JPH0899887A - Immunopotentiator - Google Patents
ImmunopotentiatorInfo
- Publication number
- JPH0899887A JPH0899887A JP7205384A JP20538495A JPH0899887A JP H0899887 A JPH0899887 A JP H0899887A JP 7205384 A JP7205384 A JP 7205384A JP 20538495 A JP20538495 A JP 20538495A JP H0899887 A JPH0899887 A JP H0899887A
- Authority
- JP
- Japan
- Prior art keywords
- enterococcus
- cells
- administration
- immunopotentiator
- microorganism belonging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、エンテロコッカス（En
terococcus）属に属する微生物の菌体又はその処理物を
有効成分として含有する、副作用のない免疫増強能、骨
髄内細胞増殖機能及びリンパ球増殖機能を有する製剤に
関するものである。BACKGROUND OF THE INVENTION The present invention relates to enterococcus (En
The present invention relates to a pharmaceutical preparation containing a bacterial cell of a microorganism belonging to the genus terococcus) or a treated product thereof as an active ingredient and having an immunopotentiating ability without side effects, an intramedullary cell proliferation function and a lymphocyte proliferation function.
【０００２】[0002]
【従来の技術】現在、生体の免疫能を高める、もしくは
免疫に関する生物活性物質（インターフェロン類、腫瘍
壊死因子（ＴＮＦ）、コロニー刺激因子等の各種サイト
カイン等）の放出能増強等の目的で生体応答調節物質
（ＢＲＭ、Biological responsemodifiers）製剤が多種
開発されており、何種類かのＢＲＭ製剤が製品化されて
いる。これらの薬剤は主として、白血球その他の癌や、
ウイルス性肝炎等の治療に使用されているが、求められ
ている効果が十分に得られないものや、効果はあっても
それ以上に重篤な副作用が生じて、投与に継続が困難に
なるものが多い。又、特定の疾患だけの使用や、短期間
の使用等、種々の制限がつけられている場合が多い。そ
のため、このようなＢＲＭ製剤を予防医学的な目的に応
じて日常生活の中で用いることは不可能である。2. Description of the Related Art At present, a biological response for the purpose of enhancing the immunopotency of a living body or enhancing the release ability of biologically active substances (interferons, tumor necrosis factor (TNF), various cytokines such as colony stimulating factor) related to immunity Various types of BRM (biological response modifiers) formulations have been developed, and several types of BRM formulations have been commercialized. These drugs are mainly white blood cells and other cancers,
It is used for the treatment of viral hepatitis, etc., but it is difficult to continue administration because it may not provide the desired effect, or it may have more serious side effects even if it is effective. There are many things. In addition, there are many cases where various restrictions are imposed such as use only for a specific disease and short-term use. Therefore, it is impossible to use such a BRM preparation in daily life for the purpose of preventive medicine.
【０００３】畜産業界では、家畜の飼料や飼育設備への
投下資本の効率的回収のため、やむなく、家畜の生態を
無視した飼育がなされている。食肉用家畜は、短期間で
の発育と飼育効率を図るために狭い畜舎の中で、運動を
制限して飼育されている場合が多い。又、養鶏場の白色
レグホンは、狭いケージで飼育されている。乳牛に対し
ては、穀物等を大量に与え、乳量増産を図っているため
に、本来の食生活とは、全くかけ離れたものとなってい
る。このように家畜をとりまく環境は過酷で、家畜はス
トレスがたまりやすくなり、疾病に対する抵抗力が落ち
ている。牧場で飼育されている家畜においても、人手不
足のため、家畜の健康管理が十分対応できていない場合
がある。家畜の羅病は畜産農家にとって重大であり、そ
ういう事態を防ぐために、家畜の飼料に抗生物質等の薬
物を添加して、疾病の予防を図っている。[0003] In the livestock industry, in order to efficiently recover the livestock feed and invested capital to the breeding equipment, the breeding of livestock is unavoidably carried out. In many cases, livestock for meat are bred with restricted movement in a narrow barn for the purpose of achieving growth and breeding efficiency in a short period of time. The white leghorns on the poultry farm are kept in narrow cages. Since cows are given large amounts of cereals and the like to increase milk production, they are completely different from the original diet. As described above, the environment surrounding livestock is harsh, and the livestock are more likely to accumulate stress and less resistant to diseases. Even for livestock bred in the ranch, the health management of livestock may not be adequate due to lack of manpower. Livestock disease is serious for livestock farmers, and in order to prevent such a situation, drugs such as antibiotics are added to the livestock feed to prevent the disease.
【０００４】乳酸菌は、抗癌作用や免疫賦活作用等、様
々な生理活性をもつことが知られているが、報告されて
いる投与例のほとんどは、腹腔内投与等の非経口投与で
ある。乳酸菌懸濁物や磨砕物等の乳酸菌処理物の非経口
投与法は、動物実験では可能であっても、臨床治療法と
しての実際の使用は困難な点が多く不可能である。ま
た、乳酸菌の生理活性効果を得るために乳酸菌そのも
の、もしくはその処理物単独の経口摂取を行う臨床上の
使用例はほとんどなく、ヨーグルト等の乳製品に含まれ
ている乳酸菌の摂取、又はケフィアのような乳酸菌の代
謝産物の飲用による生理活性効果の有無を観察している
のが実状である。[0004] Lactic acid bacteria are known to have various physiological activities such as anti-cancer action and immunostimulatory action, but most of the reported administration examples are parenteral administration such as intraperitoneal administration. Although the method of parenteral administration of a lactic acid bacterium-treated product such as a lactic acid bacterium suspension or a ground product is possible in animal experiments, practical use as a clinical treatment is difficult in many points. In addition, there are almost no clinical use examples of oral ingestion of lactic acid bacterium itself or a processed product thereof alone to obtain the physiologically active effect of lactic acid bacterium. Ingestion of lactic acid bacterium contained in dairy products such as yogurt, or of kefir The actual situation is to observe the presence or absence of the physiologically active effect of drinking the metabolites of lactic acid bacteria.
【０００５】[0005]
【発明が解決しようとする課題】免疫賦活作用を有する
薬剤は、免疫低下症例に対して専ら用いられている。又
一方では、免疫賦活剤を予防医学的に使用することが疾
病を予防する見地から希求されるが、このような場合に
は、長期にわたる使用が必要となるので、有効であるこ
とはもちろん、服用の容易さ及び副作用を示さないこと
等が重要である。A drug having an immunostimulating action is exclusively used for immunocompromised cases. On the other hand, the use of immunostimulants in preventive medicine is desired from the viewpoint of preventing diseases, but in such a case, long-term use is required, and thus it is of course effective. It is important that it is easy to take and that it does not show side effects.
【０００６】動物用薬剤、特に家畜及び家禽用薬剤は、
法的規制により、食肉や酪農産物等の出荷時に残存しな
いこととされている。しかし、抗菌剤としての抗生物質
やホルモン剤等の薬剤が、動物体に異常な変化をもたら
す可能性は否定できないし、使用されたこれら薬剤が残
存しないとは言い切れるものではない。従って、食肉や
卵及び乳製品等は、日常的に摂取されるものであるの
で、できる限りこのような薬剤を使わないようにするこ
とが重要である。[0006] Animal drugs, especially livestock and poultry drugs,
According to legal regulations, meat and dairy products do not remain when they are shipped. However, it is undeniable that drugs such as antibiotics and hormones as antibacterial agents may cause abnormal changes in the animal body, and it cannot be said that these used drugs do not remain. Therefore, since meat, eggs, dairy products and the like are ingested on a daily basis, it is important not to use such drugs as much as possible.
【０００７】そのため、ヒト及び動物とりわけ家畜類に
対して安全且つ、免疫増強等の作用により疾患を予防で
きる薬剤が求められている。[0007] Therefore, there is a need for a drug which is safe for humans and animals, especially livestock, and which is capable of preventing diseases by the action of enhancing immunity.
【０００８】[0008]
【課題を解決するための手段】本発明者らはエンテロコ
ッカス属に属する微生物に免疫賦活作用及び白血球減少
を予防する働きがあることに着目し、各種の免疫反応に
関係する細胞やその機能についての作用を詳しく調べ
た。そして、この微生物が、末梢血中の白血球数、好酸
球数、骨髄細胞の顆粒球／赤芽球比（Ｍ／Ｅ比）の増
加、リンパ球の幼若化促進、末梢血中の好中球の化学発
光能の増強など、免疫賦活に関係する細胞数の増加及び
機能の増強等の生理作用を有することを見いだし、本発
明を完成させた。又、本発明剤に使用される菌種は、健
常者の腸内およびサイレージから分離された乳酸菌の一
種であるので、副作用の無い安全な菌種である。Means for Solving the Problems The present inventors have focused on the fact that microorganisms belonging to the genus Enterococcus have an immunostimulatory action and a function to prevent leukopenia, and The effect was investigated in detail. Then, this microorganism increases the number of white blood cells in the peripheral blood, the number of eosinophils, the granulocyte / erythroblast ratio (M / E ratio) of bone marrow cells, the promotion of lymphocyte blastogenesis, The inventors have found that they have physiological effects such as an increase in the number of cells related to immunostimulation and enhancement of functions such as enhancement of chemiluminescence of neutrophils, and completed the present invention. Further, the bacterial species used in the agent of the present invention is a kind of lactic acid bacterium isolated from the intestines and silage of healthy subjects, and is therefore a safe bacterial species with no side effects.
【０００９】本発明に用いられるエンテロコッカス属に
属する微生物としてはエンテロコッカス・フェカリス
（Enterococcus faecalis）やエンテロコッカス・フェ
シウム（Enterococcus faecium）もしくはエンテロコッ
カス・エビウム（Enterococcusavium）やエンテロコッ
カス・デュランス（Enterococcus durans）、又はエン
テロコッカス・カセリフラブス（Enterococcus casseli
flavus）やエンテロコッカス・ガリナルム（Enterococc
us gallinarum）等が挙げられるが、特に有用なのは、
本発明者らにより分離された新菌株のエンテロコッカス
・フェカリス及びエンテロコッカス・カセリフラブスに
属する微生物の菌体又はその処理物である。The microorganisms belonging to the genus Enterococcus used in the present invention include Enterococcus faecalis, Enterococcus faecium, Enterococcus avium, Enterococcus durancus, or Enterococcus durancus. (Enterococcus casseli
flavus) and Enterococcus gallinarum (Enterococc)
us gallinarum) etc., but especially useful is
The present invention is a bacterial cell of a microorganism belonging to the new strains of Enterococcus faecalis and Enterococcus casei flavus isolated by the present inventors or a treated product thereof.
【００１０】エンテロコッカス・フェカリスは、ヒトの
腸内常在乳酸菌の一種である（Bergey's Manual of Sys
tematic Bacteriology. vol.2,1063(1986)）。本発明に
おいてはこの菌種に属する種々の菌株を用いることがで
きるが、特に免疫賦活作用が高い点において、ＮＦ−１
０１１菌株を用いることが好ましい。該菌株は工業技術
院微生物工業技術研究所に微工研菌寄第１２５６４号と
して寄託されている。Enterococcus faecalis is a kind of human intestinal lactic acid bacteria (Bergey's Manual of Sys).
tematic Bacteriology. vol.2,1063 (1986)). Although various strains belonging to this strain can be used in the present invention, NF-1 is particularly preferable because of its high immunostimulatory action.
It is preferred to use strain 011. The strain has been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Microorganism Research Institute No. 12564.
【００１１】エンテロコッカス・カセリフラブスは、サ
イレージ内に生息する乳酸菌の１種である（Bergey's M
anual of Systematic Bacteriology. vol.2,1063(198
6)）。本発明においてはこの菌種に属する種々の菌株を
用いることができるが、特に免疫賦活作用が高い点にお
いて、ＮＦ−１００４菌株を用いることが好ましい。該
菌株は工業技術院微生物工業技術研究所に微工研菌寄第
１４３７８号として寄託されている。Enterococcus casei flavus is one of the lactic acid bacteria that live in silage (Bergey's M
anual of Systematic Bacteriology.vol.2,1063 (198
6)). Although various strains belonging to this strain can be used in the present invention, it is preferable to use the NF-1004 strain because of its high immunostimulatory action. The strain has been deposited in the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology as Micromachine Research Institute No. 14378.
【００１２】以下にエンテロコッカス・フェカリスＮＦ
−１０１１及びエンテロコッカス・カセリフラブスＮＦ
−１００４の分離手段及び同菌株の菌学的及び生理学的
性質を示す。The following is Enterococcus faecalis NF
-1011 and Enterococcus caseiflaves NF
-1004 shows the means of isolation and the mycological and physiological properties of the strain.
【００１３】（１）エンテロコッカス・フェカリスＮＦ
−１０１１の分離手段 健常者の糞便の加熱滅菌水による１０倍希釈物を適切な
選択培地（ＫＭＮ寒天平板及びＳＦ寒天平板）に塗抹
し、好気条件下３７℃で、４８〜７２時間培養し、菌集
落を出現させた。この菌集落を別の同種平板培地に画線
塗布し、同様に培養して菌集落を再び出現させた。同様
の操作を数回繰り返し、単一の菌種だけからなる単一集
落を分離した。この新分離菌株について、菌学的（形態
的、生化学的及び血清学的）性状を調べ、エンテロコッ
カス・フェカリス（Enterococcusfaecalis）に属すると
分類同定した。 （２）エンテロコッカス・カセリフラブスＮＦ−１００
４の分離手段 サイレージの加熱滅菌水による１０倍希釈物を適切な選
択培地（ＭＲＳ寒天平板）に塗抹し、好気条件下３７℃
で、４８〜７２時間培養し、菌集落を出現させた。この
菌集落を別の同種平板培地に画線塗布し、同様に培養し
て菌集落を再び出現させた。同様の操作を数回繰り返
し、単一の菌種だけからなる単一集落を分離した。この
新分離菌株について、菌学的（形態的、生化学的及び血
清学的）性状を調べ、エンテロコッカス・カセリフラブ
ス（Enterococcus casseliflavus）に属すると分類同定
した。(1) Enterococcus faecalis NF
Separation means of 1011 A 10-fold dilution of feces of a healthy person with heat-sterilized water is smeared on an appropriate selective medium (KMN agar plate and SF agar plate), and cultured at 37 ° C under aerobic conditions for 48 to 72 hours. , A bacterial colony appeared. The bacterial colonies were streaked on another homogeneous plate medium and cultured in the same manner to reappear the bacterial colonies. The same operation was repeated several times to separate a single colony consisting of a single bacterial species. The new isolate was examined for mycological (morphological, biochemical and serological) properties and classified and identified as belonging to Enterococcus faecalis. (2) Enterococcus caseiflaves NF-100
4. Separation means 4 A 10-fold dilution of silage with heat-sterilized water is spread on an appropriate selective medium (MRS agar plate), and aerobic conditions are set at 37 ° C.
Then, the cells were cultured for 48 to 72 hours, and bacterial colonies appeared. The bacterial colonies were streaked on another homogeneous plate medium and cultured in the same manner to reappear the bacterial colonies. The same operation was repeated several times to separate a single colony consisting of a single bacterial species. The new isolate was examined for mycological (morphological, biochemical and serological) properties and classified and identified as belonging to Enterococcus casseliflavus.
【００１４】 （３）菌学的及び生理学的性質 ──────────────────────────── 性状 NF-1011 NF-1004 ──────────────────────────── グラム染色性 ＋ ＋ 菌形態 球形 球形 カタラーゼ − − 溶血性 α α 血清群 Ｄ Ｄ 増殖性 １０℃ ＋ ＋ ４５℃ ＋ ＋ ５０℃ ＋ ＋ 熱耐性 ６０℃ ３０分 ＋ ＋ 胆汁エスクリン添加培地での生育 ＋ ＋ ｐＨ９．６培地での生育 ＋ ＋ ６．５％食塩添加培地での生育 ＋ ＋ メチレンブルー染色性 ＋ ＋ ゼラチン液化 − − ０．０１％ＴＴＣ添加培地での生育 ＋ ＋ テルライト添加培地での生育 ＋ ＋ 酸生成の有無 グリセロール ＋ ＋ Ｌ−アラビノース − ＋ Ｄ−リボース ＋ ＋ Ｄ−キシロース − ＋ Ｄ−グルコース ＋ ＋ Ｄ−ガラクトース ＋ ＋ Ｄ−フラクトース ＋ ＋ Ｄ−マンノース ＋ ＋ マルトース ＋ ＋ マンニトール ＋ ＋ シュクロース ＋ ＋ Ｌ−ソルボース − − Ｄ−ソルビトール ＋ − Ｌ−ラムノース ＋ ＋ ラクトース ＋ ＋ アミグダリン ＋ ＋ エスクリン ＋ ＋ セロビオース ＋ ＋ メリビオース − ＋ イヌリン − ＋ メレジトース ＋ − ──────────────────────────── ＋；陽性、−；陰性 ＴＴＣ；２，３，５−トリフェニルテトラゾリウムクロリド(3) Bacteriological and physiological properties ──────────────────────────── Property NF-1011 NF-1004 ── ────────────────────────── Gram stain ＋ ＋ Bacterial morphology Spherical sphere Catalase − − Hemolytic αα Serogroup D D Proliferation 10 ° C + + 45 ° C + + 50 ° C + + Thermotolerance 60 ° C 30 minutes + + Growth on medium supplemented with bile esculin + + Growth on medium pH 9.6 + + + Methylene blue staining + 6.5% Sex ++ Gelatin liquefaction − − Growth in 0.01% TTC-supplemented medium ++ Growth in tellurite-supplemented medium ++ Presence / absence of acid production Glycerol + + L-arabinose − + D-ribose + + D-xylose − + D -Glucose + + D-galactose + + D-fract Source + + D-mannose + + Maltose + + Mannitol + + Sucrose + + L-sorbose − − D-sorbitol + − L-rhamnose + + Lactose + + amygdalin + + + esculin + + cellobiose in + + − + Merezitose + − ──────────────────────────── +; Positive, −; Negative TTC; 2,3,5-Triphenyl Tetrazolium chloride
【００１５】本発明に使用するエンテロコッカス・フェ
カリス及びエンテロコッカス・カセリフラブスを培養し
て得られる菌体は死菌体又は生菌体、或いは菌体を磨
砕、超音波破砕、水抽出等の処理をしたものを用いるこ
とができる。これらを製剤するにはデンプン、乳糖、大
豆蛋白等の担体、賦形剤、結合剤、崩壊剤、滑沢剤、安
定剤、矯味矯具剤等の添加物を用いて、周知の方法で錠
剤や顆粒剤にされる。The cells obtained by culturing Enterococcus faecalis and Enterococcus casei flavus used in the present invention are dead cells or viable cells, or the cells were subjected to treatments such as grinding, ultrasonic disruption, and water extraction. Any thing can be used. To formulate these, tablets are prepared by well-known methods using additives such as carriers such as starch, lactose, soybean protein, excipients, binders, disintegrating agents, lubricants, stabilizers, and corrigents. Or into granules.
【００１６】使用量は、症状、年齢等により異なるが、
有効成分として１日0.002〜0.1g/kg体重を通常成人に対
して１日１回又は数回に分けて投与することができる。The amount used depends on symptoms, age, etc.,
As an active ingredient, 0.002 to 0.1 g / kg body weight per day can be usually administered to an adult once a day or in several divided doses.
【００１７】[0017]
【実施例】以下実施例を示すが、本発明はこれらの実施
例の記載によって何ら制限されるものではない。EXAMPLES Examples will be shown below, but the present invention is not limited to the description of these Examples.
【００１８】実施例１．（エンテロコッカスの培養） エンテロコッカス・フェカリス（Enterococcus faecali
s）ＮＦ−１０１１及びエンテロコッカス・カセリフラ
ブス（Enterococcus casseliflavus） ＮＦ−１００４を、代表的培地として以下に示す組成の
ロゴサ液体培地に接種し、（菌数：１０6個/ml）、３７
℃で１０〜１６時間培養し、生菌数約１０9個/mlの培養
液を得た。得られた培養液を12,000rpmで２０分間遠心
分離して集菌し、蒸留水で２回洗浄して菌体を得た。こ
の菌体を蒸留水で懸濁し、１１０℃で１０分間加熱して
死菌体懸濁液を得た。次に、熱風乾燥法あるいは凍結乾
燥法等適当な方法で乾燥処理し、乾燥死菌体を得た。Example 1. (Cultivation of Enterococcus faecalis)
s) NF-1011 and Enterococcus casseliflavus NF-1004 were inoculated into a Rogosa liquid medium having a composition shown below as a representative medium (the number of bacteria: 10 6 cells / ml), 37
Culturing was carried out at 10 ° C. for 10 to 16 hours to obtain a culture solution containing about 10 9 viable cells / ml. The obtained culture broth was centrifuged at 12,000 rpm for 20 minutes to collect the cells, and the cells were washed twice with distilled water to obtain cells. The cells were suspended in distilled water and heated at 110 ° C for 10 minutes to obtain a dead cell suspension. Next, the cells were dried by an appropriate method such as a hot air drying method or a freeze drying method to obtain dried dead cells.
【００１９】ロゴサ液体培地の組成を示す。 トリプチケース １０ｇ 酵母エキス ５ｇ トリプトース ３ｇ リン酸一カリウム ３ｇ リン酸二カリウム ３ｇ クエン酸三アンモニウム ２ｇ ツイーン８０（界面活性剤） １ｇ グルコース ２０ｇ システイン塩酸塩 ０．２ｇ 塩類溶液（１のとおり） ５ｍｌ 蒸留水 １，０００ｍｌ （ｐＨ７．０に調整、１２１℃で１５分間加熱滅菌） （１）塩類溶液：ＭｇＳＯ4・７Ｈ2Ｏ １１．５ｇ ＦｅＳＯ4・７Ｈ2Ｏ ０．６８ｇ ＭｎＳＯ4・２Ｈ2Ｏ ２．４ｇ 蒸留水 １００ｍｌThe composition of Rogosa liquid medium is shown below. Trypticase 10 g Yeast extract 5 g Tryptose 3 g Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80 (surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as per 1) 5 ml Distilled water 1,000 ml (adjusted to pH 7.0, heat sterilized at 121 ° C. for 15 minutes) (1) Salt solution: MgSO 4 .7H 2 O 11.5 g FeSO 4 .7H 2 O 0.68 g MnSO 4 .2H 2 O 2. 4g distilled water 100ml
【００２０】実施例２． 体重１０〜１５ｋｇの健常犬
（雑種、雄性又は雌性）にＡＧＦ社製ドッグフード（乾
燥及び缶詰）及び飲料水を与え、通常の飼育環境にて飼
育した。Example 2. Healthy dogs (mongrel, male or female) having a body weight of 10 to 15 kg were fed with AGF dog food (dried and canned) and drinking water, and bred in a normal breeding environment.
【００２１】このイヌ（３匹）に実施例１で得たエンテ
ロコッカス・フェカリスＮＦ−１０１１菌体標品を生理
的食塩水（0.85％ＮａＣｌ水溶液）に溶解又は懸濁し、
１００ｍｇ／ｋｇ体重の割合になるように３ヶ月間連日
経口投与を行った。他方の群（３匹）には生理的食塩水
のみを同量投与した。投与１ヶ月後、２ヶ月後、３ヶ月
後に採血し、一般血液検査、骨髄検査（Ｍ／Ｅ比）、更
に、好中球の化学発光能、及びグルコース消費量を指標
としたリンパ球幼若化反応の程度を調べた。The Enterococcus faecalis NF-1011 bacterial cell preparation obtained in Example 1 was dissolved or suspended in physiological saline (0.85% NaCl aqueous solution) in the dogs (3 dogs),
Oral administration was performed every day for 3 months so that the ratio was 100 mg / kg body weight. The other group (3 animals) was administered with physiological saline alone in the same amount. Blood was collected 1 month, 2 months, and 3 months after administration, general blood test, bone marrow test (M / E ratio), and lymphocyte juvenile using chemiluminescent ability of neutrophils and glucose consumption as indicators. The degree of chemical reaction was examined.
【００２２】（末梢血白血球数及び分画検査）末梢血白
血球をギムザ染色し、鏡検により細胞数を数えた。ＮＦ
−１０１１菌体標品投与前の白血球数は12,680±1,429
個／μｌ（平均値±ＳＤ、以下同じ）であったが、投与
１、２、３ヶ月後のそれは各々15,993±1,285個／μ
ｌ、14,323±1,470個／μｌ、15,025±1,231個／μｌと
増加傾向を示した。又血液塗抹による血球分画検査では
好酸球の増加が見られた。(White Blood Cell Count and Fractional Examination) Peripheral blood white blood cells were stained with Giemsa and the number of cells was counted by microscopy. NF
The white blood cell count before administration of -1011 bacterial cell preparation was 12,680 ± 1,429
Number of cells / μl (mean ± SD, the same hereafter) was 15,993 ± 1,285 cells / μ after 1, 2 and 3 months after administration
1, 14,323 ± 1,470 cells / μl and 15,025 ± 1,231 cells / μl showed an increasing tendency. In addition, eosinophils were found to be increased in blood cell differential tests by blood smearing.
【００２３】（骨髄細胞Ｍ／Ｅ比）腸骨稜から採取した
骨髄液をスライドグラスに塗布し、ライト・ギムザ染色
を行った。検体をメタノールに２〜５分間浸け、固定し
た後、ライト染色液に８〜１０分間浸け、顆粒を染色し
た。その後、水でライト染色液を洗い流し、ギムザ染色
液に１５〜３０分間浸け染色した後、水洗乾燥した。染
色した標本を鏡検し、赤芽球系細胞数と顆粒球系細胞数
を数えて比率（Ｍ／Ｅ比）を求めた。(M / E Ratio of Bone Marrow Cells) Bone marrow fluid collected from the iliac crest was applied on a slide glass and Wright-Giemsa staining was performed. The sample was dipped in methanol for 2 to 5 minutes and fixed, and then dipped in Wright's staining solution for 8 to 10 minutes to stain the granules. Then, the Wright dyeing solution was washed off with water, soaked in the Giemsa dyeing solution for 15 to 30 minutes for dyeing, and then washed with water and dried. The stained sample was examined microscopically, and the ratio (M / E ratio) was calculated by counting the number of erythroid cells and granulocytes.
【００２４】骨髄細胞のＭ／Ｅ比はＮＦ−１０１１菌体
標品投与前は1.29±0.06であったが、投与１、２、３ヶ
月後のそれは各々1.80±0.06、2.34±0.23、2.53±0.27
といずれの時点でも投与前に比べて増加していた（ｐ＜
0.01）。The M / E ratio of bone marrow cells was 1.29 ± 0.06 before administration of the NF-1011 bacterial cell preparation, but it was 1.80 ± 0.06, 2.34 ± 0.23, 2.53 ± 1, 2, and 3 months after administration, respectively. 0.27
And at any time, it increased compared to before administration (p <
0.01).
【００２５】（好中球の化学発光能）好中球の化学発光
能の測定は、ケミルミネッセンス法によった。バイアル
中に５ｍＭ ＨＥＰＥＳ添加−ＭＥＭ培地で４×１０5
個／ｍｌに調整した細胞浮遊液５００μｌを注入し、ル
ミノール溶液２０ｍｌを添加して、３７℃、３０分間保
温した。化学発光測定器での基定値が安定したところで
ジモサン２０μｌを添加し、好中球化学発光能を測定し
た。ＮＦ−１０１１菌体標品投与群の発光能は対照群に
比べ、菌体投与１、２、３ヶ月後共に1.5〜1.8倍高く、
有意に増強されていた（ｐ＜0.01）。(Chemiluminescent ability of neutrophils) The chemiluminescent ability of neutrophils was measured by a chemiluminescence method. Add 5 mM HEPES in vial-4 × 10 5 in MEM medium.
500 μl of cell suspension adjusted to cells / ml was injected, 20 ml of luminol solution was added, and the mixture was kept at 37 ° C. for 30 minutes. When the standard value with a chemiluminescence measuring device became stable, 20 μl of zimosan was added to measure the chemiluminescence ability of neutrophils. The luminescence ability of the NF-1011 bacterial cell preparation-administered group was 1.5 to 1.8 times higher than that of the control group after 1, 2 and 3 months of bacterial cell administration,
It was significantly enhanced (p <0.01).
【００２６】（リンパ球幼若化反応）ヘパリン（２５〜
５０単位／ｍｌ）加末梢静脈血よりFicoll-Hypaque法に
よりリンパ球を分離した。リンパ球数を、ＨＥＰＥＳ
（２５ｍＭ）、ペニシリンＧ（１００単位／ｍｌ）、ス
トレプトマイシン（５０μｇ／ｍｌ）及び１０％健常ヒ
トＡＢ型新鮮（凍結）血清を加えたＲＰＭＩ１６４０に
て５×１０5個／ｍｌに調整し、平底マイクロプレート
（Falcon 3040）の各ウエルに０．２ｍｌずつ分注し
た。その後、幼若化反応を促進させるために、フィトヘ
マグルチニン（ＰＨＡ−Ｐ、最終濃度１５μｇ／ｍ
ｌ）、コンカナバリン Ａ（Ｃｏｎ Ａ、最終濃度５０
μｇ／ｍｌ）、リポ多糖（ＬＰＳ、最終濃度２５μｇ／
ｍｌ）を添加し、３７℃で５％ＣＯ2培養器内にて、３
日間培養した。(Lymphocyte Juvenile Reaction) Heparin (25-
Lymphocytes were isolated from peripheral venous blood (50 units / ml) by the Ficoll-Hypaque method. Lymphocyte count, HEPES
(25 mM), penicillin G (100 units / ml), streptomycin (50 μg / ml) and 10% healthy human AB fresh (frozen) serum were added to RPMI1640 to adjust to 5 × 10 5 cells / ml, and flat bottom micro 0.2 ml was dispensed to each well of the plate (Falcon 3040). After that, in order to promote the juvenile reaction, phytohemagglutinin (PHA-P, final concentration 15 μg / m
l), Concanavalin A (Con A, final concentration 50)
μg / ml), lipopolysaccharide (LPS, final concentration 25 μg /
ml) was added, and the mixture was incubated at 37 ° C. in a 5% CO 2 incubator for 3 times.
Cultured for a day.
【００２７】培養後５０％グルコース溶液をマイクロプ
レート１ウエルあたり0.02ｍｌになるように加え、３７
℃で５％ＣＯ2培養器内にて、１８時間培養した。その
後リンパ球のみを分離し、溶液中のグルコース量を測定
した。予め測定した添加前のグルコース量と培養後のグ
ルコース量の差のブランク値での補正値を、幼若化リン
パ球の取込量とした。After the culturing, 50% glucose solution was added to each well of the microplate to make 0.02 ml, and 37
Culturing was carried out at 5 ° C in a 5% CO 2 incubator for 18 hours. After that, only lymphocytes were separated, and the amount of glucose in the solution was measured. The corrected value of the blank value of the difference between the glucose amount before the addition and the glucose amount after the culture that was measured in advance was taken as the uptake amount of the immature lymphocytes.
【００２８】[0028]
【表１】 数値は刺激指数を示す。 *、***は投与前（０月）に対して有意差があること（それぞれp<0.05、p<0.001 ）を示す。[Table 1] Numerical values indicate stimulation index. *, *** indicates that there is a significant difference from before administration (0 month) (p <0.05, p <0.001 respectively).
【００２９】結果を表１に示した。リンパ球幼若化は、
ＰＨＡ−Ｐ、Ｃｏｎ Ａ及びＬＰＳのいずれの刺激剤を
用いて測定した場合も、ＮＦ−１０１１菌体標品投与前
に比較して、投与１、２、３ヶ月後のいずれにおいても
高値を示し、菌体標品投与による幼若化反応の促進作用
が認められた。The results are shown in Table 1. Lymphocyte blastogenesis is
When measured using any of the stimulants of PHA-P, Con A and LPS, it showed a high value at 1, 2 and 3 months after administration as compared with before administration of NF-1011 bacterial cell preparation. , The stimulatory effect of the juvenile response was observed by the administration of the bacterial cell preparation.
【００３０】実施例３（マウス脾細胞の幼若化活性） ５週齢、雄性のＣ３Ｈ／Ｈｅ Ｎ系マウス（日本ＳＬ
Ｃ）を平均体重が同じになるように２群（各群６匹）に
分け、一方の群には粉末ＣＥ−２（日本クレア）のみ、
もう一方の群は、実施例１で得たエンテロコッカス・カ
セリフラブスＮＦ−１００４菌体標品を５％重量配合し
た粉末ＣＥ−２をそれぞれ自由摂取させた。Example 3 (Maturation activity of mouse splenocytes) 5-week-old male C3H / He N mouse (Japan SL)
C) was divided into 2 groups (6 animals in each group) so that the average body weight was the same, and one group contained only powder CE-2 (CLEA Japan, Inc.),
The other group was allowed to freely ingest powder CE-2 containing 5% by weight of the Enterococcus casei flavus NF-1004 bacterial cell preparation obtained in Example 1.
【００３１】菌体標品投与の開始から１４日後及び３５
日後に、両群各々３匹のマウスから脾臓を摘出し、１０
％ＦＢＳ含有ＲＰＭＩ１６４０培地中ではさみで破砕
後、洗浄及びワイヤーメッシュで濾過し均一な脾臓細胞
液を得た。この細胞液を１０％ＦＢＳ含有ＲＰＭＩ１６
４０培地を用いて、５×１０6個／ｍｌに調整し、平底
マイクロプレート（Falcon 3040）の各ウエルに0.1ｍｌ
ずつ分注した。その後、幼若化反応を促進させるため
に、コンカナバリン Ａ（Ｃｏｎ Ａ、最終濃度0.1μ
ｇ／ｍｌ）、リポ多糖（ＬＰＳ、最終濃度0.01μｇ／ｍ
ｌ）を添加し、３７℃で５％ＣＯ2培養器内にて、２日
間培養した。14 days after the start of administration of the bacterial cell preparation and 35
After a day, the spleens were excised from 3 mice in each group, 10
After crushing with scissors in RPMI1640 medium containing% FBS, washing and filtration with a wire mesh were performed to obtain a uniform spleen cell liquid. RPMI 16 containing 10% FBS
Using 40 medium, adjust to 5 × 10 6 cells / ml and add 0.1 ml to each well of flat bottom microplate (Falcon 3040).
We dispensed each. After that, concanavalin A (Con A, final concentration 0.1 μm)
g / ml), lipopolysaccharide (LPS, final concentration 0.01 μg / m
1) was added and the cells were cultured at 37 ° C. in a 5% CO 2 incubator for 2 days.
【００３２】培養後、0.5％3-(4,5-Dimethylthiazol-2-
yl)-2,5-Diphenyl TetrazoliumBromide（ＭＴＴ）溶液
を0.01ｍｌ／ウエル添加し、３７℃で５％ＣＯ2培養器
内にて３時間培養した。培養後、プレートを遠心し、上
清0.1ｍｌを除去した後、0.2Ｎ ＨＣｌ含有イソプロパ
ノール溶液0.1ｍｌ添加して、ＭＴＴホルマザンを溶解
させ、波長５８８ｎｍに設定したマイクロプレートリー
ダーで吸光度を測定した。After culturing, 0.5% 3- (4,5-Dimethylthiazol-2-
yl) -2,5-Diphenyl Tetrazolium Bromide (MTT) solution was added at 0.01 ml / well, and the mixture was incubated at 37 ° C. in a 5% CO 2 incubator for 3 hours. After culturing, the plate was centrifuged, and after removing 0.1 ml of the supernatant, 0.1 ml of 0.2N HCl-containing isopropanol solution was added to dissolve MTT formazan and the absorbance was measured with a microplate reader set to a wavelength of 588 nm.
【００３３】[0033]
【表２】 数値は刺激指数を示す。 **、***は対照群に対して有意差があること（それぞれp<0.01、p<0.001）を示す 。[Table 2] Numerical values indicate stimulation index. **, *** indicates that there is a significant difference from the control group (p <0.01, p <0.001 respectively).
【００３４】結果を表２に示した。菌体標品投与群は、
Ｃｏｎ Ａ、ＬＰＳのいずれの刺激剤を用いて測定した
場合も、菌体標品投与開始から３５日後において、同週
齢の対照群と比較して高値を示し、菌体標品投与による
幼若化反応の促進作用が認められた。The results are shown in Table 2. The bacterial cell preparation administration group is
When measured using either Con A or LPS stimulants, 35 days after the start of administration of the bacterial cell preparation, the value was higher than that of the control group of the same age, and the immature body administration of the bacterial cell preparation was performed. The accelerating action of the oxidization reaction was recognized.
【００３５】実施例４ 体重１０〜１５ｋｇの健常犬（雑種、雄性又は雌性）に
本発明菌体標品を１００ｍｇ／ｋｇ体重の割合になるよ
うに３ヶ月間連日経口投与後の各犬の一般的健康状態
（発熱、活動性、食欲、嘔吐、咳、脱水症状、紫斑、血
尿、膀胱炎等の有無）を観察したところ、全実験期間を
通して、本発明菌体標品の投与による副作用及び死亡例
は見られなかった。Example 4 A healthy dog (mongrel, male or female) having a body weight of 10 to 15 kg was orally administered with the bacterial cell preparation of the present invention at a ratio of 100 mg / kg body weight for 3 months every day. Physical condition (existence of fever, activity, appetite, vomiting, cough, dehydration, purpura, hematuria, cystitis, etc.), side effects and death due to administration of the bacterial cell preparation of the present invention throughout the entire experimental period. No examples were seen.
【００３６】実施例５（製剤例） （１）実施例１で得た死菌体菌末１５０ｍｇを精製でん
ぷん末１５０ｍｇ及び乳糖７００ｍｇと混合して錠剤又
は顆粒剤にする。Example 5 (Formulation Example) (1) 150 mg of dead bacterial cell powder obtained in Example 1 is mixed with 150 mg of purified starch powder and 700 mg of lactose to give tablets or granules.
【００３７】（２）実施例１で得た死菌体菌末３００ｍ
ｇを大豆タンパク３００ｍｇ及び乳糖４００ｍｇと混合
して錠剤又は顆粒剤にする。(2) 300 m of dead bacterial cells obtained in Example 1
g is mixed with 300 mg of soy protein and 400 mg of lactose to give tablets or granules.
【００３８】[0038]
【発明の効果】本発明のエンテロコッカス属菌は、腸内
乳酸菌であるので毒性がなく、副作用もなく、末梢血白
血球数の増加作用や骨髄細胞、脾臓細胞、末梢白血球の
免疫機能活性化作用があるため、免疫力が低下して起こ
る各種疾患や、疾病による免疫力低下、更にそれに伴う
合併症に対して、予防又は治療効果が得られる。又、こ
のような特徴を有することによって、老人や乳幼児に対
しても使用可能であり、更に動物に応用することもでき
る。INDUSTRIAL APPLICABILITY Since the Enterococcus bacterium of the present invention is an intestinal lactic acid bacterium, it is non-toxic, has no side effects, and has an effect of increasing the number of peripheral blood leukocytes and an effect of activating the immune function of bone marrow cells, spleen cells, and peripheral leukocytes. Therefore, a preventive or therapeutic effect can be obtained against various diseases caused by a decrease in immunity, a decrease in immunity due to a disease, and complications associated therewith. Further, by having such characteristics, it can be used for elderly people and infants, and can also be applied to animals.
フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｃ１２Ｒ 1:01） Continuation of front page (51) Int.Cl. 6 Identification code Office reference number FI technical display area C12R 1:01)
Claims (3)
る微生物の菌体又はその処理物を含有する免疫賦活剤1. An immunostimulant containing, as an active ingredient, cells of a microorganism belonging to the genus Enterococcus or a treated product thereof.
テロコッカス・フェカリス又はエンテロコッカス・カセ
リフラブスである請求項１記載の免疫賦活剤2. The immunostimulant according to claim 1, wherein the microorganism belonging to the genus Enterococcus is Enterococcus faecalis or Enterococcus caseiflaves.
テロコッカス・フェカリスＮＦ−１０１１又はエンテロ
コッカス・カセリフラブスＮＦ−１００４である請求項
１記載の免疫賦活剤3. The immunostimulant according to claim 1, wherein the microorganism belonging to the genus Enterococcus is Enterococcus faecalis NF-1011 or Enterococcus caseriflaves NF-1004.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7205384A JPH0899887A (en) | 1994-08-01 | 1995-07-18 | Immunopotentiator |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6-199082 | 1994-08-01 | ||
| JP19908294 | 1994-08-01 | ||
| JP7205384A JPH0899887A (en) | 1994-08-01 | 1995-07-18 | Immunopotentiator |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0899887A true JPH0899887A (en) | 1996-04-16 |
Family
ID=26511331
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7205384A Pending JPH0899887A (en) | 1994-08-01 | 1995-07-18 | Immunopotentiator |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0899887A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH1029946A (en) * | 1996-07-15 | 1998-02-03 | Nichinichi Seiyaku Kk | Recovering agent of humoral immunity |
| JPH10139674A (en) * | 1996-11-11 | 1998-05-26 | Yakult Honsha Co Ltd | Production promoter of interleukin 12 |
| JPH10179142A (en) * | 1996-12-27 | 1998-07-07 | Sanei Toka Kk | New microorganism and substance derived from the new microorganism |
| JPH1192389A (en) * | 1997-09-17 | 1999-04-06 | Nichinichi Seiyaku Kk | Immunostimulator |
| JP2003113114A (en) * | 2001-10-09 | 2003-04-18 | Nichimo Co Ltd | Immunostimulator |
| US8334371B2 (en) | 2007-07-04 | 2012-12-18 | Kikkoman Corporation | Lactic acid bacteria-derived double-stranded RNA |
| WO2017082181A1 (en) * | 2015-11-10 | 2017-05-18 | キリン株式会社 | Method for enhancing immunostimulatory action of lactic acid bacteria |
| US9826770B2 (en) | 2005-03-10 | 2017-11-28 | 3M Innovative Properties Company | Antimicrobial compositions comprising esters of hydroxycarboxylic acids |
| US10471036B2 (en) | 2003-09-09 | 2019-11-12 | 3M Innovative Properties Company | Antimicrobial compositions and methods |
| US10918618B2 (en) | 2005-03-10 | 2021-02-16 | 3M Innovative Properties Company | Methods of reducing microbial contamination |
-
1995
- 1995-07-18 JP JP7205384A patent/JPH0899887A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH1029946A (en) * | 1996-07-15 | 1998-02-03 | Nichinichi Seiyaku Kk | Recovering agent of humoral immunity |
| JPH10139674A (en) * | 1996-11-11 | 1998-05-26 | Yakult Honsha Co Ltd | Production promoter of interleukin 12 |
| JPH10179142A (en) * | 1996-12-27 | 1998-07-07 | Sanei Toka Kk | New microorganism and substance derived from the new microorganism |
| JPH1192389A (en) * | 1997-09-17 | 1999-04-06 | Nichinichi Seiyaku Kk | Immunostimulator |
| JP2003113114A (en) * | 2001-10-09 | 2003-04-18 | Nichimo Co Ltd | Immunostimulator |
| US10471036B2 (en) | 2003-09-09 | 2019-11-12 | 3M Innovative Properties Company | Antimicrobial compositions and methods |
| US9826770B2 (en) | 2005-03-10 | 2017-11-28 | 3M Innovative Properties Company | Antimicrobial compositions comprising esters of hydroxycarboxylic acids |
| US10918618B2 (en) | 2005-03-10 | 2021-02-16 | 3M Innovative Properties Company | Methods of reducing microbial contamination |
| US8334371B2 (en) | 2007-07-04 | 2012-12-18 | Kikkoman Corporation | Lactic acid bacteria-derived double-stranded RNA |
| WO2017082181A1 (en) * | 2015-11-10 | 2017-05-18 | キリン株式会社 | Method for enhancing immunostimulatory action of lactic acid bacteria |
| US10774306B2 (en) | 2015-11-10 | 2020-09-15 | Kirin Holdings Kabushiki Kaisha | Method for increasing immunopotentiating activity of lactic acid bacteria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9962418B2 (en) | Lactobacillus plantarum and composition containing the same | |
| JP5718917B2 (en) | Novel Lactobacillus plantarum and composition containing the same | |
| JP5718916B2 (en) | Novel Lactobacillus plantarum and composition containing the same | |
| Kiadaliri et al. | Effects of feeding with red algae (Laurencia caspica) hydroalcoholic extract on antioxidant defense, immune responses, and immune gene expression of kidney in rainbow trout (Oncorhynchus mykiss) infected with Aeromonas hydrophila | |
| JP4667568B2 (en) | Immune enhancing composition | |
| CN101171019A (en) | Immune function modulating agents | |
| TW201023759A (en) | Method for using a bacillus subtilis strain to enhance animal health | |
| EA018687B1 (en) | Fraction of glycolipids of fermented extrats of biological food material and pharmaceutical composition comprising said fraction | |
| JP5337535B2 (en) | NK activity enhancer | |
| JP4565057B2 (en) | Novel lactic acid bacteria with high ability to induce immunoglobulin A | |
| JPH0899887A (en) | Immunopotentiator | |
| KR101075558B1 (en) | Novel Lactobacillus plantarum and compositions comprising the same | |
| LC et al. | Use of lactic acid bacteria from Nile tilapia Oreochromis niloticus as probiotics for sustainable production and improvement in fish welfare | |
| Herich et al. | The effect of Lactobacillus paracasei and Raftilose P95 upon the non-specific immune response of piglets | |
| JP2021050289A (en) | Skin indigenous bacteria lps and compound thereof | |
| JP2010077056A (en) | Infectious disease prophylactic | |
| JP2009209132A (en) | Antiallergic composition using new strain, lactobacillus crispatus kt-11, kt-23 and kt-25 | |
| JP5525180B2 (en) | Immunostimulating agent, immunostimulating composition containing the same, and immunostimulating method | |
| JP3272023B2 (en) | Leukopenia treatment | |
| KR20140048911A (en) | Novel lactobacillus plantarum and compositions comprising the same | |
| JP2015513911A (en) | Newly isolated Bacillus licheniformis and probiotics using it | |
| JPH07238024A (en) | Neutrophile function improver | |
| De et al. | Supplementation of a supranutritional dose of zinc sulfate and heat killed Bacillus firmus in early weanling pig diet: the effect on growth, neutrophil function and inflammatory cytokines | |
| CN117327606A (en) | Bifidobacterium longum strain and preparation method and application thereof | |
| CN118109353A (en) | Pediococcus acidilactici strain and application thereof |