JPS5946206B2 - Method for producing a non-pathogenic aerobic Corynebacterium culture with immunological activity - Google Patents
Method for producing a non-pathogenic aerobic Corynebacterium culture with immunological activityInfo
- Publication number
- JPS5946206B2 JPS5946206B2 JP50122197A JP12219775A JPS5946206B2 JP S5946206 B2 JPS5946206 B2 JP S5946206B2 JP 50122197 A JP50122197 A JP 50122197A JP 12219775 A JP12219775 A JP 12219775A JP S5946206 B2 JPS5946206 B2 JP S5946206B2
- Authority
- JP
- Japan
- Prior art keywords
- culture
- corynebacterium
- strain
- producing
- immunological activity
- 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.)
- Expired
Links
- 241000186216 Corynebacterium Species 0.000 title claims description 19
- 230000001717 pathogenic effect Effects 0.000 title claims description 6
- 238000004519 manufacturing process Methods 0.000 title claims description 4
- 230000001900 immune effect Effects 0.000 title description 4
- 230000003266 anti-allergic effect Effects 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 claims description 5
- 230000001093 anti-cancer Effects 0.000 claims description 4
- 230000003308 immunostimulating effect Effects 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 210000004698 lymphocyte Anatomy 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 210000005265 lung cell Anatomy 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000186064 Trueperella pyogenes Species 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 241000186246 Corynebacterium renale Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000158504 Rhodococcus hoagii Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 206010048222 Xerosis Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明は新規な抗アレルギー作用、抗ガン作用および牌
細胞ならびにリンパ球の免疫的賦活化作用を有する非病
原性好気性コリネバクテリウム培養物の製法に関するも
のである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a non-pathogenic aerobic Corynebacterium culture having a novel anti-allergic effect, anti-cancer effect and immunostimulatory effect on tile cells and lymphocytes.
近来、嫌気性コリネバクテリウム菌の培養生産物に抗ガ
ン作用および感染防御作用のあることが種々報告されて
いるが、他面、好気性コリネバクテリウム培養生産物の
かかる作用についてはいまだ報告は見られない。Recently, various reports have been made that cultured products of anaerobic Corynebacterium have anticancer and infection-preventing effects, but on the other hand, there have been no reports on such effects of cultured products of aerobic Corynebacterium. can not see.
本発明者らは非病原性好気性コリネバクテリウムに属す
るコリネバクテリウム・イクイ
（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｅｑｕｉ ） Ｋ
ｏ −８５株）コリネバクテリウム・ピオゲネス（Ｃ、
ｐｙｏｇｅｎｅｓ ）ＴｏｊｏＨａｉ株、コリｐくクテ
リウム・セロシス（Ｃ。The present inventors investigated Corynebacterium equi K, which belongs to the non-pathogenic aerobic Corynebacterium group.
o -85 strain) Corynebacterium pyogenes (C,
pyogenes) TojoHai strain, C. pyogenes (C.
ｘｅｒｏｓｉｓ ） ５３−に−１株、コリネバクテリ
ウム・レナーレ（Ｃ，ｒｅｎａｌｅ）Ｕｔｓｕｎｏｍｉ
ｙａ Ｕｓｈｉ株（これらの菌株は東京大学医科学研究
所に標準株として保存されているものであり、当該技術
分野における通常の知識を有する者が容易に入手するこ
とができるものである。xerosis) 53-ni-1 strain, Corynebacterium renale (C, renale) Utsunomi
ya Ushi strain (These strains are stored as standard strains at the Institute of Medical Science, the University of Tokyo, and can be easily obtained by those with ordinary knowledge in the technical field.
）などについて種々研究した結果、これらの好気性菌株
が免疫学的活性を有する培養物を産生じ、しかもこれら
の培養物は前述した嫌気性コリネバクテリウム培養物の
場合に比べて収率も士数倍と格段に高く得られ、免疫学
的活性度において優れているのみならず、嫌気性コリネ
バクテリウム培養物では知られていなかった抗アレルギ
ー作用を有するなど新規かつ特異なものであることを以
下に詳細に述べるごとく確認し得て本発明に到達した。), etc., it has been found that these aerobic strains produce immunologically active cultures, and these cultures also have lower yields than the aforementioned anaerobic Corynebacterium cultures. It has been shown that it is novel and unique in that it not only has an excellent immunological activity, but also has an anti-allergic effect that was not known in anaerobic Corynebacterium cultures. The present invention was achieved through confirmation as described in detail below.
本発明培養物のエールリッヒガン細胞に対する免疫治療
効果
ｄｄ系マウスにイクイ・Ｋｏ−８５株の培養物０、１１
ｎ９（乾重量）をエールリッヒガン移植前７臼目と移植
後７日目に腹腔内注射し、延命効果を観察した所、生理
食塩水にて同様に処理した対照群に比べ下表にみられる
ごとく著しい延命効果が見出された。Immunotherapeutic effect of culture of the present invention on Ehrlich cancer cells Cultures of Equi Ko-85 strain 0 and 11 in DD mice
N9 (dry weight) was injected intraperitoneally at the 7th molar before Ehrlich cancer implantation and 7 days after implantation, and the survival effect was observed, as seen in the table below compared to the control group treated in the same manner with physiological saline. A remarkable effect on prolonging life was found.
上表において対照群はエールリッヒ移植後３２日月に生
存率０％であるのに比ベイクイ・Ｋｏ−８５株処理群は
６０日目方生存率４０％を示した。In the above table, the control group had a survival rate of 0% on the 32nd day after Ehrlich transplantation, while the group treated with the Hibikui Ko-85 strain had a survival rate of 40% on the 60th day.
更に嫌気性コリネバクテリウム・Ｙ−１株の培養物で同
様の処理を施した群よりもその延命効果が優れているこ
とが見出される。Furthermore, it was found that the life prolonging effect was superior to that of a group treated with a culture of anaerobic Corynebacterium strain Y-1.
すなわち、イクイ・Ｋｏ−８５株による培養物は抗ガン
作用を有し、その作用は嫌気性コリネバクテリウム菌よ
り優れていることが確認された。That is, it was confirmed that the culture using the Ikui Ko-85 strain had an anticancer effect, and that this effect was superior to that of anaerobic Corynebacterium.
肺細胞の免疫能賦活化試験
ヤーン（Ｊｅｒｎｅ）のサイエンス（Ｓｃｉｅｎｃｅ
。Immune activation test for lung cells Jerne's Science
.
ＵＳＡ）、１４０巻、４０５．１９６３年の方法に準じ
てヒツジ赤血球で免疫したマウスの肺細胞浮遊液をその
赤血球と共に軟寒天に加え産生された溶血斑（プラーク
）により賦活化細胞数を算定した。USA), Vol. 140, 405. According to the method of 1963, a suspension of lung cells from a mouse immunized with sheep red blood cells was added to soft agar together with the red blood cells, and the number of activated cells was calculated from the hemolytic plaques produced. .
ｄｄ系マウスをヒツジ赤血球で感作する前０日、３日、
７日、１４日１にイクイ・Ｋｏ−８５株の培養物をマウ
ス当り１０μＩ静脈内投与し、肺細胞中のプラーク産生
細胞を算定すると、７日前処理群においてその産生細胞
数は生理食塩水による同様処理対照群に比べ次表２のご
とく最高５，３倍になった。Days 0 and 3 before sensitizing DD mice with sheep red blood cells.
On the 7th and 14th days, 10 μl of Ikui Ko-85 strain culture was intravenously administered per mouse, and the number of plaque-producing cells in the lung cells was calculated. As shown in Table 2 below, it increased by up to 5.3 times compared to the control group treated with the same treatment.
上表により当培養物によって牌細牌が顕著に賦活化され
たことが示される。The above table shows that this culture significantly activated the small tiles.
更に従来量もよく報告されている嫌気性コリネバクテリ
ウム・パルバムによる同様処理群よりもその賦活化能力
が高いことが判明する（パルハム処理群は最高４．８９
倍）。Furthermore, it was found that the activation ability was higher than that of the group treated with anaerobic Corynebacterium parvum, which has been well reported in conventional amounts (the group treated with Parham had a maximum of 4.89
times).
すなわち、本発明方法により得られた培養物が肺細胞の
免疫的賦活化作用を有し、その活性が嫌気性コリネバク
テリウムより高いことが前例と同様に確認される。That is, it is confirmed, as in the previous example, that the culture obtained by the method of the present invention has an immunostimulatory effect on lung cells, and its activity is higher than that of anaerobic Corynebacterium.
抗アレルギー作用
受身皮膚アナフィラキシ−反応（以下ＰＣＡ反応で示す
）が本発明のイクイＫｏ−８５株の培養物投与により阻
止されるか否かが調べられた。Anti-allergic effect It was investigated whether passive cutaneous anaphylactic reaction (hereinafter referred to as PCA reaction) could be inhibited by administration of the culture of Ikui Ko-85 strain of the present invention.
すなわち、モルモットにウサギ抗生血清アルブミン（Ｂ
ＳＡ）血清を背部に皮下注射し、２時間後にＢＳＡとエ
バルブルーで惹起（静脈内投与）することにより典型的
なＰＣＡ反応が形成されるが、この際モルモットをあら
かじめイクイ−Ｋ。That is, rabbit antibiotic serum albumin (B
A typical PCA reaction is formed by injecting serum (SA) subcutaneously into the back and 2 hours later eliciting it with BSA and Eval Blue (intravenous administration); at this time, the guinea pigs are pre-treated with Equi-K.
−８５株培養物５０μｇで腹腔内感作をし、惹起前２時
間に背部に本培養物１０μｓを皮下に注射した群と本培
養物の代りに生理食塩水を注射した群に分けてＢＳＡと
色素を静脈内投与した所、本発明培養物皮下注射群にお
いてＰＣＡ反応が阻止された。-85 strain culture was intraperitoneally sensitized, and 2 hours before challenge, 10 μs of the main culture was subcutaneously injected into the back of the group, and a group was injected with physiological saline instead of the main culture. When the dye was administered intravenously, the PCA reaction was inhibited in the subcutaneous injection group of the culture of the present invention.
上表により、腹腔自前感作に加えて惹起前に背部ＰＣＡ
反応域近傍に本発明培養物を皮下投与することによりＰ
ＣＡ反応はほぼ完全に阻止されることが示され、更に惹
起時に本培養物を静脈内に投与することによっても同程
度の阻止が示される。According to the table above, in addition to self-sensitization in the abdominal cavity, dorsal PCA should be
By subcutaneously administering the culture of the present invention near the reaction zone, P
It was shown that the CA reaction was almost completely inhibited, and the same degree of inhibition was also demonstrated by intravenous administration of the main culture at the time of challenge.
一方、本発明者らはコリネバクテリウム・イクイ・Ｋｏ
−８５株と結核菌との免疫学的共通性を見出している。On the other hand, the present inventors have discovered that Corynebacterium quii Ko
We have discovered immunological commonalities between the -85 strain and Mycobacterium tuberculosis.
すなわち、本発明培養物は広く結核菌に対する免疫を保
持している成人において抗アレルギー作用を示し得るこ
とが見出された。That is, it has been found that the culture of the present invention can exhibit antiallergic effects in a wide range of adults who have immunity to Mycobacterium tuberculosis.
これらのことから本発明培養物が特異な抗アレルギー作
用物質として実際に使用される可能性を有するものとし
て見出した。Based on these findings, the culture of the present invention was found to have the potential to be actually used as a unique anti-allergic substance.
本発明の免疫学的活性を有する非病原性好気性コリネバ
クテリウム培養物はコリネバクテリウム菌の好気性培養
に通常用いられる培養方法により得られるが、培養基も
特別なものを用いなくとも充分に目的を達成することが
でき、例えば次の一例のごときものが好適に使用される
。The non-pathogenic aerobic Corynebacterium culture having immunological activity of the present invention can be obtained by a culture method commonly used for aerobic culture of Corynebacterium, but it can be obtained without using any special culture medium. The purpose can be achieved, and for example, the following example is preferably used.
肉エキス １０ｇ ペプトン １０ｇ グルコース １（Ｂ９ 食 塩 １ｇ 再蒸溜水 １０００ｒＩＬｌ・ ＮａＯＨ液でｐＨ７，４に調整する。Meat extract 10g Peptone 10g Glucose 1 (B9 1g table salt Redistilled water 1000rILl・ Adjust the pH to 7.4 with NaOH solution.
本発明の免疫学的活性ある培養物を産生ずる菌株は非病
原性好気性コリネバクテリウム属中に分※肇布している
が、生産能の高い菌株としてはコリネバクテリウム・イ
クイ・Ｋｏ−８５株、コリネバクテリウム・ピオゲネス
・Ｔｏｊｏ Ｈａｉ株、コリネバクテリウム・セロシス
・５３−に−１株およびコリネバクテリウム・レナーレ
・Ｕ ｉ ｓｕｎｏｍｉ ｙａ −Ｕｓｈｉ株などがあ
げられ、これらのうちでもイクイ・Ｋｏ−８５株が最も
生育が優れており、従って培養物の生産能も多く好適に
用いられる。Bacterial strains that produce the immunologically active culture of the present invention are distributed in the non-pathogenic aerobic Corynebacterium genus, and strains with high productivity include Corynebacterium quii and Ko- 85 strain, Corynebacterium pyogenes Tojo Hai strain, Corynebacterium serosis strain 53-1, and Corynebacterium renale strain Ui sunomiya-Ushi, among these, Ikui. The Ko-85 strain has the best growth and therefore has a large production capacity for culture, and is therefore preferably used.
これらの菌株を培養基１０ｒＩＬｌに１白金耳量接種し
、３７℃、４８時間静置培培養液長６４０ｍμによる比
濁を測定した生育試験の結果が下表４−１に示される。The results of a growth test in which one platinum loopful of these strains was inoculated into 10 rIL of culture medium and the turbidity was measured using a static culture medium length of 640 mμ at 37° C. for 48 hours are shown in Table 4-1 below.
以下に本発明の実施の態様の例を示す。Examples of embodiments of the present invention are shown below.
実施例
Ｃ，イクイ・Ｋｏ−８５株を前記組成の培養基を用い、
３７℃、５日間培養後流速５０ｒｒＬｌ１分で１０．０
００回転／分で連続遠心し、その沈査を生理食塩水で３
回遠心洗浄し、０．５％ホルマリン含有生理食塩水に再
浮遊し、４℃で３日間静置した後生理食塩水にて遠心洗
条を３回繰返してホルマリンを除去した。Example C, Ikui Ko-85 strain using a culture medium with the above composition,
After culturing at 37°C for 5 days, the flow rate was 50rrLl for 1 minute, and the flow rate was 10.0.
Continuous centrifugation at 00 revolutions/min, and the precipitate was diluted with physiological saline for 3
After centrifugal washing, resuspension in physiological saline containing 0.5% formalin, and standing at 4° C. for 3 days, centrifugal washing was repeated three times in physiological saline to remove formalin.
こうして得られた沈査を培養物とし、凍結乾燥により粉
末状とした。The thus obtained precipitate was used as a culture, and was lyophilized to form a powder.
培養物の収量は使用培養基１０Ａ’につき１７．０ｇ（
湿量）であった。The yield of the culture was 17.0 g per 10 A' of culture medium used (
moisture).
なお、本実施例における培養菌の収率と培養時間の関係
は比濁試験の結果、次表４−２のごとくであり、培養後
約１２０時間前後において収率が最高となることが判明
した。The relationship between the yield of the cultured bacteria and the culture time in this example is as shown in Table 4-2 below as a result of the nephelometric test, and it was found that the yield was highest around 120 hours after culture. .
上記の実施例で示される本発明培養物の収量は嫌気性コ
リネバクテリウムを嫌気性菌用培地で嫌気性培養装置を
用いて得られた培養物の１７倍の収率であった。The yield of the culture of the present invention shown in the above examples was 17 times that of a culture of anaerobic Corynebacterium obtained using an anaerobic culture medium in an anaerobic culture medium.
Claims (1)
培養することを特徴とする抗アレルギー性、抗ガン作用
および牌細胞ならびにリンパ球の免疫的賦活化作用を有
する培養物を製造する方法。1. A method for producing a culture having antiallergic and anticancer effects and immunostimulatory effects on tile cells and lymphocytes, which comprises culturing a strain belonging to non-pathogenic aerobic Corynebacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP50122197A JPS5946206B2 (en) | 1975-10-08 | 1975-10-08 | Method for producing a non-pathogenic aerobic Corynebacterium culture with immunological activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP50122197A JPS5946206B2 (en) | 1975-10-08 | 1975-10-08 | Method for producing a non-pathogenic aerobic Corynebacterium culture with immunological activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5247909A JPS5247909A (en) | 1977-04-16 |
| JPS5946206B2 true JPS5946206B2 (en) | 1984-11-10 |
Family
ID=14829957
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP50122197A Expired JPS5946206B2 (en) | 1975-10-08 | 1975-10-08 | Method for producing a non-pathogenic aerobic Corynebacterium culture with immunological activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5946206B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58874B2 (en) * | 1979-08-30 | 1983-01-08 | 株式会社 目黒研究所 | Glycoprotein WENAC and its production method |
| DE3011461C2 (en) * | 1980-03-25 | 1986-10-30 | Dr. Madaus & Co, 5000 Köln | Use of propionibacteria |
| JPS57144225A (en) * | 1981-03-04 | 1982-09-06 | Tadao Kokubu | Antigenetic preparation for treating nonspecific allergic diseases and its preparation |
| JPS57163835U (en) * | 1981-04-07 | 1982-10-15 | ||
| JP2000210050A (en) * | 1998-11-20 | 2000-08-02 | Asama Kasei Kk | Decomposition product having immunoregulation activity, its production and food using the same |
-
1975
- 1975-10-08 JP JP50122197A patent/JPS5946206B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5247909A (en) | 1977-04-16 |
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