JPH026511B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a culture method for excreting various active ingredients of Streptococcus bacteria to the outside of the cells.

More specifically, the present invention involves adding penicillin or its related substances during the cultivation of Streptococcus bacteria, and culturing to expel various bacterial cell products from the bacterial cells, and adding them to the culture medium. This relates to a method of accumulating information.

Conventionally, preparations prepared by weakening viable cells of Streptococcus pyogenes have already been used as anticancer agents.

Another method is to crush the bacterial cells of Streptococcus pyogenes, extract the active ingredients with water or a salt solution, add an organic solvent, and collect the anti-seed ingredients as a precipitate (Japanese Patent Publication No. 1647-1971). , a method of lysing hemolytic streptococci with a lytic enzyme, lysozyme, cellulase, or proteolytic enzyme and fractionating the active fraction as a water-soluble fraction (British Patent No. 1163865)
etc. are 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 the Streptococcus bacteria themselves or their bacterial cell components have antitumor activity. It was nothing more than matter. If you try to isolate the active ingredient from the bacterial body or inside the bacterial body,
It was necessary to fractionate the entire bacterial body by lysing it or mechanically crushing it. Such treatment complicates purification and makes it extremely difficult to isolate the active ingredient. An example of a protein that has actually been isolated and measured as an active ingredient is known to have a molecular weight of 150,000. (Japanese Patent Publication No. 48-43841) The present inventors have discovered that if various bacterial cell products produced by Streptococcus bacteria can be excreted outside the bacterial cells during cultivation, their purification will be considerably easier. Furthermore, based on the assumption that more useful physiologically active substances could be discovered, as a result of intensive research into improving culture methods, we found that by adding penicillin during culture, we were able to remove bacterial cell products from the bacterial cells. They succeeded in ejecting it.

The present invention is characterized in that, when culturing bacteria belonging to the genus Streptococcus, penicillin or a related substance is added at an appropriate time during the culture, and various bacterial products are excreted outside the bacteria. This is a method for culturing Streptococcus bacteria.

In the method of the present invention, various bacterial cell products produced by Streptococcus bacteria are excreted outside the bacterial cells and become present in the culture solution. Isolation is quite easy since all you have to do is purify it.

Conventionally, it is known to culture Streptococcus bacteria and attenuate the resulting bacteria by treating them with penicillin. There is no known method of evacuation.

In the method of the present invention, a wide variety of bacteria of the genus Streptococcus can be used. For example, Streptococcus pyogenes ATCC21060 Streptococcus SP. ATCC21597 Streptococcus pyogenes ATCC21546 Streptococcus pyogenes ATCC21547 Streptococcus pyogenes ATCC21548
Natural media such as Streptococcus spp.) are often used, but general media containing carbon sources, nitrogen sources, etc. can also be used as long as they allow Streptococcus bacteria to grow effectively.

Cultivation conditions are selected under which bacteria of the genus Streptococcus grow, with a pH of 6.0 to 8.0, preferably 6.8 to 8.0.
7.2, it is common to perform static culture at 30 to 40°C, preferably 35 to 37°C, in an anaerobic manner, but other modified methods such as stirring culture can also be adopted.

In the present invention, by adding penicillin or a related substance at an appropriate time during culture, various useful substances produced by the bacterial cells are excreted outside the bacterial cells,
This allows it to accumulate during 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~ for penicillin G.
About 3000 units/ml, preferably 1000 units/ml of culture solution is sufficient.

The timing of addition of penicillin or related substances is 37℃.
A period of 3 to 15 hours, particularly 5 to 10 hours after the culture reaches logarithmic growth phase is preferred. By continuing the culture for 1 to 20 hours, preferably 5 to 15 hours, a large amount of bacterial cell products can be accumulated in the culture solution.

The present invention proposes a method for expelling bacterial cell products from the bacterial cells of Streptococcus bacteria.Any bacterial cell products may be discharged, and if necessary, the bacterial cell products may be discharged from the culture solution. Various useful substances are separated.

The bacterial cells are separated from the obtained culture solution by centrifugation, ammonium sulfate is added to the solution, and the active ingredient can be obtained as a precipitate.

The precipitate can be stored in a frozen state and, if necessary, purified by a purification method that combines an ion exchange resin, a gel filtration material, etc.

The products vary depending on the strain, and various substances can be separated, but by the method of the present invention,
Various bacterial cell products having antibacterial and antitumor properties are excreted and accumulated in the medium from Streptocaccus pyogenes ATCC21060. For example, SPF is a physiologically active substance with the following physical and chemical properties.
-1 was obtained.

1 Elemental analysis 2 Molecular weight When measured by gel filtration method, the molecular weight is about 500~
It is 7000.

3. Decomposition point This substance turns brown at 170℃ and turns black at 200℃ and decomposes.

4 Specific rotation [α] ²⁰ _D = +63.3 to 64.3° (C = 1.04) 5 Ultraviolet absorption spectrum The ultraviolet absorption spectrum of a 3.3% aqueous solution of this substance is shown in Figure 1, and the ultraviolet absorption spectrum of a 3.1% aqueous solution The absorption spectrum is shown in FIG. All have characteristic absorption at 257 nm, 265 nm, 280 nm, and 287 nm.

6 Infrared absorption spectrum It is shown in FIG.

7 Solubility in solvents Soluble in water, but insoluble in solvents such as methanol, ethanol, n-butanol, isobutanol, n-propanol, n-hexane, chloroform, acetone, methyl isobutyl ketone, and ethyl ether. .

8 Distinction between basic, acidic, and neutral The pH of a 0.85% aqueous solution of this substance is 6.5.

9 Color of matter It is a white powder.

10 Color reaction Ninhydrin reaction + Bieurets reaction + Mauritsch reaction - Decier reaction - Anthrone reaction - Cysteine sulfate reaction - 11 Stabilization This substance contains L-cysteine, dithiothreitol (DTT), glycerol, albumin, globulin (NH ₄ ) ₂ It is stabilized by adding SO ₄ , salt, etc.

Next, examples of the present invention will be shown.

In the Examples, the activity unit of the physiologically active substance SPF-1 was measured using the Udaka method (Journal of Antibiotics
Vol35No.10 1319-1325OCT.1982). For measurement, polymer-permeable E. coli mutant strain MP-2 was used.
(FERM−P5432) (Agric.Biol.Chem., 43, 371
(1979) to conduct a bioassay using antibacterial activity against MP-2 as an indicator.

That is, a medium (M3 medium) consisting of 1.75% Bacto Antibiotic Medium 3 (product of Difco) and _1.3 % agar was heated at 120°C for 15 minutes.
Dispense 20 ml into a shear dish and leave to prepare a plate medium.

Meanwhile, peptone 0.5%, meat extract 0.5%,
A medium consisting of 0.3% NaCl and 0.8% agar was heated at 120℃ for 15 minutes.
Leave to heat for a minute. Thereafter, the medium is kept in a constant temperature bath at 42°C, and when the temperature of the medium reaches 42°C, MP-2 bacteria that have been previously cultured at 37°C for 17 hours are added to the medium so that 10 ⁴ cells are present in 1 ml. Collect 2 ml with a pipette, add it to the surface of the _M3 medium prepared in advance, and spread it quickly and uniformly to solidify. The coating layer containing the physiologically active substance SPF-1 was diluted appropriately, and 0.05 ml of the solution was soaked into a paper disk (8 mm in diameter) (Toyo Shi). Place this paper disk on the production plate,
After culturing at 37°C for 17 hours, the size of the inhibition zone created by the physiologically active substance SPF-1 was determined. 1 unit (1u) concentration of SPF-1 that gives a diameter of inhibition circle of 10mm
It is defined as

Example 1 Meat extract 1% Polypeptone 1% NaCl 0.5% PH = 7.1 Streptococcus pyogenes ATCC21060 was inoculated into 100 ml of BHI medium and precultured by static culture at 37°C for 8 hours in A2L medium with the following composition. After inoculating 100ml of culture solution (100ml) and culturing anaerobically at 37℃ for 15 hours with stirring,
Add 1000 units/ml of penicillin G and further culture.
Continues for 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 120 units/ml of the physiologically active substance SPF-1.

Example 2 Yeast extract 3.0% BHI 1.0% Polypeptone 1.0% Maltose 0.3% KH ₂ PO ₄ 0.1% MgSO ₄ 7H ₂ O 0.02% PH = 7.2 Streptococcus pyogenes ATCC 21060 was added to B2L medium with the following composition as in Example 1. Inoculate 100ml of precultured culture solution into
After culturing anaerobically at 37°C for 15 hours with stirring, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 152 units/ml of the physiologically active substance SPF-1.

Example 3 Meat extract 1% Polypeptone 1% NaCl 0.5% Maltose 0.3% KH ₂ PO ₄ 0.1% MgSO ₄ 7H ₂ O 0.02% PH = 7.1 Streptococcus pyogenes ATCC 21060 was added in the same manner as in Example 1 to a medium C2l with the following composition. Inoculate 100ml of precultured culture solution into
After culturing anaerobically at 37°C for 15 hours with stirring, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 240 units/ml of the physiologically active substance SPF-1.

Example 4 100 ml of a culture medium with the following composition: meat extract 1.5% polypeptone 1.0% NaCl 0.5% casamino acids 0.5% yeast extract 0.2% PH = 7.1 Streptococcus pyogenes ATCC21060 was precultured in the same manner as in Example 1. After inoculating the cells stored at 20°C and anaerobically cultivating them at 37°C for 15 hours with stirring, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 108 units/ml of the physiologically active substance SPF-1.

Example 5 Meat extract 1.5% Polypeptone 1.0% NaCl 0.5% Casamino acids 0.25% Yeast extract 0.25% PH = 7.1 100 ml of the seed culture solution obtained by pre-cultivating Streptococcus pyogenes ATCC21060 in the same manner as in Example 1 was added to medium E2l with the following composition. inoculate,
After culturing anaerobically at 37°C for 15 hours with stirring, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 180 units/ml of the physiologically active substance SPF-1.

Example 6 Streptococcus pyogenes ATCC21060 was precultured in the same manner as in Example 1, and 100 ml of the culture solution stored at -20°C was inoculated into the E2l medium. After culturing anaerobically at 37°C for 15 hours with stirring, 1000 units of penicillin G/ ml and continue culturing for an additional 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 100 units/ml of the physiologically active substance SPF-1.

Example 7 Meat extract 3.0% Polypeptone 1.0% NaCl 0.5% Casamino acids 0.5% PH = 7.1 Streptococcus pyogenes ATCC21060 was precultured in the same manner as in Example 1, and 100 ml of the culture solution -20 was added to the following medium F2l.
After inoculating the culture at 37° C. and culturing anaerobically with stirring for 5.5 hours, 1000 units/ml of penicillin G was added, and the culture was continued for an additional 15 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 120 units/ml of the physiologically active substance SPF-1.

Example 8 BHI 3.7% Polypeptone 0.5% Maltose 0.3% KH ₂ PO ₄ 0.1% MgSO ₄ 7H ₂ O 0.02% PH = 7.1 Streptococcus pyogenes ATCC21060 was precultured in the same manner as in Example 1 in the following medium G2l. After inoculating 100ml of the solution and culturing anaerobically at 37℃ for 15 hours with stirring, add 1000 units/ml of penicillin G and continue culturing.
Continues for 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

The culture solution contained 300 units/ml of the physiologically active substance SPF-1.

Example 9 Streptococcus pyogenes ATCC21060 was added to the medium E3l.
Add and inoculate 300 ml of seed culture obtained by static culture at 37°C for 8 hours. After culturing anaerobically at 37°C for 15 hours with stirring, add 1000 units/ml of penicillin G and continue culturing.
Continues for 5 hours. The obtained culture solution was centrifuged to remove bacterial cells.

Culture solution 3 contains 56x the physiologically active substance SPF-1.
It contained 10 ⁴ units.

Ammonium sulfate was added to the culture solution, and a fraction with a saturation level of 50 to 80% was collected to obtain a precipitate. This precipitate contained 50×10 ⁴ units of the physiologically active substance SPF-1.

The entire amount of this precipitate was dissolved in 300 ml of stabilizer-containing buffer, and the solution was transferred to a DEAE-cellulose column (5x
70 cm), and the physiologically active substance SPF-1 was adsorbed. This is eluted stepwise using a 0.3M NaCl solution, and the active portion is separated. The activity obtained is 30×
It was 10 ⁴ units.

The active moiety is dialyzed against buffer, then
DEAE-Sephadex A-25 column (2.6×
50 cm), the active moiety is adsorbed, and the active moiety is fractionated by elution while linearly increasing the salt concentration in the phosphate buffer. The activity obtained is
It was 10.6×10 ⁴ units.

Furthermore, this eluate is concentrated and applied to a column (2 x 100 cm) of gel filtration material Toyopearl HW-50F to separate the active fraction. The activity obtained was 2.2×10 ⁴ units.

The eluate thus obtained was freeze-dried to obtain 350 mg of a white powder of the physiologically active substance SPF-1.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the physiologically active substance SPF-13.3% aqueous solution, and Figure 2 shows the same 3.1% ultraviolet absorption spectrum.
% aqueous solution, and FIG. 3 also shows the infrared absorption spectrum.

Claims (1)

[Claims] 1. When culturing bacteria belonging to the genus Streptococcus, it is necessary to
1. A method for culturing bacteria of the genus Streptococcus, which comprises adding 100 to 3000 units/ml of penicillin for culturing and expelling various bacterial cell products from the bacterial cells.