JP5261617B2 - Novel lactic acid bacteria and pharmaceuticals, foods and drinks, and feeds containing the novel lactic acid bacteria - Google Patents
Novel lactic acid bacteria and pharmaceuticals, foods and drinks, and feeds containing the novel lactic acid bacteria Download PDFInfo
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- JP5261617B2 JP5261617B2 JP2012537042A JP2012537042A JP5261617B2 JP 5261617 B2 JP5261617 B2 JP 5261617B2 JP 2012537042 A JP2012537042 A JP 2012537042A JP 2012537042 A JP2012537042 A JP 2012537042A JP 5261617 B2 JP5261617 B2 JP 5261617B2
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- lactic acid
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- mcc1375
- lactobacillus paracasei
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Classifications
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Description
本発明は、新規乳酸菌に関する。また、本発明は、新規乳酸菌を含有する医薬、飲食品、及び飼料に関する。 The present invention relates to a novel lactic acid bacterium. Moreover, this invention relates to the pharmaceutical, food-drinks, and feed containing novel lactic acid bacteria.
乳酸菌は様々な薬理作用を有することが知られており、広く利用されている。
例えば、感染症の予防作用及び治療作用を示す乳酸菌が報告されている（非特許文献１）。乳酸菌の作用は、宿主の細胞性免疫を賦活化することや、腸管や呼吸器等の粘膜からのＩｇＡの分泌を亢進させること等によるものであると報告されている。Lactic acid bacteria are known to have various pharmacological actions and are widely used.
For example, a lactic acid bacterium showing a preventive action and a therapeutic action for infectious diseases has been reported (Non-patent Document 1). It has been reported that the action of lactic acid bacteria is due to activation of cellular immunity of the host and enhancement of IgA secretion from mucous membranes such as the intestinal tract and respiratory organs.
例えば、ラクトバチルス・カゼイに属する乳酸菌には、宿主の免疫担当細胞からのＩＬ−１２（インターロイキン−１２）やＩＦＮ−γ等のサイトカイン産生を誘導することで、宿主の細胞性免疫を賦活化し、インフルエンザ・ウイルス等の感染を防御するものが報告されている（非特許文献２〜４）。ＩＬ−１２やＩＦＮ−γは、ナイーブ・ヘルパーＴ細胞をタイプ１ヘルパーＴ細胞（Ｔｈ１）へ分化誘導する作用やナチュラルキラー細胞（ＮＫ細胞）の活性化、マクロファージ等の細胞の貪食作用を亢進する作用を持つサイトカインで、ウイルスや細菌による感染防御作用や宿主の抗腫瘍効果に関わるとされる。また、ＩＬ−１２は抗腫瘍効果、抗転移効果、日和見感染症等の感染症に対する効果、喘息等のアレルギー疾患に対する予防、治療効果が知られている（非特許文献５）。
For example, lactic acid bacteria belonging to Lactobacillus casei activate cellular immunity of the host by inducing production of cytokines such as IL-12 (interleukin-12) and IFN-γ from the host immunocompetent cells. Those that protect against infections such as influenza viruses have been reported (Non-Patent
ＩＬ−１２産生誘導能を有する乳酸菌としては、ラクトバチルス・パラカゼイＫＷ３１１０（ＦＥＲＭ ＢＰ−０８６３４）が知られている（特許文献１）。ラクトバチルス・パラカゼイＫＷ３１１０のＩＬ−１２産生量は、ラクトバチルス・ラムノーサスＡＴＣＣ５３１０３株（ＧＧ株）の１．９倍であることが報告されている（非特許文献６）。また、ラクトバチルス・ラムノーサスＧＧ株は、インフルエンザ・ウイルスに対する感染予防効果が報告されている（非特許文献７）。 As a lactic acid bacterium having IL-12 production inducing ability, Lactobacillus paracasei KW3110 (FERM BP-08634) is known (Patent Document 1). It has been reported that the amount of IL-12 produced by Lactobacillus paracasei KW3110 is 1.9 times that of Lactobacillus rhamnosus ATCC 53103 strain (GG strain) (Non-patent Document 6). In addition, Lactobacillus rhamnosus GG strain has been reported to prevent infection against influenza virus (Non-patent Document 7).
このような感染防御作用を有する乳酸菌の作用は、生菌に限られるものではなく、死菌であっても効果を有することが知られている。しかしながら、乳酸菌が腸管粘膜免疫に及ぼす影響を生菌と死菌で比較した研究では、生菌の方が死菌よりも腸管粘膜免疫に及ぼす影響が強いことが報告されている（非特許文献８）。 It is known that the action of lactic acid bacteria having such an infection-protecting action is not limited to live bacteria, but is effective even when killed bacteria. However, in a study comparing the effects of lactic acid bacteria on intestinal mucosal immunity between live and dead bacteria, it is reported that live bacteria have a stronger effect on intestinal mucosal immunity than dead bacteria (Non-patent Document 8). ).
上述のように、乳酸菌は種々の好ましい薬理作用を示し、さらに、乳酸菌は、これを生菌として維持して、腸管粘膜などに到達させることが、好ましい。しかし、乳酸菌を生きたまま維持して、さらに腸管粘膜などに到達させることは、容易ではない。
例えば、乳酸菌が含まれる飲料などは、酸性の飲料であることが通常であり、この酸性条件（低ｐＨ条件）では、乳酸菌は死滅しやすい。さらに、経口投与された乳酸菌は、酸性の胃液にさらされるために、腸管粘膜の到達の前に死滅してしまいやすい。As described above, lactic acid bacteria exhibit various preferable pharmacological actions, and it is preferable that lactic acid bacteria are maintained as live bacteria and reach the intestinal mucosa. However, it is not easy to keep the lactic acid bacteria alive and reach the intestinal mucosa.
For example, beverages containing lactic acid bacteria are usually acidic beverages, and under these acidic conditions (low pH conditions), lactic acid bacteria are likely to die. Furthermore, since orally administered lactic acid bacteria are exposed to acidic gastric juice, they tend to die before reaching the intestinal mucosa.
したがって、本発明の目的は、酸性条件下の生残性の高い新規乳酸菌、及び当該乳酸菌を含有する医薬、飲食品、及び飼料を提供することにある。 Accordingly, an object of the present invention is to provide a novel lactic acid bacterium having a high survival rate under acidic conditions, and a medicine, food and drink, and feed containing the lactic acid bacterium.
本発明者らは、酸性条件下の生残性の高い新規乳酸菌を鋭意探索し、ラクトバチルス・パラカゼイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ）ＭＣＣ１３７５株（本菌株は、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ＦＥＲＭ ＢＰ−１１３１３として寄託された。）を見出し、本発明を完成するに至った。
本発明に係るラクトバチルス・パラカゼイ株は、従来の乳酸菌と比較して、酸性条件下で生残性が高いものであり、さらに、ＩＬ−１２産生を促進し、ＮＫ細胞を活性化するものであり、インフルエンザ・ウイルスの増殖を抑制し、インフルエンザ・ウイルスによる症状を予防することができるものであり、すなわち、優れた感染予防及び感染防御の作用を示すものであった。The present inventors diligently searched for a novel lactic acid bacterium having a high survival rate under acidic conditions, and the Lactobacillus paracasei MCC1375 strain (this strain is incorporated into the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology). Was deposited as FERM BP-11313.) And the present invention was completed.
The Lactobacillus paracasei strain according to the present invention has higher survival under acidic conditions than conventional lactic acid bacteria, and further promotes IL-12 production and activates NK cells. Yes, it can suppress the growth of influenza virus and prevent symptoms caused by influenza virus, that is, it exhibits excellent infection prevention and infection protection.
したがって、本発明は、次の（１）〜（１１）にもある。
（１）
ラクトバチルス・パラカゼイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ）ＭＣＣ１３７５株（ＦＥＲＭ ＢＰ−１１３１３）。
（２）
（１）に記載の株の変異株。
（３）
変異株が酸性生残性を有する、（２）に記載の株。
（４）
（１）〜（３）のいずれかに記載の株を含有する組成物。
（５）
組成物が医薬組成物である（４）に記載の組成物。
（６）
組成物が飲食品組成物である（４）に記載の組成物。
（７）
飲食品が発酵乳又は乳酸菌飲料である（６）に記載の組成物。
（８）
組成物が飼料組成物である（４）に記載の組成物。
（９）
（１）〜（３）のいずれかに記載の株を有効成分として含有するインターロイキン−１２産生促進用組成物。
（１０）
（１）〜（３）のいずれかに記載の株を有効成分として含有するＮＫ細胞活性化用組成物。
（１１）
（１）〜（３）のいずれかに記載の株を有効成分として含有する抗インフルエンザ・ウイルス用組成物。Therefore, this invention exists also in following (1)-(11).
(1)
Lactobacillus paracasei MCC1375 strain (FERM BP-11313).
(2)
A mutant of the strain described in (1).
(3)
The strain according to (2), wherein the mutant strain has acidic survival.
(4)
The composition containing the strain | stump | stock in any one of (1)-(3).
(5)
The composition according to (4), wherein the composition is a pharmaceutical composition.
(6)
The composition as described in (4) whose composition is a food-drinks composition.
(7)
The composition according to (6), wherein the food or drink is fermented milk or a lactic acid bacteria beverage.
(8)
The composition according to (4), wherein the composition is a feed composition.
(9)
A composition for promoting interleukin-12 production comprising the strain according to any one of (1) to (3) as an active ingredient.
(10)
The composition for NK cell activation containing the strain | stump | stock in any one of (1)-(3) as an active ingredient.
(11)
An anti-influenza virus composition comprising the strain according to any one of (1) to (3) as an active ingredient.
したがって、本発明は、次の（２１）〜（２６）にもある。
（２１）
ラクトバチルス・パラカゼイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ）ＭＣＣ１３７５株（ＦＥＲＭ ＢＰ−１１３１３）を含有する医薬。
（２２）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を含有するＩＬ−１２産生促進剤。
（２３）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を含有するＮＫ細胞活性化剤。
（２４）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を含有する抗インフルエンザ・ウイルス剤。
（２５）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を含有する飲食品。
（２６）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を含有する飼料。Therefore, this invention exists also in following (21)-(26).
(21)
A medicament comprising Lactobacillus paracasei MCC1375 strain (FERM BP-11313).
(22)
An IL-12 production promoter containing Lactobacillus paracasei MCC1375 strain.
(23)
NK cell activator containing Lactobacillus paracasei MCC1375 strain.
(24)
An anti-influenza virus agent containing Lactobacillus paracasei MCC1375 strain.
(25)
A food or drink containing Lactobacillus paracasei MCC1375 strain.
(26)
Feed containing Lactobacillus paracasei MCC1375 strain.
さらに、本発明は、上記の株又は組成物を有効成分として含有する、インターロイキン−１２産生促進剤、ＮＫ細胞活性化剤、抗インフルエンザ・ウイルス剤にもある。
また、酸性生残性は、例えば、ｐＨ３．０における１０℃での４週間保存後の生残率が９０％以上であること、あるいは、例えば、ｐＨ３．０における１０℃での６週間保存後の生残率が４０％以上であること、によって確認することができる。Furthermore, this invention exists also in the interleukin-12 production promoter, NK cell activator, and anti-influenza virus agent which contain said strain | stump | stock or composition as an active ingredient.
The acid survival is, for example, that the survival rate after storage for 4 weeks at 10 ° C. at pH 3.0 is 90% or more, or after storage for 6 weeks at 10 ° C. at pH 3.0, for example. It can be confirmed that the survival rate is 40% or more.
さらに、本発明は、次の（３１）〜（３４）にもある。
（３１）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を投与する工程、
を含む、ＩＬ−１２の産生を促進する方法。
（３２）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を投与する工程、
を含む、ＮＫ細胞を活性化する方法。
（３３）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を投与する工程、
を含む、インフルエンザ・ウイルスによる発症を予防する方法。
（３４）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を投与する工程、
を含む、ＮＫ細胞を活性化する方法。Furthermore, this invention exists also in following (31)-(34).
(31)
Administering Lactobacillus paracasei MCC1375 strain,
A method for promoting the production of IL-12, comprising:
(32)
Administering Lactobacillus paracasei MCC1375 strain,
A method for activating NK cells, comprising:
(33)
Administering Lactobacillus paracasei MCC1375 strain,
To prevent the onset of influenza virus.
(34)
Administering Lactobacillus paracasei MCC1375 strain,
A method for activating NK cells, comprising:
さらに、本発明は、次の（４１）〜（４４）にもある。
（４１）
ラクトバチルス・パラカゼイＭＣＣ１３７５株を含む、発酵乳製造用スターター。
（４２）
（４１）に記載の発酵乳製造用スターターを、発酵乳の原材料に添加して、発酵させる工程、
を含む、発酵乳の製造方法。
（４３）
（４２）に記載の製造方法によって製造された、発酵乳。
（４４）
ラクトバチルス・パラカゼイＭＣＣ１３７５株の、発酵乳製造用スターターとしての使用。Furthermore, this invention exists also in following (41)-(44).
(41)
A starter for producing fermented milk containing Lactobacillus paracasei MCC1375 strain.
(42)
Adding the fermented milk production starter according to (41) to the raw material of the fermented milk and fermenting it,
A method for producing fermented milk, comprising:
(43)
Fermented milk produced by the production method according to (42).
(44)
Use of Lactobacillus paracasei MCC1375 strain as a starter for producing fermented milk.
さらに、本発明は、ラクトバチルス・パラカゼイＭＣＣ１３７５株を使用して変異株を取得する方法にもある。ラクトバチルス・パラカゼイＭＣＣ１３７５株は、この株を出発材料として、公知の方法により突然変異株を取得することができる。この変異株に対して、酸性生残性（低ｐＨ生残性）を確認する工程を行うことによって、本発明の優れた特徴を有していることを確認することができる。さらに、この変異株に対して、インターロイキン−１２産生促進の性質を確認する工程、ＮＫ細胞活性化の性質を確認する工程、及び／又は、抗インフルエンザ・ウイルスの性質を確認する工程、を行うことによって、インターロイキン−１２産生促進用組成物、ＮＫ細胞活性化用組成物、及び／又は、抗インフルエンザ・ウイルス用組成物として使用できることを、確認することができる。 Furthermore, this invention exists also in the method of acquiring a mutant strain using the Lactobacillus paracasei MCC1375 strain | stump | stock. Lactobacillus paracasei MCC1375 strain can be obtained by a known method using this strain as a starting material. It can confirm that it has the outstanding characteristic of this invention by performing the process which confirms acidic survivability (low pH survivability) with respect to this mutant. Furthermore, a step of confirming the property of promoting interleukin-12 production, a step of confirming the property of NK cell activation, and / or a step of confirming the property of anti-influenza virus are performed on this mutant strain. Thus, it can be confirmed that the composition can be used as a composition for promoting interleukin-12 production, a composition for NK cell activation, and / or an anti-influenza virus composition.
本発明は、酸性条件下の生残性の高い新規乳酸菌を提供する。したがって、本発明に係る新規乳酸菌は、乳酸菌飲料などの酸性飲食品に含有させた場合でも、死滅しにくく、生菌による有益な効果を長期にわたって維持することができる。さらに、本発明に係る新規乳酸菌は、飲食品や経口製剤の形態で、経口投与された場合でも、酸性の胃液に耐えて、高い生残性で腸管粘膜に到達して、生菌による有益な効果を発揮することができる。 The present invention provides a novel lactic acid bacterium having a high survival rate under acidic conditions. Therefore, even when the novel lactic acid bacterium according to the present invention is contained in an acidic food or drink such as a lactic acid bacterium beverage, it is difficult to kill, and the beneficial effect of the live bacterium can be maintained over a long period of time. Furthermore, the novel lactic acid bacteria according to the present invention are resistant to acidic gastric juice even when orally administered in the form of foods and beverages and oral preparations, reach the intestinal mucosa with high survival, and are beneficial to live bacteria. The effect can be demonstrated.
さらに、本発明に係る新規乳酸菌は、従来知られている乳酸菌と比較して、高いＩＬ−１２産生誘導能、ＮＫ細胞活性作用、及び抗インフルエンザ・ウイルス作用を有するものであり、ＩＬ−１２産生誘導用組成物やＮＫ細胞活性化用組成物として使用することができ、さらに詳細には、抗腫瘍剤、日和見感染症の予防剤又は治療剤、アレルギー疾患予防剤、又は治療剤として広く利用することができる。 Furthermore, the novel lactic acid bacterium according to the present invention has higher IL-12 production inducing ability, NK cell activity action, and anti-influenza virus action, compared with conventionally known lactic acid bacteria, and IL-12 production It can be used as a composition for induction or a composition for NK cell activation, and more specifically, widely used as an antitumor agent, a prophylactic or therapeutic agent for opportunistic infections, a prophylactic agent for allergic diseases, or a therapeutic agent. be able to.
本発明に係る新規乳酸菌は、高い生残性を有する生菌として使用することができ、死菌体としても使用することができ、医薬、飲食品、飼料に含有して広く用いることができる。特に、ラクトバチルス・パラカゼイＭＣＣ１３７５株は、死菌体であっても、マウス由来リンパ球及びヒト由来末梢血単核球（ＰＢＭＣ）に対して高いＩＬ−１２産生誘導能を有し、マウスへ経口投与することで、ＮＫ細胞の増加を誘導し、インフルエンザ・ウイルス感染に対する予防作用を有するから、インフルエンザ・ウイルスの予防や治療のために好適に使用することができる。 The novel lactic acid bacteria according to the present invention can be used as viable bacteria having high survival properties, can also be used as dead cells, and can be widely used by being contained in medicines, foods and drinks, and feeds. In particular, Lactobacillus paracasei MCC1375 strain has a high IL-12 production-inducing ability for mouse-derived lymphocytes and human-derived peripheral blood mononuclear cells (PBMC) even if dead cells, and is orally administered to mice. When administered, it induces an increase in NK cells and has a preventive action against influenza virus infection, and thus can be suitably used for the prevention and treatment of influenza virus.
次に、本発明の実施形態について詳細に説明する。但し、本発明は以下の実施形態に限定されず、本発明の範囲内で自由に変更することができる。
本発明者らは、新規乳酸菌の菌株としてＭＣＣ１３７５株を確立した。本株は、ヒト糞便を分離源として分離された。Next, an embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely modified within the scope of the present invention.
The present inventors established MCC1375 strain as a novel lactic acid bacteria strain. This strain was isolated using human feces as the source of separation.
ＭＣＣ１３７５株の菌学的性質として、１．細胞の形態、２．運動性、３．胞子形成、４．グラム染色性、５．カタラーゼ、６．グルコースからのガスの産生、及び糖発酵性について表１に示す。糖発酵性については、細菌同定キットＡＰＩ５０ＣＨ（ｂｉｏＭｅｒｉｅｕｘ Ｊａｐａｎ製）を用い、添付のマニュアルに記載された方法に従って測定した。すなわち、一晩培養後のＭＣＣ１３７５株培養液を各糖質を含む培地に接種し、これを３７℃の嫌気グローブボックス中で培養し、培養開始から７日目に各糖質の利用性を判断した。ＭＣＣ１３７５株の菌学的性質の一覧を、次の表１に示す。 As the mycological properties of MCC1375 strain: Cell morphology, 2. 2. mobility; Sporulation, 4. 4. Gram stainability, Catalase, 6. Table 1 shows the production of gas from glucose and sugar fermentability. The sugar fermentability was measured using a bacterial identification kit API50CH (manufactured by bioMerieux Japan) according to the method described in the attached manual. That is, the culture solution of MCC1375 strain after overnight culture was inoculated into a medium containing each carbohydrate, cultured in an anaerobic glove box at 37 ° C., and the availability of each carbohydrate was judged on the seventh day from the start of the culture. did. A list of mycological properties of the MCC1375 strain is shown in Table 1 below.
ＭＣＣ１３７５株の１６ＳｒＲＮＡ遺伝子の全領域１４７６ｂｐについて公知の手法により解読を行った。
菌株の同定は、例えば、「『微生物の分類・同定実験法‐分子遺伝学・分子生物学的手法を中心に‐』、シュプリンガー・フェアラーク東京株式会社発行、２００１年９月１６日発行」に記載されている方法（p.9-14, p.48-64, p.231-285等）に従って、各種菌株の１６ＳｒＲＮＡ遺伝子の全長を解析することにより行った。
解読されたＭＣＣ１３７５株の１６ＳｒＲＮＡ遺伝子の塩基配列１４７６ｂｐが、Ｌ．ｃａｓｅｉ ＡＴＣＣ３３４T株と９９．８０％の相同性を有していたことから、ラクトバチルス・パラカゼイ又はラクトバチルス・カゼイであることが示された。
さらに、ＭＣＣ１３７５株と、ラクトバチルス・パラカゼイ標準株（ＡＴＣＣ２５３０２T）及びラクトバチルス・カゼイ標準株（ＡＴＣＣ３９３T）とのゲノムの相同性をＩｎｔ．Ｊ Ｓｙｓｔ Ｂａｃｔｅｒｉｏｌ、第３９号、第２２４−２２９ページ、に記載のＤＮＡハイブリダイゼーション試験で確認したところ、ラクトバチルス・パラカゼイ標準株との相同性が８７％であったのに対し、ラクトバチルス・カゼイ標準株との相同性が１５％であることが判明した。
上記菌学的性質、１６ＳｒＲＮＡ遺伝子の塩基配列の相同性、及びＤＮＡハイブリダイゼーション試験の結果から、ＭＣＣ１３７５株をラクトバチルス・パラカゼイに属する新規株であると判断した。The entire region 1476 bp of 16S rRNA gene of MCC1375 strain was decoded by a known method.
The identification of strains can be found, for example, in "" Microbial Classification and Identification Experiments-Focusing on Molecular Genetics and Molecular Biology-", published by Springer Fairlark Tokyo, September 16, 2001. According to the method described (p.9-14, p.48-64, p.231-285 etc.), it was performed by analyzing the full length of 16S rRNA gene of various strains.
The base sequence of 1476 bp of the 16S rRNA gene of MCC1375 strain that was decrypted is casei from ATCC334 T strain and that had a 99.80% homology, have been shown to be Lactobacillus paracasei or Lactobacillus casei.
Furthermore, the homology of the genome between the MCC1375 strain, the Lactobacillus paracasei standard strain (ATCC25302 T ), and the Lactobacillus casei standard strain (ATCC393 T ) was determined as Int. The homology with the standard strain of Lactobacillus paracasei was 87% when confirmed by the DNA hybridization test described in J Systact Bacteriol, No. 39, pp. 224-229, whereas Lactobacillus casei was 87%. It was found that the homology with the standard strain was 15%.
From the above mycological properties, the homology of the base sequence of 16S rRNA gene, and the results of the DNA hybridization test, MCC1375 strain was judged to be a new strain belonging to Lactobacillus paracasei.
ラクトバチルス・パラカゼイＭＣＣ１３７５株は、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（日本国 〒３０５−８５６６ 茨城県つくば市東１−１−１ 中央第６）に、平成２２年１１月５日より、受託番号：ＦＥＲＭ ＢＰ−１１３１３として国際寄託されている。 Lactobacillus paracasei MCC1375 strain was incorporated into the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Japan 1-1, Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture, Central 6th) from November 5, 2010 Accession number: FERM BP-11313.
ラクトバチルス・パラカゼイＭＣＣ１３７５株は、上記寄託株に制限されず、上記寄託株と実質同等の株であってもよい。上記寄託株と実質同等の株とは、ラクトバチルス・パラカゼイに属する株であって、酸性生残性を有し、好ましくは高いＩＬ−１２産生促進効果と高い抗ウイルス効果を有し、さらにその１６ＳｒＲＮＡ遺伝子の塩基配列が、上記寄託株の１６ＳｒＲＮＡ遺伝子の塩基配列に対して、好ましくは９９．８６％以上、さらに好ましくは９９．９３％以上、より好ましくは１００％の相同性を有し、且つ、好ましくは上記寄託株と同一の菌学的性質を有する株である。
また、ラクトバチルス・パラカゼイＭＣＣ１３７５株は、ＭＣＣ１３７５株を変異させた変異株を作製し、用いることができる。菌株の変異手段としては、ＵＶ等の公知の変異手段を用いて行うことができる。The Lactobacillus paracasei MCC1375 strain is not limited to the deposited strain, and may be a strain substantially equivalent to the deposited strain. The strain substantially equivalent to the deposited strain is a strain belonging to Lactobacillus paracasei, has acid survival, preferably has a high IL-12 production promoting effect and a high antiviral effect, and further The base sequence of the 16S rRNA gene preferably has 99.86% or more, more preferably 99.93% or more, more preferably 100% homology to the base sequence of the 16S rRNA gene of the deposited strain, and Preferably, it is a strain having the same mycological properties as the deposited strain.
Moreover, the Lactobacillus paracasei MCC1375 strain | stump | stock can produce and use the mutant strain which mutated MCC1375 strain | stump | stock. As a means for mutating strains, known mutation means such as UV can be used.
本発明のラクトバチルス・パラカゼイＭＣＣ１３７５は、高い生残性を有することから生菌として好適に使用することができるものであるが、死菌であっても好適に使用することができる。 The Lactobacillus paracasei MCC1375 of the present invention can be suitably used as a living bacterium because of its high survival, but even a dead bacterium can be preferably used.
本発明に用いられるラクトバチルス・パラカゼイＭＣＣ１３７５株は、例えば、上記菌株を培養することにより容易に増殖させることができる。培養する方法は、ラクトバチルス・パラカゼイＭＣＣ１３７５株が増殖できる限り特に限定されず、乳酸菌の培養に通常用いられる方法を必要により適宜修正して用いることができる。例えば、培養温度は２５℃〜５０℃でよく、３５℃〜４２℃であることが好ましい。また、培養は好気条件、嫌気条件のいずれでも行うことができる。嫌気条件としては、例えば、炭酸ガス等の嫌気ガスを通気しながら培養することができる。また、液体静置培養等の微好気条件下で培養してもよい。 The Lactobacillus paracasei MCC1375 strain used in the present invention can be easily propagated, for example, by culturing the above strain. The method of culturing is not particularly limited as long as Lactobacillus paracasei MCC1375 strain can grow, and the method usually used for culturing lactic acid bacteria can be appropriately modified as necessary. For example, the culture temperature may be 25 ° C to 50 ° C, preferably 35 ° C to 42 ° C. In addition, the culture can be performed under either aerobic conditions or anaerobic conditions. As an anaerobic condition, it can culture, for example, ventilating anaerobic gas, such as a carbon dioxide gas. Moreover, you may culture | cultivate on microaerobic conditions, such as liquid stationary culture.
ラクトバチルス・パラカゼイＭＣＣ１３７５株を培養する培地としては、特に限定されず、乳酸菌の培養に通常用いられる培地を必要により適宜修正して用いることができる。すなわち、炭素源としては、例えば、ガラクトース、グルコース、フルクトース、マンノース、セロビオース、マルトース、スクロース、トレハロース、澱粉、澱粉加水分解物、廃糖蜜等の糖類を資化性に応じて使用できる。窒素源としては、例えば、アンモニア、硫酸アンモニウム、塩化アンモニウム、硝酸アンモニウム等のアンモニウム塩類や硝酸塩類を使用できる。また、無機塩類としては、例えば、塩化ナトリウム、塩化カリウム、リン酸カリウム、硫酸マグネシウム、塩化カルシウム、硝酸カルシウム、塩化マンガン、硫酸第一鉄等を用いることができる。また、ペプトン、大豆粉、脱脂大豆粕、肉エキス、酵母エキス等の有機成分を用いてもよい。また、調製済の培地としては、例えば、ＭＲＳ培地を好適に用いることができる。 The medium for culturing Lactobacillus paracasei MCC1375 strain is not particularly limited, and a medium usually used for culturing lactic acid bacteria can be appropriately modified as necessary. That is, as the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, sucrose, trehalose, starch, starch hydrolyzate, and molasses can be used depending on the utilization. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, and nitrates can be used. Examples of inorganic salts that can be used include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Organic components such as peptone, soybean powder, defatted soybean meal, meat extract, yeast extract and the like may also be used. Moreover, as the prepared medium, for example, MRS medium can be suitably used.
ラクトバチルス・パラカゼイＭＣＣ１３７５株としては、培養後、得られた培養物をそのまま用いてもよく、希釈又は濃縮して用いてもよく、培養物から回収した菌体を用いてもよい。また、本発明の効果を損なわない限り、培養後に加熱、及び凍結乾燥等の追加操作を行うことができる。追加の操作としては、生菌の残存の割合が大きい操作が、好ましい。
ラクトバチルス・パラカゼイＭＣＣ１３７５株は、医薬、飲食品、及び飼料に広く用いることができる。また、ラクトバチルス・パラカゼイＭＣＣ１３７５株は、ＩＬ−１２産生促進剤、ＮＫ細胞活性化剤、抗インフルエンザ・ウイルス剤として用いることができる。As the Lactobacillus paracasei MCC1375 strain, after culturing, the obtained culture may be used as it is, diluted or concentrated, or cells recovered from the culture may be used. In addition, additional operations such as heating and freeze-drying can be performed after culturing as long as the effects of the present invention are not impaired. As an additional operation, an operation with a large ratio of remaining viable bacteria is preferable.
Lactobacillus paracasei MCC1375 strain can be widely used in medicine, food and drink, and feed. The Lactobacillus paracasei MCC1375 strain can be used as an IL-12 production promoter, NK cell activator, and anti-influenza virus agent.
さらに、本発明に係る新規乳酸菌の菌株は、上記ラクトバチルス・パラカゼイＭＣＣ１３７５株の突然変異株が含まれる。 Furthermore, the strains of novel lactic acid bacteria according to the present invention include mutants of the above-mentioned Lactobacillus paracasei MCC1375 strain.
好適な実施の態様において、本発明に係る新規乳酸菌の菌株は、低ｐＨ生残性（酸性生残性）であり、具体的には、例えば、ｐＨ３．０における１０℃での４週間保存後の生残率が９０％以上であり、あるいは、例えば、ｐＨ３．０における１０℃での６週間保存後の生残率が４０％以上である。これらの保存条件は、近縁と思われる菌株である、Ｌ．ラムノーサスＡＴＣＣ５３１０３株、Ｌ．パラカゼイＡＴＣＣ２５３０２T株、及びＬ．カゼイＡＴＣＣ３９３T株が、いずれも０．１％未満あるいは検出限界以下の生残率となる条件である。In a preferred embodiment, the novel strain of lactic acid bacteria according to the present invention is low pH survivability (acid survivability), specifically, for example, after storage for 4 weeks at 10 ° C. at pH 3.0. Or the survival rate after storage for 6 weeks at 10 ° C. at pH 3.0 is 40% or more. These storage conditions are determined by L. cerevisiae, a strain that appears to be closely related. Rhamnosus ATCC 53103 strain, L. Paracasei ATCC 25302 T strain and L. All of the casei ATCC 393 T strains have a survival rate of less than 0.1% or below the detection limit.
［本発明の医薬］
本発明の医薬は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を有効成分として含有する限り、特に制限されない。本発明の医薬としては、上記菌株をそのまま使用してもよく、製薬的に許容される液体又は固体の製剤担体を配合し、製剤化して使用してもよい。
また、上記菌株は生菌であっても死菌であっても使用することができる。
本発明の医薬の剤形は特に制限されず、具体的には、錠剤、丸剤、散剤、液剤、懸濁剤、乳剤、顆粒剤、カプセル剤、シロップ剤、坐剤、注射剤、軟膏剤、貼付剤、点眼剤、及び点鼻剤等を例示できる。また、製剤化にあたっては、製剤担体として通常使用される賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯味矯臭剤、希釈剤、界面活性剤、又は注射剤用溶材等の添加剤を使用することができる。[Medicine of the present invention]
The medicament of the present invention is not particularly limited as long as it contains the novel lactic acid bacterium strain according to the present invention, preferably Lactobacillus paracasei MCC1375 strain as an active ingredient. As the medicament of the present invention, the above strain may be used as it is, or a pharmaceutically acceptable liquid or solid preparation carrier may be blended and used as a preparation.
Moreover, the above strains can be used whether they are live or dead.
The pharmaceutical dosage form of the present invention is not particularly limited, and specifically, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments , Patches, eye drops, nasal drops and the like. In formulation, addition of excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, or injectable solutions that are commonly used as pharmaceutical carriers Agents can be used.
本発明の医薬における、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株の含有量は、剤形、用法、患者の年齢、性別、疾患の種類、疾患の程度、及びその他の条件等により適宜設定されるが、通常１×１０6〜１×１０12個／ｇの範囲内であることが好ましく、１×１０7〜１×１０11個／ｇの範囲内であることがより好ましい。生菌では、菌の個数をＣＦＵ（コロニー形成単位）と表してもよい。上記「個／ｇ」は生菌では「ＣＦＵ／ｇ」と表すことができる。なお、実施例の死菌体においては、１μｇが３．７５×１０6個に相当するので、この値によって換算することができる。The content of the strain of the novel lactic acid bacterium according to the present invention, preferably the Lactobacillus paracasei MCC1375 strain in the medicament of the present invention is the dosage form, usage, patient age, sex, disease type, disease severity, and other Although it is appropriately set depending on conditions and the like, it is usually preferably within a range of 1 × 10 6 to 1 × 10 12 pieces / g, and preferably within a range of 1 × 10 7 to 1 × 10 11 pieces / g. More preferred. In live bacteria, the number of bacteria may be expressed as CFU (colony forming unit). The above “pieces / g” can be expressed as “CFU / g” in living bacteria. In the dead cells of Examples, 1 μg corresponds to 3.75 × 10 6 , and can be converted by this value.
また、本発明の効果を損なわない限り、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を含有する医薬と、他の医薬、例えばＩＬ−１２産生促進剤、ＮＫ細胞活性化剤、抗ウイルス剤等を併用してもよい。 In addition, as long as the effect of the present invention is not impaired, a drug containing the strain of the novel lactic acid bacterium according to the present invention, preferably the Lactobacillus paracasei MCC1375 strain, and other drugs such as IL-12 production promoter, NK cell activation Agents, antiviral agents and the like may be used in combination.
本発明の医薬の投与時期は特に限定されず、対象となる疾患の治療方法に従って、適宜投与時期を選択することができる。また、予防的に投与してもよく、維持療法に用いてもよい。また、投与形態は製剤形態、患者の年齢、性別、その他の条件、患者の症状の程度等に応じて決定されることが好ましい。なお、本発明の医薬は、いずれの場合も１日１回又は複数回に分けて投与することができ、また、数日又は数週間に１回の投与としてもよい。 The administration time of the medicament of the present invention is not particularly limited, and the administration time can be appropriately selected according to the treatment method for the target disease. It may be administered prophylactically or used for maintenance therapy. The dosage form is preferably determined according to the dosage form, the patient's age, sex, other conditions, the degree of symptoms of the patient, and the like. In any case, the pharmaceutical of the present invention can be administered once or a plurality of times a day, or can be administered once every several days or weeks.
［ＩＬ−１２産生促進用組成物］
本発明の医薬は、好適にはＩＬ−１２産生促進用組成物として使用することができる。本発明のＩＬ−１２産生促進用組成物は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を有効成分として含有するものであり、生体内のＩＬ−１２を顕著に産生促進することができる。増加したＩＬ−１２は、細胞性免疫を活性化させる。
従って、本発明のＩＬ−１２産生促進用組成物は細胞性免疫を強化することができるため、アレルギー症状、具体的には食物アレルギー、気管支喘息、蕁麻疹、鼻炎、花粉症、アナフィラキシーショック等の緩和の他、感染症に対する抵抗力の増進、癌の予防、進行の防止等が挙げられる。
また、本発明のＩＬ−１２産生促進用組成物は、ＩＬ−１２産生誘導用組成物として使用することができ、さらに詳細には、抗腫瘍剤、日和見感染症の予防剤又は治療剤、アレルギー疾患予防剤、又は治療剤として広く利用することができる。[Composition for promoting IL-12 production]
The medicament of the present invention can be suitably used as a composition for promoting IL-12 production. The composition for promoting production of IL-12 of the present invention contains the novel lactic acid bacteria strain according to the present invention, preferably Lactobacillus paracasei MCC1375 strain as an active ingredient, and remarkably produces in vivo IL-12. Can be promoted. Increased IL-12 activates cellular immunity.
Therefore, since the composition for promoting IL-12 production of the present invention can enhance cellular immunity, allergic symptoms such as food allergy, bronchial asthma, urticaria, rhinitis, hay fever, anaphylactic shock, etc. In addition to mitigation, there are increased resistance to infectious diseases, prevention of cancer, prevention of progression, and the like.
Further, the IL-12 production promoting composition of the present invention can be used as a composition for inducing IL-12 production, and more specifically, an antitumor agent, a prophylactic or therapeutic agent for opportunistic infections, an allergy, It can be widely used as a disease preventive or therapeutic agent.
本発明のＩＬ−１２産生促進用組成物におけるラクトバチルス・パラカゼイＭＣＣ１３７５株の含有量は、剤形、用法、患者の年齢、性別、疾患の種類、疾患の程度、及びその他の条件等により適宜設定されるが、通常１×１０6〜１×１０12個／ｇの範囲内であることが好ましく、１×１０7〜１×１０11個／ｇの範囲内であることがより好ましい。
また、本発明の効果を損なわない限り、本願のＩＬ−１２産生促進用組成物と、他のＩＬ−１２産生促進用組成物等を併用してもよい。
本発明のＩＬ−１２産生促進用組成物の投与時期は特に限定されず、対象となる疾患の治療方法に従って、適宜投与時期を選択することができる。また、予防的に投与してもよく、維持療法に用いてもよい。また、投与形態は製剤形態、患者の年齢、性別、その他の条件、患者の症状の程度等に応じて決定されることが好ましい。なお、本発明の医薬は、いずれの場合も１日１回又は複数回に分けて投与することができ、また、数日又は数週間に１回の投与としてもよい。
ＩＬ−１２産生促進効果は、ＩＬ−１２産生量を測定して確認することができる。ＩＬ−１２産生量はＥＬＩＳＡ法で測定することが好ましい。The content of the Lactobacillus paracasei MCC1375 strain in the IL-12 production promoting composition of the present invention is appropriately set depending on the dosage form, usage, patient age, sex, disease type, disease level, and other conditions. However, it is usually preferably in the range of 1 × 10 6 to 1 × 10 12 pieces / g, and more preferably in the range of 1 × 10 7 to 1 × 10 11 pieces / g.
Moreover, unless the effect of this invention is impaired, you may use together the composition for IL-12 production promotion of this application, the composition for other IL-12 production promotion, etc.
The administration time of the IL-12 production promoting composition of the present invention is not particularly limited, and the administration time can be appropriately selected according to the treatment method for the target disease. It may be administered prophylactically or used for maintenance therapy. The dosage form is preferably determined according to the dosage form, the patient's age, sex, other conditions, the degree of symptoms of the patient, and the like. In any case, the pharmaceutical of the present invention can be administered once or a plurality of times a day, or can be administered once every several days or weeks.
The IL-12 production promoting effect can be confirmed by measuring the amount of IL-12 production. The IL-12 production amount is preferably measured by the ELISA method.
［ＮＫ細胞活性化用組成物］
本発明の医薬は、別の好適な態様として、ＮＫ細胞（ナチュラルキラー細胞）活性化用組成物として使用することができる。
ＮＫ細胞は、種々の腫瘍細胞やウイルス感染細胞、及び主要組織適合抗原の異なる細胞に対して細胞障害活性を有する細胞群で、主として自己、非自己を認識するレセプターによって活性化され、または抑制されている。
本発明のＮＫ細胞活性化用組成物は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を有効成分として含有するものであり、生体内のＮＫ細胞を顕著に活性化することにより、ＮＫ細胞の増殖によって予防され得る疾患の予防、治療のために用いることができる。
ＮＫ細胞の増殖によって予防され得る疾患とは、具体的にはアレルギー症状、具体的には食物アレルギー、気管支喘息、蕁麻疹、鼻炎、花粉症、アナフィラキシーショック等の緩和の他、感染症に対する抵抗力の増進、癌の予防、進行の防止等が挙げられる。
また、本発明のＮＫ細胞活性化用組成物は、さらに詳細には、抗腫瘍剤、日和見感染症の予防剤又は治療剤、アレルギー疾患予防剤、又は治療剤として広く利用することができる。
ＮＫ細胞活性化効果は、例えばマウスの脾細胞に占めるＮＫ細胞の割合を測定して確認することができる。ＮＫ細胞の割合の測定は、フローサイトメトリーで測定することが好ましい。[Composition for NK cell activation]
In another preferred embodiment, the medicament of the present invention can be used as a composition for activating NK cells (natural killer cells).
NK cells are a group of cells having cytotoxic activity against various tumor cells, virus-infected cells, and cells with different major histocompatibility antigens, and are mainly activated or suppressed by receptors that recognize self and non-self. ing.
The composition for NK cell activation of the present invention contains the strain of the novel lactic acid bacterium according to the present invention, preferably Lactobacillus paracasei MCC1375 as an active ingredient, and remarkably activates NK cells in vivo. Therefore, it can be used for the prevention and treatment of diseases that can be prevented by the proliferation of NK cells.
Diseases that can be prevented by proliferation of NK cells are specifically allergic symptoms, specifically food allergies, bronchial asthma, urticaria, rhinitis, hay fever, anaphylactic shock, etc., as well as resistance to infectious diseases Improvement, prevention of cancer, prevention of progression, and the like.
In addition, the composition for activating NK cells of the present invention can be widely used as an antitumor agent, a prophylactic or therapeutic agent for opportunistic infections, an allergic disease preventive agent, or a therapeutic agent.
The NK cell activation effect can be confirmed, for example, by measuring the proportion of NK cells in mouse spleen cells. The ratio of NK cells is preferably measured by flow cytometry.
本発明のＮＫ細胞活性化用組成物におけるラクトバチルス・パラカゼイＭＣＣ１３７５株の含有量は、剤形、用法、患者の年齢、性別、疾患の種類、疾患の程度、及びその他の条件等により適宜設定されるが、通常１×１０6〜１×１０12個／ｇの範囲内であることが好ましく、１×１０7〜１×１０11個／ｇの範囲内であることがより好ましい。
また、本発明の効果を損なわない限り、本願のＮＫ細胞活性化用組成物と、他のＮＫ細胞活性化用組成物等を併用してもよい。
本発明のＮＫ細胞活性化用組成物の投与時期は特に限定されず、対象となる疾患の治療方法に従って、適宜投与時期を選択することができる。また、予防的に投与してもよく、維持療法に用いてもよい。また、投与形態は製剤形態、患者の年齢、性別、その他の条件、患者の症状の程度等に応じて決定されることが好ましい。なお、本発明の医薬は、いずれの場合も１日１回又は複数回に分けて投与することができ、また、数日又は数週間に１回の投与としてもよい。The content of the Lactobacillus paracasei MCC1375 strain in the composition for activating NK cells of the present invention is appropriately set depending on the dosage form, usage, patient age, sex, disease type, disease severity, and other conditions. However, it is usually preferably in the range of 1 × 10 6 to 1 × 10 12 pieces / g, and more preferably in the range of 1 × 10 7 to 1 × 10 11 pieces / g.
Moreover, as long as the effect of this invention is not impaired, you may use together the composition for NK cell activation of this application, the composition for other NK cell activation, etc.
The administration time of the composition for activating NK cells of the present invention is not particularly limited, and the administration time can be appropriately selected according to the treatment method for the target disease. It may be administered prophylactically or used for maintenance therapy. The dosage form is preferably determined according to the dosage form, the patient's age, sex, other conditions, the degree of symptoms of the patient, and the like. In any case, the pharmaceutical of the present invention can be administered once or a plurality of times a day, or can be administered once every several days or weeks.
［抗インフルエンザ・ウイルス用組成物］
本発明の医薬は、別の好適な態様として、抗ウイルス用組成物として使用することができ、特に抗インフルエンザ・ウイルス用組成物として使用することができる。本発明の抗インフルエンザ・ウイルス用組成物は、インフルエンザ・ウイルスに起因する疾患の予防又は治療に用いることができる。
本発明の抗インフルエンザ・ウイルス用組成物は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を有効成分として含有するものであり、生体内のインフルエンザ・ウイルス数を顕著に減少させることができる。また、本発明の抗インフルエンザ・ウイルス剤は、生体内のインフルエンザ・ウイルス数の増殖を顕著に抑制することができ、インフルエンザ・ウイルスの感染による症状を予防及び／又は改善することができる。
従って、本発明の抗インフルエンザ・ウイルス用組成物は、インフルエンザ・ウイルスの増殖によって生じる感染症の予防、治療のために用いることができる。[Anti-influenza / virus composition]
In another preferred embodiment, the medicament of the present invention can be used as an antiviral composition, particularly as an anti-influenza virus composition. The composition for anti-influenza virus of the present invention can be used for prevention or treatment of diseases caused by influenza virus.
The anti-influenza virus composition of the present invention contains the novel lactic acid bacterium strain according to the present invention, preferably the Lactobacillus paracasei MCC1375 strain as an active ingredient, and significantly reduces the number of influenza viruses in vivo. Can be made. In addition, the anti-influenza virus agent of the present invention can remarkably suppress the proliferation of the number of influenza viruses in the living body, and can prevent and / or improve symptoms caused by influenza virus infection.
Therefore, the anti-influenza virus composition of the present invention can be used for the prevention and treatment of infectious diseases caused by the proliferation of influenza virus.
本発明の抗ウイルス用組成物におけるラクトバチルス・パラカゼイＭＣＣ１３７５株の含有量は、剤形、用法、患者の年齢、性別、疾患の種類、疾患の程度、及びその他の条件等により適宜設定されるが、通常１×１０6〜１×１０12個／ｇの範囲内であることが好ましく、１×１０7〜１×１０11個／ｇの範囲内であることがより好ましい。
また、本発明の効果を損なわない限り、本願の抗インフルエンザ・ウイルス用組成物と、他の抗インフルエンザ・ウイルス用組成物等を併用してもよい。
本発明の抗インフルエンザ・ウイルス用組成物の投与時期は特に限定されず、対象となる疾患の治療方法に従って、適宜投与時期を選択することができる。また、予防的に投与してもよく、維持療法に用いてもよい。また、投与形態は製剤形態、患者の年齢、性別、その他の条件、患者の症状の程度等に応じて決定されることが好ましい。なお、本発明の医薬は、いずれの場合も１日１回又は複数回に分けて投与することができ、また、数日又は数週間に１回の投与としてもよい。
抗インフルエンザ・ウイルス効果は、マウスに本発明の抗ウイルス用組成物を投与したあとに、インフルエンザ・ウイルスを鼻腔より接種した感染モデルについて、マウスの状態を観察してスコア化した感染スコアの測定、感染後の体重減少率、感染後のマウスの肺中ウイルス濃度の測定等を総合評価して判断することができる。
マウスの肺中ウイルス濃度は、ＭＤＣＫ細胞（イヌ腎由来細胞）を用いたプラークアッセイ法で測定することが好ましい。The content of Lactobacillus paracasei MCC1375 strain in the antiviral composition of the present invention is appropriately set depending on the dosage form, usage, patient age, sex, disease type, disease severity, and other conditions. Usually, it is preferably in the range of 1 × 10 6 to 1 × 10 12 pieces / g, and more preferably in the range of 1 × 10 7 to 1 × 10 11 pieces / g.
Moreover, unless the effect of this invention is impaired, you may use together the composition for anti-influenza viruses of this application, the composition for other anti-influenza viruses, etc.
The administration time of the anti-influenza virus composition of the present invention is not particularly limited, and the administration time can be appropriately selected according to the treatment method for the target disease. It may be administered prophylactically or used for maintenance therapy. The dosage form is preferably determined according to the dosage form, the patient's age, sex, other conditions, the degree of symptoms of the patient, and the like. In any case, the pharmaceutical of the present invention can be administered once or a plurality of times a day, or can be administered once every several days or weeks.
The anti-influenza virus effect is measured by measuring the infection score obtained by observing the state of the mouse and scoring the infection model in which the influenza virus was inoculated from the nasal cavity after administering the antiviral composition of the present invention to the mouse. The weight loss rate after infection, the measurement of the virus concentration in the lungs of mice after infection, etc. can be comprehensively evaluated and judged.
It is preferable to measure the virus concentration in the mouse lung by a plaque assay method using MDCK cells (canine kidney-derived cells).
［飲食品］
本発明の飲食品は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を含有する限り特に制限されず、飲食品としては、清涼飲料、炭酸飲料、栄養飲料、果汁飲料、及び乳酸菌飲料等の飲料（これらの飲料の濃縮原液及び調製用粉末を含む）；アイスクリーム、シャーベット、かき氷等の氷菓；飴、チューインガム、キャンディー、ガム、チョコレート、錠菓、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、及び焼き菓子等の菓子類；加工乳、乳飲料、発酵乳、ドリンクヨーグルト、及びバター等の乳製品；パン；経腸栄養食、流動食、育児用ミルク、スポーツ飲料；その他機能性食品等が例示される。また、飲食品は、サプリメントであってもよく、例えばタブレット状のサプリメントであってもよい。サプリメントである場合には、一日当たりの食事量及び摂取カロリーについて他の食品に影響されることなく、ラクトバチルス・パラカゼイＭＣＣ１３７５株を摂取できる。[Food and Drink]
The food / beverage product of the present invention is not particularly limited as long as it contains the strain of the novel lactic acid bacterium according to the present invention, preferably Lactobacillus paracasei MCC1375 strain, and food / beverage products include soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, And beverages (including concentrated concentrates and powders for preparation of these beverages); ice confectionery such as ice cream, sherbet and shaved ice; rice cake, chewing gum, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, Confectionery such as jam, cream, and baked confectionery; processed milk, dairy drinks, fermented milk, drink yogurt, butter and other dairy products; bread; enteral nutrition, liquid food, infant milk, sports drink; other functions Examples include sex foods. The food and drink may be a supplement, for example, a tablet-like supplement. In the case of a supplement, the Lactobacillus paracasei MCC1375 strain can be ingested without being influenced by other foods in terms of the daily meal amount and calorie intake.
本発明の飲食品は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を、飲食品の原料に添加することにより製造することができ、上記菌株を添加すること以外は、通常の飲食品と同様にして製造することができる。菌株の添加は、飲食品の製造工程のいずれの段階で行ってもよい。また、添加した菌株による発酵工程を経て、飲食品が製造されてもよい。食品又は飲料の原料としては、通常の飲料や食品に用いられている原料を使用することができる。製造された飲食品は、経口的に摂取することが可能である。 The food and drink of the present invention can be produced by adding the strain of the novel lactic acid bacterium according to the present invention, preferably Lactobacillus paracasei MCC1375, to the raw material of the food and drink, except that the above strain is added, It can be produced in the same manner as ordinary food and drink. The addition of the strain may be performed at any stage of the production process of the food or drink. Moreover, food-drinks may be manufactured through the fermentation process by the added strain. As a raw material for food or beverage, a raw material used for a normal beverage or food can be used. The manufactured food and drink can be taken orally.
本発明の飲食品におけるラクトバチルス・パラカゼイＭＣＣ１３７５株の含有量は、飲食品の形状、摂取量、摂取者の年齢、性別及びその他の条件等により適宜設定されるが、死菌体又は生菌が、通常１×１０6〜１×１０12個／ｇの範囲内であることが好ましく、１×１０7〜１×１０11個／ｇの範囲内であることがより好ましい。したがって、生菌では、通常１×１０6〜１×１０12ＣＦＵ／ｇの範囲内であることが好ましく、１×１０7〜１×１０11ＣＦＵ／ｇの範囲内であることがより好ましいということができる。
本発明の飲食品は、ＩＬ−１２産生促進効果、ＮＫ細胞活性化効果、抗ウイルス効果を利用する種々の用途に用いることができる。The content of the Lactobacillus paracasei MCC1375 strain in the food and drink of the present invention is appropriately set according to the shape of the food and drink, the amount of intake, the age of the intaker, sex and other conditions, etc. Usually, it is preferably in the range of 1 × 10 6 to 1 × 10 12 pieces / g, and more preferably in the range of 1 × 10 7 to 1 × 10 11 pieces / g. Therefore, in the case of viable bacteria, it is usually preferably in the range of 1 × 10 6 to 1 × 10 12 CFU / g, more preferably in the range of 1 × 10 7 to 1 × 10 11 CFU / g. be able to.
The food / beverage products of this invention can be used for the various uses using an IL-12 production promotion effect, an NK cell activation effect, and an antiviral effect.
本発明の飲食品は、ＩＬ−１２産生促進、ＮＫ細胞活性化、抗ウイルスの用途を表示した飲食品として販売することが可能である。すなわち、本発明の飲食品には、「ＩＬ−１２産生促進用」、「ＮＫ細胞活性化用」、「抗ウイルス用」、「抗インフルエンザ・ウイルス用」、「抗アレルギー用」、「感染症予防用」、「免疫活性化用」、「免疫賦活用」等の表示をすることができる。これ以外でも、上記のＩＬ−１２産生促進、ＮＫ細胞活性化、抗ウイルスなどの効果や用途を表す文言であれば表示に使用できる。
前記「表示」とは、需要者に対して上記用途を知らしめるための全ての行為を意味し、上記用途を想起・類推させうるような表示であれば、表示の目的、表示の内容、表示する対象物及び媒体等の如何に拘わらず、全て本発明の「表示」に該当する。しかしながら、需要者が上記用途を直接的に認識できるような表現により表示することが好ましい。具体的には、本発明の飲食品に係る商品又は商品の包装に上記用途を記載する行為、商品又は商品の包装に上記用途を記載したものを譲渡し、引渡し、譲渡若しくは引渡しのために展示し、輸入する行為、商品に関する広告、価格表若しくは取引書類に上記用途を記載して展示し、若しくは頒布し、又はこれらを内容とする情報に上記用途を記載して展示し、若しくは頒布し、又はこれらを内容とする情報に上記用途を記載して電磁的方法（インターネット等）により提供する行為等が例示でき、特に包装、容器、カタログ、パンフレット、ＰＯＰ等の販売現場における宣伝材、その他の書類等への表示が好ましい。
また、表示としては、行政等によって許可された表示（例えば、行政が定める各種制度に基づいて認可を受け、そのような認可に基づいた態様で行う表示）であることが好ましい。例えば、健康食品、機能性食品、経腸栄養食品、特別用途食品、栄養機能食品、医薬用部外品等としての表示を例示することができ、その他厚生労働省によって認可される表示、例えば、特定保健用食品、これに類似する制度にて認可される表示を例示することができる。後者の例としては、特定保健用食品としての表示、条件付特定保健用食品としての表示、身体の構造や機能に影響を与える旨の表示、疾病リスク低減表示等を例示することができる。さらに詳細には、健康増進法施行規則（平成１５年４月３０日、日本国厚生労働省令第８６号）に定められた特定保健用食品としての表示（特に保健の用途の表示）、及びこれに類する表示等を例示することができる。The food / beverage products of the present invention can be sold as food / beverage products displaying IL-12 production promotion, NK cell activation, and antiviral uses. That is, the food and drink of the present invention includes “for IL-12 production promotion”, “for NK cell activation”, “for antiviral”, “for anti-influenza virus”, “for antiallergy”, “infectious disease” “Prevention”, “immunity activation”, “immunostimulation”, and the like can be displayed. In addition to this, any wording that expresses the effects and uses of the above-mentioned IL-12 production promotion, NK cell activation, antiviral, etc. can be used for display.
The “display” means all acts for informing the consumer of the use, and if it is a display that can recall and analogize the use, the purpose of the display, the content of the display, the display Regardless of the object and medium to be used, all of them correspond to the “display” of the present invention. However, it is preferable to display in such an expression that the consumer can directly recognize the application. Specifically, the act of describing the above-mentioned use in the product or product packaging relating to the food or drink of the present invention, the product or product packaging describing the above-mentioned use is transferred, and displayed for delivery, transfer or delivery And display or distribute the above uses in the acts of importing, advertising about products, price lists or transaction documents, or displaying or distributing the above uses in the information containing them, Or, it is possible to exemplify the act of providing the above uses in the information containing the contents and providing them by electromagnetic methods (Internet etc.), especially advertising materials at sales sites such as packaging, containers, catalogs, pamphlets, POP, etc. Display on documents is preferable.
In addition, the display is preferably a display permitted by the government or the like (for example, a display which is approved based on various systems determined by the government and is performed in a mode based on such approval). For example, indications such as health foods, functional foods, enteral nutrition foods, special purpose foods, functional nutrition foods, quasi-drugs, and other indications approved by the Ministry of Health, Labor and Welfare, such as specific Examples of indications approved by health foods and similar systems. Examples of the latter include a display as a food for specified health use, a display as a food for specific specified health use, a display that affects the structure and function of the body, a display for reducing disease risk, and the like. In more detail, the indication as food for specified health (especially the indication of the use of health) stipulated in the Enforcement Regulations of the Health Promotion Act (April 30, 2003, Ministry of Health, Labor and Welfare Ordinance No. 86), and this A display similar to the above can be exemplified.
［乳酸菌飲料］
本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株は、低ｐＨ（酸性）において顕著な生残性を有することから、乳酸菌飲料及び／又は発酵乳に好適に使用することができる。
ラクトバチルス・パラカゼイＭＣＣ１３７５株は低ｐＨにおける生残性が顕著に高いという性質を有している。この性質により、本菌株を用いた乳酸菌飲料は従来達成できなかった程度の低いｐＨに調整することが可能である。本発明の乳酸菌飲料のｐＨは特に制限されることなく利用可能であるが、ラクトバチルス・パラカゼイＭＣＣ１３７５株の特性を最大限に活用するためには、ｐＨ７以下が好ましく、ｐＨ６以下がさらに好ましく、ｐＨ５．５以下がさらに好ましく、ｐＨ５以下がさらに好ましく、ｐＨ４以下がさらに好ましく、ｐＨ３．５以下がさらに好ましく、ｐＨ３．０以下がさらに好ましい。ｐＨの下限は特に制限はないが、例えば、ｐＨ１．０以上、ｐＨ１．５以上、ｐＨ２．０以上、ｐＨ２．５以上、ｐＨ３．０以上とすることが出来る。[Lactic acid bacteria beverage]
The strain of the novel lactic acid bacterium according to the present invention, preferably Lactobacillus paracasei MCC1375 strain, has remarkable survival at low pH (acidic), and therefore can be suitably used for lactic acid bacteria beverages and / or fermented milk. .
The Lactobacillus paracasei MCC1375 strain has the property that the survival at low pH is remarkably high. Due to this property, a lactic acid bacteria beverage using this strain can be adjusted to a pH as low as previously unattainable. The pH of the lactic acid bacteria beverage of the present invention can be used without any particular limitation, but in order to make full use of the characteristics of Lactobacillus paracasei MCC1375 strain, the pH is preferably 7 or less, more preferably 6 or less, and
乳酸菌を添加する飲料としては、みかん、オレンジ、レモン、リンゴ、ブドウ、ナシ、グレープフルーツ、ザクロ、イチジクその他の果汁飲料、清涼飲料水、牛乳、ココア等の乳飲料、コーヒー等に使用することができる。
本発明の乳酸菌飲料は、例えば、１０℃、１ヶ月の保存後においても、１０6ｃｆｕ／ｍｌ程度の乳酸菌生菌数を維持することができる。この保存条件は、近縁と思われる菌株である、Ｌ．ラムノーサスＡＴＣＣ５３１０３株、Ｌ．パラカゼイＡＴＣＣ２５３０２T株、及びＬ．カゼイＡＴＣＣ３９３T株では、有効な生存率がほとんど期待できない条件である。Beverages to which lactic acid bacteria are added can be used for mandarin oranges, oranges, lemons, apples, grapes, pears, grapefruits, pomegranates, figs and other juice drinks, soft drinks, milk drinks such as milk and cocoa, coffee, etc. .
The lactic acid bacteria beverage of the present invention can maintain a viable lactic acid bacteria count of about 10 6 cfu / ml even after storage at 10 ° C. for 1 month, for example. This storage condition is that of L. cerevisiae, which is a closely related strain. Rhamnosus ATCC 53103 strain, L. Paracasei ATCC 25302 T strain and L. In casei ATCC393 T strain, effective survival rate is hardly expected.
［飼料］
本発明の飼料としては、ペットフード、家畜飼料、及び養魚飼料等が例示される。本発明の飼料は、一般的な飼料、例えば、穀類、粕類、糠類、魚粉、骨粉、油脂類、脱脂粉乳、ホエー、鉱物質飼料、又は酵母類等にラクトバチルス・パラカゼイＭＣＣ１３７５株を混合することにより製造することができる。また、例えばサイレージのように、添加したラクトバチルス・パラカゼイＭＣＣ１３７５株による発酵工程を経て、飼料が製造されてもよい。製造された飼料は、一般的な哺乳動物、家畜類、養魚類、及び愛玩動物等に経口的に投与することが可能である。[feed]
Examples of the feed of the present invention include pet food, livestock feed, and fish feed. The feed of the present invention is mixed with Lactobacillus paracasei MCC1375 in general feed such as cereals, potatoes, potatoes, fish meal, bone meal, fats and oils, skim milk powder, whey, mineral feed, yeasts, etc. Can be manufactured. Moreover, a feed may be manufactured through the fermentation process by the added Lactobacillus paracasei MCC1375 strain | stump | stock like silage, for example. The produced feed can be orally administered to general mammals, livestock, fish farms, pets and the like.
本発明の飼料におけるラクトバチルス・パラカゼイＭＣＣ１３７５株の含有量は、対象動物の種類、対象動物の年齢、性別、摂取量、及びその他の条件等により適宜設定されるが、通常１×１０6〜１×１０12個／ｇの範囲内であることが好ましく、１×１０7〜１×１０11個／ｇの範囲内であることがより好ましい。The content of the Lactobacillus paracasei MCC1375 strain in the feed of the present invention is appropriately set depending on the type of the target animal, the age, sex, intake, and other conditions of the target animal, but usually 1 × 10 6 to 1 It is preferably in the range of × 10 12 pieces / g, and more preferably in the range of 1 × 10 7 to 1 × 10 11 pieces / g.
［発酵乳製造用スターター］
本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株は、酵乳製造用スターターとして使用することができる。
好適な実施の態様において、この酵乳製造用スターターを、発酵乳の原材料に添加して、発酵させることによって、発酵乳を製造することができる。発酵乳の原材料としては、特に制限することなく、公知の原材料を使用することができる。本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株は、酸性条件下でも生残性が高いことから、通常の発酵乳よりもより低いｐＨ範囲まで、発酵を継続することができる点において、優れた酵乳製造用スターターである。さらに、製造された発酵乳は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を含むものであるために、従来の乳酸菌に比較して、上述したような有利な作用を有するものである。[Starter for fermented milk production]
The novel lactic acid bacteria strain according to the present invention, preferably Lactobacillus paracasei MCC1375 strain, can be used as a starter for producing fermented milk.
In a preferred embodiment, fermented milk can be produced by adding the fermented milk production starter to the raw material of the fermented milk and fermenting it. As a raw material of fermented milk, a well-known raw material can be used without a restriction | limiting in particular. Since the strain of the novel lactic acid bacterium according to the present invention, preferably Lactobacillus paracasei MCC1375, has high survival even under acidic conditions, it can continue fermentation to a lower pH range than normal fermented milk. In that respect, it is an excellent starter for producing fermented milk. Furthermore, since the produced fermented milk contains the strain of the novel lactic acid bacterium according to the present invention, preferably the Lactobacillus paracasei MCC1375 strain, it has the advantageous effects as described above compared to the conventional lactic acid bacterium. It is.
また、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株は、製造された発酵乳に後から添加することもできる。製造された後に添加された場合でも、この発酵乳は、本発明に係る新規乳酸菌の菌株、好ましくはラクトバチルス・パラカゼイＭＣＣ１３７５株を含むものとなるために、従来の乳酸菌に比較して、上述したような有利な作用を有するものである。 Moreover, the strain of the novel lactic acid bacterium according to the present invention, preferably the Lactobacillus paracasei MCC1375 strain, can be added later to the produced fermented milk. Even when added after being manufactured, this fermented milk contains the strain of the novel lactic acid bacterium according to the present invention, preferably the Lactobacillus paracasei MCC1375 strain. It has such an advantageous action.
［ラクトバチルス・パラカゼイＭＣＣ１３７５株の突然変異株］
本発明は、低ｐＨ生残性を有するラクトバチルス・パラカゼイＭＣＣ１３７５株の突然変異株にもある。
ＭＣＣ１３７５株の突然変異株は、ＭＣＣ１３７５株を変異させた変異株を作製し、用いることができる。菌株の変異手段としては、ＵＶ等の公知の変異手段を用いて行うことができる。[Lactobacillus paracasei MCC1375 mutant]
The present invention also resides in a mutant strain of Lactobacillus paracasei MCC1375 strain having low pH survival.
As a mutant strain of MCC1375 strain, a mutant strain obtained by mutating MCC1375 strain can be prepared and used. As a means for mutating strains, known mutation means such as UV can be used.
好適な実施の態様において、このような突然変異株は、例えば、ＭＣＣ１３７５株の１６ＳｒＲＮＡ遺伝子の塩基配列（第１１塩基から第１４８６塩基までの長さ１４７６塩基）に対して、塩基配列の同一性が、好ましくは９９．８６％以上、さらに好ましくは９９．９３％以上、より好ましくは１００％である。しかし、ＭＣＣ１３７５株の突然変異株は、このような塩基配列の同一性を有するものに限られるものではない。 In a preferred embodiment, such a mutant strain has, for example, a base sequence identity to the base sequence of the 16S rRNA gene of MCC1375 strain (length 1476 from base 11 to base 1486). , Preferably 99.86% or more, more preferably 99.93% or more, more preferably 100%. However, the mutant strain of MCC1375 strain is not limited to those having such nucleotide sequence identity.
ＭＣＣ１３７５株の突然変異株に対して、例えば、酸性生残性（低ｐＨ生残性）を確認することができる。具体的には、例えば、ｐＨ３．０における１０℃での４週間保存後の生残率が９０％以上であり、あるいは、例えば、ｐＨ３．０における１０℃での６週間保存後の生残率が４０％以上であることが好ましい。これらの保存条件は、近縁と思われる菌株である、Ｌ．ラムノーサスＡＴＣＣ５３１０３株、Ｌ．パラカゼイＡＴＣＣ２５３０２T株、及びＬ．カゼイＡＴＣＣ３９３T株が、いずれも０．１％未満あるいは検出限界以下の生残率となる条件である。しかし、ＭＣＣ１３７５株の突然変異株は、これらの生存条件を有するものに限られるものではない。
ＭＣＣ１３７５株の突然変異株は、本発明のＭＣＣ１３７５株と同様に本発明の医薬の有効成分として含有させることができ、本発明の飲食品の有効成分として含有させることができ、本発明の飼料の有効成分として含有させることができる。
ＭＣＣ１３７５株の突然変異株を含有する医薬としては、ＩＬ−１２産生促進用組成物として使用することが好ましく、ＮＫ細胞活性化用組成物として使用することが好ましく、抗インフルエンザ・ウイルス用組成物として使用することが好ましい。
ＭＣＣ１３７５株の突然変異株を含有する飲食品としては、ＭＣＣ１３７５株の突然変異株は、乳酸菌飲料の有効成分として使用することが好ましい。For example, acidic survival (low pH survival) can be confirmed against the mutant strain of MCC1375 strain. Specifically, for example, the survival rate after storage for 4 weeks at 10 ° C. at pH 3.0 is 90% or more, or the survival rate after storage for 6 weeks at 10 ° C. at pH 3.0, for example. Is preferably 40% or more. These storage conditions are determined by L. cerevisiae, a strain that appears to be closely related. Rhamnosus ATCC 53103 strain, L. Paracasei ATCC 25302 T strain and L. All of the casei ATCC 393 T strains have a survival rate of less than 0.1% or below the detection limit. However, the mutant strain of MCC1375 strain is not limited to those having these survival conditions.
The mutant strain of MCC1375 strain can be contained as an active ingredient of the medicament of the present invention, like the MCC1375 strain of the present invention, and can be contained as an active ingredient of the food and drink of the present invention. It can be contained as an active ingredient.
As a medicament containing a mutant of MCC1375 strain, it is preferably used as a composition for promoting IL-12 production, preferably used as a composition for NK cell activation, and as a composition for anti-influenza virus. It is preferable to use it.
As a food or drink containing a mutant strain of MCC1375 strain, the mutant strain of MCC1375 strain is preferably used as an active ingredient of a lactic acid bacteria beverage.
以下に実施例を挙げて本発明をより具体的に説明する。なお、本発明は以下の実施例に限定されるものではない。本発明の試験例ではラクトバチルス・パラカゼイＭＣＣ１３７５株の死菌体を使用するが、生菌体を使用しても同様の効果が得られる。 Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited to a following example. In the test example of the present invention, dead cells of Lactobacillus paracasei MCC1375 strain are used, but the same effect can be obtained even if live cells are used.
［試験例１］マウス脾細胞を用いたＩＬ−１２産生誘導能の評価
［試験方法］
ＭＣＣ１３７５株を含む乳酸菌群を対象に、マウス脾細胞を用いてＩＬ−１２産生誘導能の評価を行った。乳酸菌群には、インフルエンザ・ウイルスによる感染予防効果の知られるラクトバチルス・ラムノーサスＡＴＣＣ５３１０３株（ＧＧ株）、ラクトバチルス・ロイテリＪＣＭ１１１２株、エンテロコッカス・フェカリスＪＣＭ５８０３株、ビフィドバクテリウム・ロンガムＡＴＣＣ１５７０７株、ラクトバチルス・ガセリＪＣＭ５３４３株が含まれている。[Test Example 1] Evaluation of IL-12 production inducing ability using mouse spleen cells [Test method]
IL-12 production inducing ability was evaluated using mouse spleen cells for the group of lactic acid bacteria including MCC1375 strain. The lactic acid bacteria group includes Lactobacillus rhamnosus ATCC 53103 strain (GG strain), Lactobacillus reuteri JCM1112 strain, Enterococcus faecalis JCM5803 strain, Bifidobacterium longum ATCC 15707 strain, Lactobacillus・ Gasseri JCM5343 strain is included.
初めに、乳酸菌の各菌株をＭＲＳ（de Man Rogasa Sharpe）培地（Difco社製）で１６時間培養した物を、ＰＢＳ（phosphate buffered saline）で２回洗浄し、さらに蒸留水で２回洗浄した後に蒸留水で懸濁し、凍結乾燥を行った。凍結乾燥したものをPBSで懸濁し、１００℃にて３０分間の加熱処理を行い、乳酸菌死菌体試料（３．７５×１０12個／ｇ）とした。
実験動物には６週齢のオスBALB/cマウス（チャールズリバー社より入手）を用い、７−９週齢時に解剖し脾臓を採取した。採取した脾臓より脾細胞を採取し、赤血球溶血液（０．１４４Ｍ塩化アンモニウム、１７ｍＭトリスアミノメタン、ｐＨ７．６５）にて３分間処理し、赤血球を除去したものを調製した。調製した脾細胞を、ＲＰＭＩ１６４０（ＳＩＧＭＡ社）に１０％ＦＣＳ（Ｇｉｂｃｏ社製）、１００ＩＵ／ｍｌペニシリン、０．１ｍｇ／ｍｌストレプトマイシンを加えた培地で、２×１０6ｃｅｌｌｓ／ｍｌになるように懸濁し、乳酸菌死菌体試料を終濃度１μｇ／ｍｌ（３．７５×１０6個／ｍｌ）になるように添加し、９６穴のマイクロプレート（ＦＡＬＣＯＮ社製）にて３７℃、５％のＣＯ2存在下で培養した。２日後に培養上清を回収し、培養上清中のＩＬ−１２ｐ７０の濃度をＲ＆Ｄ ｓｙｓｔｅｍｓ社製、ＤｕｏＳｅｔ（ＤＹ４１９）を使用して測定した。
試験は３回行い、その平均値を表２に示す。First, after culturing each strain of lactic acid bacteria in MRS (de Man Rogasa Sharpe) medium (Difco) for 16 hours, it was washed twice with PBS (phosphate buffered saline) and further washed twice with distilled water. It was suspended in distilled water and lyophilized. The lyophilized product was suspended in PBS and heat-treated at 100 ° C. for 30 minutes to obtain a lactic acid bacterium dead cell sample (3.75 × 10 12 cells / g).
As experimental animals, 6-week-old male BALB / c mice (obtained from Charles River) were dissected at 7-9 weeks of age and spleens were collected. Spleen cells were collected from the collected spleen and treated with erythrocyte lysate (0.144M ammonium chloride, 17 mM trisaminomethane, pH 7.65) for 3 minutes to prepare a erythrocyte-removed product. The prepared spleen cells were suspended in RPMI1640 (SIGMA) in a medium containing 10% FCS (Gibco), 100 IU / ml penicillin, 0.1 mg / ml streptomycin to 2 × 10 6 cells / ml. After turbidity, a lactic acid bacterium dead cell sample was added to a final concentration of 1 μg / ml (3.75 × 10 6 cells / ml). Cultured in the presence of 2 . Two days later, the culture supernatant was collected, and the concentration of IL-12p70 in the culture supernatant was measured using DuoSet (DY419) manufactured by R & D systems.
The test was performed three times, and the average value is shown in Table 2.
［結果］
表２は、各乳酸菌株によるＩＬ−１２産生量の平均値を示している。
ラクトバチルス・パラカゼイＭＣＣ１３７５株を使用したときのＩＬ−１２産生量は２５４ｐｇ／ｍｌとなり、試験した乳酸菌の中で最も高いＩＬ−１２産生誘導能を示した。
一方、ラクトバチルス・ラムノーサスＡＴＣＣ５３１０３株のＩＬ−１２産生量は８７ｐｇ／ｍｌであった。
従って、ＩＬ−１２産生量では、本発明のラクトバチルス・パラカゼイＭＣＣ１３７５株は、ラクトバチルス・ラムノーサスＡＴＣＣ５３１０３株の２．９倍であった。
その他、ラクトバチルス・ロイテリＪＣＭ１１１２株のＩＬ−１２産生量は４８ｐｇ／ｍｌ、エンテロコッカス・フェカリスＪＣＭ５８０３株のＩＬ−１２産生量は２５ｐｇ／ｍｌ、ビフィドバクテリウム・ロンガムＡＴＣＣ１５７０７T株のＩＬ−１２産生量は１４ｐｇ／ｍｌ、ラクトバチルス・ガセリＪＣＭ５３４３株のＩＬ−１２産生量は０．７ｐｇ／ｍｌであった。
このように、ラクトバチルス・パラカゼイＭＣＣ１３７５株は、対比した従来の乳酸菌の菌株と比較して、顕著なＩＬ−１２産生促進の効果を有していた。[result]
Table 2 shows the average value of IL-12 production by each lactic acid strain.
When the Lactobacillus paracasei MCC1375 strain was used, the IL-12 production amount was 254 pg / ml, indicating the highest IL-12 production inducing ability among the tested lactic acid bacteria.
On the other hand, the amount of IL-12 produced by the Lactobacillus rhamnosus ATCC 53103 strain was 87 pg / ml.
Therefore, in the amount of IL-12 produced, the Lactobacillus paracasei MCC1375 strain of the present invention was 2.9 times the Lactobacillus rhamnosus ATCC53103 strain.
In addition, the IL-12 production amount of Lactobacillus reuteri JCM1112 strain was 48 pg / ml, the IL-12 production amount of Enterococcus faecalis JCM5803 strain was 25 pg / ml, and the IL-12 production amount of Bifidobacterium longum ATCC15707 T strain Was 14 pg / ml, and the amount of IL-12 produced by Lactobacillus gasseri strain JCM5343 was 0.7 pg / ml.
Thus, the Lactobacillus paracasei MCC1375 strain had a remarkable effect of promoting IL-12 production as compared with the conventional strain of lactic acid bacteria.
［試験例２］ヒト由来末梢血単核球（ＰＢＭＣ）を用いたＩＬ−１２産生誘導能の評価
［試験方法］
ラクトバチルス・パラカゼイＭＣＣ１３７５株のＩＬ−１２産生誘導能をヒト由来ＰＢＭＣを用いて検証した。
ラクトバチルス・パラカゼイＭＣＣ１３７５株の死菌体を試験例１と同様に調製した。
健常人のＰＢＭＣ（Lonza Walkersville Inc社より入手）を、Ｘ-ＶＩＶＯ (Lonza社製)に１０％ＦＣＳ（Gibco社製）、１００ＩＵ／ｍｌペニシリン、０．１ｍｇ／ｍｌストレプトマイシンを加えた培地で、２×１０6ｃｅｌｌｓ／ｍｌになるように懸濁し、ＭＣＣ１３７５株の菌体試料を終濃度０．１−１０μｇ／ｍｌ（３．７５×１０5〜３．７５×１０7）になるように添加し、９６穴のマイクロプレート（ＦＡＬＣＯＮ社製）にて３７℃、５％ＣＯ2存在下で培養した。２日後に培養上清を回収し、培養上清中のＩＬ−１２ｐ４０の濃度をＲ＆Ｄ ｓｙｓｔｅｍｓ社製、ＤｕｏＳｅｔ（ＤＹ１２４０）を使用して測定した。試験結果を図１に示す。[Test Example 2] Evaluation of IL-12 production inducing ability using human-derived peripheral blood mononuclear cells (PBMC) [Test method]
The ability to induce IL-12 production of Lactobacillus paracasei MCC1375 strain was verified using human-derived PBMC.
A dead cell of Lactobacillus paracasei MCC1375 strain was prepared in the same manner as in Test Example 1.
A healthy person's PBMC (obtained from Lonza Walkersville Inc) was added to X-VIVO (Lonza) with 10% FCS (Gibco), 100 IU / ml penicillin, 0.1 mg / ml streptomycin. Suspend to × 10 6 cells / ml, and add a cell sample of MCC1375 strain to a final concentration of 0.1-10 μg / ml (3.75 × 10 5 to 3.75 × 10 7 ). The cells were cultured in a 96-well microplate (manufactured by FALCON) at 37 ° C. in the presence of 5% CO 2 . Two days later, the culture supernatant was collected, and the concentration of IL-12p40 in the culture supernatant was measured using DuoSet (DY1240) manufactured by R & D systems. The test results are shown in FIG.
［結果］
図１において、縦軸はＩＬ−１２産生量を示し、横軸はＭＣＣ１３７５株菌体の添加量を示す。
ＭＣＣ１３７５株死菌体の濃度が０．１μｇ／ｍｌのとき、ＩＬ−１２産生量は１１３９ｐｇ／ｍｌであった。濃度が０．４μｇ／ｍｌのとき、ＩＬ−１２産生量は１４７６ｐｇ／ｍｌであった。濃度が１．１μｇ／ｍｌのとき、ＩＬ−１２産生量は１５９５ｐｇ／ｍｌであった。また、濃度が３．３μｇ／ｍｌのとき、ＩＬ−１２産生量は２０２０ｐｇ／ｍｌであった。さらに、濃度が１０．０μｇ／ｍｌのときのＩＬ−１２産生量は２１０４ｐｇ／ｍｌであった。
このように、ＭＣＣ１３７５株の死菌体は、ヒト由来ＰＢＭＣから濃度依存的にＩＬ−１２産生を誘導した。また、本発明のＭＣＣ１３７５株のＩＬ−１２産生誘導作用は、ヒトの免疫担当細胞に対して有効であることが明らかになった。[result]
In FIG. 1, the vertical axis represents the amount of IL-12 production, and the horizontal axis represents the amount of MCC1375 strain cells added.
When the concentration of MCC1375 strain dead cells was 0.1 μg / ml, IL-12 production was 1139 pg / ml. When the concentration was 0.4 μg / ml, IL-12 production was 1476 pg / ml. When the concentration was 1.1 μg / ml, IL-12 production was 1595 pg / ml. In addition, when the concentration was 3.3 μg / ml, the IL-12 production amount was 2020 pg / ml. Furthermore, the IL-12 production amount when the concentration was 10.0 μg / ml was 2104 pg / ml.
Thus, dead cells of the MCC1375 strain induced IL-12 production from human-derived PBMC in a concentration-dependent manner. In addition, it was revealed that the IL-12 production inducing action of the MCC1375 strain of the present invention is effective against human immunocompetent cells.
［試験例３］ＭＣＣ１３７５株投与がマウスのＮＫ細胞の割合に及ぼす影響
［試験方法］
ＭＣＣ１３７５投与がＮＫ細胞に及ぼす影響を、マウスを用いて調べた。
ＭＣＣ１３７５株をＭＲＳ培地で１６時間培養した物を、ＰＢＳで２回洗浄し、さらに蒸留水で２回洗浄した後に蒸留水で懸濁し、１００℃にて３０分間の加熱処理を行った。加熱処理したものを凍結乾燥処理し、ＭＣＣ１３７５株死菌体（３．７５×１０12個／ｇ）とした。
調製したＭＣＣ１３７５株死菌体０．０１％を含むＡＩＮ９３−Ｇ飼料（ＭＣＣ１３７５群）または菌体を含まない飼料（対照群）を、８週齢の雄性ＢＡＬＢ／ｃマウス （ＳＬＣ）に１０日間投与した。投与後にマウスの脾細胞を調製し、蛍光標識した抗マウスＣＤ３抗体と抗マウスＤＸ５抗体で染色し、ＣＤ３陰性ＤＸ５陽性細胞をＮＫ細胞として、脾細胞中のＮＫ細胞の割合をフローサイトメトリー(機種名：ＦＡＣＳＣａｎｔｏ、べクトン・ディッキンソン社製)で測定した。試験は８回行い、平均値を求めた。[Test Example 3] Effect of MCC1375 strain administration on the proportion of NK cells in mice [Test method]
The effect of MCC1375 administration on NK cells was examined using mice.
A product obtained by culturing MCC1375 strain in MRS medium for 16 hours was washed twice with PBS, further washed twice with distilled water, suspended in distilled water, and heat-treated at 100 ° C. for 30 minutes. The heat-treated product was freeze-dried to obtain dead cells of MCC1375 strain (3.75 × 10 12 cells / g).
The prepared AIN93-G diet (MCC1375 group) containing 0.01% dead cells of MCC1375 strain or a diet containing no cells (control group) was administered to 8-week-old male BALB / c mice (SLC) for 10 days. did. After administration, mouse spleen cells were prepared, stained with fluorescently labeled anti-mouse CD3 antibody and anti-mouse DX5 antibody, CD3 negative DX5 positive cells were used as NK cells, and the ratio of NK cells in spleen cells was determined by flow cytometry (model). Name: FACSCanto, manufactured by Becton Dickinson). The test was performed 8 times, and the average value was obtained.
［結果］
結果を図２に示す。有意差検定はｔ検定を用いた。図中の＊は両群間の統計学的有意差を示す。図中のｃｏｎｔｒｏｌは、対照群を示す。
脾細胞中のＮＫ細胞の割合は、対照群が６．８％であったのに対し、ＭＣＣ１３７５株では７．８％となり、有意にＮＫ細胞の増加が見られた。
このように、ＭＣＣ１３７５株がＮＫ細胞増殖促進効果を有することが明らかになった。[result]
The results are shown in FIG. The t-test was used for the significant difference test. * In the figure indicates a statistically significant difference between the two groups. In the figure, control indicates a control group.
The proportion of NK cells in the spleen cells was 6.8% in the control group compared with 7.8% in the MCC1375 strain, and a significant increase in NK cells was observed.
Thus, it was revealed that the MCC1375 strain has an NK cell proliferation promoting effect.
［試験例４］インフルエンザ・ウイルス感染予防効果の確認
［試験方法］
ＭＣＣ１３７５株のインフルエンザ・ウイルス感染予防作用をマウスで確認した。
ＭＣＣ１３７５株をＭＲＳ培地で１６時間培養した物を、ＰＢＳで２回洗浄し、さらに蒸留水で２回洗浄した後に蒸留水で懸濁し、凍結乾燥を行った。
凍結乾燥したＭＣＣ１３７５株を生理食塩水で５ｍｇ／ｍｌ（１．８８×１０10個／ｍｌ）に懸濁し、１００℃にて３０分間の加熱処理を行ったものをＭＣＣ１３７５株死菌体の懸濁液とした。
ＢＡＬＢ／ｃマウスに０．２ｍｌのＭＣＣ１３７５株懸濁液（ＭＣＣ１３７５群）または生理食塩水（対照群）を１４日間経口投与した後、インフルエンザ・ウイルス(Ａ／ＰＲ８／３４（Ｈ１Ｎ１）株)を鼻腔より接種し、感染させた。各群のマウスの数は１０匹（ｎ＝１０）とした。
感染後の状態については、感染後の６日間の間に、マウスの状態を観察してスコア化した症状スコアにより評価した。
症状スコアは、１．マウスの眼（開瞼と眼瞼の程度）、２．被毛（毛並みと立毛）、３．呼吸（呼吸不整）、４．行動（自発運動の程度）、の各項目を１点刻みで０〜４とするスコア化を行った。
評価の基準は、正常を０、軽度の感染を１、中程度の感染を２、重度の感染を３、死亡を４とした。
各項目の数値の合計を項目数（４）で割り、マウスの個体ごとのスコアとした。
さらに、マウス１０匹の症状スコアの合計を、マウスの数（ｎ＝１０）で割って症状スコアとした。
また、感染前と感染６日後の体重を測定し、感染６日後の体重減少率を測定した。
さらに、感染６日後に肺を摘出してＭＤＣＫ細胞（イヌ腎由来細胞）を用いたプラークアッセイ法で肺中ウイルス濃度を測定した。[Test Example 4] Confirmation of influenza virus infection prevention effect [Test method]
The prevention of influenza virus infection of MCC1375 strain was confirmed in mice.
A product obtained by culturing MCC1375 strain in MRS medium for 16 hours was washed twice with PBS, further washed twice with distilled water, suspended in distilled water, and freeze-dried.
The lyophilized MCC1375 strain was suspended in physiological saline at 5 mg / ml (1.88 × 10 10 cells / ml) and subjected to heat treatment at 100 ° C. for 30 minutes. A liquid was used.
BALB / c mice were orally administered with 0.2 ml of MCC1375 strain suspension (MCC1375 group) or physiological saline (control group) for 14 days, and then influenza virus (A / PR8 / 34 (H1N1) strain) was administered to the nasal cavity. More inoculated and infected. The number of mice in each group was 10 (n = 10).
The state after infection was evaluated by a symptom score obtained by observing the mouse state during the 6 days after infection.
The symptom score is: 1. Mouse eye (degree of eyelid and eyelid) 2. Covered hair (hair and raised hair); 3. Breathing (breathing irregularity) Each item of behavior (degree of spontaneous movement) was scored from 0 to 4 in 1 point increments.
The evaluation criteria were 0 for normal, 1 for mild infection, 2 for moderate infection, 3 for severe infection and 4 for death.
The total of the numerical values of each item was divided by the number of items (4) to obtain a score for each mouse.
Furthermore, the total of the symptom scores of 10 mice was divided by the number of mice (n = 10) to obtain a symptom score.
Moreover, the body weight before infection and 6 days after infection was measured, and the
Furthermore, 6 days after the infection, the lungs were removed and the virus concentration in the lungs was measured by a plaque assay method using MDCK cells (canine kidney-derived cells).
［結果］
試験結果を図３に示す。図３の（ａ）は症状スコアの変化を示すグラフである。横軸は感染後の日数、縦軸は症状スコアを示す。図３の（ｂ）は体重減少率を示すグラフである。縦軸は感染前の体重と比較した体重減少率である。図３の（ｃ）はマウスの肺中のウイルス濃度を示すグラフである。また、図３の（ａ）〜（ｃ）の有意差検定はｔ検定を用いた。図中の＊は両群間の統計学的有意差を示す。また、図中の＋は両群間の統計学的な傾向を示す。図中のｃｏｎｔｒｏｌは、対照群を示す。[result]
The test results are shown in FIG. FIG. 3A is a graph showing changes in symptom scores. The horizontal axis represents the number of days after infection, and the vertical axis represents the symptom score. FIG. 3B is a graph showing the weight loss rate. The vertical axis is the weight loss rate compared to the weight before infection. FIG. 3 (c) is a graph showing the virus concentration in the lungs of mice. In addition, the t-test was used for the significant difference test of (a) to (c) in FIG. * In the figure indicates a statistically significant difference between the two groups. Moreover, + in a figure shows the statistical tendency between both groups. In the figure, control indicates a control group.
図３の（ａ）には感染後１〜６日目の症状スコア変化が示されている。ＭＣＣ１３７５群では、感染から１日目が０．００、２日目が０．００、３日目が０．０８、４日目が０．０８、５日目が０．２８、６日目が０．４８であった。
一方、対照群では、感染から１日目が０．０３、２日目が０．２０、３日目が０．４８、４日目が０．６８、５日目が１．００、６日目が１．３５であった。
このように、ＭＣＣ１３７５群の２、３、４、６日目の症状スコアは対照群と比較して有意に症状スコアの上昇抑制がみられた。また、ＭＣＣ１３７５群の５日目の症状スコアは対照群と比較して症状の抑制傾向がみられた。FIG. 3 (a) shows changes in symptom scores on
On the other hand, in the control group, the first day after infection was 0.03, the second day was 0.20, the third day was 0.48, the fourth day was 0.68, the fifth day was 1.00, and the sixth day. The eyes were 1.35.
Thus, as for the symptom score of the 2, 3, 4, and 6th day of the MCC1375 group, the rise suppression of the symptom score was significantly seen compared with the control group. Moreover, the symptom score of the 5th day of the MCC1375 group showed the tendency for suppression of the symptom compared with the control group.
図３の（ｂ）には感染後の体重減少率が示されている。ＭＣＣ１３７５群が４．８％であるのに対し、対照群が１３．２％であり、ＭＣＣ１３７５群が有意に感染後の体重減少率を抑制することが明らかになった。 FIG. 3 (b) shows the weight loss rate after infection. The MCC1375 group was 4.8%, whereas the control group was 13.2%, and the MCC1375 group was found to significantly suppress the rate of weight loss after infection.
図３の（ｃ）にはマウス肺中のウイルス濃度が示されている。ＭＣＣ１３７５群が１．２１×１０6ｐｆｕ／ｇであるのに対し、対照群が０．５７×１０6ｐｆｕ／ｇであり、ＭＣＣ１３７５群が有意に感染後のウイルス濃度が抑制することが明らかになった。FIG. 3 (c) shows the virus concentration in the mouse lung. The MCC1375 group is 1.21 × 10 6 pfu / g, whereas the control group is 0.57 × 10 6 pfu / g, and it is clear that the MCC1375 group significantly suppresses the virus concentration after infection. became.
本試験の結果より、ＭＣＣ１３７５株の投与により、インフルエンザ・ウイルス投与による症状スコアの悪化及び体重減少が改善され、さらには肺中ウイルス濃度も減少することが明らかになった。
これらの結果から、ＭＣＣ１３７５株の投与がインフルエンザ・ウイルス感染の予防に有効であることが示された。From the results of this study, it was clarified that administration of the MCC1375 strain improved the worsening of symptom scores and weight loss due to influenza virus administration, and also decreased the virus concentration in the lungs.
From these results, it was shown that administration of MCC1375 strain is effective in preventing influenza virus infection.
［試験例５］低ｐＨにおける生残性の評価
本試験は、本発明のラクトバチルス・パラカゼイＭＣＣ１３７５株が、低ｐＨ環境下においても顕著な生残性を有することを確認することを目的とした。
なお、本試験において使用したピーチジュースに添加する乳酸菌の量は僅かである。従って、最終的な乳酸菌飲料におけるｐＨは、殺菌ベースにおけるｐＨとほぼ等しい。また、本試験における「％」は乳酸菌の菌数の百分率である。
本試験では、対照株として同種又は近縁の株である、ラクトバチルス・ラムノーラスＡＴＣＣ５３１０３株、ラクトバチルス・パラカゼイＡＴＣＣ２５３０２T株、ラクトバチルス・カゼイＡＴＣＣ３９３T株を使用した。[Test Example 5] Evaluation of survival at low pH The purpose of this test was to confirm that the Lactobacillus paracasei MCC1375 strain of the present invention has remarkable survival even in a low pH environment. .
The amount of lactic acid bacteria added to the peach juice used in this test is very small. Thus, the pH in the final lactic acid bacteria beverage is approximately equal to the pH in the sterilization base. Moreover, “%” in this test is a percentage of the number of lactic acid bacteria.
In this test, Lactobacillus rhamnolaus ATCC 53103 strain, Lactobacillus paracasei ATCC 25302 T strain, and Lactobacillus casei ATCC 393 T strain were used as control strains.
［試験方法］
殺菌済のピーチジュース（ｐＨ３．０）１０００ｍｌに、ラクトバチルス・パラカゼイＭＣＣ１３７５株、ラクトバチルス・ラムノーラスＡＴＣＣ５３１０３株、ラクトバチルス・パラカゼイＡＴＣＣ２５３０２T株、ラクトバチルス・カゼイＡＴＣＣ３９３T株のカルチャーをそれぞれの菌数が約１０7ｃｆｕ／ｍｌ前後となるように添加し、４種類の試験液を調製した。
次いで、各試験液を１００ｍｌずつプラスチック容器に分注し、１０℃以下で６週間保存し、調製直後、保存後２週間後、４週間後および６週間後における乳酸菌数を測定し、生残率を求めた。
乳酸菌数の測定は、BCP加プレートカウント寒天培地（栄研社製）平板で行った。生残率とは、試験液調製直後の乳酸菌数を基準とした、保存期間後の乳酸菌数を百分率を示した数値である。結果を表３に示す。[Test method]
The culture of Lactobacillus paracasei MCC1375 strain, Lactobacillus rhamnolaus ATCC53103 strain, Lactobacillus paracasei ATCC25302 T strain, Lactobacillus casei ATCC393 T strain was added to 1000 ml of sterilized peach juice (pH 3.0). Four kinds of test solutions were prepared by adding about 10 7 cfu / ml.
Next, 100 ml of each test solution was dispensed into a plastic container and stored at 10 ° C. or lower for 6 weeks. The number of lactic acid bacteria was measured immediately after preparation, 2 weeks after storage, 4 weeks and 6 weeks, and the survival rate. Asked.
The number of lactic acid bacteria was measured on a BCP-added plate count agar medium (manufactured by Eiken). The survival rate is a numerical value indicating the percentage of the number of lactic acid bacteria after the storage period based on the number of lactic acid bacteria immediately after preparation of the test solution. The results are shown in Table 3.
［結果］
ラクトバチルス・パラカゼイＭＣＣ１３７５株は、２週間後、４週間後、６週間後の生残率がそれぞれ１０２％、９３％、４１％となった。
一方、他の３種の乳酸菌は、２週間後には生残率がほぼ０％となり、４週間後、６週間後には、全く検出されなかった。表３のなかのn.d.は、検出限界以下であったことを示す。
この結果から、本発明の乳酸菌が同種、近縁の菌株と比較して、低ｐＨの環境下において顕著な生残性を有していることが明らかとなった。[result]
Lactobacillus paracasei MCC1375 strains had survival rates of 102%, 93% and 41% after 2 weeks, 4 weeks and 6 weeks, respectively.
On the other hand, the remaining three lactic acid bacteria had a survival rate of almost 0% after 2 weeks, and were not detected at all after 4 weeks or 6 weeks. Nd in Table 3 indicates that it was below the detection limit.
From these results, it was revealed that the lactic acid bacteria of the present invention have remarkable survival in a low pH environment as compared with the same and related strains.
［実施例１］
還元脱脂粉乳１０％、グルコース１％、酵母エキス０．１％を含む培地１０００ｍＬを９０℃で３０分間殺菌し、ラクトバチルス・パラカゼイＭＣＣ１３７５株のシードカルチャーを３０ｍＬ接種し、３７℃１６時間培養した。
これとは別に、脱脂粉乳、全粉乳、ペクチン、及び蔗糖からなる原料を混合溶解して得られた、乳脂肪０．５％、無脂乳固形分８．０％、蔗糖５．０％、ペクチン０．２％からなるベース５０Ｌを、９０℃で１０分間殺菌し、４０℃に冷却した。殺菌したベースに前記の培養したラクトバチルス・パラカゼイＭＣＣ１３７５株のカルチャー５０ｍＬを接種し、３７℃１６時間培養して発酵乳を得た。前記発酵乳を直ちに攪拌冷却し、冷却した発酵乳を１５ＭＰａの圧力で均質化し、２００ｍＬ容量ガラス容器に充填し、密封し、ドリンクヨーグルト２５０個を得た。[Example 1]
1000 mL of a medium containing 10% reduced skim milk powder, 1% glucose, and 0.1% yeast extract was sterilized at 90 ° C. for 30 minutes, inoculated with 30 mL of a seed culture of Lactobacillus paracasei MCC1375 strain, and cultured at 37 ° C. for 16 hours.
Separately, skim milk powder, whole milk powder, pectin, and sucrose raw materials were mixed and dissolved, 0.5% milk fat, 8.0% non-fat milk solids, 5.0% sucrose, 50 L of base consisting of 0.2% pectin was sterilized at 90 ° C. for 10 minutes and cooled to 40 ° C. The sterilized base was inoculated with 50 mL of the cultured Lactobacillus paracasei MCC1375 culture and cultured at 37 ° C. for 16 hours to obtain fermented milk. The fermented milk was immediately stirred and cooled, and the cooled fermented milk was homogenized at a pressure of 15 MPa, filled in a 200 mL capacity glass container, and sealed to obtain 250 drink yogurts.
［実施例２］
肉エキス５０ｇ、酵母エキス１００ｇ、ペプトン１００ｇ、グルコース２００ｇ、Ｋ2ＨＰＯ4５０ｇ、ＫＨ2ＰＯ4 １０ｇ、シスチン４ｇ及び水９．５Ｌの組成からなる培地で３７℃１６時間前培養したラクトバチルス・パラカゼイＭＣＣ１３７５株のシードカルチャー５００ｍＬを、前記培地と同一組成の培地１０Ｌに接種し、３７℃、１６時間培養した。更に、前記培地と同一組成の培地２００Ｌに、前記培養液全量（１０．５Ｌ）を接種し、３７℃１６時間培養した。
次いで、シャープレス型遠心分離機を用いて、遠心分離（１５，０００ｒｐｍ）により菌体を集め、培地と同量の生理食塩水に再懸濁し、前記と同様遠心分離して再度集菌した。集めた菌体を、脱脂粉乳１０％、蔗糖１％、グルタミン酸ソーダ１％からなる溶液２０Ｌに懸濁し、常法に従って凍結乾燥し、同量のスターチで倍散した。３．７５×１０12ＣＦＵ／ｇのラクトバチルス・パラカゼイＭＣＣ１３７５株を含む粉末約５．０ｋｇを得た。[Example 2]
Lactobacillus paracasei pre-cultured at 37 ° C. for 16 hours in a medium comprising 50 g of meat extract, 100 g of yeast extract, 100 g of peptone, 200 g of glucose, 50 g of K 2 HPO 4 , 10 g of KH 2 PO 4 , 4 g of cystine and 9.5 L of water. 500 mL of seed culture of MCC1375 strain was inoculated into 10 L of medium having the same composition as the above medium and cultured at 37 ° C. for 16 hours. Further, 200 L of the medium having the same composition as the medium was inoculated with the whole culture solution (10.5 L) and cultured at 37 ° C. for 16 hours.
Subsequently, the cells were collected by centrifugation (15,000 rpm) using a sharpless centrifuge, resuspended in the same amount of physiological saline as the medium, and centrifuged again to collect the cells. The collected cells were suspended in 20 L of a solution comprising 10% skim milk powder, 1% sucrose, and 1% sodium glutamate, lyophilized according to a conventional method, and triturated with the same amount of starch. About 5.0 kg of powder containing 3.75 × 10 12 CFU / g Lactobacillus paracasei MCC1375 strain was obtained.
［実施例３］
乾燥殺菌した澱粉１４ｋｇ及び乳糖６ｋｇに、実施例２で得られたラクトバチルス・パラカゼイＭＣＣ１３７５株を含む粉末２０ｇを加えて均一に混合し、３．７５×１０9ＣＦＵ／ｇのラクトバチルス・パラカゼイＭＣＣ１３７５株を含むＩＬ−１２産生誘導用組成物約２０ｋｇを得た。[Example 3]
To 14 kg of dry-sterilized starch and 6 kg of lactose, 20 g of the powder containing Lactobacillus paracasei MCC1375 strain obtained in Example 2 was added and mixed uniformly to obtain 3.75 × 10 9 CFU / g Lactobacillus paracasei MCC1375. About 20 kg of an IL-12 production-inducing composition containing the strain was obtained.
［実施例４］
乾燥殺菌した澱粉７ｋｇ及び乳糖３．５ｋｇに、実施例２で得られたラクトバチルス・パラカゼイＭＣＣ１３７５株を含む粉末１１ｇを加えて均一に混合し、３．９２×１０9ＣＦＵ／ｇのラクトバチルス・パラカゼイＭＣＣ１３７５株を含むＮＫ細胞活性化用組成物約１０ｋｇを得た。[Example 4]
To 7 kg of dry sterilized starch and 3.5 kg of lactose, 11 g of the powder containing Lactobacillus paracasei MCC1375 strain obtained in Example 2 was added and mixed uniformly, and 3.92 × 10 9 CFU / g of Lactobacillus. About 10 kg of a composition for NK cell activation containing Paracasei MCC1375 strain was obtained.
［実施例５］
乾燥殺菌した澱粉１０ｋｇ及び乳糖４．３ｋｇに、実施例２で得られたラクトバチルス・パラカゼイＭＣＣ１３７５株を含む粉末１４ｇを加えて均一に混合し、３．６７×１０9ＣＦＵ／ｇのラクトバチルス・パラカゼイＭＣＣ１３７５株を含む抗インフルエンザ・ウイルス用組成物約１４ｋｇを得た。[Example 5]
To 10 kg of dry sterilized starch and 4.3 kg of lactose, 14 g of powder containing Lactobacillus paracasei MCC1375 strain obtained in Example 2 was added and mixed uniformly, and 3.67 × 10 9 CFU / g of Lactobacillus. About 14 kg of an anti-influenza virus composition containing Paracasei MCC1375 strain was obtained.
［実施例６］
澱粉１４ｋｇ、脱脂粉乳１３．５ｋｇ、植物油脂３０ｋｇ、大豆粕１３ｋｇ、小麦粉１１ｋｇ、ビタミン混合物９ｋｇ、ミネラル混合物２ｋｇ、セルロース３ｋｇを均一に混合し、さらに実施例２で得られたラクトバチルス・パラカゼイＭＣＣ１３７５株を含む粉末２．５ｋｇを添加して均一に混合し、９．５７×１０10ＣＦＵ／ｇのラクトバチルス・パラカゼイＭＣＣ１３７５株を含む飼料組成物約９８ｋｇを得た。[Example 6]
14 kg starch, skim milk powder 13.5 kg, vegetable oil and fat 30 kg, soybean meal 13 kg, wheat flour 11 kg, vitamin mixture 9 kg,
本発明の新規乳酸菌、特にラクトバチルス・パラカゼイＭＣＣ１３７５株は、酸性条件下の生残性の高いものであり、さらに、死菌体であっても高いＩＬ−１２産生誘導能を有し、ＮＫ細胞の増加を誘導し、インフルエンザ・ウイルス感染に対する予防作用を示すものであるので、これらの用途の医薬品、特に経口投与製剤、これらの用途の機能性の飲食品、特に酸性乳酸菌飲料などにおいて、産業上広く利用できるものである。 The novel lactic acid bacteria of the present invention, particularly the Lactobacillus paracasei MCC1375 strain, has high survival under acidic conditions, and has high IL-12 production-inducing ability even in dead cells, NK cells In the pharmaceuticals for these uses, especially oral preparations, functional foods and drinks for these uses, especially acidic lactic acid bacteria beverages, etc. It can be widely used.
ＦＥＲＭ ＢＰ−１１３１３ FERM BP-11313
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| CN103070287B (en) * | 2013-02-26 | 2014-10-08 | 湖南省微生物研究所 | Lactobacillus paracasei fermented feed and preparation method thereof, fermenting equipment and application of lactobacillus paracasei fermented feed |
| AU2014288647B8 (en) | 2013-07-12 | 2015-10-29 | Morinaga Milk Industry Co., Ltd. | Novel Lactic Acid Bacterium, Drug, Food or Drink, and Feed which Contain the Novel Lactic Acid Bacterium |
| BR122022016606B1 (en) | 2013-07-12 | 2023-01-24 | Morinaga Milk Industry Co., Ltd | LACTOBACILLUS PARACASEI DEAD BY HEATING, DRUG, FOOD OR DRINK, FEED, AGENT PROMOTING IL-12 PRODUCTION AND USE |
| JP6590330B2 (en) * | 2013-11-11 | 2019-10-16 | キッコーマン株式会社 | Oral immune tolerance enhancing substance screening method and oral immune tolerance enhancing composition |
| US11457641B2 (en) * | 2015-02-17 | 2022-10-04 | Kabushiki Kaisha Yakult Honsha | Method for producing fermented milk food |
| KR101791850B1 (en) * | 2015-06-29 | 2017-11-01 | (주)진바이오텍 | Total mixed ration and silage fermentation accelerator for ruminant |
| CA3032572C (en) | 2017-06-09 | 2020-04-21 | Asahi Kohsan Corporation | Lactobacillus paracasei bacterium and use thereof |
| JP6523579B2 (en) | 2017-06-09 | 2019-06-05 | 旭興産株式会社 | Extracellular polysaccharide of lactic acid bacteria and use thereof |
| TWI727293B (en) * | 2018-04-25 | 2021-05-11 | 日商曾根農場股份有限公司 | Composition for Type IV Allergy |
| CN114657100B (en) * | 2022-04-01 | 2023-07-25 | 江南大学 | Lactobacillus pentosus capable of relieving pathological characteristics of influenza virus infected mice and application thereof |
| WO2024004139A1 (en) * | 2022-06-30 | 2024-01-04 | 森永乳業株式会社 | Fermented milk and production method for fermented milk |
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| WO2000053201A1 (en) * | 1999-03-11 | 2000-09-14 | Societe Des Produits Nestle S.A. | Lactic acid bacteria strains capable of preventing diarrhoea |
| WO2004096246A1 (en) * | 2003-03-13 | 2004-11-11 | Kirin Beer Kabushiki Kaisha | Antiallergic composition |
| WO2007040445A1 (en) * | 2005-10-06 | 2007-04-12 | Probi Ab | Use of lactobacillus for treatment of virus infections |
| WO2009130423A2 (en) * | 2008-04-18 | 2009-10-29 | Compagnie Gervais Danone | Novel strain of lactobacillus paracasei subspecies paracasei having antimicrobial and immunomodulatory properties |
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