JP6053466B2 - Stress diarrhea inhibitor - Google Patents
Stress diarrhea inhibitor Download PDFInfo
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- JP6053466B2 JP6053466B2 JP2012252422A JP2012252422A JP6053466B2 JP 6053466 B2 JP6053466 B2 JP 6053466B2 JP 2012252422 A JP2012252422 A JP 2012252422A JP 2012252422 A JP2012252422 A JP 2012252422A JP 6053466 B2 JP6053466 B2 JP 6053466B2
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Description
本発明は、ある種のラクトバチルス・ガセリ株又はその処理物を有効成分として含む、ストレス性下痢の抑制剤に関する。 The present invention relates to an inhibitor of stress diarrhea containing a certain type of Lactobacillus gasseri strain or a processed product thereof as an active ingredient.
本発明はまた、上記抑制剤を含有する、食品添加剤若しくは動物飼料添加剤、或いは、食品又は医薬組成物に関する。 The present invention also relates to a food additive or animal feed additive, or a food or pharmaceutical composition containing the inhibitor.
ラクトバチルス（Lactobacillus）属の乳酸菌にストレス抑制作用や胃腸疾患に有効である可能性を示す先行技術文献が、以下に例示するようにいくつか散見される。 Several prior art documents showing the possibility of Lactobacillus lactic acid bacteria being effective for stress-suppressing action and gastrointestinal diseases are found as shown below.
例えば、特開2009-102292号公報（特許文献１）には、ラクトバチルス・パラカゼイ・サブスピシズ・パラカゼイ(Lactobacillus paracasei subsp. paracasei)である乳酸菌FERM P-19169を有効成分とし、サイトカイン産生調節作用、感染防御作用、肥育作用又は下痢抑制作用に基づくストレス抑制剤が記載されている。この文献では、ストレス抑制とは、ストレスを引き起こす外部刺激であるストレッサーにより生じた生体の非特異的反応を抑制することによって、食欲減退、消化不良、不眠、体調不良、感染症などの症状を緩和することができると記載されている。 For example, JP 2009-102292 A (Patent Document 1) includes Lactobacillus paracasei subsp. Paracasei lactic acid bacteria FERM P-19169 as an active ingredient, cytokine production regulating action, infection A stress suppressor based on a protective action, fattening action or diarrhea inhibiting action is described. In this document, stress suppression refers to alleviating symptoms such as loss of appetite, indigestion, insomnia, poor physical condition, and infection by suppressing nonspecific reactions in the body caused by stressors, which are external stimuli that cause stress. It is stated that you can.
特開2009-100692号公報（特許文献2）及び特開2008-212140号公報（特許文献3）には、ストレス軽減作用を有するラクトバチルス属微生物を含有することを特徴とする哺乳動物用乳酸菌製剤が記載されており、特定の乳酸菌を、哺乳動物に投与することにより、優れたストレス軽減効果を発揮し、ストレスに起因する下痢症などの消化器疾患を予防及び治療し得ると記載されている。 JP 2009-100692 (Patent Document 2) and JP 2008-212140 (Patent Document 3) contain a lactobacillus microorganism having a stress-reducing action, and a lactic acid bacteria preparation for mammals It is described that, by administering a specific lactic acid bacterium to a mammal, it exhibits an excellent stress-reducing effect and can prevent and treat digestive diseases such as diarrhea caused by stress. .
日本特許第4350170号公報（特許文献4）には、ヒト及び動物における胃腸障害の予防及び/又は処置に必要な薬学的調製物であって、寄託番号NCIMB 40564を有するラクトバチルス・カゼイ・ラムノーサス(Lactobacillus caseisubsp. rhamnosus)LB21の生存可能な微生物株を、微生物がその生存性を維持している少なくとも１つの薬学的に受容可能なキャリア媒体に含むことを特徴とする、薬学的調製物が記載されている。 Japanese Patent No. 4350170 (Patent Document 4) describes a pharmaceutical preparation required for the prevention and / or treatment of gastrointestinal disorders in humans and animals, which is Lactobacillus casei rhamnosus having the deposit number NCIMB 40564 ( Ractobacillus caseisubsp. Rhamnosus) LB21 viable microbial strain is described in at least one pharmaceutically acceptable carrier medium in which the microorganism maintains its viability. ing.
特開2011-200211号公報（特許文献5）には、ラクトバチルス・ガセリMCC1183株を含有する抗潰瘍剤が記載されており、この薬剤は、ヘリコバクター・ピロリに起因する胃潰瘍又は十二指腸潰瘍の予防又は治療に用いられると記載されている。 JP 2011-200211 A (Patent Document 5) describes an anti-ulcer agent containing Lactobacillus gasseri MCC1183 strain, which is used for the prevention or prevention of gastric ulcer or duodenal ulcer caused by Helicobacter pylori. It is described as being used for treatment.
特表2009-511471号公報（特許文献6）には、自己免疫疾患の治療及び/又は予防のための薬学的組成物の製造のための、乳酸菌から選択された少なくとも1種のプロバイオティクス細菌 (Probiotic bacteria) 株の使用が記載されており、該自己免疫が、多発性硬化症(MS)、アレルギー、乾癬、関節リウマチ（rhematoid arthritis）、クローン病（Crohn’s disease）、潰瘍性大腸炎、I型糖尿病、炎症性腸疾患、及び全身性エリテマトーデスなどの臓器特異的自己免疫（organ specific autoimmunity）からなる群から選ばれる、また、乳酸菌株が、ラクトバチルス・プランタラム（Lactobacillus plantarum）、ラクトバチルス・ラムノーサス（Lactobacillus rhamnosus）、ラクトバチルス・ファーメンタム（Lactobacillus fermentum）、ラクトバチルス・パラカゼイ（Lactobacillus paracasei）、及びラクトバチルス・ガセリ（Lactobacillus gasseri）からなる群より選ばれると記載されている。
JP-T 2009-511471 (Patent Document 6) discloses at least one probiotic bacterium selected from lactic acid bacteria for the production of a pharmaceutical composition for the treatment and / or prevention of autoimmune diseases. (Probiotic bacteria) strains are described and the autoimmunity may be multiple sclerosis (MS), allergies, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, I Selected from the group consisting of organ specific autoimmunity such as
日本特許4853986号公報（特許文献7）には、ラクトバチルス・ガセリSBT2055(LG2055という)(FERM P-15535)の菌体又はその発酵産物を有効成分とする炎症性腸疾患及び/又は過敏性腸症候群の予防及び治療剤が記載されており、また、一般に過敏性腸症候群の治療について、心理的ストレスのコントロールを行うとともに、整腸薬、緩下薬から抗精神病薬まで、症状に応じた段階的な薬物治療が施されていると記載されている。 Japanese Patent No. 4853986 (Patent Document 7) discloses an inflammatory bowel disease and / or irritable intestine containing Lactobacillus gasseri SBT2055 (referred to as LG2055) (FERM P-15535) or its fermentation product as an active ingredient. Syndrome prophylaxis and treatment agents are described. In general, psychological stress control is performed for the treatment of irritable bowel syndrome, and stages ranging from intestinal and laxatives to antipsychotics are applied according to symptoms. It is described that a typical drug treatment is given.
澤田大輔ら、第13回腸内細菌学会(平成21年)、東京、日本（非特許文献1）には、ラクトバチルス・ガセリCP2305株の生菌体によるストレス緩和作用、例えばストレス性腹痛を改善することが記載されている。 Daisuke Sawada et al., 13th Society of Intestinal Bacteriology (2009), Tokyo, Japan (Non-patent Document 1) improved the stress relieving action of Lactobacillus gasseri CP2305 strain by living cells, such as stress abdominal pain It is described.
Daisuke Sawada et al., The 10th Syｍposium on Lactic acid bacteria （2011年）、オランダ国、Federation of European Microbiological Societies (FEMS)と Netherlands Society for Microbiology (NVvM)（非特許文献2)には、過敏性腸症候群（IBS）患者にCP2305株生菌体を投与したことが記載されているが下痢症状緩和に関する記載はない。 Daisuke Sawada et al., The 10th Symposium on Lactic acid bacteria (2011), Netherlands, Federation of European Microbiological Societies (FEMS) and Netherlands Society for Microbiology (NVvM) (IBS) It is described that CP2305 strain cells were administered to patients, but there is no description about relief of diarrhea symptoms.
特開2003-95963号公報（特許文献8）には、ラクトバチルス・ガセリの菌体又はその発酵産物による炎症性腸疾患又は過敏性腸症候群の予防及び治療剤が記載されている。 Japanese Unexamined Patent Publication No. 2003-95963 (Patent Document 8) describes a preventive and therapeutic agent for inflammatory bowel disease or irritable bowel syndrome caused by Lactobacillus gasseri cells or fermentation products thereof.
乳酸菌の一種であるラクトバチルス・ガセリ株が、炎症性腸疾患、過敏性腸症候群（IBS）などの胃腸障害の予防又は治療に有効であるという記載はある。また、そのような胃腸障害には、ストレスとの関係を推定する記載もある。しかし、従来、ラクトバチルス・ガセリ株が、ストレスを抑制して過剰な腸運動を正常に保持しながら、ストレス性下痢症状を有意に改善することについての記載がない。 There is a description that a Lactobacillus gasseri strain, which is a kind of lactic acid bacteria, is effective in preventing or treating gastrointestinal disorders such as inflammatory bowel disease and irritable bowel syndrome (IBS). In addition, such gastrointestinal disorders also have a description for estimating the relationship with stress. However, conventionally, there is no description that the Lactobacillus gasseri strain significantly improves stress diarrhea symptoms while suppressing excessive stress and normally maintaining excessive intestinal motility.
そして、従来、ラクトバチルス・ガセリ株が、脳内ストレスホルモンが分泌されるようなストレスに曝露された状況下で、過剰な腸運動を正常に保持しながら、ストレス性下痢症状を有意に改善することについての記載がない。特に、IBS患者と区別される健常人に対するストレス負荷によるストレス性下痢症状の抑制（軽減もしくは緩和）に関する記載はない。 Conventionally, Lactobacillus gasseri strains significantly improve stress diarrhea symptoms while maintaining excessive intestinal motility under conditions exposed to stress that secretes stress hormones in the brain. There is no description about this. In particular, there is no description regarding the suppression (reduction or alleviation) of stress-related diarrhea symptoms caused by stress load on healthy individuals who are distinguished from IBS patients.
本発明は、以下の特徴を包含する。
(1) ラクトバチルス・ガセリCP2305株（FERM BP-11331）、その菌体処理物、或いはそれらの混合物、を有効成分として含有することを特徴とする、ストレス性下痢の抑制剤。
(2) 前記CP2305株が、生菌体又は殺菌体である、上記（1)に記載の抑制剤。
The present invention includes the following features.
(1) An inhibitor of stress diarrhea, comprising Lactobacillus gasseri CP2305 strain (FERM BP-11331), a treated product thereof, or a mixture thereof as an active ingredient.
(2) The inhibitor according to (1) above, wherein the CP2305 strain is a viable cell or a sterilized cell.
(3) 前記菌体処理物が、乾燥菌体、破壊菌体、破砕菌体又は発酵乳である、上記(1)又(2)に記載の抑制剤。
(4) ストレスによる骨盤（副交感）神経活動の抑制を阻害する作用を有する、上記(1)〜（3）のいずれかに記載の抑制剤。
(3) The inhibitor according to (1) or (2) above, wherein the processed microbial cell product is a dried microbial cell, a broken microbial cell, a crushed microbial cell, or fermented milk.
(4) The inhibitor according to any one of (1) to (3) above, which has an action of inhibiting suppression of pelvic (parasympathetic) nerve activity due to stress.
(5) 大腸クロライドチャネルを抑制することよって水分分泌を抑制する作用を有する、上記(1)〜(4)のいずれかに記載の抑制剤。
(6) 上記(1)〜(5)のいずれかに記載の抑制剤を動物飼料成分と配合して、ストレス性下痢抑制効果を有する動物飼料を製造することを含む、動物飼料の製造方法。
(5) The inhibitor according to any one of (1) to (4) above, which has an action of suppressing water secretion by inhibiting a colon chloride channel.
(6) A method for producing animal feed, comprising producing an animal feed having an effect of suppressing stress diarrhea by blending the inhibitor according to any one of (1) to (5) above with an animal feed component.
(7) 上記(1)〜(5)のいずれかに記載の抑制剤を含む動物用飼料。
(8) 上記(1)〜(5)のいずれかに記載の抑制剤を含有する食品添加剤又は動物飼料添加剤。
(9) 上記(1)〜(5)のいずれかに記載の抑制剤を食品用の担体、賦形剤又は希釈剤と混合し、場合により食品添加物をさらに混合して、ストレス性下痢抑制効果を有する機能性飲食品を製造することを含む、機能性飲食品の製造方法。
(7) Animal feed containing the inhibitor according to any one of (1) to (5) above.
(8) A food additive or animal feed additive containing the inhibitor according to any one of (1) to (5) above.
(9) The inhibitor according to any one of the above (1) to (5) is mixed with a food carrier, excipient or diluent, and optionally further mixed with a food additive to suppress stress diarrhea The manufacturing method of functional food / beverage products including manufacturing functional food / beverage products which have an effect.
(10) ラクトバチルス・ガセリCP2305株（FERM BP-11331）、その菌体処理物、及びそれらの混合物、を有効成分として含有することを特徴とする、ストレス性下痢を予防、抑制又は治療するための医薬組成物。
(11) 上記(1)〜(5)のいずれかに記載の抑制剤と、薬学的に許容されうる担体、賦形剤又は希釈剤とを含む、ストレス性下痢を予防、抑制又は治療するための医薬組成物。
(10) To prevent, suppress or treat stress diarrhea characterized by containing Lactobacillus gasseri CP2305 strain (FERM BP-11331), a treated product thereof, and a mixture thereof as active ingredients Pharmaceutical composition.
(11) To prevent, suppress or treat stress diarrhea, comprising the inhibitor according to any one of (1) to (5) above and a pharmaceutically acceptable carrier, excipient or diluent. Pharmaceutical composition.
本発明のラクトバチルス・ガセリCP2305株（FERM BP-11331）、その菌体処理物、又はその混合物は、ストレス誘発性骨盤（副交感）神経活動の抑制を阻害して腸運動を正常に保持すること、並びに、被験体のストレス性下痢を効果的に抑制するため、下痢型ストレス性腸障害の下痢症状の予防、軽減又は治療に有効である。 Lactobacillus gasseri CP2305 strain (FERM BP-11331) of the present invention, a treated product thereof, or a mixture thereof inhibits the suppression of stress-induced pelvic (parasympathetic) nerve activity and maintains intestinal motility normally. In addition, in order to effectively suppress the stress diarrhea of the subject, it is effective for the prevention, reduction or treatment of diarrhea symptoms of diarrhea type stress bowel disorder.
本発明をさらに具体的に説明する。
＜ストレス性下痢の抑制剤＞
本発明は、第1の態様により、ラクトバチルス・ガセリCP2305株（FERM BP-11331）（以下、単に「CP2305株」とも称する。）、その菌体処理物、或いはその混合物、を有効成分として含有することを特徴とする、被験体のストレス性下痢の抑制剤を提供する。
CP2305株は、生菌体又は殺菌体のいずれでもよい。
The present invention will be described more specifically.
<Suppressor of stress diarrhea>
The present invention comprises, according to the first aspect, Lactobacillus gasseri CP2305 strain (FERM BP-11331) (hereinafter also simply referred to as “CP2305 strain”), a treated product thereof, or a mixture thereof as an active ingredient. An agent for suppressing stress diarrhea in a subject is provided.
The CP2305 strain may be either a living cell or a sterilized cell.
本明細書中の「被験体」は、哺乳類及び鳥類に属する動物、好ましくはヒトなどの霊長類、ウシ、ウマ、ヒツジ、ヤギ、ブタ、ニワトリ、シチメンチョウなどの家畜動物、ウマなどの競技用動物、イヌ、ネコなどの愛玩動物などである。 The “subject” in this specification refers to animals belonging to mammals and birds, preferably primates such as humans, domestic animals such as cows, horses, sheep, goats, pigs, chickens, and turkeys, and competition animals such as horses. , Pets such as dogs and cats.
ラクトバチルス・ガセリCP2305株は、本出願人により、ブダペスト条約の規定に基づく国際寄託機関である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（〒305-8566日本国茨城県つくば市東１丁目１番地１中央第６）に、2007年9月11日付でFERM BP-11331として国際寄託されている。
The Lactobacillus gasseri CP2305 strain was obtained by the applicant from the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center, an international depositary organization based on the provisions of the Budapest Treaty (1-1, Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, 305-8566, Japan). It has been deposited internationally as FERM BP-11331 as of September 11, 2007, at the center of
CP2305株の派生株もまた、親株と同等の、腸管での水分過剰分泌の抑制効果を有する（図１参照）。派生株は、親株に紫外線を照射すること、変異原性物質の存在下で親株を培養すること、至適温度以外の温度で培養すること、有機溶剤存在下で培養することなどの、親株にとって非一般的な条件下に親株を置くことによって作製可能である。 A derivative of the CP2305 strain also has the same effect of suppressing excessive water secretion in the intestine as the parent strain (see FIG. 1). Derivative strains are used for parent strains such as irradiating the parent strain with ultraviolet light, culturing the parent strain in the presence of a mutagenic substance, culturing at a temperature other than the optimum temperature, and culturing in the presence of an organic solvent. It can be created by placing the parent strain under non-generic conditions.
関連する公知ラクトバチルス・ガセリ株に関して、炎症性腸疾患又は過敏性腸症候群の予防・治療効果があるラクトバチルス・ガセリSBT 2055株(FERM P-15535)が知られている（特開2003-95963号公報）。この菌は、日本人の腸から分離・培養された乳酸菌であり、DDS誘発潰瘍性大腸炎モデルを使用した生菌での試験では、対照群に比べて血便や下血が抑制されたと報告されている。また、腸内菌叢を改善して下痢の発症を低減するラクトバチルス・ガセリOLL2716株(FERM BP-6999)が知られている（特開2004-305128号公報）。その実施例では、仔牛の飼育の間に下痢の発生率（試験牧場に導入後2週間以内に下痢を発生した率）が低下したと記載されている。しかし、これらの公知ラクトバチルス・ガセリ株は、以下に記載するように、本発明のCP2305株と明らかに異なる株である。 Regarding a related known Lactobacillus gasseri strain, Lactobacillus gasseri SBT 2055 strain (FERM P-15535) having a prophylactic / therapeutic effect on inflammatory bowel disease or irritable bowel syndrome is known (JP 2003-95963 A). Issue gazette). This bacterium is a lactic acid bacterium isolated and cultured from the Japanese intestine. In a test using live bacteria using the DDS-induced ulcerative colitis model, it was reported that blood stool and melena were suppressed compared to the control group. ing. Also known is Lactobacillus gasseri OLL2716 strain (FERM BP-6999) which improves the gut microbiota and reduces the occurrence of diarrhea (Japanese Patent Laid-Open No. 2004-305128). In the Examples, it is described that the incidence of diarrhea (rate of occurrence of diarrhea within 2 weeks after introduction into the test ranch) decreased during the breeding of calves. However, these known Lactobacillus gasseri strains are clearly different from the CP2305 strain of the present invention, as described below.
本発明のCP2305株の生菌体及び殺菌体、並びに該株の菌体処理物は、意外にも腸管からの水分過剰分泌を抑制する作用を有し、ストレス性下痢を著しく抑制する効果を有している（図5及び図6参照）。この作用は、本発明のラクトバチルス・ガセリCP2305株の殺菌発酵乳を、公知の寄託ラクトバチルス・ガセリ株、L. acidophilus、B. pseudocatenulatum、L. delbrueckii、S. thermophilus及びL. kefirgranumの殺菌発酵乳と比較した場合であっても、格別有意である（図4参照）。腸管からの水分過剰分泌の抑制は、CRF投与ラットの結腸組織での遺伝子発現の網羅的解析により、CP2305株が、大腸クロライドチャネルを抑制し、水分の移動を制御していることによることが立証された（図3参照）。このCP2305株はまた、今回、ストレス誘発性骨盤（副交感）神経活動の抑制を阻害する作用を有することが、下部大腸を神経支配する骨盤（副交換）神経の電気活動を測定することによって証明された。CP2305株の投与によって、被験体のストレス時の腸管機能が正常に回復し維持される。このため、CP2305株は、下痢型ストレス性腸障害の下痢症状を有意に軽減するという格別の効果を有する。 Surviving cells and bactericides of the CP2305 strain of the present invention, and treated cells of the strain, surprisingly have an effect of suppressing excessive secretion of water from the intestinal tract, and have an effect of significantly suppressing stress diarrhea. (See Fig. 5 and Fig. 6). This action is achieved by sterilizing fermented milk of Lactobacillus gasseri CP2305 strain of the present invention with known deposited Lactobacillus gasseri strains, L. acidophilus, B. pseudocatenulatum, L. delbrueckii, S. thermophilus and L. kefirgranum. Even when compared to milk, it is particularly significant (see Figure 4). Inhibition of excessive water secretion from the intestinal tract is based on a comprehensive analysis of gene expression in the colon tissue of CRF-treated rats, and it is proved that CP2305 suppresses colonic chloride channels and regulates water movement. (See Figure 3). This CP2305 strain is also proved by measuring the electrical activity of the pelvic (collateral exchange) nerve that innervates the lower colon, which has the effect of inhibiting the suppression of stress-induced pelvic (parasympathetic) neural activity. It was. Administration of the CP2305 strain restores and maintains normal intestinal function during stress in the subject. For this reason, the CP2305 strain has a special effect of significantly reducing diarrhea symptoms of diarrhea-type stress bowel disorders.
本発明のラクトバチルス・ガセリCP2305株は、乳酸菌の培養に通常用いられる培地を使用して、適当な条件下で培養することにより調製することができる。培養に用いる培地は、炭素源、窒素源、無機塩類等を含有し、乳酸菌の培養を効率的に行うことができる培地であれば、天然培地、合成培地のいずれを用いてもよく、当業者であれば使用する菌株に適切な公知の培地を適宜選ぶことができる。炭素源としてはラクトース、グルコース、スクロース、フルクトース、ガラクトース、廃糖蜜などを使用することができる。また、窒素源としてはカゼインの加水分解物、乳漿タンパク質加水分解物、大豆タンパク質加水分解物等の有機窒素含有物を使用することができる。無機塩類としては、リン酸塩、ナトリウム、カリウム、マグネシウムなどを用いることができる。乳酸菌の培養に適した培地としては、例えばMRS液体培地、GAM培地、BL培地、Briggs Liver Broth、獣乳、脱脂乳、乳性ホエーなどが挙げられる。好ましくは、滅菌されたMRS培地を使用することができる。 The Lactobacillus gasseri CP2305 strain of the present invention can be prepared by culturing under suitable conditions using a medium usually used for culturing lactic acid bacteria. As long as the medium used for culture contains a carbon source, a nitrogen source, inorganic salts, and the like and can culture lactic acid bacteria efficiently, either a natural medium or a synthetic medium may be used. If so, a known medium suitable for the strain to be used can be appropriately selected. As the carbon source, lactose, glucose, sucrose, fructose, galactose, molasses, etc. can be used. Further, as the nitrogen source, organic nitrogen-containing materials such as casein hydrolyzate, whey protein hydrolyzate, and soy protein hydrolyzate can be used. As inorganic salts, phosphates, sodium, potassium, magnesium and the like can be used. Examples of the medium suitable for culturing lactic acid bacteria include MRS liquid medium, GAM medium, BL medium, Briggs Liver Broth, animal milk, skim milk, and milky whey. Preferably, a sterilized MRS medium can be used.
また、本発明のCP2305株の培養は、20℃〜50℃、好ましくは25℃〜42℃、より好ましくは約37℃において、嫌気的培養条件下で行う。温度条件は、恒温槽、マントルヒーター、ジャケットなどにより調整することができる。ここで、嫌気条件下とは、菌が増殖可能な程度の低酸素環境下のことであり、例えば嫌気チャンバー、嫌気ボックス又は脱酸素剤を入れた密閉容器若しくは袋などを使用することにより、あるいは単に培養容器を密閉することにより、嫌気条件とすることができる。培養は、静置培養、振とう培養、タンク培養などのいずれの培養様式でもよい。また、培養時間は3時間〜100時間又はそれ以上、好ましくは約10時間〜50時間とすることができる。培養開始時の培地のpHは3.5〜8.0に維持することが好ましい。 In addition, the CP2305 strain of the present invention is cultured at 20 ° C. to 50 ° C., preferably 25 ° C. to 42 ° C., more preferably about 37 ° C. under anaerobic culture conditions. The temperature condition can be adjusted by a thermostatic bath, a mantle heater, a jacket, or the like. Here, the anaerobic condition means a hypoxic environment where bacteria can grow, for example, by using an anaerobic chamber, an anaerobic box, a sealed container or bag containing an oxygen scavenger, or the like. Anaerobic conditions can be achieved by simply sealing the culture vessel. The culture may be any culture mode such as stationary culture, shaking culture, tank culture and the like. Further, the culture time can be 3 hours to 100 hours or more, preferably about 10 hours to 50 hours. It is preferable to maintain the pH of the medium at the start of the culture at 3.5 to 8.0.
培養後、培養液から濾過、遠心分離等の分離技術により分離した生菌体、或いは生菌体含有培養液を得ることができる。また、これらの生菌体又はその培養液を、オートクレーブ等による公知の殺菌処理にかけることによって、殺菌体を得ることができる。また、生菌体又は生菌体培養液から菌体処理物を作製し、必要に応じて殺菌処理にかけることができる。 After culturing, viable bacterial cells separated from the culture solution by a separation technique such as filtration and centrifugation, or a viable cell-containing culture solution can be obtained. Moreover, a sterilized body can be obtained by subjecting these live cells or a culture solution thereof to a known sterilization treatment using an autoclave or the like. Moreover, a microbial cell processed material can be produced from a living microbial cell or a living microbial cell culture solution, and can be subjected to a sterilization treatment as necessary.
菌体処理物は、菌体を、乾燥処理、破壊又は破砕処理、発酵処理などの物理的、化学的、生物学的処理等にかけることによって得られるものである。したがって、本発明で使用する菌体処理物の例は、乾燥菌体、破壊菌体、破砕菌体、発酵処理物（例えば、発酵乳）などである。 The treated microbial cell product is obtained by subjecting the microbial cell to physical, chemical, biological treatment such as drying treatment, destruction or crushing treatment, and fermentation treatment. Therefore, examples of the processed microbial cell product used in the present invention include dried microbial cells, disrupted microbial cells, crushed microbial cells, fermented processed products (for example, fermented milk), and the like.
乾燥菌体は、上記培養液から分離した菌体を、必要であれば殺菌処理にかけられた菌体を、乾燥処理にかけることによって得ることができる。乾燥処理としては、例えばドラム乾燥、噴霧乾燥、真空乾燥、凍結乾燥などを挙げることができる。 The dried microbial cells can be obtained by subjecting the microbial cells separated from the above culture solution to a sterilizing treatment if necessary, and subjecting the microbial cells to a drying treatment. Examples of the drying process include drum drying, spray drying, vacuum drying, freeze drying, and the like.
破壊菌体又は破砕菌体は、菌体を、破砕、磨砕、酵素処理、薬品処理、溶解などによって処理することによって得ることができる。破壊菌体又は破砕菌体は、破壊又は破砕された菌体の固体成分及び可溶性成分のすべてから本質的になり、例えば破壊物又は破砕物をそのまま凍結乾燥にかけることによって得ることができる。破壊又は破砕は、公知の手法、例えば物理的破砕、酵素溶解処理、薬品処理、等によって行うことができる。物理的破砕は、湿式又は乾式のいずれで行ってもよく、ホモゲナイザー、ボールミル、ビーズミル、ダイノミル、遊星ミル等を使用した撹拌により、ジェットミル、フレンチプレス、細胞破砕機、等を使用して行うことができる。酵素溶解処理は、例えばリゾチームなどの酵素を用いて、菌の細胞構造を破壊することによって行うことができる。薬品処理は、グリセリン脂肪酸エステル、ダイズリン脂質などの界面活性剤を使用して、菌の細胞構造を破壊することによって行うことができる。好ましくは、物理的破砕である。 The disrupted cells or disrupted cells can be obtained by treating the cells by crushing, grinding, enzyme treatment, chemical treatment, dissolution, or the like. The disrupted or disrupted cells consist essentially of all the solid and soluble components of the disrupted or disrupted cells, and can be obtained, for example, by subjecting the disrupted or disrupted product to lyophilization as it is. Destruction or crushing can be performed by a known method such as physical crushing, enzyme dissolution treatment, chemical treatment, and the like. Physical crushing may be carried out either wet or dry, with stirring using a homogenizer, ball mill, bead mill, dyno mill, planetary mill, etc., using a jet mill, French press, cell crusher, etc. Can do. The enzyme dissolution treatment can be performed by destroying the cell structure of the fungus using an enzyme such as lysozyme. The chemical treatment can be carried out by destroying the cell structure of the fungus using a surfactant such as glycerin fatty acid ester and soybean phospholipid. Preferably, physical crushing.
破砕物を調製するための具体的な方法は、例えば、菌の懸濁液を、公知のダイノミル細胞破砕機（DYNO-MILL破砕装置など）において、ガラスビーズを使用して、周速10.0〜20.0m/s（例えば約14.0m/s）、処理流速0.1〜10L/10min（例えば約1L/10min）にて、破砕槽温度10〜30℃（例えば約15℃）で1〜7回（例えば3〜5回）処理することによって、菌体を破砕することを含む。また例えば、菌の懸濁液を、公知の湿式ジェットミル細胞破砕機（JN20 ナノジェットパルなど）において、吐出圧力50〜1000Mpa（例えば270MPa）、処理流速50〜1000（例えば300ml／min）にて、1〜30回（例えば10回）処理することによって、菌体を破砕することができる。また、公知の乾式遊星ミル細胞破砕機（GOT5 ギャラクシー5など）において、菌体粉末を各種ボール（例えばジルコニア製10mmボール、ジルコニア製5mmボール、アルミナ製1mmボール）共存下で、回転数50〜10,000rpm（例えば240rpm、190rpm）で30分〜20時間（例えば5時間）処理することによって、菌体を破砕することも可能である。菌体粉末を公知の乾式ジェットミル細胞破砕機（ジェットOマイザーなど）において、供給速度0.01〜10000g/min（例えば0.5g/min）、吐出圧力1〜1000kg/cm2（例えば6kg/cm2）の圧力にて、複数回処理することによって、菌体を破砕してもよい。
A specific method for preparing the crushed material is, for example, using a glass bead in a known dynomill cell crusher (such as a DYNO-MILL crushing device), and a peripheral speed of 10.0 to 20.0. 1-7 times (for example, 3 times) at a crushing tank temperature of 10 to 30 ° C. (for example, about 15 ° C.) at a m / s (for example, about 14.0 m / s), a processing flow rate of 0.1 to 10 L / 10 min (for example, about 1 L / 10 min). ~ 5 times) by crushing the cells by treatment. In addition, for example, the suspension of the bacterium is discharged with a discharge pressure of 50 to 1000 MPa (for example, 270 MPa) and a processing flow rate of 50 to 1000 (for example, 300 ml / min) in a known wet jet mill cell crusher (such as JN20 Nanojet Pal). The cells can be crushed by treating 1 to 30 times (for example, 10 times). In addition, in a known dry planetary mill cell crusher (
発酵乳は、生乳、脱脂粉乳又は豆乳などに菌株を接種し、乳酸菌発酵条件にて発酵を行うことによって得ることができる。得られる発酵産物は、必要に応じて、濾過、希釈、濃縮などの処理にかけてもよい。 Fermented milk can be obtained by inoculating a raw milk, skim milk powder or soy milk with a strain and performing fermentation under lactic acid bacteria fermentation conditions. The obtained fermentation product may be subjected to treatments such as filtration, dilution, and concentration as necessary.
上記の菌体及び菌体処理物は、そのまま使用するか、或いは、殺菌処理にかけて殺菌体としうる。殺菌処理は、例えば高エネルギー線照射による殺菌、加熱殺菌、加圧殺菌、オートクレーブ殺菌などの公知の処理を含む。好ましい殺菌法は、オートクレーブ殺菌である。この方法は、オートクレーブ（高圧蒸気滅菌器）内で、生菌又は生菌含有培養液を、例えば約10分〜１時間にわたり、加圧水蒸気下で例えば110℃〜140℃の温度で殺菌することを含む。 The above microbial cells and microbial cell processed products can be used as they are or can be sterilized by sterilization. The sterilization treatment includes, for example, known treatments such as sterilization by high energy ray irradiation, heat sterilization, pressure sterilization, and autoclave sterilization. A preferred sterilization method is autoclave sterilization. This method involves sterilizing a living bacterium or a bacterium-containing culture solution in an autoclave (high pressure steam sterilizer) at a temperature of, for example, 110 ° C. to 140 ° C. under pressurized steam for, for example, about 10 minutes to 1 hour. Including.
上記の特性のために、本発明のCP2305株又はその菌体処理物は、下痢を伴うストレス性下痢の症状の悪化の予防、改善、又は治療に有効である。 Due to the above characteristics, the CP2305 strain of the present invention or a treated product thereof is effective in preventing, improving, or treating worsening of symptoms of stress diarrhea accompanied by diarrhea.
明細書中「ストレス性下痢」とは、脳内ストレスホルモンが分泌されるようなストレスに曝露された状況下、すなわちストレスに起因する異常な腸管での水分分泌を伴う症状である。 In the specification, “stress diarrhea” is a symptom accompanied by abnormal secretion of water in the intestinal tract under a situation where the brain is exposed to stress such that stress hormones are secreted.
したがって、特に、健常人に対するストレスに起因するストレス性下痢に有効である点において、明細書中「ストレス性下痢」は、ストレスの有無に関係なくQOLの低い過敏性腸症候群(IBS)の患者と区別される。IBSの診断基準は、RomeIIIに規定されており、ストレス環境の有無に関係なくQOLの低い患者である。 Therefore, particularly in the point that it is effective for stress diarrhea caused by stress on healthy individuals, `` stress diarrhea '' in the specification refers to patients with irritable bowel syndrome (IBS) having low QOL regardless of the presence or absence of stress. Differentiated. The diagnostic criteria for IBS are defined in Rome III and are patients with low QOL regardless of the presence or absence of stress environment.
本発明の抑制剤は、CP2305株、その菌体処理物、又はその混合物からなる有効成分を、以下のものに限定されないが、該抑制剤あたり、例えば約0.1重量％〜100重量％、好ましくは約10重量％〜約90重量％含有する。 The inhibitor of the present invention is not limited to the following active ingredients comprising the CP2305 strain, its treated cell, or a mixture thereof, but for example, about 0.1 wt% to 100 wt% per inhibitor, preferably Contains from about 10% to about 90% by weight.
上記抑制剤は、上記有効成分の他に、賦形剤、希釈剤又は担体、必要に応じてその他の添加物を含むことができる。その形態は、固体形態又は液体形態のいずれでもよい。固体形態は、例えば粉末、顆粒、タブレット、ペレットなどの任意の形状からなる。また、液体形態は、例えば懸濁液、乳濁液、発酵液（例えば発酵乳）などの形状である。 In addition to the active ingredient, the inhibitor may contain an excipient, a diluent or a carrier, and other additives as necessary. The form may be either a solid form or a liquid form. A solid form consists of arbitrary shapes, such as a powder, a granule, a tablet, a pellet, for example. The liquid form is, for example, a shape such as a suspension, an emulsion, a fermented liquid (for example, fermented milk).
賦形剤、希釈剤又は担体は、以下に述べるような食品、動物飼料又は医薬品で通常使用されうる物質である。他の添加物は、必ずしも必要ないが、添加する場合、以下に記載されるような、例えば保存料、pH調整剤、乳化剤、酸化防止剤、着色料などを挙げることができる。 Excipients, diluents or carriers are substances that can normally be used in foods, animal feeds or pharmaceuticals as described below. Other additives are not necessarily required, but when added, examples include preservatives, pH adjusters, emulsifiers, antioxidants, and colorants as described below.
＜食品又は動物飼料＞
本発明は、第2の態様により、上記抑制剤を含有する食品添加剤又は動物飼料添加剤を提供する。
<Food or animal feed>
According to the second aspect of the present invention, there is provided a food additive or animal feed additive containing the inhibitor.
上記抑制剤が、有効成分の他に、賦形剤、希釈剤又は担体、及びその他の添加物を含む場合、食品添加剤又は動物飼料添加剤として使用することができる。これらの食品添加剤又は動物飼料添加剤は、固体形態又は液体形態のいずれでもよい。固体形態は、例えば粉末、顆粒、タブレット、ペレット、ゲル、カプセルなどの任意の形状からなる。また、液体形態は、例えば懸濁液、乳濁液、発酵液（例えば発酵乳）などの形状である。賦形剤、希釈剤又は担体は、食品又は動物飼料で通常使用されうる物質であり、また他の添加物は、例えば保存料、pH調整剤、乳化剤、着色料、栄養補助剤、（天然若しくは人工）甘味料、調味料、香料などを挙げることができる。 When the said inhibitor contains an excipient | filler, a diluent or a carrier, and another additive other than an active ingredient, it can be used as a food additive or an animal feed additive. These food additives or animal feed additives may be in solid or liquid form. The solid form consists of any shape such as powder, granule, tablet, pellet, gel, capsule and the like. The liquid form is, for example, a shape such as a suspension, an emulsion, a fermented liquid (for example, fermented milk). Excipients, diluents or carriers are substances that can normally be used in food or animal feed, and other additives include, for example, preservatives, pH adjusters, emulsifiers, colorants, nutritional supplements (natural or Artificial) sweeteners, seasonings, flavors and the like.
食品添加剤又は動物飼料添加剤は、上記抑制剤を、以下のものに限定されないが、添加剤あたり、例えば約1重量％〜約50重量％、好ましくは約5重量％〜約20重量％含有することができる。 The food additive or animal feed additive contains, but is not limited to, the above-mentioned inhibitors, for example, about 1% to about 50% by weight, preferably about 5% to about 20% by weight, per additive. can do.
食品添加剤又は動物飼料添加剤は、食品又は動物飼料に添加して、被験体のストレス性下痢を抑制するために、下痢症状を軽減するために、使用することができる。 A food additive or animal feed additive can be used to reduce diarrhea symptoms in order to add to food or animal feed to suppress stress diarrhea in the subject.
本発明はさらに、上記の抑制剤を含む動物飼料、或いは、上記の抑制剤を動物飼料成分と配合して、被験体のストレス性下痢を抑制する、或いは、ストレス性下痢の下痢症状を軽減する効果を有する動物飼料を製造することを含む、動物飼料の製造方法を提供する。 The present invention further includes an animal feed containing the above-mentioned inhibitor, or the above-mentioned inhibitor is combined with an animal feed ingredient to suppress stress diarrhea in a subject or reduce diarrhea symptoms of stress diarrhea. An animal feed production method comprising producing an animal feed having an effect is provided.
すなわち、動物飼料を製造するための飼料成分に、本発明の抑制剤を有効量配合し、必要であれば任意の剤型に製剤化することによって動物飼料を製造することができる。 That is, an animal feed can be produced by blending an effective amount of the inhibitor of the present invention with a feed component for producing an animal feed, and if necessary, formulating it into an arbitrary dosage form.
本発明はさらに、上記の抑制剤を含む機能性食品、或いは、上記の抑制剤を食品用担体、賦形剤又は希釈剤と混合し、場合により食品添加物をさらに混合して、被験体のストレス性下痢を抑制する下痢症状を軽減する効果を有する機能性飲食品を製造することを含む、機能性飲食品の製造方法を提供する。 The present invention further includes a functional food containing the above-mentioned inhibitor, or the above-mentioned inhibitor mixed with a food carrier, excipient or diluent, optionally further mixed with a food additive, Provided is a method for producing a functional food or drink, which comprises producing a functional food or drink having an effect of reducing diarrheal symptoms that suppress stress diarrhea.
動物飼料又は機能性飲食品において、上記抑制剤の配合量は、飼料や飲食品の形態や求められる効果（すなわち、被験体のストレス性下痢を抑制する、或いは、下痢症状を軽減する効果）を考慮して、当業者が適宜定めることができる。動物飼料又は機能性飲食品中のCP2305株及び/又は菌体処理物の総量が、以下のものに限定されないが、該菌の生菌量として、105〜1012cfu/g、好ましくは106〜約1012cfu/g、より好ましくは107〜1012cfu/gに相当する量であるか、或いは、該菌の量として、例えば0.001重量％〜10重量％、好ましくは0.01〜5重量％となるような量である。その有効量は、添加量と下痢症状の軽減効果との関係に基づいて、当業者が適宜設定することが可能である。 In animal feeds or functional foods and drinks, the amount of the above inhibitor is the form of the feed or food or drink and the required effect (that is, the effect of suppressing stress diarrhea in the subject or reducing the symptoms of diarrhea). In consideration, it can be appropriately determined by those skilled in the art. The total amount of CP2305 strain and / or bacterial cell processed product in animal feed or functional food or drink is not limited to the following, but the amount of viable bacteria is 10 5 to 10 12 cfu / g, preferably 10 The amount corresponds to 6 to about 10 12 cfu / g, more preferably 10 7 to 10 12 cfu / g, or the amount of the bacteria is, for example, 0.001% to 10% by weight, preferably 0.01 to 5%. The amount is such that the weight%. The effective amount can be appropriately set by those skilled in the art based on the relationship between the added amount and the effect of reducing diarrhea symptoms.
動物飼料の剤型は、固体形態の場合、例えば粉末、顆粒、タブレット、ペレットなどの任意の形状からなり、また、液体形態の場合、例えば懸濁液、発酵乳などの形状である。 The animal feed dosage form has an arbitrary shape such as powder, granule, tablet, pellet and the like in the solid form, and is in the form of suspension, fermented milk and the like in the liquid form.
機能性食品の剤型は、固体形態の場合、例えば粉末、顆粒、タブレット、ペレット、ゲル、カプセルなどの任意の形状からなり、また、液体形態の場合、例えば懸濁液、乳濁液、発酵乳などの形状である。 The functional food dosage form may be of any shape such as powder, granule, tablet, pellet, gel, capsule, etc. in the solid form, and in the liquid form, such as suspension, emulsion, fermentation, etc. The shape is milk.
上記の剤型中、タブレット、ペレット及びカプセルは、必要であれば、腸溶性となるように腸溶性物質で単層又は多層にコーティングすることができる。腸溶性物質は、胃液等の酸性域のpHで溶けず、中性域のpHで溶解するものであり、例えばヒプロメロースフタル酸エステル、セラック、ゼイン、ラクトフェリン、ヒドロキシプロピルメチルセルロースフタレート、ヒドロキシプロピルメチルセルロースアセテートサクシネート、カルボキシメチルエチルセルロース、酢酸フタル酸セルロースなどを挙げることができる。 In the above dosage form, tablets, pellets and capsules can be coated in a single layer or multiple layers with an enteric substance so as to be enteric if necessary. Enteric substances are those that do not dissolve at pH in the acidic range such as gastric juice but dissolve at pH in the neutral range, such as hypromellose phthalate, shellac, zein, lactoferrin, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose. Examples thereof include acetate succinate, carboxymethyl ethyl cellulose, and cellulose acetate phthalate.
賦形剤、希釈剤又は担体は、以下のものに限定されないが、水、乳発酵液、炭酸水、エタノール、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、アルギン酸ナトリウム、水溶性デキストラン、水溶性デキストリン、カルボキシメチルスターチナトリウム、カルボキシメチルセルロース、スターチ、ペクチン、キサンタンガム、アラビアゴム、カゼイン、ゼラチン、寒天、グリセリン、プロピレングリコール、ポリエチレングリコール、ワセリン、パラフィン、ステアリルアルコール、マンニトール、ソルビトール、ラクトースなどを挙げることができる。 Excipients, diluents or carriers are not limited to the following: water, milk fermentation liquid, carbonated water, ethanol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, Examples thereof include sodium carboxymethyl starch, carboxymethyl cellulose, starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, mannitol, sorbitol, and lactose.
動物飼料又は機能性飲食品の製造においては、飼料又は飲食品に慣用的に使用されるような各種添加物を使用しうる。添加物としては、以下のものに限定するものではないが、着色料（植物色素、合成色素等）、香料（天然香料等）、甘味料（ステビア、アステルパーム等）、保存料（酢酸ナトリウム、ソルビン酸等）、乳化剤（コンドロイチン硫酸ナトリウム、プロピレングリコール脂肪酸エステル等）、酸化防止剤（EDTA二ナトリウム、ビタミンC等）、pH調整剤（クエン酸等）、化学調味料（イノシン酸ナトリウム、グルタミン酸ナトリウム等）、増粘剤（キサンタンガム等）、膨張剤（炭酸カルシウム等）、消泡剤（リン酸カルシウム）、結着剤（ポリリン酸ナトリウム等）、栄養強化剤（カルシウム強化剤、ビタミンA等）、等が挙げられる。その他、各種栄養素、各種ビタミン、ミネラル、食物繊維、酸味料、安定剤、フレーバー、天然物エキスなどを配合しうる。配合量は、添加物としての効果が発揮される量であり、適宜選択可能である。 In the production of animal feed or functional food or drink, various additives that are conventionally used for feed or food or drink can be used. Additives are not limited to the following, but coloring agents (plant pigments, synthetic pigments, etc.), flavoring agents (natural flavoring agents, etc.), sweeteners (stevia, aster palm, etc.), preservatives (sodium acetate, Sorbic acid, etc.), emulsifiers (sodium chondroitin sulfate, propylene glycol fatty acid ester, etc.), antioxidants (disodium EDTA, vitamin C, etc.), pH adjusters (citric acid, etc.), chemical seasonings (sodium inosinate, sodium glutamate) Etc.), thickeners (xanthan gum, etc.), swelling agents (calcium carbonate, etc.), antifoaming agents (calcium phosphate), binders (sodium polyphosphate, etc.), nutrient enhancers (calcium fortifier, vitamin A, etc.), etc. Is mentioned. In addition, various nutrients, various vitamins, minerals, dietary fiber, acidulants, stabilizers, flavors, natural product extracts, and the like can be blended. The blending amount is an amount that exhibits an effect as an additive, and can be appropriately selected.
＜医薬組成物＞
本発明はさらに、ラクトバチルス・ガセリCP2305株（FERM BP-11331）、その菌体処理物、或いはその混合物、を有効成分として含有することを特徴とする、被験体のストレス性下痢、とりわけ下痢型ストレス性腸障害のストレス性下痢、を予防、抑制（軽減）又は治療するための医薬組成物を提供する。
<Pharmaceutical composition>
The present invention further comprises Lactobacillus gasseri CP2305 strain (FERM BP-11331), a treated product thereof, or a mixture thereof as an active ingredient, characterized by stress diarrhea of a subject, especially diarrhea type Provided is a pharmaceutical composition for preventing, suppressing (reducing) or treating stress diarrhea due to stress bowel disorder.
或いは、本発明は、上記の抑制剤と、薬学的に許容されうる担体、賦形剤又は希釈剤とを含む、被験体のストレス性下痢、のを予防、抑制（軽減）又は治療するための医薬組成物を提供する。 Alternatively, the present invention is for preventing, inhibiting (reducing) or treating stress diarrhea in a subject comprising the above-mentioned inhibitor and a pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutical composition is provided.
いずれの医薬組成物においても、本発明の有効成分は、ストレス性下痢の下痢症状を抑制する効果を有する。 In any pharmaceutical composition, the active ingredient of the present invention has an effect of suppressing diarrhea symptoms of stress diarrhea.
CP2305株又はその菌体処理物は、上記のとおり、ストレス誘発性骨盤（副交感）神経抑制を阻害する作用、すなわち抗ストレスにより腸管運動を正常に維持する働きを有すること、及び、大腸クロライドチャネルを抑制し、それによって腸管での水分過剰分泌を抑制する作用を有し、ストレス性下痢を抑制、軽減又は改善する。ストレスは、ストレスに過敏な胃腸障害をもたらし、該障害の症状を悪化させる。本発明のCP2305株は、ストレス性下痢、の頻発の下痢症状を抑制するため、ストレス性下痢の予防、抑制（軽減）又は治療のために有効である。この効果は、市販されるいずれのラクトバチルス・ガセリ株製剤と比較しても高いことが確認された。 As described above, the CP2305 strain or a treated product thereof has an action of inhibiting stress-induced pelvic (parasympathetic) nerve suppression, that is, a function of maintaining intestinal motility normally by anti-stress, and a colorectal chloride channel. It suppresses, thereby suppressing the excessive secretion of water in the intestinal tract, and suppresses, reduces or improves stress diarrhea. Stress leads to stress-sensitive gastrointestinal disorders and exacerbates the symptoms of the disorders. Since the CP2305 strain of the present invention suppresses frequent diarrheal symptoms of stress diarrhea, it is effective for prevention, suppression (reduction) or treatment of stress diarrhea. This effect was confirmed to be higher than any commercially available Lactobacillus gasseri strain preparation.
医薬組成物は、上記効果を発揮するかぎりいずれの剤型に製剤化しうる。例えば、錠剤、カプセル剤、顆粒剤、散剤、粉剤、シロップ剤、ドライシロップ剤、液剤、懸濁剤、乳濁剤、発酵乳剤などであり、経口製剤、坐剤など、特に経口製剤が好ましい。 The pharmaceutical composition can be formulated into any dosage form as long as it exhibits the above effects. For example, tablets, capsules, granules, powders, powders, syrups, dry syrups, solutions, suspensions, emulsions, fermentation emulsions and the like, and oral preparations, suppositories, etc., particularly oral preparations are preferred.
本発明の有効成分に、薬学的に許容される通常用いられる賦形剤、担体、希釈剤、崩壊剤、結合剤、湿潤剤、安定剤、緩衝剤、滑沢剤、保存剤、界面活性剤、甘味料、矯味剤、芳香剤、酸味料、着色剤などの添加物を剤型に応じて配合し、常法に従って製造することができる。これらの物質の例は、上記の物質である。 As the active ingredient of the present invention, pharmaceutically acceptable commonly used excipients, carriers, diluents, disintegrants, binders, wetting agents, stabilizers, buffers, lubricants, preservatives, surfactants Additives such as sweeteners, flavoring agents, fragrances, acidulants, and coloring agents can be blended according to the dosage form, and can be produced according to conventional methods. Examples of these substances are those mentioned above.
医薬組成物中のCP2305株及び/又は菌体処理物の総量が、以下のものに限定されないが、該菌の生菌量として、105〜1012cfu/g、好ましくは106〜約1012cfu/g、より好ましくは107〜1012cfu/gに相当する量であるか、或いは、該菌の量として、例えば0.001重量％〜10重量％、好ましくは0.01〜5重量％となるような量である。その有効量は、添加量と下痢症状の軽減効果との関係に基づいて、当業者が適宜設定することが可能である。
好ましい投与法は、経口投与又は坐剤投与である。
The total amount of CP2305 strain and / or cell-treated product in the pharmaceutical composition is not limited to the following, but the amount of viable bacteria is 10 5 to 10 12 cfu / g, preferably 10 6 to about 10 The amount corresponds to 12 cfu / g, more preferably 10 7 to 10 12 cfu / g, or the amount of the bacteria is, for example, 0.001% to 10% by weight, preferably 0.01 to 5% by weight. It is an amount like this. The effective amount can be appropriately set by those skilled in the art based on the relationship between the added amount and the effect of reducing diarrhea symptoms.
The preferred administration method is oral administration or suppository administration.
本発明の医薬組成物は、ストレス性下痢における下痢症状、或いはストレス時の下痢症状の悪化、の予防、抑制（軽減）又は治療に有効である。 The pharmaceutical composition of the present invention is effective in preventing, suppressing (reducing) or treating diarrhea symptoms in stress diarrhea or worsening of diarrhea symptoms during stress.
以下に実施例によって本発明をさらに具体的に説明するが、本発明の範囲は、これらの実施例に制限されないものとする。 Examples The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited to these examples.
［実施例１］
SD雄ラット（４週齢、日本チャールス・リバー社製）を12時間毎の明暗周期下（8時より12時間点灯）で１週間飼育後、試験菌体配合餌を以下のように3週間自由摂取させた。
a)餌は、オリエンタル酵母株式会社のCRF-1粉末餌を使用した。
b)上記餌（CRF−１）25ｇに対し、菌数2.0×1010個が含まれる試験菌体の凍結乾燥粉末を配合（餌CRF-1：25gに対し、凍結乾燥粉末0.25g）し、試験菌体配合餌とした。
c)試験菌体配合餌を３週間自由摂取させた。
d)この試験による菌体摂食量はおおよそ2.0×1010個/日と推定された。
e)各群19匹に摂取させた。
3週間後、CRF（Corticotropin Releasing Factor、アナスペック社製、USA）溶液を125μg/kg腹腔内投与し、ストレス性下痢を誘発させ、CRF投与後の100分間の糞便状況を以下に記載する下痢スコアにて評価し、試験菌体のストレス性下痢抑制を評価した。
[Example 1]
SD male rats (4 weeks old, manufactured by Charles River Japan, Inc.) are reared for 1 week under a light-dark cycle every 12 hours (lights up for 12 hours from 8 o'clock), and then the test cell combination diet is free for 3 weeks as follows: Ingested.
a) As a bait, a CRF-1 powder bait from Oriental Yeast Co., Ltd. was used.
b) A lyophilized powder of test cells containing 2.0 × 10 10 bacteria is mixed with 25 g of the above-mentioned bait (CRF-1) (freeze-dried powder 0.25 g for bait CRF-1: 25 g), A test bacillus mixed feed was used.
c) A diet containing test cells was ingested freely for 3 weeks.
d) Bacteria consumption in this study was estimated to be approximately 2.0 × 10 10 cells / day.
e) Ingested by 19 animals in each group.
Three weeks later, CRF (Corticotropin Releasing Factor, Anaspec Corp., USA) solution was intraperitoneally administered 125 μg / kg to induce stress diarrhea, and the diarrhea score described below is 100-minute stool status after CRF administration And the suppression of stress-induced diarrhea of the test cells was evaluated.
下痢スコアの定義は、以下のとおりである。
(a)CRF投与後の100分間の糞便を10分間隔で10回採取する。
(b)各回で採取した糞便の重量をg単位で測定する(各回の糞便重量(i値))。
The definition of diarrhea score is as follows.
(a) Collect stool for 100 minutes after
(b) The weight of stool collected at each time is measured in g (feces weight at each time (i value)).
例えば、ある回の糞便重量が0.4gであった場合は、i値=0.4とする。
(c)各回で採取した糞便について、糞便の水分状況及び形状から図8に示す糞便スコア基準により、各回の糞便スコアとして数値化する（各回の糞便スコア（ii値））
(d)各回の糞便重量（i値）×各回の糞便スコア（ii値）を求める。
(e)各回で求めた、糞便重量（i値）×糞便スコア（ii値）の10回分の総和を「下痢スコア」と定義する。
(f)これによって、CRF投与後100分間の糞便状況を評価した。
For example, if the stool weight at a certain time is 0.4 g, i value = 0.4.
(c) The stool collected at each time is quantified as the stool score of each time according to the stool score standard shown in FIG. 8 from the water status and shape of the stool (the stool score (ii value) of each time)
(d) Obtain the stool weight (i value) x stool score (ii value) each time.
(e) The total of 10 stool weight (i value) x stool score (ii value) obtained in each round is defined as “diarrhea score”.
(f) This evaluated the stool condition for 100 minutes after CRF administration.
その結果、本発明のラクトバチルス・ガセリCP2305株の生菌体及びラクトバチルス・ガセリCP2305株又はその派生株の殺菌体は、プラセボ（CRF-1のみ）と比較して、有意にストレス性下痢を抑制した（図１）。 As a result, the viable cell of Lactobacillus gasseri CP2305 strain and the fungus of Lactobacillus gaseri CP2305 strain or derivative thereof of the present invention have significantly stress diarrhea compared with placebo (CRF-1 only). Suppressed (FIG. 1).
［実施例２］
比較対象とした公知寄託菌株A菌体の結果は、CP2305株と同菌数投与した結果である。また、市販生菌製剤Bの結果は、ヒト1日分の摂取用量の重量をラットの体重換算で、単位体重あたり同量となるよう投与した結果である（図2）。公知寄託菌株A（L. gasseri）及び市販生菌製剤B（L. acidophilus 含む）ではストレス性下痢が抑制されず、CP2305株（生菌体）のみ有意なストレス性下痢の抑制を確認した（図2）。
[Example 2]
The result of the known deposited strain A as a comparison target is the result of administration of the same number of bacteria as the CP2305 strain. In addition, the results of the commercially available viable bacterial preparation B are the results of administration so that the weight of the daily intake for humans is equivalent to the weight per unit body weight in terms of the body weight of rats (FIG. 2). The stress deposited diarrhea was not suppressed by the publicly known deposited strain A (L. gasseri) and the commercially available live bacterial preparation B (including L. acidophilus), and only the CP2305 strain (live cell body) was confirmed to suppress significant stress diarrhea (Fig. 2).
［実施例３］
さらにまた、CRF投与4時間後に解剖を実施し、結腸組織をISOGEN(ニッポンジーン)およびRNeasy Mini Kit(Qiagen,USA)にて総RNAを抽出後、遺伝子発現をRat whole genome ver. 3 (G4847B,Agilent,USA) で網羅的に解析を行った。その結果、プラセボ群でCRF投与後に増加するクロライド・チャネル関連遺伝子を、CP2305株投与群では抑制することを確認した（図3）。従って、CP2305株はクロライドチャネルを抑制し、水分の移動を制御していると考えられる。
[Example 3]
Furthermore, dissection was performed 4 hours after administration of CRF, and colon tissues were extracted with total RNA using ISOGEN (Nippon Gene) and RNeasy Mini Kit (Qiagen, USA), and then gene expression was determined using Rat whole genome ver. 3 (G4847B, Agilent , USA). As a result, it was confirmed that the chloride channel-related gene increased after CRF administration in the placebo group was suppressed in the CP2305 strain administration group (FIG. 3). Therefore, it is considered that the CP2305 strain suppresses chloride channels and controls water movement.
［実施例４］
SDラット（体重380〜400g、日本SLC社製）を麻酔後、断頭、放血屠殺し、解剖学的基準により遠位結腸(distal colon)を摘出し、実態顕微鏡下で輪走筋および縦走筋層を剥離し、粘膜下神経叢を含む粘膜−粘膜下神経叢標本を作製した。標本を測定面積0.64cm2のUssing chamber(CHM2, WPI, FL, USA)に装着し、標本の粘膜側−漿膜側間に生じる電位差及び短絡電流short-circuit current(ISC)を測定した。1分間に3秒間10mVのコマンドパルスを組織に与え、短絡電流の変化量から膜コンダクタンス（Gt）を算出した。basel ISC およびGtが安定したところで粘膜下神経に電気刺激(25V, duration of 0.5ms, 5Hz, 120s)を行い組織の状態を確認した。その後、試験乳酸菌の菌体または発酵乳の希釈溶液(500μg/ml)を粘膜側に投与し、ISCおよびGtの変化を測定し、神経刺激誘発により発生するCl−/HCO3 −分泌に対する試験物質の影響を評価した。
[Example 4]
SD rat (weight: 380-400 g, manufactured by Japan SLC) was anesthetized, decapitated and exsanguinated, and the distal colon was removed according to anatomical criteria. And the mucosa-submucosa plexus specimen containing the submucosal plexus was prepared. The specimen was mounted in a Ussing chamber (CHM2, WPI, FL, USA) having a measurement area of 0.64 cm 2 and the potential difference generated between the mucosa side and the serosa side of the specimen and the short-circuit current (I SC ) were measured. A 10 mV command pulse was applied to the tissue for 3 seconds per minute, and the membrane conductance (Gt) was calculated from the amount of change in the short-circuit current. When basel I SC and Gt were stabilized, electrical stimulation (25 V, duration of 0.5 ms, 5 Hz, 120 s) was performed on the submucosal nerve to confirm the tissue condition. Then, test lactic acid bacteria or fermented milk diluted solution (500 μg / ml) is administered to the mucosa side, changes in I SC and Gt are measured, and tests for Cl − / HCO 3 − secretion generated by neural stimulation induction The effect of the substance was evaluated.
各種乳酸菌の殺菌発酵乳の影響を評価した結果、CP2305株の殺菌発酵乳は、公知寄託菌株A（L. gasseri）及び他の5菌株（B〜F）よりも強くイオン分泌を抑制し、水分分泌を制御し、下痢抑制に寄与していることが示唆された（図4）。 As a result of evaluating the effect of sterilized fermented milk of various lactic acid bacteria, the sterilized fermented milk of CP2305 strain suppressed ion secretion more strongly than the known deposited strain A (L. gasseri) and other 5 strains (BF), It was suggested that it regulates secretion and contributes to diarrhea suppression (Fig. 4).
［実施例５］
さらに、ラット大腸（遠位結腸）の神経刺激誘発のイオン輸送に及ぼす菌体のみの影響を評価するために、各種乳酸菌の殺菌体を調製し、投与した結果、CP2305株殺菌体は、公知寄託菌株A（L. gasseri）殺菌体及びL. acidophilus殺菌体と比較し、有意にイオン分泌を顕著に抑制し、下痢抑制に強く寄与していることが示唆された（図5）。
[Example 5]
Furthermore, in order to evaluate the effect of bacterial cells alone on nerve stimulation-induced ion transport in the rat large intestine (distal colon), sterilized lactic acid bacteria were prepared and administered. Compared with bacterial strains A (L. gasseri) and L. acidophilus, it was suggested that it significantly suppressed ion secretion and strongly contributed to diarrhea suppression (FIG. 5).
さらにまた、ラット大腸（遠位結腸）の神経刺激誘発のイオン輸送に及ぼすCP2305株の生菌体、生菌発酵乳、殺菌体、殺菌発酵乳の投与による影響を調べた結果、そのすべての形態で、とりわけCP2305株殺菌体で、下痢抑制に強く寄与していることが示された（図6）。 Furthermore, as a result of investigating the effects of CP2305 strain administration on the nerve stimulation-induced ion transport in the rat large intestine (distal colon) by the administration of live cells, live fermented milk, pasteurized milk, and pasteurized fermented milk, all its forms In particular, it was shown that CP2305 strain bactericides contributed strongly to diarrhea suppression (Fig. 6).
［実施例６］
SD系雄ラット（４週齢、株式会社紀和動物製）を12時間毎の明暗周期下（8時より12時間点灯）で１週間飼育後、CP2305株配合餌もしくは未配合対照粉餌を3週間自由摂食させた。実験当日はラットを3時間絶食させた後、ウレタン麻酔し、頚静脈に静脈投与用のカニューレを挿入し、その後、下部大腸を神経支配する骨盤（副交感）神経を銀電極で吊上げ、その電気活動を測定した。測定値が安定した時期にCRF溶液（1μg/kg）を頚静脈投与し骨盤神経の電気活動(PNA)の変化を電気生理学的に測定した。データは5分間毎の5秒あたりの発火頻度の平均値にて解析し、頚静脈投与直前の値（0分値）を100％として百分率で示した。その結果、ストレスホルモン(CRF）投与することで、ラット大腸を神経支配する骨盤（副交感）神経活動が抑制されることを確認した。更にはCP2305株の投与がストレス誘発の骨盤(副交感)神経抑制を阻害することを確認した（図7）。従って、CP2305株の投与により、ストレス時の腸管機能が正常に保たれるものと考えられる。
[Example 6]
SD male rats (4 weeks old, manufactured by Kiwa Animal Co., Ltd.) are bred for 12 weeks under a light / dark cycle (lights on for 12 hours from 8 o'clock) for 1 week, and then CP2305 mixed diet or uncontained control powder diet for 3 weeks Ad libitum. On the day of the experiment, rats were fasted for 3 hours, then anesthetized with urethane, a cannula for intravenous administration was inserted into the jugular vein, and then the pelvic (parasympathetic) nerve that innervates the lower colon was lifted with a silver electrode, and its electrical activity Was measured. When the measured value was stable, the CRF solution (1 μg / kg) was administered to the jugular vein, and the change in pelvic nerve electrical activity (PNA) was measured electrophysiologically. The data was analyzed by the average value of the firing frequency per 5 seconds every 5 minutes, and the value immediately before the administration of the jugular vein (0 minute value) was taken as 100% and expressed as a percentage. As a result, it was confirmed that pelvic (parasympathetic) nerve activity that innervates the rat large intestine is suppressed by administration of stress hormone (CRF). Furthermore, it was confirmed that administration of the CP2305 strain inhibits stress-induced pelvic (parasympathetic) nerve suppression (FIG. 7). Therefore, administration of CP2305 strain is considered to maintain normal intestinal function during stress.
本発明のラクトバチルス・ガセリCP2305株（FERM BP-11331）、その菌体処理物、或いはその混合物は、ストレス誘発性骨盤（副交感）神経活動の抑制を阻害して腸運動を正常に保持するとともに、被験体のストレス性下痢、の下痢症状、を予防、抑制（軽減）又は改善することができる。 The Lactobacillus gasseri CP2305 strain (FERM BP-11331) of the present invention, a treated product thereof, or a mixture thereof inhibits the suppression of stress-induced pelvic (parasympathetic) nerve activity and maintains intestinal motility normally. It is possible to prevent, suppress (reduce) or ameliorate stress diarrhea and symptoms of diarrhea in a subject.
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| JP2012252422A JP6053466B2 (en) | 2012-11-16 | 2012-11-16 | Stress diarrhea inhibitor |
| EP13854909.2A EP2923704B1 (en) | 2012-11-16 | 2013-11-15 | Stress-induced bowel disorder-relieving agent comprising specific lactobacillus gasseri strain or treatment product thereof |
| US14/443,089 US20150283187A1 (en) | 2012-11-16 | 2013-11-15 | Agent for alleviating stress-induced bowel disorder containing specific lactobacillus gasseri strain or treated product thereof |
| CN201380059938.4A CN104797265A (en) | 2012-11-16 | 2013-11-15 | Stress-induced bowel disorder-relieving agent comprising specific lactobacillus gasseri strain or treatment product thereof |
| PCT/JP2013/080930 WO2014077365A1 (en) | 2012-11-16 | 2013-11-15 | Stress-induced bowel disorder-relieving agent comprising specific lactobacillus gasseri strain or treatment product thereof |
| CA2890507A CA2890507A1 (en) | 2012-11-16 | 2013-11-15 | Agent for alleviating stress-induced bowel disorder containing specific lactobacillus gasseri strain or treated product threof |
| BR112015010978A BR112015010978A2 (en) | 2012-11-16 | 2013-11-15 | stress-induced bowel disorder relief agent containing specific lactobacillus gasseri strain or product treated therewith |
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