JP2004357703A - Method for culturing lactobacillus belonging to genus enterococcus and method for producing killed lactobacillus belonging to genus enterococcus - Google Patents
Method for culturing lactobacillus belonging to genus enterococcus and method for producing killed lactobacillus belonging to genus enterococcus Download PDFInfo
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本発明は、エンテロコッカス属に属する乳酸菌の培養方法、及びエンテロコッカス属に属する乳酸菌死菌体の製造方法に関する。 The present invention relates to a method for culturing lactic acid bacteria belonging to the genus Enterococcus and a method for producing dead lactic acid bacteria belonging to the genus Enterococcus.
近年における日本人一人当りの動物性脂肪の消費量増大と食生活の欧米化の進展に伴い、生活習慣病、成人病の一つである高脂血症及びこれによる動脈硬化症疾患者の数が増加している。 With the recent increase in consumption of animal fat per person in Japan and the progress of westernization of eating habits, the number of patients with lifestyle-related diseases, hyperlipidemia, which is one of adult diseases, and arteriosclerosis due to this disease Has increased.
このため、現在において多くの種類の高脂血症治療薬が実用に供されている。しかし、これらの疾患抑制に用いられている薬剤は、いずれも服用後の胃の不快感、嘔吐、肝機能異常、急性腎不全、脱力感等の副作用が報告されており、さらに、他の薬剤を投与中の者はその服用を控えなければならない場合もある。 For this reason, many types of therapeutic drugs for hyperlipidemia are currently in practical use. However, all of the drugs used to suppress these diseases have been reported to have side effects such as stomach discomfort, vomiting, liver dysfunction, acute renal failure, and weakness after taking these drugs. In some cases, those who are taking this drug should refrain from taking it.
本願の発明者は、新規坑動脈硬化剤の研究の結果、乳酸菌、特にエンテロコッカス属に属する各種微生物の生菌体及び死菌体が、血中コレステロール値及びトリグリセリド値を著しく低下せしめるものであることを発見し、且つ当該微生物の起源が元来人間の体内に存在している腸内細菌であることから、これを服用しても無毒であり、従来の坑動脈硬化剤や高指血症の治療薬のような副作用がないことを知見したのである(例えば、特許文献1)。 As a result of researches on novel anti-arteriosclerotic agents, the inventor of the present application shows that lactic acid bacteria, particularly living and dead cells of various microorganisms belonging to the genus Enterococcus, significantly reduce blood cholesterol levels and triglyceride levels. Since the origin of the microorganism is an intestinal bacterium originally present in the human body, it is non-toxic even if it is taken. It has been found that there is no side effect like a therapeutic drug (for example, Patent Document 1).
さらに、これらの微生物は種々の生理活性を有するが、その生菌体のみならず所定の条件下において処理された死菌体においても、その生理活性の例として、コレステロール値及びトリグリセリド値を著しく低下せしめる活性を有していることを知見の一例として示したのである。
しかし、このようにコレステロール値及びトリグリセリド値を著しく低下させる活性を有するエンテロコッカス属に属する乳酸菌(乳酸球菌)であっても、その培養方法によっては、当該乳酸菌の活性が低下し期待される効果が得られなくなることが判明した。また、エンテロコッカス属に属する乳酸菌死菌体もその生菌体と同様に、コレステロール値及びトリグリセリド値を著しく低下させることが知られているが、その生菌体から死菌体を得る処理の仕方によっては、期待される効果を喪失することが判明した。 However, even in the case of a lactic acid bacterium (Lactococcus) belonging to the genus Enterococcus having an activity of remarkably reducing the cholesterol level and the triglyceride level in this way, depending on the culturing method, the activity of the lactic acid bacterium is reduced and an expected effect is obtained. It turned out to be impossible. In addition, lactic acid bacteria killed cells belonging to the genus Enterococcus are known to remarkably reduce cholesterol and triglyceride levels as well as live cells, but depending on the method of obtaining dead cells from the live cells. Was found to lose the expected effect.
本発明は、血中コレステロール及びトリグリセライドを低下させる効果を有するエンテロコッカス属に属する乳酸菌の活性を喪失させることがない培養方法、及びエンテロコッカス属に属する乳酸菌死菌体の製造方法に関する。 The present invention relates to a culture method that does not lose the activity of lactic acid bacteria belonging to the genus Enterococcus having the effect of reducing blood cholesterol and triglycerides, and a method for producing dead lactic acid bacteria belonging to the genus Enterococcus.
このため、本願は、エンテロコッカス属に属する乳酸菌の培養方法であって、エンテロコッカス属に属する乳酸菌を、(i)pH:5.5乃至7.5、(ii)攪拌方法:1分間当たり5乃至40回、(iii)温度条件:摂氏30乃至40度、
(iv)培養時間:6乃至24時間、の培養条件下において好気的に培養し、培養液から生菌体を採取することにより成るコレステロール及びトリグリセリドを低下させる活性を有するエンテロコッカス属に属する乳酸菌の培養方法を提供するものである。
Therefore, the present application is a method for culturing lactic acid bacteria belonging to the genus Enterococcus, wherein the lactic acid bacteria belonging to the genus Enterococcus are (i) pH: 5.5 to 7.5, (ii) Stirring method: 5 to 40 per minute. Times (iii) temperature conditions: 30-40 degrees Celsius,
(Iv) Cultivation time: lactic acid bacteria belonging to the genus Enterococcus having an activity of lowering cholesterol and triglycerides obtained by aerobic culture under culture conditions of 6 to 24 hours and collecting viable cells from the culture solution A culture method is provided.
ここで、前記培養における培地組成は、(i)グルコース:1乃至4%と、(ii)酵母エキス:0.3乃至6%と、(iii)ペプトン:0.1乃至5%と、(iv)リン酸二カリウム(K2HPO4):1乃至5%と、を含むことを特徴とする。 Here, the culture medium composition in the culture is (i) glucose: 1 to 4%, (ii) yeast extract: 0.3 to 6%, (iii) peptone: 0.1 to 5%, (iv ) Dipotassium phosphate (K 2 HPO 4 ): 1 to 5%.
ところで、前記エンテロコッカス属に属する乳酸菌は、エンテロコッカス・フェカリス、エンテロコッカス・フェシウム、エントロコッカス・ボービス、エンテロコッカス・エビウム、エンテロコッカス・デュランス、エンテロコッカス・サリバリウス、エンテロコッカス・ミティス、及びエンテロコッカス・イクイヌスより成る群から1種又は2種以上の生菌体である。 By the way, the lactic acid bacterium belonging to the genus Enterococcus is composed of Enterococcus faecalis, Enterococcus faecium, Entrococcus bovis, Enterococcus ebium, Enterococcus dulans, Enterococcus salivarius, Enterococcus mittis, and Enterococcus equinos 1 Two or more types of viable cells.
本発明は、さらに、エンテロコッカス属に属する乳酸菌死菌体の製造方法であって、前記乳酸菌を、(1)培養条件(i)pH:5.5乃至7.5、(ii)攪拌方法:1分間当たり5乃至40回、(iii)温度条件:摂氏30乃至40度、(iv)培養時間:6乃至24時間、において好気的に培養し、(2)前記生菌体を含有する菌液を生理食塩水で洗浄し、(3)生理食塩水溶液に懸濁して得られる菌液を、(i)温度80乃至125°C、(ii)圧力5乃至25ポンドの環境下に、(iii)5乃至25分間置くことによる、コレステロール及びトリグリセリドを低下させる活性を有するエンテロコッカス属に属する乳酸菌死菌体の製造方法を提供するものである。 The present invention further relates to a method for producing dead lactic acid bacteria belonging to the genus Enterococcus, wherein the lactic acid bacteria are (1) cultured conditions (i) pH: 5.5 to 7.5, (ii) stirring method: 1 5 to 40 times per minute, (iii) temperature condition: 30 to 40 degrees Celsius, (iv) culture time: 6 to 24 hours, aerobically cultured, (2) bacterial solution containing the living cells (3) A bacterial solution obtained by suspending in a physiological saline solution is subjected to (i) a temperature of 80 to 125 ° C. and (ii) a pressure of 5 to 25 pounds in an environment (iii) The present invention provides a method for producing dead lactic acid bacteria belonging to the genus Enterococcus having an activity of lowering cholesterol and triglycerides by leaving for 5 to 25 minutes.
ここで、前記(1)の培養における培地組成は、(i)グルコース:1乃至4%、(ii)酵母エキス:0.3乃至6%、(iii)ペプトン:0.1乃至5%、及び(iv)リン酸二カリウム(K2HPO4):1乃至5%を含むことを特徴とする。 Here, the medium composition in the culture of (1) is (i) glucose: 1 to 4%, (ii) yeast extract: 0.3 to 6%, (iii) peptone: 0.1 to 5%, and (Iv) It is characterized by containing dipotassium phosphate (K 2 HPO 4 ): 1 to 5%.
以下、本発明に係るエンテロコッカス属に属する乳酸菌の培養方法及びエンテロコッカス属に属する乳酸菌死菌体の製造方法において使用される微生物の種類、当該微生物の培養方法、培養された微生物の死菌体の製造方法、及びその結果得られた乳酸菌及び乳酸菌死菌体の薬理作用について詳細に説明する。 Hereinafter, the type of microorganisms used in the method for culturing lactic acid bacteria belonging to the genus Enterococcus and the method for producing dead lactic acid bacteria belonging to the genus Enterococcus according to the present invention, the method for culturing the microorganisms, and the production of dead cells of the cultured microorganisms The method and the pharmacological action of the resulting lactic acid bacteria and lactic acid bacteria killed cells will be described in detail.
1.微生物
エンテロコッカス属に属する乳酸菌が使用され、この中で特に、エンテロコッカス・フェカリス、エンテロコッカス・フェシウム、エントロコッカス・ボービス、エンテロコッカス・エビウム、エンテロコッカス・デュランス、エンテロコッカス・サリバリウス、エンテロコッカス・ミティス、及びエンテロコッカス・イクイヌスより成る群から1種又は2種以上の生菌体が好適なものとして例示される。尚、上記エンテロコッカス属に属する乳酸菌は、例えば、ATCC(American Type Culture Collection)における寄託番号「ATCC700802」として入手可能である。
1. Lactic acid bacteria belonging to the genus Enterococcus are used, among which Enterococcus faecalis, Enterococcus faecium, Entrococcus bovis, Enterococcus ebium, Enterococcus dulans, Enterococcus salivaius, Enterococcus mittis, and Enterococcus eucus One type or two or more types of viable cells from the group are exemplified as preferable examples. The lactic acid bacteria belonging to the genus Enterococcus are available, for example, under the deposit number “ATCC7000080” in ATCC (American Type Culture Collection).
2.生菌体製造条件
前記生菌体の培養条件は、以下のとおりである。
(i)培養組成の水素イオン指数(pH):5.5乃至7.5の範囲とする。
(ii)攪拌方法:1分間に5回乃至40回ゆるやかに攪拌する。
(iii)培養時間:6乃至24時間の範囲とする。
(iv)培地組成:グルコース;1乃至4%、酵母エキス;0.3乃至6%、ペプトン;0.1乃至5%、リン酸ニカリウム(K2HPO4);1乃至5%、を含み、
(v)温度:30乃至45℃の温度環境下において、
(vi)好気的に培養するが特に酸素の付加は必要ない、ものとする。
2. Live Cell Production Conditions The culture conditions for the live cell bodies are as follows.
(I) Hydrogen ion index (pH) of the culture composition: The range is 5.5 to 7.5.
(Ii) Stirring method: Gently stir 5 to 40 times per minute.
(Iii) Incubation time: 6 to 24 hours.
(Iv) medium composition: glucose; 1 to 4%, yeast extract; 0.3 to 6%, peptone; 0.1 to 5%, dipotassium phosphate (K 2 HPO 4 ); 1 to 5%,
(V) Temperature: In a temperature environment of 30 to 45 ° C.
(Vi) Cultivate aerobically, but no additional oxygen is required.
3.死菌体製造条件
上記2の生菌体製造条件に基づいて得られた生菌体培養液を、さらに生理食塩水で1乃至2回程度洗浄した後、生理食塩水溶液に懸濁して得られる菌液を、以下の条件下において加熱/圧力処理を行う。
(i)温度:80乃至125°C
(ii)圧力:5乃至25ポンド
(iii)時間:5乃至25分
上記条件による加熱/高圧処理により前記微生物の活性ある死菌体を得ることができる。
3. Dead Cell Production Conditions Bacteria obtained by washing the live cell culture solution obtained based on the above-mentioned 2 live cell production conditions with physiological saline and then suspending in a physiological saline solution. The liquid is heated / pressure treated under the following conditions.
(I) Temperature: 80 to 125 ° C
(Ii) Pressure: 5 to 25 pounds (iii) Time: 5 to 25 minutes By the heating / high pressure treatment under the above-mentioned conditions, dead cells with active microorganisms can be obtained.
4.薬理作用
後記各実験例に示す通り、本発明による死菌体製造方法により、乳酸菌特にエンテロコッカス属の微生物が、血中コレステロール及びトリグリセリド値を極めて効果的に低下せしめるものであることを示した。
4). Pharmacological action As shown in the following experimental examples, the method for producing dead cells according to the present invention showed that lactic acid bacteria, particularly microorganisms of the genus Enterococcus, can significantly reduce blood cholesterol and triglyceride levels.
(1)実験例1
上記1に示したエンテロコッカス属に属する各種微生物の生菌体と加熱、加圧処理菌体をマウス(雄18週令、平均体重20g:各群10匹)に4週間生菌体1010個/日、死菌体1010個/日相当量を、経口的に連日投与し、血清中のコレステロールの低下率を測定した。当該実験の結果を表1に示す。
(1) Experimental example 1
Heating the viable cells of various microorganisms belonging to the genus Enterococcus shown above 1, the pressure treated cells mice (male 18 weeks old, average weight 20 g: each group 10 rats) for 4 weeks Probiotic 10 10 at / The daily dose of 10 10 dead cells / day was orally administered daily and the serum cholesterol reduction rate was measured. The results of this experiment are shown in Table 1.
(2)実験例2
上記実験例1と同様にしてエンテロコッカス属各種微生物の生菌体を加熱、加圧処理死菌体を通常ラット(雄18週令、平均体重250g:各群7匹)4週間、生菌体1010個/日、死菌体1010個/日相当量を経口的に連日、投与し血清中のコレステロールの低下率を測定した。当該実験の結果を表2に示す。
(2) Experimental example 2
In the same manner as in Experimental Example 1, live cells of various microorganisms belonging to the genus Enterococcus were heated, and pressure-treated dead cells were treated with normal rats (male 18 weeks old, average body weight 250 g: 7 mice in each group) for 4 weeks. An amount equivalent to 10 cells / day and 10 10 dead cells / day was orally administered daily, and the reduction rate of serum cholesterol was measured. The results of the experiment are shown in Table 2.
(3)実験例3
上記した生菌体調整例及び死菌体調整例に従って調整されたエンテロコッカス属各種、微生物の生菌体と加熱、加圧処理死菌体を通常マウス(18週令、平均体重20g:各群10匹)に4週間、生菌体1010個/日、死菌体1010個/日相当量を経口的に連日投与し、血清中のトリグリセリドの各低下率を測定した。当該実験の結果を表3に示す。
(3) Experimental example 3
Various Enterococcus species prepared according to the above-mentioned examples of live cell preparation and dead cell preparation, live cells of microorganisms, and heated and pressurized treated dead cells are usually used for mice (18 weeks old, average body weight 20 g: each group 10 The animals were orally administered daily for 10 weeks with 10 10 live cells / day and 10 10 dead cells / day, and the respective reduction rates of triglycerides in the serum were measured. The results of the experiment are shown in Table 3.
(4)実験例4
上記実験例3と同様にして調整したエンテロコッカス属各種微生物の生菌体と加熱、加圧処理死菌体を通常ラット(雄18週令、平均体重250g:各群7匹)に4週間、生菌体1010個/日、死菌体1010個/日相当量を経口的に連日投与し血清中のトリグリセリドの低下率を測定した。当該実験の結果を表4に示す。
(4) Experimental example 4
Live cells of various microorganisms of the genus Enterococcus prepared in the same manner as in Experimental Example 3 above, and heated and pressurized treated dead cells were cultured in normal rats (male 18 weeks old, average body weight 250 g: 7 mice in each group) for 4 weeks. An amount equivalent to 10 10 cells / day and 10 10 dead cells / day was orally administered daily, and the reduction rate of triglycerides in serum was measured. The results of this experiment are shown in Table 4.
(5)エンテロコッカス属に属する微生物の安全性について
急性毒性試験において経口で可能な限りの量のエンテロコッカス・フェカリス・K−23加熱、加圧処理菌体をラット(雄18週令、平均体重250g:1群10匹)に経口投与したが(5600mg/kg体重)何ら異常が認められず、経口投与においては事実上、無毒性であった。また腹腔内(マウス雄18週令、体重20g:各群10匹)投与でも802mg/kg体重と高値であった。
当該安全性に関する実験の結果を表5に示す。
(5) About the safety of microorganisms belonging to the genus Enterococcus Enterococcus faecalis K-23 heated and pressurized treated cells in an acute toxicity test as much as possible by rat (male 18 weeks old, average body weight 250 g: 10 mice per group) were orally administered (5600 mg / kg body weight), but no abnormality was observed, and the oral administration was virtually nontoxic. Moreover, even when intraperitoneally (mouse male 18 weeks old, body weight 20 g: 10 mice in each group) was administered, it was as high as 802 mg / kg body weight.
Table 5 shows the results of the safety experiment.
上記したとおり、本発明のエンテロコッカス属に属する乳酸菌の培養方法においては、エンテロコッカス属に属する乳酸菌を、(i)pH:5.5乃至7.5、(ii)攪拌方法:1分間当たり5乃至40回、(iii)温度条件:摂氏30乃至40度、(iv)培養時間:6乃至24時間、の培養条件下において好気的に培養し、培養液から生菌体を採取する。 As described above, in the method for culturing lactic acid bacteria belonging to the genus Enterococcus according to the present invention, lactic acid bacteria belonging to the genus Enterococcus are (i) pH: 5.5 to 7.5, (ii) Stirring method: 5 to 40 per minute. (Iii) Temperature conditions: 30 to 40 degrees Celsius, (iv) Culture time: 6 to 24 hours, aerobically cultured, and viable cells are collected from the culture solution.
また、上記のように培養された生菌体を培養し、前記生菌体を含有する菌液を生理食塩水で洗浄し、生理食塩水溶液に懸濁して得られる菌液を、(i)温度80乃至125°C、(ii)圧力5乃至25ポンドの環境下に、(iii)5乃至25分間置くことによって当該乳酸菌の死菌体を得る。 In addition, by culturing the viable cells cultured as described above, washing the bacterial solution containing the viable cells with physiological saline, and suspending it in a physiological saline solution, A dead cell of the lactic acid bacterium is obtained by placing (iii) for 5 to 25 minutes in an environment of 80 to 125 ° C. and (ii) a pressure of 5 to 25 pounds.
これによって、本発明は、血中コレステロール及びトリグリセライドを低下させる効果を有するエンテロコッカス属に属する乳酸菌の培養方法及びその死菌体の製造方法を確立したのである。 Thus, the present invention has established a method for culturing lactic acid bacteria belonging to the genus Enterococcus having the effect of reducing blood cholesterol and triglycerides and a method for producing dead cells thereof.
本発明は、エンテロコッカス属に属する乳酸菌の生理活性を維持する培養方法及びエンテロコッカス属に属する乳酸菌死菌体の製造方法を提供するものであり、産業上の利用可能性を有する。 The present invention provides a culture method for maintaining the physiological activity of lactic acid bacteria belonging to the genus Enterococcus and a method for producing dead lactic acid bacteria belonging to the genus Enterococcus, which have industrial applicability.
Claims (6)
エンテロコッカス属に属する乳酸菌を
(i)pH:5.5乃至7.5、
(ii)攪拌方法:1分間当たり5乃至40回、
(iii)温度条件:摂氏30乃至40度、
(iv)培養時間:6乃至24時間、の培養条件下において好気的に培養し、
培養液から生菌体を採取することにより成るエンテロコッカス属に属する乳酸菌の培養方法。 A method for culturing lactic acid bacteria belonging to the genus Enterococcus,
Lactic acid bacteria belonging to the genus Enterococcus (i) pH: 5.5 to 7.5,
(Ii) Stirring method: 5 to 40 times per minute,
(Iii) Temperature conditions: 30 to 40 degrees Celsius,
(Iv) culture time: aerobically cultured under culture conditions of 6 to 24 hours,
A method for culturing lactic acid bacteria belonging to the genus Enterococcus, which comprises collecting viable cells from a culture solution.
(i)グルコース:1乃至4%と、
(ii)酵母エキス:0.3乃至6%と、
(iii)ペプトン:0.1乃至5%と、
(iv)リン酸二カリウム(K2HPO4):1乃至5%と、
を含む請求項1に記載の培養方法。 The medium composition in the culture is
(I) glucose: 1 to 4%;
(Ii) Yeast extract: 0.3 to 6%,
(Iii) peptone: 0.1 to 5%;
(Iv) dipotassium phosphate (K 2 HPO 4 ): 1 to 5%;
The culture method according to claim 1, comprising:
前記乳酸菌を、
(1)培養条件(i)pH:5.5乃至7.5、(ii)攪拌方法:1分間当たり5乃至40回、(iii)温度条件:摂氏30乃至40度、(iv)培養時間:6乃至24時間、において好気的に培養し、
(2)前記生菌体を含有する菌液を生理食塩水で洗浄し、
(3)生理食塩水溶液に懸濁して得られる菌液を、(i)温度80乃至125°C、(ii)圧力5乃至25ポンドの環境下に、(iii)5乃至25分間置くことによるエンテロコッカス属に属する乳酸菌死菌体の製造方法。 A method for producing dead lactic acid bacteria belonging to the genus Enterococcus,
The lactic acid bacteria,
(1) Culture conditions (i) pH: 5.5 to 7.5, (ii) Stirring method: 5 to 40 times per minute, (iii) Temperature conditions: 30 to 40 degrees Celsius, (iv) Culture time: Aerobically cultured for 6 to 24 hours,
(2) The bacterial solution containing the living cells is washed with physiological saline,
(3) Enterococcus by placing a bacterial solution obtained by suspending in a physiological saline solution in an environment of (i) a temperature of 80 to 125 ° C. and (ii) a pressure of 5 to 25 pounds (iii) for 5 to 25 minutes A method for producing dead lactic acid bacteria belonging to the genus.
(i)グルコース:1乃至4%、
(ii)酵母エキス:0.3乃至6%、
(iii)ペプトン:0.1乃至5%、及び
(iv)リン酸二カリウム(K2HPO4):1乃至5%を含む、請求項4に記載の製造方法。 The medium composition in the culture of (1) above is:
(I) glucose: 1 to 4%,
(Ii) Yeast extract: 0.3 to 6%,
The production method according to claim 4, comprising (iii) peptone: 0.1 to 5%, and (iv) dipotassium phosphate (K 2 HPO 4 ): 1 to 5%.
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JPS58131917A (en) * | 1982-02-01 | 1983-08-06 | Advance Res & Dev Co Ltd | Agent for counteracting arteriosclerosis |
JPS5927833A (en) * | 1982-08-06 | 1984-02-14 | Advance Res & Dev Co Ltd | Novel microorganism belonging to genus streptococcus |
JPS5984825A (en) * | 1982-11-05 | 1984-05-16 | Yakult Honsha Co Ltd | Antilipemic agent |
JPS63280027A (en) * | 1987-05-11 | 1988-11-17 | Nichinichi Shokuhin Kk | Antihyperlipemic |
JPH08259450A (en) * | 1995-03-17 | 1996-10-08 | Nichinichi Seiyaku Kk | Enhancer for production of interferon |
JP2000256202A (en) * | 1999-03-04 | 2000-09-19 | Bhph Co Ltd | Novel organism depuration active form lactic acid bacterium preparation |
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JPS58131917A (en) * | 1982-02-01 | 1983-08-06 | Advance Res & Dev Co Ltd | Agent for counteracting arteriosclerosis |
JPS5927833A (en) * | 1982-08-06 | 1984-02-14 | Advance Res & Dev Co Ltd | Novel microorganism belonging to genus streptococcus |
JPS5984825A (en) * | 1982-11-05 | 1984-05-16 | Yakult Honsha Co Ltd | Antilipemic agent |
JPS63280027A (en) * | 1987-05-11 | 1988-11-17 | Nichinichi Shokuhin Kk | Antihyperlipemic |
JPH08259450A (en) * | 1995-03-17 | 1996-10-08 | Nichinichi Seiyaku Kk | Enhancer for production of interferon |
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