JP2023002622A - Periodontal disease-preventing composition - Google Patents
Periodontal disease-preventing composition Download PDFInfo
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- JP2023002622A JP2023002622A JP2022163283A JP2022163283A JP2023002622A JP 2023002622 A JP2023002622 A JP 2023002622A JP 2022163283 A JP2022163283 A JP 2022163283A JP 2022163283 A JP2022163283 A JP 2022163283A JP 2023002622 A JP2023002622 A JP 2023002622A
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- periodontal disease
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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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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。 TECHNICAL FIELD 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 occurring in periodontal tissue caused by periodontal pathogens such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. When the periodontal tissue inflammation becomes chronic and the periodontal disease progresses, the alveolar bone is finally absorbed, resulting in tooth loss. In addition, it has been reported that periodontal disease is associated not only with the destruction of periodontal tissue due to chronic inflammation, but also with the onset of lifestyle-related diseases such as arteriosclerosis and diabetes. It is important not only for oral health, but also for improving quality of life.
Traditionally, periodontal disease has been prevented and improved by methods such as direct sterilization of periodontal disease bacteria using mouthwashes containing bactericidal and antibacterial agents, surgical techniques to directly remove plaque formed by periodontal disease bacteria, or It has been performed by methods such as drug administration by taking antibiotics.
Patent Document 1 aims to provide an oral composition that causes coaggregation of major oral bacteria that form biofilms. Disclosed are oral compositions for prophylactic and/or therapeutic use. Application to chewing gum, lozenges, candies, etc. is also disclosed.
Patent Document 2 is a lactic acid bacterium useful for caries prevention, periodontal disease prevention/treatment, bad breath improvement/prevention, and intraoral biofilm formation suppression in the oral cavity during normal times (other than when eating), or this lactic acid bacterium. The object of the present invention is to provide cultures, culture supernatants, neutralized products thereof, compositions containing these, and the like. A lactic acid bacterium and a culture-derived product thereof are disclosed that have the property of suppressing the growth of the oral bacterium and/or the formation of a biofilm formed by the oral bacterium when co-cultivated in a free medium.
Patent Document 3 aims to provide an oral disease preventive agent that can prevent periodontal disease by a simple method without needing to keep it in the oral cavity, and belongs to the genus Lactobacillus as a solution. It discloses an oral disease preventive agent containing Lactobacillus gasseri SBT2055 as an active ingredient, which prevents oral diseases by the antibacterial peptide produced in the oral cavity by the establishment of lactic acid bacteria in the intestinal tract.
However, none of the documents disclose or suggest the solution provided by the present application.
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 containing Lactobacillus helveticus SBT2171 (FERM BP-5445) as an active ingredient.
(2) A periodontal disease-preventing food, drink, periodontal disease-preventing agent, or periodontal disease-preventing feed comprising the periodontal disease-preventing composition according to (1).
(3) A composition for promoting antimicrobial peptide production containing Lactobacillus helveticus SBT2171 (FERM BP-5445) as an active ingredient.
(4) The composition for promoting antimicrobial peptide production according to (3), wherein the antimicrobial peptide is β-defensin.
(5) Antimicrobial peptide production-enhancing food or drink, antimicrobial peptide production-enhancing agent, or antimicrobial peptide production-enhancing feed comprising the antimicrobial peptide production-enhancing composition 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 is expected that Porphyromonas gingivalis in the oral cavity will be reduced and periodontal disease preventive effects will be 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 periodontal disease preventive composition of the present invention is described in detail below.
The present invention provides Lactobacillus helveticus SBT2171 (FERM BP-5445) or a mutant strain substantially equivalent to Lactobacillus helveticus SBT2171 (FERM BP-5445) as a strain that exhibits a periodontal disease preventive effect by oral ingestion. use.
A substantially equivalent mutant strain exhibits a periodontal disease preventive effect by oral ingestion, and has a 16S rRNA gene base sequence that is 98% or more, preferably 99%, that of the 16S rRNA gene base sequence of Lactobacillus helveticus SBT2171. % or more, more preferably 100% homology and preferably the same mycological properties as Lactobacillus helveticus SBT2171. Furthermore, as long as the effects of the present invention are not impaired, strains bred from Lactobacillus helveticus SBT2171 or substantially equivalent strains by mutation treatment, genetic recombination, selection of natural mutant strains, etc. may be used. .
This Lactobacillus helveticus SBT2171 has been reported as a novel strain of the genus Lactobacillus (Japanese Patent Laid-Open No. 7-274949), and has been deposited as FERM BP-5445 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. ing. In addition, production of novel aminopeptidases and production of novel endopeptidases have been reported (JP-A-8-9973, JP-A-8-298987, JP-A-9-206074). It has also been reported that it produces polysaccharides and is suitable for producing low-fat hard natural cheese (Japanese Patent Application Laid-Open No. 11-155481).
Although the effect of oral ingestion of the composition for preventing periodontal disease of the present invention on exhibiting a periodontal disease-preventing effect is not necessarily clear, the ingested Lactobacillus helveticus SBT2171 has an antibacterial peptide production-promoting effect against periodontal disease bacteria in vivo. It is thought that it strengthened the infection defense function such as and suppressed the growth of periodontal disease bacteria.
（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 cells 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 resulting culture from the resulting culture by means of harvesting means such as centrifugation. Compositions containing Lactobacillus helveticus SBT2171 can be prepared using not only purely isolated bacterial cells, but also bacterial cell-containing substances such as cultures and suspensions, as well as dried products, concentrates, pastes, etc. thereof. 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 and drink for preventing periodontal disease of the present invention may be any form as long as it does not interfere with the effect of preventing periodontal disease. As a form of the food and drink for preventing periodontal disease of the present invention, fermented milk, cheese, etc. prepared using bacteria containing Lactobacillus helveticus SBT2171, a culture containing the bacteria, and lactic acid bacteria containing Lactobacillus helveticus SBT2171. Fermented foods, furthermore, the above bacteria, cultures containing bacteria, fermented milk, breads containing fermented foods such as cheese, snacks, cakes, pudding beverages, noodles, processed foods such as sausages, various powdered milk, Infant foods, supplements and the like can be exemplified.
（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。
(Pharmaceuticals for periodontal disease prevention, animal feed for periodontal disease prevention)
Forms of pharmaceuticals and animal feeds, which are one aspect of the composition for preventing periodontal disease of the present invention, will be described.
When preparing pharmaceuticals, excipients, stabilizers, corrigents, etc. that are permitted for formulation can be appropriately mixed and used, and excipients can be added to the extent that the effects of the present invention are not impaired. . Dosage forms include tablets, capsules, granules, powders, powders, syrups and the like, and these are orally administered. When it is included in animal feed, similar effects can be expected for animals that have ingested 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 exhibit the periodontal disease preventive effect of the present invention, it is desirable for adults to take 1 to 1,000 mg/day of lactic acid bacteria body weight.
When preparing a food or drink for preventing periodontal disease containing Lactobacillus helveticus SBT2171 of the present invention, the content of Lactobacillus helveticus SBT2171 is not particularly limited, and the content of Lactobacillus helveticus SBT2171 is not particularly limited. It may be adjusted accordingly. For example, when the form is liquid, it is preferably 1×10 5 cells/ml to 1×10 10 cells/ml, and when it is solid, it is 1×10 5 cells/g to 1×10 10 cells/ml. g is preferred.
The above-mentioned composition for preventing periodontal disease can also be used as a composition for promoting the production of antibacterial peptides due to its action. The composition for antimicrobial peptide production shall be read as a composition for promoting antimicrobial peptide production.
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。 Examples of the present invention will be described in detail below, but the present invention is not limited to these.
〔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 the inhibitory effect of the lactic acid bacteria of the present invention on mouse periodontitis 1. Preparation of Lactobacillus helveticus SBT2171 Cells A preparation example of Lactobacillus helveticus SBT2171 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, the cells were collected by centrifugation (8,000×g, 4° C., 10 minutes), and sterilized twice with physiological saline. After washing once with MilliQ water, cells were obtained.
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. Suppressive effect of lactic acid bacteria on mouse experimental periodontitis 2-1. Method for Inducing Experimental Periodontitis Seven-week-old female BALB/c mice were divided into two test groups A and B, and test group A was intragastrically administered with a 25% trehalose solution alone. In test group B, Lactobacillus helveticus SBT2171 (10 9 cfu/200 μl) mixed with 25% trehalose aqueous solution was forcibly administered into the stomach every day for 3 weeks using a sonde. Then, in order to experimentally induce periodontitis in the mice of each of the two test groups, Porphyromonas gingivalis strain 381 solution (10 9 cfu/100 μl) prepared with a 5% carboxymethylcellulose solution was added to the mice. It was forcibly inoculated into the oral cavity every day for 2 weeks. During the 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 received 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. Resorption amount of alveolar bone (1) Evaluation method In both test groups, the mice were euthanized with carbon dioxide on the 30th day after oral inoculation with Porphyromonas gingivalis bacteria, and the skulls were heated under 2 atmospheres for 10 minutes, and then treated for 3 minutes. Soft tissues were removed by immersion in % sodium hypochlorite solution, alveolar bone was stained with 1% methylene blue solution, dried, and observed with a digital HD microscope. The observation results are shown in FIG.
In addition, the distance from the cement-enamel boundary to the alveolar bone crest in the mandibular molar region was measured at 7 points, and the measured values were averaged to obtain the alveolar bone resorption amount per individual. The results are shown in FIG.
(2) Evaluation Results FIG. 1 is a photograph of a digital HD microscope, showing test group A on the top and test group B on the bottom. Compared to the alveolar bone of test group A, the alveolar bone of test group B has fewer stained parts, so administration of Lactobacillus helveticus SBT2171 significantly suppresses inflammation and alveolar bone resorption due to Porphyromonas gingivalis infection. I know it was done.
In FIG. 2, the vertical axis represents the average amount of alveolar bone resorption (μm). Test group A is on the left and test group B is on the right. Since test group B had a lower average bone resorption amount than test group A, administration of Lactobacillus helveticus SBT2171 markedly suppressed inflammation and alveolar bone resorption due to Porphyromonas gingivalis infection. 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 tissues (1) Evaluation method On day 30 after oral inoculation with P. gingivalis, gingival tissue was collected from mice of both test groups, and Porphyromonas in the tissue was collected. - The DNA of L. gingivalis was amplified by PCR and detected by agarose gel electrophoresis. The results are shown in FIG.
(2) Evaluation results Test group B has a thinner band derived from the DNA of Porphyromonas gingivalis than Test group A. It can be seen that the number of Lactobacillus gingivalis 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 expression level of TNFα-specific mRNA in tissues (1) Evaluation method On day 1 after oral inoculation with Porphyromonas gingivalis, gingival tissues were collected from mice of both test groups and treated with Trizol (registered trademark) Reagent ( Total RNA was extracted using Thermo Fisher Scientific). cDNA was synthesized from the extracted total RNA solution using GeneAmp RNA PCR Kit (Takara Bio). The resulting reverse transcription reaction solution was subjected to real-time PCR as a template cDNA.
AmpliTaq Gold (Life Technologies) was used for real-time PCR. Primers were TNFα (Forward: 5'-GGCAGTCAGATCATCTTCTCGAA-3', Reverse: 5'-GAAGGCCTAAGGTCCACTTGTGT-3'), Gapdh (Forward: 5'-TGTGTCCGTCGTGGATCTGA-3', Reverse: 5'-TTGCTGTGTAGGAGCAGT3') was used. . A Thermal Cycler Dice (registered trademark) real-time PCR system was used for the reaction. FIG. 4 shows the results of TNFα-specific mRNA expression level measurement in tissues.
(2) Evaluation results Since the expression of TNFα was lower in test group B than in test group A, administration of Lactobacillus helveticus SBT2171 suppressed gingival inflammation due to Porphyromonas gingivalis infection. 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 Gingival tissue was collected from the cells and total RNA was extracted from the cells using the RNeasy Mini Kit (Qiagen). The concentration of the extracted total RNA was measured with NanoDrop2000 (Thermo Fisher Scientific).
cDNA was synthesized from the extracted total RNA solution 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 a template cDNA.
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。 A 384-well plate (Life Technologies, Cat#4309849) was used for the reaction, and ViiA ™ 7 (Life Technologies) was used. Gene expression was evaluated as a relative expression level to test group A by the ΔΔCt method.
FIG. 5 shows the results of β-defensin (Defb4, Defb14)-specific mRNA expression level measurement in tissues.
(2) Evaluation results Since the expression of β-defensin is higher in test group B than in test group A, administration of Lactobacillus helveticus SBT2171 significantly enhances β-defensin production, resulting in periodontal infection It can be seen that the symptoms have been 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 in food)
To 100 mg of Lactobacillus helveticus SBT2171 cells, 30 g of skimmed 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 cornstarch and lactose were added and mixed. The mixture was packed in a stick-shaped bag to produce a stick-shaped 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０．２
ｋｇ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 in feed)
20 g of Lactobacillus helveticus SBT2171 cells were suspended in 3980 g of deionized water, heated to 40° C., and stirred and mixed at 3,600 rpm for 20 minutes with a TK homomixer (MARK II 160; manufactured by Tokushu Kika Kogyo Co., Ltd.). to obtain a 2 g/4 kg bacterial cell solution. 1 kg of soybean meal, 1 kg of skimmed milk powder, 0.4 kg of soybean oil, 0.2 kg of corn oil, 2.3 kg of palm oil, 1 kg of corn starch, 0.9 kg of wheat flour, 0.2 kg of wheat bran, and 0.2 kg of vitamin mixture were added to 2 kg of this bacterial cell solution. 5 kg, cellulose 0.3 kg, mineral mixture 0.2
kg and heat sterilized at 120° C. for 4 minutes to produce 10 kg of the 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 in pharmaceuticals)
A liquid culture of Lactobacillus helveticus SBT2171 was centrifuged at 4° C. and 7,000 rpm for 15 minutes, and then washed with sterilized water and centrifuged three times to obtain washed cells. The washed cells were freeze-dried to obtain a powder of cells. 4 parts of powdered skim milk was mixed with 1 part of this bacterial powder, and 1 g of this mixed powder was tableted by a conventional method 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。 By taking into the body a material containing Lactobacillus helveticus SBT2171 as an active ingredient, it can be expected to obtain a periodontal disease preventive action.
Claims (3)
組成物。 The composition for promoting antimicrobial peptide production according to claim 1, wherein the antimicrobial peptide is a β-defensin.
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