JP2014227355A - Biofilm formation inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new dental caries prevention agent that has no influence over oral indigenous bacteria.SOLUTION: By a biofilm formation inhibitor comprising one or more of natto, isolated Bacillus natto, or a culture filtrate of isolated Bacillus natto as an active ingredient, provided is a new dental caries prevention agent that has no influence over oral indigenous bacteria.

Description

  The present invention relates to a biofilm formation inhibitor. More specifically, the present invention relates to a biofilm formation inhibitor containing as an active ingredient natto, natto isolate, or natto isolate culture solution.

Caries (decayed teeth) are cariogenic bacteria Streptococcus mutans (hereinafter sometimes referred to as S. mutans) and Streptococcus sobrinus (hereinafter sometimes referred to as S. sobrinus) adhere to and grow on the tooth surface. It is caused by forming a caries-induced biofilm.
In recent years, diseases caused by dental caries such as aspiration pneumonia have increased in elderly people and the like, and provision of a useful agent for prevention of dental caries is desired.

  Conventionally, bactericides and antibacterial agents against cariogenic bacteria have been used as caries preventive agents. However, these bactericides and antibacterial agents have a problem that they also act on useful oral resident bacteria that exhibit immune functions against opportunistic bacterial infections and the like. Therefore, it is desired to provide a new caries preventive agent that does not affect oral bacteria.

S. cariogenic bacteria For example, in Patent Document 1, beans that are pulverized with beans such as cowpea, red beans, purple flower beans, white flower beans, black bean, kintokimame, and the like are extracted with water, a hydrophilic organic solvent, or the like. Extract of S. It is listed as a substance that controls mutans quorum sensing (bacterial density-dependent gene expression control system). Inhibiting biofilm formation by mutans is disclosed.
In Patent Document 2, a strain of lactic acid bacteria isolated from the human oral cavity and pickles is designated as S. cerevisiae. It has been disclosed that the composition for oral cavity that suppresses the growth of mutans and biofilm formation, the biofilm formation inhibitor for oral cavity, and the like.

Patent Document 3 discloses that nattokinase has an effect of improving bad breath by removing not only mutans (S. mutans) but also a plurality of oral pathogenic bacterial species, and nattokinase containing nattokinase. It has been disclosed that an oral composition comprising an extract and polyoxyethylene hydrogenated castor oil as essential components has a high tartar removal effect and a bad breath improving effect.
In Patent Document 4, the stickiness of natto produced by fermenting beans with natto bacteria and its components are S. cerevisiae. Although it is described that the growth of mutans (mutans bacteria), acid production, tooth erosion by the produced acid, calculus formation ability, etc. are suppressed, these specific effects are not disclosed in this document. .

  As shown in these documents, various components are cariogenic bacteria. Although it is disclosed as acting specifically on mutans, it is desired to provide an agent capable of preventing caries more safely and effectively.

JP 2012-67018 A JP 2007-117064 A Japanese Patent Application Laid-Open No. 2011-126819 JP 2010-124772 A

  It is an object to provide a new caries preventive agent that does not affect oral bacteria.

  As a result of intensive research to solve the above problems, the present inventors have suppressed the formation of biofilms by cariogenic bacteria by acting natto, natto isolate, or natto isolate culture filtrate. The present inventors have found that this can be done and have completed the present invention. This makes it possible to prevent and treat caries caused by caries and caries safely and effectively.

That is, the present invention relates to the following biofilm formation inhibitors (1) to (4).
(1) A biofilm formation inhibitor comprising as an active ingredient at least one selected from the group consisting of natto, natto isolates, and natto isolate culture filtrate.
(2) The biofilm formation inhibitor as described in (1) above, wherein the active ingredient is a low molecular fraction of less than 10 kDa contained in the natto isolate bacterial culture filtrate.
(3) The biofilm formation inhibitor according to (1) or (2), which suppresses biofilm formation by cariogenic bacteria.
(4) The biofilm formation inhibitor according to (3), wherein the cariogenic bacterium is Streptococcus mutans or Streptococcus sobrinus.

  In accordance with the present invention, S.M. By providing a biofilm formation inhibitor that specifically acts on cariogenic bacteria such as mutans, it becomes possible to prevent and treat caries and diseases caused by caries. The biofilm formation inhibitor of the present invention has high safety and can be provided at low cost because the active ingredient is derived from food.

It is the figure which showed the biofilm formation inhibitory effect by a biofilm formation inhibitor (natto AE) (Test Example 1). It is the figure which showed the biofilm formation inhibitory effect by a biofilm formation inhibitor (natto isolate bacteria ae) (Test Example 1). It is the figure which showed the biofilm formation inhibitory effect by a biofilm formation inhibitor (culture filtrate ae) (Test Example 1). It is the figure which showed the biofilm formation inhibitory effect by the biofilm formation inhibitor (low molecular fraction e) (test example 1). It is the figure which showed the growth inhibitory effect of the cariogenic microbe (S. mutans) by the biofilm formation inhibitor (culture filtrate ae) (Test Example 2). It is the figure which showed the formation inhibitory effect of the biofilm by nattokinase (Test Example 3). It is the figure which showed the growth inhibitory effect of the cariogenic microbe (S. mutans) by nattokinase (Test example 3).

The “biofilm formation inhibitor” of the present invention refers to an agent that suppresses the formation of caries-induced biofilms formed by cariogenic bacteria.
Here, the cariogenic bacteria include S. cerevisiae. mutans, S.M. and sobrinus. The “biofilm formation inhibitor” of the present invention may be any agent that suppresses the formation of biofilms due to these cariogenic bacteria and oral flora containing cariogenic bacteria.
The “biofilm formation inhibitor” of the present invention may be any agent that suppresses the formation of this biofilm, but has the effect of suppressing the growth of cariogenic bacteria as well as the effect of suppressing the formation of biofilm. It may be.

  The “biofilm formation inhibitor” of the present invention may be any agent containing at least one selected from natto, natto isolates, and natto isolate culture filtrate, and a combination of two or more of these active components May be included. Moreover, the agent which uses as an active ingredient the low molecular fraction of less than 10 kDa contained in a natto isolate bacterial culture filtrate may be sufficient. Furthermore, in addition to these active ingredients, other ingredients that can be safely ingested by animals such as humans, pharmaceutically acceptable carriers, and the like may be included.

  Such a “biofilm formation inhibitor” of the present invention can be used as it is as a food, medicine or the like, or can be used as an active ingredient such as food or medicine. Further, it can be used in various forms such as liquid, powder, paste, liquid and solid. For example, it can be prepared into dentifrices, mouthwashes, tablets, mouth fresheners, chewing gums, wrinkles, etc., and also in dentifrices, liquid dentifrices, powder dentifrices, etc. it can.

  EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated further in detail, this invention is not limited to these.

1. Preparation of biofilm formation inhibitor Natto 5 kinds (3 companies) of commercially available natto A to E were added to each sterilized distilled water so as to be 10 w / v%, and stirred for 10 minutes with a blender (Waring Commercial Blender (Model 31BL92)). It was set as the film formation inhibitor.

2. Natto isolates above 1. The biofilm formation inhibitors (5 types) prepared in step 1 were appropriately diluted with sterilized distilled water and applied to a BHI agar medium (Brain Heart Infusion medium, BD). After culturing at 37 ° C. for 2 days under aerobic conditions, the grown colonies were picked and suspended in a BHI liquid medium (Brain Heart Infusion medium, BD), and cultured under the same conditions for 2 days. After that, similarly applied to BHI agar medium, cultured, and the grown colonies were picked and cultured in BHI liquid medium and purified twice repeatedly to separate each natto in the state contained in BHI liquid medium. Bacteria (live bacteria) were made into natto isolates a to e according to the type of natto (natto A to E) from which they were derived, and were used as biofilm formation inhibitors.

3. Culture filtrate Above 2. The natto isolates a to e prepared in Step 1 were suspended in a BHI liquid medium (BRAIN HEART INFUSION medium, BD), further cultured overnight at 37 ° C. and 5% CO ₂ , and then centrifuged (6,000 RPM, 15 MIN), and the supernatant was treated with a sterilizing filter (pore size 0.2 MM) to completely remove the cells. It was set as culture filtrate ae according to the kind (natto AE) of natto derived from this, and it was set as the biofilm formation inhibitor, respectively.

4). Low molecular fraction 3. 12 ml of each of the culture filtrates a to e prepared in the above were applied to ultrafiltration membranes (Amicon ultra-15 Filter Unit 10,000 NMWL, Millipore) and centrifuged. Each of the fractions of less than 10 kDa after ultrafiltration was made up to 12 ml with sterilized water, and the low molecular fractions a to e were used as biofilm formation inhibitors.
Each fraction of 10 kDa or more after ultrafiltration was also made up to 12 ml with sterilized water, and used as a sample in the following test example.

[Test Example 1]
Examination of effect of biofilm formation inhibitor Each biofilm formation inhibitor (natto A to E, natto isolates a to e, culture filtrate a to e, low molecular fraction a to e prepared in the same manner as the above examples. ) The effect of the biofilm formation inhibitor was examined by a co-culture test with cariogenic bacteria (S. mutans).

1. Co-culture test with natto and natto isolates Each biofilm formation inhibitor (any one of natto A to E or natto isolates a to e) and cariogenic bacteria (S. mutans) prepared in the same manner as in the above examples In the co-culture test, the presence or absence of biofilm formation by cariogenic bacteria was examined.
Example 1 Example 2 Each biofilm formation inhibitor (any one of natto A to E or natto isolates a to e) prepared by the same method was used. B. B. subtilis culture medium containing BHI liquid medium at 37 ° C. under aerobic conditions and stationary phase cells after overnight culture. Subtilis broth was used.
C. cariogenic bacteria (S. mutans) include S. mutans, including stationary phase cells after overnight culture at 37 ° C. with 5% CO ₂ in BHI liquid medium (BRAIN HEART INFUSION medium, BD). Mutans broth was used.
As a medium for co-culture, a double volume of Tryptic soy broth with outextrose medium (BD) containing 0.5% glucose was used.
160 μL of medium for co-culture, S. 20 μL of mutans culture solution and 20 μL of biofilm formation inhibitor (Natto A to E or Natto isolates a to e) are mixed and dispensed into a 96-well plate at 37 ° C. with 5% CO ₂ . For 20 hours.
In place of the biofilm formation inhibitor, B.I. A subtilis culture solution or sterilized distilled water was used as Control.
This was washed twice with 150 μL of distilled water and stained with 50 μL of safranin for 10 minutes. Thereafter, the plate was further washed twice with 150 μL of distilled water, dye extraction was performed with 70% EtOH, absorbance (492 nm) was measured with a plate reader, and the amount of biofilm was evaluated based on the absorbance value.

FIG. 1 shows the biofilm formation inhibitory effect when co-cultured with biofilm formation inhibitors (natto A to E). Moreover, the biofilm formation inhibitory effect at the time of carrying out a coculture with the biofilm formation inhibitor (natto isolation bacteria ae) in FIG. 2 was shown.
As a result, as shown in FIG. 1 and FIG. 2, any biofilm formation inhibitor (natto A to E, natto isolates a to e) is a biofilm produced by cariogenic bacteria (S. mutans). It was confirmed that the formation was significantly suppressed.

2. Co-culture test with culture filtrate and low molecular fractions Each biofilm formation inhibitor (any one of culture filtrates a to e or low molecular fractions a to e) and cariogenic bacteria prepared in the same manner as in the above examples. The presence or absence of biofilm formation by cariogenic bacteria was examined by a co-culture test with (S. mutans).
Example 3 Example 4 Each biofilm formation inhibitor (any one of culture filtrates a to e or low molecular fractions a to e) prepared by the same method as above was used. B. B. subtilis culture medium containing BHI liquid medium at 37 ° C. under aerobic conditions and stationary phase cells after overnight culture. Subtilis broth was used.
C. cariogenic bacteria (S. mutans) include S. mutans, including stationary phase cells after overnight culture at 37 ° C. with 5% CO ₂ in BHI liquid medium (BRAIN HEART INFUSION medium, BD). Mutans broth was used.
As a medium for co-culture, a double volume of Tryptic soy broth with outextrose medium (BD) containing 0.5% glucose was used.
80 μL of medium for co-culture, S. 20 μL of mutans culture solution and 100 μL of biofilm formation inhibitor (any one of culture filtrates a to e or low molecular fractions a to e) were mixed and dispensed into a 96-well plate, followed by 37 ° C., 5% CO Co-cultured for 20 hours under the conditions of ₂ .
In place of the biofilm formation inhibitor, B.I. Subtilis broth, which was used in Example 4 A fraction using a fraction less than 10 kDa that was ultrafiltered in the same manner as described above and a fraction using 10 kDa or more, or using sterile distilled water was defined as Control.
This was washed twice with 150 μL of distilled water and stained with 50 μL of safranin for 10 minutes. Thereafter, the plate was further washed twice with 150 μL of distilled water, dye extraction was performed with 70% EtOH, absorbance (492 nm) was measured with a plate reader, and the amount of biofilm was evaluated based on the absorbance value.

FIG. 3 shows the biofilm formation inhibitory effect when co-cultured with biofilm formation inhibitors (culture filtrates a to e). For comparison, the culture filtrates a to e heated at 95 ° C. for 15 minutes were subjected to the above 2. The results when co-cultured in the same manner as in (Fig. 3, Heat) are also shown.
As a result, as shown in FIG. 3, it was confirmed that any of the biofilm formation inhibitors (culture filtrates a to e) significantly suppressed the formation of biofilms by cariogenic bacteria (S. mutans). did it. On the other hand, the culture filtrates a to e heated at 95 ° C. for 15 minutes also have a lower biofilm formation inhibitory effect than the non-heated biofilm formation inhibitor (culture filtrate a to e), but compared with the control. It was confirmed that the biofilm formation inhibitory effect was exhibited.

FIG. 4 shows the biofilm formation inhibitory effect when co-cultured with the biofilm formation inhibitor (low molecular fraction e) among the biofilm formation inhibitors (low molecular fractions a to e). For comparison, the culture filtrate e, which is a biofilm formation inhibitor, and the polymer fraction of 10 kDa or higher fractionated from the culture filtrate e are prepared in the above 2. The results when co-cultured in the same manner as above are also shown.
As a result, as shown in FIG. 4, the biofilm formation inhibitor (low molecular fraction e) is biodegradable by cariogenic bacteria (S. mutans) in the same manner as the biofilm formation inhibitor (culture filtrate e). It was confirmed that the film formation was significantly suppressed. On the other hand, it was confirmed that the polymer fraction of 10 kDa or higher fractionated from the culture filtrate e had no biofilm formation inhibitory effect.
In addition, other biofilm formation inhibitors (low molecular fractions a to d) also significantly formed biofilms with cariogenic bacteria (S. mutans) in the same manner as biofilm formation inhibitors (culture filtrate e). It was confirmed that it was suppressed. On the other hand, it was confirmed that none of the polymer fractions of 10 kDa or higher fractionated from the culture filtrates a to d had a biofilm formation inhibitory effect.

[Test Example 2]
Examination of growth inhibitory effect of cariogenic bacteria by biofilm formation inhibitor Co-combination of biofilm formation inhibitors (culture filtrates a to e) and cariogenic bacteria (S. mutans) prepared in the same manner as in the above examples. The growth inhibitory effect of cariogenic bacteria was examined by a culture test.
Example 3 Each biofilm formation inhibitor (culture filtrates a to e) prepared by the same method was used. C. cariogenic bacteria (S. mutans) include S. mutans, including stationary phase cells after overnight culture at 37 ° C. with 5% CO ₂ in BHI liquid medium (BRAIN HEART INFUSION medium, BD). Mutans broth was used. As a medium for growth measurement for co-culture, a double volume of Tryptic soy broth outdextrose medium (BD) with 0.5% glucose added was used.
80 μl of growth measuring medium, 100 μl of biofilm inhibitor, 20 μl of mutans culture solution was mixed, dispensed into a 96-well plate, and cultured at 37 ° C. and 5% CO ₂ for 16 hours. After cultivation, the amount of growth was evaluated by measuring the absorbance value at 600 nm. What used sterilized distilled water instead of the biofilm formation inhibitor was set as Control.

As shown in FIG. 5, the biofilm formation inhibitor (culture filtrate c) not only acts on the suppression of biofilm formation by cariogenic bacteria, but also grows cariogenic bacteria (S. mutans). It was confirmed that it also acts on suppression.
On the other hand, biofilm formation inhibitors (culture filtrates a, b, d, e) do not suppress the growth of cariogenic bacteria (S. mutans) and therefore do not act on the growth of cariogenic bacteria. It was confirmed that the agent acts specifically on the formation of biofilms by cariogenic bacteria.

[Test Example 3]
Examination of the effect of nattokinase on cariogenic bacteria According to the same method as in Test Example 1 and Test Example 2, whether nattokinase has a biofilm formation inhibitory effect on protozoal cariogens or a growth inhibitory effect on protozoal cariogens investigated. Nattokinase was from Wako Pure Chemical Industries.

FIG. 6 shows the biofilm formation inhibitory effect of nattokinase.
As a result, as shown in FIG. 6, when nattokinase was added so that the final concentration was 2 mg / ml or 200 μg / ml, biofilm formation by cariogenic bacteria (S. mutans) was prevented. Although significantly suppressed, it was confirmed that biofilm formation could not be suppressed at a low concentration such as 20 μg / ml.
Moreover, although examination of the growth inhibitory effect of the cariogenic bacterium by nattokinase was shown in FIG. 7, it was confirmed that the growth of the cariogenic bacterium was not suppressed at any concentration.

  S. of the present invention. By providing a biofilm formation inhibitor that specifically acts on cariogenic bacteria such as mutans, it becomes possible to prevent and treat caries and diseases caused by caries. The biofilm formation inhibitor of the present invention has high safety and can be provided at low cost because the active ingredient is derived from food.

Claims (4)

A biofilm formation inhibitor comprising as an active ingredient at least one selected from the group consisting of natto, natto isolate, and natto isolate culture filtrate. The biofilm formation inhibitor of Claim 1 which uses as an active ingredient the low molecular fraction of less than 10 kDa contained in natto isolate bacterial culture filtrate. The biofilm formation inhibitor according to claim 1 or 2, which inhibits biofilm formation by cariogenic bacteria. The biofilm formation inhibitor according to claim 3, wherein the cariogenic bacterium is Streptococcus mutans or Streptococcus sobrinus.

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