JPH0630567B2 - Antibiotic production medium - Google Patents

Description

TECHNICAL FIELD The present invention relates to a medium for culturing a microorganism having an antibiotic-producing ability to produce and accumulate an antibiotic in the culture (hereinafter referred to as an antibiotic-producing medium). .

2. Description of the Related Art Antibiotics are produced by microorganisms and inhibit the growth of microorganisms and other cells, or have specific pharmacological and enzyme inhibitory effects (Nobuo Tanaka, Shoshiro Nakamura, Antibiotics Comprehension No. 2 Edition, page 1, 1977, published by The University of Tokyo), and is now widely used in medicine, agricultural chemicals, animal feed additives, anthelmintic agents and the like.

Examples of known media that have been modified to increase antibiotic production are as follows.

(1) Medium containing an effective precursor for antibiotic biosynthesis [J. Am. Chem. Soc., Vol. 68, p. 1669 (1946)] (2 ) A medium containing a buffer solution effective for keeping the pH of the culture solution in the optimum range [Domain (Demain) Archive of Microbiology, Vol. 115, No. 169]
Page (1977)] (3) Medium containing an effective surfactant to suppress foaming, or to favor the outflow of antibiotics from inside the cells of antibiotic-producing microorganisms to the outside (Yoshio Ueno, Satoshi Omura) (Edited by Microbial Chemical Chemistry, p. 176, 1979, Nankodo) (4) Select effective nutrients (carbon source, nitrogen source, phosphate root, etc.) from various substances to optimize the concentration of each. Medium to be contained (same as above) A medium suitable for the production of each antibiotic is determined by combining such various compositions. However, this usually requires enormous labor and a long time, and there are few cases where a medium that is advantageous for the production of one antibiotic is disadvantageous to the production of another antibiotic. . Therefore, it has been desired to provide a culture medium for producing antibiotics, which has applicability to a wide range of antibiotic production and high productivity.

The present inventor has previously found that the production of a large number of antibiotics can be markedly increased by producing antibiotics by a conventional method using a medium containing zeolite (JP-A-57-206).
No. 380).

Problems to be Solved by the Invention The object of the present invention is to easily and easily control the amount of antibiotics produced and accumulated as a medium for culturing a microorganism having an antibiotic production ability by a conventional method to produce and accumulate antibiotics. It is to provide a medium that can be increased.

Means for Solving the Problems The medium of the present invention is characterized by containing a silica-alumina gel in addition to the composition contained in the conventionally used medium. When a microorganism capable of producing an antibiotic is cultivated by a conventional method using the medium according to the present invention, its production amount can generally be increased several times, for example, two times or more.

The present invention will be described in detail below.

The microorganism used for producing an antibiotic using the medium of the present invention may be any microorganism having the ability to produce an antibiotic. For example, it may be a wild strain, or a strain having a specific auxotrophy or resistance or sensitivity to a specific substance. Alternatively, it may or may not have the property of decomposing or inactivating a specific substance.
Therefore, it is possible to use a wide variety of known microorganisms having a capability of producing antibiotics, including molds, yeasts, bacteria, and actinomycetes. Also, the microorganisms are in the natural world, such as soil,
It may be present in river water, seawater, air, or the like, or may be previously grown in a medium other than the medium of the present invention.

The silica-alumina gel used in the present invention is a kind of ore that naturally exists in the form of clay, and its scientific name is Allophene (Allophene).
ane) is called.

Mineralogically, such clays result from natural weathering from ores of crystalline silica-alumina metal salts. Therefore, kaolinite and zeolite (Zeolit)
It is clearly distinguished from e) etc. because it is amorphous.

Alofuen is a major component of colloidal clay called Kanuma soil, millet soil, and miso soil in Japan, but it is also found in Europe and America. Alofuen is a hydrous silica-alumina gel produced by leaching of metal salts from volcanic glassy rocks due to natural weathering for a long period of time, and many very small pores are left due to the leaching of these leached molecules. In many cases, the fine pores have excellent adsorption properties and are used as an adsorbent. Also, although the composition is hydrous silica alumina,
Its molecular structure is amorphous, such as mullite (3Al ₂ O ₃
Unlike crystalline minerals such as 2SiO ₂ ) kaolinite [Al ₄ (OH) ₈ Si ₄ O ₁₀ ), they are chemically very active such that they easily react with hydrochloric acid to form gelatin. The silica-alumina gel used in the present invention is a natural allophene,
Alternatively, it can be purified before use.

The natural silica-alumina gel has a slightly different color tone, chemical composition, and eventually physicochemical properties, probably because the type and amount of contaminants differ depending on the place of origin and the state of existence. As an example,
Table 1 shows the properties of allophene, which is one of the silica-alumina gels used in the examples.

It is known that allophane has the ability to adsorb inorganic acids and water in the air, and it is conventionally used as a dehumidifying agent, but by adding it to an antibiotic production medium, the production amount of antibiotics can be increased. It was unknown that it could be increased. The silica-alumina gel used in the medium of the present invention may be used alone, or may be used in combination with the zeolite etc. taught by the present inventors.

As the silica-alumina gel used in the present invention, a commercially available powder product may be used as it is, but if it is used after being heat treated at 110 ° -500 ° C for several hours, it is likely to give good results in antibiotic production.

The concentration of the silica-alumina gel to be contained in the medium should be determined in consideration of the particle size, microorganisms used and culture conditions, but a concentration of 0.1-5% is usually preferable.

Various conditions such as optimum temperature, time and pH for the production of a certain antibiotic may be in accordance with known culture of the antibiotic production medium.

The medium of the present invention, in addition to the silica-alumina gel, appropriately contains a carbon source, a nitrogen source, an inorganic substance, and other necessary nutrients that can be utilized by the microorganism used, following an example of a conventionally used antibiotic production medium. The medium of the present invention may be either a natural medium or a synthetic medium, and its form may be a liquid medium or a solid medium. When the medium of the present invention shows in detail the nutrients contained in addition to the silica-aluminous gel, examples of carbon sources include glucose, glycerol, fructose, maltose, mannitol, xylose, galactose, lactose, ribose, starch or hydrolysis thereof. Various carbohydrates such as a thing can be used. The concentration is usually preferably 0.1-5% (glucose equivalent) based on the medium. Also gluconic acid, pyruvic acid,
Various organic acids such as lactic acid and acetic acid, glycine, glutamic acid,
Various amino acids such as alanine, alcohols such as methanol and ethanol, various non-aromatic hydrocarbons such as normal paraffin, and various vegetable or animal fats and oils can also be used.

As a nitrogen source, ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate,
Ammonium salts of various inorganic or organic acids such as ammonium lactate, urea, peptone, NZ-amine, meat extract, yeast extract, dry yeast, corn sleep liquor, casein hydrolyzate, fish meal or its digest, soybean powder or its Nitrogen-containing organic substances such as digested material, defatted soybean or its digested material, pupa hydrolyzate, and various amino acids such as glycine, glutamic acid, and alanine can be used.

As the inorganic substance, various phosphates, magnesium sulfate, sodium chloride and the like, and a trace amount of heavy metal salts are used.

Also, when using a mutant strain that exhibits an auxotrophy, it is of course necessary to add a substance that satisfies the auxotrophy to the medium, but this type of nutrient is especially added when using a medium containing a natural product. May not be needed.

Examples of known medium compositions that can be applied to the present invention are as follows.

(1) Glucose 2%, peptone 0.5%, yeast extract 0.3
%, Meat extract 0.5%, NaCl 0.3%, CaCO ₃ 0.3% (pH 7.0) (2) Glucose 1%, peptone 0.3%, meat extract 0.5%, N
aCl 0.3%, agar 1.2% (pH7.2) (3) Glycerin 1%, Glucose 0.2%, Soybean flour 1%, Na
Cl 0.3% (pH7.5) (4 ) Starch 2%, corn steep liquor _{2%, MgSO 4 · 7H 2} O
0.1% (pH7.5) (5) Dextrin 2%, Peptone 0.5%, Soybean flour 1%, KH
_{2 PO 4 0.05%, MgSO 4} · 7H 2 O 0.05% (pH7.0) (6) glucose _{4%, (NH 4) 2} SO 4 0.5%, KH 2 PO 4 0.02%,
MgSO ₄ 0.1%, FeSO ₄ / 7H ₂ O 0.01%, CuSO ₄ / 5H ₂ O 0.001
_{%, ZnSO 4 · 7H 2 O} 0.001%, MnSO 4 · 4H 2 O 0.001%, CaCl 2 ·
2H ₂ O 0.001%, CaCO ₃ 0.3% (pH 6.5) (7) Glucose 2%, Casamino acid 1%, K ₂ HPO ₄ 0.1%, Mg
_{SO 4 · 7H 2 O 0.1%} , KCl 0.05%, ZnSO 4 · 7H 2 O 0.01%, MnS
O ₄ / 5H ₂ O 0.005%, FeSO ₄ / 7H ₂ O 0.005%, CaCO ₃ 0.3% (p
H7.5) When culturing a microorganism using the medium of the present invention, the culturing conditions are according to a conventional method. That is, the pH of the culture solution is usually in the range of 3-9, and the culture temperature is usually in the range of 20 ° C to 40 ° C. The culture period is usually 1-10 days. Of course, other conditions, such as microorganisms that grow more vigorously under acidic or alkaline conditions, or
When using a microorganism that particularly prefers a low temperature of 20 ° C or lower or a high temperature of 40 ° C or higher, cultivate in a desirable environment.

Further, various additives such as effective precursors, various surfactants, various solvents, saturated or unsaturated compounds are added for the purpose of further increasing the production amount of antibiotics at an appropriate time from the start of culture to the end of culture. Fatty acids and the like may be added to the medium.

When culturing a microorganism using the medium of the present invention, regularity cannot be confirmed at this stage as to what kind of composition the medium of the present invention is most suitable for. That is, when a microorganism having an antibiotic-producing ability is cultivated using the medium of the present invention having a specific composition, the production amount of the antibiotic is generally higher than that in the case where the conventional medium is used in the conventional culture method. However, it is a fact that an increase in the production amount may not be observed depending on the microorganism used. However, this cannot be said to impair the practical value of the present invention. This is because the conditions such as the suitability of the medium according to the present invention for a specific microorganism, the type and the amount of the silica-aluminous gel, and the like can be determined very easily by a simple experimental culture as described below. Because. Those skilled in the art can easily understand that this type of experimental culture is far simpler than the complicated trial-and-error method required for determining the optimal medium composition.

Experimental Example As a strain, Streptomyces rosa subsp.
rosa subsp.notoensis) ATCC31135, a tylosin-producing bacterium Streptomyces fradi
ae) ATCC 19609 was used.

As the medium for culturing these strains to produce antibiotics, the following mediums I to V that are commonly used for culturing actinomycetes were selected as the experimental medium. Medium containing silica-alumina gel, 0.5% of Sekado (Shinagawa White Brick Co., Ltd., 48 mesh) in each medium, and 0.5% of kaolin (Kanto Chemical Co., Inc.) in medium I. Prepare 6 kinds of media and add 10 of each media to the Sakaguchi flask.
The solution was dispensed at 0 m and cultured at 27 ° C. for 3 days for production of nanaomycin and 5 days for production of tylosin by reciprocal shaking as usual.

Medium composition I. Glycerol 2%, Bactosoyton 1%, NaCl 0.3
% (PH 7.0) II. Glycerol 2%, soybean flour 2%, NaCl 0.3% (pH 7.
0) III. Glucose 2%, peptone 0.5%, dry yeast 0.3
%, Meat extract 0.5%, NaCl 0.3%, CaCO _0.3 % (pH 6.0) IV. Glucose 1%, starch 2%, peptone 0.5%,
Yeast extract 0.5%, CaCO ₃ 0.4%, L-aspartic acid 0.3
% (PH 7.2) V. Starch 2%, glucose 0.5%, ammonium sulfate 0.2%, sodium lactate 0.3%, K ₂ HPO ₄ 0.05%, MgSO ₄
・ 7H ₂ O 0.05%, CaCO ₃ 0.3%, trace metal salts (iron, manganese, zinc, copper, cobalt) 3 mg / (pH 7.0) after completion of culture, measure the amount of antibiotics produced and accumulated in the culture solution As a result, the results shown in Table 2 were obtained. For comparison, the results obtained by similarly culturing in a medium containing no secard or kaolin are also shown.

From Table 2, it can be easily seen that the medium of I was suitable for the production of nanaomycin, and the medium of IV was suitable for the production of tylosin, each containing 0.5% secard. It is also clear from Table 2 that the medium according to the invention produces significantly more antibiotics than the known medium. It can be seen that Securd used in the present invention is suitable for addition to the medium I.

Examples of antibiotics suitable for production in the medium according to the present invention include nanaomycin, tylosin, protyronolide, streptomycin, dientamicin, kanamycin, neomycin, butyrosine, penicillin, cephalosporin, cefamycin, thienamycin, chlortetracycline, vancomycin, ristomycin. , Canyandecidin, nystatin, polymyxin, and other industrially important macrolides, polyene macrolides, aminoglycosides, β-lactams, tetracyclines, quinones, and peptide antibiotics.

The details of how the silica-alumina gel used in the present invention increases the production of antibiotics are not clear at present. However, we have
In the course of research to complete the present invention, the fact that the effect of reducing the concentration of free phosphate roots in the culture solution of silica-alumina gel and the increase in antibiotic production are closely related was investigated. I stopped. The test example is shown below.

Test example Streptomyces as a test bacterium Streptomyces rosa subsp. Notoensis (Streptomyces)
rosa subsp.notoensis) ATCC31135 was used. Also,
The following composition was used as a basal medium for nanaomycin production.

Glycerol 2%, Bactosoyton 1%, NaCl 0.3%
(PH7.0) 0 to one of the silica-alumina gel, Secard (manufactured by Shinagawa White Brick Co., Ltd., 48 mesh or less) is added to the basal medium of this composition
Or 0.5 and KH ₂ PO ₄ was added in 4 combinations of 0 or 100 μg / m to prepare 4 types of production medium. The test strain was inoculated into a 2-volume Sakaguchi flask containing 300 m of each of the four types of production medium and shake-cultured at 27 ° C. for 3 days by a conventional method. At this time, nanaomycin was produced and accumulated in the culture medium as shown in Table 3.

It is clear from Table 3 that:

First, the production of nanaomycin is inhibited by phosphate roots (comparison with experiment numbers 1 and 3).

Secondly, when Securd was added, the concentration of phosphate radicals decreased and the amount of nanaomycin produced increased (Experiment Nos. 1 and 2).
Comparison with).

Thirdly, even under the condition that the addition of phosphate root inhibits the production of nanaomycin, the concentration of phosphate root decreases and the production of nanaomycin is restored by further addition of secard (comparison between experiment numbers 3 and 4).

Fourth, in the presence of secard, the addition of 100 μg / m phosphate does not result in a substantial increase in phosphate and does not cause a substantial decrease in nanaomycin production (Experiment Nos. 2 and 4). Comparison).

This is an example of nanaomycin production,
Similar results can be obtained by testing other antibiotics as an example.

The fact that the concentration of free phosphate radicals does not increase remarkably despite the addition of phosphoric acid to the medium is due to the phosphate adsorption (or binding) action of Securd, after adding Securd to phosphoric acid water separately and shaking. This was confirmed by the fact that the free phosphate group concentration of No. 1 was clearly decreased.

On the other hand, it is known that the production of many antibiotics including tylosin as well as nanaomycin is remarkably reduced under the condition that free phosphate radicals above a certain concentration are present in the culture solution. Regarding this, for example, Martin (JFMartin), Domain (ALDemain), Bacteriological Review (No. 44)
Vol. 230, page 1980, etc. The concentration of free phosphate roots, which begins to show inhibition of antibiotic production, varies depending on the producing bacteria and medium. In addition, the detailed mechanism of inhibition is often unclear. Examples of known that the production of antibiotics is inhibited by the presence of phosphate roots include nanaomycin, tylosin, streptomycin, penicillin, candicidin, polymyxin, etc. There are many overlaps with antibiotics suitable for the production of From these facts and the results of test examples, it is because the decrease in free phosphate roots in the medium of the present invention substantially alleviates the inhibition of antibiotic production caused by free phosphate roots in the ordinary medium. I understand.

Example 1 Stanatomy-producing strain Streptomyces as a seed strain
rosa subsp.notoensis) ATCC31135 was used.

Seed medium consisting of glucose 2%, peptone 0.5%, meat extract 0.5%, dry yeast 0.3, NaCl 0.5%, CaCO ₃ 0.3% (pH
7.0) A 500 m Sakaguchi flask containing 100 m was inoculated with the inoculum and cultured with shaking at 27 ° C for 2 days.

Glycerol 2%, Bactosoyton 1%, NaCl 0.3%,
6m of the seed culture solution obtained as described above was inoculated into a 2-volume Sakaguchi flask containing 300m of a production medium (pH 7.0) consisting of 0.5% of Secard (48 Messyu, manufactured by Shinagawa White Brick Co., Ltd.)
After reciprocal shaking culture for one day, nanaomycin was produced and accumulated in the culture medium as shown in Table 4.

Example 2 The tylosin-producing strain Streptomyces fradiae ATCC19609 was used as the seed strain.

Seed medium consisting of glucose 2%, peptone 0.5%, meat extract 0.5%, dry yeast 0.3%, NaCl 0.5%, CaCO ₃ 0.3% (p
A 50 m large test tube (2 x 20 cm) containing 10 m of H7.0) was inoculated with the inoculum and shake-cultured at 27 ° C for 2 days.

Production medium (pH7.2) consisting of glucose 1%, starch 2%, peptone 0.5%, yeast extract 0.5%, CaCO ₃ 0.4%, L-aspartic acid 0.3%, Secard (Shinagawa White Brick Co., Ltd., 48 mesh). ) Inoculate the 500m Sakaguchi flask containing 100m with 2m of the seed culture obtained as described above, and incubate at 27 ° C for 3
After reciprocal shaking culture for 1 day, tylosin was produced and accumulated in the culture solution as shown in Table 4.

EFFECTS OF THE INVENTION By culturing a microorganism capable of producing an antibiotic using the medium according to the present invention, the production amount can be remarkably increased.

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Claims (2)

[Claims] 1. A medium for culturing a microorganism capable of producing an antibiotic to produce and accumulate the antibiotic in the culture, the medium containing an effective amount of allophane. 2. The medium according to claim 1, which contains 0.1-5% (by weight) of the allophane with respect to the medium.