JP7410250B2 - Composition for preventing periodontal disease - Google Patents
Composition for preventing periodontal disease Download PDFInfo
- Publication number
- JP7410250B2 JP7410250B2 JP2022163283A JP2022163283A JP7410250B2 JP 7410250 B2 JP7410250 B2 JP 7410250B2 JP 2022163283 A JP2022163283 A JP 2022163283A JP 2022163283 A JP2022163283 A JP 2022163283A JP 7410250 B2 JP7410250 B2 JP 7410250B2
- Authority
- JP
- Japan
- Prior art keywords
- periodontal disease
- composition
- lactobacillus helveticus
- helveticus sbt2171
- bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000203 mixture Substances 0.000 title claims description 39
- 208000028169 periodontal disease Diseases 0.000 title description 43
- 230000003405 preventing effect Effects 0.000 title description 22
- 240000002605 Lactobacillus helveticus Species 0.000 claims description 40
- 235000013967 Lactobacillus helveticus Nutrition 0.000 claims description 40
- 229940054346 lactobacillus helveticus Drugs 0.000 claims description 40
- 230000001737 promoting effect Effects 0.000 claims description 15
- 108050002883 beta-defensin Proteins 0.000 claims description 13
- 102000012265 beta-defensin Human genes 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 235000013305 food Nutrition 0.000 claims description 9
- 239000004480 active ingredient Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 description 27
- 241000894006 Bacteria Species 0.000 description 25
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 20
- 230000001580 bacterial effect Effects 0.000 description 16
- 241000605862 Porphyromonas gingivalis Species 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- 230000014509 gene expression Effects 0.000 description 11
- 239000004310 lactic acid Substances 0.000 description 10
- 235000014655 lactic acid Nutrition 0.000 description 10
- 230000002554 disease preventive effect Effects 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 208000006386 Bone Resorption Diseases 0.000 description 7
- 230000024279 bone resorption Effects 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 210000000988 bone and bone Anatomy 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 5
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 5
- 230000011641 antimicrobial peptide production Effects 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 201000001245 periodontitis Diseases 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 208000025157 Oral disease Diseases 0.000 description 4
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000006806 disease prevention Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 210000000214 mouth Anatomy 0.000 description 4
- 208000030194 mouth disease Diseases 0.000 description 4
- 230000003239 periodontal effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000031729 antibacterial peptide production Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 235000013351 cheese Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 206010006326 Breath odour Diseases 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 101100447432 Danio rerio gapdh-2 gene Proteins 0.000 description 2
- 101150052772 Defb14 gene Proteins 0.000 description 2
- 101150033466 Defb4 gene Proteins 0.000 description 2
- 101150112014 Gapdh gene Proteins 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 229940099112 cornstarch Drugs 0.000 description 2
- 235000015140 cultured milk Nutrition 0.000 description 2
- 208000002925 dental caries Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 235000021107 fermented food Nutrition 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- 241000606749 Aggregatibacter actinomycetemcomitans Species 0.000 description 1
- 102000004400 Aminopeptidases Human genes 0.000 description 1
- 108090000915 Aminopeptidases Proteins 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 241000186606 Lactobacillus gasseri Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 241000605894 Porphyromonas Species 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 208000008312 Tooth Loss Diseases 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000008452 baby food Nutrition 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 235000012970 cakes Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 235000021552 granulated sugar Nutrition 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 235000004213 low-fat Nutrition 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000011812 mixed powder Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 235000011962 puddings Nutrition 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- Fodder In General (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
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, the present invention 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 bacteria such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. When periodontal tissue inflammation becomes chronic and periodontal disease progresses, alveolar bone is eventually resorbed, leading to tooth loss. In addition, it has been reported that periodontal disease is not only associated 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.
Conventionally, methods for preventing and improving periodontal disease include methods to directly sterilize periodontal disease bacteria using gargles containing bactericidal and antibacterial agents, surgical methods that directly remove plaque formed by periodontal disease bacteria, or This has been done 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, and as a means of solving the problem, a composition for treating oral diseases containing lactic acid bacteria of the genus Leuconostoc is proposed. Oral compositions for use in prevention and/or treatment are disclosed. Applications to chewing gum, troches, candies, etc. are also disclosed.
Patent Document 2 discloses that lactic acid bacteria, or this lactic acid bacterium, which is useful for caries prevention, periodontal disease prevention/treatment, bad breath improvement/prevention, and inhibition of biofilm formation in the oral cavity during normal times (other than during feeding) Our goal is to provide cultures, culture supernatants, neutralized products of these, and compositions containing these, and as a means of solving these problems, we aim to provide sugar-coated bacteria that cause caries, periodontal disease, and bad breath. Disclosed are lactic acid bacteria and culture-derived products thereof that have the property of suppressing the growth of the oral bacteria and/or the formation of a biofilm formed by the oral bacteria in co-culture in a non-containing medium.
Patent Document 3 aims to provide an oral disease preventive agent that does not need to be kept in the oral cavity and can prevent periodontal disease in a simple way, and as a means of solving the problem, it uses a drug belonging to the genus Lactobacillus. Discloses an oral disease preventive agent containing Lactobacillus gasseri SBT2055 as an active ingredient, which prevents oral diseases with antibacterial peptides produced in the oral cavity when lactic acid bacteria colonize in the intestinal tract.
However, the solution provided by the present application is neither disclosed nor suggested in any of the documents.
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 food or drink for preventing periodontal disease, an agent for preventing periodontal disease, or a feed for preventing periodontal disease, comprising the composition for preventing periodontal disease according to (1).
(3) A composition for promoting antibacterial 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) A food or drink for promoting antimicrobial peptide production, an antimicrobial peptide production promoter, or a feed for promoting antimicrobial peptide production, comprising the composition for promoting antimicrobial 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 to reduce Porphyromonas gingivalis in the oral cavity and obtain a periodontal disease preventive effect.
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 explained in detail below.
The present invention uses Lactobacillus helveticus SBT2171 (FERM BP-5445) or a mutant strain substantially equivalent to Lactobacillus helveticus SBT2171 (FERM BP-5445) as a bacterial strain that exhibits periodontal disease preventive effects when ingested orally. use
A substantially equivalent mutant strain is one that exhibits a periodontal disease preventive effect when ingested orally, and whose 16S rRNA gene base sequence is 98% or more, preferably 99% or more, of the 16S rRNA gene base sequence of Lactobacillus helveticus SBT2171. % or more, more preferably 100% homology, and preferably has 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 a strain substantially equivalent thereto by mutation treatment, genetic recombination, selection of natural mutants, etc. may be used. .
This Lactobacillus helveticus SBT2171 has been reported as a new Lactobacillus strain (Japanese Patent Application Laid-Open No. 7-274949), and has been deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary as FERM BP-5445. ing. In addition, it has been reported that a novel aminopeptidase or a novel endopeptidase is produced (Japanese Patent Application Laid-open No. 8-9973, JP-A-8-298987, and JP-A-9-206074). It has also been reported that it produces polysaccharides and is suitable for producing low-fat hard natural cheese (Japanese Unexamined Patent Publication No. 11-155481).
Although the effects of oral ingestion of the composition for preventing periodontal disease of the present invention to prevent periodontal disease are not necessarily clear, the ingested Lactobacillus helveticus SBT2171 has an effect of promoting the production of antibacterial peptides against periodontal disease bacteria in vivo. It is thought to have strengthened the infection defense function such as bacteria 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 bacterial 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 by bacterial collection means such as centrifugation. Compositions containing Lactobacillus helveticus SBT2171 can be prepared using not only purely isolated bacterial cells, but also bacterial cell-containing materials such as cultures and suspensions, as well as dried products, concentrates, pastes, etc. 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 beverages for preventing periodontal disease)
The form of the food/beverage product which is one embodiment of the composition for preventing periodontal disease of the present invention will be explained.
The food and drink for periodontal disease prevention of the present invention may have any form as long as it does not interfere with the periodontal disease preventive effect. The form of the food and drink for periodontal disease prevention of the present invention includes fermented milk, cheese, etc. prepared using Lactobacillus helveticus SBT2171 bacteria or a culture containing the bacteria, and lactic acid bacteria containing Lactobacillus helveticus SBT2171. Fermented foods, as well as the above-mentioned bacterial cells, cultures containing bacterial cells, fermented milk, breads containing fermented foods such as cheese, snacks, cakes, pudding drinks, noodles, processed foods such as sausages, and various milk powders, Examples include infant foods and supplements.
（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。
(Drugs for preventing periodontal disease, animal feed for preventing periodontal disease)
The forms of pharmaceuticals and animal feed that are one embodiment of the composition for preventing periodontal disease of the present invention will be explained.
When preparing a pharmaceutical product, excipients, stabilizers, flavoring agents, etc. that are permitted in the formulation may be appropriately mixed and used. , a binder, a disintegrating agent, a lubricant, a flavoring agent, a suspending agent, a coating agent, and other arbitrary agents may be mixed to form a formulation. Possible dosage forms include tablets, capsules, granules, powders, powders, and syrups, which are administered orally. If it is included in animal feed, animals that ingest it can expect similar effects.
（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, it is desirable for adults to ingest 1 to 1,000 mg/day of lactic acid bacteria by weight.
When preparing foods and drinks for periodontal disease prevention containing Lactobacillus helveticus SBT2171 of the present invention, the content ratio of Lactobacillus helveticus SBT2171 is not particularly limited, and depends on ease of production, preferred daily dosage, etc. It may be adjusted accordingly. For example, if the shape is liquid, it is preferably 1×10 5 cells/ml to 1×10 10 cells/ml, and if it is solid, it is preferably 1×10 5 cells/g to 1×10 10 cells/ It is preferable to set it to g.
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 promoting antibacterial peptide production shall be read as the composition for promoting antibacterial 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 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 the inhibitory effect of the lactic acid bacteria of the present invention on mouse periodontitis 1. Preparation of Lactobacillus helveticus SBT2171 cells An example of preparing 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 bacterial cells were collected by centrifugation (8,000 x g, 4°C, 10 minutes), and sterilized twice with physiological saline. The cells were washed once with MilliQ water to obtain bacterial 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 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 administered only a 25% trehalose solution intragastrically. In test group B, Lactobacillus helveticus SBT2171 (10 9 cfu/200 μl) mixed with a 25% trehalose aqueous solution was forcibly administered into the stomach using a probe every day for 3 weeks. Thereafter, in order to experimentally induce periodontitis in the mice of each of the two test groups, a bacterial solution of Porphyromonas gingivalis strain 381 (10 9 cfu/100 μl) prepared with a 5% carboxymethyl cellulose solution was added. Inoculations were forcefully administered intraorally every day for two 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. Amount of alveolar bone resorption (1) Evaluation method For both test groups, mice were euthanized with carbon dioxide gas on the 30th day after oral inoculation with Porphyromonas gingivalis bacteria, and the skulls were heated under 2 atm for 10 minutes. The soft tissue was removed by immersion in a 1% sodium hypochlorite solution, and the alveolar bone was stained and dried with a 1% methylene blue solution. The dried sample was observed using a digital HD microscope. The observation results are shown in Figure 1.
In addition, the distance from the cement-enamel border of the mandibular molar region to the alveolar bone crest was measured at seven locations, and the measured values were averaged to determine the amount of alveolar bone resorption per individual. The results are shown in Figure 2.
(2) Evaluation results Figure 1 is a photograph taken with a digital HD microscope, with the upper part showing test group A and the lower part showing test group B. Since the alveolar bone of test group B had less stained area compared to the alveolar bone of test group A, administration of Lactobacillus helveticus SBT2171 significantly suppressed inflammation and alveolar bone resorption caused by Porphyromonas gingivalis infection. I know what happened.
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. Test group B had a lower average bone resorption amount than test group A, indicating that administration of Lactobacillus helveticus SBT2171 significantly suppressed inflammation and alveolar bone resorption caused by 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 Thirty days after oral inoculation with Porphyromonas gingivalis, gingival tissue was collected from mice in both test groups, and Porphyromonas in the tissue was detected. - DNA of P. gingivalis was amplified by PCR and detected by agarose gel electrophoresis. The results are shown in Figure 3.
(2) Evaluation results In test group B, the band derived from the DNA of Porphyromonas gingivalis was weaker than in test group A. It can be seen that the number of Gingivalis bacteria 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 the expression level of TNFα-specific mRNA in tissues (1) Evaluation method Gingival tissues were collected from mice in both test groups on the first day after oral inoculation with Porphyromonas gingivalis bacteria, 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 obtained reverse transcription reaction solution was used as template cDNA for real-time PCR.
AmpliTaq Gold (Life Technologies) was used for real-time PCR. The primers are TNFα (Forward: 5'-GGCAGTCAGATCATCTTCTCGAA-3', Reverse: 5'-GAAGGCCTAAGGTCCACTTGTGT-3'), Gapdh (Forward: 5'-TGTGTCCGTCGTGGATCTGA- 3', Reverse: 5'-TTGCTGTTGAAGTCGCAGGAG-3') was used . Thermal Cycler Dice (registered trademark) real-time PCR system was used for the reaction. The results of measuring the expression level of TNFα-specific mRNA in tissues are shown in FIG.
(2) Evaluation results Since test group B had lower expression of TNFα than test group A, administration of Lactobacillus helveticus SBT2171 suppressed inflammation in the gums caused by infection with Porphyromonas gingivalis bacteria. I understand that.
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 After 3 weeks of administration of Lactobacillus helveticus SBT2171 or trehalose aqueous solution, and before oral inoculation with Porphyromonas gingivalis bacteria, mice of both test groups were Gingival tissues were 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 using 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 obtained reverse transcription reaction solution was used as template cDNA for real-time PCR.
For real-time PCR, TaqMan® Fast Advanced Master Mix (Life Technologies, Cat #4444556) and TaqManGene Expression Assay (Defb4: Mm00731768_m1, Defb14: Mm00806979_m1, Gapdh: Mm99999915_g1) (Life Technologies) were used. 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 the relative expression level to test group A by the ΔΔCt method.
The results of measuring β-defensin (Defb4, Defb14)-specific mRNA expression levels in tissues are shown in FIG.
(2) Evaluation results Test group B had higher expression of β-defensin than test group A, so administration of Lactobacillus helveticus SBT2171 significantly promoted 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 combination in food)
To 100 mg of Lactobacillus helveticus SBT2171 cells were added and mixed 30 g of skim milk powder, 40 g of a mixture of equal amounts of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of a mixture of equal amounts of cornstarch and lactose. The mixture was packed into 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 into feed)
Suspend 20 g of Lactobacillus helveticus SBT2171 cells in 3980 g of deionized water, heat to 40°C, and stir and mix at 3,600 rpm for 20 minutes using a TK homomixer (MARK II 160 model; manufactured by Tokushu Kika Kogyo Co., Ltd.). A 2 g/4 kg bacterial cell solution was obtained. To 2 kg of this bacterial cell solution, 1 kg of soybean meal, 1 kg of skim 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 bran, and 0.0 kg of vitamin mixture. 5kg, cellulose 0.3kg, mineral mixture 0.2
kg was blended and heat sterilized at 120° C. for 4 minutes to produce 10 kg of the periodontal disease preventive feed 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 combination in pharmaceuticals)
A liquid culture of Lactobacillus helveticus SBT2171 was centrifuged at 4° C. and 7,000 rpm for 15 minutes, and washing with sterile water and centrifugation were repeated three times to obtain washed bacterial cells. The washed bacterial cells were freeze-dried to obtain bacterial powder. 1 part of this bacterial powder was mixed with 4 parts of skim milk powder, and the mixed powder was compressed into 1 g portions using a tablet machine in a conventional 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。 By ingesting a material containing Lactobacillus helveticus SBT2171 as an active ingredient, it can be expected to have a preventive effect on periodontal disease.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022163283A JP7410250B2 (en) | 2018-11-29 | 2022-10-11 | Composition for preventing periodontal disease |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018223460A JP7160652B2 (en) | 2018-11-29 | 2018-11-29 | Periodontal disease preventive composition |
| JP2022163283A JP7410250B2 (en) | 2018-11-29 | 2022-10-11 | Composition for preventing periodontal disease |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018223460A Division JP7160652B2 (en) | 2018-11-29 | 2018-11-29 | Periodontal disease preventive composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2023002622A JP2023002622A (en) | 2023-01-10 |
| JP7410250B2 true JP7410250B2 (en) | 2024-01-09 |
Family
ID=70904408
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018223460A Active JP7160652B2 (en) | 2018-11-29 | 2018-11-29 | Periodontal disease preventive composition |
| JP2022163283A Active JP7410250B2 (en) | 2018-11-29 | 2022-10-11 | Composition for preventing periodontal disease |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2018223460A Active JP7160652B2 (en) | 2018-11-29 | 2018-11-29 | Periodontal disease preventive composition |
Country Status (1)
| Country | Link |
|---|---|
| JP (2) | JP7160652B2 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015083547A (en) | 2013-10-25 | 2015-04-30 | 雪印メグミルク株式会社 | Preventive agent for oral diseases |
| JP2016531089A (en) | 2013-07-05 | 2016-10-06 | インテグラ・メディカル・インコーポレーテッド | Oral composition |
| WO2017022560A1 (en) | 2015-08-04 | 2017-02-09 | 雪印メグミルク株式会社 | Plasmacytoid dendritic cell proliferation promoter |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3476516B2 (en) * | 1993-01-25 | 2003-12-10 | 鐘淵化学工業株式会社 | New antibacterial substance produced by lactic acid bacteria |
| NL9301525A (en) * | 1993-09-03 | 1995-04-03 | Snow Brand Europ Research Lab | Novel Lactobacillus strains, proteins and sequences thereof, as well as methods for the use of these strains, proteins and sequences. |
| JP4203855B2 (en) | 2002-03-29 | 2009-01-07 | 株式会社フレンテ・インターナショナル | Live bacterial preparations containing lactic acid bacteria as active ingredients and foods containing lactic acid bacteria |
| JP4469792B2 (en) | 2005-02-25 | 2010-05-26 | 明治乳業株式会社 | Fermented milk for preventing and / or treating oral diseases, and fermented milk for preventing and / or suppressing bad breath |
| JP5544234B2 (en) | 2010-07-23 | 2014-07-09 | 森永乳業株式会社 | Composition for inhibiting periodontal disease growth |
| AU2012293231B2 (en) | 2011-08-05 | 2017-04-13 | Kabushiki Kaisha Yakult Honsha | Prophylactic or therapeutic agent for oral diseases |
-
2018
- 2018-11-29 JP JP2018223460A patent/JP7160652B2/en active Active
-
2022
- 2022-10-11 JP JP2022163283A patent/JP7410250B2/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016531089A (en) | 2013-07-05 | 2016-10-06 | インテグラ・メディカル・インコーポレーテッド | Oral composition |
| JP2015083547A (en) | 2013-10-25 | 2015-04-30 | 雪印メグミルク株式会社 | Preventive agent for oral diseases |
| WO2017022560A1 (en) | 2015-08-04 | 2017-02-09 | 雪印メグミルク株式会社 | Plasmacytoid dendritic cell proliferation promoter |
Non-Patent Citations (4)
| Title |
|---|
| Bhagat Singh & Renu Singh，Milchwissenschaft，2011年，Vol. 66, No. 3，pages 262-265 |
| Prasant Kumar Jena et al.，Applied Biochemistry and Biotechnology，2013年，Vol. 169，pages 2088-2100 |
| 綾部時芳 他，家畜感染症学会誌，2015年，Vol. 4, No. 4，pages 133-142 |
| 雪印メグミルク研究報告，No.2，2016年，pages 1-61 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2023002622A (en) | 2023-01-10 |
| JP2020080785A (en) | 2020-06-04 |
| JP7160652B2 (en) | 2022-10-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5982376B2 (en) | Preventive or therapeutic agent for oral diseases | |
| KR101353692B1 (en) | Compositions comprising probiotic and prebiotic components and mineral salts, with lactoferrin | |
| CN101056972B (en) | Means and methods for preventing and/or treating caries | |
| EP2485743B1 (en) | Lactobacillus plantarum strains as hypocholesterolemic agents. | |
| DK2478910T3 (en) | ANTI-ADIPOSITY AGENT, ANTI-ADIPOSITA NUTRITION OR DRINK, GLUCOSE TOLERANCE EFFECTIVE AGENT, AND NUTRITION OR DRINK FOR IMPROVING GLUCOSE TOLERANCE | |
| CN103298924A (en) | Probiotic composition for oral health | |
| JP2010521136A (en) | Composition for improving intestinal flora | |
| KR102001074B1 (en) | Lactobacillus having anticariogenic activities and composition comprising the same | |
| JP6285687B2 (en) | Oral disease preventive agent | |
| WO2019112053A1 (en) | Novel bifidobacterium bacteria and composition including novel bifidobacterium bacteria | |
| AU2011314299B2 (en) | Compositions and methods for augmenting kidney function | |
| JP2008054556A (en) | New lactobacillus having immunity activation activity, and/or allergy suppression activity, and also having gastric juice resistance | |
| KR101062779B1 (en) | Bifidobacterium adolescentis inhibiting the growth of streptococcus mutans and foodstuffs and pharmaceutical composition for preventing tooth decay containing bifidobacterium adolescentis or its culture | |
| JP2019097544A (en) | Novel bacteria belonging to bifidobacterium and compositions comprising the same | |
| TWI705135B (en) | Anti-caries agent and anti-caries composition | |
| KR102230517B1 (en) | Lactobacillus salivarius having anticariogenic activities and composition comprising the same | |
| JP7410250B2 (en) | Composition for preventing periodontal disease | |
| KR102210092B1 (en) | Lactobacillus reuteri MG505 having anticariogenic activities and composition comprising the same | |
| JP2021180619A (en) | Fermented tea composition for controlling intestinal function and method of producing the same | |
| JP6671950B2 (en) | Functional gastrointestinal amelioration agent | |
| Pavithra et al. | Probiotics–A Miracle in Periodontal Therapy | |
| JP7557519B2 (en) | Composition for promoting sugar absorption | |
| WO2024117190A1 (en) | Composition for maintaining health of intraoral environment or gums | |
| KR100930251B1 (en) | Bifidobacterium longum having the ability of reducing cholesterol in blood | |
| JP5017271B2 (en) | Kidney function improving agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20221013 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230815 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230925 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20230925 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20231108 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20231220 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20231221 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7410250 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |