tetramethylbenzidine, OPD (o-Phenylenediamine), and ABTS (2,2' acid); and fluorescent labeling substances, for example, peroxidase fluorogenic substrate (10-acetyl-3,7-dihydroxyphenoxazine). That is, the present invention may be characterized by diagnosing by colorimetric or fluorescence using active oxygen generated by the catalytic reaction of tin fluoride as a substrate. Through this, biofilms in the oral cavity can be selectively labeled, and at the same time, biofilm formation and pathogens can be specifically inhibited.

[0024] Another embodiment of the present invention provides a composition for the simultaneous inhibition and targeting of oral pathogens comprising tin fluoride (SnF2), a commensal strain, hydrogen peroxide (H2O2), and a colorimetric / fluorescent labeling agent.

[0025] Another embodiment of the present invention provides a method for specifically labeling oral pathogens using a composition comprising tin fluoride (SnF2), a commensal strain, hydrogen peroxide (H2O2), and a colorimetric / fluorescent labeling agent.

[0026]

[0027] According to the present invention, in an oral environment where a commensal strain producing hydrogen peroxide and pathogens forming circular structures coexist, tin with hydrogen peroxide as a substrate is used as a catalyst to produce active oxygen, thereby specifically inhibiting pathogens, and selective detection of a virulent biofilm in which pathogens in the form of circular structures are dominant and create an acidic environment inside is possible.

[0028]

[0029] Figure 1 is a schematic diagram of an ecological condition experiment using an oral biofilm model.

[0030] Figure 2 shows the results of confirming the pathogen-inhibiting effect of oral commensal bacteria in an oral biofilm model.

[0031] Figure 3 shows the results of confirming the reaction of tin fluoride + hydrogen peroxide + substrate according to pH.

[0032] Figure 4 shows the results of pathogen labeling and inhibition according to the difference in hydrogen peroxide secretion in an oral biofilm model, which is an oral environment simulation model reflecting the ecological conditions within the oral cavity, composed of S. mutans (Sm), a late-attached bacterium that is a pathogen; Streptococcus oralis (So), an early-attached bacterium and commensal strain that secretes hydrogen peroxide; and Actinomyces naeslundii (An), an early-attached bacterium that grows in a neutral pH environment, with Sm + An + S.o1 (low concentration of hydrogen peroxide secretion) and Sm + An + J22 (high concentration of hydrogen peroxide secretion).

[0033] Figure 5 shows the identification of a colorimetric label in the presence of pathogens, tin fluoride, and hydrogen peroxide.

[0034] Figure 6 shows the results of confirming the pathogen-inhibiting effect of tin fluoride on an oral biofilm model.

[0035] Figure 7 shows the results of comparing the absorbance at a wavelength of 652 nm for each reaction group depending on whether tin fluoride and hydrogen peroxide were included.

[0036] Figure 8 is a reaction rate measurement result showing the curve of the change in absorbance of the reaction group over time.

[0037] Figure 9 is a Michaelis-Menten Kinetics reaction curve measured based on the initial reaction rate according to hydrogen peroxide concentration, which is the result of quantitatively analyzing the catalytic activity of tin fluoride.

[0038] Figure 10 shows the results of confirming the color development of natural pigments that rapidly label biofilms according to the H₂O₂ catalytic reaction of SnF₂.

[0039]

[0040] The present invention will be described in detail below.

[0041]

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0043] The present invention focuses on the catalytic role of tin (Tin) among tin fluoride (SnF2) approved for oral use from a clinical perspective. In the oral cavity, there are commensal strains that maintain a relatively high proportion and have a competitive advantage over pathogens. Tin produces reactive oxygen species (ROS) using hydrogen peroxide secreted by commensal strains as a substrate, and the generated reactive oxygen species have the effect of specifically inhibiting pathogens. Furthermore, the invention is characterized by the fact that when this catalytic reaction of tin is reacted with ROS as a substrate using the colorimetric label TMB (3,3',5,5'-Tetramethylbenzidine), it is labeled blue (other substrates are labeled purple, violet, yellow, or fluorescent), making it visible to the naked eye.

[0044] When oral health is poor or sugar intake increases, pathogenic microorganisms form a biofilm with a circular architectural structure on the tooth surface. Many pathogenic microorganisms exist within this biofilm, and as they produce acid through sugar metabolism, an acidified microenvironment is formed within the circular structure. The circular architectural form is attributed to the formation of extracellular polysaccharides using sucrose as a substrate by glucosyltransferase (primarily derived from S. mutans) secreted by bacteria. These extracellular polysaccharides contribute to the formation of a spatial structure in which pathogens exist at a high density within the biofilm, which resembles a circular structure, while commensal strains form a band around the outside (coexisting around the circular structure).

[0045] Under these spatial structures and acidified microenvironment conditions, ROS is generated through a tin-catalyzed reaction using hydrogen peroxide produced by the resident strain as a substrate, and at the same time, when TMB, a substance that labels ROS, is used, a change from colorless to blue can be visually observed.

[0046] The present invention is a strategy for the simultaneous labeling and inhibition of pathogens that reflects the specific structure (circular structure) of oral biofilms and their ecological characteristics (reaction using hydrogen peroxide produced by commensal strains as a substrate), using clinically approved tin fluoride as a catalyst, while also expecting a remineralization effect of fluoride.

[0047] That is, for the simultaneous labeling and control of pathogens, the present invention is characterized by 1) mimicking the presence of hydrogen peroxide-secreting symbiotic bacteria (Streptococcus of the mitis group such as S. oralis) within a biofilm ecosystem, 2) reflecting the biological hydrogen peroxide secretion concentration and the clinically used concentration, 3) exposing to tin fluoride (fluoride concentration of 250 ppm) for 1 minute, and 4) simultaneously treating with a fluorescent labeling substance capable of labeling reactive oxygen species secretion in real time. Such a composition for the simultaneous labeling and control of oral pathogens may include tin fluoride and a labeling substance (any one or a complex of natural pigments (purple sweet potato pigment or cochineal pigment), TMB, OPD (o-Phenylenediamine), ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), peroxidase fluorogenic substrate (10-acetyl-3,7-dihydroxyphenoxazine)).

[0048] In the present invention, to manufacture an oral biofilm model reflecting the ecological conditions within the oral cavity, S. mutans (Sm), a pathogenic and late-attaching bacterium; S. oralis (So), an early-attaching bacterium and commensal strain that secretes hydrogen peroxide; and A. naeslundii (An), an early-attaching bacterium that grows in a neutral pH environment were used.

[0049] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are non-toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art, such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc.

[0050] In the present invention, the term "prevention" may be included without limitation as long as it is any act capable of blocking symptoms related to oral diseases or inhibiting or delaying their progression using the composition of the present invention, and the term "treatment" refers to a series of activities performed to alleviate and improve the intended disease.

[0051]

[0052] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0053]

[0054] Example 1. Preparation of an oral biofilm model and confirmation of the pathogen-inhibitory effect of oral commensal strains

[0055] 1-1. Preparation of Oral Biofilm Model

[0056] As part of the oral environment simulation model, an oral biofilm model reflecting the ecological conditions within the oral cavity was constructed by appropriately mixing and culturing early-attached strains and changing the sugar concentration to reflect changes in their relative distribution (Fig. 1).

[0057] Specifically, S. mutans is 10 3 (CFU / ml),S. oralis10 7 (CFU / ml),A. naeslundii10 6Initial attachment was induced in a low molecular-weight medium containing 0.1% sucrose by inoculation at (CFU / ml). Next, a medium containing 1% sucrose was supplied as a condition for inducing dental caries to create an environment in which S. mutans was dominant over S. oralis and A. naeslundii. The interaction of these three strains is influenced by changes in pH, as well as by the extracellular polysaccharides formed by S. mutans in response to changes in hydrogen peroxide secretion and sugar concentration.

[0058]

[0059] 1-2. Confirmation of pathogen inhibition in an oral biofilm model using commensal strains

[0060] Using the oral biofilm model prepared above, the inhibitory effect of resident strains on pathogens according to sugar concentration, time elapsed, and strain combinations was confirmed.

[0061] The pathogen S. mutans (Sm), the initial attached strain A. naeslundii (An), the commensal strain S. oralis (S.o1, S.o5), and the commensal strain J22 were combined to establish conditions of S.m+An (Model 1, control), S.m+A.n+S.o1 (Model 2), S.m+A.n+S.o5 (Model 3), and S.m+A.n+J22 (Model 4). As a result, after 43 hours (1% sucrose), almost no initial attached strains were detected, and after 67 hours (1% sucrose), the pathogens in Models 1 to 3 increased compared to the 29-hour (0.1% sucrose) time, whereas no pathogens were detected in Model 4, which included the commensal strain J22.

[0062]

[0063] In addition, when the pH was checked over time in the four models, the pH was approximately 6 in all four models at 19 and 29 hours, whereas it was approximately 4 in all four models after 43 hours, indicating that the microorganisms inside the biofilm produce acid during the sugar metabolism process over time (Fig. 2).

[0064] In addition, the total biomass was measured by dry weight, and the extracellular polysaccharides were classified into water-soluble polysaccharides (WSP) and alkaline-soluble polysaccharides (ASP) to determine their content (Fig. 2). As a result, compared to the control group (Model 1), the biomass decreased under co-culture conditions with a resident strain that secretes hydrogen peroxide (Models 2, 3, 4), and the content of water-soluble and alkaline-soluble polysaccharides also decreased significantly.

[0065]

[0066] Example 2. Confirmation of pathogen labeling and inhibition by tin fluoride

[0067] Using the oral biofilm model prepared above, upon exposure to tin fluoride, the presence of the hydrogen peroxide-producing commensal strain S. oralis (reflecting the difference in its production content) was used to simultaneously confirm the inhibition of the dominance of the pathogen S. mutans and the inhibition of biofilm structure formation, such as circular structures, and the presence of a label.

[0068]

[0069] 2-1. Confirmation of Labeling by Tin Fluoride, Hydrogen Peroxide, and Labeling Substances

[0070] First, the reaction of tin fluoride + hydrogen peroxide + substrate was verified according to pH. This involved verifying the catalytic activity of tin fluoride using TMB, a labeling agent (substrate of ROS), for the generation of ROS resulting from a catalytic reaction with hydrogen peroxide as a substrate under different pH conditions (acidic (pH 4.5), weakly acidic (pH 5.5), neutral (pH 6.5)) based on a 0.1 M sodium acetate (NaOAc) buffer. As a result, it was confirmed that ROS was generated by the catalytic reaction of tin fluoride under acidic conditions of pH 4.5, and a blue color was observed due to the labeling agent (Fig. 3).

[0071] In addition, changes in the oral biofilm model were examined in the cases containing TMB and TMB+H2O2 in Sm + An + S.o1 (Model 2) and Sm + An + J22 (Model 4), respectively (Fig. 4). The formation of biofilms resembling circular structures is influenced by the difference in the competitive inhibitory activity (amount of hydrogen peroxide secretion) of S. oralis S.o1 and J22 against the pathogen S. mutans in response to ecological conditions (sugar concentration). In this model, J22 showed strong competitive inhibitory activity against S. mutans due to high hydrogen peroxide secretion regardless of sugar concentration, whereas in the case of S.o1, the amount of hydrogen peroxide secreted changed according to changes in sugar concentration (a decrease in secretion in the presence of high sugar), creating an acidified environment and biofilms resembling circular structures due to relatively weak competitive inhibitory activity against S. mutans. In this embodiment, to verify a method for rapidly labeling the transition from the initial formation of a biofilm to a biofilm structure in the form of a circular building and to a highly virulent biofilm resulting from the creation of an acidified microenvironment, 1% (v / v) of hydrogen peroxide was added to enhance the catalytic reaction of tin fluoride. This enabled rapid labeling through the reaction between the ROS generated and TMB. As a result, rapid labeling in the form of a circular building was possible in Model 2, which was dominated by S. mutans. Although color appears partially in Model 4, it is clearly distinguished from Model 2 because it exhibits a thin, carpet-like spread rather than a structure like a circular building.

[0072] Furthermore, by utilizing purple sweet potato and cochineal pigments, which are edible colorings with ensured oral safety, rapid labeling was possible due to the purple and violet coloration in the form of circular structures dominated by S. mutans, as shown in the example above. This offers advantages for human application compared to compounds well-known as ROS substrates, such as TMB, and enables faster labeling than TMB.

[0073] Next, labeling by fluorescent substances was confirmed in the presence of pathogens, tin fluoride, and hydrogen peroxide. G1-G4 were set as G1: control (NaOAc buffer, pH 4.5), G2: tin fluoride (SnF2), G3: hydrogen peroxide (H2O2), and G4: SnF2+H2O2 treatment group, respectively, and S. mutansUA159 10 was applied to all columns. 5 ..., the fluorescent substance Alexa Fluor 647-dextran conjugate was added and reacted under 1% sugar conditions. At this time, Alexa Fluor 647-dextran conjugate does not stain bacteria, while the bound dextran acts as a primer for alpha-linkage glucan synthesis by glucosyltransferase (using sucrose as a substrate), being utilized in the synthesis of extracellular polysaccharides and simultaneously labeled with fluorescence. After 19 hours, the samples were centrifuged and washed once with NaOAc buffer, after which G2 and G4 were treated with SnF 20.1% for 1 minute (Fig. 5). As a result, in G4 (SnF2 + H2O2), ROS were generated by a catalytic reaction using hydrogen peroxide as a substrate, and the generated ROS degraded the extracellular polysaccharides labeled with Alexa Fluor 647. The fluorescence intensity of the fluorescently labeled polysaccharides reduced by the degradation of the polysaccharides was measured using a Fluorescence spectrophotometer (Hidex, Finland).

[0074]

[0075] 2-2. Confirmation of Pathogen Inhibition by Tin Fluoride in an Oral Biofilm Model

[0076] Using the above-mentioned oral biofilm model based on ecological conditions, the inhibitory effect of tin fluoride on pathogens according to sugar concentration and strain combinations was confirmed. Specifically, S. mutans 10 3 (CFU / ml),S. oralis10 7 (CFU / ml),A. naeslundii106 Initial attachment to saliva-coated hydroxyapatite discs was induced by inoculating at (CFU / ml) and culturing in a low molecular-weight medium containing 0.1% sucrose (from inoculation of the strain until 19 hours). Next, from 19 to 29 hours, a new medium containing 0.1% sucrose in a low molecular-weight medium was supplied. From 29 hours, a medium containing 1% sucrose was supplied under conditions inducing dental caries, and at 43 and 53 hours, the medium was replaced with a new medium containing 1% sucrose and cultured until 67 hours, while comparing the patterns of change among the three strains (measuring viable cell counts by obtaining biofilms formed on the disc surface at 29, 43, and 67 hours).

[0077] Sample treatment (treated identically with SnF2 at a concentration of 0.1% (w / v), NaF at 0.05% (w / v), and fluoride ions at a concentration of 250 ppm) was performed by immersing the disc in each treatment solution for 1 minute. The disc was exposed after being coated with saliva before bacterial inoculation (hour 0), after 6 hours had passed since bacterial inoculation, and before changing the medium (hours 19, 29, 43, and 53). However, the immediate treatment was omitted to measure the viable cell count of the biofilm attached to the disc at hours 29, 43, and 67 following the sample treatment.

[0078] Combinations of pathogen S. mutans (Sm), early-attached strain A. naeslundii (An), commensal strain S. oralis (S.o1, S.o5), and commensal strain J22 were used to produce S.m+A.n+S.o1 (control), S.m+A.n+S.o5 (control), and S.m+A.n+J22 (control) without a catalyst, S.m+A.n+S.o1 (SnF2), S.m+A.n+S.o5 (SnF2), and S.m+A.n+J22 (SnF2) using SnF2 as a catalyst, and SnF2's Identical F as a positive control- Conditions were established for S.m+A.n+S.o1(NaF), S.m+A.n+S.o5(NaF), and S.m+A.n+J22(NaF) using NaF at an ion concentration of 250 ppm. As a result, it was confirmed that pathogens disappeared after 43 hours when SnF2 was used as a catalyst (Fig. 6).

[0079]

[0080] Example 3. Evaluation of the catalytic activity of tin fluoride

[0081] In this example, to evaluate whether tin fluoride (SnF2) has catalytic activity for generating ROS using hydrogen peroxide (H2O2) as a substrate, absorbance analysis using a chromogenic label (TMB) and measurement of the reaction rate curve over time were performed.

[0082]

[0083] 3-1. Absorbance Measurement Experiment

[0084] To evaluate the peroxidase-like activity of SnF₂ (0.1% w / v = 250 ppm fluoride ions), a mixed sample of SnF₂ (0.1%) and H₂O₂ (0.1%) (or alone) and TMB (1 mg / ml) were mixed in a 0.1 M sodium acetate (NaOAc) buffer solution (pH 4.5) and reacted at 37°C. To determine whether the catalytic activity of SnF₂ varied with pH, ​​the experiment was repeated under the same conditions at pH 4.5, 5.5, and 6.5. After the optimal pH condition was identified, the experiment was conducted to compare and analyze peroxidase-like activities by evaluating the reactions of SnF₂ alone, H₂O₂ alone, and the SnF₂+H₂O₂ combination at the corresponding condition (pH 4.5).

[0085] As the absorbance bar graph of the SnF₂ + H₂O₂ treatment group is significantly higher than that of SnF₂ alone or H₂O₂ alone, SnF₂ generates ROS through a catalytic reaction using H₂O₂ as a substrate, and the blue color indicates the reaction with TMB (nanozyme chromogenic agent) (oxidation of TMB by ROS). The inner bar graph suggests that this catalytic activity is activated at an acidic pH (an environment that promotes mineral dissolution due to the acidified environment of caries-inducing virulence factors / biofilms) (Fig. 7).

[0086]

[0087] 3-2. Experiment on Measuring Reaction Rate Curves

[0088] The experiment was conducted by measuring the absorbance at 652 nm for about 10 minutes at 30-second intervals starting immediately after the start of the reaction for each reaction group at the optimal pH of 4.5 confirmed in the absorbance measurement experiment above.

[0089] The curve in Fig. 8 represents the SnF₂ + H₂O₂ + TMB treatment group, showing a stable saturation state after a rapid increase in absorbance. This indicates that TMB is continuously oxidizing, and this This suggests that continuous ROS (reactive oxygen species) generation is occurring through the redox cycle. The curves of the control groups (SnF₂ alone, H₂O₂ alone, TMB alone (control)) remained low, confirming that the catalytic effect occurs only in the combination of SnF₂ + H₂O₂ (Fig. 8).

[0090]

[0091] Example 4. Quantitative analysis of SnF₂ catalytic activity (Michaelis-Menten kinetic analysis)

[0092] To quantitatively evaluate the catalytic activity of tin fluoride (SnF₂), the initial reaction rate was measured according to the concentration of hydrogen peroxide (H₂O₂), and analyzed by applying the Michaelis-Menten kinetic model as follows.

[0093]

[0094] : Initial reaction rate (e.g., rate of change of TMB absorbance at 652 nm)

[0095] : Maximum reaction rate (when the catalyst is saturated)

[0096] : Michaelis constant, Substrate concentration exhibiting half the reaction rate; the lower the value, the higher the affinity for the substrate.

[0097] : Substrate concentration

[0098] The reagents used in the experiment were TMB (1 mg / mL), SnF₂ (0.1% (w / v)), and H₂O₂ (range 0-1.0 mM), and the buffer solution used was NaOAc (0.1 M) with a pH of 4.5.

[0099] Through this experiment, it was confirmed that tin fluoride (SnF₂) has catalytic activity similar to that of horseradish peroxidase (HRP) (Fig. 9).

[0100]

[0101] Example 5. Application of a natural pigment for rapid labeling of biofilms via the H₂O₂ catalytic reaction of SnF₂

[0102] In the case of TMB or OPD, biofilms can be labeled by developing color through substrates that react with ROS generated by catalytic reactions, but their application in the human body is limited. Therefore, among natural pigments whose safety as food materials has been established, pigments that react with ROS at low pH were selected.

[0103] Control is S. The mutans-only biofilm culture was treated with natural pigments by dip-washing in 0.1 M NaOAc to remove the culture medium, treating with 0.1 M NaOAc for 1 minute, and then dip-washing again in 0.1 M NaOAc; the SnF2 group was treated with natural pigments by dip-washing in 0.1 M NaOAc, treating with SnF2 at a concentration of 0.1% (w / v) for 1 minute, and then dip-washing again in 0.1 M NaOAc; the H2O2 group was treated with natural pigments by dip-washing in 0.1 M NaOAc, treating with 0.1 M NaOAc for 1 minute, and then dip-washing again in 0.1 M NaOAc; and the SnF2+H2O2 group was treated with natural pigments by dip-washing in 0.1 M NaOAc, treating with SnF2 at a concentration of 0.1% (w / v) for 1 minute, and then again in 0.1 M It was treated with 1% (v / v) H2O2 and natural pigments by dip-washing in NaOAc. At this time, the pigment was applied at a concentration of 1 mg / ml.

[0104] The biofilm markings of circular structures are purple or due to the treatment of natural pigments in SnF2 + H2O2. It was confirmed that a distinct purple color reaction appeared (Fig. 10).

[0105]

[0106] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition for inhibiting oral pathogens comprising tin fluoride (SnF2).

2. In Paragraph 1, The above composition is a composition for inhibiting pathogens in the oral cavity, wherein tin fluoride produces active oxygen (ROS) through a catalytic reaction using hydrogen peroxide (H2O2) present in the oral cavity as a substrate.

3. In Paragraph 1, The above composition is a composition for inhibiting oral pathogens, comprising a commensal strain that produces hydrogen peroxide.

4. In Paragraph 3, A composition for inhibiting oral pathogens, wherein the above-mentioned commensal strain produces hydrogen peroxide under conditions of 0.1 to 2% (w / v) sucrose or 0.2 to 2% (w / v) glucose.

5. In Paragraph 3, A composition for inhibiting oral pathogens, wherein the above-mentioned commensal strain is Streptococcus of the mitis group.

6. In Paragraph 5, A composition for inhibiting oral pathogens, wherein the above-mentioned commensal strain is Streptococcus oralis, which exhibits no change in the inhibitory ability against Streptococcus mutans even in the presence of sugar and shows a high amount of hydrogen peroxide secretion.

7. In Paragraph 1, The above composition is a composition for inhibiting oral pathogens, having antibacterial activity against Streptococcus mutans.

8. A composition for labeling oral pathogens comprising tin fluoride (SnF2) and a labeling substance.

9. In Paragraph 8, A composition for labeling oral pathogens, wherein the above-mentioned labeling substance is selected from the group consisting of natural pigments such as purple sweet potato pigment and cochineal pigment; colorimetric labeling substances such as TMB (3,3',5,5'-Tetramethylbenzidine, OPD (o-Phenylenediamine), ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); and fluorescent labeling substances such as peroxidase fluorogenic substrate (10-acetyl-3,7-dihydroxyphenoxazine).

10. In Paragraph 8, The above composition is a composition for labeling pathogens in the oral cavity, wherein tin fluoride produces active oxygen (ROS) through a catalytic reaction using hydrogen peroxide (H2O2) present in the oral cavity as a substrate.

11. In Paragraph 8, A composition for labeling oral pathogens, wherein the above-mentioned labeling substance develops color using active oxygen generated by the catalytic reaction of tin fluoride as a substrate.

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