JP2015157768A - Oral composition, periodontal disease risk prediction device, and animal periodontal disease treatment method - Google Patents
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Abstract
【課題】口腔内細菌を活用し、体内を循環する物質との相互作用によって、持続的かつ安全的に、歯周病原菌に対して特異的な殺菌効果がある口腔用組成物を提供する。【解決手段】Actinomyces属やRothia属の細菌が、硝酸還元菌として作用し、口腔善玉菌として働き、歯周病原菌を殺菌することを見出した。すなわち、口腔用組成物は、アクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌と、硝酸塩あるいは硝酸イオンを含有してなる。Actinomyces属やRothia属の細菌が、硝酸還元菌として作用し、硝酸塩存在下で一酸化窒素（ＮＯ）を生成することによって、歯周病原菌の殺菌および歯周病の予防や治療を可能とする。口腔用組成物は、口腔内に適用したときに、唾液のｐＨを５．５〜６．５の範囲に下降させることが好ましく、また、硝酸濃度が２〜１０ｍＭであることが好ましい。【選択図】図１To provide an oral composition having a specific bactericidal effect against periodontal pathogens by utilizing oral bacteria and interacting with substances circulating in the body in a continuous and safe manner. It has been found that bacteria of the genus Actinomyces and Rothia act as nitrate-reducing bacteria, work as oral good bacteria, and kill periodontal pathogens. That is, the composition for oral cavity contains bacteria belonging to the genus Actinomyces (Actinomyces) or Russia (Rothia), and nitrate or nitrate ion. Bacteria from the genus Actinomyces and Rothia act as nitrate-reducing bacteria and produce nitric oxide (NO) in the presence of nitrate, thereby enabling the sterilization of periodontal pathogens and the prevention and treatment of periodontal diseases. When applied to the oral cavity, the oral composition preferably lowers the pH of saliva to a range of 5.5 to 6.5, and the nitric acid concentration is preferably 2 to 10 mM. [Selection] Figure 1
Description
本発明は、口腔用組成物、歯周病疾患リスク予測装置および動物の歯周病治療方法に関するものである。 The present invention relates to an oral composition, a periodontal disease risk prediction apparatus, and a method for treating periodontal disease in animals.
歯周病を予防するためには、歯周病の原因であるプラーク（歯垢）を除去することが重要であり、プラークを取り除くことやプラークをつきにくくするプラークコントロールによる歯周病菌の除去が行われている。
また、歯周病のリスクファクター（危険因子）を少なくすることも重要である。
昨今、歯周病予防剤として薬用歯磨剤（練り歯磨き、液体歯磨き）が多数商品化されており、プラーク除去を目的として、薬用成分としてデキストラナーゼ（酵素）を含み、プラークの分解を促進させている。
In order to prevent periodontal disease, it is important to remove plaque (plaque) that is the cause of periodontal disease, and removal of periodontal bacteria by plaque control that makes it difficult to remove plaque or plaque. Has been done.
It is also important to reduce the risk factor (risk factor) of periodontal disease.
Recently, a number of medicinal dentifrices (paste toothpaste and liquid toothpaste) have been commercialized as preventive agents for periodontal disease. For the purpose of removing plaque, dextranase (enzyme) is included as a medicinal ingredient to promote plaque degradation. ing.
歯周病（歯肉炎・歯周炎）の予防を目的としては、出血防止効果があるトラネキサム酸、抗炎症作用があるε-アミノカプロン酸、グリチルリチン酸ジカリウムまたはβ-グリチルレチン酸、殺菌作用があるＩＰＭＰ（イソプロピルメチルフェノール）、塩化セチルピリジニウム（ＣＰＣ）またはトリクロサン、収れん作用がある塩化ナトリウム、血行作用がある酢酸トコフェロール（ビタミンＥ）が薬用成分として知られている。 To prevent periodontal disease (gingivitis and periodontitis), tranexamic acid with anti-bleeding effect, ε-aminocaproic acid with anti-inflammatory action, dipotassium glycyrrhizinate or β-glycyrrhetinic acid, IPMP with bactericidal action (Isopropylmethylphenol), cetylpyridinium chloride (CPC) or triclosan, sodium chloride with an astringent action, and tocopherol acetate (vitamin E) with a blood circulation action are known as medicinal ingredients.
また、むし歯予防を目的としては、モノフルオロリン酸ナトリウム（ＭＦＰ）やフッ化ナトリウム（ＮａＦ）が薬用成分として知られており、これらの成分は歯質強化、再石灰化の促進、酸産生の抑制の働きがある。
また、歯石予防を目的として、歯石形成の抑制効果があるポリリン酸ナトリウム、ピロリン酸ナトリウムが薬用成分として知られている。
この他、冷たいものが歯にしみるのを防ぐのを目的として、乳酸アルミニウムや硝酸カリウムが薬用成分として知られており、これらは刺激の伝達を防ぐ働きを担っている。
Also, for the purpose of preventing caries, sodium monofluorophosphate (MFP) and sodium fluoride (NaF) are known as medicinal ingredients, and these ingredients enhance tooth quality, promote remineralization, and promote acid production. There is a control action.
For the purpose of preventing calculus, sodium polyphosphate and sodium pyrophosphate which are effective in suppressing calculus formation are known as medicinal ingredients.
In addition to this, aluminum lactate and potassium nitrate are known as medicinal ingredients for the purpose of preventing cold objects from getting into the teeth, and these have a function to prevent transmission of stimuli.
また、薬用歯磨剤の他、歯周病予防や歯周病治療として、歯周病巣に殺微生物的有効量の金属イオンを投入する技術（特許文献１）、口腔細菌の菌体外水溶性低分子物質を含有する口腔用組成物の技術（特許文献２）、有効量のカテキンを含有する口腔常在菌叢調整剤の技術（特許文献３）、生きた乳酸菌とキシリトールを含有する口腔内疾患の予防剤の技術（特許文献４）が知られている。
また、有効塩素濃度が５０〜７００ｐｐｍ、ｐＨが６．３〜８であって、次亜塩素酸及び炭酸水素ナトリウムを含んでなり、歯周病原菌を殺菌する技術（特許文献５）が知られている。
In addition to medicated dentifrices, as a periodontal disease prevention and periodontal disease treatment, a technique for injecting a microbicidal effective amount of metal ions into periodontal lesions (Patent Document 1) Techniques for oral compositions containing molecular substances (Patent Document 2), techniques for regulating oral resident flora containing effective amounts of catechins (Patent Document 3), oral diseases containing living lactic acid bacteria and xylitol The technique of the preventive agent of this (patent document 4) is known.
In addition, a technique (Patent Document 5) is known that has an effective chlorine concentration of 50 to 700 ppm, a pH of 6.3 to 8, and contains hypochlorous acid and sodium hydrogen carbonate to sterilize periodontal pathogens. Yes.
歯周病の病原菌として、嫌気性のグラム陰性桿菌が主に知られている。具体的には、ポルフィロモナス・ジンジバリス(Porphyromonas gingivalis)、プレボテラ・インターメディア(Prevotella intermedia)、エイケネラ・コローデンス(Eikenella corrodens)、カンピロバクター・レクタス(Campylobacter rectus)である。
歯周病の予防法や治療法は以前から多くの方法があるが、いずれも根本的な治療方法を見出せていないのが現状である。これは、歯周病原菌の殆どが、浮遊菌ではなく、自ら産生した菌体外多糖からなるバイオフィルムで保護されており、このバイオフィルムで抗生剤等を遮断しながら、緩やかに増殖を続けるためである。
Anaerobic gram-negative bacilli are mainly known as pathogens of periodontal disease. Specifically, Porphyromonas gingivalis, Prevotella intermedia, Eikenella corrodens, Campylobacter rectus.
There have been many methods for preventing and treating periodontal disease, but none of them have found a fundamental treatment method. This is because most of the periodontal pathogens are protected by biofilms made of exopolysaccharides produced by themselves rather than planktonic bacteria, and this biofilm keeps growing slowly while blocking antibiotics etc. It is.
このため、歯周病原菌の殺菌には、持続的に効果が続くことが期待でき、体内に安全で、さらに、歯周病原菌に対して特異的な殺菌効果があり他の常在菌（共生菌）を殺菌しないことが望まれる。
かかる状況に鑑みて、本発明は、口腔内細菌を活用し、体内を循環する物質との相互作用によって、持続的かつ安全的に、歯周病原菌に対して特異的な殺菌効果がある口腔用組成物を提供することを目的とする。
For this reason, it can be expected that sterilization of periodontal pathogens will continue to be effective, safe in the body, and has a specific bactericidal effect against periodontal pathogens. ) Is not sterilized.
In view of such a situation, the present invention utilizes oral bacteria and has a bactericidal effect specific to periodontal pathogens, continuously and safely, by interaction with substances circulating in the body. An object is to provide a composition.
本発明者らは、口腔内細菌を鋭意検討した結果、口腔内細菌の内、Actinomyces属やRothia属の細菌が、硝酸塩存在下で、歯周病原菌を殺菌できることの知見を得た。すなわち、Actinomyces属やRothia属の細菌が、硝酸還元菌として作用し、口腔善玉菌として働くこと、ならびに歯周病原菌の殺菌の仕組みを見出した。 As a result of intensive studies on oral bacteria, the present inventors have found that among oral bacteria, bacteria belonging to the genus Actinomyces or Rothia can kill periodontal pathogens in the presence of nitrate. That is, the present inventors have found that bacteria belonging to the genus Actinomyces and Rothia act as nitrate-reducing bacteria, work as oral good bacteria, and sterilize the periodontal pathogens.
すなわち、本発明の口腔用組成物は、アクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌と、硝酸塩あるいは硝酸イオンを含有してなることを特徴とする。
Actinomyces属やRothia属の細菌が、硝酸還元菌として作用し、硝酸塩存在下で一酸化窒素（ＮＯ）を生成することによって、歯周病原菌の殺菌および歯周病の予防や治療を可能とする。
具体的な細菌は、例えば、Actinomyces oris MG-1菌株、Actinomyces naeslundii ATCC12104菌株、Rothia aeria JCM11412菌株、Rothia mucilaginosa DY-18菌株、Rothia dentocariosa ATCC17931菌株から選ばれる菌株から得られる。
That is, the composition for oral cavity of the present invention is characterized by containing bacteria belonging to the genus Actinomyces or Rothia, and nitrate or nitrate ion.
Bacteria from the genus Actinomyces and Rothia act as nitrate-reducing bacteria and produce nitric oxide (NO) in the presence of nitrate, thereby enabling the sterilization of periodontal pathogens and the prevention and treatment of periodontal diseases.
Specific bacteria are obtained from strains selected from, for example, Actinomyces oris MG-1 strain, Actinomyces naeslundii ATCC12104 strain, Rothia aeria JCM11412 strain, Rothia mucilaginosa DY-18 strain, and Rothia dentocariosa ATCC17931 strain.
本発明の口腔用組成物は、口腔内に適用したときに、唾液のｐＨを５．５〜６．５の範囲に下降させることが好ましい。歯周病原菌の硝酸塩存在下での殺菌は、ｐＨが６．５以下になると著しく効果が現れる。ｐＨが５．５より下回っても殺菌効果は現れるが、他の副作用を生じさせるリスクを回避すべく上記の範囲とする。
また、本発明の口腔用組成物は、口腔内に適用したときに、硝酸濃度が２〜１０ｍＭであることが好ましい。硝酸濃度が２ｍＭ以上とすることで著しく殺菌効果が現れる。硝酸濃度が１０ｍＭを超えても殺菌効果は現れるが、他の副作用を生じさせるリスクを回避すべく上記の範囲とする。
When the composition for oral cavity of the present invention is applied to the oral cavity, the pH of saliva is preferably lowered to the range of 5.5 to 6.5. Sterilization of periodontal pathogens in the presence of nitrates is very effective when the pH is 6.5 or lower. Although the bactericidal effect appears even when the pH is lower than 5.5, the above range is used to avoid the risk of causing other side effects.
The oral composition of the present invention preferably has a nitric acid concentration of 2 to 10 mM when applied to the oral cavity. When the nitric acid concentration is 2 mM or more, the bactericidal effect appears remarkably. Although the bactericidal effect appears even if the nitric acid concentration exceeds 10 mM, the above range is set to avoid the risk of causing other side effects.
硝酸塩は、具体的には、硝酸ナトリウム（ＮａＮＯ３）または硝酸カリウム（ＫＮＯ３）である。硝酸ナトリウム（ＮａＮＯ３）または硝酸カリウム（ＫＮＯ３）は、本来的に食品に含まれる硝酸塩であり、葉菜類(ホウレンソウ、春菊、レタス)に多く含まれる。これらの硝酸塩であれば口腔内に投与しても安全である。 The nitrate is specifically sodium nitrate (NaNO 3 ) or potassium nitrate (KNO 3 ). Sodium nitrate (NaNO 3 ) or potassium nitrate (KNO 3 ) is a nitrate that is inherently contained in foods, and is abundant in leaf vegetables (spinach, spring chrysanthemum, lettuce). These nitrates are safe for oral administration.
本発明の口腔用組成物は、Porphyromonas gingivalis及びPrevotella intermediaの歯周病原菌を殺菌できる。
Porphyromonas gingivalis及びPrevotella intermediaは、代表的な歯周病原菌であり、後述する実施例において、これらの歯周病原菌が硝酸依存的に殺菌されることが確認できている。
The oral composition of the present invention can sterilize periodontopathic bacteria of Porphyromonas gingivalis and Prevotella intermedia.
Porphyromonas gingivalis and Prevotella intermedia are typical periodontal pathogens, and it has been confirmed that these periodontal pathogens are sterilized in a nitrate-dependent manner in the examples described below.
本発明の口腔用組成物は、歯磨剤、洗口剤、歯肉マッサージクリーム、トローチ、チューイングガム、タブレットまたはジュースの形態で提供できる。 The oral composition of the present invention can be provided in the form of a dentifrice, mouthwash, gingival massage cream, troche, chewing gum, tablet or juice.
次に、本発明の歯周病疾患リスク予測装置について説明する。
本発明の歯周病疾患リスク予測装置は、口腔内細菌叢（フローラ）におけるアクチノマイセス（Actinomyces）属およびロシア（Rothia）属の細菌の存在割合を測定する手段を備え、歯周病疾患の発生リスクを予測する。
Actinomyces属やRothia属の細菌が、硝酸還元菌として作用し、硝酸塩存在下で一酸化窒素（ＮＯ）を生成して歯周病原菌を殺菌するので、口腔内細菌叢（フローラ）におけるアクチノマイセス（Actinomyces）属およびロシア（Rothia）属の細菌の存在割合を測定することで、歯周病疾患の発生リスクを予測することができる。
Next, the periodontal disease risk prediction apparatus of the present invention will be described.
The device for predicting periodontal disease risk of the present invention comprises means for measuring the abundance of bacteria belonging to the genus Actinomyces and Russian in the oral flora (flora), Predict the risk of occurrence.
Actinomyces and Rothia spp. Act as nitrate-reducing bacteria, producing nitric oxide (NO) in the presence of nitrate to sterilize periodontal pathogens, so Actinomyces in the oral flora (flora) ( The risk of developing periodontal disease can be predicted by measuring the proportion of bacteria belonging to the genus Actinomyces and Russia.
また、本発明の歯周病疾患リスク予測装置は、口腔内における硝酸還元能を測定する手段を備え、歯周病疾患の発生リスクを予測する。例えば、唾液成分の硝酸還元能の測定、その他口腔内における硝酸還元能を測定することにより、硝酸塩存在下で一酸化窒素（ＮＯ）を生成して歯周病原菌を殺菌できる能力を測定するのである。 Moreover, the periodontal disease risk prediction apparatus of this invention is equipped with the means to measure the nitrate reduction ability in an oral cavity, and estimates the generation | occurrence | production risk of a periodontal disease. For example, by measuring the nitrate reducing ability of saliva components and other nitrate reducing ability in the oral cavity, it measures the ability to sterilize periodontal pathogens by producing nitric oxide (NO) in the presence of nitrate. .
次に、本発明の歯周病治療方法について説明する。
本発明の歯周病治療方法は、硝酸塩水溶液を歯肉炎下へ注入することにより、人を除く動物の歯周病を治療する。硝酸塩水溶液を歯肉炎下へ注入することにより、口腔内に元々存在するアクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌が、硝酸を還元して亜硝酸を生成する。亜硝酸は、口腔内の弱酸性環境によって還元され、一酸化窒素（ＮＯ）を生成するので歯周病原菌が殺菌される。なお、硝酸塩水溶液を歯肉炎下へ注入することにより、人の歯周病を治療することも可能である。
また、歯周病の治療や予防目的で、硝酸塩水溶液を口腔内に投与することも効果的である。例えば、野菜ジュース程度の硝酸濃度（５ｍＭ）でも歯周病原菌の十分な殺菌効果がある。なお、過剰摂取した硝酸イオンは尿として排出され、人体に害となる亜硝酸や一酸化窒素が過剰になることはない。
Next, the method for treating periodontal disease of the present invention will be described.
The method for treating periodontal disease of the present invention treats periodontal disease in animals other than humans by injecting an aqueous nitrate solution under gingivitis. By injecting an aqueous nitrate solution under gingivitis, bacteria belonging to the genus Actinomyces or Russian genus that originally exist in the oral cavity reduce nitric acid to produce nitrous acid. Nitrous acid is reduced by a weakly acidic environment in the oral cavity and produces nitric oxide (NO), so that periodontal pathogens are sterilized. It is also possible to treat human periodontal disease by injecting an aqueous nitrate solution under gingivitis.
It is also effective to administer a nitrate aqueous solution into the oral cavity for the purpose of treating or preventing periodontal disease. For example, a nitric acid concentration (5 mM) that is about the same as vegetable juice has a sufficient bactericidal effect on periodontal pathogens. In addition, excessively ingested nitrate ions are excreted as urine, and nitrous acid and nitric oxide, which are harmful to the human body, do not become excessive.
ここで、硝酸塩水溶液は、アクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌が混入されているのが好ましい。口腔内に元々存在するアクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌の量を増加して、硝酸還元能を高めるためである。
混入されている細菌は、具体的には、Actinomyces oris MG-1菌株、Actinomyces naeslundii ATCC12104菌株、Rothia aeria JCM11412菌株、Rothia mucilaginosa DY-18菌株、Rothia dentocariosa ATCC17931菌株から選ばれる菌株から得られる。
また、本発明の歯周病治療方法において、硝酸塩水溶液のｐＨを５．５〜６．５の範囲に調製することが好ましい。弱酸性環境に保つことにより、亜硝酸が還元されやすく、一酸化窒素（ＮＯ）が生成される量が多くなるからである。また、本発明の歯周病治療方法において、硝酸塩水溶液の濃度が２〜１０ｍＭであること好ましい。
Here, the nitrate aqueous solution is preferably mixed with bacteria belonging to the genus Actinomyces or the genus Rothia. This is to increase the nitrate reduction ability by increasing the amount of bacteria belonging to the genus Actinomyces or Rothia originally present in the oral cavity.
Specifically, the contaminated bacteria are obtained from a strain selected from Actinomyces oris MG-1 strain, Actinomyces naeslundii ATCC12104 strain, Rothia aeria JCM11412 strain, Rothia mucilaginosa DY-18 strain, and Rothia dentocariosa ATCC17931 strain.
Moreover, in the periodontal disease treatment method of this invention, it is preferable to adjust pH of nitrate aqueous solution to the range of 5.5-6.5. This is because maintaining a weakly acidic environment facilitates reduction of nitrous acid and increases the amount of nitric oxide (NO) produced. Moreover, in the periodontal disease treatment method of this invention, it is preferable that the density | concentration of nitrate aqueous solution is 2-10 mM.
本発明の口腔用組成物によれば、歯周病原菌を硝酸依存的に殺菌することができ、また特異的に歯周病原菌を殺菌でき、歯周病の予防や治療が期待できる。 According to the composition for oral cavity of the present invention, periodontopathic bacteria can be sterilized in a nitrate-dependent manner, and periodontopathic bacteria can be specifically sterilized, and prevention and treatment of periodontal disease can be expected.
以下、本発明の実施形態の一例を、図面を参照しながら詳細に説明していく。なお、本発明の範囲は、以下の実施例や図示例に限定されるものではなく、幾多の変更及び変形が可能である。 Hereinafter, an example of an embodiment of the present invention will be described in detail with reference to the drawings. The scope of the present invention is not limited to the following examples and illustrated examples, and many changes and modifications can be made.
まず、本発明者らが見出した歯周病原菌の殺菌の仕組みについて説明する。
図１は、アクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌が、硝酸銀存在下で、歯周病原菌を殺菌する仕組みを示している。アクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌が善玉菌として働き、硝酸イオン（ＮＯ３ −）を亜硝酸イオン（ＮＯ２ −）に還元する。弱酸性環境下では亜硝酸イオン（ＮＯ２ −）から一酸化窒素（ＮＯ）への還元過程が自発的に起きる。歯周病原菌（P. intermediaなど）は、一酸化窒素（ＮＯ）に非常に弱い細菌であるため、一酸化窒素によって歯周病原菌を特異的に殺菌できるのである。
First, the mechanism of sterilization of periodontal pathogens found by the present inventors will be described.
FIG. 1 shows a mechanism by which bacteria belonging to the genus Actinomyces or the genus Rothia kill periodontal pathogens in the presence of silver nitrate. Bacteria belonging to the genus Actinomyces (Actinomyces) or Russia (Rothia) act as good bacteria and reduce nitrate ions (NO 3 − ) to nitrite ions (NO 2 − ). In a weakly acidic environment, a reduction process from nitrite ions (NO 2 − ) to nitric oxide (NO) occurs spontaneously. Periodontal pathogens (such as P. intermedia) are bacteria that are very sensitive to nitric oxide (NO), and therefore nitric oxide can specifically kill periodontal pathogens.
次に、歯周病原菌を殺菌する仕組みを検証する方法について、図２を参照して説明する。
図２に示すように、歯周病原菌（P. intermediaなど）と、善玉菌であるアクチノマイセス（Actinomyces）属またはロシア（Rothia）属の細菌を共培養する。共培養は、硝酸塩（硝酸イオン）を添加する場合と非添加の場合の２通りの条件で行い、それぞれ３７℃、嫌気条件で数分から数時間振盪培養する。培養開始時および培養中における生菌数の算出は、段階希釈した培養液を寒天培地上へ塗抹して、生じたコロニー数からコロニー形成単位（ＣＦＵ：Colony Forming Unit）を求めることで行う。
Next, a method for verifying a mechanism for sterilizing periodontal pathogens will be described with reference to FIG.
As shown in FIG. 2, periodontopathic bacteria (such as P. intermedia) and bacteria of the genus Actinomyces or Russian genus which are good bacteria are co-cultured. The co-culture is performed under the two conditions of adding and not adding nitrate (nitrate ions), and each is cultured with shaking at 37 ° C. and anaerobic conditions for several minutes to several hours. The number of viable cells at the start of the culture and during the culture is calculated by smearing the serially diluted culture solution on the agar medium and obtaining a colony forming unit (CFU) from the number of colonies formed.
後述する実施例で使用した菌株、実験方法、共培養と共存率の算出方法について、以下に説明する。
＜アクチノマイセス（Actinomyces）属の口腔内細菌＞
・Actinomyces odontolyticus ATCC17929（以下、A. odontolyticus）
・Actinomyces oris MG-1（以下、A. oris）
・Actinomyces oris TN9011 (narG)
・Actinomyces oris TN9012 (nirK)
・Actinomyces naeslundii ATCC12104（以下、A. naeslundii）
＜ロシア（Rothia）属の口腔内細菌＞
・Rothia aeria JCM11412（以下、R. aeria）
・Rothia mucilaginosa DY-18（以下、R. mucilaginosa）
・Rothia dentocariosa ATCC17931（以下、R. dentocariosa）
＜歯周病原菌＞
・Porphyromonas gingivalis ATCC33277（以下、P. gingivalis）
・Prevotella intermedia ATCC25611（以下、P. intermedia）
The strains used in the examples described later, experimental methods, and methods for calculating co-culture and coexistence rates are described below.
<Oral bacteria of the genus Actinomyces>
・ Actinomyces odontolyticus ATCC17929 (hereinafter A. odontolyticus)
・ Actinomyces oris MG-1 (hereinafter A. oris)
・ Actinomyces oris TN9011 (narG)
・ Actinomyces oris TN9012 (nirK)
・ Actinomyces naeslundii ATCC12104 (hereinafter A. naeslundii)
<Oral bacteria of the Russian genus>
・ Rothia aeria JCM11412 (hereinafter R. aeria)
・ Rothia mucilaginosa DY-18 (hereinafter R. mucilaginosa)
・ Rothia dentocariosa ATCC17931 (hereinafter R. dentocariosa)
<Periodontal pathogen>
・ Porphyromonas gingivalis ATCC33277 (hereinafter P. gingivalis)
・ Prevotella intermedia ATCC25611 (P. intermedia)
（方法、共培養と共存率の算出方法について）
R. mucilaginosa，R. aeria，R. dentocariosa， A. naeslundiiは、ハートインフュージョン培地（ＨＩＢ：Heart Infusion Broth，Ｄｉｆｃｏ社製）に接種し、３７℃、好気条件で一晩静置培養した。また、A. orisは、ＨＩＢに接種し，３７℃、好気条件で一晩振盪培養した。
また、歯周病原菌のP. gingivalis，P. intermediaは、変法ＧＡＭ培地（ニッスイ社製）で一晩嫌気培養した。
培養後の細菌細胞は嫌気培養装置内での遠心操作によりＨＩＢで２回洗浄した。ＨＩＢに懸濁したロシア（Rothia）属の細菌あるいはアクチノマイセス（Actinomyces）属の細菌を２００、歯周病原菌のP. gingivalisあるいはP. intermediaを１の容量比で混合した。
(How to calculate the co-culture and coexistence rate)
R. mucilaginosa, R. aeria, R. dentocariosa, A. naeslundii were inoculated into a heart infusion medium (HIB: Heart Infusion Broth, manufactured by Difco) and statically cultured overnight at 37 ° C. under aerobic conditions. A. oris was inoculated into HIB and cultured overnight at 37 ° C. under aerobic conditions.
Periodontal pathogens P. gingivalis and P. intermedia were anaerobically cultured overnight in modified GAM medium (Nissui).
The cultured bacterial cells were washed twice with HIB by centrifugation in an anaerobic culture apparatus. 200 Russian rotogen or Actinomyces bacterium suspended in HIB and P. gingivalis or P. intermedia of periodontal pathogen were mixed at a volume ratio of 1.
必要に応じて、終濃度０．１Ｍのリン酸ナトリウム緩衝液（和光純薬工業製）でｐＨを７．２，６．５，６．０に調整したＨＩＢ、終濃度０．１Ｍのクエン酸ナトリウム緩衝液（和光純薬工業製）でｐＨを５．５に調整したＨＩＢを用いた。
また、必要に応じて硝酸ナトリウム（和光純薬工業製），硝酸カリウム（和光純薬工業製），ＰＴＩＯ（2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl（東京化成工業社製）を加えた。
HIB adjusted to pH 7.2, 6.5, 6.0 with 0.1 M sodium phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.) with final concentration of 0.1 M, citric acid with final concentration of 0.1 M HIB whose pH was adjusted to 5.5 with sodium buffer (manufactured by Wako Pure Chemical Industries, Ltd.) was used.
If necessary, sodium nitrate (manufactured by Wako Pure Chemical Industries), potassium nitrate (manufactured by Wako Pure Chemical Industries), PTIO (2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (Tokyo) Kasei Kogyo Co., Ltd.) was added.
なお、ＰＴＩＯを用いる際は、ＨＩＢではなくリン酸緩衝生理食塩水（ＰＢＳ，タカラバイオ社製）で洗浄した細菌細胞を用いた。それぞれの共培養液は、３７℃嫌気条件で数分から数時間振盪培養した。培養開始時、培養中における生菌数の算出は、段階希釈した培養液を寒天培地上へ塗抹し、生じたコロニー数からコロニー形成単位（ＣＦＵ）を求めることで行った。
歯周病原菌（P. gingivalis，P. intermedia）のＣＦＵの算出は、段階希釈液を２５ｍｇ／Ｌのゲンタマイシン（和光純薬工業製）を添加した変法ＧＡＭ培地へ塗抹して、嫌気培養することにより求めた。また、ロシア（Rothia）属の細菌あるいはアクチノマイセス（Actinomyces）属の細菌のＣＦＵの算出は、段階希釈液をＨＩＢ寒天培地へ塗抹して好気培養することにより求めた。
共培養開始時（０時間）のそれぞれの菌種のＣＦＵを１００％として、経時的に得たＣＦＵから生存率（％）を求めた。なお、全ての実験は独立した３系統で行っており、平均と標準誤差を求めて図に示すグラフにプロットした。
When PTIO was used, bacterial cells washed with phosphate buffered saline (PBS, manufactured by Takara Bio Inc.) instead of HIB were used. Each of the co-cultures was cultured with shaking under anaerobic conditions at 37 ° C. for several minutes to several hours. At the start of the culture, the number of viable cells during the culture was calculated by smearing a serially diluted culture solution on an agar medium and obtaining a colony forming unit (CFU) from the number of colonies generated.
CFU of periodontopathic bacteria (P. gingivalis, P. intermedia) is calculated by coating the serially diluted solution on a modified GAM medium supplemented with 25 mg / L gentamicin (manufactured by Wako Pure Chemical Industries, Ltd.) and anaerobic culture. Determined by Further, the CFU of the Russian genus or the Actinomyces genus was calculated by smearing the serially diluted solution on the HIB agar medium and aerobic culture.
The survival rate (%) was determined from the CFU obtained over time, with the CFU of each bacterial species at the start of co-culture (0 hour) as 100%. All experiments were performed in three independent systems, and the average and standard error were obtained and plotted on the graph shown in the figure.
ロシア（Rothia）属のR. mucilaginosaによる歯周病原菌（P. gingivalis）の硝酸依存的な殺菌効果について調べた結果を説明する。
まず、R. mucilaginosaとP. gingivalisをＨＩＢに懸濁し、３７℃で３時間インキュベートした。培養は、終濃度１０ｍＭとなるよう硝酸ナトリウム（ＮａＮＯ３）を添加した場合（＋）と非添加の場合（−）で行い、培養開始時のP. gingivalisのＣＦＵ（３．４×１０７）を１００％とし、経時的に測定したＣＦＵからP. gingivalisの生存率を求めた。
The results of a study on the nitrate-dependent bactericidal effect of periodontopathic bacteria (P. gingivalis) by R. mucilaginosa of the genus Russian (Rothia) will be explained.
First, R. mucilaginosa and P. gingivalis were suspended in HIB and incubated at 37 ° C. for 3 hours. Culturing is performed with sodium nitrate (NaNO 3 ) added to a final concentration of 10 mM (+) and without (−), and P. gingivalis CFU at the start of the culture (3.4 × 10 7 ). The survival rate of P. gingivalis was determined from the CFU measured over time.
R. mucilaginosaとP. gingivalisを共培養した際のP. gingivalisの生存率を図３に示す。
P. gingivalisの生存率は、硝酸ナトリウム（ＮａＮＯ３）非添加条件では時間経過に関わらずほぼ１００％で推移したのに対して、硝酸ナトリウム（ＮａＮＯ３）添加条件では、経時的に著しい生存率の低下がみられ、３時間後では約９５％のP. gingivalisが殺菌されていた。
一方、培養開始時のR. mucilaginosaのＣＦＵ（２．８×１０７）を１００％とし、培養３時間後のＣＦＵからR. mucilaginosaの生存率を求めた。R. mucilaginosaの生存率を図４に示す。R. mucilaginosaは、P. gingivalisと異なり、硝酸ナトリウムの添加、非添加の条件に関わらず、生存率の低下はみられなかった。
図３および図４に示す生存率の結果から、P. gingivalisとR. mucilaginosaを共培養した場合、硝酸ナトリウム依存的にP. gingivalisの殺菌がみられることが理解できる。
FIG. 3 shows the survival rate of P. gingivalis when R. mucilaginosa and P. gingivalis were co-cultured.
The survival rate of P. gingivalis was almost 100% regardless of the passage of time when sodium nitrate (NaNO 3 ) was not added, whereas the survival rate was remarkable over time when sodium nitrate (NaNO 3 ) was added. After about 3 hours, about 95% of P. gingivalis was sterilized.
On the other hand, the CFU (2.8 × 10 7 ) of R. mucilaginosa at the start of the culture was taken as 100%, and the survival rate of R. mucilaginosa was determined from the CFU after 3 hours of culture. The survival rate of R. mucilaginosa is shown in FIG. Unlike P. gingivalis, R. mucilaginosa did not show a decrease in survival rate regardless of whether or not sodium nitrate was added.
From the results of the survival rate shown in FIG. 3 and FIG. 4, it can be understood that when P. gingivalis and R. mucilaginosa are co-cultured, sterilization of P. gingivalis is observed depending on sodium nitrate.
また、P. gingivalis単独培養時の生存率を確認するため、P. gingivalisをＨＩＢに懸濁し、３７℃で３時間インキュベートした。培養は、終濃度１０ｍＭとなるよう硝酸ナトリウム（ＮａＮＯ３）を添加した場合（＋）と非添加の場合（−）で行い、培養開始時のP. gingivalisのＣＦＵ（２．０×１０７）を１００％とし、培養３時間後のＣＦＵから生存率を求めた。P. gingivalis単独培養時の生存率を図５に示す。
図５から、培養液にR. mucilaginosa を加えず、P. gingivalis単独での培養を行った場合、硝酸依存的な殺菌は見られなかった。
In order to confirm the survival rate when P. gingivalis was cultured alone, P. gingivalis was suspended in HIB and incubated at 37 ° C. for 3 hours. Culturing is performed with sodium nitrate (NaNO 3 ) added to a final concentration of 10 mM (+) and without (−), and P. gingivalis CFU at the start of culture (2.0 × 10 7 ). The survival rate was calculated from CFU after 3 hours of culture. The survival rate when P. gingivalis is cultured alone is shown in FIG.
From FIG. 5, when R. mucilaginosa was not added to the culture solution and P. gingivalis was cultured alone, nitrate-dependent sterilization was not observed.
次に、硝酸カリウム（ＫＮＯ３）添加時のP. gingivalisの生存率を調べた。
R. mucilaginosaとP. gingivalisをＨＩＢに懸濁し、３７℃で３時間インキュベートした。培養は、終濃度１０ｍＭとなるよう硝酸カリウムを添加した場合（＋）と非添加の場合（−）で行い、培養開始時のP. gingivalisのＣＦＵ（３．８×１０７）を１００％とし、培養３時間後のＣＦＵからP. gingivalisの生存率を求めた。
Next, the survival rate of P. gingivalis when potassium nitrate (KNO 3 ) was added was examined.
R. mucilaginosa and P. gingivalis were suspended in HIB and incubated at 37 ° C. for 3 hours. Cultivation is performed with potassium nitrate added to a final concentration of 10 mM (+) and with no potassium nitrate added (−), and P. gingivalis CFU (3.8 × 10 7 ) at the start of the culture is taken as 100%. The survival rate of P. gingivalis was determined from CFU after 3 hours of culture.
硝酸カリウム（ＫＮＯ３）添加時のP. gingivalisの生存率を図６に示す。硝酸カリウム（ＫＮＯ３）添加時も、硝酸ナトリウム（ＮａＮＯ３）の場合と同様に、経時的に著しい生存率の低下がみられ、３時間後では約９０％のP. gingivalisが殺菌されていた。
一方、培養開始時のR. mucilaginosaのＣＦＵ（３．０×１０７）を１００％とし、培養３時間後のＣＦＵからR. mucilaginosaの生存率を求めたが、硝酸カリウムの添加、非添加の条件に関わらず、生存率の低下はみられなかった。
FIG. 6 shows the survival rate of P. gingivalis upon addition of potassium nitrate (KNO 3 ). When potassium nitrate (KNO 3 ) was added, as in the case of sodium nitrate (NaNO 3 ), the survival rate decreased significantly with time, and about 90% of P. gingivalis was sterilized after 3 hours.
On the other hand, the survival rate of R. mucilaginosa was determined from the CFU after 3 hours of culture, with the CFU of R. mucilaginosa at the start of culture being 100% (3.0 × 10 7 ). Regardless, there was no decline in survival.
すなわち、硝酸カリウムを用いて共培養した場合でも、硝酸ナトリウムを用いた場合と同様の結果が得られた。実施例１および実施例２の結果から、R. mucilaginosaとP. gingivalisの共培養系でのP. gingivalisの殺菌は、R. mucilaginosaと硝酸イオン依存的であることが確認できた。 That is, even when co-cultured with potassium nitrate, the same results as when sodium nitrate was used were obtained. From the results of Example 1 and Example 2, it was confirmed that the sterilization of P. gingivalis in the co-culture system of R. mucilaginosa and P. gingivalis was dependent on R. mucilaginosa and nitrate ions.
R. mucilaginosaとP. gingivalisの共培養系でみられたP. gingivalisの殺菌は、R. mucilaginosaが硝酸イオンを還元した際に生じる一酸化窒素が原因となっている可能性があり、これを調べるために、共培養系に一酸化窒素スカベンジャー（捕捉剤）を添加して、硝酸イオン依存的な殺菌が抑圧されるか確認した。
実験は、ＰＢＳで洗浄したR. mucilaginosaとP. gingivalisをＨＩＢに懸濁し、３７℃、４時間でインキュベートした。培養は、終濃度１０ｍＭとなるよう硝酸ナトリウム（ＮａＮＯ３）を添加した場合（＋）と非添加の場合（−）、また、終濃度１ｍＭとなるようＰＴＩＯを添加した場合（＋）と非添加の場合（−）で行った。
培養開始時のP. gingivalisのＣＦＵ（２．０×１０７）を１００％とし、経時的に測定したＣＦＵから生存率を求めた。また、培養開始時のR. mucilaginosaのＣＦＵ（２．１×１０７）を１００％とし、経時的に測定したＣＦＵから生存率を求めた。
The sterilization of P. gingivalis observed in the co-culture system of R. mucilaginosa and P. gingivalis may be caused by nitric oxide produced when R. mucilaginosa reduces nitrate ions. In order to investigate, a nitric oxide scavenger (scavenger) was added to the co-culture system to confirm whether nitrate ion-dependent sterilization was suppressed.
In the experiment, R. mucilaginosa and P. gingivalis washed with PBS were suspended in HIB and incubated at 37 ° C. for 4 hours. In the culture, sodium nitrate (NaNO 3 ) is added to a final concentration of 10 mM (+) and not added (−), and PTIO is added to a final concentration of 1 mM (+) and not added. In the case of (-).
The PFU gingivalis CFU (2.0 × 10 7 ) at the start of the culture was taken as 100%, and the survival rate was determined from the CFU measured over time. In addition, R. mucilaginosa CFU (2.1 × 10 7 ) at the start of culture was defined as 100%, and the survival rate was determined from CFU measured over time.
結果を図７に示す。R. mucilaginosaとP. gingivalisの共培養系に一酸化窒素スカベンジャー（捕捉剤）であるＰＴＩＯを加えた際のP. gingivalisの生存率の変化を調べた。硝酸ナトリウム非添加の場合、培養液へのＰＴＩＯの添加の有無に関わらず、培養４時間後のP. gingivalisの生存率はほぼ１００％であった。
一方、硝酸ナトリウムを添加した場合、ＰＴＩＯ非添加では著しい生存率の減少がみられたが、ＰＴＩＯ添加によって生存率の減少の程度が抑圧されたことが確認できた。このことから、P. gingivalisの殺菌は、R. mucilaginosaによる硝酸イオン還元の結果生じた一酸化窒素によるものであると理解できる。
The results are shown in FIG. The change in the survival rate of P. gingivalis when PTIO, a nitric oxide scavenger (capture agent), was added to the co-culture system of R. mucilaginosa and P. gingivalis. When sodium nitrate was not added, the survival rate of P. gingivalis after 4 hours of culture was almost 100% regardless of whether PTIO was added to the culture medium.
On the other hand, when sodium nitrate was added, a significant decrease in the survival rate was observed when PTIO was not added, but it was confirmed that the decrease in the survival rate was suppressed by the addition of PTIO. From this, it can be understood that the sterilization of P. gingivalis is caused by nitric oxide produced as a result of nitrate ion reduction by R. mucilaginosa.
硝酸還元により生じた亜硝酸イオンは、酸性側ｐＨ環境下でその一部が自発的に一酸化窒素に変換する。従って、一酸化窒素が殺菌効果に関与しているのであれば、ｐＨ環境の違いでP. gingivalisの生存率が異なるはずであり、それを調べた。
R. mucilaginosaとP. gingivalisをリン酸ナトリウム緩衝液（終濃度０．１Ｍ）でｐＨ７．２，６．５，６．０に調整したＨＩＢに懸濁し、３７℃で２時間インキュベートした。また、これらの菌をクエン酸ナトリウム緩衝液（終濃度０．１Ｍ）でｐＨ５．５に調整したＨＩＢにも懸濁し、３７℃で１時間インキュベートした。培養は、終濃度１０ｍＭの硝酸ナトリウムを添加した場合（＋）と非添加の場合（−）で行った。培養開始時のP. gingivalisのＣＦＵ（ｐＨ７．２とｐＨ６．５では２．３×１０７、ｐＨ６．０では２．６×１０７、ｐＨ５．５では４．４×１０７）を１００％とし、経時的に測定したＣＦＵから生存率を求めた。また、培養開始時のR. mucilaginosaのＣＦＵ（ｐＨ７．２とｐＨ６．５では２．０×１０７、ｐＨ６．０では２．７×１０７、ｐＨ５．５では３．０×１０７）を１００％とし、経時的に測定したＣＦＵから生存率を求めた。
A part of the nitrite ions generated by the reduction of nitric acid is spontaneously converted into nitric oxide under an acidic pH environment. Therefore, if nitric oxide was involved in the bactericidal effect, the survival rate of P. gingivalis should be different depending on the pH environment, and this was investigated.
R. mucilaginosa and P. gingivalis were suspended in HIB adjusted to pH 7.2, 6.5, 6.0 with sodium phosphate buffer (final concentration 0.1 M) and incubated at 37 ° C. for 2 hours. These bacteria were also suspended in HIB adjusted to pH 5.5 with a sodium citrate buffer (final concentration 0.1 M) and incubated at 37 ° C. for 1 hour. Incubation was performed with (+) and (-) not adding sodium nitrate having a final concentration of 10 mM. 100% of C. of P. gingivalis at the start of culture (2.3 × 10 7 at pH 7.2 and pH 6.5, 2.6 × 10 7 at pH 6.0, 4.4 × 10 7 at pH 5.5) The survival rate was determined from the CFU measured over time. In addition, CFU of R. mucilaginosa at the start of culture (2.0 × 10 7 at pH 7.2 and pH 6.5, 2.7 × 10 7 at pH 6.0, 3.0 × 10 7 at pH 5.5) The survival rate was determined from the CFU measured over time with 100%.
結果を図８に示す。R. mucilaginosaとP. gingivalisを異なるｐＨに調整されたＨＩＢに懸濁し、３７℃でインキュベートした。培養２時間後のP. gingivalisの生存率を求めたところ、ｐＨ７．２とｐＨ６．５に調製した培養液を用いた場合では、硝酸ナトリウム添加時においてもほとんど生存率の低下はみられなかった。一方、ｐＨ６．０の培養液を用いた場合，生存率が１％以下に減少した。さらに、ｐＨ５．５の培養液を用いた場合、１時間で生存率が１％以下に減少した。このことから、中性より酸性側ｐＨの方が、P. gingivalisの殺菌効果が高いことがわかった。 The results are shown in FIG. R. mucilaginosa and P. gingivalis were suspended in HIB adjusted to different pH and incubated at 37 ° C. When the viability of P. gingivalis after 2 hours of culture was determined, there was almost no decrease in the viability even when sodium nitrate was added when using culture solutions prepared at pH 7.2 and pH 6.5. . On the other hand, when the culture solution with pH 6.0 was used, the survival rate decreased to 1% or less. Furthermore, when a culture solution having a pH of 5.5 was used, the survival rate decreased to 1% or less in 1 hour. From this, it was found that acidic pH was higher than P. gingivalis than neutral.
硝酸イオン濃度と殺菌効果を調べた。R. mucilaginosaとP. gingivalisをリン酸ナトリウム緩衝液（終濃度０．１Ｍ）でｐＨ６．０に調整したＨＩＢに懸濁し、３７℃で２時間インキュベートした。培養は、終濃度１０ｍＭの硝酸ナトリウムを添加した場合（＋）と非添加の場合（−）で行った。培養開始時のP. gingivalisのＣＦＵ（１．６×１０７）を１００％とし、経時的に測定したＣＦＵから生存率を求めた。なお、培養開始時のR. mucilaginosaのＣＦＵは、２．０×１０７であったが、生存率の変化はなかった。 Nitrate ion concentration and bactericidal effect were investigated. R. mucilaginosa and P. gingivalis were suspended in HIB adjusted to pH 6.0 with sodium phosphate buffer (final concentration 0.1 M) and incubated at 37 ° C. for 2 hours. Incubation was performed with (+) and (-) not adding sodium nitrate having a final concentration of 10 mM. P. gingivalis CFU (1.6 × 10 7 ) at the start of the culture was taken as 100%, and the survival rate was determined from the CFU measured over time. The CFU of R. mucilaginosa at the start of the culture was 2.0 × 10 7 , but there was no change in the survival rate.
結果を図９に示す。R. mucilaginosaとP. gingivalisを異なる硝酸ナトリウム濃度に調整されたＨＩＢに懸濁し、３７℃でインキュベートした。培養２時間後のP. gingivalisの生存率を求めたところ、硝酸ナトリウムの濃度依存的にP. gingivalisの生存率の減少が確認された。 The results are shown in FIG. R. mucilaginosa and P. gingivalis were suspended in HIB adjusted to different sodium nitrate concentrations and incubated at 37 ° C. When the survival rate of P. gingivalis after 2 hours of culture was determined, a decrease in the survival rate of P. gingivalis was confirmed depending on the concentration of sodium nitrate.
次に、R. mucilaginosa以外にも硝酸還元活性を持つとされるActinomycesでも硝酸イオン依存的にP. gingivalisを殺菌できるか調べた。具体的には、硝酸還元酵素をコードする遺伝子（ｎａｒＧ）、亜硝酸還元酵素をコードする遺伝子（ｎｉｒＫ）の欠損株（亜硝酸還元酵素の欠損株）において、P. gingivalisの殺菌効果が消失するか確認した。
A. oris（ＭＧ−１）、その硝酸還元酵素欠損株（ＴＮ９０１１，ｎａｒＧ）、亜硝酸還元酵素欠損株（ＴＮ９０１２，ｎｉｒＫ）のいずれかと、P. gingivalisをリン酸ナトリウム緩衝液（終濃度０．１Ｍ）で、ｐＨ６．０に調整したＨＩＢに懸濁し、３７℃で８時間インキュベートした。培養は、終濃度１０ｍＭの硝酸ナトリウムを添加した場合（＋）と非添加の場合（−）で行った。培養開始時のP. gingivalisのＣＦＵ（ＭＧ−１，ＴＮ９０１１，ＴＮ９０１２との培養では、それぞれ４．１×１０７，４．５×１０７，４．４×１０７）を１００％とし、経時的に測定したＣＦＵから生存率を求めた。
Next, in addition to R. mucilaginosa, it was investigated whether Actinomyces, which is said to have nitrate reduction activity, could kill P. gingivalis in a nitrate ion-dependent manner. Specifically, the bactericidal effect of P. gingivalis disappears in a gene (narG) encoding nitrate reductase and a strain (nirK) deficient in a gene encoding nitrite reductase (nitrite reductase deficient strain). I confirmed.
A. oris (MG-1), its nitrate reductase-deficient strain (TN9011, narG), nitrite reductase-deficient strain (TN9012, irK), and P. gingivalis in sodium phosphate buffer (final concentration 0. 1M) and suspended in HIB adjusted to pH 6.0 and incubated at 37 ° C. for 8 hours. Incubation was performed with (+) and (-) not adding sodium nitrate having a final concentration of 10 mM. The CFU of P. gingivalis at the beginning of the culture (4.1 × 10 7 , 4.5 × 10 7 and 4.4 × 10 7 in the culture with MG-1, TN9011, and TN9012, respectively) was taken as 100%, The survival rate was determined from the CFU measured automatically.
結果を図１０、図１１に示す。図１０に示すように、A. orisとP. gingivalisを共培養したところ、P. gingivalisの生存率は硝酸依存的に著しく減少した。しかし、硝酸還元酵素ｎａｒＧ欠損株ＴＮ９０１１を添加した場合では、P. gingivalisの生存率が減少しておらず、硝酸依存的殺菌効果が消失したことを確認できた。このことは硝酸イオンの還元反応の結果生じた一酸化窒素が、P. gingivalisを殺菌しているということと矛盾しない。
一方、亜硝酸還元酵素ｎｉｒＫ欠損株ＴＮ９０１２を添加した場合では、P. gingivalisの生存率は著しく減少し、硝酸依存的殺菌効果は消失しなかったことを確認できた。これは、酸性ｐＨ環境では亜硝酸から一酸化窒素への還元過程が自発的に起こることから説明できる。
なお、図１１に示すように、A. orisとP. gingivalisを共培養において、A. orisの生存率はいずれの場合も変化しなかった。
The results are shown in FIGS. As shown in FIG. 10, when A. oris and P. gingivalis were co-cultured, the survival rate of P. gingivalis significantly decreased in a nitrate-dependent manner. However, when the nitrate reductase narG deficient strain TN9011 was added, the survival rate of P. gingivalis was not decreased, and it was confirmed that the nitrate-dependent bactericidal effect was lost. This is consistent with the fact that nitric oxide resulting from the reduction reaction of nitrate ions sterilizes P. gingivalis.
On the other hand, when the nitrite reductase irK-deficient strain TN9012 was added, the survival rate of P. gingivalis was remarkably reduced, and it was confirmed that the nitrate-dependent bactericidal effect was not lost. This can be explained by the spontaneous reduction of nitrous acid to nitric oxide in an acidic pH environment.
In addition, as shown in FIG. 11, when A. oris and P. gingivalis were cocultured, the survival rate of A. oris did not change in any case.
上述の実施例で、R. mucilaginosaやA. orisが、硝酸依存的にP. gingivalisを殺菌することを示したが、これら以外の口腔に存在するRothia属の細菌またはActinomyces属の細菌も、硝酸依存的にP. gingivalisを殺菌することができるか調べた。
具体的には、口腔内のRothia属の細菌（R. dentocariosa，R. aeria）、あるいは口腔内のActinomyces属の細菌（A. naeslundii）のいずれかと、P. gingivalisを共培養した際のP. gingivalisの生存率を調べた。培養開始時のP. gingivalisのＣＦＵを１００％とし、３時間後のＣＦＵとの比較から生存率を求めた。培養は、終濃度０．１Ｍのリン酸ナトリウム緩衝液でｐＨを６．０に調整したＨＩＢを用い、終濃度１０ｍＭの硝酸ナトリウムを添加した場合（＋）と非添加の場合（−）で行った。培養開始時の培養液には、２．７×１０７ＣＦＵのP. gingivalisと１．０×１０９ＣＦＵのR. dentocariosa，２．９×１０７ＣＦＵのP. gingivalisと１．３×１０８ＣＦＵのR. aeria，２．８×１０７ＣＦＵのP. gingivalisと１．０×１０８ＣＦＵのA. naeslundiiが含まれていた。
In the above examples, it was shown that R. mucilaginosa and A. oris sterilize P. gingivalis in a nitrate-dependent manner, but other bacteria in the genus Rothia or Actinomyces also exist in the oral cavity. It was investigated whether P. gingivalis could be sterilized in a dependent manner.
Specifically, when P. gingivalis is co-cultured with either an oral Rothia bacterium (R. dentocariosa, R. aeria) or an oral Actinomyces bacterium (A. naeslundii). The survival rate of gingivalis was examined. P. gingivalis CFU at the start of culture was taken as 100%, and the survival rate was determined from comparison with CFU after 3 hours. Cultivation was performed using HIB adjusted to pH 6.0 with a final concentration of 0.1 M sodium phosphate buffer, with or without the addition of 10 mM sodium nitrate (−). It was. The culture solution at the start of the culture was 2.7 × 10 7 CFU of P. gingivalis and 1.0 × 10 9 CFU of R. dentocariosa, 2.9 × 10 7 CFU of P. gingivalis and 1.3 × 10. 8 CFU R. aeria, 2.8 × 10 7 CFU P. gingivalis and 1.0 × 10 8 CFU A. naeslundii.
結果を図１２に示す。図１２のグラフの横軸において、ＲｄはR. dentocariosa、ＲａはR. aeria、ＡｎはA. naeslundiiを表す。
P. gingivalisと口腔内のRothia属の細菌（R. aeria，R. dentocariosa）あるいは口腔内のActinomyces属の細菌（A. naeslundii）とを共培養したところ、硝酸ナトリウム依存的にP. gingivalisの生存率の低下が確認できた。このことから、上述の実施例で示されたR. mucilaginosaやA. orisにおよる硝酸依存的なP. gingivalisの殺菌は、口腔内の主要なRothia属の細菌やActinomyces属の細菌でもみられる共通の特徴であることがわかった。すなわち、硝酸依存的殺菌効果は、Actinomyces属，Rothia属細菌で一般化できることが確認できた。
The results are shown in FIG. In the horizontal axis of the graph of FIG. 12, Rd represents R. dentocariosa, Ra represents R. aeria, and An represents A. naeslundii.
When P. gingivalis was co-cultured with bacteria of the genus Rothia (R. aeria, R. dentocariosa) or bacteria of the genus Actinomyces (A. naeslundii), the survival of P. gingivalis was dependent on sodium nitrate. A decrease in rate was confirmed. Therefore, the nitrate-dependent killing of P. gingivalis by R. mucilaginosa and A. oris shown in the above examples is common to the bacteria of the genus Rothia and Actinomyces in the oral cavity. It turned out to be a feature of In other words, it was confirmed that the nitrate-dependent bactericidal effect can be generalized with bacteria belonging to the genus Actinomyces and Rothia.
次に、P. gingivalisと並び、歯周病原菌として知られるP. intermediaにおいても、R. mucilaginosaとの共培養で硝酸ナトリウム依存的に殺菌されるかを調べた。
R. mucilaginosaとP. intermediaをリン酸ナトリウム緩衝液（終濃度０．１Ｍ）でｐＨ６．０に調整したＨＩＢに懸濁し、３７℃で２時間インキュベートした。培養は、終濃度１０ｍＭの硝酸ナトリウムを添加した場合（＋）と非添加の場合（−）で行い、培養開始時のP. intermediaのＣＦＵ（２．１×１０７）を１００％とし、経時的に測定したＣＦＵから生存率を求めた。なお、培養開始時のR. mucilaginosaのＣＦＵは、４．１×１０７であったが、生存率の変化はなかった。
Next, P. gingivalis and P. intermedia, known as a periodontal pathogen, were examined to be killed in a sodium nitrate-dependent manner by co-culture with R. mucilaginosa.
R. mucilaginosa and P. intermedia were suspended in HIB adjusted to pH 6.0 with sodium phosphate buffer (final concentration 0.1 M) and incubated at 37 ° C. for 2 hours. Cultivation is performed with (+) and without (-) the addition of sodium nitrate at a final concentration of 10 mM, and the P. intermedia CFU (2.1 × 10 7 ) at the start of the culture is taken as 100%. The survival rate was determined from the CFU measured automatically. The CFU of R. mucilaginosa at the start of culture was 4.1 × 10 7 , but there was no change in the survival rate.
結果を図１３に示す。R. mucilaginosaとP. intermediaを共培養したところ、硝酸ナトリウム存在下において著しいP. intermediaの生存率の減少が確認できた。R. mucilaginosaによる硝酸ナトリウム依存的殺菌効果は、歯周病原菌のP. gingivalisだけではなく、P. intermediaにも有効であることがわかった。 The results are shown in FIG. When R. mucilaginosa and P. intermedia were co-cultured, a significant decrease in the survival rate of P. intermedia was confirmed in the presence of sodium nitrate. The sodium nitrate-dependent bactericidal effect of R. mucilaginosa was found to be effective not only for P. gingivalis, a periodontal pathogen, but also for P. intermedia.
本発明は、歯周病菌殺菌剤、歯周病菌予防剤または歯周病菌治療剤として、歯磨剤、サプリメント、食品（タブレット、ジュース）に有用である。
INDUSTRIAL APPLICABILITY The present invention is useful for dentifrices, supplements, and foods (tablets and juices) as a periodontal fungicide, a preventive agent for periodontal bacteria, or a therapeutic agent for periodontal bacteria.
Claims (14)
The method of treating periodontal disease for treating periodontal disease in animals other than humans according to any one of claims 10 to 13, wherein the concentration of the aqueous nitrate solution is 2 to 10 mM.
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