JP2012025699A - Composition for inhibiting growth of periodontal disease bacteria - Google Patents

Abstract

The present invention provides a composition for inhibiting the growth of periodontal disease bacteria, which is highly safe and exhibits an effect in a relatively short time after intraoral administration.
SOLUTION: Lactobacillus delbrucky subspecies lactis TL6 strain (FERM BP-10758) is used as an active ingredient of a composition for inhibiting periodontal disease growth.
[Selection] Figure 1

Description

  The present invention relates to a composition for inhibiting periodontal disease growth, a preventive and / or therapeutic agent for periodontal disease and / or bad breath, and a food and drink for preventing and / or treating periodontal disease and / or bad breath. About.

  Periodontal disease is the second most common disease after caries as a problem in the oral cavity. Periodontal disease destroys the periodontal tissues such as gingiva, periodontal ligament, cementum, and alveolar bone and affects its function. Periodontal disease causing periodontal disease includes Gram-negative bacteria, Porphyromonas gingivalis, Treponema denticola, Tannerrella forsythia, Actinobacillus actinomycetemcomi Oral bacteria such as stance (Actinobacillus actinomycetemcomitans) are known (for example, Non-Patent Document 1). It has been revealed that such periodontal disease bacteria are also involved in the development of halitosis.

  In general, microorganisms that work beneficially to hosts such as humans and animals are called probiotics, and the effects of probiotics via the digestive tract such as intestinal regulation and immunomodulation are known. In recent years, the usefulness of probiotics has been verified even in the oral cavity, and an inhibitory effect on periodontal disease, bad breath, caries, etc. has been reported (for example, Non-Patent Document 2).

  Among these, there are the following reports on the use of lactic acid bacteria for the prevention and treatment of periodontal disease.

  According to Non-Patent Documents 3 and 4, lactic acid bacteria isolated from the oral cavity of humans, when orally administered for several days to several weeks, settle to the oral cavity to some extent even after discontinuation of administration, and reduce periodontal disease bacteria. Has been reported.

Patent Document 1 discloses a viable bacterial preparation or a lactic acid bacteria-containing food for preventing or treating periodontal disease, which comprises Lactobacillus salivarius TI2711 strain isolated from the oral cavity of a healthy person as an active ingredient.
In the same literature, in an in vitro antibacterial activity test, when inoculated with Lactobacillus salivaius TI2711 strain having the same number of viable bacteria as 1 × 10 ⁶ cfu / ml periodontal disease bacteria in a culture medium, It is described that the survival rate of periodontal disease bacteria decreased to 0.002% after time culture.

In addition, according to a report (for example, Non-Patent Document 5) of examining the antibacterial activity of this Lactobacillus sarivarius TI2711 strain against periodontal disease bacteria over time by changing the number of inoculated bacteria, it is 1 × 10 ⁸ cfu in the medium. / Ml of periodontal disease bacteria and 1 × 10 ⁶ cfu / ml of Lactobacillus salivarius TI2711 strain, which is 1/100 of the number of viable bacteria, and then inoculated the periodontal disease bacteria after culturing at 37 ° C. for 12 hours. It is said that the number has decreased below the detection limit. Moreover, when 1 × 10 ⁸ cfu / ml of Lactobacillus salivarius strain TI2711 having the same number of viable bacteria as 1 × 10 ⁸ cfu / ml of periodontal disease bacteria was mixed and inoculated in the medium, It is said that the viable count of periodontal disease bacteria has decreased below the detection limit.
That is, in the same document, Lactobacillus sarivarius TI2711 strain sterilizes periodontopathic bacteria 100 times the number of viable bacteria within 12 hours in a mixed culture with periodontal pathogens. It can be said that sterilization was reported within 9 hours.

Patent Document 2 describes fermented milk for oral disease prevention and / or treatment containing Lactobacillus delbruecki subspecies bulgaricus OLL1255 strain and Streptococcus thermophilus OLS3294 strain. This document shows that fermented milk obtained using Lactobacillus delbruecki sub-species Bulgarix OLL1255 strain and Streptococcus thermophilus OLS3294 strain inhibited the growth of periodontal disease bacteria.

Patent Document 3 discloses a preventive or therapeutic agent for periodontal disease characterized by containing bacteria belonging to bifidobacteria, lactic acid bacteria or butyric acid bacteria, and sugars that can be assimilated by these bacteria. In the same document, in mixed culture, 1 × 10 ⁸ cfu / ml periodontal disease bacteria and 5 × 10 ⁶ cfu / ml each of 3 types of lactic acid bacteria were mixed and inoculated, and after culturing at 37 ° C. for 24 hours, It is described that the growth of periodontal disease bacteria is suppressed to some extent, but no strong antibacterial activity was observed.

Patent Document 4 discloses a composition for eradication of Porphyromonas gingivalis and / or protection against infection, which contains a lactic acid bacterium belonging to Enterococcus faecium and / or a culture thereof used in the production of fermented milk. Has been. In the same document, Enterococcus faecium (1 × 10 ⁶ cfu / ml) is about 10 times the number of viable bacteria (1 × 10 ⁷ cfu / ml) in periodontal disease bacteria. It is described that after 24 hours of culture, growth was suppressed, but no strong antibacterial activity was observed.

  Patent Document 5 discloses a composition for preventing and / or treating food poisoning or oral disease containing, as an active ingredient, Lactobacillus salivarius WB21 strain isolated from a human digestive tract and selected as a highly acid-resistant strain. Has been.

  Patent Document 6 describes a growth inhibitor of Porphyromonas gingivalis and / or Fusobacterium nucleatum comprising a specific strain belonging to the genus Lactococcus.

  In addition, there are reports on the suppression of the growth of lactic acid bacteria by substances (for example, proteins) produced by periodontal disease bacteria, and it is known that lactic acid bacteria and periodontal disease bacteria compete with each other (Non-patent Document 7).

  By the way, as described in Patent Document 7, Lactobacillus delbrucky subspecies Lactis TL6 strain is known to be used as an active ingredient of a composition for preventing dental caries.

  Although periodontal disease and caries are both infections caused by oral bacteria, the characteristics of the causative bacteria and the mechanism of pathogenesis differ greatly. Caries are caused by decalcification of the tooth by acid produced by cariogenic bacteria such as Streptococcus mutans and Streptococcus sobrinus, which are Gram-positive. These cariogenic bacteria form plaque together with water-insoluble glucan, and produce an acid that causes caries in plaque (for example, Non-Patent Document 1). In fact, it has been reported that even substances exhibiting strong antibacterial activity against cariogenic bacteria have weak antibacterial activity against periodontal disease bacteria (for example, Non-Patent Document 6).

Japanese Patent No. 4203855 Japanese Patent No. 4469792 JP 2003-171292 A JP 2006-328052 A Japanese Patent No. 3944676 JP 2010-124772 A JP 2008-237198A

Oral Hygiene 2010, Takashi Matsukubo, Ken Yaegaki, Masao Maeno, Issei Publishing, Tokyo, 2010, 160-184 Periodontology 2000, Denmark, Vol. 48, 2008, p. 111-147 Applied and Environmental Microbiology, USA, Vol. 72, 2006, p. 3050-3053 Japanese Journal of Periodontology, Japan, Vol. 48, 2006, p. 315-324 Japanese Journal of Periodontology, Japan, Vol. 46, 2004, p. 118-126 Journal of Medical Microbiology, UK, Vol. 52, 2003, p. 1083-1093 Research in Microbiology, France, Vol. 154, p. 669-675

Among the lactic acid bacteria that are said to be effective in inhibiting the growth of periodontal disease bacteria, lactic acid bacteria isolated from human specimens are relatively easy to settle in the oral cavity of humans and are expected to have a sustained growth-inhibiting effect on periodontal disease bacteria. The
However, such lactic acid bacteria may cause side effects due to long-term administration or ingestion depending on the person, and there is a problem that it is difficult to ensure safety when used for oral compositions and foods.
On the other hand, for example, lactic acid bacteria isolated from the natural world other than human specimens and used for producing fermented milk are highly practical from the viewpoint of safety.
However, there is a problem that although it is temporarily present in the oral cavity after administration and ingestion of lactic acid bacteria, it is poorly fixed in the oral cavity over a long period of time.

Therefore, in order to apply lactic acid bacteria isolated from the natural world other than the human oral cavity as probiotics in the oral cavity, it is necessary to select a strain that exerts an effect while the lactic acid bacteria remain in the oral cavity, It is necessary to select a strain that exhibits an effect in a relatively short time after ingestion.
Then, this invention makes it a subject to provide the periodontal disease microbe growth suppression composition with high safety | security, and an effect is exhibited in a comparatively short time after administration to an intraoral area. Moreover, the prevention and / or treatment agent of periodontal disease or bad breath which is highly safe and excellent in effect, and the food and drink for the prevention and / or treatment of periodontal disease or bad breath containing the composition It is an issue to provide.

  In light of the above background art, the present inventors have conducted extensive searches on the physiological functions of lactic acid bacteria. As a result, specific lactic acid strains isolated from the natural world other than the human oral cavity have strong antibacterial activity against periodontal disease bacteria. Found to show.

That is, this invention which solves the said subject is a composition for periodontal disease bacteria growth suppression which uses the Lactobacillus delbrucky subspecies lactis TL6 strain | stump | stock (FERM BP-10758) as an active ingredient.

  In a preferred embodiment of the present invention, the composition for inhibiting periodontal disease growth is such that the Lactobacillus delbruecki subspecies Lactis TL6 strain has a bacterial count of 1% or more with respect to the number of periodontal disease bacteria. Be administered.

  In particular, the composition for inhibiting periodontal disease growth of the present invention is suitable for inhibiting the growth of periodontal disease bacteria belonging to Porphyromonas gingivalis.

  The present invention also provides a preventive and / or therapeutic agent for periodontal disease or bad breath containing the above-mentioned composition for inhibiting the growth of periodontal disease bacteria, and a preventive and / or therapeutic agent for periodontal disease or bad breath containing the composition. Or provide food and drink for treatment.

The composition of the present invention exerts the effect of suppressing the growth of periodontal disease bacteria in a short time and has high safety as compared with a composition containing a lactic acid strain isolated from the natural world other than the human oral cavity.
Therefore, the preventive and / or therapeutic agent for periodontal disease or bad breath of the present invention and the food and drink for the prevention and / or treatment of periodontal disease or bad breath containing the composition are It is effective for prevention and treatment and has high safety.

It is a figure which shows the viable count of the periodontal disease microbe in the test example 2. FIG. It is a figure which shows the number of living bacteria of the lactic acid bacteria in Test Example 2.

  Next, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention.

The composition for inhibiting the growth of periodontal disease bacteria of the present invention comprises Lactobacillus delbruecki subspecies lactis TL6 strain (herein simply referred to as “TL6 strain”) as an active ingredient. The TL6 strain was transferred to the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (Tsukuba Center Tsukuba Center 6th, Tsukuba City, Ibaraki Prefecture) from January 17, 2007 under the accession number FERM BP-10758. It has been deposited.
As disclosed in Japanese Patent Application Laid-Open No. 2008-237198, when the strain is cultured in combination with Streptococcus thermophilus in a reduced skim milk medium from among strains isolated from various samples collected from nature, It is a strain obtained by selecting a strain having a fermentability capable of coagulating, and further selecting a strain having a growth inhibitory property against Streptococcus mutans.
The mycological properties of the TL6 strain are as disclosed in JP 2008-237198 A. Moreover, as described in the same document, the TL6 strain is a strain suitable for the production of fermented milk and is safe to the human body.

The composition for inhibiting periodontal pathogen growth of the present invention has a periodontal pathogen growth inhibitory effect equal to or higher than that of the TL6 strain, and uses a mutant of the TL6 strain as long as it is safe when administered to the oral cavity. Also good. Whether or not a certain mutant strain has a “periodontal fungus growth inhibitory effect” with the TL6 strain is determined by, for example, a method as shown in a test example to be described later. This change can be confirmed by comparing it with that of the TL6 strain. Such a mutant strain may be constructed by introducing an artificial mutation into the TL6 strain. Alternatively, mutations may be constructed by introducing mutations into the TL6 strain by treatment with a mutagen such as UV.
Mutants may be constructed by introducing mutations into the L6 strain.
The mutant strain of the TL6 strain described above is a strain derived from the TL6 strain as a parent strain, and is limited to strains belonging to the Lactobacillus delbruecki subspecies lactis species. Moreover, the mutant strain of the above-mentioned TL6 strain does not include any conventionally known lactic acid strains that have been confirmed to have preventive and therapeutic effects on periodontal disease bacteria.
In the following description, the TL6 strain and the above-described mutant strain may be collectively referred to as “TL6 strain” in some cases.

  The TL6 strain can be cultured under culture conditions usually used for culture of bacteria belonging to the genus Lactobacillus. For example, the culture temperature is usually about 25 to 50 ° C, preferably about 35 to 42 ° C. Moreover, as culture conditions, microaerobic conditions or anaerobic conditions are preferable.

  The medium for culturing the TL6 strain is not particularly limited, and a medium usually used for culturing bacteria belonging to the genus Lactobacillus can be used. That is, as the carbon source, for example, saccharides such as glucose, galactose, lactose, arabinose, mannose, sucrose, starch, starch hydrolyzate, and molasses can be used depending on utilization. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean powder, defatted soybean meal, meat extract, yeast extract and the like may also be used.

The composition for inhibiting periodontal disease bacteria growth of the present invention may be the whole of the culture of TL6 strain or a part containing the cells. For example, the culture obtained after culturing can be centrifuged, and the cells can be recovered and used.
The whole or a part of the culture obtained as described above is preferably dried from the viewpoints of storage stability and handleability. As a drying method, freeze-drying is preferable.
As will be described later, the composition for inhibiting the growth of periodontal disease bacteria of the present invention can be used as an active ingredient in agents and foods and drinks for the purpose of preventing and treating periodontal disease and bad breath.

  The concentration of the TL6 strain in the composition for inhibiting the growth of periodontal pathogens of the present invention can be adjusted within a range in which the growth of periodontal pathogens can be suppressed depending on the method of use, the amount of use, and the like.

In addition, the composition for inhibiting periodontal pathogen growth of the present invention is used in such a form that the TL6 strain is administered so that the bacterial count is 1% or more, preferably 100% or more, relative to the number of periodontal pathogens. It is preferred that
As shown in the test examples described later, when TL6 strain and periodontal disease bacteria are mixed and cultured, even if TL6 strain is about 1% of periodontal disease bacteria, growth of periodontal disease bacteria is suppressed in a short time of 9 hours. To do. Furthermore, from the results of the test examples described later, when the TL6 strain is about 100% of the periodontal disease bacteria, it is estimated that the growth of the periodontal disease bacteria is suppressed in a short time of 3 hours.

In addition to the TL6 strain, which is an active ingredient, the composition for inhibiting periodontal disease growth of the present invention includes other components that exhibit beneficial effects in the oral cavity, such as sugar alcohols such as xylitol and sorbitol, polyphenols such as catechin, lactoferrin, It is also preferable to include proteins such as lysozyme, lactoperoxidase, and immunoglobulin.
The composition for inhibiting the growth of periodontal disease bacteria of the present invention can contain other components that are usually used in compositions administered into the oral cavity.

  The composition for inhibiting the growth of periodontal disease bacteria of the present invention can be used as a food additive for imparting an effect of inhibiting the growth of periodontal disease bacteria to various foods.

  The composition for inhibiting the growth of periodontal disease bacteria of the present invention is a gram-negative bacterium, Porphyromonas gingivalis, Treponema denticola, Tannerrella forsythia, Actinobacillus actinomycetem Effective in inhibiting the growth of periodontal pathogens such as Comitans (Actinobacillus actinomycetemcomitans). In particular, it is effective in suppressing the growth of Porphyromonas gingivalis.

The preventive and / or therapeutic agent for periodontal disease and / or halitosis according to the present invention includes a composition for suppressing periodontal disease growth. The preventive and / or therapeutic agent for periodontal disease and / or bad breath of the present invention includes a pharmaceutical form.
The dosage form is not particularly limited as long as it can be administered into the oral cavity, but is preferably in a form suitable for staying in the oral cavity for a certain period of time (several minutes or more), and examples include a washing solution, a troche tablet, a chewing gum, and a paste. .
The preventive and / or therapeutic agent for periodontal disease and / or bad breath can be produced by formulating the TL6 strain together with any additive such as a pharmaceutically acceptable excipient. In formulating, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, and diluents can be used.

The concentration of the TL6 strain in the preventive and / or therapeutic agent for periodontal disease and / or halitosis according to the present invention is adjusted within a range in which the growth of periodontal disease bacteria can be suppressed depending on the method of use, the amount used, and the like. can do.
As a standard of the content of the TL6 strain, it is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass.
As the measure of the content of the TL6 strain, normally ^{10 7} ~10 10 cfu / g, preferably 10 ⁸ ~10 ⁹ cfu / g.

In addition, the preventive and / or therapeutic agent for periodontal disease and / or bad breath of the present invention is such that the TL6 strain has 1% or more, preferably 100% or more of the number of bacteria of periodontal disease bacteria. It is preferably used in an administered form.
For example, periodontal disease bacteria of about 10 ⁸ to 10 ¹⁰ cfu are present in the oral cavity of a human periodontal disease group or periodontal patient. Therefore, the number of bacteria administered once in the preventive and / or therapeutic agent for periodontal disease and bad breath of the present invention is usually 10 ⁷ cfu or more, preferably 10 ⁸ cfu or more, more preferably 10 ⁹ cfu or more.

  The preventive and / or therapeutic agent for periodontal disease and / or bad breath of the present invention is administered into the oral cavity. The form of administration into the oral cavity may be any of a form discharged from the oral cavity after administration into the oral cavity and a form swallowed after administration into the oral cavity. Examples include troches, powders, liquids, toothpastes, and the like. Among these, a form like a troche that easily stays in the oral cavity is preferable. The preventive and / or therapeutic agent for periodontal disease and / or bad breath of the present invention is preferably administered a plurality of times a day, preferably 2 to 3 times, every 3 to 4 hours. In particular, administration within 30 minutes after meal is preferred.

The food and drink for preventing and / or treating periodontal disease and / or halitosis according to the present invention includes a composition for inhibiting periodontal disease growth.
Examples of the form include fermented foods such as yogurt, confectionery such as chocolate, candy, and chewing gum. Among these, fermented foods that allow easy intake of a sufficient amount of TL6 strain are preferred.

The concentration of TL6 strain in the food and drink for preventing and / or treating periodontal disease and / or bad breath according to the present invention is a range in which the growth of periodontal bacteria can be suppressed according to the form of the food and drink. Can be adjusted to.
As a standard of the content of the TL6 strain, it is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass.
As the measure of the content of the TL6 strain, usually ^{10 6} ~10 10 cfu / g, preferably 10 ⁷ ~10 ⁹ cfu / g.

In the food and drink for preventing and / or treating periodontal disease and bad breath according to the present invention, the TL6 strain has 1% or more, preferably 100% or more of the number of bacteria of periodontal disease bacteria. It is preferable to be used in a form to be administered.
As described above, in consideration of the number of periodontal disease bacteria in the human oral cavity, the number of bacteria taken at one time of the food for preventing and / or treating periodontal disease and bad breath of the present invention is usually 10 ⁷ cfu or more. , Preferably 10 ⁸ cfu or more, more preferably 10 ⁹ cfu or more.

  The food and drink for preventing and / or treating periodontal disease and / or halitosis according to the present invention is preferably taken a plurality of times a day, preferably 2 to 3 times, every 3 to 4 hours. In particular, in the form of candy, chewing gum or the like, it is preferably taken after a meal or between meals.

  The food and drink for preventing and / or treating periodontal disease and bad breath according to the present invention is sold as a food and drink displaying the use as "for preventing and / or treating periodontal disease and / or bad breath". It is preferable.

  The “indication” means all acts for informing the consumer of the use, and the act of describing the use in the product or product packaging related to the food or drink of the present invention. Transfer, display, display for distribution, transfer, or delivery of the above-mentioned use on the packaging, display or distribute the above-mentioned use in the act of importing, advertising about products, price lists or transaction documents, Or the act which describes the said use in the information which contains these, and provides with an electromagnetic method etc. can be illustrated.

  However, the display is preferably a display approved by the government or the like (for example, a display that is approved based on various systems determined by the government and is performed in a mode based on such approval). The indication (particularly the indication of the use of health) as the food for food (the Health Promotion Act Enforcement Regulations (Ministry of Health, Labor and Welfare Ordinance No. 86 of April 30, 2003)) is most suitable.

  The wording used for the above indication is not limited to the wording “for prevention and / or treatment of periodontal disease and / or bad breath”, but other words. If it is a word suggesting the effect of prevention or treatment of periodontal disease and / or bad breath, or a word suggesting a secondary effect due to prevention or treatment of periodontal disease and / or bad breath, use Needless to say, it can be done.

Next, a test example is shown and the antibacterial action with respect to periodontal disease bacteria of TL6 strain | stump | stock is demonstrated in detail.
[Test Example 1]
This test was conducted to select lactic acid bacteria having antibacterial action against periodontal disease bacteria.
(1) Preparation of Periodontal Bacterial Pre-culture Solution Porphyromonas gingivalis strain JCM8525 (distributed from RIKEN), a periodontal disease fungus, was added with 5 mg / L hemin, 1 mg / L menadione, and 7 g / L glucose added. Inoculate 4 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) and incubate at 37 ° C for 1 day in an anaerobic chamber (manufactured by Coy) filled with 85% nitrogen gas, 5% hydrogen gas and 10% carbon dioxide gas. This was used as a preculture solution for periodontal disease bacteria.

(2) Preparation of Lactobacillus Test Bacteria 13 Lactobacillus reference strains (distributed from RIKEN or American Type Culture Collection) or 28 stored strains of Lactobacillus 5 mg / L hemin, 1 mg / L menadione And 4 ml of GAM broth supplemented with 7 g / L glucose, and cultured at 37 ° C. for 1 day in an anaerobic chamber filled with 85% nitrogen gas, 5% hydrogen gas, and 10% carbon dioxide gas. This culture solution was diluted 100 times with phosphate buffered saline to obtain a lactic acid bacteria test bacterial solution.

(3) Test method 40 μL of periodontopathic bacteria preculture solution and 40 μL of lactic acid bacteria test culture solution are added to a 15 ml tube containing 4 ml of GAM bouillon supplemented with 5 mg / L hemin, 1 mg / L menadione, and 7 g / L glucose. And stirred. This mixed culture was mixed and cultured at 37 ° C. in an anaerobic chamber filled with 85% nitrogen gas, 5% hydrogen gas, and 10% carbon dioxide gas. In order to selectively detect only periodontal disease bacteria by 20 μl each by collecting 100 μL of the culture solution at 8, 9, and 10 hours after the start of the mixed culture and preparing a 10-fold dilution series with phosphate buffered saline. Agar medium (5% equine defibrinated blood, blood agar basic medium NO.2 (Oxoid) supplemented with 5 mg / L hemin, 1 mg / L menadione, 75 mg / L neomycin, and 3.5 mg / L vancomycin) After coating and culturing at 37 ° C. for 5 days in an anaerobic chamber, the number of colonies formed on the agar medium was measured. From the detected number of bacterial colonies, the logarithmic value (log ₁₀ cfu / ml) of the number of periodontal disease viable bacteria per 1 ml of the above test mixture was determined. As a control experiment, the number of viable periodontal bacteria was examined under the condition that no lactic acid bacteria test bacterial solution was added.

(4) Test results Tables 1 and 2 show the results of this test.

As shown in Table 1, among the lactic acid bacteria reference strains, L. gasseri
JCM1131 strain reduces periodontal disease bacteria to 1/100 or less (4.70 log ₁₀ cfu / ml) of the initial bacterial count (about 7.0 log ₁₀ cfu / ml) 10 hours after the start of mixed culture, Bactericidal activity was observed. On the other hand, in the other 12 lactic acid bacteria reference strains, the viable count of periodontal disease bacteria was hardly affected as compared with the control without addition of lactic acid bacteria until 10 hours in the mixed culture.

  As shown in Table 2, among the preserved strains, the TL6 strain decreased the periodontal disease bacteria count below the detection limit after 9 hours, and bactericidal activity was observed. Moreover, Lactobacillus salivarius (L. salivarius) 102-1-1 strain reduced the periodontal disease bacteria count below the detection limit after 10 hours, and bactericidal activity was observed. On the other hand, the 26 strains other than the above two strains had almost no effect on the viable count of periodontal disease bacteria as compared to the control without addition of lactic acid bacteria until 10 hours in the mixed culture. Therefore, it was found that among the tested reference strains and preserved strains, the TL6 strain exhibited antibacterial activity against periodontal disease bacteria in the shortest time.

[Test Example 2]
This test was performed to examine the strength of the antibacterial activity, particularly the time required to sterilize the periodontal disease bacteria, for the TL6 strain in which antibacterial activity against periodontal disease bacteria was observed in Test Example 1.

(1) Preparation of Periodontal Bacterial Pre-culture Solution A periodontal disease pre-culture solution was prepared in the same manner as in Test Example 1 above.

(2) Preparation of lactic acid bacteria test bacterial solution The TL6 strain was cultured in the same manner as in Test Example 1 to prepare a lactic acid bacteria test bacterial solution.

(3) Test method Periodontal disease bacteria and lactic acid bacteria were mixed and cultured in the same manner as in Test Example 1 above. However, the number of viable bacteria of periodontal disease immediately after the start of mixed culture (0 hour), 3, 6, 7, 8, 9, and 10 hours was measured by the same method as in Test Example 1. Furthermore, 20 μL of a 10-fold dilution series prepared with phosphate buffered saline was applied to a BCP-added plate count agar medium (manufactured by Eiken Chemical Co., Ltd.) and 37 ° C. in an incubator filled with 10% carbon dioxide gas. After culturing for 3 days, the number of colonies formed on the agar medium was counted, and the number of living lactic acid bacteria was measured.

(4) Test results The results of this test are shown in FIGS. FIG. 1 shows the results of the viable count of periodontal disease bacteria (◯: no lactic acid bacteria added, ●: lactic acid bacteria added). In addition, FIG. 2 shows the results of the viable count of lactic acid bacteria (◯: no lactic acid bacteria added, ●: lactic acid bacteria added). As a result, periodontal bacteria and viable cell count of lactic acid bacteria, the mixed culture initiation time (after 0 hours) were respectively 7.44log ₁₀ cfu / ml, and 4.96log ₁₀ cfu / ml. In the mixed culture with lactic acid bacteria, the viable count of periodontal disease bacteria increased until 6 hours after the start of mixed culture, and an almost constant viable count was observed until 8 hours, whereas in the control without addition of lactic acid bacteria, The viable count of periodontal disease increased until 10 hours after the start of culture. In addition, the number of living lactic acid bacteria increased until 10 hours after the start of mixed culture. Nine hours after the start of the mixed culture with lactic acid bacteria, the number of viable bacteria of periodontal disease bacteria fell below the detection limit (<2.70 log ₁₀ cfu / ml), and bactericidal activity was observed. Therefore, it was clarified that the TL6 strain sterilizes periodontal disease bacteria having about 100 times the number of viable bacteria within 9 hours in a mixed culture using a growth medium of periodontal disease bacteria.
In addition, the number of bacteria of TL6 strain increased to 7.48 log ₁₀ cfu / ml 6 hours after the start of mixed culture, and reached approximately the same number of viable bacteria as at the start of mixed culture of periodontal disease bacteria (after 0 hours). . Based on this time point (6 hours after the start of mixed culture), periodontal disease bacteria were sterilized to the detection limit or less after 3 hours (9 hours after the start of mixed culture). Therefore, it was speculated that the TL6 strain sterilizes periodontal disease bacteria having the same number of viable bacteria within at least 3 hours.

  From the above test, it was found that the TL6 strain has higher antibacterial activity against periodontal pathogens than conventional lactic acid strains, and can kill periodontal pathogens in a short time.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
Lactate pre-cultured for 16 hours at 25 ° C. in a medium comprising 50 g of meat extract, 100 g of yeast extract, 100 g of peptone, 200 g of lactose, 50 g of dipotassium hydrogen phosphate, 10 g of potassium dihydrogen phosphate, 4 g of cystine and 9.5 L of water. 500 mL of a seed culture of Bacillus delbruecki sub-species Lactis TL6 strain was inoculated into 10 L of the medium having the same composition as the above medium, and cultured at 25 ° C. for 16 hours. Further, 200 L of the medium having the same composition as the medium sterilized at 90 ° C. for 30 minutes was inoculated with the whole culture solution (10.5 L) and cultured at 25 ° C. for 16 hours. The viable cell count after the culture was 3.0 × 10 ⁹ cfu / mL.
Next, using a sharpless centrifuge, the cells are collected by centrifugation (15,000 rpm), resuspended in the same amount of physiological saline (sterilized at 90 ° C. for 30 minutes) as the medium, and centrifuged as described above. Separated and collected again. The collected cells are suspended in 20 L of a solution comprising 10% skim milk powder, 1% sucrose, and 1% sodium glutamate (sterilized at 90 ° C. for 30 minutes), freeze-dried according to a conventional method, and 8.5 × 10 ¹⁰ cfu / About 2.2 kg of powder containing g of Lactobacillus delbrucky subspecies Lactis TL6 strain was obtained.

  This powder is the composition for inhibiting periodontal disease bacteria growth of the present invention, and the preventive and / or therapeutic agent for periodontal disease and / or bad breath of the present invention, or for the prevention of periodontal disease and / or bad breath of the present invention. And / or can be used as a raw material for food and drink for treatment.

[Example 2]
To 14 kg of reduced indigestible dextrin and 6 kg of sorbitol, 20 g of powder containing Lactobacillus delbruecki subspecies lactis TL6 strain obtained in Example 3 was added and mixed uniformly. -About 20 kg of viable bacterial agent containing bacterial powder of Lactis TL6 strain was obtained.

  This powder is the preventive and / or therapeutic agent for periodontal disease and / or bad breath of the present invention, and is expected to be effective for the prevention and treatment of periodontal disease and bad breath.

  The application of the composition for inhibiting periodontal disease bacteria growth of the present invention to foods and drinks is safe and effective as a means for ensuring the hygiene of the oral cavity. In particular, since it is suitable for long-term daily intake, it is highly practical.

Claims (5)

Lactobacillus delbruecki subspecies lactis TL6 strain (FERM
BP-10758) as an active ingredient, a composition for inhibiting periodontal fungus growth.   The periodontal according to claim 1, wherein the Lactobacillus delbruecki sub-species Lactis TL6 strain is administered so as to have a bacterial count of 1% or more with respect to the bacterial count of periodontal disease bacteria. A composition for suppressing the growth of disease bacteria.   The composition for periodontal pathogen growth inhibition according to claim 1 or 2, wherein the periodontal pathogen is Porphyromonas gingivalis.   A preventive and / or therapeutic agent for periodontal disease or bad breath containing the composition according to any one of claims 1 to 3.   The food-drinks for prevention and / or treatment of periodontal disease or bad breath containing the composition as described in any one of Claims 1-3.

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