TWI620576B - Oral composition - Google Patents

Abstract

The present invention provides a periodontal disease preventive agent with a novel viewpoint compared with existing antibacterial agents and the like, which has the advantages of low risk of occurrence of resistant bacteria and the like. Oral composition having a biofilm formation inhibitory effect containing cold water extract of watercress.

Description

Oral composition

The present invention relates to an oral composition having an excellent periodontal disease improving effect on an oral biofilm that causes oral diseases.

The so-called periodontal disease is a group of diseases found in periodontal tissues. In the narrow sense, it is equivalent to gingivitis, periodontitis, and occlusive trauma. Periodontal disease is an oral infection caused by dental plaque as the main cause. There are more than 700 kinds of bacteria in the human oral cavity. In healthy oral cavity, the initial attached bacteria of the genus Streptococcus or Actinomyces belong to the tooth surface. Among them, Actinomyces naeslundii is known as the cause of hemorrhagic gingivitis, and A.naeslundii, which is initially attached, forms a biofilm, forms plaque, and causes inflammation of the gums. The report revealed that lipoproteins on the bacterial membrane of the gingival inflammation system caused by Actinomyces are caused by the production of IL-8 or TNF-α through the TLR2 of gingival epithelial cells or macrophages. If the gums cause inflammation, periodontal sacs are formed and accompanied by bleeding or exudate of gingival sulcus exudates, known as Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans, Treponema Denticola and others are settled in the environment of periodontal sac. In addition, A.naeslundii not only adheres to the tooth surface, but also co-aggregates with many oral bacteria, becoming a scaffold for periodontal pathogenic bacteria to settle on plaque. From these facts, A. naeslundii has attracted attention in recent years as an important bacterium related to the occurrence of periodontal disease because plaque in the early stage is transformed into a plaque in the late stage (biofilm of periodontal disease).

Traditional periodontal disease prevention systems have sterilized periodontal pathogenic bacteria such as P.gingivalis and suppressed periodontal disease. However, because periodontal pathogenic bacteria and biofilms coexist deep in the periodontal sac, Antibacterial substances are difficult to penetrate, and most of them fail to obtain the desired effect.

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

Because periodontal disease is exacerbated by gingivitis, preventing gingivitis can prevent periodontal disease more effectively. Therefore, an effective periodontal disease prevention effect can be expected by inhibiting A.naeslundii from forming a biofilm material.

The present inventors confirmed that the formation of A.naeslundii biofilm was promoted by acid pressure, and confirmed that the extracts of five cruciferous plants such as Brassica chinensis or Komatsuki and ice flowers reduced the amount of acid-derived biofilms of A.naeslundii. To 50 to 90% of activity (Patent Document 2).

In this application, water extract (water dish, Brassica, Brassica rapa var.nipposinica) is a candidate material, and the detailed activity evaluation and the identification of the properties of the active ingredients are reviewed in detail.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] JP 2008-174542

[Patent Document 2] Japanese Patent Application Laid-Open No. 2011-196315

Periodontal pathogenic bacteria coexist with the biofilm in the deep part of the periodontal sac. Using the traditional periodontal disease suppression method with periodontal pathogenic bacteria as the target antibacterial agent, the oral biofilm hinders the penetration of the antibacterial agent. Produces the expected periodontal disease suppression effect. In addition, the use of an antibacterial agent is not suitable because of the high risk of the appearance of resistant bacteria. Therefore, it is considered to be a safer and more effective method for the prevention of periodontal disease than to control the periodontal pathogenic bacteria with an antibacterial agent and to perform a biofilm formation system for controlling periodontal pathogenic bacteria at an early stage.

As a result of diligent research by the present inventors, it has been found that the watercress extract has an inhibitory effect on the film formation of Actinomyces naeslundii derived from acid. The suppression effect is that the higher the extraction temperature is, the lower the temperature is. As a result of separating the active ingredients of the watercress 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 more than 80% of the ingredients contained in the active division are proteins, and the present invention has been completed. Also, because the water vegetable extract is wrong A. naeslundii proliferation affects, so it is believed that the mechanism of action is different from the antibacterial effect.

Actinomyces naeslundii is a Gram-positive bacilli found in gingivitis or root erosions, and is known as the primary periodontal pathogenic bacteria. Because it co-aggregates with streptococci or periodontal pathogenic bacteria, in order to grasp the bacteria that are key to the bacterial plaque migration to periodontal disease, controlling A.naeslundii is associated with the prevention of periodontal disease. Studies by the present inventors have confirmed that acids such as butyric acid produced by periodontal pathogenic bacteria increase biofilm formation.

Since the oral composition containing the hydroponic extract of the present invention significantly inhibits the formation of a biofilm of periodontal pathogenic bacteria at an early stage, it is effective as a method for preventing periodontal disease that is safer and more effective than an antibacterial agent.

The best form to implement the invention

The present invention relates to a composition for oral cavity containing a hydroponic extract.

Furthermore, the present invention relates to an oral composition in which the aforementioned water vegetable extract is a cold water extract.

Furthermore, the present invention relates to a periodontal disease biofilm inhibitor containing a hydroponic extract.

Furthermore, the present invention relates to an acid-derived biofilm inhibitor in which the aforementioned water vegetable extract is a cold water extract.

Furthermore, the present invention relates to a mouthwash, a toothpaste, an inhalant, a lozenge, and a food made of the composition for oral cavity.

Hereinafter, the present invention will be described more specifically with reference to examples. In addition, the invention of this application is not limited by these examples.

[Figure 1] Comparison of biofilm formation inhibitory activity by extraction temperature

[Figure 2] Comparison of biofilm formation inhibitory activity according to extraction temperature

[Figure 3] Biofilm formation inhibitory activity of cold water extract of hydroponics on hydroxyphosphate lime

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

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

[Figure 5] Inhibitory activity of cold water extracts from watercress on biofilm formation in clinical isolates

[Figure 6] Inhibition activity of biofilm formation from cold water extracts of water vegetables in a flow cell

[Figure 7] Observation diagram of biofilm by conjugate focus laser microscope

[Figure 8] Comparison of biofilm formation inhibitory activity of cold water extract and cold water extract of diarrhea by dialysis treatment

[Fig. 9] Results of anion exchange chromatography for dialysis of internal fluids of cold water extracts of watercress

[Fig. 10] Biofilm formation inhibitory activity of ammonium sulfate separation zone for dialysis of internal liquid of cold water extract

[Figure 11] Ammonium sulfate separation zone for dialysis of internal fluids from cold water extracts of water vegetables Of anion exchange chromatography

[Figure 12] Inhibition activity of chewing gum mixed with cold water extract of hydroponic vegetables

[Example] (Example 1) Preparation method of water vegetable extract:

A commercially available water dish (from Ibaraki Prefecture) was purchased, and dried leaves of the water dish were prepared by freeze-drying. The dried vegetable leaf was finely pulverized, and extracted at 70 ° C, room temperature, and 4 ° C for 2 hours at a ratio of 50 ml of deionized distilled water to 1 g of the crushed dried vegetable leaf. The obtained extract was suction-filtered to 13,000 × g. After centrifugation for 10 minutes, the supernatant was lyophilized as a water vegetable extract for test.

(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 culture medium at 37 ° C. overnight to a stationary phase at 1,100 × g. Centrifuge for 10 minutes to collect bacteria. Under the same conditions, the cells were centrifuged and washed three times with PBS, and those with PBS adjusted to an OD of _{660 nm} = 0.3 were used as test bacteria suspensions for the test system.

Biofilm formation was performed using a 96-well microtiter plate. Add 100 μl of 2 × Trypticase Soy Broth (TSB) medium 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% in each well. Under the condition of CO ₂ , the culture is performed for 16 to 20 hours.

(Example 2-2)

Quantification of biofilm formation using a 96-well microtiter plate

The culture supernatant cultured according to Example 2-1 was decanted, and each well was washed with 200 μl of PBS, and then 100 μl of a 0.25% safranine solution (Nissui Pharmaceutical) was added, and left to stand for 15 minutes to stain the biofilm. . After decanting the safran 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 saffron. Using a microplate analyzer, the absorbance at 492 nm was used to quantify the amount of biofilm. .

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

Therefore, is it possible to use cold-water extracts of hydroponics as candidate materials for Actinomyces biofilm inhibition materials to show activity in human oral cavity? And further review.

(Example 2-3)

Biofilm formation on hydroxyphosphate lime (HA)

The HA sheet is made of cow teeth covered with enamel to form a 7mm × 7mm × 1.5mm shape. After the HA tablets were sterilized in an autoclave, they were equilibrated with PBS at room temperature for 1 hour, 400 μl of human saliva was taken aseptically, and they were left at room temperature for 1 hour to form a thin film (pellicle). After washing with PBS, A 5 mg / ml BSA solution was blocked at room temperature for 30 minutes, and used in the test.

Biofilm formation was performed using a 24-well microtiter plate. Add 400 μl of 2 × TSB medium 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 to each well, and place HA slices in each well, according to Example 2 -1 for culture.

(Example 2-4)

Quantification of biofilm formation on HA

After culturing according to Example 2-3, the HA pieces were taken out with nickel seeds and immersed in PBS once to wash. The washed HA sheet was put into a new well, and 600 μl of a saffron solution was added, and left to stand for 15 minutes to stain the biofilm. Take out HA with nickel, wash it with deionized distilled water, put it into a new well, add 600 μl of 70% ethanol, shake for 30 minutes to dissolve saffron, and transfer 300 μl of this eluate to a 96-well microtiter plate. According to Example 2-2, Amount of biofilm.

According to the above examples, the results of evaluating the biofilm formation inhibitory activity of cold water extracts of water vegetables on the hydroxyphosphate lime forming a pellicle are shown in FIG. 3. The cold water extract of watercress was on hydroxyphosphate lime, and it showed the inhibitory activity of A.naeslundii biofilm formation in the same way as in 96 wells.

(Example 3)

Isolation of Actinomyces clinical isolates

(Example 3-1)

PCR / Multiplex PCR (multiple primer polymerase chain reaction)

For PCR, 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 were performed in a PCR tube. The multiple primer polymerase chain reaction was performed by changing the above composition to three 50 μM forward primers, three Reverse primers, and 16.75 μl H ₂ O each. The reaction conditions were heat-treated at 95 ° C for 10 minutes, and then performed at 95 ° C. 30 seconds, 50 ° C. 30 seconds, 72 ° C. 30 cycles of 30 seconds, and finally 72 ° C, 7 minutes, to complete the extension reaction completely. The annealing temperature of a plurality of primer polymerase chain reactions was performed at 53 ° C.

(Example 3-2)

Isolation of clinical isolates

7 healthy men and women from any choice (Male: 3, Female: 4 Human). Plaque was collected and cultured in Actinomyces selection medium (CFAT agar medium). The formed colonies were Gram-stained and then observed under a microscope to screen for Gram-positive bacilli.

Genes were extracted from each Gram-positive bacterium with a gene extraction reagent set (sigma), and PCR was performed according to Example 3-1 to increase the upper end of the 16SrRNA gene by about 500 bp. The sequences used for PCR are shown in Table 1.

The PCR products were purified using the PCR Clean-Up reagent set (promega), and the analysis of the base sequence was commissioned by an external (stock) Macrogen-japan company. The obtained base sequence of the 16SrRNA gene was searched for the identity with the database on GenBank to estimate the bacteria. According to Example 3-1, the above-mentioned bacterial line of Actinomyces genus was used to increase atpA by a plurality of pairs of primer polymerase chain reactions, and similarly commissioned an external (stock) Macrogen-japan company to perform a salt-based sequence analysis. The primer sequences used are shown in Table 1. The obtained base sequence was systematically analyzed together with the base sequence of atpA on the database for species identification. The obtained A.naeslundii and A.oris were Actinomyces spp. Clinical isolates.

According to the above examples, the clinical isolates were isolated from the oral cavity of humans, and a total of 9 Actinomyces clinical isolates (A. naeslundii: 4 strains and A.oris: 5 strains) were successfully isolated from 7 people. Further, for 7 of the clinical isolates of Actinomyces isolates, when evaluating the biofilm formation inhibitory activity of cold water extracts of watercress, although the amount of biofilm formation differed by strains, the cold water extracts of watercress showed that for all clinical isolates, Biofilm inhibitory activity (Figure 5). When the results of FIG. 3 are combined, it is shown that the cold water extract of watercress is highly likely to show the activity of inhibiting biofilm formation even in a human oral cavity.

(Example 4)

Biofilm Inhibition Evaluation of Cold Water Extracts from Water Vegetables Using a Flow Cell

The above examples were evaluated by a stationary system using a 96-well plate, and showed that cold water extracts of watercress have Actinomyces naeslundii biofilm inhibitory activity. However, when the actual oral cavity is considered, saliva is continuously secreted, and there is a flow of saliva in the oral cavity. Therefore, in order to evaluate whether the cold water extract of aquatic plants showed A.naeslundii biofilm inhibitory activity even in the oral cavity, a flow-type processing tank simulating the oral environment was used to evaluate the A.naeslundii biofilm inhibitory activity of cold water extracts of vegetable.

assessment method (Example 4-1) Evaluation strain

Actinomyces naeslundii ATCC19039 strain was used.

(Example 4-2) Preparation of water vegetable extract

A commercially available water dish was purchased, and the dried leaves of the water dish were prepared by freeze-drying. Extraction was performed at 4 ° C. for 2 hours at a ratio of 50 ml of deionized distilled water to 1 g of the crushed hydrophyte dried leaves. The supernatant of the water vegetable residue was removed by suction filtration and centrifugation, and the water vegetable extract was recovered.

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

A.naeslundii was cultured in 5 ml of BHI medium overnight, and the bacteria were collected by centrifugation, followed by centrifugation and washing with PBS. The BHI medium was prepared to have an OD of _{660 nm} = 0.4, and this was used as the test bacterial solution. Inoculate 400 μl of the test bacterial solution in a flow processing tank chamber (ACCFL0001: STOVALL LIFE SCIENCE). After the biofilm forming surface of the chamber is on the lower side, let it 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. A 60 mM butyric acid TSB medium was cultured under 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 watercress is to add the cold water extract of watercress to the aforementioned culture medium so that the final concentration becomes 1 mg / ml, and the evaluation is performed after the same culture.

(Example 4-4) Observation of biofilm

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

According to the above examples, when the biofilm formation inhibitory activity of cold water extracts of cold water extracts under the conditions of a flow system using a flow processing tank was evaluated, the cold water extracts of cold water extracts inhibited the biofilm formation of A.naeslundii (Figures 6 and 7). In addition, the observation by a conjugate coke laser microscope showed that the occupancy ratio of the biofilm-based dead bacteria formed under the condition that the cold-water extract of water vegetable was added (FIG. 7).

In more detail, in the evaluation using a flow processing tank, if butyric acid is added, the biofilm formation of A.naeslundii is increased, and the biofilm system has the same degree of bacterial and dead bacteria. The ratio of dead bacteria constituting the biofilm is higher than when no butyric acid is added. In the presence of cold water extract of 1 mg / ml water vegetables, the biofilm formation of A.naeslundii was inhibited, especially the amount of dead bacteria attached was reduced. Therefore, it is shown that the cold water extract of Brassica napus inhibits the formation of biofilms of A. naeslundii dependent on butyric acid.

(Example 5)

Separation of cold water extracts from water vegetables

(Example 5-1)

Dialysis

The cold water extract of water vegetable was dissolved in deionized distilled water, centrifuged at 13,000 × g for 10 minutes, and the supernatant was recovered. The cellulose was placed in a dialysis cellulose tube (as-1) with a molecular weight of 10 kDa in a separation zone. Dialysis was performed indoors for 2 days. Freeze-dried part of the inner and outer dialysis fluids Close.

In order to estimate the molecular weight of cold-water extracts of water vegetables as described above, the dialysis of cold-water extracts of water vegetables was evaluated by separating the dialysis cellulose tube with a molecular weight of 10 kDa, and the activity of dialysis internal and external liquids was evaluated. . The molecular weight of the active ingredient was 10 kDa or more (Fig. 8).

(Example 5-2)

Ion exchange chromatography of dialysis internal fluid of cold water extract

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

Anion exchange chromatography was used to separate the dialysis fluid from the cold water extract of watercress, and the activity was evaluated depending on which component was extracted (Figure 9). As a result, the activity is in the first peak to the second peak of the protein, and the activity is shown depending on the extraction mode of the protein. The above shows the possibility that the active ingredient is a protein.

(Example 5-3)

Division of ammonium sulfate separation

A dialysis internal fluid sample of the cold water extract of the water vegetable prepared according to Example 5-1 was dissolved in PBS, and the concentration of ammonium sulfate was gradually increased to 30%, 45%, 60%, 75%, the precipitates of each concentration were recovered by centrifugation at 13,000 × g for 15 minutes. The recovered precipitation division was dissolved in deionized distilled water, and then lyophilized and recovered after dialysis. In addition, 75% of the unprecipitated area was also freeze-dried and recovered after dialysis.

As mentioned above, if the biofilm formation inhibitory active ingredient is a protein, ammonium sulfate separation is considered effective. The ammonium sulfate separation zone is used to divide the dialysis internal solution of cold water extracts of vegetables, and the biofilm formation inhibitory activity of the precipitation zone at each concentration is evaluated. (Figure 10). It can be seen that the biofilm formation inhibitory activity is highest in the precipitation zone with ammonium sulfate concentration of 45 ~ 60%.

(Example 5-4)

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

According to Example 5-3, the precipitation division with ammonium sulfate concentration of 45 to 60% was prepared. According to Example 5-2, the results of the separation division by ion exchange chromatography showed that two biofilm formation inhibition Active peak (Figure 11).

(Example 6)

Contained ingredients

(Example 6-1)

Protein quantification

The protein was quantified using the BCA method.

(Example 6-2)

Sugar ration

The sugar was quantified using the phenol-sulfuric acid method.

(Example 6-3)

Polyphenol quantification

Polyphenols were quantified using the Folin-ciocalteu method.

As mentioned above, Table 2 summarizes the results of refining the separation results of cold water extracts of watercress by dialysis, ammonium sulfate separation, and anion exchange chromatography. As the refining stage progresses, the specific activity per weight becomes higher, and the refining of the active ingredient is progressing. In addition, more than 80% of the content of the active zone of ion exchange chromatography is a protein, indicating that the active ingredient is a protein.

(Example 7)

Evaluation of Biofilm Formation Inhibitory Activity of Chewing Gum Blended with Hydroponic Extract

(Example 7-1)

Production of chewing gum mixed with water vegetables

Based on the third composition, a cold water extract chewing gum with mixed vegetables is prepared.

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

BFI-02: After 12 minutes of kneading, add cold water extract of water vegetable, and then knead for about 3 minutes.

(Example 7-2)

Preparation of chewing gum extract

To 5 g of chewing gum, add 25 ml of PBS warmed to 37 ° C, mash in a mortar for 5 minutes, extract, centrifuge at 1,100 × g for 15 minutes, recover the supernatant, and perform a sterilizing filter (0.2 μm). The sterilized product was used as a gum extract.

(Example 7-3)

Biofilm formation inhibitory activity of chewing gum extract

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

(Example 7-4)

Evaluation of dissolution rate of water vegetable extract from chewing gum

The absorbance (Abs 280 nm) of a wavelength of 280 nm was measured, and the dissolution rate of the water vegetable extract was evaluated from the following calculation formula.

(A1) Abs 280nm of a water-soluble vegetable extract dissolved in a control chewing gum extract at a concentration of 2000 ppm

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

(A3) Abs280nm of control chewing gum

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

According to the above embodiment, regarding the biofilm formation inhibitory activity of chewing gum cold water extract blended with cold water extract, when reviewing, chewing gum cold liquid extract mixed with cold water extract inhibited the biofilm formation of A.naeslundii (Figure 12). In addition, there was no decrease in the activity caused by mixing cold water extracts of hydroponics with chewing gum. In addition, the dissolution rate of cold water extracts of water vegetables was 83.0% for BFI-01 and 85.7% for BFI-02.

The cold water extract of Actinidia esculenta showed clinical inhibitory activity against Actinomyces clinical strains. In addition, it was treated with hydroxyphosphate lime and flow-through. In the evaluation of the tank, because the biofilm formation is inhibited, the possibility that the cold-water extract of water vegetable shows the biofilm formation inhibitory activity even in the human oral cavity is shown.

According to the separation of active ingredients in cold water extracts of water vegetables, the possibility of proteins as active ingredients is shown. On the other hand, although the data is not shown, it is believed that because no biofilm inhibitory activity of BSA or milk protein preparations is seen, not all proteins are active, but specific Actinomyces are seen in the proteins contained in cold water extracts of water vegetables. Biofilm formation inhibitory activity.

Actinomyces biofilm formation inhibitory activity was found in cold water extracts of watercress, and it was effective even on clinical isolates of Actinomyces. The cold water extract of watercress on hydroxyphosphate lime, and the evaluation using a flow treatment tank, also showed the biofilm formation inhibitory activity, showing the possibility of showing activity in the human mouth. In addition, chewing gum with cold water extract of hydroponics showed A.naeslundii biofilm formation inhibitory activity.

When the cold-water extract of water vegetable was separated by dialysis, ammonium sulfate separation, and anion exchange chromatography, the active ingredient was a protein with a molecular weight of 10 kDa or more.

Next, a mouthwash, a toothpaste, a bad breath spray, a lozenge, a hard candy, a lozenge, a softener containing the biofilm formation inhibitor containing the cold-water extract of the present invention and a product other than the above-mentioned chewing gum are manufactured by a conventional method. Sugar, drinks. The following shows these formulations. In addition, the scope of the present invention is not limited by these.

(Example 8)

Mouthwash was manufactured according to the following formulation.

(Example 9)

Toothpaste was manufactured according to the following formulation.

(Example 10)

A bad breath spray was produced according to the following formulation.

(Example 11)

Oral tablets were produced according to the following formulation.

(Example 12)

Hard candies were produced according to the following recipe.

(Example 13)

Ingots were produced according to the following recipe.

(Example 14)

Fudge was produced according to the following recipe.

(Example 15)

A beverage was manufactured according to the following recipe.

[Industrial availability]

The oral composition containing the cold-water extract of hydroponic vegetable of the present invention has a new view because it has a different effect from the conventional antibacterial agent for periodontal pathogenic bacteria and has a periodontal disease prevention effect showing an inhibitory effect on biofilm formation. Point of periodontal disease preventive. Therefore, compared with existing antibacterial agents and the like, it is considered that they have advantages such as a low risk of occurrence of resistant bacteria, and can be applied to various products.

Claims (9)

A composition for oral cavity, which is characterized by containing cold water extract of cold water extract extracted at 4 ° C, and separating and dividing the active ingredients of the cold water extract of cold water extract. The active ingredient is a component with a molecular weight of 10 kDa or more, and the active zone More than 80% of the contained ingredients are protein. A biofilm formation inhibitor, which is characterized by containing cold water extract of cold water extract extracted at 4 ° C, and separating and dividing active ingredients of the cold water extract of cold water extract. The active ingredient is a component with a molecular weight of 10 kDa or more, and the active zone More than 80% of the contained ingredients are protein. For example, the biofilm formation inhibitor of the second patent application range, wherein the biofilm formation inhibitor is a periodontal disease biofilm inhibitor. For example, the biofilm formation inhibitor of the second patent application range, wherein the biofilm formation inhibitor is an acid-derived biofilm inhibitor. A mouthwash, which is characterized by containing the composition for oral cavity as described in claim 1 of the scope of patent application. A toothpaste is characterized by containing the composition for oral cavity as described in the first patent application. An inhalant comprising an oral composition as described in claim 1 of the scope of patent application. An oral lozenge is characterized in that it contains an oral composition as described in item 1 of the patent application. A food product characterized by containing an oral composition as described in claim 1 of the scope of patent application.