TW201431565A - Composition for oral cavity - Google Patents

Abstract

Provided is a novel periodontal disease-preventing agent having the advantage of having low risk of the emergence of resistant bacteria, in comparison to existing antibacterial agents. An oral cavity composition containing a cold water-extract of Brassica rapa var. nipposinica, and having a biofilm-formation suppression action.

Description

Oral composition

The present invention relates to an oral composition excellent in the effect of improving periodontal disease of an oral biofilm for oral diseases.

The so-called periodontal disease system is a general term for disease groups found in periodontal tissues, and is narrowly equivalent to diseases of gingivitis, periodontitis, and occlusal trauma. Dental plaque in the periodontal disease is the main cause of oral infection. There are more than 700 kinds of bacteria in the human mouth. In the healthy oral cavity, the initial adherent bacteria of the genus Streptococcus or Actinomyces adhere to the tooth surface. Among them, Actinomyces naeslundii is called a causative agent of hemorrhagic gingivitis. The initially attached A.naeslundii forms a biofilm, which forms plaque and causes inflammation of the gums. The report reveals that lipoproteins on the membrane of gingival inflammatory cells caused by actinomyces are caused by the production of IL-8 or TNF-α via TLR2 of gingival epithelial cells or macrophages. If the gums cause inflammation, a periodontal capsular bag is formed, and accompanied by bleeding or exudation of the gingival effusion, Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans, Treponema, known as periodontal pathogenic bacteria. Denticola and other preparations are fixed in the environment of the periodontal pocket. In addition, A. naeslundii not only adheres to the tooth surface, but also coagulates with many oral bacteria, and becomes a scaffold for periodontal pathogenic bacteria to settle on plaque. As a result, A. naeslundii has become an important bacterium for the pathogenesis of periodontal disease, and has attracted attention in recent years because A.naeslundii has transformed the initial plaque into a late plaque (a biofilm of periodontal disease).

The traditional periodontal disease prevention system is mainly to sterilize periodontal pathogenic bacteria such as P.gingivalis and to suppress periodontal disease. However, since periodontal pathogenic bacteria and biofilm coexist in the deep part of the periodontal capsular bag, Antibacterial substances are difficult to penetrate, and most of them do not achieve the desired results.

In order to improve this, Patent Document 1 discloses the quality of (A)/(B) by blending (A) N-methyl sarcosine or a salt thereof, and (B) benzyl isothiocyanate. The ratio is 0.5~20, which shows the antibacterial effect of oral biofilm and the improvement effect of gingivitis. However, in Patent Document 1, the risk of occurrence of resistant bacteria is still high.

Since periodontal disease is exacerbated by the onset of gingivitis, prevention of gingivitis can prevent periodontal disease more effectively. Therefore, it is expected that an effective periodontal disease prevention effect can be expected by inhibiting the formation of a biofilm material by A. naeslundii.

The present inventors confirmed that the biofilm formation of A. naeslundii was promoted by acid pressure, and confirmed that the extracts of five cruciferous plants and ice flowers, such as watercress or Komatsu, resulted in a low formation of the acid-derived biofilm of A. naeslundii. Up to 50 to 90% of activity (Patent Document 2).

This application is the most active and easily available vegetable extract in plant extracts considered to have biofilm formation inhibitory activity (water dish, Brassica). Rapa var.nipposinica) is a candidate material, detailed review of detailed activity assessment and identification of traits of its active ingredients.

[Previous Technical Literature] [Patent Document]

[Patent Document 1] JP-A-2008-174542

[Patent Document 2] JP-A-2011-196315

Periodontal pathogenic bacteria and biofilm coexist in the deep part of the periodontal capsular bag, and the periodontal disease inhibition method using the antibacterial agent which has been traditionally labeled as periodontal pathogenic bacteria, the oral biofilm hinders the penetration of the antibacterial agent, and it is difficult Produces the expected periodontal disease inhibition effect. Moreover, the use of an antibacterial agent poses a high risk of resistant bacteria and is not suitable. Therefore, it is considered that a periodontal disease prevention method for controlling a biofilm formation system of an early periodontal pathogenic bacterium is safer and more effective than control by an antibacterial agent for periodontal pathogenic bacteria.

As a result of intensive studies by the present inventors, it was found that the watercress extract has an inhibitory effect on the formation of a film of Actinomyces naeslundii derived from an acid. The suppression effect is that the extraction temperature is higher at a lower temperature. As a result of separating the active ingredient of the water-sweet extract, it is estimated that the active ingredient is a component having a molecular weight of 10 kDa or more, and it is found that 80% or more of the components contained in the active partition are proteins, and the present invention has been completed. In addition, because the watercress extract is wrong The proliferation of A.naeslundii has an effect, so the mechanism of action is considered to be different from the antibacterial effect.

Actinomyces naeslundii is a Gram-positive bacterium found in gingivitis or root eclipse, and is called an early periodontal pathogenic bacterium. Because it is coagulated with streptococcus or periodontal pathogenic bacteria, it is considered that controlling A.naeslundii is associated with prevention of periodontal disease in order to grasp the key bacteria that migrate to the plaque of periodontal disease. The inventors of the present invention have confirmed that an acid such as butyric acid produced by periodontal pathogenic bacteria increases the formation of a biofilm.

Since the oral composition containing the watercress extract of the present invention remarkably inhibits the formation of a biofilm of the early periodontal pathogenic bacteria, it is effective as a method for preventing periodontal disease which is safer and more effective than the antibacterial agent.

The best form for implementing the invention

The invention of the present application relates to an oral composition containing a watercress extract.

Further, the present invention relates to an oral composition in which the watercress extract is a cold water extract.

Further, the present invention relates to a periodontal disease biofilm inhibitor containing a watercress extract.

Further, the invention of the present application relates to an acid-derived biofilm inhibitor of the aforementioned watercress extract which is a cold water extract.

Further, the present invention relates to a mouthwash, a toothpaste, an inhalant, a buccal tablet, and a food obtained from the above oral composition.

Hereinafter, the present invention will be more specifically described by way of examples. Further, the invention of the present application is not limited by the embodiments.

[Fig. 1] Comparison of biofilm formation inhibitory activity depending on extraction temperature

[Fig. 2] Comparison of biofilm formation inhibitory activity depending on extraction temperature

[Fig. 3] Biofilm formation inhibitory activity of cold water extract of watercress on hydroxyphosphorus lime

[Fig. 4A] Systematic analysis of clinical isolates in the oral cavity

[Fig. 4B] Systematic analysis of clinical isolates in the oral cavity

[Fig. 5] Inhibitory activity of cold water extract of Chinese cabbage on biofilm formation of clinical isolates

[Fig. 6] Biofilm formation inhibitory activity of watercress cold water extract in flow cell

[Fig. 7] Biofilm observation chart by conjugated focal laser microscope

[Fig. 8] Comparison of biofilm formation inhibitory activity of lyophilized aqueous solution and dialyzed external solution of lyophilized cold water extract and water vegetable cold water extract by dialysis treatment

[Fig. 9] Results of anion exchange chromatography of dialysis liquid of water-cooled vegetable extract

[Fig. 10] Biofilm formation inhibitory activity of ammonium sulfate separation zone of dialysis solution of water-cooled vegetable extract

[Fig. 11] Separation zoning of ammonium sulfate in dialysis liquid of water vegetable cold water extract Results of anion exchange chromatography

[Fig. 12] Biofilm formation inhibitory activity of blended cold water extract chewing gum

[Examples] (Example 1) Method of preparation of watercress extract:

Commercially available water vegetables (produced in Ibaraki Prefecture) were purchased, and dried leaves were prepared by freeze-drying. The dried leaves of the dried cabbage were finely pulverized, and extracted at 70 ° C, room temperature, and 4 ° C for 2 hours with respect to 1 g of the dried dried cabbage leaves and 50 ml of deionized distilled water. The resulting extract was suction filtered to 13,000 x g. After centrifugation for 10 minutes, the supernatant was lyophilized as a water lily extract for testing.

(Example 2)

Biofilm formation test

(Example 2-1)

Biofilm formation using a 96-well microtiter plate

A. naeslundii ATCC19039 or Actinomyces spp. clinical isolates were cultured in 5 ml of Brain Heart Infusion (BHI) liquid medium at 37 ° C for one night until the stationary phase, 1,100 × g. Centrifuge the bacteria for 10 minutes. Under the same conditions, the cells were washed three times with PBS, and PBS was adjusted to OD _{660 nm} = 0.3 as a test bacterial suspension, and supplied to the test system.

Biofilm formation was performed using a 96-well microtiter plate. 100 μl of 2×Trypticase Soy Broth (TSB) medium supplemented with 0.5% sucrose, 50 μl of test sample, 20 μl of 125 mM butyric acid, 20 μl of test bacterial suspension, 10 μl of PBS at 37 ° C, 5% were added to each well. Under the conditions of CO ₂ , culture is carried out for 16 to 20 hours.

(Example 2-2)

Quantification of biofilm formation using a 96-well microtiter plate

The culture supernatant cultured in accordance with Example 2-1 was decanted, and each well was washed with 200 μl of PBS, and then 100 μl of a 0.25% safranin solution (Nisshui Pharmaceutical Co., Ltd.) was added, and allowed to stand for 15 minutes to stain the biofilm. . After decanting the safranin solution, it was washed twice with deionized distilled water. After drying, 100 μl of 70% alcohol was added and shaken for 30 minutes to dissolve the safranin. The amount of biofilm was quantified by absorbance at 492 nm using a microplate analyzer. .

According to the above examples, the specific activity of the cold water extract extracted at 4 ° C was highest when the specific activity per weight of each water extract extracted from the water vegetable at 70 ° C, room temperature and 4 ° C was evaluated. (Figure 1 and Figure 2). In addition, it was shown that the cold water extract of Chinese cabbage had no effect on the proliferation of A. naeslundii.

Therefore, the cold water extract of Chinese cabbage is used as a candidate material for Actinomyces biofilm inhibition material, and it is possible to show activity in human oral cavity. Sexuality, further review.

(Example 2-3)

Forming a biofilm on hydroxyphosphorus lime (HA)

The HA sheet was used to cover the surface with enamel to form a shape of 7 mm × 7 mm × 1.5 mm. After sterilizing the HA piece in a sterilizer, it was equilibrated by PBS at room temperature for 1 hour, and 400 μl of human saliva was aseptically taken and allowed to stand at room temperature for 1 hour to form a pellicle, which was washed with PBS. The test was carried out by blocking the BSA solution at 5 mg/ml for 30 minutes at room temperature.

Biofilm formation was performed using a 24-well microtiter plate. 400 μl of 2×TSB medium supplemented with 0.5% sucrose, 200 μl of test sample, 80 μl of 125 mM butyric acid, 80 μl of test bacterial suspension, 40 μl of PBS were added to each well, and a HA piece was placed in each well, according to Example 2. -1 was cultured.

(Example 2-4)

Quantification of the amount of biofilm formed on HA

After culturing according to Example 2-3, the HA piece was taken out with nickel and immersed in PBS once to wash. The washed HA piece was placed in a new hole, 600 μl of a Safranin solution was added, and allowed to stand for 15 minutes to stain the biofilm. The HA was taken out with nickel, washed with deionized distilled water, placed in a new well, 600 μl of 70% ethanol was added, shaken for 30 minutes to dissolve the saffron, and 300 μl of the eluate was transferred to a 96-well microtiter plate. According to the embodiment 2-2, The amount of biofilm is measured.

According to the above examples, the results of evaluating the biofilm formation inhibitory activity of the cold water extract of the water vegetable on the hydroxyphosphorus lime forming the pellicle are shown in Fig. 3. The cold water extract of Chinese cabbage was hydrolyzed on hydroxyphosphorus lime, and the A. naeslundii biofilm formation inhibitory activity was similarly exhibited on 96-well.

(Example 3)

Isolation of Actinomyces clinical isolates

(Example 3-1)

PCR/Multiplex PCR (multiple pairs of primers polymerase chain reaction)

The PCR system was carried out by adding 2.5 μl of 10×Ex Taq buffer, 1 μl of DNA template, 2 μl of dNTP, 0.025 μl of each primer, 0.125 μl of Ex Taq, 2 μl of MgCl ₂ , and 16.88 μl of H ₂ O to a PCR tube. The multi-pair primer polymerase chain reaction reaction was carried out by changing the above composition into three 0.1 μl 50 μM Forward primers, three reverse primers, and 16.75 μl H ₂ O. The reaction conditions were heat treated at 95 ° C for 10 minutes and then at 95 ° C. 30 seconds, 50 ° C. 30 seconds, 72 ° C. The 30 second cycle was performed 30 times, and finally 72 ° C, 7 minutes, to completely complete the extension reaction. The annealing temperature of the multi-pair primer polymerase chain reaction was carried out at 53 °C.

(Example 3-2)

Separation of clinical isolates

From the arbitrarily chosen healthy person 7 men and women (male: 3, female: 4 Human), plaque was taken, cultured in Actinomyces selection medium (CFAT agar medium), colonies formed, Gram stain, and Gram-positive bacilli were screened by microscopic observation.

From each Gram-positive bacillus, the gene was extracted with a gene extraction reagent set (sigma), and PCR was carried out according to Example 3-1 to increase the upper end of the 16S rRNA gene by about 500 bp. The causative sequences used in PCR are shown in Table 1.

The PCR product was purified by PCR Clean-Up reagent group (promega), and the base sequence analysis was carried out by external (stock) Macrogen-japan. The strain was obtained by searching for the identity of the obtained base sequence of the 16SrRNA gene and the database on GenBank. The above-mentioned strains of the genus Actinomyces were estimated to increase the atpA by a chain reaction of a plurality of pairs of primers according to Example 3-1, and the external base was also subjected to a base sequence analysis by Macrogen-japan. The primer sequences used are shown in Table 1. The obtained salt base sequence was systematically analyzed together with the base sequence of atpA on the database, and the obtained species were identified. The obtained A. naeslundii and A. oris were clinical isolates of Actinomyces spp.

According to the above examples, a total of 9 strains of Actinomyces clinical isolates (A. naeslundii: 4 strains, A. oris: 5 strains) were successfully isolated from 7 humans in isolation from clinical isolates. Further, for the 7 strains of the isolated clinical strains of Actinomyces, when the biofilm formation inhibitory activity of the cold water extract of the water lily was evaluated, the amount of biofilm formation varied depending on the strain, but the cold water extract of the water lily showed for all clinical isolates. Biofilm inhibition activity (Figure 5). When the results of Fig. 3 were combined, it was revealed that the cold water extract of the water vegetable has a high possibility of exhibiting biofilm formation inhibitory activity even in human oral cavity.

(Example 4)

Biofilm inhibition evaluation of watercress cold water extract using flow cell

The above examples show that the water-repellent cold water extract has Actinomyces naeslundii biofilm inhibitory activity by evaluation using a 96-well plate stationary system. However, when considering the actual oral cavity, saliva is continuously secreted, and there is a flow of saliva in the oral cavity. Therefore, since the cold water extract of the water vegetable was evaluated for the A. naeslundii biofilm inhibitory activity even in the oral cavity, the A. naeslundii biofilm inhibitory activity of the cold water extract of the watercress was evaluated using a flow treatment tank simulating the oral environment.

evaluation method (Example 4-1) Evaluation of strains

Actinomyces naeslundii ATCC 19039 strain was used.

(Example 4-2) Preparation of watercress extract

Commercially available water vegetables were purchased, and dried leaves were prepared by freeze-drying. The extraction was carried out at 4 ° C for 2 hours at a ratio of 50 ml of deionized distilled water relative to 1 g of the dried water-dried leaves. The supernatant of the water residue is removed by suction filtration and centrifugation to freeze and dry, and the water lily extract is recovered.

(Example 4-3) Biofilm formation test using a flow treatment tank

A. naeslundii was cultured in 5 ml of BHI medium for one night, centrifuged, and washed with PBS, and adjusted to OD _{660 nm} = 0.4 with BHI medium, and this was used as a test bacterial solution. Inoculate 400 μl of the test bacterial solution in a flow-through tank (ACCFL0001: STOVALL LIFE SCIENCE), and the chamber is oriented to the lower side of the biofilm formation surface, and then allowed to stand at 37 ° C for 3 hours to allow the bacteria to adhere to the film. surface. After standing, the orientation of the cabin is based on the biofilm formation surface, and 0.25% sucrose will be added. The TSB medium of 60 mM butyric acid was cultured by a Peristaltic Pump at a flow rate of 3 ml/hour for 48 hours to form a biofilm. The biofilm inhibiting activity of the cold water extract of R. serrata was added to the above culture medium by adding a cold water extract of R. sylvestris L. to a final concentration of 1 mg/ml, and the same culture was carried out for evaluation.

(Example 4-4) Observation of biofilm

The formed biofilm was washed with deionized distilled water, and subjected to Live/Dead staining by LIVE/DEAE BIOFILM VIABILITY KIT (invitrogen), and the biofilm was observed by a conjugated laser microscope. membrane.

According to the above examples, when the biofilm formation inhibitory activity of the cold water extract of R. serrata under the flow system conditions of the flow treatment tank was evaluated, the cold water extract of R. sinensis inhibited the biofilm formation of A. naeslundii (Fig. 6, 7). Further, the ratio of the amount of dead cells of the biofilm formed by the addition of the cold water extract of the watercress was reduced by observation by a conjugated-focus laser microscope (Fig. 7).

More specifically, in the evaluation using the flow treatment tank, when butyric acid is added, the formation of a biofilm of A. naeslundii is increased, and the biofilm is present to the same extent as the dead bacteria. The ratio of dead cells constituting the biofilm is higher than when no butyric acid is added. In the presence of 1 mg/ml of cold water extract of water lily, the amount of biofilm formed by A. naeslundii was inhibited, especially the amount of adhesion of dead bacteria was reduced. Therefore, it was shown that the cold water extract of the water vegetable inhibits the formation of a biofilm which is dependent on butyric acid A. naeslundii.

(Example 5)

Separation division of cold water extract of watercress

(Example 5-1)

Dialysis

The cold water extract of water lily was dissolved in deionized distilled water, centrifuged at 13,000 × g for 10 minutes, and the supernatant was recovered, and the dialysis cellulose tube (as-1) having a molecular weight of 10 kDa was separated and separated, and deionized distilled water was used at a low temperature. Dialysis was performed indoors for 2 days. Freeze part of the dialysis inner and outer liquids back Received.

In order to estimate the molecular weight of the cold water extract of the water vegetable, the activity of the dialysis liquid is high by separating the dialysis cellulose tube having a molecular weight of 10 kDa, dialysis the cold water extract of the water vegetable, and evaluating the biofilm inhibitory activity of the dialysis liquid and the external liquid. . The molecular weight of the active ingredient was shown to be 10 kDa or more (Fig. 8).

(Example 5-2)

Ion exchange chromatography of dialysate in water extract of cold water

The sample was dissolved in 10 mM potassium phosphate buffer solution (pH 7.4), and the supernatant was centrifuged and applied to DEAE-TOYOPEARL 650M ( 1.6 × 70cm). After washing with the same buffer solution, it was washed with a linear gradient of 0-0.5 M NaCl, and 5 ml of each fraction was fractionated. In addition, all operations were carried out at a flow rate of 1 ml/min.

The dialyzed solution of the cold water extract of the Chinese cabbage was separated by anion exchange chromatography, and it was evaluated which component was motivated to exhibit activity (Fig. 9). As a result, the activity is based on the first to second peaks of the protein, and the activity is exhibited depending on the elution mode of the protein. This shows the possibility that the active ingredient is a protein.

(Example 5-3)

Ammonium sulfate separation zone

The dialysis liquid sample of the cold water extract of the water vegetable prepared according to the example 5-1 was dissolved in PBS, and the concentration of the ammonium sulfate was gradually increased to 30%. 45%, 60%, and 75%, and the precipitates of each concentration were collected by centrifugation at 13,000 × g for 15 minutes. The precipitated portion is recovered, dissolved in deionized distilled water, dialyzed, and freeze-dried and recovered. In addition, 75% of the unprecipitated sections were also lyophilized and recovered by freeze drying.

As described above, if the biofilm formation inhibiting active ingredient is a protein, it is considered that the ammonium sulfate separation zone is effective, and the biofilm formation inhibitory activity of the precipitation fraction of each concentration is evaluated by separating the dialysis liquid of the water extract of the water lily extract by ammonium sulfate separation. (Figure 10). It can be seen that the precipitation zone of the ammonium sulfate concentration of 45-60% has the highest biofilm formation inhibitory activity.

(Example 5-4)

Ion exchange chromatography of ammonium sulfate separation zone of watercress cold water extract

According to Example 5-3, the precipitation fraction with ammonium sulfate concentration of 45-60% was used, and according to Example 5-2, the results of separation by ionic exchange chromatography showed that two biofilm formation inhibitions were observed. Active peaks (Figure 11).

(Example 6)

Quantitative component

(Example 6-1)

Protein quantification

The quantification of proteins was carried out using the BCA method.

(Example 6-2)

Sugar quantification

The quantification of sugar is carried out using a phenol-sulfuric acid method.

(Example 6-3)

Polyphenol quantification

The quantification of polyphenols was carried out using the Folin-ciocalteu method.

As described above, regarding the purification, the results of separating the division of the cold water extract of the water vegetable by dialysis, ammonium sulfate separation and anion exchange chromatography are summarized in Table 2. As the refining stage progresses, the specific activity per weight becomes higher, and the refining of the active ingredient progresses. Further, 80% or more of the components contained in the active partition portion of the ion exchange chromatography are proteins, indicating that the active ingredient is a protein.

(Example 7)

Evaluation of biofilm formation inhibition activity of chewing gum blended with watercress extract

(Example 7-1)

Production of chewing gum mixed with watercress

The cold water extract chewing gum was mixed with the third composition.

BFI-01: Add water lily cold water extract and mix for 12 minutes.

BFI-02: After 12 minutes of kneading, the water lily cold water extract was added, and then kneaded for 3 minutes.

(Example 7-2)

Modulation of chewing gum extract

To 5 g of chewing gum, 25 ml of PBS heated to 37 ° C was added, and the mixture was immersed in a mortar for 5 minutes, extracted, centrifuged at 1,100 × g for 15 minutes, and the supernatant was collected and sterilized (0.2 μm). The sterilized treatment is used as a chewing gum extract.

(Example 7-3)

Biofilm formation inhibitory activity of chewing gum extract

This was done using a 96-well microtiter plate. Add 50 μl of 4×TSB medium supplemented with 1% sucrose, 100 μl of chewing gum extract, 20 μl of 125 mM butyric acid, 20 μl of test bacterial suspension, 10 μl of PBS to each well, at 37 ° C, 5% CO ₂ Next, culture for 16 to 20 hours. The amount of biofilm formation was quantified according to Example 2-2.

(Example 7-4)

Evaluation of dissolution rate of watercress extract from chewing gum

The absorbance at a wavelength of 280 nm (Abs 280 nm) was measured, and the dissolution rate of the watercress extract was evaluated by the following calculation formula.

(A1) The watercress extract was dissolved in a concentration of 2000 ppm in Abs 280 nm of the control chewing gum extract.

(A2) Abs280nm blended with cold water extract chewing gum extract of watercress

(A3) Abs280nm against chewing gum

Dissolution rate (%) = ((A2-A3) / (A1-A3)) × 100

According to the above examples, the biofilm formation inhibitory activity of the cold water extract chewing gum extract mixed with watercress was examined, and when the review was carried out, the cold water extract chewing gum extract was mixed to inhibit the formation of the biofilm of A. naeslundii (Fig. 12). In addition, there was no decrease in the activity caused by blending the cold water extract of Chinese cabbage with chewing gum. In addition, the dissolution rate of the cold water extract of Chinese cabbage was 83.0% for BFI-01 and 85.7% for BFI-02.

Watercress cold water extract also shows biofilm formation inhibitory activity against Actinomyces clinical strains. In addition, hydroxyphosphorus lime and flow treatment are used. In the evaluation of the tank, since the formation of the biofilm was suppressed, it was revealed that the cold water extract of the water vegetable showed the possibility of inhibiting the activity of biofilm formation even in the human mouth.

According to the separation zone of the active ingredient of the cold water extract of the water vegetable, the possibility of the protein being the active ingredient is shown. On the other hand, although the data is not shown, it is considered that since there is no biofilm inhibitory activity of BSA or milk protein preparation, not all proteins are active, but the proteins contained in the cold water extract of water lily are visible with specific Actinomyces. The biofilm forms an inhibitory activity.

The cold water extract of Chinese cabbage has the inhibitory activity of Actinomyces biofilm formation, and it is effective even for clinical isolates of Actinomyces. The evaluation of the biofilm formation inhibitory activity of the cold water extract of the water vegetable on the hydroxyphosphorus lime and the evaluation using the flow treatment tank showed the possibility of exhibiting activity in the human oral cavity. In addition, the blended water lily extract of the water lily showed a biofilm formation inhibitory activity of A. naeslundii.

When the cold water extract of the water vegetable is separated by dialysis, ammonium sulfate separation, and anion exchange chromatography, the protein having an active ingredient molecular weight of 10 kDa or more is shown.

Next, a mouthwash, a toothpaste, a spray for bad breath, a mouth-containing lozenge, a hard candy, an ingot, a soft, a biofilm formation inhibitor containing the cold water extract of the water vegetable of the present invention, which is a product other than the chewing gum, is produced by a conventional method. Sugar, drink. The following formulas represent these. In addition, the scope of the invention is not limited by the above.

(Example 8)

A mouthwash is prepared according to the following formulation.

(Example 9)

Toothpaste was made according to the following formulation.

(Embodiment 10)

A spray for bad breath was prepared according to the following formulation.

(Example 11)

The ingot was prepared according to the following formulation.

(Embodiment 12)

Hard candy was made according to the following formulation.

(Example 13)

The ingots were made according to the following formulation.

(Example 14)

The soft candy was made according to the following formula.

(Example 15)

Beverages were made according to the following formula.

[industrial use]

The oral composition containing the cold water extract of sauerkraut of the present invention has a different effect on the antibacterial agent against periodontal pathogenic bacteria, and has a preventive effect on the formation of a biofilm, thereby preventing the periodontal disease. Point periodontology preventive agent. Therefore, compared with the existing antibacterial agents and the like, it is considered that the resistant bacteria have a low risk and the like, and can be applied to various products.

Claims (11)

An oral composition characterized by a cold water extract of a cruciferous plant. The oral composition of claim 1, wherein the cold water extract of the aforementioned cruciferous plant is a cold water extract of water lily. A biofilm formation inhibitor characterized by a cold water extract of a cruciferous plant. The biofilm formation inhibitor of claim 3, wherein the extract of the aforementioned cruciferous plant is a cold water extract of water lily. A periodontal disease biofilm inhibitor characterized by a biofilm formation inhibitor according to claim 3 of the patent application. An acid-derived biofilm inhibitor characterized by a biofilm formation inhibitor according to claim 3 of the patent application. A mouthwash comprising the oral composition of claim 1 or 2 of the patent application. A toothpaste comprising the oral composition of claim 1 or 2 of the patent application. An inhalant characterized by being formed into an oral composition according to item 1 or item 2 of the patent application. A buccal tablet comprising the oral composition of claim 1 or 2 of the patent application. A food product characterized by containing an oral composition according to item 1 or item 2 of the patent application.