JP2020080785A - Periodontal disease preventive composition - Google Patents
Periodontal disease preventive composition Download PDFInfo
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- JP2020080785A JP2020080785A JP2018223460A JP2018223460A JP2020080785A JP 2020080785 A JP2020080785 A JP 2020080785A JP 2018223460 A JP2018223460 A JP 2018223460A JP 2018223460 A JP2018223460 A JP 2018223460A JP 2020080785 A JP2020080785 A JP 2020080785A
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- periodontal disease
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Fodder In General (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
本発明は、歯周病予防用組成物に関する。特にラクトバチルス・ヘルベティカスを有効成分とする歯周病予防用組成物に関する。 The present invention relates to a composition for preventing periodontal disease. In particular, it relates to a composition for preventing periodontal disease containing Lactobacillus helveticus as an active ingredient.
歯周病は、ポルフィロモナス・ジンジバリスやアグリゲイティバクター・アクチノミセテムコミタンスなどの歯周病菌により引き起こされる歯周組織に発生する炎症性疾患の総称である。歯周組織の炎症が慢性化し、歯周病が進行すると、最終的には歯槽骨が吸収されることで歯牙の喪失を招く。また、歯周病は慢性炎症による歯周組織の破壊のみならず、動脈硬化や糖尿病などの生活習慣病の発症とも関連していることが報告されており、歯周病を予防することは、単に口腔内の健康だけでなく、生活の質を向上させるためにも重要である。
従来、歯周病の予防および改善には、殺菌・抗菌剤を含有するうがい薬などを用いて歯周病菌を直接殺菌する方法や、歯周病菌が形成するプラークを直接取り除く外科的手法、または抗生物質の服用による薬物投与法などの方法により行われてきた。
特許文献１は、バイオフィルムを形成する主要な口腔内細菌に対して共凝集を引き起こす口腔用組成物を提供することを課題とし、その解決手段としてロイコノストック属の乳酸菌を含有する口腔疾患の予防および／または治療のために用いられる口腔用組成物を開示している。また、チューインガム、トローチ、キャンディーなどへの応用が開示されている。
特許文献２は、通常時（摂食時以外）の口腔内で、う蝕予防、歯周病予防・治療、口臭改善・予防、口腔内のバイオフィルム形成抑制に有用な乳酸菌、又はこの乳酸菌の培養物、培養上清、これらの中和物、及びこれらを含有する組成物等を提供することを課題とし、その解決手段としてう蝕、歯周病又は口臭の原因となる口腔細菌との糖不含培地での共培養において、上記口腔細菌の生育及び／又は上記口腔細菌によって形成されたバイオフィルムの形成を抑制する性質を有する乳酸菌およびその培養由来物を開示している。
特許文献３は、口腔内に留めておく必要がなく、簡便な方法で歯周病を予防することができる口腔内疾患予防剤を提供することを課題とし、その解決手段としてラクトバチルス属に属する乳酸菌が腸管内で定着することで、口腔内で産生した抗菌ペプチドにより口腔内疾患を予防する、ラクトバチルス・ガセリＳＢＴ２０５５を有効成分とする口腔内疾患予防剤を開示している。
しかしながら、本願の提供する解決手段はいずれの文献にも開示も示唆もされていない。
Periodontal disease is a general term for inflammatory diseases that occur in periodontal tissues caused by periodontal disease bacteria such as Porphyromonas gingivalis and Agrigateibacter actinomycetemcomitans. When inflammation of periodontal tissues becomes chronic and periodontal disease progresses, alveolar bone is eventually absorbed, resulting in tooth loss. In addition, it is reported that periodontal disease is associated not only with the destruction of periodontal tissue due to chronic inflammation but also with the development of lifestyle-related diseases such as arteriosclerosis and diabetes, and preventing periodontal disease is It is important not only for oral health but also for improving quality of life.
Conventionally, for the prevention and improvement of periodontal disease, a method of directly sterilizing periodontal disease bacteria using a mouthwash containing a bactericidal/antibacterial agent, or a surgical method of directly removing plaque formed by periodontal disease bacteria, or It has been carried out by methods such as drug administration by taking antibiotics.
Patent Document 1 aims to provide an oral composition that causes coaggregation with respect to major oral bacteria forming a biofilm, and as a means for solving the problem, an oral composition containing a lactic acid bacterium belonging to the genus Leuconostoc Disclosed are oral compositions used for prevention and/or treatment. Further, application to chewing gum, troche, candy, etc. is disclosed.
Patent Document 2 is a lactic acid bacterium useful for preventing dental caries, preventing/treating periodontal disease, improving/preventing halitosis, and suppressing biofilm formation in the oral cavity in the oral cavity during normal times (other than eating), or It is an object of the present invention to provide a culture, a culture supernatant, a neutralized product thereof, a composition containing the same, etc., and a means for solving the problem is caries, periodontal disease, and sugars with oral bacteria that cause halitosis. Disclosed is a lactic acid bacterium having a property of suppressing the growth of the above-mentioned oral bacteria and/or the formation of a biofilm formed by the above-mentioned oral bacteria in a co-culture in a culture medium and a product derived from the culture thereof.
Patent Document 3 aims to provide an oral disease preventive agent that can prevent periodontal disease by a simple method without having to keep it in the oral cavity, and belongs to the genus Lactobacillus as a means for solving the problem. Disclosed is a preventive agent for oral diseases containing Lactobacillus gasseri SBT2055 as an active ingredient, which prevents oral diseases by the antibacterial peptide produced in the oral cavity when lactic acid bacteria settle in the intestinal tract.
However, the solution provided by the present application is neither disclosed nor suggested in any document.
本発明の課題は、ラクトバチルス・ヘルベティカスＳＢＴ２１７１を有効成分とする新規な歯周病予防用組成物を提供することである。 An object of the present invention is to provide a novel composition for preventing periodontal disease containing Lactobacillus helveticus SBT2171 as an active ingredient.
上記課題を解決するため、本発明には以下の構成が含まれる。
（１）ラクトバチルス・ヘルベティカスＳＢＴ２１７１（ＦＥＲＭ ＢＰ−５４４５）を有効成分とする歯周病予防用組成物。
（２）（１）に記載の歯周病予防用組成物を含む歯周病予防用飲食品、歯周病予防剤又は歯周病予防用飼料。
（３）ラクトバチルス・ヘルベティカスＳＢＴ２１７１（ＦＥＲＭ ＢＰ−５４４５）を有効成分とする抗菌ペプチド産生促進用組成物。
（４）抗菌ペプチドがβディフェンシンである（３）に記載の抗菌ペプチド産生促進用組成物。
（５）（３）又は（４）に記載の抗菌ペプチド産生促進用組成物を含む抗菌ペプチド産生促進用飲食品、抗菌ペプチド産生促進剤又は抗菌ペプチド産生促進用飼料。
In order to solve the above problems, the present invention includes the following configurations.
(1) A composition for preventing periodontal disease, which contains Lactobacillus helveticus SBT2171 (FERM BP-5445) as an active ingredient.
(2) A food/beverage for preventing periodontal disease, a preventive agent for periodontal disease, or a feed for preventing periodontal disease, which comprises the composition for preventing periodontal disease according to (1).
(3) A composition for promoting antibacterial peptide production, which contains Lactobacillus helveticus SBT2171 (FERM BP-5445) as an active ingredient.
(4) The composition for promoting antibacterial peptide production according to (3), wherein the antibacterial peptide is β-defensin.
(5) A food or drink for promoting antibacterial peptide production, an antibacterial peptide production promoter or a feed for promoting antibacterial peptide production, which comprises the composition for promoting antibacterial peptide production according to (3) or (4).
ラクトバチルス・ヘルベティカスＳＢＴ２１７１菌体を含む組成物を摂取することにより、口腔内のポルフィロモナス・ジンジバリスを減少させ、歯周病予防効果を得ることが期待できる。 By ingesting a composition containing Lactobacillus helveticus SBT2171 cells, it can be expected that Porphyromonas gingivalis in the oral cavity is reduced and a periodontal disease preventive effect is obtained.
本発明は、ラクトバチルス・ヘルベティカスＳＢＴ２１７１を有効成分として含む新規な歯周病予防用組成物を提供するものである。本発明の歯周病予防用組成物について以下に詳細に説明する。
本発明は、経口摂取により歯周病予防効果を示す菌株として、ラクトバチルス・ヘルベティカスＳＢＴ２１７１（ＦＥＲＭ ＢＰ−５４４５）、またはラクトバチルス・ヘルベティカスＳＢＴ２１７１（ＦＥＲＭ ＢＰ−５４４５）と実質的に同等の変異株を用いる。
実質的に同等の変異株とは、経口摂取により歯周病予防効果を示し、その１６Ｓ ｒＲＮＡ遺伝子の塩基配列が、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の１６Ｓ ｒＲＮＡ遺伝子の塩基配列と９８％以上、好ましくは９９％以上、より好ましくは１００％の相同性を有し、且つ、好ましくはラクトバチルス・ヘルベティカスＳＢＴ２１７１と同一の菌学的性質を有するものである。さらに、本発明の効果が損なわれない限り、ラクトバチルス・ヘルベティカスＳＢＴ２１７１又はそれと実質的に同等の菌株から、変異処理、遺伝子組換え、自然変異株の選択等によって育種された菌株であってもよい。
このラクトバチルス・ヘルベティカスＳＢＴ２１７１は新規なラクトバチルス属の菌株として報告されており（特開平７−２７４９４９号公報）、ＦＥＲＭ ＢＰ−５４４５として、独立行政法人産業技術総合研究所特許生物寄託センターに寄託されている。また、新規アミノペプチダーゼを産生することや新規エンドペプチダーゼを産生すること等が報告されている（特開平８−９９７３号公報、特開平８−２９８９８７号公報、特開平９−２０６０７４号公報）。また、多糖を産生し、低脂肪硬質ナチュラルチーズの製造に適していることが報告されている（特開平１１−１５５４８１号公報）。
本発明の歯周病予防用組成物の経口摂取により歯周病予防効果を示す作用は、必ずしも明らかではないが、摂取したラクトバチルス・ヘルベティカスＳＢＴ２１７１が生体内において歯周病菌に対する抗菌ペプチド産生促進作用などの感染防御機能を強化し、歯周病菌の増殖を抑制したと考えられる。
The present invention provides a novel composition for preventing periodontal disease containing Lactobacillus helveticus SBT2171 as an active ingredient. The composition for preventing periodontal disease of the present invention will be described in detail below.
The present invention, as a bacterial strain showing a periodontal disease preventive effect by oral ingestion, a mutant strain substantially equivalent to Lactobacillus helveticus SBT2171 (FERM BP-5445) or Lactobacillus helveticus SBT2171 (FERM BP-5445). To use.
The substantially equivalent mutant strain shows a periodontal disease preventive effect by oral ingestion, and its 16S rRNA gene nucleotide sequence is 98% or more, preferably 99% or more, with the nucleotide sequence of the 16S rRNA gene of Lactobacillus helveticus SBT2171. % Or more, more preferably 100%, and preferably has the same mycological properties as Lactobacillus helveticus SBT2171. Furthermore, as long as the effect of the present invention is not impaired, it may be a strain cultivated from Lactobacillus helveticus SBT2171 or a strain substantially equivalent thereto by mutation treatment, gene recombination, selection of natural mutant strain, or the like. ..
This Lactobacillus helveticus SBT2171 has been reported as a novel strain of the genus Lactobacillus (Japanese Patent Application Laid-Open No. 7-274949), and has been deposited as FERM BP-5445 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center. ing. Further, production of a novel aminopeptidase, production of a novel endopeptidase, and the like have been reported (JP-A-8-9973, JP-A-8-298987, JP-A-9-206074). Further, it has been reported that it produces a polysaccharide and is suitable for producing a low-fat hard natural cheese (JP-A-11-155481).
Although the action showing the periodontal disease preventive effect by oral ingestion of the composition for preventing periodontal disease of the present invention is not necessarily clear, the ingested Lactobacillus helveticus SBT2171 has an in vivo action of promoting antibacterial peptide production against periodontal disease bacteria. It is thought that the infection defense function such as was strengthened and the growth of periodontal disease bacteria was suppressed.
（歯周病予防用組成物の調製）
ラクトバチルス・ヘルベティカスＳＢＴ２１７１の菌体は、乳酸菌培養の常法に従ってラクトバチルス・ヘルベティカスＳＢＴ２１７１を培養し、得られた培養物から遠心分離等の集菌手段によって分離することにより得ることができる。ラクトバチルス・ヘルベティカスＳＢＴ２１７１を含む組成物は、純粋に分離された菌体だけでなく、培養物や懸濁物といった菌体含有物、さらにこれらの乾燥物、濃縮物、ペースト状物等を用いて調製することができる。
(Preparation of composition for preventing periodontal disease)
The bacterium of Lactobacillus helveticus SBT2171 can be obtained by culturing Lactobacillus helveticus SBT2171 according to a conventional method for culturing lactic acid bacteria, and separating the obtained culture by a cell-collecting means such as centrifugation. A composition containing Lactobacillus helveticus SBT2171 is prepared by using not only purely isolated cells but also cell-containing materials such as cultures and suspensions, as well as dried products, concentrates and pastes thereof. It can be prepared.
（歯周病予防用飲食品）
本発明の歯周病予防用組成物の一態様である飲食品の形態について説明する。
本発明の歯周病予防用飲食品の形態は歯周病予防効果を妨げないものであればどのようなものでもよい。本発明の歯周病予防用飲食品の形態として、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の菌体や菌体を含む培養物、およびラクトバチルス・ヘルベティカスＳＢＴ２１７１を含む乳酸菌を用いて調製した発酵乳やチーズ等の発酵食品、さらに、上記の菌体、菌体を含む培養物、発酵乳、チーズ等の発酵食品を含むパン、スナック菓子、ケーキ、プリン飲料、麺類、ソーセージ等の加工食品、さらには、各種粉乳、乳幼児食品、サプリメント等を例示することができる。
(Food and drink for periodontal disease prevention)
The form of food and drink, which is one aspect of the composition for preventing periodontal disease of the present invention, will be described.
The form of the food or drink for preventing periodontal disease of the present invention may be any one as long as it does not interfere with the effect of preventing periodontal disease. As a form of the food or drink for preventing periodontal disease of the present invention, a bacterium containing Lactobacillus helveticus SBT2171 or a culture containing the bacterium, and fermented milk or cheese prepared using lactic acid bacteria containing Lactobacillus helveticus SBT2171 Fermented foods, further, the above-mentioned bacterial cells, cultures containing bacterial cells, fermented milk, bread containing fermented foods such as cheese, snacks, cakes, pudding beverages, noodles, processed foods such as sausages, and various powdered milk, Examples include baby foods, supplements, and the like.
（歯周病予防用医薬品、歯周病予防用動物飼料）
本発明の歯周病予防用組成物の一態様である医薬品及び動物飼料の形態について説明する。
医薬品を調製する場合は、製剤化に際しては製剤上許可されている賦型剤、安定剤、矯味剤などを適宜混合して用いることができ、本発明の効果を妨げない範囲で、賦型剤、結合剤、崩壊剤、滑沢剤、矯味矯臭剤、懸濁剤、コーティング剤、その他の任意の薬剤を混合して製剤化することもできる。剤形としては、錠剤、カプセル剤、顆粒剤、散剤、粉剤、シロップ剤などが可能であり、これらを経口的に投与する。動物飼料に含まれる場合は、これを摂取した動物も同様の効果が期待できる。
(Medicines for preventing periodontal disease, animal feed for preventing periodontal disease)
The forms of the medicinal product and animal feed, which are one aspect of the composition for preventing periodontal disease of the present invention, will be described.
In the case of preparing a pharmaceutical product, a formulation-approved excipient, stabilizer, flavoring agent or the like may be appropriately mixed and used in the formulation, and the excipient may be added within a range that does not impair the effects of the present invention. , A binder, a disintegrating agent, a lubricant, a flavoring agent, a suspending agent, a coating agent, and other arbitrary agents can be mixed to prepare a formulation. The dosage form can be tablets, capsules, granules, powders, powders, syrups, etc., which are orally administered. When included in animal feed, the same effect can be expected in animals that ingest it.
（摂取量）
本発明の歯周病予防効果を発揮させるためには、成人の場合、乳酸菌体重量で１〜１,０００ｍｇ／日摂取することが望ましい。
本発明のラクトバチルス・ヘルベティカスＳＢＴ２１７１を配合した歯周病予防用飲食品などを調製する場合、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の含有割合は特に限定されず、製造の容易性や好ましい一日投与量等に合わせて適宜調節すればよい。例えば形状が液体の場合には、１×１０５ｃｅｌｌｓ／ｍｌ〜１×１０１０ｃｅｌｌｓ／ｍｌとすることが好ましく、固体の場合には、１×１０５ｃｅｌｌｓ／ｇ〜１×１０１０ｃｅｌｌｓ／ｇとすることが好ましい。
上記歯周病予防用組成物は、その作用から抗菌ペプチド産生促進用組成物としても利用することができ、その場合、上記調製方法、摂取量、飲食品、医薬品、動物飼料において歯周病予防用組成物を抗菌ペプチド産生促進用組成物と読み替えるものとする。
(Intake)
In order to exert the periodontal disease preventive effect of the present invention, in the case of an adult, it is desirable to take 1 to 1,000 mg/day by weight of lactic acid bacteria.
When preparing foods and drinks for preventing periodontal disease containing the Lactobacillus helveticus SBT2171 of the present invention, the content ratio of Lactobacillus helveticus SBT2171 is not particularly limited, and the ease of production and the preferred daily dose etc. It may be adjusted as appropriate. For example, when the shape is a liquid, preferably to 1 × 10 5 cells / ml~1 × 10 10 cells / ml, in the case of solid, 1 × 10 5 cells / g~1 × 10 10 cells / It is preferably g.
The composition for preventing periodontal disease can also be used as a composition for promoting antibacterial peptide production from its action, and in that case, in the above-mentioned preparation method, intake, food and drink, pharmaceuticals, animal feed, periodontal disease prevention The composition should be read as an antimicrobial peptide production promoting composition.
以下、本発明の実施例を詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, examples of the present invention will be described in detail, but the present invention is not limited thereto.
〔試験例１〕本発明乳酸菌のマウス歯周炎に対する抑制効果の確認
１．ラクトバチルス・ヘルベティカスＳＢＴ２１７１菌体の調製
本試験に用いたラクトバチルス・ヘルベティカスＳＢＴ２１７１菌体の調製例を以下に示す。
ラクトバチルス・ヘルベティカスＳＢＴ２１７１をＭＲＳ液体培地１００ｍＬで３７℃、１６時間培養後、遠心分離（８，０００×ｇ、４℃、１０分間）にて菌体を回収し、生理食塩水で２回、滅菌ＭｉｌｌｉＱ水で１回洗浄し、菌体を得た。
[Test Example 1] Confirmation of inhibitory effect of the lactic acid bacterium of the present invention on mouse periodontitis 1. Preparation of Lactobacillus helveticus SBT2171 bacterial cells An example of preparation of Lactobacillus helveticus SBT2171 bacterial cells used in this test is shown below.
After culturing Lactobacillus helveticus SBT2171 in 100 mL of MRS liquid medium at 37° C. for 16 hours, cells were collected by centrifugation (8,000×g, 4° C., 10 minutes) and sterilized twice with physiological saline. The cells were washed once with MilliQ water to obtain cells.
２．マウス実験的歯周炎に対する乳酸菌の抑制効果
２−１．実験的歯周炎の惹起方法
生後７週齢の雌性ＢＡＬＢ/ｃマウスを２試験群Ａ、Ｂに分け、試験群Ａには２５％トレハロース溶液のみを胃内投与した。試験群Ｂには２５％トレハロース水溶液に混合したラクトバチルス・ヘルベティカスＳＢＴ２１７１（１０９ｃｆｕ／２００μｌ）を、ゾンデを用いて３週間連日胃内に強制投与した。その後、２試験群それぞれのマウスに対して実験的に歯周炎を惹起させるために、５％カルボキシメチルセルロース溶液で調製したポルフィロモナス・ジンジバリス３８１株の菌液（１０９ｃｆｕ／１００μｌ）を、２週間連日口腔内に強制接種した。ポルフィロモナス・ジンジバリス菌の投与中も、試験群Ｂには２５％トレハロース溶液に混合したラクトバチルス・ヘルベティカスＳＢＴ２１７１（１０９ｃｆｕ／２００μｌ）の投与を続け、試験群Ａには２５％トレハロース溶液のみを胃内投与した。
2. Inhibitory effect of lactic acid bacteria on experimental periodontitis in mice 2-1. Method of Inducing Experimental Periodontitis Female BALB/c mice aged 7 weeks were divided into 2 test groups A and B, and 25% trehalose solution alone was intragastrically administered to test group A. In the test group B, Lactobacillus helveticus SBT2171 (10 9 cfu/200 μl) mixed with a 25% trehalose aqueous solution was forcibly administered intragastrically for 3 weeks using a sonde. Thereafter, in order to experimentally induce periodontitis in each mouse of the 2 test groups, a bacterial solution of Porphyromonas gingivalis strain 381 strain (10 9 cfu/100 μl) prepared with a 5% carboxymethylcellulose solution was added, It was forcibly inoculated into the oral cavity for 2 consecutive days. During administration of Porphyromonas gingivalis, test group B continued to receive Lactobacillus helveticus SBT2171 (10 9 cfu/200 μl) mixed with 25% trehalose solution, and test group A contained only 25% trehalose solution. Was administered intragastrically.
２−２．歯槽骨の吸収量
（１）評価方法
両試験群共にポルフィロモナス・ジンジバリス菌の口腔接種後３０日目にマウスを炭酸ガスにて安楽死させ、頭蓋骨を２気圧下で１０分間加熱後、３％次亜塩素酸ナトリウム溶液に浸漬して軟組織を除去し、１％ メチレンブルー溶液で歯槽骨を染色乾燥させた試料をデジタルＨＤマイクロスコープにて観察した。観察結果を図１に示す。
また、下顎臼歯部のセメントエナメル境から歯槽骨頂までの距離を７カ所測定し、測定値を平均し個体当たりの歯槽骨吸収量とした。結果を図２に示す。
（２）評価結果
図１はデジタルＨＤマイクロスコープの写真であり、上は試験群Ａを下は試験群Ｂを示す。試験群Ｂの歯槽骨は試験群Ａの歯槽骨と比較し、染色部が少ないことから、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の投与により、ポルフィロモナス・ジンジバリス菌感染による炎症及び歯槽骨吸収が顕著に抑制されたことが分かる。
図２は、縦軸に平均歯槽骨吸収量（μｍ）を表した。左側が試験群Ａで右側が試験群Ｂである。試験群Ｂは、試験群Ａと比較し平均骨吸収量が低いことから、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の投与により、ポルフィロモナス・ジンジバリス菌感染による炎症及び歯槽骨吸収が顕著に抑制されたことが分かる。
2-2. Alveolar bone absorption (1) Evaluation method In both test groups, 30 days after oral inoculation of Porphyromonas gingivalis, mice were euthanized with carbon dioxide gas, and the skull was heated under 2 atm for 10 minutes, then 3 % Sodium hypochlorite solution to remove the soft tissue, and the alveolar bone was dyed and dried with a 1% methylene blue solution. The sample was observed with a digital HD microscope. The observation result is shown in FIG.
In addition, the distance from the cement enamel boundary of the mandibular molar to the alveolar bone crest was measured at 7 locations, and the measured values were averaged to obtain the alveolar bone resorption amount per individual. The results are shown in Figure 2.
(2) Evaluation Results FIG. 1 is a photograph of a digital HD microscope, in which the upper part shows the test group A and the lower part shows the test group B. Since the alveolar bone of test group B has less stained parts than the alveolar bone of test group A, administration of Lactobacillus helveticus SBT2171 significantly suppressed inflammation and alveolar bone resorption caused by infection with Porphyromonas gingivalis. You can see that it was done.
In FIG. 2, the vertical axis represents the average alveolar bone resorption amount (μm). The test group A is on the left side and the test group B is on the right side. Since the average bone resorption amount of the test group B was lower than that of the test group A, it was found that the administration of Lactobacillus helveticus SBT2171 significantly suppressed the inflammation and the alveolar bone resorption caused by the infection with Porphyromonas gingivalis. I understand.
２−３．組織中のポルフィロモナス・ジンジバリス菌のＤＮＡの検出
（１）評価方法
ポルフィロモナス・ジンジバリス菌の口腔接種後３０日目に両試験群のマウスから歯肉組織を採取し、組織中のポルフィロモナス・ジンジバリス菌のＤＮＡをＰＣＲで増幅し、アガロースゲル電気泳動にて検出した。結果を図３に示す。
（２）評価結果
試験群Ｂは、試験群Ａと比較し、ポルフィロモナス・ジンジバリス菌のＤＮＡに由来するバンドが薄いことから、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の投与により、口腔内のポルフィロモナス・ジンジバリス菌が減少したことが分かる。
2-3. Detection of Porphyromonas gingivalis DNA in Tissue (1) Evaluation Method 30 days after oral inoculation of Porphyromonas gingivalis, gingival tissue was collected from mice in both test groups, and Porphyromonas in the tissue was collected. -The DNA of Gingivalis was amplified by PCR and detected by agarose gel electrophoresis. Results are shown in FIG.
(2) Evaluation results Since the test group B has a thinner band derived from the DNA of Porphyromonas gingivalis than that of the test group A, the administration of Lactobacillus helveticus SBT2171 results in the oral administration of Porphyromonas It can be seen that the Gingivalis bacterium has decreased.
２−４．組織中のＴＮＦα特異的ｍＲＮＡの発現量の測定
（１）評価方法
ポルフィロモナス・ジンジバリス菌の口腔接種後１日目に両試験群のマウスから歯肉組織を採取し、Ｔｒｉｚｏｌ(登録商標） Ｒｅａｇｅｎｔ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を用いてトータルＲＮＡを抽出した。抽出したトータルＲＮＡ溶液から、ＧｅｎｅＡｍｐ ＲＮＡ ＰＣＲ Ｋｉｔ（タカラバイオ）を用いてｃＤＮＡを合成した。得られた逆転写反応液をｔｅｍｐｌａｔｅ ｃＤＮＡとしてリアルタイムＰＣＲに供した。
リアルタイムＰＣＲにはＡｍｐｌｉＴａｑ Ｇｏｌｄ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）を使用した。プライマーはＴＮＦα（Ｆｏｒｗａｒｄ：５’−ＧＧＣＡＧＴＣＡＧＡＴＣＡＴＣＴＴＣＴＣＧＡＡ−３’、Ｒｅｖｅｒｓｅ： ５’−ＧＡＡＧＧＣＣＴＡＡＧＧＴＣＣＡＣＴＴＧＴＧＴ−３’）、Ｇａｐｄｈ（Ｆｏｒｗａｒｄ：５’−ＴＧＴＧＴＣＣＧＴＣＧＴＧＧＡＴＣＴＧＡ−３’、Ｒｅｖｅｒｓｅ： ５’−ＴＴＧＣＴＧＴＴＧＡＡＧＴＣＧＣＡＧＧＡＧ−３’）を使用した。反応にはＴｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ(登録商標） ｒｅａｌ−ｔｉｍｅ ＰＣＲ ｓｙｓｔｅｍを使用した。組織中のＴＮＦα特異的ｍＲＮＡ発現量測定結果を図４に示す。
（２）評価結果
試験群Ｂが試験群Ａと比較してＴＮＦαの発現が低いことから、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の投与により、ポルフィロモナス・ジンジバリス菌の感染による歯肉での炎症が抑制されたことが分かる。
2-4. Measurement of TNFα-Specific mRNA Expression Level in Tissue (1) Evaluation Method One day after oral inoculation with Porphyromonas gingivalis, gingival tissues were collected from mice in both test groups, and Trizol (registered trademark) Reagent ( Total RNA was extracted using Thermo Fisher Scientific). From the extracted total RNA solution, cDNA was synthesized using GeneAmp RNA PCR Kit (Takara Bio). The resulting reverse transcription reaction solution was subjected to real-time PCR as template cDNA.
AmpliTaq Gold (Life Technologies) was used for real-time PCR. Primer TNFα (Forward: 5'-GGCAGTCAGATCATCTTCTCGAA-3 ', Reverse: 5'-GAAGGCCTAAGGTCCACTTGTGT-3'), Gapdh (Forward: 5'-TGTGTCCGTCGTGGATCTGA-3 ', Reverse: 5'-TTGCTGTTGAAGTCGCAGGAG-3') were used .. For the reaction, a Thermal Cycler Dice (registered trademark) real-time PCR system was used. The results of measuring the amount of TNFα-specific mRNA expression in the tissue are shown in FIG.
(2) Evaluation results Since the test group B had a lower expression of TNFα than the test group A, administration of Lactobacillus helveticus SBT2171 suppressed inflammation in the gingiva due to infection with Porphyromonas gingivalis. I understand.
２−５．組織中のβ−ディフェンシン濃度特異的ｍＲＮＡ発現量の測定
（１）評価方法
ラクトバチルス・ヘルベティカスＳＢＴ２１７１もしくはトレハロース水溶液の３週間投与後、ポルフィロモナス・ジンジバリス菌の口腔接種前に、両試験群のマウスから歯肉組織を採取し、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（Ｑｉａｇｅｎ）を用いて細胞からトータルＲＮＡを抽出した。抽出したトータルＲＮＡは、ＮａｎｏＤｒｏｐ２０００（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）で濃度を測定した。
抽出したトータルＲＮＡ溶液から、ＲｅｖｅｒＴｒａ Ａｃｅ ｑＰＣＲ ＲＴ Ｍａｓ ｔｅｒ Ｍｉｘ ｗｉｔｈ ｇＤＮＡ Ｒｅｍｏｖｅｒ（東洋紡）を用いてｃＤＮＡを合成した。逆転写反応には、ＴａＫａＲａ ＰＣＲ Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ(登録商標）Ｇｒａｄｉｅｎｔ（タカラバイオ）を使用した。得られた逆転写反応液をｔｅｍｐｌａｔｅ ｃＤＮＡとしてリアルタイムＰＣＲに供した。
リアルタイムＰＣＲにはＴａｑＭａｎ(登録商標）Ｆａｓｔ Ａｄｖａｎｃｅｄ Ｍａｓｔｅｒ Ｍｉｘ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ、Ｃａｔ＃４４４４５５６）およびＴａｑＭａｎＧｅｎｅ Ｅｘｐｒｅｓｓｉｏｎ Ａｓｓａｙ（Ｄｅｆｂ４：Ｍｍ００７３１７６８＿ｍ１、Ｄｅｆｂ１４：Ｍｍ００８０６９７９＿ｍ１、Ｇａｐｄｈ：Ｍｍ９９９９９９１５＿ｇ１）（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）を使用した。反応には３８４ウェルプレート（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ、Ｃａｔ＃４３０９８４９）を用い、ＶｉｉＡTM７（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ）を使用した。遺伝子発現はΔΔＣｔ法によって試験群Ａに対する相対発現量として評価した。
組織中のβ−ディフェンシン（Ｄｅｆｂ４、Ｄｅｆｂ１４）特異的ｍＲＮＡ発現量測定結果を図５に示す。
（２）評価結果
試験群Ｂが試験群Ａと比較してβ−ディフェンシンの発現が高いことから、ラクトバチルス・ヘルベティカスＳＢＴ２１７１の投与により、β−ディフェンシン産生が顕著に亢進し、歯周病菌感染による症状を抑制したことがわかる。
2-5. Measurement of β-defensin concentration-specific mRNA expression level in tissues (1) Evaluation method Lactobacillus helveticus SBT2171 or an aqueous trehalose solution was administered for 3 weeks, and before oral administration of Porphyromonas gingivalis to mice in both test groups. Gingival tissue was collected from the cells, and total RNA was extracted from the cells using RNeasy Mini Kit (Qiagen). The concentration of the extracted total RNA was measured with NanoDrop2000 (Thermo Fisher Scientific).
From the extracted total RNA solution, cDNA was synthesize|combined using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). TaKaRa PCR Thermal Cycler Dice (registered trademark) Gradient (Takara Bio) was used for the reverse transcription reaction. The resulting reverse transcription reaction solution was subjected to real-time PCR as template cDNA.
For the real-time PCR, TaqMan (registered trademark) Fast Advanced Master Mix (Life Technologies, Cat#444556) and TaqMan Gene Expression Lis99_99m99_99m99m15m9Gm, Defb4:Mm00731768_m1:Mm00731768_m1:m0080, Defb were used. 384-well plates (Life Technologies, Cat#4309849) were used for the reaction, and ViiA ™ 7 (Life Technologies) was used. The gene expression was evaluated as the relative expression level with respect to the test group A by the ΔΔCt method.
FIG. 5 shows the measurement results of β-defensin (Defb4, Defb14)-specific mRNA expression levels in tissues.
(2) Evaluation results Since the expression of β-defensin in test group B is higher than that in test group A, administration of Lactobacillus helveticus SBT2171 markedly enhanced β-defensin production, resulting in infection with periodontal disease bacteria. It can be seen that the symptoms were suppressed.
（食品への配合例）
ラクトバチルス・ヘルベティカスＳＢＴ２１７１菌体１００ｍｇに、脱脂粉乳３０ｇ、ビタミンＣとクエン酸の等量混合物４０ｇ、グラニュー糖１００ｇ、コーンスターチと乳糖の等量混合物６０ｇを加えて混合した。混合物をスティック状袋に詰め、本発明の歯周病予防用スティック状健康食品を製造した。
(Example of blending with food)
To 100 mg of Lactobacillus helveticus SBT2171 cells, 30 g of skim milk powder, 40 g of an equal mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equal mixture of corn starch and lactose were mixed. The mixture was packed in a stick bag to produce the stick health food for preventing periodontal disease of the present invention.
（飼料への配合例）
ラクトバチルス・ヘルベティカスＳＢＴ２１７１菌体２０ｇを３９８０ｇの脱イオン水に懸濁し、４０℃まで加熱後、ＴＫホモミクサー（ＭＡＲＫ ＩＩ １６０型；特殊機化工業社製）にて、３，６００ｒｐｍで２０分間撹拌混合して２ｇ／４ｋｇの菌体溶液を得た。この菌体溶液２ｋｇに大豆粕１ｋｇ、脱脂粉乳１ｋｇ、大豆油０．４ｋｇ、コーン油０．２ｋｇ、パーム油２．３ｋｇ、トウモロコシ澱粉１ｋｇ、小麦粉０．９ｋｇ、ふすま０．２ｋｇ、ビタミン混合物０．５ｋｇ、セルロース０．３ｋｇ、ミネラル混合物０．２ｋｇを配合し、１２０℃、４分間加熱殺菌して、本発明の歯周病予防用飼料１０ｋｇを製造した。
(Example of blending with feed)
20 g of Lactobacillus helveticus SBT2171 cells were suspended in 3980 g of deionized water, heated to 40° C., and then stirred and mixed at 3,600 rpm for 20 minutes with a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.). Then, a bacterial cell solution of 2 g/4 kg was obtained. Soybean meal 1 kg, skim milk powder 1 kg, soybean oil 0.4 kg, corn oil 0.2 kg, palm oil 2.3 kg, corn starch 1 kg, wheat flour 0.9 kg, bran 0.2 kg, vitamin mixture 0. 5 kg, 0.3 kg of cellulose and 0.2 kg of a mineral mixture were blended and sterilized by heating at 120° C. for 4 minutes to produce 10 kg of a feed for preventing periodontal disease of the present invention.
（医薬品への配合例）
ラクトバチルス・ヘルベティカスＳＢＴ２１７１の液体培養物を、４℃、７，０００ｒｐｍで１５分間遠心分離した後、滅菌水による洗浄と遠心分離を３回繰り返して行い、洗浄菌体を得た。この洗浄菌体を凍結乾燥処理して菌体粉末を得た。この菌体粉末１部に脱脂粉乳４部を混合し、この混合粉末を打錠機により１ｇずつ常法により打錠して、本発明の歯周病予防用錠剤を調製した。
(Example of compounding into pharmaceutical products)
A liquid culture of Lactobacillus helveticus SBT2171 was centrifuged at 4° C. and 7,000 rpm for 15 minutes, followed by washing with sterile water and centrifugation three times to obtain washed bacterial cells. The washed bacterial cells were freeze-dried to obtain bacterial cell powder. 1 part of this bacterial cell powder was mixed with 4 parts of skim milk powder, and 1 g of this mixed powder was tabletted by a tableting machine in a usual manner to prepare a tablet for preventing periodontal disease of the present invention.
ラクトバチルス・ヘルベティカスＳＢＴ２１７１を有効成分とする素材を体内に摂取することにより、歯周病予防作用を得ることが期待できる。
Ingestion of a material containing Lactobacillus helveticus SBT2171 as an active ingredient can be expected to have a periodontal disease preventive effect.
Claims (5)
A food or drink for promoting antibacterial peptide production, an antibacterial peptide production promoting agent or a feed for promoting antibacterial peptide production, comprising the composition for promoting antibacterial peptide production according to claim 3 or 4.
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