JPS62164628A - Spf 10-11: antitumor component and its preparation - Google Patents

Abstract

NEW MATERIAL:An antitumor component: SPF 10-11 [elementary analyses: C 37.2-39.4%, H 5.6-6.5%, N 0.1-2.3%, O 56.9-46.3%, Ash 0.2-2.5%; decomposition point: browning at 165 deg.C and darkening at 240 deg.C with decomposition; specific rotatory power [alpha]<20>D=+95 deg.-+130 deg. (C=1.00); pH is 6.2-6.8 in 0.1% aqueous solution; color: white (freeze-dried); color reactions: positive to the Rowry reaction, negative to orcinol-hydrochloric acid reaction; molecular weight is more than 20X10<4> (according to the ultracentrifugation method); solubility: soluble in water, sparingly soluble or insoluble in methanol, etc.] USE:Antitumor agent. PREPARATION:A microorganism in Streptococcus, capable of producing an antitumor, SPF 10-11, is cultured and penicillin or its relating substance is added to the culture mixture in an appropriate period and cultivation is continued. The cell bodies are centrifuged off, the filtrate is concentrated and purified to give the objective antitumor agent, SPF 10-11.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antitumor component SPF10-11 and a method for producing the same.

Conventionally, preparations prepared by attenuating viable cells of hemolytic streptococcus (hereinafter referred to as hemolytic streptococci) have been used as anticancer agents.

In addition, there is a method of crushing the cells of hemolytic streptococci, extracting the active ingredients with water or a salt solution, adding an organic solvent, and collecting the antitumor components as precipitates (Special Publication of Publication No. 38-1647). , a method in which cells are lysed using cellulase or proteolytic enzymes, and the active fraction is fractionated as a water-soluble fraction (British Patent No. 1163865); the cells of hemolytic streptococcus are crushed, the condensate-insoluble material is collected, and the cells are lysed using nucleases or proteolytic enzymes. A method of treating with an enzyme (Japanese Unexamined Patent Publication No. 55-7014) is known.

As described above, it is widely known that Streptococcus bacteria themselves or their bacterial cell components have antitumor activity, but what was previously known was that Streptococcus bacteria themselves or their cell components have antitumor activity. It was just that. In order to isolate the active ingredient from the bacterial cells or inside the bacterial cells, the entire bacterial cell had to be fractionated by lysis or mechanical crushing.

Such treatments complicate purification and isolation of active ingredients is extremely difficult. In an example that was actually separated and measured as an active ingredient, a protein with a molecular weight of 200,000 (
Japanese Patent Publication No. 48-43841, Japanese Patent Publication No. 51-44617) and a glycoprotein with a molecular weight of 150,000 (Japanese Patent Publication No. 58-22
026) is known.

The present inventors have previously conducted extensive research on a method for eluating antitumor components into the culture solution of streptococcus, and found that by adding penicillin or related substances during culture, the antitumor component was released into the culture solution. Unexamined Japanese Patent Publication No. 1983-1989 discovered that elution occurs.
No. 30677), physiologically active substance 5PF-1 from culture solution
This led to the separation of the two.

(Unexamined Japanese Patent Publication No. 60-30689) The present inventors further conducted research for the purpose of separating more effective components from the culture filtrate of hemolytic streptococcus. cell L
We have isolated the antitumor component SPF10-11, which is characterized by inhibiting the growth of 1210) and being a positive color reaction fraction by the Anthrone sulfuric acid method.

The antitumor component SPF10-11 of the present invention is considered to be a substance whose main component is sugar and partially contains peptides based on elemental analysis, color reaction, etc.; A substance is something that is recognized as a different and new substance.

The present invention provides a method for producing the antitumor component SPF10-11, which is characterized by culturing antitumor component SPF10-11-producing bacteria belonging to the genus Streptococcus and collecting the antitumor component SPF10-11 from the culture. It is inclusive.

In the present invention, bacteria that produce the antitumor component SPF10-11 belonging to the genus Streso1hecoticus can be widely used6.
Next, the antitumor component 5PF10-11 producing bacteria will be described.

Culture media include meat extract medium, yeast extract medium, plain,
Although natural media such as bar 1 infusion medium ([1tl I medium) are often used, any medium can be used as long as it is suitable for the growth of Streptococcus bacteria.

The culture is carried out at a pH of 5.0 to 8.0, preferably 6.1 to 7.
．． 2, and the culture temperature is 30 to 40°C, preferably
The temperature is 35 to 37°C, and static culture or stirring culture can be performed anaerobically.

In the present invention, adding penicillin or its related substances at an appropriate time during culture plays an important role in collecting the antitumor component SPF10-11.

When should penicillin or related substances be added?

Culture at 35-37°C, after entering the logarithmic growth phase, 3-3
A period of 20 hours, especially 5 to 10 hours is preferred. then 1
By continuing the culture for ~20 hours, preferably 3 to 15 hours, a large amount of the antitumor component SPF10-11 can be accumulated during the culture.

Penicillin or its related substances may be any known related substances that have similar effects to penicillin, but penicillin G is commonly used.

The amount added is 100-7000 units/mQ of penicillin G.
, preferably about 300 to 5000 units/m of culture solution is sufficient.

A culture solution obtained by culturing Streptococcus bacteria with the addition of penicillin or its related substances is centrifuged to remove bacterial cells to obtain a filtrate.

Ammonium sulfate is added to the filtrate and a fraction having a saturation level of 50 to 90% is collected, or the filtrate is concentrated using an ultrafiltration membrane.

The obtained ammonium sulfate precipitate containing the antitumor component SPF10-11 can also be stored in a frozen state.

To extract the antitumor component SPF10-11 from this ammonium sulfate salt precipitate, the salt precipitate is dissolved in a buffer solution and used.

Further, a concentrated solution obtained using an ultrafiltration membrane can also be used as it is. This aqueous solution was adsorbed onto a DEAE cellulose column and eluted stepwise using a phosphate buffer to collect an active fraction that inhibits the growth of cultured cell L 1210. Adsorb onto a column, elute while linearly increasing the sodium chloride concentration in the phosphate buffer, separate the non-adsorbed portion and the low sodium chloride concentration portion, and dialyze this fractionated portion against the buffer. , adsorbed on gel filtration material TOYOPEARL HW-50F column, 0. Elute with phosphate buffer containing LM NaCQ, and check for color reaction and activity against cultured cells L 1210 by the Anthrone sulfuric acid method. Here, a fraction showing strong activity against cultured cells L 1210, that is, a fraction forming a peak in the color reaction by the Anthrone sulfuric acid method near the exclusion limit (hereinafter abbreviated as Vo) of Toyopearl 11W50F is obtained.

This fraction was concentrated and adsorbed onto a 1-Copal 1Ili160F column. Elute with phosphate buffer containing IM NaCQ.

SPF10-11 is Toyopa/L/IIIJ60F
Force = / 28 near vO in muchromatography
It can be obtained as a fraction that exhibits very weak absorption at 00m and exhibits strong coloration using the Anthrone sulfuric acid method.

Next, the antitumor component SPF10-1 obtained in Example 1
The freeze-dried specimen of No. 1 exhibits the following physical and chemical properties.

1. Element Origin Prayer C37, 2-39.4% H5, 6-6
．． 5% N 0.1-2.3% 8 56.9-49.3% Ash 0.2-2.5% 2. Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes.

3. Specific optical rotation [α) 3' = +95' to +130' (C
= 1.00) 4. Ultraviolet absorption spectrum of this substance 0.1
% aqueous solution is shown in FIG.

5. Infrared absorption spectrum shown in Figure 2.

Absorption is observed at 840 cm-' and is characteristic.

6. Distinction between basic, acidic and neutral The p++ of a 0.1% aqueous solution of this substance is 6.2 to 6.8.

7. Color of substance White (freeze-dried product) 8. Color reaction Lowry reaction 10 Buret reaction 10 Ninhydrin reaction 10 Anthrone sulfuric acid reaction 10 Molish reaction 10 Cystine sulfuric acid reaction 10 Orsine hydrochloric acid reaction - 99 molecules fiRayleigh interference optical system It is determined to be 20x104 or more by the ultracentrifugation method used.

10. Solubility in solvents: Soluble in water.

It is sparingly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone 5-ethyl ether, n-hexane, chloroform, and ethyl acetate.

Next, we will show a method for measuring antitumor activity against cultured cells L 1210 in vitro and a color reaction method using the anthrone sulfuric acid method. (IR%) was measured.

L 1210 cells in RPMI 164 with 10% FBS
0 medium (5 mg, containing /Q kanamycin).

Pour 0.5 nl of this culture solution into a Falcon 2058 tube, and adjust the number of cells to 1 x 10' cells/tu.
I made it to be. Next, 0.5 mQ of a predetermined amount of the preparation (anti-Il! □ 48 hours after adding the preparation, staining with trypan blue was performed, and IR (%) was calculated using the following formula.

Here, (A) indicates the number of viable cells in the control group.

A control was performed at the same time using 0.5 mQ of culture solution containing no standard.

Color reaction by Anthrone sulfuric acid method 2mf in 1mQ of sample dissolved in deionized water to the desired concentration
l of Anthrone reagent (0.20 gr of Anthrone
After 30 minutes of mixing, 21iQ was added to deionized water IIIQ in place of 1mQ of the standard product.
620 nm using the solution injected with Anthrone reagent as a control.
Measure the absorption. The quantitative value is obtained from a calibration formula obtained by measuring in the same manner using glucose.

Next, examples of the present invention will be shown.

Example 1 Streptococcus pyogenes
A seed culture solution obtained by mixing ATCC21060 in 1 BIII medium and 10 volumes was inoculated into the medium AIQ shown in Table 1, and preculture was performed anaerobically under the same conditions as the seed culture.

Table 1 Medium A Maltose 0.25% Meat Extract
1.0% Polypeptone 1.0% Yeast extract 0.25% Acid monobasic potassium phosphate 0.1% Magnesium sulfate 0.05% PH = 6.8 Pour medium A8Q into a 10Q jar fermenter.
After heat sterilization at 20°C for 110 minutes, cool to 37°C.
Inoculate 1Q of preculture solution and incubate at 37°C for 15.5 hours, p++
Cultivate anaerobically with stirring at 6,8,300 rpm. Then penicillin 0 1000111th position/m f
1 culture solution, and culture was continued for an additional 5 hours.

The resulting culture solution was centrifuged to remove bacterial cells.

The culture filtrate was concentrated to 1.5Ω using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Romicon, USA).

This concentrated solution was applied to a DEAE cellulose column (5
70cm), washed with a 10mM, pH 7.0 phosphate buffer, and then eluted stepwise using the above phosphate buffer containing 0.3 sodium achloride, followed by the Anthrone sulfuric acid method. The active fraction that showed a positive color reaction and inhibited the growth of cultured cells L1210 was collected.

This active fraction was adsorbed on a DEAE Sephadex A-25 column (7.5 x 50 cm), then washed with the above phosphate buffer, elution was performed by linearly increasing the sodium chloride concentration, and cultured cells L 1210 were eluted. Collect the active part. This elution curve is shown in FIG.

In FIG. 3, the dotted line portion is the non-adsorbed portion, and the solid line portion is the sodium chloride added portion.

A is the cultured cell L 1210 growth inhibiting activity fraction, MP-
2 (JP-A-6O-30689) is a growth-inhibiting non-active fraction, B is a growth-inhibiting non-active fraction of cultured cells L 1210 /4E, and B is a growth-inhibiting active fraction of MP-2.

The eluate of the active fraction of A is concentrated to 30 mQ using an ultrafiltration membrane with a molecular weight cutoff of 10,000 (Millibore). This concentrated solution was transferred to a Toyopearl Hυ50 F column (5, OX 10
0 cm) and then eluted with a solution containing 0.1M sodium chloride in the above phosphate buffer, and fractions with a positive color reaction by the Anthrone sulfuric acid method were collected. This elution curve is shown in FIG. In FIG. 4, the solid line shows the overall elution curve, and the dotted line shows the elution curve of the positive color reaction portion by the Anthrone sulfuric acid method. Here, C is a positive color reaction fraction obtained by the Anthrone sulfuric acid method, but the growth inhibiting activity against cultured cells L 1210 is examined to obtain the D fraction, which forms a peak near Vo, where the activity is strong, by the Anthrone sulfuric acid method.

Fraction D is lyophilized. This freeze-dried sample was dissolved in phosphate buffer, and Toyopearl 1 (160F column (2, 6
x 100cm) and then Toyopearl HW50
Elute with the same eluent as for F.

This elution curve is shown in FIG. In FIG. 5, the solid line shows the entire elution curve, and the solid line with black circles shows the elution curve of the positive color reaction area by the Anthrone sulfuric acid method.

Here, the F fraction that shows strong coloration in the color reaction by the Anthrone sulfuric acid method is designated as SPF10-11, and the G fraction is designated as SPF10-11.
The score was 10-12.

The F fraction was lyophilized. This freeze-dried specimen was an antitumor component SPF10-11, and 0.32 gr was obtained.

Example 2 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at 300 units/IIIQ culture solution, and the culture was continued for an additional 10 hours. Ammonium sulfate is added to this culture filtrate, and a fraction having a saturation level of 50 to 90% is collected. Ammonium sulfate was removed by dialysis and purified in the same manner as in Example 1 to obtain 15 gr of antitumor component SPF10-110.

Example 3 Streptococcus pyogenes AC21060 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a rate of 3000 units/lan culture solution, and the culture was continued for an additional 3 hours.

This culture filtrate was purified in the same manner as in Example 1 to obtain 37 gr of antitumor component SPF10-110.

Example 4 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using medium B shown in Table 2. In this case, penicillin G is at position 10001/mj2
The mixture was added to form a culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 20 gr of antitumor component SPF10-110.

Table 2 Medium B Maltose 0.1% Meat Extract
0.5% polypeptone l・0% yeast extract 0.25% sodium chloride 0.1% pH=7.2 Example 5 Same as Example 1 using Streptococcus pyogenes ATCC21060 in medium C shown in Table 3. to 35℃
It was cultured in In this case, penicillin G is 1000 units/
The mixture was added to form an mQ culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 21 gr of antitumor component SPF10-110.

Table 3 Medium C Maltose 0.1% Meat Extract
0.5% polypeptone 0.5% casamino acids 0.3% yeast extract
0.5% Acidic potassium monophosphate 0.1% Magnesium sulfate 0.05%-pH=6
．． 5 Example 6 Streptococcus pyogenes ATCC21060 was cultured in the same manner as in Example 1 using the medium shown in Table 4. In this case, penicillin G was added at a concentration of 1000 units/mQ culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain anti-j! 1ffj
A sexual component SPF10-110, 12gr was obtained.

Table 4 Medium D Maltose 0.25% Casamino Acid
0.3% Yeast extract 1.0% Acid monobasic potassium phosphate 0.1% &'! Magnesium acid 0.05%pH=6
．． 9 Example 7 Streptococcus sp. ATCC21597 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 1000 units/mQ culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SP.
F10-110, 27 gr was obtained.

Example 8 Streptococcus pyogenes ATCC21546 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G was added at a concentration of 1000 units/n+Q culture solution, and the culture was further continued for 5 hours.

This culture filtrate was purified in the same manner as in Example 1 to obtain anti-IIIT!
Ulcerative component SPF], 0-110.29 gr was obtained.

Example 9 Streptococcus pyogenes ATCC215/17
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is 1000 units/llI
n culture solution was added, and the culture was further continued for 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SPF10-110, 26gr.

Example 10 Streptococcus pyogenes ATCC21548 was cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, penicillin G is 1000 units/IIIQ
The mixture was added to form a culture solution, and the culture was continued for an additional 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain 23 gr of antitumor component SPF10-110.

Example 11 Streptococcus pyogenes ATCC21,060
were cultured in the same manner as in Example 1 using medium A shown in Table 1. In this case, cephalosporin C was 600 μg/m
12 culture solutions were added, and the culture was further continued for 5 hours. This culture filtrate was purified in the same manner as in Example 1 to obtain the antitumor component SPF10-11.0.28gr.

Experimental Example 1 An in vitro antitumor activity test of the antitumor component SPF10-11 obtained in Example 1 was conducted using the method for measuring antitumor activity described in the text, and the results are shown in Table 5.

Table 5 Results of antitumor activity of SPF10-11 SPF1
0-11 (mg/mQ) IR%1.0
53.4 0.5 21.3 0.25 8.5 Experimental Example 2 The in vivo antitumor activity test of the antitumor component SPF10-11 obtained in Example 1 showed that Crj; CD-
] (ICR strain, female, 7 weeks old).

Sarcoms-180 ascites cancer cells were used as tumor cells, suspended in physiological saline, and intraperitoneally injected into mice at 5 x 105ce1. ls/mouse was inoculated.

SP 101 times for 5 consecutive days starting 24 hours after cancer cell inoculation
F10-11 was administered intraperitoneally and the number of survivors was observed, and the results are shown in Table 6.

Table 6 Antitumor activity of SPF10-11 - Days of survival of mice Number 0 10
1.5 20 25 30 control
8/86/80/80/80/80/8SPFIO-1
1 (0.3mg/mouse) 8/8 8/8 6
/8 6/8 4/8 3/8 Experimental Example 3 The antitumor activity test of SPF10-11 obtained in Example 2 against solid tumors was carried out using Crj;CD-1 (ICR strain, female 5 weeks old) mice. It was used and implemented.

As solid tumor cells, Sarcoma 180 was used,
5×10 5 cells/mouse were inoculated subcutaneously into the left thigh of the mouse.

5PF10-11 was administered once a day starting 24 hours after cancer cell inoculation.
Mfl was administered intravaginally for 5 consecutive days, and cancer cells were inoculated 3 times.
On the second day, the solid tumor was excised and its weight was measured.

The average weight values are shown in Table 7.

Table 7 Effect of SPF10-11 on solid cancer Solid tumor weight Suppression rate control 7.96g SPF10-II 2mg/k (H1, 5181,
0% Experimental Example 4 Ehrlich with SPF 10-11 obtained in Example 1
The influence on cell morphology changes was determined as follows.

Ehrlich cells were grown in the peritoneal cavity of Crj;
(containing Q kanamycin), 1 x 10'cell
After adjusting to ls/mQ, add 0.5mM to Falcon 20
58 tube.

SPF10-11 was dissolved in the above medium and 0.45
Filtered with a membrane filter of pm, and its 0.5 m
After mixing fi with the above cells, culture is performed at 37° C. in the presence of 5% CO2 for 20 hours. Morphological changes were measured under a microscope by comparing cells with significant swelling, forming intracellular cavities, and counting cells leaking intracellular components.

The results are shown in Table 8.

Table 8 Effect of SPF10-11 on Ehrlich cells: mnt O-/L/12.2X10
'0.45X10' 3.7%SPF1
0-110,05mg/+n12 13.2XlO'
5.9 XIO' 44.7% Experimental Example 5 The TNF-inducing activity of SPF10-11 obtained in Example 2 was measured with reference to Katsuyuki Haranaka; , proceeded as follows.

Mice (Crj; CD-1, ICR strain, female, 7 weeks old) were intraperitoneally inoculated with BCG (Japan Besiege Manufacturing Association) 5B (primary sensitization), and 2 weeks later, SPF10-11
Two hours after administration of SPF10-11, blood was collected from the heart and serum was adjusted.

Test cells were grown in RPMI 1640 medium supplemented with 10% FBS (
L cultured for 3 to 4 days with 5mg/Q kanamycin
929 cells are used.

For L929 cells, a 0.5 mM suspension of I x 10' cells/mQ is injected into a Falcon 24 vessel.

Here, the above serum was mixed with the above RPMI 1640 medium for 20 minutes.
Pour 0.5mM of diluted solution and heat at 37°C, 5% CO.
Culture in the presence of 2 for 48 hours.

To measure the TNF-inducing activity, L929 cells were stained with trivan blue, and the number of viable cells was counted using a dye exclusion method.

The results are shown in Table 9.

Table 9 Experimental Example 61 of TNF-inducing activity of SPF10-11 Measurement of the deforming activity of SPF10-11 obtained in Example 1 on cultured cells J-774-1 (hereinafter abbreviated as J-774-1) , proceeded as follows.

J-774-1 is RPMI 1640 with 10% FBS added.
After culturing in a medium (containing 5 mg IQ kanamycin) for 3 to 4 days, the concentration was adjusted to 10 x 104 cells/mQ.

5PF10-11 was 5mg/mQ in physiological saline,
Filter with a 0.22 μm sterilization filter, and dispense 10 μQ each into 96 νwells by 2-fold stepwise dilution method.

100 μQ of the above J-774-1 suspension is added to each well, and cultured at 37° C. and 5% CO2 for 96 hours.

To measure the deformation activity, each well was examined under an inverted microscope.
The minimum concentration that induces a morphological change (from spherical to elongated and elongated) was determined. The results are shown in Table 10.

Table 10 Deformation-inducing minimum concentration of 5PF10-11 for J-774-1

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the antitumor component SPF10-11, and FIG. 2 similarly shows the infrared absorption spectrum. Figure 3 shows the DEAE of the active fraction in Example 1.
A diagram showing the elution curve of a Sephadex A-25 column,
Figure 4 shows the elution curve of this A fraction adsorbed onto a Toyopearl 150F column and a phosphate buffer containing 0.IM NaCQ, and Figure 5 shows the elution curve of this D fraction adsorbed onto a Toyopearl H116OF column. , 0, IM Shows the elution curve using a phosphate buffer containing NaCQ.Representative: Patent Attorney To: 1) Parent: Figure 3 Figure 4 Figure 5

Claims (4)

[Claims] (1) Antitumor component SPF with the following physical and chemical properties
10-11. 1. Elemental prayer C 37.2-39.4% H 5.6-6.5% N 0.1-2.3% O 56.9-49.3% Ash 0.2-2.5% 2. Decomposition point This substance turns brown at 165°C and turns black at 240°C and decomposes. 3. Specific optical rotation [α] ^2^0_D = +95° ~ +130
°(C=1.00)4, Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 0.1% aqueous solution of this substance is shown in FIG. 5. Infrared absorption spectrum shown in Figure 2. Absorption is observed at 840 cm^-^1, which is characteristic. 6. Distinction between basic, acidic, and neutral The pH of a 0.1% aqueous solution of this substance is 6.2 to 6.8. 7. Color of substance White (freeze-dried product) 8. Color reaction Lowry reaction + Buret reaction + Ninhydrin reaction + Anthrone sulfuric acid reaction + Molisch reaction + Cysteine sulfuric acid reaction + Orsine hydrochloric acid reaction - 9. Molecular weight Rayleigh interference optical system It is determined to be 20×10^4 or more by the ultracentrifugation method used. 10. Solubility in solvents It is soluble in water, but poorly soluble or insoluble in solvents such as methanol, ethanol, n-propanol, acetone, ethyl ether, n-hexane, chloroform, and ethyl acetate. (2) Antitumor component SP belonging to the genus Streptococcus
A method for producing an antitumor component SPF10-11, which comprises culturing F10-11 producing bacteria and collecting the antitumor component SPF10-11 from the culture. (3) Antitumor component SP belonging to the genus Streptococcus
Antitumor component SPF10-11 according to claim 2, characterized in that when culturing the F10-11 producing bacteria, penicillin or a related substance is added at an appropriate time during the culture. manufacturing method. (4) Antitumor component SP belonging to the genus Streptococcus
When culturing F10-11-producing bacteria and collecting the antitumor component SPF10-11 from the culture, cultured cells L12
The antitumor component SPF10-1 according to claim 2, wherein the antitumor component SPF10-1 inhibits the growth of SPF10 and/or collects a positive color reaction fraction by anthrone sulfuric acid method.
1 manufacturing method.