JPWO2014118829A1 - Oral composition - Google Patents

Abstract

Providing a new preventive agent for periodontal disease that has advantages such as lower risk of appearance of resistant bacteria than existing antibacterial agents. The composition for oral cavity which has a biofilm formation inhibitory effect containing a mizuna cold water extract.

Description

  The present invention relates to an oral composition excellent in periodontal disease improving effect on an oral biofilm which is a cause of oral diseases.

  Periodontal disease is a general term for a group of diseases found in periodontal tissues, and in a narrow sense, it is a disease to which gingivitis, periodontitis, and occlusal trauma are equivalent. Periodontal disease is an oral infection caused mainly by dental plaque. There are more than 700 kinds of bacteria in the human oral cavity, and in the healthy oral cavity, early-adherent bacteria such as Streptococcus and Actinomyces are attached to the tooth surface. Among them, Actinomyces naeslundii is said to be a causative agent of hemorrhagic gingivitis, and A. naeslundii that initially adheres forms a biofilm and makes plaque, which causes inflammation in the gums. It has been reported that inflammation of the gingiva caused by Actinomyces is caused by the production of IL-8 and TNF-α through TLR2 of gingival epithelial cells and macrophages by lipoproteins on the cell membrane. When inflammation occurs in the gums, periodontal pockets are formed, and bleeding and gingival crevicular fluid exudate. Is arranged. A. naeslundii not only adheres to the tooth surface but also co-aggregates with many oral bacteria, providing a scaffold for periodontopathic bacterial colonization. From these facts, A. naeslundii has recently attracted attention as an important bacterium involved in the onset of periodontal disease since it shifts from early plaque to late plaque (periodontal disease biofilm).

  In the conventional periodontal disease prevention, the idea of controlling periodontal disease by killing periodontopathic bacteria such as P. gingivalis was the mainstream, but periodontopathic bacteria are biofilms deep in the periodontal pocket. In many cases, antibacterial substances are difficult to penetrate, and the desired effect is often not obtained.

  In order to improve this point, Patent Document 1 includes (A) N-acyl sarcosine or a salt thereof and (B) benzylisothiocyanate, and the mass ratio of (A) / (B) is 0. It is disclosed that an oral biofilm antibacterial effect and a gingivitis improving effect are exhibited by being .5-20. However, even in Patent Document 1, the risk of appearance of resistant bacteria is still high.

  Since periodontal disease progresses from developing gingivitis, preventing gingivitis can prevent periodontal disease more effectively. Therefore, an effective periodontal disease prevention effect can be expected from a material that suppresses biofilm formation of A. naeslundii.

  The present inventors confirmed that the biofilm formation of A. naeslundii was promoted by acid stress, and the acid-inducibility of A. naeslundii was extracted from five kinds of cruciferous plants such as mizuna and komatsuna and ice plant extracts. The activity of reducing the amount of biofilm formed by 50 to 90% has been confirmed (Patent Document 2).

  In this application, among the plant extracts recognized to have biofilm formation inhibitory activity, the most active and easily available mizuna (Mizuna, Brassica rapa var. Nipposinica) is used as a candidate material. The properties of the ingredients were examined in detail.

JP 2008-174542 A JP 2013-056855 A

  Periodontopathic bacteria are present in the deep part of the periodontal pocket along with biofilms. In the conventional periodontal disease control method using antibacterial agents targeting periodontopathic bacteria, oral biofilms are antibacterial agents. It was difficult to prevent periodontal disease as intended and prevent the penetration of Also, the use of antibacterial agents is not preferred because of the high risk of resistant bacteria appearing. Therefore, it is considered that the control of the initial periodontal pathogenic bacteria biofilm formation is a safer and more effective periodontal disease prevention method than the control of periodontopathic bacteria with antibacterial agents.

  As a result of diligent research by the present inventors, it has been found that the extract of mizuna has an inhibitory effect on acid-induced biofilm formation of Actinomyces naeslundii. This inhibitory effect was higher as the extraction temperature was lower. As a result of fractionating the active ingredients of mizuna extract, the active ingredient is estimated to be 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 fraction are proteins, and the present invention is completed. did. The mizuna extract did not affect the growth of A. naeslundii, so the mechanism of action was considered to be different from the antibacterial action.

  Actinomyces naeslundii is a Gram-positive gonococci found in gingivitis and root caries sites and is said to be an early periodontopathogenic bacterium. Since it coaggregates with streptococci and periodontopathic bacteria, it is the key to the transition of flora to periodontal disease plaques, and control of A. naeslundii is thought to lead to periodontal disease prevention. In our study, we have confirmed that biofilm formation is increased by acids such as butyric acid produced by periodontopathic bacteria.

  The composition for oral cavity containing the mizuna extract of the present invention remarkably suppresses biofilm formation of early periodontal pathogenic bacteria, and is useful as a safer and more effective periodontal disease prevention method than antibacterial agents. It is.

Comparison of biofilm formation inhibitory activity with different extraction temperatures Comparison of biofilm formation inhibitory activity with different extraction temperatures Biofilm formation inhibitory activity of mizuna cold water extract on hydroxyapatite Phylogenetic analysis of oral clinical isolates Phylogenetic analysis of oral clinical isolates Biofilm formation inhibitory activity of clinical extract of mizuna cold water extract Biofilm formation inhibitory activity of mizuna cold water extract in flow cell Biofilm observation with confocal laser microscope Comparison of biofilm formation inhibitory activity of dialysis treatment of mizuna cold water extract and mizuna cold water extract by dialysis treatment Results of anion exchange chromatography of dialysis internal solution of mizuna cold water extract Biofilm formation inhibitory activity of ammonium sulfate fraction in dialysis internal solution of mizuna cold water extract Results of Anion Exchange Chromatography of Ammonium Sulfate Fractionation in Mizuna Cold Water Extract Dialysis Internal Solution Biofilm formation inhibitory activity of chewing gum containing mizuna cold water extract

This invention relates to the composition for oral cavity containing a mizuna extract.
Furthermore, this invention relates to the composition for oral cavity whose said mizuna extract is a cold water extract.
Furthermore, this invention relates to the periodontal disease biofilm formation inhibitor containing a mizuna extract.
The present invention further relates to an acid-induced biofilm formation inhibitor, wherein the mizuna extract is a cold water extract.
Furthermore, the present invention relates to a mouthwash, a toothpaste, an inhalant, a troche, and a food comprising the oral composition.

  Hereinafter, the present invention will be specifically described by way of examples. The present invention is not limited to these examples.

Example 1
Preparation method of mizuna extract:
Commercially available mizuna (produced in Ibaraki Prefecture) was purchased and freeze-dried to prepare dried mizuna leaves. The dried mizuna leaf was finely pulverized, and 1 g of the crushed dried mizuna leaf was extracted at a rate of 50 ml of deionized distilled water at 70 ° C., room temperature, and 4 ° C. for 2 hours. The obtained extract was subjected to suction filtration, centrifuged at 13,000 × g for 10 minutes, and the supernatant was freeze-dried and used for the test as a mizuna extract.

(Example 2)
Biofilm formation test

(Example 2-1)
Biofilm formation using 96-well microtiter plates
A. naeslundii ATCC19039 or Actinomyces spp. Clinical isolates were cultured in 5 ml of Brain Heart Infusion (BHI) liquid medium under anaerobic conditions at 37 ° C. overnight until stationary phase and centrifuged at 1,100 × g for 10 minutes. Under the same conditions, the plate was centrifuged 3 times with PBS, and prepared with PBS to an OD _{660 nm of} 0.3 was used as a test bacterial suspension in the test system.
Biofilm formation was performed using 96 well microtiter plates. To each well, 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 the test bacterial suspension, 10 μl of PBS, 37 ° C., 5% CO ₂ The culture was performed for 16 to 20 hours under the conditions.

(Example 2-2)
Quantification of amount of biofilm formed in 96-well microtiter plate Decant the culture supernatant cultured according to Example 2-1, wash each well with 200 μl of PBS and add 100 μl of 0.25% safranin solution (Nissui Pharmaceutical). The biofilm was stained by adding and allowing to stand for 15 minutes. The safranine solution was decanted and washed twice with deionized distilled water. After drying, 100 μl of 70% ethanol was added, and the mixture was shaken for 30 minutes to elute safranin, using a microplate reader at an absorbance of 492 nm. The amount of biofilm was quantified.
When the specific activity per weight of each mizuna extract extracted from mizuna under the conditions of 70 ° C., room temperature, and 4 ° C. was evaluated according to the above-mentioned examples, the cold water extract extracted under the condition of 4 ° C. Specific activity was the highest (FIGS. 1 and 2). Mizuna cold water extract did not affect the growth of A. naeslundii.
Therefore, we investigated further whether mizuna cold water extract may be active in human oral cavity as a candidate material for Actinomyces biofilm suppression material.

(Example 2-3)
Biofilm Formation on Hydroxyapatite (HA) The HA disk used was a bovine tooth molded into a 7 mm x 7 mm x 1.5 mm shape so that the surface was covered with enamel. The HA disk was sterilized in an autoclave, equilibrated with PBS for 1 hour at room temperature, and 400 μl of aseptically collected human saliva was allowed to stand at room temperature for 1 hour to form a pellicle. After washing with PBS, 5 mg / ml A BSA solution blocked at room temperature for 30 minutes was used for the test.
Biofilm formation was performed using 24-well microtiter plates. To each well was added 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 the test bacterial suspension, and 40 μl of PBS, and the HA disk was placed in each well. Example 2-1 Incubated according to

(Example 2-4)
Quantification of biofilm formation on HA After culturing according to Example 2-3, the HA disk was taken out with tweezers and washed by immersing it once in PBS. The washed HA disk was placed in a new well, 600 μl of safranin solution was added, and the biofilm was stained by allowing to stand for 15 minutes. Remove HA with tweezers, place it in a new well after washing with deionized distilled water, add 600 μl of 70% ethanol, shake for 30 minutes to elute safranin, and add 300 μl of the eluate to a 96-well microtiter plate. The amount of biofilm was quantified according to Example 2-2.
FIG. 3 shows the results of evaluating the biofilm formation inhibitory activity of the mizuna cold water extract on the hydroxyapatite on which the pellicle was formed according to the above example. The mizuna cold water extract showed A. naeslundii biofilm formation inhibitory activity on hydroxyapatite as well as on 96 wells.

(Example 3)
Actinomyces clinical isolate isolation

(Example 3-1)
PCR / multiplex PCR
PCR was performed by adding 2.5 μl of 10 × Ex Taq buffer, 1 μl of DNA template, 2 μl of dNTP, each primer 0.025 μl, Ex taq 0.125 μl, MgCl ₂ 2 μl, H ₂ O 16.88 μl to the PCR tube. Multiplex PCR was performed by changing the above composition to three 50 μM Forward primers and three Reverse primers to 0.1 μl each and 16.75 μl H ₂ O. The reaction conditions were 95 ° C for 30 minutes, 95 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, and finally the extension reaction was completed completely at 72 ° C for 7 minutes. It was. Multiplex PCR was performed at an annealing temperature of 53 ° C.

(Example 3-2)
Separation of clinical isolates Plaques were collected from 7 healthy males and females (man: 3 and female: 4) selected arbitrarily, cultured in Actinomyces selective medium (CFAT agar medium), and colonies formed were gram-stained Thereafter, Gram-positive rods were selected by microscopic observation.
A genome was extracted from each Gram-positive rod using a genome extraction kit (sigma), and PCR was performed according to Example 3-1, to amplify about 500 bp upstream of the 16SrRNA gene. The primer sequences used for PCR are shown in Table 1.
The PCR product was purified with a PCR Clean-Up kit (promega), and nucleotide sequence analysis was outsourced to Macrogen Japan. Bacteria were estimated by homology search of the obtained 16S rRNA gene base sequence with a database on GenBank. The bacteria presumed to belong to the genus Actinomyces were amplified atpA by multiplex PCR according to Example 3-1, and were similarly outsourced to Macrogen Japan Co., Ltd. for base sequence analysis. The primer sequences used are shown in Table 1. Species were identified by systematic analysis of the obtained nucleotide sequence together with the nucleotide sequence of atpA in the database, and the obtained A. naeslundii and A. oris were used as Actinomyces spp. Clinical isolates.

  As a result of isolating clinical isolates from the human oral cavity according to the above example, a total of 9 Actinomyces clinical isolates (A. naeslundii: 4 strains, A. oris: 5 strains) were successfully isolated from 7 people. . Furthermore, when the biofilm formation inhibitory activity of mizuna cold water extract was evaluated for 7 isolates of Actinomyces clinical isolates, the amount of biofilm formation varied depending on the strain, but mizuna cold water extract was found in all clinical isolates. On the other hand, the biofilm inhibitory activity was shown (FIG. 5). When combined with the results in FIG. 3, it was suggested that the extract of cold water from mizuna is highly likely to exhibit biofilm formation inhibitory activity even in the human oral cavity.

Example 4
Evaluation of Biofilm Inhibition of Mizuna Cold Water Extract Using Flow Cell The above examples showed that Mizuna cold water extract has Actinomyces naeslundii biofilm suppression activity in a static system evaluation using a 96-well plate. However, considering the actual oral cavity, saliva is constantly secreted, and there is a flow of saliva in the oral cavity. Therefore, in order to evaluate whether the mizuna cold water extract shows the biofilm inhibitory activity of A. naeslundii even in the oral cavity, the A. naeslundii biofilm inhibitory activity of the mizuna cold water extract was evaluated using a flow cell system that mimics the oral environment. evaluated.
Evaluation method (Example 4-1) Evaluation strain
Actinomyces naeslundii ATCC19039 strain was used.
(Example 4-2) Preparation of mizuna extract Commercially available mizuna was purchased, and dried leaves of mizuna were prepared by freeze-drying. Extraction was performed for 2 hours at 4 ° C. with 50 ml of deionized distilled water per 1 g of finely ground dried mizuna leaves. The supernatant from which the mizuna residue was removed by suction filtration and centrifugation was lyophilized to recover the mizuna cold water extract.
(Example 4-3) Biofilm formation test using flow cell
A. naeslundii was cultured overnight in 5 ml of BHI medium, collected by centrifugation, centrifuged and washed with PBS, and O.B. D. It adjusted to _660nm = 0.4, and this was made into the test microbe liquid. Inoculate 400 μl of the test bacterial solution into a flow cell chamber (ACCFL0001: STOVALL LIFE SCIENCE), set the direction of the chamber with the biofilm formation side down, and leave it at 37 ° C. for 3 hours to form a biofilm. Bacteria were attached to the surface. After standing, the biofilm was cultured for 48 hours while flowing the TSB medium with 0.25% sucrose and 60 mM butyric acid added at a flow rate of 3 ml / hour with a peristaltic pump. Formed. The biofilm inhibitory activity of the mizuna cold water extract was evaluated by adding the mizuna cold water extract having a final concentration of 1 mg / ml to the medium and culturing in the same manner.
(Example 4-4) Observation of biofilm The formed biofilm was washed with deionized distilled water and subjected to Live / Dead staining with LIVE / DEAE BIOFILM VIABILITY KIT (Invitrogen), followed by a confocal laser microscope. The biofilm was observed.
When the biofilm formation inhibitory activity of the mizuna cold water extract was evaluated under the flow system conditions using the flow cell according to the above-described examples, the mizuna cold water extract suppressed the biofilm formation of A. naeslundii (FIG. 6, 7). Moreover, from the observation with a confocal laser microscope, in the biofilm formed under the mizuna cold water extract addition conditions, the ratio for which dead bacteria occupied decreased (FIG. 7).
More specifically, in the evaluation using the flow cell, the addition of butyric acid increased the biofilm formation of A. naeslundii, and the biofilm was present with the same number of live and dead bacteria. The proportion of dead bacteria constituting the biofilm was higher than when no butyric acid was added. In the presence of 1 mg / ml mizuna cold water extract, the amount of A. naeslundii biofilm formed was suppressed, especially the amount of dead bacteria attached. Therefore, it was revealed that the mizuna cold water extract suppresses biofilm formation of A. naeslundii depending on butyric acid.

(Example 5)
Fractionation of mizuna cold water extract

(Example 5-1)
Dialysis Mizuna cold water extract is dissolved in deionized distilled water, centrifuged at 13,000 xg for 10 minutes, the supernatant is recovered, put into a cellulose tube for dialysis with a molecular weight cut off of 10 kDa (As One), and against deionized distilled water Dialysis was performed in a cold room for 2 days. A portion of the dialyzed solution and the dialyzed solution were lyophilized and collected.
In order to estimate the molecular weight of the mizuna cold water extract as described above, the mizuna cold water extract was dialyzed with a cellulose tube for dialysis with a molecular weight cut off of 10 kDa, and the biofilm formation inhibitory activity of the dialysis internal solution and external solution was evaluated. The activity of the dialysis internal solution was higher. It was suggested that the molecular weight of the active ingredient is 10 kDa or more (FIG. 8).

(Example 5-2)
Ion exchange chromatography of dialysis internal solution of mizuna cold water extract The sample was dissolved in 10 mM potassium phosphate buffer (pH 7.4), and the supernatant after centrifugation was applied to DEAE-TOYOPEARL 650M (ψ1.6 × 70 cm). . After washing with the same buffer, elution was performed with a 0-0.5M NaCl linear gradient, and 5 ml was fractionated into each fraction. All operations were performed at a flow rate of 1 ml / min.
The dialysis internal solution of the mizuna cold water extract was fractionated by anion exchange chromatography, and it was evaluated which component shows the activity depending on the elution (FIG. 9). As a result, the activity was shown to depend on the protein elution pattern from the first peak to the second peak of the protein. These facts suggested that the active ingredient may be a protein.

(Example 5-3)
Ammonium sulfate fraction The dialysis internal solution sample of the extract of cold water extract prepared according to Example 5-1 was dissolved in PBS, and the ammonium sulfate concentration was increased stepwise to 30%, 45%, 60%, and 75%. The precipitate was collected by centrifugation at 13,000 × g for 15 minutes. The collected precipitate fraction was dissolved in deionized distilled water, lyophilized after dialysis, and collected. The 75% unprecipitated fraction was also collected by lyophilization after dialysis.
As described above, if the biofilm formation inhibitory active ingredient is protein, ammonium sulfate fractionation is considered to be effective, and the dialysis internal solution of mizuna cold water extract is fractionated with ammonium sulfate, and the precipitate fraction bio at each concentration is separated. The film formation inhibitory activity was evaluated (FIG. 10). The highest biofilm formation inhibitory activity was observed in the precipitate fraction at an ammonium sulfate concentration of 45-60%.

(Example 5-4)
Ion Exchange Chromatography of Ammonium Sulfate Fraction of Mizuna Cold Water Extract Precipitate fraction prepared according to Example 5-3 at an ammonium sulfate concentration of 45-60% was fractionated by anion exchange chromatography according to Example 5-2. As a result, two biofilm formation inhibitory activity peaks were observed (FIG. 11).

(Example 6)
Content determination

(Example 6-1)
Protein quantification Protein quantification was performed using the BCA method.

(Example 6-2)
Sugar quantification Sugar was quantified using the phenol-sulfuric acid method.

(Example 6-3)
Determination of polyphenols Polyphenols were determined using the Folin-ciocalteu method.
Regarding the above-mentioned purification, Table 2 shows a summary of the results of fractionation of mizuna cold water extract by dialysis, ammonium sulfate fractionation and anion exchange chromatography. As the purification step progressed, the specific activity per weight increased, and the purification of the active ingredient progressed. In addition, 80% or more of the components contained in the active fraction of ion exchange chromatography are proteins, suggesting the possibility that the active components are proteins.

(Example 7)
Evaluation of biofilm formation inhibitory activity of chewing gum with mizuna extract

(Example 7-1)
Preparation of Mizuna Mixed Chewing Gum Mizuna extract mixed chewing gum was prepared with the composition shown in Table 3.

BFI-01: Mizuna cold water extract was added and kneaded for 12 minutes.
BFI-02: After brewing for 12 minutes, the mizuna cold water extract is added and then kneaded for about 3 minutes.

(Example 7-2)
Preparation of chewing gum extract solution 25 ml of PBS heated to 37 ° C is added to 5 g of chewing gum, extracted by crushing in a mortar for 5 minutes, centrifuged at 1,100 xg for 15 minutes, the supernatant is recovered, and a sterile filter The product sterilized with (0.2 μm) was used as a gum extract.

(Example 7-3)
Evaluation of biofilm inhibitory activity of chewing gum extract A 96-well microtiter plate was used. To each well, 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 the test bacterial suspension, 10 μl of PBS was added at 37 ° C. under 5% CO ₂ condition. Culture was performed for 20 hours. The amount of biofilm formed was determined according to Example 2-2.

(Example 7-4)
Evaluation of dissolution rate of mizuna extract from chewing gum Absorbance at a wavelength of 280 nm (Abs 280 nm) was measured, and the dissolution rate of mizuna extract was evaluated by the following formula.
(A1) Abs 280 nm of mizuna extract dissolved in control gum extract at a concentration of 2000 ppm
(A2) Abs 280 nm of mizuna cold water extract mixed chewing gum extract
(A3) Abs 280 nm of control gum
Elution rate (%) = ((A2-A3) / (A1-A3)) × 100

  According to the above examples, the biofilm formation inhibitory activity of the mizugam cold water extract-containing tune gum extract was examined. The mizuna cold water extract-containing chewing gum extract suppressed the biofilm formation of A. naeslundii (FIG. 12). Moreover, the fall of activity by mix | blending a mizuna cold water extract with chewing gum was not seen. In addition, the elution rate of Mizuna cold water extract was 83.0% for BFI-01 and 85.7% for BFI-02.

Mizuna cold water extract also showed biofilm formation inhibitory activity against Actinomyces clinical strains, and also suppressed biofilm formation on hydroxyapatite and flow cell evaluation. The possibility of exhibiting biofilm formation inhibitory activity was suggested.
The fractionation of the active ingredient in the extract of cold water extract of mizuna suggested that protein may be the active ingredient. On the other hand, although not shown in the data, since BSA and milk protein preparations did not show biofilm inhibitory activity, not all proteins showed activity, and they were specific to the protein contained in mizuna extract. Actinomyces Biofilm formation inhibitory activity was considered.

The mizuna cold water extract showed the activity of inhibiting the formation of Actinomyces biofilm and was also effective against Actinomyces clinical isolates. Mizuna cold water extract showed biofilm formation inhibitory activity on hydroxyapatite and in evaluation using a flow cell, suggesting the possibility of activity in the human oral cavity. In addition, chewing gum containing mizuna cold water extract showed A. naeslundii biofilm formation inhibitory activity.
The mizuna cold water extract was fractionated by dialysis, ammonium sulfate fractionation, and anion exchange chromatography, suggesting that the active ingredient is a protein having a molecular weight of 10 kDa or more.

  Next, it is a product other than the above-mentioned chewing gum, a dressing containing a biofilm formation inhibitor containing the mizuna cold water extract of the present invention, toothpaste, spray for halitosis, troche, candy, tablet confectionery, gummy jelly, Beverages were produced in a conventional manner. Their formulations are shown below. Note that the scope of the present invention is not limited by these.

(Example 8)
A mouthwash was prepared according to the following formulation.
Ethanol 2.0% by weight
Mizuna cold water extract 1.0
Fragrance 1.0
Water remaining
100.0

Example 9
A toothpaste was produced according to the following formulation.
Calcium carbonate 50.0% by weight
Glycerin 19.0
Mizuna cold water extract 1.0
Carboxymethylcellulose 2.0
Sodium ralyl sulfate 2.0
Fragrance 1.0
Saccharin 0.1
Chlorhexidine 0.01
Water remaining
100.0

(Example 10)
A spray for halitosis was produced according to the following formulation.
Ethanol 10.0% by weight
Glycerin 5.0
Mizuna cold water extract 1.0
Fragrance 0.05
Coloring 0.001
Water remaining
100.0

(Example 11)
A lozenge was produced according to the following formulation.
Mizuna cold water extract 92.3% by weight
Gum arabic 6.0
Fragrance 1.0
Sodium monofluorophosphate 0.7
100.0

(Example 12)
Candy was manufactured according to the following prescription.
51.0% by weight sugar
Reduced water candy 32.0
Citric acid 1.0
Fragrance 0.2
L-Menthol 1.0
Mizuna cold water extract 0.4
Water remaining
100.0

(Example 13)
Tablet confectionery was produced according to the following prescription.
74.7% by weight sugar
Lactose 18.9
Mizuna cold water extract 2.0
Sucrose fatty acid ester 0.15
Water 4.25
100.0

(Example 14)
Gummy jelly was manufactured according to the following prescription.
Gelatin 60.0% by weight
Reduced water candy 32.4
Mizuna cold water extract 0.5
Vegetable oil 4.5
Malic acid 2.0
Fragrance 0.5
100.0

(Example 15)
A beverage was produced according to the following formulation.
Orange juice 30.0% by weight
Mizuna cold water extract 0.5
Citric acid 0.1
Vitamin C 0.04
Fragrance 0.1
Water remaining
100.0

  The composition for oral cavity containing the mizuna cold water extract of the present invention has a periodontal disease preventive action showing a biofilm formation inhibitory action, which is an action different from that of the conventional antibacterial agent against periodontal pathogenic bacteria. It is a preventive agent for periodontal disease in terms of eyes. Therefore, there are merits such that the risk of appearance of resistant bacteria is low compared to existing antibacterial agents and the like, and application to various products is possible.

This application claims priority from Japanese Patent Application No. 2013-018728 filed on Feb. 1, 2013, the contents of which are incorporated herein by reference.

Claims (11)

  An oral composition containing a cold water extract of a cruciferous plant.   The composition for oral cavity according to claim 1, wherein the cold water extract of the cruciferous plant is a mizuna cold water extract.   Biofilm formation inhibitor containing cold water extract of cruciferous plant.   The biofilm formation inhibitor according to claim 3, wherein the cruciferous plant extract is a mizuna cold water extract.   The biofilm formation inhibitor according to claim 3 is a periodontal disease biofilm inhibitor.   The biofilm formation inhibitor according to claim 3 is an acid-derived biofilm inhibitor.   A mouthwash comprising the oral composition according to claim 1 or 2.   A toothpaste comprising the oral composition according to claim 1 or 2.   An inhalant comprising the oral composition according to claim 1 or 2.   A lozenge comprising the oral composition according to claim 1. A food containing the composition for oral cavity according to claim 1 or 2.