JP2011217610A - In-vitro biofilm model - Google Patents

Abstract

An in vitro biofilm model capable of obtaining an evaluation result similar to a biofilm formed in a human oral cavity and a method for evaluating a biofilm removal effect of a test liquid using the same.
The following oral bacteria (A), (B), (C) and (D):
(A) a bacterium that produces a water-insoluble glucan as an extracellular polysaccharide,
(B) a bacterium that produces water-soluble glucan as an extracellular polysaccharide,
(C) a bacterium that produces fructan as an exopolysaccharide,
(D) A mixed bacterium comprising a combination of (A), (B) and (C), or (D) and (B) among bacteria selected from bacteria that produce water-insoluble glucan and fructan as extracellular polysaccharides An in vitro biofilm model produced by culturing a glass body or a synthetic resin body on the surface.
[Selection figure] None

Description

  The present invention relates to an in vitro biofilm model and a method for evaluating a biofilm dispersion removal effect using the in vitro biofilm model.

  Intraoral biofilms include dental plaque that causes caries and periodontal disease, tongue coating that causes bad breath, denture plaques that form dentures and cause infections, etc. Inhibition of formation and sterilization / removal of biofilms are required for oral hygiene products and oral compositions. That is, it is considered that it is very important to target biofilms for the search and evaluation of active ingredients for prevention and treatment of oral diseases.

  For the purpose of searching for and evaluating this active ingredient, animal experiments, human clinical trials, and in vitro evaluation models using a single or a plurality of bacteria have been proposed. However, methods based on animal experiments are difficult due to ethical problems and labor, and in human clinical trials, pathogenic bacterial flora varies among individuals and appropriate comparison may be difficult. Furthermore, in clinical trials using humans, there are problems that impose a heavy burden on subjects due to the accumulation of dental plaque and the like, and because there is a need to test many subjects, it takes a lot of time and money.

  On the other hand, in vitro biofilm models have been studied, for example, mutans streptococci batch-cultured in a sucrose-containing medium (see Non-Patent Document 1), two or more Streptococcus species and one or more genus Streptococcus In vitro biofilm (patent document 1) that causes decalcification of crystals containing hydroxyapatite, including oral bacteria including Actinomyces sp. And sugars of 1.3 gf or more, and bacteria living under the gingival margin There has been reported an in vitro biofilm (Patent Document 2) in which collagen or the like is adhered to the surface of hydroxyapatite and continuously formed on the surface of hydroxyapatite.

JP 2003-116516 A JP 2005-117926 A

Infect Immu. 1975 Dec. 12 (6) Page 1415-1425

Since the in vitro model described in Non-Patent Document 1 is for culturing only a single bacterial species, even if the active ingredient is evaluated by this model, it can be applied to biofilms such as plaque in the human oral cavity and tongue coating. It may be different from evaluation. On the other hand, those obtained by culturing a plurality of bacterial species in order to approximate the biofilm in the human oral cavity, the in vitro biofilm model described in Patent Document 1 has cariogenicity, The in vitro biofilm model described in Patent Document 2 reproduces the pathogenicity of subgingival plaque and can be said to be close to the biofilm in the human oral cavity, but these models are comparable to human gargle There was a problem that it could be easily removed by ultrasonic cleaning. In other words, biofilms in the human oral cavity are not easily removed even if they are garnished, but when evaluating components for removing biofilms, the effectiveness of these in vitro biofilm models is evaluated. Has a problem that it does not always match the evaluation when applied to the biofilm in the human oral cavity.
Accordingly, an object of the present invention is to provide an in vitro biofilm model capable of obtaining an evaluation similar to that in a human oral biofilm for a component for removing a biofilm, and a biofilm such as dental plaque in a human oral cavity. It is an object of the present invention to provide a means for searching for evaluation of components useful for the removal of odor.

  Therefore, the present inventor studied to create a model that can be evaluated in the same way as a biofilm in human oral cavity, which cannot be easily removed with a normal degree of gargle, using a plurality of oral bacteria. As a result, surprisingly, mixed bacteria mixed with bacteria that produce three types of exopolysaccharide among oral bacteria living in the oral cavity are not hydroxyapatite, but on the surface of a glass body or a synthetic resin body. It was found that an in vitro biofilm formed by culturing cannot be easily removed at a normal moistness level, shows a removal behavior similar to that of a human oral biofilm, and is useful as an in vitro biofilm model.

That is, the present invention provides the following oral bacteria (A), (B), (C) and (D):
(A) a bacterium that produces a water-insoluble glucan as an extracellular polysaccharide,
(B) a bacterium that produces water-soluble glucan as an extracellular polysaccharide,
(C) Bacteria producing fructan as extracellular polysaccharide (D) Among bacteria selected from bacteria producing water-insoluble glucan and fructan as extracellular polysaccharide, (A), (B) and (C), or An in vitro biofilm model produced by culturing mixed bacteria containing the combination of (D) and (B) on the surface of a glass body or a synthetic resin body is provided.

  Further, the present invention provides a biofilm removal effect of a test component, wherein the mixture of the biofilm and the test component-containing liquid by the in vitro biofilm model is subjected to ultrasonic treatment, and the amount of biofilm before and after the treatment is used as an index. An evaluation method is provided.

  By using the in vitro biofilm model of the present invention, it becomes possible to obtain an evaluation similar to a biofilm in a human oral cavity for a test component having a biofilm removal effect, It is effective in the evaluation search for components useful for biofilm removal.

It is a figure which shows the measurement site | part of the biofilm amount and plaque amount in a biofilm removal test.

  The in vitro biofilm model of the present invention comprises (A) a water-insoluble glucan-producing bacterium, (B) a water-soluble glucan-producing bacterium, and (C) a fructan-producing bacterium as an oral bacterium, or (D) a water-insoluble glucan. And mixed bacteria containing fructan-producing bacteria and (B) water-soluble glucan-producing bacteria can be produced by culturing on the surface of a glass body or a synthetic resin body.

  The (A) water-insoluble glucan-producing bacterium in the present invention is a bacterium that mainly produces water-insoluble glucan as an exopolysaccharide, and is a Streptococcus mutans (hereinafter referred to as “S. mutans”) that has been detected in human oral cavity. And Streptococcus sobrinus (hereinafter referred to as “S. sobrinus”) and the like, and one or more of these can be used. Of these bacteria, S.mutans and S.sobrinus are preferable from the viewpoint of ease of handling. A bacterium that mainly produces water-insoluble glucan is a bacterium in which the most produced polysaccharide among the exopolysaccharide produced is a water-insoluble glucan.

  As (B) water-soluble glucan-producing bacteria in the present invention, Streptococcus gordonii (hereinafter referred to as “S. gordonii”), Streptococcus oraris (hereinafter referred to as “S. oraris”), which has been detected in human oral cavity, Streptococcus mitis (hereinafter referred to as “S. mitis”), Streptococcus sanguinis (hereinafter referred to as “S. sanguinis”), Streptococcus intermedius (hereinafter referred to as “S. intermedius”) and the like. More than seeds can be used. Among these bacteria, S. gordonii and S. oraris are preferable from the viewpoint of ease of handling, and S. gordonii is preferable from the viewpoint of evaluating the biofilm removal effect.

  The (C) fructan-producing bacterium in the present invention is a bacterium that is known to mainly produce fructan as an exopolysaccharide, and is an actinomyces viscosus (hereinafter referred to as “A. viscosus”) that has been successfully detected in the human oral cavity. )), Actinomyces naeslundii (hereinafter “A. naeslundii”), Actinomyces oris (hereinafter “A.oris”), Actinomyces israelii (hereinafter “A. israelii”), Streptococcus Salivarius (hereinafter “S. salivarius”), etc. 1 type or 2 types or more selected from these can be used. Among these bacteria, A.viscosus and A.naeslundii are preferable from the viewpoint of ease of handling. For example, S. salivarius is known as a fructan-producing bacterium, but there is a report that water-insoluble glucan was detected as an extracellular polysaccharide. However, the exopolysaccharide produced by S. salivarius alone has less water-insoluble glucan production than, for example, the polysaccharide produced by S. mutans, and the most produced exopolysaccharide is fructan. Therefore, S. Salivarius is regarded as a fructan-producing bacterium.

  Among the water-insoluble glucan-producing bacteria (A), S. mutans produces the most water-insoluble glucan, but also produces the next largest amount of fructan. Therefore, when this (D) water-insoluble glucan and fructan-producing bacterium is used, this may be combined with (B) a water-soluble glucan-producing bacterium.

  In addition, since the bacteria of said (A), (B), (C) and (D) are used for the search and evaluation of the active ingredient for oral disease prevention treatments, such as a dental caries and periodontal disease. Anaerobic bacteria are preferred.

  In the present invention, mixed bacteria containing a combination of (A), (B) and (C), or mixed bacteria containing a combination of (D) and (B) are used. These mixed bacteria may be cultured on the surface of the glass body or the synthetic resin body, that is, these mixed bacteria may be cultured in a state where they are attached to the surface of the glass body or the synthetic resin body. Specifically, these bacterial mixtures may be added to a glass body or a synthetic resin body and cultured. The mixing ratio of (A) water-insoluble glucan-producing bacteria and (B) water-soluble glucan-producing bacteria in the bacterial mixture is 1: 2 to 1.5 in terms of volume ratio from the viewpoint of biofilm removal effect and bactericidal evaluation. 1 is preferable, and 1: 1.5 to 1.2: 1 is more preferable. The mixing ratio of (A) water-insoluble glucan-producing bacterium and (C) fructan-producing bacterium in the bacterial mixture is 1: 2 to 1.5: 1 in volume ratio from the viewpoint of biofilm removal effect and bactericidal evaluation. The ratio is more preferably 1: 1.5 to 1.2: 1. The mixing ratio of (D) water-insoluble glucan and fructan producing bacteria and (B) water-soluble glucan producing bacteria is 1: 2 to 1.5: 1 in volume ratio from the viewpoint of biofilm removal effect and bactericidal evaluation. Is more preferable, and 1: 1.5 to 1.5: 1 is more preferable. The mixing ratio of (A) water-insoluble glucan-producing bacterium, (B) water-soluble glucan-producing bacterium, and (C) fructan-producing bacterium is a volume ratio from the viewpoint of biofilm removal effect and bactericidal evaluation. (B + C) is preferably 1: 4 to 1.5: 2, more preferably 1: 3 to 1: 2. These volume ratios are the volume ratios when the bacterial concentrations are made equal, that is, the volume ratios when the optical density (OD: Optical Density) of the mixture of each cultivated bacterium and the culture solution is made equal. Thus, OD can be made equal by adjusting the culture solution, for example. OD is preferably equal at an arbitrary transmittance between 0.4 and 1.0, more preferably 0.6 to 0.9, and particularly preferably 0.7 to 0.9. OD can be measured in a cell having a wavelength of 600 nm and an optical path length of 10 mm, and can be measured, for example, with a spectrophotometer (BioSpec-mini manufactured by Shimadzu Corporation).

  These mixed bacteria are prepared by culturing each of (A) a water-insoluble glucan-producing bacterium, (B) a water-soluble glucan-producing bacterium, (C) a fructan-producing bacterium, (D) a water-insoluble glucan and a fructan-producing bacterium. It is preferable to do this. Specifically, for example, it can be cultured together with a culture solution in an agar medium or a liquid medium. Agar media include wilkins-chalgren anaerobe agar, schaedler anaerobe agar, brain heart infusion (BHI) agar, Anaero Columbia Agar with Rabbit Blood and PEA Center for Disease Control Anaerobe Blood Agar, Trypticase Soy (SCD) agar heart infusion agar, brucella Medium, west wilkins agar medium, Muller Hinton agar medium, modified medium thereof and the like can be used. As the liquid medium, wilkins-chalgren medium, schaedler medium, SCD medium, BHI medium, heart infusion medium, Mueller hint medium, west wilkins medium, modified medium thereof, and the like can be used. From the viewpoint of improving the culture amount of bacteria, a liquid medium is preferable, and it can be cultured in a liquid medium after culturing on an agar medium. The medium or liquid medium (culture medium) preferably contains a yeast extract or a saccharide such as sucrose, glucose, fructose, or glucose, and particularly contains one or more selected from sucrose, glucose, and yeast extract. Those are preferred. The culture condition is preferably an environment (room temperature) of 36 to 38 ° C., and is cultured under anaerobic conditions. The anaerobic condition is a condition in which the oxygen concentration is extremely low, and generally refers to an oxygen concentration of 1% or less, preferably 0.1% or less.

  The bacterial mixture may be added directly to the glass body or the synthetic resin body, or may be added to the glass body or the synthetic resin body to which human saliva or saliva component-containing liquid has been applied in advance. Human saliva is healthy human saliva, and saliva component-containing liquid is saliva mucin, proline-rich glycoprotein, secretory IgA, lactoferrin, α-amylase, phosphate protein, lysozyme, which are components of healthy human saliva Those containing one or more selected from albumin are preferred, and those containing two or more are preferred.

  The substrate for in vitro biofilm formation according to the model of the present invention is a glass body or a synthetic resin body. When hydroxyapatite is used as a substrate, a biofilm that can be easily removed by gargle or the like is formed. As a glass body or a synthetic resin body, a bacterial mixture can be added as long as it can be maintained on the surface of the glass body or the synthetic resin body. May be added as a glass body or a synthetic resin body. As the synthetic resin body, a thermoplastic resin is preferable. Examples of the thermoplastic resin include polyethylene, polypropylene, methacrylic resin, polyvinyl chloride, polyethylene terephthalate, polyacetal, polystyrene, and copolymers thereof, such as ethylene / propylene copolymer, ethylene butene copolymer, acrylonitrile. / Styrene resin and acrylonitrile / butadiene / styrene resin (ABS resin). The surface of the glass body or the synthetic resin body may be a flat surface or an uneven surface, but preferably has a flat surface or a smooth surface.

  The culture of the mixed bacteria on the surface of the glass body or the synthetic resin body is preferably performed for 20 to 30 hours under anaerobic conditions of 36 to 38 ° C. By such culturing, an in vitro biofilm having dispersion removal resistance similar to that in the human oral cavity, which cannot be easily removed by ordinary gargle, can be formed on the surface of the glass body or the synthetic resin body.

  The effect of removing the biofilm using the in vitro biofilm model of the present invention can be evaluated by quantifying the in vitro biofilm before and after the treatment with the liquid containing the test component. The removability of the biofilm in the present invention includes the dispersibility of removing the biofilm by dispersing it. As the treatment with the test component-containing liquid, the in vitro biofilm is brought into contact with the test component-containing liquid to evaluate the bactericidal property, or the in vitro biofilm and the test component-containing liquid are mixed, The method of sonication is mentioned. This sonication is preferably sonication similar to human gargle, and sonication with a frequency of 40-60 kHz is preferred. For example, using an ultrasonic cleaner AW5800 (manufactured by Citizen), the treatment can be performed for 30 seconds at a frequency of 42 kHz and at room temperature (20 ° C.). In addition, it is preferable that the time for sonication is a general human moistening time, preferably 10 seconds to 1 minute, and more preferably 20 seconds to 50 seconds. The effect of removing the biofilm with the test component-containing liquid can be determined using the amount of biofilm before and after the treatment with the test component-containing liquid as an index. The amount of biofilm can be quantified by the phenol-sulfuric acid method. The bactericidal property can be based on the number of live bacteria before and after the treatment. The number of viable bacteria can be quantified by dispersing the biofilm, culturing it, and counting the number of colonies.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these illustrations.

(Materials and methods)
1) Culture of oral bacteria
S. mutans ATCC 25175, S. sobrinus ATCC 33478, A. viscosus ATCC 43146, A. naeslundii ATCC 12104, S. gordonii ATCC10558 strain and S. oraris ATCC35037 strain were added to 50 mL of culture solution (SCD-YE medium) in which 0.5% Yeast Extract (Difco) was added to SCD medium (Soybean-Casein Digest Broth, manufactured by Nippon Pharmaceutical Co., Ltd.) Platinum ears were inoculated and cultured at 37 ° C. under anaerobic conditions (N ₂ 80%, CO ₂ 20%) until the logarithmic growth phase.

2) Saliva From healthy men without oral disease, the saliva secreted by chewing paraffin wax is collected, and the oral mucosa and the like peeled off at the time of saliva collection are removed by centrifugation, and this is removed into a glass base dish (IWAKI). φ27mm) (hereinafter referred to as “glass”), polystyrene dish (IWAKI φ27mm) (hereinafter referred to as “polystyrene”), or hydroxyapatite plate (apatite pellets APP-100, manufactured by PENTAX) (hereinafter referred to as “HAP plate”) It was dispensed at a rate of 2 mL / dish and allowed to stand at 4 ° C. for 24 hours to allow human saliva to be adsorbed on glass, polystyrene and HAP plates. In Examples 1-6 and Comparative Examples 2-6, human saliva was adsorbed on glass, and in Examples 1b-4b, human saliva was adsorbed on polystyrene. In Comparative Examples 7 to 10, human saliva was adsorbed on the HAP plate placed on the bottom of the glass base dish.

3-1) Production of in vitro biofilm model The bacteria cultured in 1) above are cultured with a spectrophotometer (BioSpec-mini, manufactured by Shimadzu Corporation) so that the OD of 600 nm is 0.7 with an optical path length of 10 mm. Prepared in a liquid (SCD-YE medium), inoculated with the volumes shown in Table 1, Table 2, Table 3, and Table 4, and mixed with 50 mL of the culture solution at a rate of 2 mL / dish in Examples 1 to 6 and the glass to which the saliva of Comparative Examples 2 to 6 is attached, the HAP plate to which the saliva of Comparative Examples 7 to 10 is attached, the glasses of Examples 1a to 4a, and the saliva of Examples 1b to 4b and Example 7 are attached. The resultant was added to the polystyrene and cultured for 24 hours to form a biofilm. Here, the culture solution used was added with yeast 0.5% (Yeast Extract, manufactured by Difco), Glucose 1.0%, and Sucrose 2.0%.

3-2) Human plaque For human plaque, healthy men who have no oral disease were prohibited from brushing and wrinkling for 24 hours, and plaque adhered to the tooth surface was used.

4) Sterilization test As a chemical | medical agent of a sterilization test, the chemical | medical agent containing 100 ppm cetyl pyridinium chloride in purified water was used. In the sterilization test, human plaque collected from the in vitro biofilms of Examples and Comparative Examples and the human oral cavity was washed twice with purified water, then 1 mL of the drug was added and allowed to act for 30 seconds. The drug was then removed and further washed twice with purified water. The sterilization rate was determined by measuring the number of bacteria in the biofilm before and after treatment by the method of 5) described later, and was defined as (the number of bacteria before treatment-the number of bacteria after treatment) / the number of bacteria before treatment.

5) Bacterial count 2 mL of physiological saline was added to the in vitro biofilm and human plaque on the substrates of Examples and Comparative Examples, and sonication was performed until the in vitro biofilm and plaque were dispersed. The ultrasonic treatment in this case was performed at room temperature (20 ° C.) using a SONIFIER CELL DISTORPTOR 200 manufactured by BRASON at full power (frequency of about 27 KHz) for 15 seconds. In addition, the frequency of ultrasonic treatment for dispersing the biofilm in a short time is preferably 25 to 30 KHz. Next, the dispersed in vitro biofilm and plaque are collected and smeared on an agar medium containing the blood of Anero Columbia rabbits, and the temperature is 37 ° C. and anaerobic conditions (N ₂ 80%, CO ₂ 20%). Incubate for hours. After culturing, the number of colonies was measured to determine the number of viable bacteria.

6) Removal test 1 mL of physiological saline was dropped on the in vitro biofilms on the substrates of Examples and Comparative Examples, and sonicated for 30 seconds at room temperature (20 ° C.). The sonication of the removal test was performed with an ultrasonic cleaning machine AW5800 (manufactured by Citizen), which was designed with an ultrasonic frequency of 42 KHz in order to obtain a level similar to that of human gargle. After the treatment, the peeled in vitro biofilm amount (A) and the remaining in vitro biofilm amount (B) were measured by the phenol sulfate method. The remaining in vitro biofilm was stripped by adding 1 mL of 2N sodium hydroxide and neutralized by adding 1 mL of 2N hydrochloric acid. Similar to the peeled in vitro biofilm, the amount of biofilm was measured by the phenol sulfuric acid method. In the phenol sulfuric acid method, 20 μL of biofilm is dispensed into a 96-well microplate reader, 20 μL of 5% (W / V) phenol aqueous solution is added, and 100 μL of 98% concentrated sulfuric acid is added and mixed. Absorbance was measured. The calibration curve used the absorbance of 0.0078 to 0.125% glucose solution to quantify the amount of biofilm. The biofilm removal rate was determined as in vitro biofilm amount (A) / (in vitro biofilm amount (A) + in vitro biofilm amount (B)), and is shown in Tables 1 and 2. The removal rate of human plaque was quantified by rinsing with physiological saline for 30 seconds and measuring the amount of plaque before and after the inclusion. For the measurement of the amount of plaque, the measurement site in the human oral cavity is made up of 16 teeth, 4 each in the top, bottom, left and right (1st, 4th, 6th, 7th teeth), and the measurement location is as shown in FIG. There were a total of 10 locations, 4 locations in between and 6 locations in the smooth surface. As shown in FIG. 1, the amount of plaque was quantified by the height from the gingiva in the area where plaque adhered to the teeth as shown in FIG. The removal rate of human plaque is (the amount of plaque before gargle-the amount of plaque after gargle) / the amount of plaque before gargle, and the measurement results are shown in Tables 1 to 4.

7) Biofilm removal test using erythritol Erythritol was used as a test component, and mixed with purified water so as to be 10% by mass of erythritol to prepare a test component-containing solution. In the test, 1 mL of a test component-containing solution (containing 10% by mass of erythritol) was dropped in place of the physiological saline in the removal test of 6), and the same removal test as 6) was performed to obtain the removal rate. The removal rates are shown in Tables 1 and 3.

  As shown in Table 1, in vitro biofilm formation was not observed in Comparative Examples 4 to 6 prepared from (B) water-soluble glucan-producing bacteria and (C) fructan-producing bacteria. On the other hand, the human plaque of Comparative Example 1 has a biofilm removal rate of about 20% due to the rinsing of physiological saline, and it is recognized that the plaque is garnished and difficult to remove, but (A) a water-insoluble glucan-producing bacterium ( C) The removal rate of the in vitro biofilm of Comparative Example 2 produced by fructan-producing bacteria is about 77%, which is considered easier to remove than human plaque. In contrast, the removal rates of the in vitro biofilm models of Examples 1 to 6 produced on the glass by the bacteria of (A), (B) and (C) were almost the same as the human plaque of Comparative Example 1. there were. Moreover, the biofilm removal rate in the case of the erythritol containing liquid which uses erythritol as a test component is higher than the removal rate with physiological saline. In this evaluation, the removal of the in vitro biofilms of Examples 1-6 The rate was almost the same as that of human plaque in Comparative Example 1. The removal rate of the in vitro biofilms of Comparative Examples 7 to 10 formed on the HAP plate by the bacteria (A), (B) and (C) was about 66 to 80%, and the human plaque of Comparative Example 1 It is thought that it is easier to remove than

  As shown in Table 2, the sterilization rate and removal rate of the in vitro biofilm prepared on the glass not coated with human saliva of Examples 1a to 4a are the same as those of Examples 1 to 4 prepared on glass coated with human saliva. Was found to be. Moreover, as shown in Table 3, it was confirmed that the in vitro biofilms produced on the polystyrenes of Examples 1b to 4b have the same degree of sterilization and removal as human plaque.

  As shown in Table 4, the sterilization rate and removal rate of the in vitro biofilm of Example 7 by (D) S. mutans that produces water-insoluble glucan and fructan, and (B) S. gordonii that produces water-soluble glucan are It was found to be similar to human plaque.

Claims (10)

The following oral bacteria (A), (B), (C) and (D):
(A) a bacterium that produces a water-insoluble glucan as an extracellular polysaccharide,
(B) a bacterium that produces water-soluble glucan as an extracellular polysaccharide,
(C) a bacterium that produces fructan as an exopolysaccharide,
(D) A mixed bacterium comprising a combination of (A), (B) and (C), or (D) and (B) among bacteria selected from bacteria that produce water-insoluble glucan and fructan as extracellular polysaccharides An in vitro biofilm model produced by culturing a glass body or a synthetic resin body on the surface.   The volume ratio of the mixed bacteria when mixed is (A) a water-insoluble glucan-producing bacterium and (B) a water-soluble glucan-producing bacterium is a volume ratio (A: B) of 1: 2 to 1.5: 1, (A) Volume ratio (A: C) of water-insoluble glucan-producing bacteria and (C) fructan-producing bacteria is 1: 2 to 1.5: 1, or (D) water-insoluble glucan and fructan-producing bacteria and (B) water-soluble glucan The in vitro biofilm model according to claim 1, wherein the volume ratio (D: B) with the producing bacteria is 1: 2 to 1.5: 1.   (B) The in vitro biofilm model according to claim 1 or 2, wherein the water-soluble glucan-producing bacterium is selected from Streptococcus gordonii, Streptococcus oraris, Streptococcus mitis, Streptococcus sanguinis, and Streptococcus intermedius.   (A) The in vitro biofilm model according to any one of claims 1 to 3, wherein the water-insoluble glucan-producing bacterium is selected from Streptococcus mutans and Streptococcus sobrinus.   (C) The in vitro biofilm model according to any one of claims 1 to 4, wherein the fructan-producing bacterium is selected from Actinomyces viscosus, Actinomyces naeslundii, Actinomyces oris, Actinomyces israelii and Streptococcus salivarius.   (D) The in vitro biofilm model according to any one of claims 1 to 3, wherein the water-insoluble glucan and fructan-producing bacterium are Streptococcus mutans.   The in vitro biofilm model according to any one of claims 1 to 6, wherein human saliva or saliva component-containing liquid is attached to the surface of the glass body or synthetic resin body, and the mixed bacteria are added and cultured.   A mixture of the biofilm and the test component-containing liquid by the in vitro biofilm model according to any one of claims 1 to 8 is subjected to ultrasonic treatment, and the amount of biofilm before and after the treatment is used as an index. Evaluation method of biofilm removal effect of components. The following oral bacteria (A ′), (B ′) and (C ′):
(A ′) a bacterium selected from Streptococcus mutans and Streptococcus sobrinus,
(B ′) a bacterium selected from Streptococcus gordonii, Streptococcus oraris, Streptococcus mitis, Streptococcus sanguinis and Streptococcus intermedius,
(C ′) An in vitro biofilm model produced by culturing mixed bacteria containing bacteria selected from Actinomyces viscosus, Actinomyces naeslundii, Actinomyces oris, Actinomyces israelii and Streptococcus salivarius on the surface of a glass body or a synthetic resin body.   (D ') Streptococcus mutans and (B') Streptococcus gordonii, Streptococcus oraris, Streptococcus mitis, Streptococcus sanguinis, and mixed bacteria containing bacteria selected from Streptococcus intermedius were produced on the surface of a glass body or a synthetic resin body. In vitro biofilm model.