JP6836298B1 - Composition containing lactic acid bacteria and natto bacteria - Google Patents
Composition containing lactic acid bacteria and natto bacteria Download PDFInfo
- Publication number
- JP6836298B1 JP6836298B1 JP2020139609A JP2020139609A JP6836298B1 JP 6836298 B1 JP6836298 B1 JP 6836298B1 JP 2020139609 A JP2020139609 A JP 2020139609A JP 2020139609 A JP2020139609 A JP 2020139609A JP 6836298 B1 JP6836298 B1 JP 6836298B1
- Authority
- JP
- Japan
- Prior art keywords
- natto
- composition
- lactobacillus brevis
- cells
- promoting
- 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 abstract description 57
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 49
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 49
- 241000894006 Bacteria Species 0.000 title claims description 41
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title description 44
- 235000014655 lactic acid Nutrition 0.000 title description 22
- 239000004310 lactic acid Substances 0.000 title description 22
- 235000013957 Lactobacillus brevis Nutrition 0.000 claims abstract description 51
- 240000001929 Lactobacillus brevis Species 0.000 claims abstract description 51
- 230000001737 promoting effect Effects 0.000 claims abstract description 32
- 235000013557 nattō Nutrition 0.000 claims abstract description 30
- 235000013305 food Nutrition 0.000 claims abstract description 29
- 230000019734 interleukin-12 production Effects 0.000 claims abstract description 19
- 210000004027 cell Anatomy 0.000 claims description 47
- 230000001580 bacterial effect Effects 0.000 claims description 15
- 210000000822 natural killer cell Anatomy 0.000 claims description 7
- 230000004663 cell proliferation Effects 0.000 claims description 6
- 230000006052 T cell proliferation Effects 0.000 claims description 5
- 108010065805 Interleukin-12 Proteins 0.000 abstract description 29
- 102000013462 Interleukin-12 Human genes 0.000 abstract description 27
- 230000009471 action Effects 0.000 abstract description 19
- 238000004519 manufacturing process Methods 0.000 abstract description 16
- 238000010586 diagram Methods 0.000 abstract description 3
- 241000193830 Bacillus <bacterium> Species 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 description 17
- 238000000034 method Methods 0.000 description 16
- 229940117681 interleukin-12 Drugs 0.000 description 13
- 239000002609 medium Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 108020004999 messenger RNA Proteins 0.000 description 9
- 240000006024 Lactobacillus plantarum Species 0.000 description 8
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 229940072205 lactobacillus plantarum Drugs 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 241001468192 Leuconostoc citreum Species 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 230000008520 organization Effects 0.000 description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 235000021109 kimchi Nutrition 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 230000003266 anti-allergic effect Effects 0.000 description 3
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000010195 expression analysis Methods 0.000 description 3
- 235000013376 functional food Nutrition 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 235000001497 healthy food Nutrition 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000006872 mrs medium Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 241000194032 Enterococcus faecalis Species 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 239000012979 RPMI medium Substances 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000002924 anti-infective effect Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940032049 enterococcus faecalis Drugs 0.000 description 2
- 235000021107 fermented food Nutrition 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 235000019629 palatability Nutrition 0.000 description 2
- 235000021110 pickles Nutrition 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 235000015067 sauces Nutrition 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 235000013618 yogurt Nutrition 0.000 description 2
- GUAHPAJOXVYFON-ZETCQYMHSA-N (8S)-8-amino-7-oxononanoic acid zwitterion Chemical compound C[C@H](N)C(=O)CCCCCC(O)=O GUAHPAJOXVYFON-ZETCQYMHSA-N 0.000 description 1
- 241000726096 Aratinga Species 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 235000008636 Bacillus subtilis subsp natto Nutrition 0.000 description 1
- 244000075779 Bacillus subtilis subsp natto Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 230000006051 NK cell activation Effects 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 210000000447 Th1 cell Anatomy 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- -1 acidulants Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 239000007894 caplet Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000008975 immunomodulatory function Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 230000012121 regulation of immune response Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000019710 soybean protein Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Non-Alcoholic Beverages (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
【課題】ＩＬ−１２の産生を促進する作用を有し、かつ日常的に摂取可能な飲食品等に簡便に利用できるＩＬ−１２産生促進用組成物の提供。【解決手段】ラクトバチルス・ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓｂｒｅｖｉｓ）及び納豆菌（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓｖａｒ． ｎａｔｔｏ）を含む、組成物。ラクトバチルス・ブレビスと、納豆菌との含有比率が菌体数比（ラクトバチルス・ブレビス：納豆菌）で１：１０〜１０：１である、該組成物。【選択図】図２PROBLEM TO BE SOLVED: To provide a composition for promoting IL-12 production, which has an action of promoting the production of IL-12 and can be easily used for foods and drinks that can be ingested on a daily basis. A composition comprising Lactobacillus brevis and Bacillus subtilisvar. Natto. The composition in which the content ratio of Lactobacillus brevis and Bacillus natto is 1:10 to 10: 1 in terms of the number of cells (Lactobacillus brevis: Bacillus natto). [Selection diagram] Fig. 2
Description
本発明は、乳酸菌及び納豆菌を含む組成物に関する。 The present invention relates to a composition containing lactic acid bacteria and natto bacteria.
細菌、ウイルス等の微生物の感染、腫瘍、細胞傷害等に対して生体は免疫反応によって対応するが、その免疫反応は、免疫担当細胞間の直接的又は間接的な相互作用により調節されている。そして、免疫応答の調節にはリンパ球、マクロファージ等が産生するインターロイキン、腫瘍壊死因子α等のサイトカインが重要な役割を果たしている。 The living body responds to infections of microorganisms such as bacteria and viruses, tumors, cytotoxicity, etc. by an immune response, and the immune response is regulated by direct or indirect interaction between immunocompetent cells. Cytokines such as interleukins produced by lymphocytes and macrophages and tumor necrosis factor α play an important role in the regulation of immune response.
インターロイキンに属するサイトカインのひとつであるインターロイキン１２（以下、「ＩＬ−１２」ともいう。）は、Ｔ細胞やナチュラルキラー細胞に対して細胞増殖の促進、細胞傷害活性誘導、ＩＦＮ−γ産生誘導、ＬＡＫ細胞誘導の作用を有することが確認されている。また、ＩＬ−１２は、ナイーブヘルパーＴ細胞（Ｔｈ０細胞）を、Ｔｈ１細胞に分化させＴｈ１／Ｔｈ２バランスを細胞性免疫のＴｈ１側にシフトさせる免疫調節機能を有することが確認されている。
上記の作用・機能を有することにより、ＩＬ−１２は感染症、腫瘍、アレルギー等、多くの疾患の治療に利用可能になるものと期待されている。
Interleukin 12 (hereinafter, also referred to as “IL-12”), which is one of the cytokines belonging to interleukin, promotes cell proliferation, induces cytotoxic activity, and induces IFN-γ production in T cells and natural killer cells. , It has been confirmed that it has an action of inducing LAK cells. In addition, it has been confirmed that IL-12 has an immunomodulatory function of differentiating naive helper T cells (Th0 cells) into Th1 cells and shifting the Th1 / Th2 balance to the Th1 side of cell-mediated immunity.
By having the above-mentioned actions and functions, IL-12 is expected to be usable for the treatment of many diseases such as infectious diseases, tumors and allergies.
プロバイオティクスによりＩＬ−１２の産生を誘導する方法としては、ラクトバチルス・アシドフィルス等の乳酸菌の菌体を用いることが知られている（特許文献１参照）。 As a method for inducing the production of IL-12 by probiotics, it is known to use cells of lactic acid bacteria such as Lactobacillus acidophilus (see Patent Document 1).
ここで、乳酸菌は、生理活性を有する機能性成分として注目されている。乳酸菌が有する生理活性としては、整腸作用、抗アレルギー作用、免疫賦活作用、コレステロール低減作用、脂質改善作用、糖代謝改善作用、血圧降下作用、美肌作用、安眠作用等が報告されている。このような生理活性を期待して、乳酸菌を簡便に日常的に摂取するために、乳酸菌を含有する様々な食品が市販されている。乳酸菌を含有する食品の代表例としては、例えばキムチやヨーグルト等の発酵食品が挙げられる。 Here, lactic acid bacteria are attracting attention as functional components having physiological activity. As the physiological activities of lactic acid bacteria, intestinal regulation action, antiallergic action, immunostimulatory action, cholesterol lowering action, lipid improving action, glucose metabolism improving action, blood pressure lowering action, skin beautifying action, restful sleep action and the like have been reported. In anticipation of such physiological activity, various foods containing lactic acid bacteria are commercially available in order to easily and daily ingest lactic acid bacteria. Typical examples of foods containing lactic acid bacteria include fermented foods such as kimchi and yogurt.
また、大豆を納豆菌で発酵させた納豆は、日本古来の発酵食品であり、大豆タンパク質を豊富に含み栄養価が高い。また、栄養価に加えて、プロバイオティクス作用、抗菌作用、機能性成分等による各種健康増進効果があることが報告されており、益々需要が期待されている食品である。 In addition, natto obtained by fermenting soybeans with natto bacteria is an ancient Japanese fermented food, which is rich in soybean protein and has high nutritional value. In addition to nutritional value, it has been reported that it has various health-promoting effects due to probiotic action, antibacterial action, functional ingredients, etc., and it is a food that is expected to be in increasing demand.
特許文献２には、納豆とそれに添付されるタレにエンテロコッカス・フェカリス（乳酸菌）が含有された納豆製品が記載されている。 Patent Document 2 describes a natto product containing Enterococcus faecalis (lactic acid bacterium) in natto and a sauce attached thereto.
しかしながら、特許文献１に記載の方法によっては、ＩＬ−１２の産生を促進する作用が十分とはいえない。また、特許文献２に記載の納豆製品では、エンテロコッカス・フェカリスと納豆菌とを混合して培養することにより、ＩＬ−１２誘導能が失活するため、流通、販売、飲食等の場面で制限がある。 However, depending on the method described in Patent Document 1, the action of promoting the production of IL-12 cannot be said to be sufficient. Further, in the natto product described in Patent Document 2, the IL-12 inducibility is inactivated by culturing a mixture of Enterococcus faecalis and Bacillus natto, so that there are restrictions in distribution, sales, eating and drinking, etc. is there.
そこで、本発明が解決しようとする課題は、ＩＬ−１２の産生を促進する作用を有し、かつ日常的に摂取可能な飲食品等に簡便に利用できるＩＬ−１２産生促進用組成物を提供することにある。 Therefore, the problem to be solved by the present invention is to provide a composition for promoting IL-12 production, which has an action of promoting the production of IL-12 and can be easily used for foods and drinks that can be ingested on a daily basis. To do.
本発明者は、上記課題を解決するために鋭意研究を積み重ねた結果、驚くべきことに、様々な菌の組み合わせの中から、特に、ラクトバチルス・ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）と、納豆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ｖａｒ． ｎａｔｔｏ）との組み合わせは、乳酸菌単体及び納豆菌単体よりもＩＬ−１２の産生を促進することができることを見出した。本発明はこれらの知見及び成功例に基づいて完成するに至った発明である。 As a result of diligent research to solve the above problems, the present inventor surprisingly among various combinations of bacteria, in particular, Lactobacillus brevis and Bacillus subtilis. It was found that the combination with var. Natto) can promote the production of IL-12 more than lactic acid bacteria alone and Bacillus natto alone. The present invention has been completed based on these findings and successful examples.
したがって、本発明は以下に関する。
［１］ラクトバチルス・ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）及び納豆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ｖａｒ． ｎａｔｔｏ）を含む、組成物。
［２］ 前記ラクトバチルス・ブレビスがラクトバチルス・ブレビス ミヤビスＬＢ２７株である［１］に記載の組成物。
［３］ 前記ラクトバチルス・ブレビスと、納豆菌との含有比率が菌体数比（ラクトバチルス・ブレビス：納豆菌）で１：１０〜１０：１である、［１］〜［２］のいずれか１項に記載の組成物。
［４］前記組成物がインターロイキン１２産生促進用である、［１］〜［３］のいずれか１項に記載の組成物。
［５］前記組成物がナチュラルキラー細胞増殖促進用である、［１］〜［３］のいずれか１項に記載の組成物。
［６］前記組成物がＴ細胞増殖促進用である、［１］〜［３］のいずれか１項に記載の組成物。
［７］前記組成物が飲食品用である、［１］〜［６］のいずれか１項に記載の組成物。
Therefore, the present invention relates to the following.
[1] A composition containing Lactobacillus brevis and Bacillus subtilis var. Natto.
[2] The composition according to [1], wherein the Lactobacillus brevis is a Lactobacillus brevis Miyabis LB27 strain.
[3] Any of [1] to [2], wherein the content ratio of the Lactobacillus brevis and the natto bacterium is 1:10 to 10: 1 in terms of the number of bacterial cells (Lactobacillus brevis: natto bacterium). The composition according to item 1.
[4] The composition according to any one of [1] to [3], wherein the composition is for promoting the production of interleukin 12.
[5] The composition according to any one of [1] to [3], wherein the composition is for promoting natural killer cell proliferation.
[6] The composition according to any one of [1] to [3], wherein the composition is for promoting T cell proliferation.
[7] The composition according to any one of [1] to [6], wherein the composition is for food and drink.
本発明の組成物は、ＩＬ−１２の産生を促進する作用を有し、かつ日常的に摂取可能な飲食品等に簡便に利用できる。また、ＩＬ−１２の産生を促進する作用に基づき、ナチュラルキラー細胞増殖促進作用又はＴ細胞増殖促進作用を有する。 The composition of the present invention has an action of promoting the production of IL-12 and can be easily used for foods and drinks that can be ingested on a daily basis. In addition, it has a natural killer cell proliferation promoting action or a T cell proliferation promoting action based on the action of promoting the production of IL-12.
そして、上記作用に関連して、抗ウイルス効果、抗アレルギー効果、抗感染症効果、抗腫瘍効果及び抗癌効果などを奏することが期待できる。 Then, in relation to the above-mentioned action, it can be expected to exert an antiviral effect, an antiallergic effect, an antiinfectious disease effect, an antitumor effect, an anticancer effect and the like.
以下、本発明の一態様である組成物の詳細について説明するが、本発明の技術的範囲は本項目の事項によってのみに限定されるものではなく、本発明はその目的を達成する限りにおいて種々の態様をとり得る。 Hereinafter, the details of the composition which is one aspect of the present invention will be described, but the technical scope of the present invention is not limited to the matters of the present item, and the present invention varies as long as the object is achieved. Can be taken.
〔組成物〕
本発明の一態様の組成物は、ラクトバチルス・ブレビス及び納豆菌を含む。
〔Composition〕
The composition of one aspect of the invention comprises Lactobacillus brevis and Bacillus natto.
本明細書における各用語は、別段の定めがない限り、当業者により通常用いられている意味で使用され、不当に限定的な意味を有するものとして解釈されるべきではない。
例えば、「組成物」は、通常用いられている意味のものとして特に限定されないが、例えば、２種以上の物質が組み合わさってなるものであり、具体的には、有効成分と別の物質とが組み合わさってなるもの、有効成分の２種以上が組み合わさってなるもの等が挙げられ、より具体的には、有効成分の１種以上と固形担体又は溶媒の１種以上とが組み合わさってなる固形組成物及び液性組成物等が挙げられる。
Unless otherwise specified, each term in the present specification is used in the meaning commonly used by those skilled in the art and should not be construed as having an unreasonably limited meaning.
For example, the "composition" is not particularly limited as having a commonly used meaning, but is, for example, a combination of two or more kinds of substances, specifically, an active ingredient and another substance. Examples thereof include those obtained by combining two or more kinds of active ingredients, and more specifically, one or more kinds of active ingredients and one or more kinds of solid carriers or solvents are combined. Examples thereof include solid compositions and liquid compositions.
本明細書における「ＩＬ−１２産生促進作用」とは、例えば、ＩＬ−１２遺伝子の発現量を促進すること及びＩＬ−１２タンパク質の翻訳量を増大することのうち少なくともいずれか１つの作用をいう。
「ＩＬ−１２産生促進作用」の評価方法は、特に限定されないが、例えば、後述する実施例に記載したように、腸管上皮細胞にラクトバチルス・ブレビス及び納豆菌を添加して２４時間共培養し、次いで培養後のｍＲＮＡの発現量を定量ＰＣＲで測定すること等により評価することができる。
「ＩＬ−１２産生促進作用」の程度は、特に限定されないが、例えば、後述する実施例に記載したように、菌単体を含む組成物よりも多くなる程度等が挙げられる。
As used herein, the term "IL-12 production-promoting action" refers to, for example, at least one of the actions of promoting the expression level of the IL-12 gene and increasing the translation level of the IL-12 protein. ..
The evaluation method of "IL-12 production promoting action" is not particularly limited, but for example, as described in Examples described later, Lactobacillus brevis and Bacillus natto are added to intestinal epithelial cells and co-cultured for 24 hours. Then, the expression level of mRNA after culturing can be evaluated by measuring it by quantitative PCR or the like.
The degree of the "IL-12 production promoting action" is not particularly limited, but for example, as described in Examples described later, the degree of the "IL-12 production promoting action" may be greater than that of the composition containing a simple substance.
本発明におけるラクトバシルス・ブレビスと、納豆菌との組み合わせが、ＩＬ−１２の産生を促進する理由については定かではないが、後述する実施例に記載があるとおり、ラクトバチルス・ブレビス及び納豆菌の組み合わせの組成物を添加して細胞を培養した場合は、それぞれの菌単体のみを添加した場合よりも、インターロイキン１２の産生が促進する結果が得られたことから、本発明のラクトバチルス・ブレビスと納豆菌との組み合わせは、互いのＩＬ−１２の産生作用を阻害・抑制することがない組み合わせであり、ＩＬ−１２の産生を惹起する作用機序が異なるものであると推測される。 The reason why the combination of Lactobacillus brevis and Bacillus natto in the present invention promotes the production of IL-12 is not clear, but as described in Examples described later, the combination of Lactobacillus brevis and Bacillus natto When the cells were cultured by adding the composition of the above, the result was that the production of interleukin 12 was promoted as compared with the case where only each bacterium was added. Therefore, the Lactobacillus brevis of the present invention was obtained. The combination with Bacillus natto is a combination that does not inhibit or suppress each other's IL-12 production action, and it is presumed that the mechanism of action that induces IL-12 production is different.
〔ラクトバチルス・ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）〕
ラクトバチルス・ブレビスを入手する方法としては、特に限定されないが、例えば、市販や寄託されている乳酸菌（例えば、ラクトバチルス・ブレビス ミヤビスＬＢ２７株）を利用する方法、キムチ等の漬物、乳酸菌飲料等の乳酸菌含有物から単離株として分離して利用する方法等が挙げられる。
[ Lactobacillus brevis ]
The method for obtaining Lactobacillus brevis is not particularly limited, but for example, a method using commercially available or deposited lactic acid bacteria (for example, Lactobacillus brevis Miyabis LB27 strain), pickles such as kimchi, lactic acid bacteria beverages, etc. Examples thereof include a method of separating and using it as an isolated strain from a lactic acid bacterium-containing substance.
ラクトバチルス・ブレビスを分離する方法は、特に限定されないが、例えば、常法に従って、乳酸菌含有物の抽出液を適宜希釈して、ＭＲＳ培地やＭ１７培地等の公知の乳酸菌培養用培地を用いて、１５℃〜４０℃程度で培養することを含む方法等が挙げられる。 The method for separating Lactobacillus brevis is not particularly limited, but for example, an extract of a lactic acid bacterium-containing substance is appropriately diluted according to a conventional method, and a known lactic acid bacterium culture medium such as MRS medium or M17 medium is used. Examples thereof include a method including culturing at about 15 ° C. to 40 ° C.
ラクトバチルス・ブレビスの形態は、特に限定されないが、生菌体又は加熱菌体（死菌体）のいずれでもよく、又凍結乾燥したものであってもよく、或いはこれらを含む培養物として利用することもできる。保存性の観点から加熱菌体（死菌体）であることが好ましい。 The form of Lactobacillus brevis is not particularly limited, but may be either live cells or heated cells (dead cells), may be freeze-dried, or may be used as a culture containing these. You can also do it. From the viewpoint of storage stability, it is preferably a heated cell (killed cell).
ラクトバチルス・ブレビスの菌体は、例えば、ラクトバチルス・ブレビスの培養物を遠心分離等の通常知られている固液分離手段を用いて培地を除去することにより得られる菌体等が挙げられる。 Examples of the cells of Lactobacillus brevis include cells obtained by removing the medium by using a commonly known solid-liquid separation means such as centrifugation of the culture of Lactobacillus brevis.
ラクトバチルス・ブレビスの培養物は、例えば、ラクトバチルス・ブレビスを培養して得た培養液そのものが挙げられる。 Examples of the culture of Lactobacillus brevis include the culture solution itself obtained by culturing Lactobacillus brevis.
ラクトバチルス・ブレビスの培養物又は菌体を得る具体例としては、ラクトバチルス・ブレビスをＭＲＳ培地に植菌し、１５℃〜４０℃ の範囲にて８〜７２時間培養することによりラクトバチルス・ブレビスの培養物を得ることができる。培養は振盪培養、撹拌培養又は静置培養で行うことができる。次いで得られた培養物から遠心濃縮機によって培地を除去して菌体を回収し、得られた菌体をＰＢＳ（リン酸緩衝生理食塩水：Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ）といった緩衝液などで洗浄することにより、ラクトバチルス・ブレビスの菌体を得ることが挙げられる。 As a specific example of obtaining a culture or cells of Lactobacillus brevis, Lactobacillus brevis is inoculated into an MRS medium and cultured at a temperature range of 15 ° C. to 40 ° C. for 8 to 72 hours to obtain Lactobacillus brevis. Cultures can be obtained. The culture can be carried out by shaking culture, stirring culture or static culture. Next, the medium was removed from the obtained culture by a centrifugal concentrator to collect the cells, and the obtained cells were washed with a buffer solution such as PBS (Phosphate buffered saline). , Obtaining cells of Lactobacillus brevis.
ラクトバチルス・ブレビスは、１種単独で含有されていてもよいし、２種以上組み合わせて含有されていてもよい。 Lactobacillus brevis may be contained alone or in combination of two or more.
〔納豆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ｖａｒ． ｎａｔｔｏ）〕
納豆菌を入手する方法としては、特に限定されないが、例えば、市販や寄託されている納豆菌（例えば、納豆菌 宮城野株、納豆菌 成瀬株、納豆菌 高橋株等）を利用する方法、納豆等の納豆菌含有物から単離株として分離して利用する方法等が挙げられる。
[ Bacillus subtilis var. Natto]
The method for obtaining natto bacteria is not particularly limited, but for example, a method using commercially available or deposited natto bacteria (for example, natto bacterium Miyagino strain, natto bacterium Naruse strain, natto bacterium Takahashi strain, etc.), natto, etc. Examples thereof include a method of separating and using as an isolated strain from the natto bacterium-containing substance of.
納豆菌を分離する方法は、特に限定されないが、例えば、常法に従って、納豆菌含有物の抽出液を適宜希釈して、トリプトソーヤブイヨン（日水製薬株式会社）やＬＢ培地等の公知の納豆菌培養用培地を用いて、３０℃〜５０℃程度で培養することを含む方法等が挙げられる。 The method for separating Bacillus natto is not particularly limited, but for example, the extract of the Bacillus natto-containing substance is appropriately diluted according to a conventional method, and known such as tryptosome bouillon (Nissui Pharmaceutical Co., Ltd.) and LB medium are known. Examples thereof include a method including culturing at about 30 ° C. to 50 ° C. using a medium for culturing Bacillus natto.
納豆菌の形態は、特に限定されないが、生菌体又は加熱菌体（死菌体）のいずれでもよく、又凍結乾燥したものであってもよく、或いはこれらを含む培養物として利用することもできる。 The form of Bacillus natto is not particularly limited, but it may be either live cells or heated cells (killed cells), it may be freeze-dried, or it may be used as a culture containing these. it can.
納豆菌の菌体は、例えば、納豆菌の培養物を遠心分離等の通常知られている固液分離手段を用いて培地を除去することにより得られる菌体等が挙げられる。 Examples of the bacterial cells of Bacillus natto include bacterial cells obtained by removing the medium by using a commonly known solid-liquid separation means such as centrifugation of a culture of Bacillus natto.
納豆菌の培養物は、例えば、納豆菌を培養して得た培養液そのものが挙げられる。 Examples of the culture of Bacillus natto include the culture solution itself obtained by culturing Bacillus natto.
納豆菌の培養物又は菌体を得る具体例としては、納豆菌をトリプトソーヤブイヨン培地に植菌し、３０℃〜５０℃ の範囲にて１２〜７２時間培養することにより培養物を得ることができる。培養は振盪培養、撹拌培養又は静置培養で行うことができる。次いで得られた培養物から遠心濃縮機によって培地を除去して菌体を回収し、得られた菌体をＰＢＳといった緩衝液などで洗浄することにより、納豆菌の菌体を得ることが挙げられる。 As a specific example of obtaining a culture or cells of Bacillus natto, the culture is obtained by inoculating Bacillus natto in a tryptosawyer bouillon medium and culturing in a range of 30 ° C to 50 ° C for 12 to 72 hours. Can be done. The culture can be carried out by shaking culture, stirring culture or static culture. Next, the medium is removed from the obtained culture by a centrifugal concentrator to collect the cells, and the obtained cells are washed with a buffer solution such as PBS to obtain the cells of Bacillus natto. ..
納豆菌は、１種単独で含有されていてもよいし、２種以上組み合わせて含有されていてもよい。 The natto bacterium may be contained alone or in combination of two or more.
本発明の一態様の組成物において、ラクトバチルス・ブレビスと、納豆菌との含有比率は、期待する嗜好性や機能性が認められる程度の量であれば特に限定されないが、例えば、各菌の菌体数比（ラクトバチルス・ブレビス：納豆菌）で、１：１０〜１０：１が好ましい。 In the composition of one aspect of the present invention, the content ratio of Lactobacillus brevis and Bacillus natto is not particularly limited as long as the expected palatability and functionality are recognized, but for example, of each bacterium. The cell count ratio (Lactobacillus brevis: Bacillus natto) is preferably 1:10 to 10: 1.
また、本発明の一態様の組成物は、インターロイキン１２の産生促進作用を有することからインターロイキン１２産生促進用組成物である。また、Ｔ細胞及びナチュラルキラー細胞は、インターロイキン１２によって細胞増殖が促進する。したがって、本発明の組成物は、インターロイキン１２の産生促進作用に基づきＴ細胞増殖促進作用及びナチュラルキラー細胞増殖促進作用を有することから、本発明の一態様の組成物は、Ｔ細胞増殖促進作用組成物及びナチュラルキラー細胞増殖促進作用組成物である。 Moreover, since the composition of one aspect of the present invention has an interleukin 12 production promoting action, it is an interleukin 12 production promoting composition. In addition, T cells and natural killer cells are promoted by interleukin 12. Therefore, since the composition of the present invention has a T cell proliferation promoting action and a natural killer cell proliferation promoting action based on the production promoting action of interleukin 12, the composition of one aspect of the present invention has a T cell proliferation promoting action. The composition and the natural killer cell proliferation promoting action composition.
本発明の一態様の組成物は、その摂取方法については特に限定されないが、例えば、経口的又は非経口的に適用される。
非経口的な適用としては、皮内、皮下、静脈内、筋肉内投与等による注射及び注入；経皮；鼻、咽頭等の粘膜からの吸入等が挙げられるが、これらに限定されない。
The composition of one aspect of the present invention is not particularly limited in its ingestion method, but is applied, for example, orally or parenterally.
Parenteral applications include, but are not limited to, injection and injection by intradermal, subcutaneous, intravenous, intramuscular administration, etc .; transdermal; inhalation through mucosa such as the nose and pharynx.
本発明の一態様の組成物の摂取個体は、特に限定されないが、例えば、動物、中でも哺乳類が挙げられ、哺乳類としてはヒト、イヌ、ネコ、ウシ、ウマ、ブタ、ヒツジ等が挙げられ、鳥類ではニワトリ、インコ等があげられ、これらの中でもヒトであることが好ましい。 The individual ingesting the composition of one aspect of the present invention is not particularly limited, and examples thereof include animals, especially mammals, and mammals include humans, dogs, cats, cows, horses, pigs, sheep, and birds. Examples include chickens and parakeets, and among these, humans are preferable.
本発明の一態様の組成物は、ラクトバチルス・ブレビス及び納豆菌に追加して、本発明の目的を達成し得る限り、その他の成分を含有することができる。その他の成分の含有量は、本発明の課題の解決を妨げない限り特に限定されず、適宜設定される。 The composition of one aspect of the present invention may contain other components in addition to Lactobacillus brevis and Bacillus natto as long as the object of the present invention can be achieved. The content of other components is not particularly limited as long as it does not interfere with the solution of the problem of the present invention, and is appropriately set.
他の成分としては、例えば、糖質甘味料、安定化剤、乳化剤、澱粉、澱粉加工物、澱粉分解物、食塩、着香料、着色料、酸味料、風味原料、栄養素、果汁、賦形剤、増量剤、結合剤、増粘剤、香油、保存剤、緩衝剤等の通常の食品加工や医薬品加工に使用される添加物、動植物性食材、加工食品等を挙げることができる。他の成分の使用量は、本発明の課題の解決を妨げない限り特に限定されず、適宜設定される。 Other ingredients include, for example, sugar sweeteners, stabilizers, emulsifiers, starches, processed starch products, starch decomposition products, salt, flavoring agents, coloring agents, acidulants, flavor ingredients, nutrients, fruit juices, excipients. , Additives used in ordinary food processing such as bulking agents, binders, thickeners, perfume oils, preservatives, buffers, and pharmaceutical processing, animal and vegetable foodstuffs, processed foods, and the like. The amount of the other component used is not particularly limited as long as it does not interfere with the solution of the problem of the present invention, and is appropriately set.
本発明の一態様の組成物は、通常用いられる形態であれば特に限定されないが、例えば、固形状、液状、ゲル状、懸濁液状、クリーム状、シート状、スティック状、粉状、粒状、顆粒状、錠状、棒状、板状、ブロック状、ペースト状、カプセル状、カプレット状等の各形態が採用される。 The composition of one aspect of the present invention is not particularly limited as long as it is in a commonly used form, and for example, solid, liquid, gel, suspension, cream, sheet, stick, powder, granule, etc. Granules, tablets, rods, plates, blocks, pastes, capsules, caplets and the like are used.
本発明の一態様の組成物が飲食品用組成物である場合は、あらゆる飲食品の形態をとり得るが、例えば、ヨーグルトやチーズ等の乳製品、キムチ等の漬物、納豆等の大豆製品、飲料、調味料等が挙げられる。 When the composition of one aspect of the present invention is a composition for food and drink, it can take any form of food and drink, for example, dairy products such as yogurt and cheese, pickles such as kimchi, and soybean products such as natto. Beverages, seasonings and the like can be mentioned.
本発明の一態様の組成物が飲食品用組成物である場合の具体的な形態としては、例えば、ラクトバチルス・ブレビスを含むキムチと、納豆菌を含む納豆とを混合した形態、納豆菌を含む納豆と、当該納豆に添付されるタレにラクトバチルス・ブレビスが含有されたものとからなる形態等が挙げられる。このように、本発明の一態様の組成物は、キムチや納豆に各菌を含有させることで、ＩＬ−１２産生促進作用を確保しながら、嗜好性を阻害することなく日常的に摂取している飲食品に簡便に適用することができる。 As a specific form when the composition of one aspect of the present invention is a composition for food and drink, for example, a form in which kimchi containing Lactobacillus brevis and natto containing natto bacteria are mixed, natto bacteria. Examples thereof include a form consisting of natto containing natto and a sauce attached to the natto containing lactobacillus brevis. As described above, the composition of one aspect of the present invention is ingested on a daily basis without impairing palatability while ensuring the IL-12 production promoting action by containing each bacterium in kimchi or natto. It can be easily applied to foods and drinks.
本発明の組成物の摂取量は特に限定されず、摂取者の体格や組成物の形態に合わせて適宜設定することができ、例えば、菌体数で１食分あたり１００億個以上、好ましくは２５０億個以上となるように摂取することができる。 The amount of the composition of the present invention to be ingested is not particularly limited and can be appropriately set according to the physique of the ingestor and the form of the composition. For example, the number of cells per serving is 10 billion or more, preferably 250. It can be ingested to be 100 million or more.
飲食品用組成物の具体的な一態様は、例えば、生体に対して一定の機能性を有する飲食品である機能性飲食品である。機能性飲食品は、例えば、特定保健用飲食品、機能性表示飲食品、栄養機能飲食品、保健機能飲食品、特別用途飲食品、栄養補助飲食品、健康補助飲食品、サプリメント、美容飲食品等のいわゆる健康飲食品に加えて、乳児用飲食品、妊産婦用飲食品、高齢者用飲食品等の特定者用飲食品を包含する。さらに機能性飲食品は、コーデックス（ＦＡＯ／ＷＨＯ合同食品規格委員会）の食品規格に基づく健康強調表示（Ｈｅａｌｔｈ ｃｌａｉｍ）が適用される健康飲食品を包含する。 A specific aspect of the composition for food and drink is, for example, a functional food and drink which is a food and drink having a certain functionality with respect to a living body. Functional foods and drinks include, for example, foods and drinks for specified health use, foods and drinks with functional claims, foods and drinks with nutritional functions, foods and drinks with health functions, foods and drinks for special purposes, foods and drinks for nutritional supplements, healthy foods and drinks, supplements, beauty foods and drinks. In addition to so-called healthy foods and drinks such as, etc., foods and drinks for specific persons such as foods and drinks for infants, foods and drinks for pregnant women, and foods and drinks for the elderly are included. Further, functional foods and drinks include healthy foods and drinks to which a health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) is applied.
〔製造方法〕
本発明の別の一態様は、本発明の一態様の組成物の製造方法である。本発明の一態様の製造方法は、ラクトバチルス・ブレビスと、納豆菌とを混合することにより、ＩＬ−１２産生促進用組成物を得る工程を含む。ＩＬ−１２産生促進用組成物の原料としては、上記した本発明の一態様のＩＬ−１２産生促進用組成物の具体例や成分を特に限定せずに挙げることができる。
〔Production method〕
Another aspect of the present invention is a method for producing a composition according to another aspect of the present invention. The production method of one aspect of the present invention includes a step of obtaining a composition for promoting IL-12 production by mixing Lactobacillus brevis and Bacillus natto. As the raw material of the composition for promoting IL-12 production, specific examples and components of the composition for promoting IL-12 production according to the above-described aspect of the present invention can be mentioned without particular limitation.
本発明の一態様の製造方法では、本発明の目的を達成し得る限り、上記した工程の前段若しくは後段又は工程中に、種々の工程や操作を加入することができる。 In the production method of one aspect of the present invention, various steps and operations can be added to the pre-stage or the post-stage or the process of the above-mentioned steps as long as the object of the present invention can be achieved.
以下、本発明を実施例により更に詳細に説明するが、本発明はこれら実施例に限定されるものではなく、本発明の課題を解決し得る限り、本発明は種々の態様をとることができる。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples, and the present invention can take various aspects as long as the problems of the present invention can be solved. ..
［例１．乳酸菌と納豆菌との組み合わせによるインターロイキン１２の産生促進試験］
乳酸菌としてラクトバチルス・ブレビス、ラクトバチルス・プランタラム、リューコノストック・シトレウムと納豆菌とを使用し、インターロイキン１２産生促進試験を実施した。
[Example 1. Interleukin 12 production promotion test by combination of lactic acid bacteria and natto bacteria]
An interleukin 12 production promotion test was carried out using Lactobacillus brevis, Lactobacillus plantarum, Leuconostoc citreum and Bacillus natto as lactic acid bacteria.
（１）菌体及び細胞
乳酸菌としてラクトバチルス・ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ） ミヤビスＬＢ２７株、ラクトバチルス・プランタラム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）及びリューコノストック・シトレウム（Ｌｅｕｃｏｎｏｓｔｏｃ ｃｉｔｒｅｕｍ）を使用した。本出願人は、該乳酸菌株を下記の条件で寄託した。
(1) Lactobacillus brevis as bacteria and cells lactobacilli (Lactobacillus brevis) Miyabisu LB27 strain, were used Lactobacillus plantarum (Lactobacillus plantarum) and Leuconostoc, citreum (Leuconostoc citreum). The applicant deposited the lactic acid bacterium strain under the following conditions.
〔ラクトバチルス・ブレビス〕
（ｉ） 寄託機関名： 独立行政法人製品評価技術基盤機構 バイオテクノロジーセンター 特許微生物寄託センター
（ｉｉ） 連絡先： 住所 〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８
電話番号 ０４３８−２０−５５８０
（ｉｉｉ） 受託番号：ＮＩＴＥ Ｐ−０３２３７
（ｉｖ） 識別の表示：ＬＢ
（ｖ） 通知年月日：２０２０年７月１５日
（ｖｉ） 通知番号：２０２０−０２１８
〔ラクトバチルス・プランタラム〕
（ｉ） 寄託機関名： 独立行政法人製品評価技術基盤機構 バイオテクノロジーセンター 特許微生物寄託センター
（ｉｉ） 連絡先： 住所 〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８
電話番号 ０４３８−２０−５５８０
（ｉｉｉ） 受託番号：ＮＩＴＥ Ｐ−０３２３８
（ｉｖ） 識別の表示：ＬＰ
（ｖ） 通知年月日：２０２０年７月１５日
（ｖｉ） 通知番号：２０２０−０２１９
〔リューコノストック・シトレウム〕
（ｉ） 寄託機関名： 独立行政法人製品評価技術基盤機構 バイオテクノロジーセンター 特許微生物寄託センター
（ｉｉ） 連絡先： 住所 〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８
電話番号 ０４３８−２０−５５８０
（ｉｉｉ） 受託番号：ＮＩＴＥ Ｐ−０３２３９
（ｉｖ） 識別の表示：ＬＣ
（ｖ） 受領日：２０２０年７月１５日
（ｖｉ） 通知番号：２０２０−０２２０
[Lactobacillus Brevis]
(I) Depositary organization name: Independent Administrative Institution Product Evaluation Technology Infrastructure Organization Biotechnology Center Patented Microbial Deposit Center (ii) Contact: Address 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(Iii) Contract number: NITE P-03237
(Iv) Identification display: LB
(V) Notification date: July 15, 2020 (vi) Notification number: 2020-0218
[Lactobacillus plantarum]
(I) Depositary organization name: Independent Administrative Institution Product Evaluation Technology Infrastructure Organization Biotechnology Center Patented Microbial Deposit Center (ii) Contact: Address 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(Iii) Contract number: NITE P-03238
(Iv) Identification display: LP
(V) Notification date: July 15, 2020 (vi) Notification number: 2020-0219
[Leuconostoc Citreum]
(I) Depositary organization name: Independent Administrative Institution Product Evaluation Technology Infrastructure Organization Biotechnology Center Patented Microbial Deposit Center (ii) Contact: Address 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(Iii) Contract number: NITE P-03239
(Iv) Identification display: LC
(V) Date of receipt: July 15, 2020 (vi) Notification number: 2020-0220
納豆菌としては、有限会社宮城野納豆製造所より入手した納豆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ｖａｒ． ｎａｔｔｏ）宮城野株を使用した。
細胞は、ヒト大腸癌細胞ＣＯＬＯ−３２０（理化学研究所 バイオリソース研究センターより入手）を使用した。
As the natto bacterium, a natto bacterium ( Bacillus subtilis var. Natto) Miyagino strain obtained from Miyagino Natto Factory Co., Ltd. was used.
As cells, human colon cancer cell COLO-320 (obtained from RIKEN BioResource Research Center) was used.
（２）菌液の調製
乳酸菌については、各乳酸菌をＭＲＳ培地（日本ベクトン・ディッキンソン社製）を用いて、３０℃で２４時間静置培養した。培養した菌液全量を３０００ｒｐｍで５分間遠心し、上清を除去して、集菌した。次いで、ＰＢＳに懸濁して７５００ｒｐｍで５分間遠心後、上清を除去する洗浄操作を２回繰り返し、最後にＰＢＳに懸濁して、１２１℃で２０分間のオートクレーブ処理をした後、各乳酸菌の菌液を得た。
納豆菌については、トリプトソーヤブイヨン（日水製薬株式会社）を用いて、３７℃で４８時間振盪培養した。その後の操作は、乳酸菌と同様の操作を行い、納豆菌の菌液を得た。
(2) Preparation of bacterial solution For lactic acid bacteria, each lactic acid bacterium was statically cultured at 30 ° C. for 24 hours using an MRS medium (manufactured by Becton Dickinson, Japan). The entire amount of the cultured bacterial solution was centrifuged at 3000 rpm for 5 minutes, the supernatant was removed, and the cells were collected. Next, the cells were suspended in PBS, centrifuged at 7500 rpm for 5 minutes, and the washing operation for removing the supernatant was repeated twice. Finally, the cells were suspended in PBS, autoclaved at 121 ° C. for 20 minutes, and then the bacteria of each lactic acid bacterium. I got the liquid.
Bacillus natto was cultured with shaking at 37 ° C. for 48 hours using trypto-soya bouillon (Nissui Pharmaceutical Co., Ltd.). Subsequent operations were the same as for lactic acid bacteria to obtain a bacterial solution of Bacillus natto.
（３）細胞培養
インターロイキン１２産生促進試験は、ＣＯＬＯ−３２０細胞におけるインターロイキン１２ｍＲＮＡの発現量を指標として評価した。ＣＯＬＯ−３２０細胞を、２４ウェルプレートにＲＰＭＩ（ロズウェルパーク記念研究所：Ｒｏｓｗｅｌｌ Ｐａｒｋ Ｍｅｍｏｒｉａｌ Ｉｎｓｔｉｔｕｔｅ）培地（ナカライテスク株式会社製：１０％ＮＣＳ，１％アンピシリン・ストレプトマイシン添加）中１．０×１０６ｃｅｌｌｓ／ｍｌの濃度となるように播種し、３７℃、５％ＣＯ２下で２４時間培養して定着させ、さらに新鮮なＲＰＭＩ培地に交換後、３７℃、５％ＣＯ２下で２４時間前培養した。次いで、表１に記載の菌体濃度（菌体数１０７個／ｍＬ）に従って菌液を添加して調製した各ＲＰＭＩ培地 １ｍＬに交換して、３７℃、５％ＣＯ２下で２４時間前培養した。
(3) Cell culture The interleukin 12 production promotion test was evaluated using the expression level of interleukin 12 mRNA in COLO-320 cells as an index. COLO-320 cells were placed in a 24-well plate in RPMI (Roswell Park Memorial Institute) medium (manufactured by Nakaraitesk Co., Ltd .: 10% NCS, 1% ampicillin streptomycin added) 1.0 × 10 6 cells. Seed to a concentration of / ml, cultured at 37 ° C. under 5% CO 2 for 24 hours to settle, and after replacement with fresh RPMI medium, pre-cultured at 37 ° C. under 5% CO 2 for 24 hours. did. Then, replace each RPMI medium 1mL prepared by adding bacterial liquid according cell concentration described (cell number 107 cells / mL) in Table 1, 37 ℃, 5% CO 2 under 24 hours ago It was cultured.
（４）顕微鏡観察
各菌液の菌体及び菌液を混合した混合液の菌体に対してグラム染色を行い、位相差顕微鏡を用いて菌体の形態を確認した。
(4) Microscopic observation Gram stain was performed on the cells of each bacterial solution and the cells of the mixed solution in which the bacterial solutions were mixed, and the morphology of the bacterial cells was confirmed using a phase-contrast microscope.
（５）遺伝子発現解析
培養した細胞からＴＲＩｚｏｌ Ｒｅａｇｅｎｔ試薬（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）を用いて、試薬の製造会社の推奨の操作方法に従ってｔｏｔａｌＲＮＡを回収し、ｔｏｔａｌＲＮＡ ２μｇをＳｕｐｅｒＳｃｒｉｐｔ ＶＩＬＯ ＭａｓｔｅｒＭｉｘ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）を用いて、試薬の製造会社の推奨の操作方法に従ってｃＤＮＡを合成し、得られたｃＤＮＡを鋳型として、インターロイキン１２遺伝子に対するプライマー（北海道システム・サイエンス株式会社）及びＫＡＰＡ ＳＹＢＲ ＦＡＳＴ ｑＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ （２Ｘ） Ｋｉｔ（カパ・バイオシステムズ社）を用いて定量リアルタイムＰＣＲを行い、インターロイキン１２のｍＲＮＡ発現量を測定した。内在性コントロールとして、ＧＡＰＤＨプライマー（北海道システム・サイエンス株式会社）を用いて、ＧＡＰＤＨのｍＲＮＡ発現量を測定した。インターロイキン１２のｍＲＮＡの発現量は、内在性コントロールのＧＡＰＤＨのｍＲＮＡの発現量に対する相対発現量とし、さらに菌体を含まない培地で培養した細胞（対照）のインターロイキン１２のｍＲＮＡの発現量を１．０として示した。
(5) Gene expression analysis Using a TRIzol Reagent reagent (Thermo Fisher Scientific) from the cultured cells, totalRNA was recovered according to the operation method recommended by the reagent manufacturer, and 2 μg of totalRNA was collected from SuperScript VILO MasterMix (ThersimSterMix) The cDNA was synthesized according to the procedure recommended by the reagent manufacturer, and the obtained cDNA was used as a template as a primer for the interleukin 12 gene (Hokkaido System Science Co., Ltd.) and KAPA SYBR FAST qPCR Master Mix (2X). ) Quantitative real-time PCR was performed using Kit (Kapa Biosystems) to measure the mRNA expression level of interleukin 12. As an endogenous control, GAPDH primer (Hokkaido System Science Co., Ltd.) was used to measure the mRNA expression level of GAPDH. The expression level of interleukin 12 mRNA is defined as the relative expression level of the endogenous control GAPDH mRNA expression level, and the expression level of interleukin 12 mRNA expression in cells (control) cultured in a cell-free medium is used. It is shown as 1.0.
（６）結果
（ｉ）菌体の顕微鏡画像
各菌液及び混合液の菌体についての菌体の形態の画像を図１に示す。図１に示すとおり、ラクトバチルス・ブレビス（ＬＢ）、リューコノストック・シトレウム（ＬＣ）、ラクトバチルス・プランタラム（ＬＰ）及び納豆菌（ＢＳ）は菌体の形態が保持されていることが示された。
(6) Results (i) Microscopic image of bacterial cells Fig. 1 shows an image of the morphology of the bacterial cells of each bacterial solution and the mixed solution. As shown in FIG. 1, it is shown that Lactobacillus brevis (LB), Leuconostoc citreum (LC), Lactobacillus plantarum (LP) and Bacillus natto (BS) retain their morphology. Was done.
（ｉｉ）遺伝子発現解析の結果
遺伝子発現解析の結果を図２に示す。図２に示すとおり、納豆菌とラクトバチルス・ブレビスとを組み合わせた実施例１〜４は、納豆菌単体である比較例１及び２並びにラクトバチルス・ブレビス単体である比較例３及び４と比較して、インターロイキン１２の相対発現量が多かった。すなわち、納豆菌とラクトバチルス・ブレビスとを組み合わせは菌単体よりインターロイキン１２のｍＲＮＡの発現量を促進した。
(Ii) Results of gene expression analysis The results of gene expression analysis are shown in FIG. As shown in FIG. 2, Examples 1 to 4 in which Bacillus natto and Lactobacillus brevis are combined are compared with Comparative Examples 1 and 2 which are natto bacteria alone and Comparative Examples 3 and 4 which are Lactobacillus brevis alone. Therefore, the relative expression level of interleukin 12 was high. That is, the combination of Bacillus natto and Lactobacillus brevis promoted the expression level of interleukin 12 mRNA rather than the bacterium alone.
一方、ラクトバチルス・プランタラム又はリューコノストック・シトレウムと納豆菌とを組み合わせた比較例５及び６は、インターロイキン１２の相対発現量が納豆菌単独の場合と同等かそれ以下であった。 On the other hand, in Comparative Examples 5 and 6 in which Lactobacillus plantarum or Leuconostoc citreum was combined with Bacillus natto, the relative expression level of interleukin 12 was equal to or less than that of Bacillus natto alone.
以上の結果から、納豆菌とラクトバチルス・ブレビスとを組み合わせは、インターロイキン１２のｍＲＮＡの発現量を相乗的に促進する作用を有することが示された。 From the above results, it was shown that the combination of Bacillus natto and Lactobacillus brevis has an action of synergistically promoting the expression level of interleukin 12 mRNA.
本発明の一態様の組成物は、ＩＬ−１２産生促進作用を通じて、ナチュラルキラー細胞賦活効果、細胞性免疫賦活効果、免疫賦活効果、抗ウイルス効果、抗アレルギー効果、抗感染症効果、抗腫瘍効果、抗癌効果等が期待され、経口的又は非経口的に摂取可能であり、飲食品をはじめ、医薬品、医薬部外品、化粧品、サプリメント等に有用である。 The composition of one aspect of the present invention has a natural killer cell activation effect, a cell-mediated immunization activation effect, an immunostimulation effect, an antiviral effect, an antiallergic effect, an antiinfectious disease effect, and an antitumor effect through an IL-12 production promoting action. It is expected to have anti-cancer effects and can be taken orally or parenterally, and is useful for foods and drinks, pharmaceuticals, quasi-drugs, cosmetics, supplements, and the like.
Claims (5)
The composition according to any one of claims 1 to 4, wherein the composition is for food and drink.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020139609A JP6836298B1 (en) | 2020-08-20 | 2020-08-20 | Composition containing lactic acid bacteria and natto bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020139609A JP6836298B1 (en) | 2020-08-20 | 2020-08-20 | Composition containing lactic acid bacteria and natto bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP6836298B1 true JP6836298B1 (en) | 2021-02-24 |
| JP2022035360A JP2022035360A (en) | 2022-03-04 |
Family
ID=74661728
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020139609A Active JP6836298B1 (en) | 2020-08-20 | 2020-08-20 | Composition containing lactic acid bacteria and natto bacteria |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6836298B1 (en) |
-
2020
- 2020-08-20 JP JP2020139609A patent/JP6836298B1/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2022035360A (en) | 2022-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6685974B2 (en) | Novel lactic acid bacterium having IgA production promoting action and use thereof | |
| EP2360237B1 (en) | Novel lactobacillus plantarum and composition containing the same | |
| TWI645854B (en) | Lactobacillus plantarum TCI378 and its application in reducing fat and improving gastrointestinal function | |
| JP7179343B2 (en) | Novel lactic acid strain and immunostimulant containing the same | |
| KR101271379B1 (en) | Lactic acid bacterium having immunoregulatory activity derived from moromi for wine fermentation | |
| KR102543494B1 (en) | Novel probiotics and use thereof | |
| JP5337535B2 (en) | NK activity enhancer | |
| US20120301505A1 (en) | Immunostimulating Agent and Method for Production Thereof | |
| TW200538047A (en) | A composition having immunoregulating activities | |
| JP6796299B2 (en) | Immunostimulatory composition and cytokine production promoting composition | |
| JP7516053B2 (en) | Composition, food and drink composition, nutritional composition and formula | |
| CN108795823B (en) | It is a kind of improve women pregnant and lying-in women's intestinal flora probiotics cultural method and application | |
| TW201609120A (en) | Intestinal barrier function enhancer containing lactic acid bacteria | |
| WO2020067422A1 (en) | Composition for activating t cells | |
| JP6836298B1 (en) | Composition containing lactic acid bacteria and natto bacteria | |
| JP6061530B2 (en) | NASH preventive and therapeutic agent | |
| JP2006076961A (en) | Immunopotentiator composition | |
| JP2023023981A (en) | LACTIC ACID BACTERIA FORMULATION FOR PROMOTING IN VIVO IgA AND IFN-γ PRODUCTION AND ORAL COMPOSITION | |
| JP7331310B2 (en) | Composition for suppressing decrease in immunostimulatory activity due to passage of storage time of lactic acid bacteria having immunostimulatory activity or processed product thereof | |
| WO2024071336A1 (en) | Acidic bacterial exopolysaccharide having immunopotentiating activity | |
| JP2019089733A (en) | Intestinal bacterial flora improver | |
| JP5481702B2 (en) | Novel lactic acid bacteria having immunostimulatory action and gastric juice resistance | |
| JP2024128968A (en) | Composition for promoting ghrelin secretion | |
| TW202335676A (en) | Intestine immunostimulant and IgA generation promoter for ensuring Apilactobacillus lactobacillus having IgA generation promotion effect and immunostimulation function | |
| JP5851242B2 (en) | Lactic acid bacteria having thioredoxin-inducing activity, and foods and beverages and pharmaceuticals for preventing and / or improving biological injury mediated by thioredoxin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20200821 |
|
| A871 | Explanation of circumstances concerning accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A871 Effective date: 20200821 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20200821 |
|
| A975 | Report on accelerated examination |
Free format text: JAPANESE INTERMEDIATE CODE: A971005 Effective date: 20200916 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20201006 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20201027 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20201201 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20201208 |
|
| 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: 20210105 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210122 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6836298 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |