JP5204977B2 - Microorganisms for eradication of periodontal disease-causing bacteria, and pharmaceutical compositions, foods or food additives for the prevention or treatment of periodontal disease containing the microorganisms - Google Patents

Description

  The present invention belongs to the technical field of periodontal disease prevention and periodontal disease treatment. Specifically, the present invention relates to bifidobacteria useful for periodontal disease prevention and periodontal disease treatment.

  Periodontal disease is divided into gingivitis that causes inflammation of the gums and periodontitis that destroys the alveolar bone that supports the teeth. So-called alveolar pyorrhea is classified as adult periodontitis. Gingivitis is caused by supragingival plaque, periodontitis is caused by subgingival plaque, and the pathogenic bacteria involved in these are Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythensis (Tf) It is said that there are several types of bacteria. In particular, Porphyromonas gingivalis (P.g.) is said to be the main cause.

  Among the various enzymes produced by Porphyromonas gingivalis (Pg), trypsin-like cysteine protease (gingipain) has a strong trypsin-like activity as a catalytic activity, causing host protein degradation and host cell damage, and periodontal activity. It is thought to produce various pathologies related to the disease. Therefore, inhibiting the action of gingipain protease as a means for preventing or treating periodontal disease is considered to be a very effective means. A protease inhibitor characterized by using a plant extract as an active ingredient has also been reported (Patent Document 1: JP-A-62-25000).

  In addition, there are preventive agents or foods by lactic acid bacteria that have a prophylactic effect on periodontal disease. For example, a composition for preventing / treating oral diseases including dental caries and a probiotic product containing a bacterial body belonging to lactic acid bacteria (Lactobacillus salivarius) as an active ingredient (Patent Document 2: WO2002 / 016554) Furthermore, compositions with lactic acid bacteria or their filtrates or treated products and various sugar / plant extracts (Patent Document 3: WO2002 / 080946) have been developed.

  On the other hand, Patent Document 4 (Japanese Patent Application Laid-Open No. 2003-171292) describes a preventive or therapeutic agent for periodontal disease containing lactic acid bacteria having an effect of preventing periodontal disease and saccharides that can be assimilated by these.

  Patent Document 5 (Japanese Patent Application Laid-Open No. 2003-299480) describes production of a food containing yogurt and a lactic acid bacterium (Reuteri bacterium) having a caries (caries) prevention effect. Reuteri bacteria are known to produce antibiotics (Patent Document 6: US Pat. No. 5,352,586).

  Probiotics mean useful microorganisms that can improve the bacterial flora in the gastrointestinal tract and bring about beneficial effects on the host, and their growth promoting substances.

Japanese Patent Laid-Open No. 6-25000 WO2002 / 016554 WO2002 / 080946 JP2003-171292 JP2003-299480 U.S. Pat.No. 5,352,586

  Although there are preventive agents or foods by lactic acid bacteria that have a prophylactic effect on periodontal disease, there is actually little scientific data showing that probiotic strains are effective for periodontal disease. In addition, there are still many unclear points about the role of lactic acid bacteria in the oral cavity, and the lactic acid bacteria species specific to the healthy oral cavity have not been fully elucidated. To date, there is Lactobacillus salivarius TI2711 as a probiotic with relatively basic data. Lactic acid is the periodontal pathogen control mechanism of this strain, and probiotic L. salivarius Caries have also been reported.

  In view of the present state of the art, the present invention has an object to develop a microorganism having a probiotic function capable of suppressing periodontal pathogens by a mechanism other than lactic acid, which is concerned about the risk of caries. And Another object of the present invention is to develop a microorganism having a probiotic function that is useful for the prevention and / or treatment of periodontal disease, with or without caries (caries).

  The present inventors suppress the growth of P. gingivalis by competing with vitamin K, which is a growth factor of P. gingivalis, using a microorganism (Bifidobacterium) belonging to the genus Bifidobacterium that requires vitamin K. Found that you can.

  The present invention comprises (1) a pharmaceutical composition such as a prophylactic and / or therapeutic agent that inhibits the growth of P. gingivalis containing vitamin K-requiring bifidobacteria as an active ingredient, and (2) a vitamin K-requiring bifido. Provided is a food or food additive having a growth-inhibiting effect of P. gingivalis containing a fungus as an active ingredient.

  The present invention has an excellent effect that periodontal disease can be prevented and / or treated using microorganisms that have no or little caries (caries) property and have been confirmed to be safe and effective for the human body. .

1. 1. Introduction The inventors of the present application tried to find bacteria having an inhibitory effect on periodontal disease bacteria from bacteria that compete with periodontal disease bacteria and essential nutrients. Therefore, we first elucidated and compared the oral flora of normal healthy people and periodontal disease patients. Next, among the microorganisms that contribute to periodontal disease, bacterial species belonging to the genus Porphyromonas and Prevotella are known, and most of them are vitamin K-requiring strains. We decided to investigate suppression of K competition.

  Reference 1 (Glick, MC, Zilliken, F. and Gyorgy, P., Supplementary growth promoting effect of 2-methyl-1,4-naphthoquinone on Lactobacillus bifidus var. Pennsylvanicus, J. Bacteriol., 77, 230-236 (1959 )) The bifidobacteria described (former name: Lactobacillus bifidus var. Pennsylvanicus) was also known to be vitamin K-requiring, but there was no information on vitamin K-requiring for other bacterial species and strains.

  Accordingly, the inventors of the present invention have examined the vitamin K requirement of various Bifidobacterium and found the presence of a vitamin K requirement strain among the bifidobacteria isolated from humans. Furthermore, the inventors of the present application have shown that vitamin K-requiring Bifidobacterium competes with vitamin K, which is a growth factor for periodontal pathogen P. gingivalis, and can suppress the growth of P. gingivalis by an action mechanism other than lactic acid. I found it for the first time.

2. Distribution of oral bacteria in healthy individuals and periodontal patients Conventionally, it has been reported that young healthy individuals have higher detection of Streptococcus and Veillonella ssp. Than in periodontal patients (Kumar, PS, Griffen, AL, Moeschberger, ML and Leys, EJ, Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J. Clin. Microbiol., 43, 3944-3955 (2005)).

  The inventors of the present application focus on Lactobacillus microorganisms and Bifidobacterium microorganisms that are effective in the intestines as microorganisms having a probiotic function, and a group of young healthy individuals, former periodontal disease patients In the present study, we compared oral bacteria in the group with the well-managed oral environment and the periodontal disease patient group.

  In microorganisms belonging to the genus Lactobacillus, there were many detectors of three strains of L. (Lactobacillus) salivarius, L. gasseri, and L. fermentum, but the number and detection rate of all strains (three species) There was no big difference.

  On the other hand, in microorganisms belonging to the genus Bifidobacterium, B. (Bifidobacterium) adolescentis, B. longum, and B. urinalis confirmed the detector in the young healthy group, but in the periodontal patient group could not.

3. The anti-periodontal effect of microorganisms present in healthy individuals The inventors of the present application have abundant bifidobacteria species in the oral cavity of young healthy individuals, and the smallest number of bifidobacteria in periodontal disease patients Therefore, we thought that bifidobacteria were useful in the oral cavity. In addition, the growth of P. gingivalis is suppressed by competing and consuming vitamin K, a growth-promoting factor of P. gingivalis, a major periodontal disease fungus, by microorganisms belonging to the genus Bifidobacterium. We hypothesized that we could do it and examined it experimentally.

  Specifically, we evaluated the vitamin K requirement of microorganisms belonging to the genus Bifidobacterium that had not been studied in detail until now, and that these microorganisms differ in vitamin K requirement depending on the species, more strictly strains It was found that the requirement for vitamin K varies depending on the type.

  Veillonella was also considered as a microorganism that supplies vitamin K to P. gingivalis. In contrast, the inventors of the present application have clarified that the culture supernatant of Bayonella bacteria promotes the growth of microorganisms belonging to the genus P. gingivalis and Bifidobacterium. That is, we found a phenomenon suggesting that Veillonella is producing vitamin K, which is necessary for the growth of microorganisms belonging to the genus P. gingivalis and Bifidobacterium in the oral cavity.

  Specifically, menaquinone-7 (MK-7), the main vitamin K produced by Veillonella, promotes the growth of microorganisms belonging to the genus Bifidobacterium, in which case it belongs to the genus Bifidobacterium It was experimentally verified that microorganisms consumed MK-7 in the culture solution. From these experimental results, it was theoretically found that microorganisms belonging to the genus Bifidobacterium can reduce MK-7 (vitamin K) in the environment necessary for the growth of P. gingivalis (such as in the oral cavity).

  Next, Bifidobacterium adolescentis OLB6398, a representative strain of the genus Bifidobacterium that has high vitamin K requirement and high vitamin K consumption ability, coexisted with P. gingivalis to significantly proliferate P. gingivalis. I found it to be suppressed.

B. adolescentis OLB6398 used in the present invention was deposited under the following conditions.
(1) Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Receipt number: NITE AP-294
(4) Accession number: NITE P-294
(5) Display for identification: Bifidobacterium adolescentis OLB6398
(6) Date of deposit (date of receipt): December 22, 2006

  This strain is obtained after anaerobic culture at 37 ° C. for 18 hours in the case where the culture supernatant of Bayonella is added to the medium at 1% (v / v) and in the case where the culture supernatant of Bayonella is not added. The difference in absorbance (OD) at a wavelength of 600 nm was 0.58.

4). Pharmaceutical composition (preventive and therapeutic agent for periodontal disease) containing vitamin K-requiring bifidobacteria as an active ingredient
The periodontal disease preventive agent and therapeutic agent of the inventors of the present application are characterized by containing vitamin K-requiring bifidobacteria. Examples of Bifidobacteria that require vitamin K include B. adolescentis, B. longum, B. breve, B. bifidum, B. urinalis, and B. lactis. Furthermore, bifidobacteria selected by the screening method described in “6. Method for Screening Microorganisms Useful for Prevention or Treatment of Periodontal Diseases” below may be mentioned, and preferably B. adolescentis, B. longum, and B. bifidum. Specifically, for example, the aforementioned Bifidobacterium adolescentis OLB6398 (NITE P-294) can be mentioned.

  The periodontal disease preventive agent and therapeutic agent according to the present invention may take any form. Examples include tablets, powders, granules, pastes, pills, chewables, gargles, troches or patches, yogurts, etc., preferably tablets, powders, granules, pastes, chewables, A mouthwash and a lozenge are used.

  For formulation, adjuvants commonly used for formulation, such as excipients, binders, disintegrants and lubricants, can be added. Examples of excipients include starch, lactose, sucrose, methylcellulose, carboxymethylcellulose, sodium alginate, calcium hydrogen phosphate, synthetic aluminum silicate, microcrystalline cellulose, polyvinylpyrrolidone (PVP), and hydroxypropyl starch (HPS). is there. Examples of the binder include starch, microcrystalline cellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP), gum arabic powder, gelatin, glucose, sucrose, and aqueous / ethanol solutions thereof. Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, microcrystalline cellulose, hydroxypropyl starch, and calcium phosphate. Lubricants include carnauba wax, light anhydrous silicic acid, synthetic aluminum silicate, natural aluminum silicate, synthetic magnesium silicate, hardened oil, hardened vegetable oil derivative (Sterotex HM), sesame oil, white beeswax, titanium oxide, dry water Aluminum oxide gel stearic acid, calcium stearate, magnesium stearate, talc, calcium hydrogen phosphate, and sodium lauryl sulfate.

5. Foods or food additives for preventing or treating periodontal disease comprising vitamin K-requiring bifidobacteria as an active ingredient For preventing or treating periodontal diseases comprising vitamin K-requiring bifidobacteria of the present invention as an active ingredient The food may be solid (powder, granule, etc.), paste, liquid or suspension, and supplements of appropriate dosage forms such as powders, granules, tablets, capsules, liquids, etc. You can also. The present invention can be used as a food additive for preparing a food for preventing periodontal disease, a food for treatment, a food for special use or a dietary supplement.

6). Method for Screening Microorganisms Useful for Prevention or Treatment of Periodontal Disease The present invention further includes a method for screening Bifidobacteria useful for prevention or treatment of periodontal disease. Specifically, in the present invention, vitamin K, quinones or naphthalenes are added to the medium at 10 ⁻⁶ M to 10 ⁻² M, and vitamin K, quinones or naphthalenes are not added to the medium The method of screening for microorganisms useful for the prevention or treatment of periodontal disease, which comprises selecting bifidobacteria requiring vitamin K by comparing the number of bacteria, is included. Here, the comparison of the number of bacteria can be carried out by counting the number of bacteria with a microscope, but it can also be measured by a difference in absorbance at a wavelength of about 600 nm.

For example, selecting a vitamin K-requiring microorganism showing a difference in the number of bacteria after anaerobic culture at 30 to 45 ° C. for 12 to 72 hours with or without adding vitamin K, quinones or naphthalenes to the medium it can. Specifically, anaerobic culture can be exemplified at 36 to 38 ° C. for 18 to 20 hours, preferably 37 ° C. for 18 hours. And, in the difference in the number of bacteria after anaerobic culture under these conditions, the ratio of the number of bacteria is preferably 10 ¹ or more, more preferably 5 × 10 ¹ or more, and further preferably 10 ² or more. The difference in absorbance is preferably 0.1 or more, more preferably 0.2 or more, and still more preferably 0.3 or more. That is, by selecting microorganisms exhibiting these states, vitamin K-requiring microorganisms can be screened. Examples of quinones and naphthalenes include α-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, 2,3-dichloro-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, phylloquinone, Menaquinone-n, menadione, 4-amino-2-methyl-1-naphthol, 1-naphthoic acid, 2-naphthoic acid, 1,4-dihydroxy-2-naphthoic acid, 1-hydroxy-2-naphthoic acid, 1, 4-Naphthalenedicarboxylic acid, 1-naphthylacetic acid, α-naphthylamine, α-naphthylaldehyde, and β-naphthylaldehyde can be used.

[Example 1] Composition of strains of lactobacilli and bifidobacteria in the oral cavity of healthy individuals and periodontal patients (subjects)
The subjects agreed with the gist of the experiment, (1) 16 healthy young people (6 men, 10 women) with good oral hygiene aged 19 to 27 years old (hereinafter referred to as healthy people group), (2) Periodontal disease patients from 37 to 70 years old (6 men, 10 women) (hereinafter referred to as periodontal disease group), (3) 47 to 76 years old after periodontal disease treatment There were 14 former periodontal patients (4 men, 10 women) (hereinafter referred to as the maintenance group). Each subject has not been administered antibiotics for at least one month prior to the study and is instructed not to eat foods that contain lactobacilli such as fermented milk or pickles or bifidobacteria. It was. Oral findings (such as plaque adhesion, swelling of gingiva, periodontal pocket depth, etc.) were confirmed by dentists for each subject, which was significantly higher in the periodontal disease group compared to the healthy group and the maintenance group. Unfortunately, there was no difference between the healthy group and the maintenance group.

(Separation and identification of lactobacilli and bifidobacteria)
Saliva was collected before breakfast in the morning before breakfast, and diluted appropriately with GAM broth (Nissui Pharmaceutical). The diluted solution is inoculated into Lactobacillus selective medium (LBS agar, BBL) and Bifidobacterium selective medium (Beerens agar), and these mediums are placed in an anaerobic glove box (N ₂ : CO ₂ : O ₂ = 8: 1: Anaerobic culture was performed at 37 ° C for 2 days in 1). After culturing, colonies that grew on the medium were picked and subcultured in BL agar (Nissui Pharmaceutical). For the strain cultured in this manner, after observing the Gram stainability, colony and cell morphology, PCR method using species-specific primers (Song, Y., Kato, N., Liu, C., Matsumiya, Y., Kato, H., and Watanabe, K., Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group-and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Micro biol. Lett., 187, 167-173 (2000) and Matsuki, T., Watanabe, K., Tanaka, R., Fukuda, M., Takada, T. and Oyaizu, H., Distribution of bifidobacterial species in human intestinal microfrola examined with 16S rRNA-gene-targeted species-specific primers. Appl. Environ. Microbiol., 65, 4506-4512 (1999) method) or 16S rDNA homology analysis did. The 16S rDNA sequence of each isolate was identified as the same species having 98% similarity or more compared to the sequence of the bacterial species registered in the DNA Data Bank of Japan.

(Composition of Lactobacillus and Bifidobacterium species)
Table 1 and Table 2 show the bacterial species composition for each group of Lactobacilli and Bifidobacteria. As shown in Table 1, 11 strains of lactobacilli were detected in the human oral cavity. In the microorganisms belonging to the genus Lactobacillus, the detection rate was large in the three bacterial species L. salivarius, L. gasseri, and L. fermentum, but in any bacterial species (three bacterial species), the group of young healthy individuals and periodontal There was no significant difference in the number of bacteria or the number of detectors in the disease group. In other words, specific bacterial species could not be found in the healthy group, periodontal disease patient group, and maintenance group.

  On the other hand, as shown in Table 2, in the microorganisms belonging to the genus Bifidobacterium, the number of detectors was high in the young healthy group of B. (Bifidobacterium) dentium, but the number of detectors was also high in the periodontal disease patient group. . However, in B. adolescentis, B. longum, and B. urinalis, the detector was confirmed in the young healthy group, but the detector was not confirmed in the periodontal patient group. The total number of bifidobacteria was smaller in the periodontal disease patient group than in the maintenance group.

Example 2 Effect of Vitamin K and Bayonella Culture Supernatant on Bifidobacteria Growth Bayonella (Veillonella parvula ATCC 17745) was anaerobically cultured at 37 ° C. for 24 hours in Brain Heart Infusion (BBL). After centrifuging the culture, the supernatant was sterilized by filtration through a 0.45 μm filter to obtain a culture supernatant of Bayonella. The strains described as ATCC in these strain names are reference strains obtained from the American Type Culture Collection (ATCC) which is a cell / microorganism / gene bank.

  Number 1 medium (Kaneko et al., J. Dairy Sci., 77, 393-404 (1994)) served as a control. Then, each medium was prepared by adding vitamin K (menadione) to 1 μg / ml or Bayonella culture supernatant to 1% (v / v). Microorganisms (Bifidobacteria) belonging to the genus Bifidobacterium isolated from the oral cavity were inoculated into the above-mentioned three types of medium and anaerobically cultured (Aneropack, Mitsubishi Gas Chemical Company) at 37 ° C. for 18 hours. After culturing, OD (optical density, absorbance at a wavelength of 600 nm) of each culture was measured using a spectrophotometer (U-2000, Hitachi). The influence of the culture supernatant of vitamin K and Bayonella on the growth of bifidobacteria was evaluated by the value obtained by subtracting the O.D. of the culture broth as a control (ΔA 600 nm) from the O.D. of each culture broth. That is, the larger ΔA600 nm, the higher the growth promoting effect of bifidobacteria. Specifically, it is considered that ΔA600nm is 0.1 and the effect of promoting the growth of bifidobacteria is high, but ΔA600nm is 0.2 and the effect is higher, and ΔA600nm is 0.3 and the effect is higher.

  As shown in Table 3, with the exception of B. urinalis, microorganisms belonging to B. dentium, B. adolescentis, and B. longum, the growth was promoted by the culture supernatant of vitamin K and bayoella. And as shown in FIG. 1, the sensitivity (vitamin K requirement) of the bifidobacteria with respect to the culture supernatant of vitamin K and Bayonella bacteria differed also strictly according to the strain. A positive correlation (rs = 0.46, P <0.01) was observed between the growth-promoting effect of vitamin K and the growth-promoting effect of Bayonella culture supernatant. It was speculated that it is a substance that promotes the growth of bifidobacteria contained in.

[Example 3] Relationship between Vitamin K Concentration and Bifidobacteria Growth Bayonella is considered to be a microorganism that supplies vitamin K to P. gingivalis, and the main vitamin K produced by Bayonella is menaquinone. -7 (MK-7). Therefore, in order to verify the contents described above, MK-7 (Wako Pure Chemical Industries) was added to Number 1 medium at 0 to 10.0 μg / ml (0 to 1.5 × 10 ⁻⁴ M (mol / l)). Bifidobacteria were cultured and their growth (OD) was compared. As the activation culture, bifidobacteria were anaerobically cultured at 37 ° C. for 18 hours using a GAM medium, and were appropriately taken into the test system at 0.1 to 1% by weight. Unless otherwise noted, the bifidobacteria were similarly activated in other examples. In this experiment, only B. adolescentis OLB6398 (NITE P-294) was selected from the microorganisms belonging to the genus Bifidobacterium (Bifidobacteria) used in Example 2 for the convenience of work. Further, L. paracasei JCM 1181 was used as a lactobacilli as a control for bifidobacteria, and P. gingivalis ATCC33277 was used as a periodontal disease. In addition, the strain described as JCM in these strain names is a reference strain obtained from the Microbial Materials Development Department of RIKEN BioResource Center.

  As shown in FIG. 2 and Table 4, the growth of B. adolescentis OLB6398 was promoted by the addition of MK-7 (a kind of vitamin K) and proliferated depending on vitamin K. As described above, the growth of B. adolescentis OLB6398 was also promoted by menadione (a kind of vitamin K) and the culture supernatant of Bayonella. The growth of P. gingivalis was also promoted by MK-7. However, L. paracasei JCM 1181 was not promoted by MK-7, suggesting that lactobacilli are not affected by vitamin K.

[Example 4] Study on vitamin K consumption ability of bifidobacteria
Menaquinone-4 (MK-4, Wako Pure Chemical Industries, Ltd.) or menaquinone-7 (MK-7, Wako Pure Chemical Industries, Ltd.) is added to Number 1 medium at 5.0 μg / ml, and various strains are cultured. The concentration of menaquinone in the culture supernatant was compared. B. adolescentis OLB6398-containing microorganism belonging to the genus Bifidobacterium (B. adolescentis 61 strains, B. longum 9 strains, B. bifidum 1 strain) and lactobacilli (Lactobacillus paracasei JCM1181) Was anaerobically cultured at 37 ° C. for 18 hours. After culturing, the concentration of vitamin K remaining in the culture supernatant was measured and compared with various strains. The concentration of vitamin K was measured using HPLC according to the method of Kojima et al. (Kojima et al., Acta. Paediatr. 93, 457-463 (2004)).

  If vitamin K can be reduced from the environment inhabiting P. gingivalis, it is thought that the growth of P. gingivalis can be suppressed. Therefore, the ability of Bifidobacterium to consume vitamin K (vitamin K requirement) is an extremely important factor in the present invention. Therefore, the concentration of vitamin K remaining after culturing bifidobacteria was measured and evaluated.

  According to the results, all of the bifidobacteria used in this experiment had a lower concentration of vitamin K remaining in the medium (culture supernatant) than lactobacilli. The concentrations of MK-4 remaining in the culture supernatants of B. adolescentis, B. longum, and B. bifidum were 1.4 ± 0.9 μg / ml, 2.0 ± 1.2 μg / ml, and 1.0 μg / ml, respectively. It was decreased compared to μg / ml. As shown in FIG. 3, the concentration of MK-7 remaining in the culture supernatant of B. adolescentis OLB6398 was 0.7 μg / ml, which was reduced compared to the original 5.0 μg / ml. Thus, bifidobacteria have high vitamin K consumption ability (vitamin K requirement).

  On the other hand, the concentration of MK-7 remaining in the culture supernatant of Lactobacillus (L. paracasei JCM1181) was unchanged from the original 5.0 μg / ml, and Lactobacilli had no vitamin K consumption (vitamin K requirement) . From FIG. 2, it was found that lactobacilli were not promoted by vitamin K, and from FIG. 3, lactobacilli did not consume vitamin K.

  From the above examination, since microorganisms belonging to the genus Bifidobacterium have vitamin K requirement (vitamin K consumption ability), they can consume and compete with vitamin K which is a growth promoting factor of P. gingivalis. It was thought that proliferation could be suppressed. On the other hand, Lactobacillus has no requirement for vitamin K, and therefore, it is thought that vitamin K, which is a growth promoting factor of P. gingivalis, cannot compete and be consumed, and the growth of P. gingivalis cannot be suppressed.

[Example 5] Growth inhibitory action of periodontal disease bacteria by bifidobacteria (1)
The suitability of the screening method for microorganisms shown in Examples 2 to 4 was examined. That is, it was confirmed whether bifidobacteria selected by a screening method for a strain having vitamin K requirement or vitamin K consumption ability actually inhibits the growth of P. gingivalis.

  B. adolescentis OLB6398, which is one of the strains used in Example 2, was mixed (co-) cultured with P. gingivalis, and the growth was compared with the case where P. gingivalis was cultured alone. A GAM medium (Nissui Pharmaceutical Co., Ltd.) was placed in a neutralization culture apparatus (Shibata Kagaku) and sterilized by autoclaving. A solution of MK-7 was aseptically added to the sterilized medium described above to a concentration of 5.0 μg / ml. Then, B. adolescentis OLB6398 and P. gingivalis were inoculated together, and only P. gingivalis was inoculated at 37 ° C. for 72 hours.

  At this time, the pH of the medium may be lowered by lactic acid or acetic acid produced by B. adolescentis OLB6398, which may inhibit the growth of P. gingivalis. Therefore, the culture was performed using 4N-NaOH and 2N-HCl while automatically controlling the pH of the medium in the range of 6.8 to 7.1. Then, sampling was performed over time, and the number of various microorganisms was measured.

As shown in FIG. 4, when B. adolescentis OLB6398 was mixed and cultured with P. gingivalis, the final bacterial count of P. gingivalis was 4.7 × 10 ⁷ . In contrast, when P. gingivalis was cultured alone, the final bacterial count of P. gingivalis was 2.4 × 10 ¹⁰ . Bifidobacteria (B. adolescentis OLB6398) were mixed with periodontal bacteria (P. gingivalis), so that Bifidobacterium could suppress the growth (growth) of periodontal bacteria. In addition, when L. paracasei JCM1181 was mixed and cultured with P. gingivalis, the final bacterial count of P. gingivalis was 3.6 × 10 ¹⁰ . Therefore, even when lactobacilli (L. paracasei JCM1181) was mixed with periodontal bacteria (P. gingivalis), lactobacilli could not suppress the growth (growth) of periodontal bacteria.

[Example 6] Growth inhibitory action of periodontal disease bacteria by bifidobacteria (2)
Bayonella is the dominant bacteria group in the oral cavity and is considered to be a microorganism that supplies vitamin K to P. gingivalis. Inventors of the present application revealed that the culture supernatant of Veillonella parvula ATCC17745 actually promotes the growth of bifidobacteria in Example 2. In this experiment, we confirmed whether bifidobacteria selected by the screening method described above actually suppressed the growth of periodontal disease bacteria (P. gingivalis) under conditions close to the microbial ecosystem in the human oral cavity.

A part of the same neutralization culture apparatus as Example 5 was improved so that continuous culture was possible, and Bayonella bacteria (Veillonella parvula ATCC17745), periodontitis bacteria (P. gingivalis), bifidobacteria (B. adolescentis OLB6398) Were mixed and cultured. In this mixed culture, continuous culture was performed, and the dilution rate was set to 0.06 h ⁻¹ , and the pH of the medium was controlled to 6.7 to 7.1. Then, sampling was performed over time, and the number of various microorganisms was measured. The continuous culture is a culture method in which a fresh medium is supplied to the culture system at a constant flow rate (rate) and at the same time, the same amount of culture medium is discharged. The inventors of the present application considered that this culturing method is a model close to the oral cavity where saliva always flows in and out.

  As shown in FIG. 5 (a), when B. adolescentis OLB6398 was inoculated prior to P. gingivalis, the growth of P. gingivalis was suppressed. Specifically, after Veillonella parvula ATCC17745 was cultured alone, B. adolescentis OLB6398 was inoculated 1 day later, and P. gingivalis was inoculated 5 days later. P. gingivalis has been found not to be isolated from the oral cavity of patients who are regularly maintained by healthy and dentists, or a very low number of bacteria even when isolated. Therefore, when bifidobacteria is administered to healthy subjects or patients undergoing maintenance, it is considered that the initial infection and reinfection of P. gingivalis can be prevented by the competitive action of vitamin K consumption ability (vitamin K requirement).

  On the other hand, as shown in FIG. 5B, when P. gingivalis was inoculated prior to B. adolescentis OLB6398, the growth of P. gingivalis was not suppressed. Specifically, after Veillonella parvula ATCC17745 was cultured alone, P. gingivalis was inoculated 1 day later, and B. adolescentis OLB6398 was inoculated 5 days later. Therefore, in order to apply bifidobacteria to the treatment of periodontal disease, bifidobacteria alone may not be effective enough, and it is considered necessary to combine it with normal periodontal treatment. .

  The present invention can be used in the field of periodontal bacteria prevention or / and treatment and in the industrial field of pharmaceutical or food production therefor.

The result of having compared the sensitivity (vitamin K requirement) of the bifidobacteria with respect to the culture supernatant of a vitamin K and a bayoella bacteria is shown. The results of comparing the growth (growth) of periodontal disease P. gingivalis, Bifidobacterium B. adolescentis, and Lactobacillus L. paracasei by OD by changing the concentration of vitamin K (MK-7) in the medium Show. The results of anaerobic culture of B. adolescentis OLB6398 (deposited strain NITE P-294) and L. paracasei JCM1181 and comparison of the remaining vitamin K concentrations are shown. Bars indicate standard deviation (± SD) of three measurements. The result of trying mixed neutralization culture of B. adlescentis OLB6398 (deposited strain NITE P-294) and P. gingivalis is shown. The horizontal axis represents the culture time, and the vertical axis represents the number of viable cells (cfu / ml). The result of trying mixed continuous culture of B. adlescentis OLB6398 (deposited strain NITE P-294), P. gingivalis, and V. parvula ATCC17745 is shown. The horizontal axis represents the culture time, and the vertical axis represents the number of viable cells (cfu / ml). (A) shows the result of mixed continuous culture when B. adolescentis OLB6398 was inoculated before P. gingivalis, and (b) shows the result of mixed continuous culture when P. gingivalis was inoculated before B. adolescentis OLB6398. Show.

Claims (4)

  Periodontal disease preventive agent or therapeutic agent for periodontal disease containing B. adolescentis OLB6398 (NITE P-294) as an active ingredient which is vitamin K-requiring B. adolescentis.   A food or food additive for preventing periodontal disease or treating periodontal disease comprising B. adolescentis OLB6398 (NITE P-294), which is vitamin K-requiring B. adolescentis as an active ingredient.   The periodontal disease preventive agent or periodontal disease therapeutic agent of Claim 1 aiming at mix | blending with a foodstuff or a food additive. Bifidobacterium adolescentis OLB639
8 (NITE P-294).

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