JPS6350991B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a medium for culturing microorganisms capable of producing antibiotics to produce and accumulate antibiotics in the culture (hereinafter referred to as an antibiotic production medium). Antibiotics are substances produced by microorganisms that inhibit the growth of microorganisms and other cells, or have specific pharmacological or enzyme inhibitory effects (Nobuo Tanaka,
Shoshiro Nakamura, Compendium of Antibiotics, 2nd edition, page 1,
(University of Tokyo Press, 1977) and is currently engaged in pharmaceutical,
It is widely used in pesticides, animal feed additives, anthelmintics, etc. Examples of known media that have been modified to increase antibiotic production are as follows. (1) Medium containing effective precursors for antibiotic biosynthesis [Journal of American Chemical Society (J.Am.Chem.Soc.) Vol. 68,
No. 1669 (1946)] (2) A medium containing a buffering agent effective to maintain the pH of the culture medium within the optimum range [Demain Archives of Microbiology (Arch.
Microbiol.) Vol. 115, p. 169 (1977)] (3) Contains an effective surfactant to suppress foaming or facilitate extravasation of antibiotics from inside to outside the cells of antibiotic-producing microorganisms. (edited by Yoshio Ueno and Satoshi Omura, Microbial Pharmaceutical Chemistry, p. 176)
(1979, Nankodo) (4) A culture medium that selects effective substances from among various substances as nutrients (carbon source, nitrogen source, phosphate roots, etc.) and contains each in the optimum concentration (same as above). A medium suitable for producing each antibiotic is determined by combining the compositions of the following. However, this usually requires a huge amount of labor and time, and in many cases, a culture medium that is advantageous for the production of one antibiotic is disadvantageous for the production of another antibiotic. . Therefore, it has been desired to provide a culture medium for antibiotic production that has high productivity and applicability to a wide range of antibiotic production. Previously, the present inventor has discovered that the production of many antibiotics can be significantly increased when antibiotics are produced by a conventional method using a medium containing some phosphoric acids such as tertiary magnesium phosphate. (Japanese Patent Application No. 54-153792), it has now been discovered that the production of many antibiotics can be easily and significantly increased by adding zeolite to a known culture medium for antibiotic production. Therefore, an object of the present invention is to use a medium for culturing microorganisms capable of producing antibiotics in a conventional manner to produce and accumulate antibiotics, which can simply and easily increase the amount of antibiotics produced and accumulated. The goal is to provide a culture medium. The medium of the present invention is characterized in that it contains zeolite in addition to the compositions contained in conventionally used medium. When microorganisms capable of producing antibiotics are cultured in a conventional manner using the medium according to the present invention, the production amount can generally be increased several times, for example, by more than two times. The present invention will be explained in detail below. Any microorganism can be used to produce antibiotics using the culture medium of the present invention as long as it has the ability to produce antibiotics. For example, it may be a wild strain or a strain having 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, a variety of microorganisms capable of producing antibiotics can be used, including a wide range of known molds, yeasts, bacteria, and actinomycetes. Further, the microorganism may exist in nature, for example, in soil, river water, seawater, or air, or may be grown in advance in a medium other than the medium of the present invention. The zeolite used in the present invention is a mineral called zeolite in Japan, and has the following formula: Mx/n [(AlO ₂ ) _x (SiO ₂ ) _y ] _z H ₂ O [However, in the above formula, M is sodium, potassium, calcium, etc. n is the number of charges on the metal ion, i.e., 1 for potassium and 2 for calcium; It is an alkali metal salt of aluminosilicate that has the following composition, contains zeolite water, and has a three-dimensional structure.
); FA Kotton, GA Wilkinson, translated by Masaru Nakahara, “Inorganic Chemistry (Part 1)”, published by Baifukan (1976)
Ryoichi Kiriyama, "Structural Inorganic Chemistry," Kyoritsu Zensho (1978), pp. 226-238]. Naturally occurring zeolites include analzeite, craziite, chabazite, turbidite, yugawara zeolite, diabolite, fasciolite, clinotyl zeolite, halite, mordenite,
It is known as soda zeolite, and is often a mixture of several types. Examples of synthetic zeolites include zeolact A, C, G, L, T, S,
W, X, etc. are known. The culture medium according to the present invention contains natural zeolite, which has the ability to increase antibiotic production.
Any synthetic zeolite can be used. Zeolite is known to have ammonia nitrogen adsorption properties and is used for sewage treatment, etc., but by adding it to antibiotic production media, it is possible to significantly increase the production amount of antibiotics. is not known. The zeolite used in the culture medium of the present invention may be used alone or in combination with magnesium phosphate, etc., as previously taught by the present inventors.
If desired, various industrial wastes containing zeolite or various industrial products containing, adsorbing, incorporating or bonding zeolite can be used. Commercially available zeolites may be used as they are. Alternatively, it may be used after being washed in advance with one or more solutions of substances shown in the following (1) to (6). (1) Acids such as hydrochloric acid, sulfuric acid, and formic acid (2) Alkalies such as caustic soda, caustic potash, and ammonia (3) One or more of the compositions of known antibiotic production media such as common salt, ammonium sulfate, and magnesium chloride. (4) Production medium for the target antibiotic (5) Organic solvents such as methanol, ethanol, acetone, chloroform, and benzene (6) Water The concentration of zeolite produced in the medium depends on its particle size, the microorganism used, and the culture. The concentration should be determined taking into consideration the conditions, but a concentration of 0.01-5% is usually suitable.
Optimal temperature, time, and PH for the production of certain antibiotics
The conditions may be the same as those for the known antibiotic production medium. In addition to the zeolite in the culture medium of the present invention, it contains carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients suitable for the microorganisms used, following the example of conventionally used antibiotic production media. The type of medium of the present invention may be either a natural medium or a synthetic medium, and its form may be either a liquid medium or a solid medium. In detail, the nutrients contained in the culture medium of the present invention in addition to zeolite include, for example, as a carbon source:
Various carbohydrates can be used, such as glucose, glycerol, fructose, maltose, mannite, xylose, galactose, lactose, ribose, starch or its hydrolysates. The concentration is usually preferably 0.1-5% (calculated as glucose) based on the medium. In addition, various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, various amino acids such as glycine, glutamic acid, and alanine, alcohols such as methanol and ethanol, various non-aromatic hydrocarbons such as normal paraffin, and plants. Various oils and fats of natural or animal origin can also be used. Nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, ammonium lactate, urea, peptone, NZ-amine, meat extract, yeast extract, dried yeast, corn steep. Nitrogen-containing organic substances such as liquor, casein hydrolyzate, fat meal or its digested product, soybean flour or its digested product, defatted soybean or its digested product, pupa hydrolysate, and various amino acids such as glycine, glitamic acid, and alanine. is available. As inorganic substances, various phosphates, magnesium sulfate, common salt, etc., and trace amounts of heavy metal salts are used. Furthermore, when using a mutant strain that exhibits auxotrophy, it is of course necessary to add substances that satisfy the auxotrophy to the medium, but it is especially important to add this kind of nutrients when using a medium containing natural substances. may not be necessary. Examples of known culture 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% (PH7.0) (2) Glucose 1%, peptone 0.3%, meat extract
0.5%, NaCl 0.3%, agar 1.2% (PH7.2) (3) Glycerin 1%, glucose 0.2%, soy flour 1%, NaCl 0.3% (PH7.5) (4) Starch 2%, corn steep 2% liquor,
MgSO ₄ 7H ₂ O 0.1% (PH 7.5) (5) Dextrin 2%, peptone 0.5%, soy 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%,
_{KH2PO4 0.1} %, _MgSO4 0.1%, _FeSO4・
_7H2O0.01 %, _CuSO4・_5H2O0.001 %, _ZnSO4・
_7H2O0.001 %, _MnSO4・_4H2O0.001 %,
CaCl ₂ 2H ₂ O 0.001%, CaCO ₃ 0.3% (PH6.5) (7) Glucose 2%, Casamino acid 1%,
_{K2HPO4 0.1} %, _MgSO4・_7H2O0.1 %, KCl0.05
%, _ZnSO4・_7H2O0.01 %, _MnSO4・
_5H2O0.005 %, _FeSO4・_7H2O0.005 %,
CaCO ₃ 0.3% (PH7.5) When culturing microorganisms using the culture medium of the present invention, the culture conditions are according to conventional methods. That is,
The pH of the culture is usually in the range of 3-9, and the culture temperature is usually
A range of 20°C-40°C may give good results. The culture period is usually 1-10 days. Of course, if you want to use microorganisms that grow more vigorously under other conditions, such as acidic or alkaline conditions, or microorganisms that particularly prefer low temperatures below 20°C or high temperatures above 40°C, it is necessary to use microorganisms that grow more vigorously under other conditions, such as under the desired environment. Cultivate. In addition, various additives such as effective precursors, various surfactants, various solvents, and saturated or unsaturated additives may be added at appropriate times from the start of cultivation to the completion of cultivation to further increase the production of antibiotics. Fatty acids and the like may be added to the medium. When culturing microorganisms using the culture medium of the present invention, no regularity can be confirmed at this stage regarding which composition of the culture medium of the present invention is optimal for what type of microorganism. In other words, when a microorganism capable of producing antibiotics is cultured using the medium of the present invention having a specific composition, the amount of antibiotic produced by a conventional culture method is generally lower than when using a conventional culture medium. However, it is true that depending on the microorganism used, there may be cases where no increase in production is observed. However, this cannot be said to impair the practical value of the present invention. because,
This is because conditions such as the suitability of a particular culture medium according to the present invention for a particular microorganism, the type of zeolite and the amount used can be determined very easily by a simple experimental culture as described below. Those skilled in the art will readily understand that this type of experimental culture is much simpler than the complex trial-and-error approach required for conventional determination of optimal medium composition. Experimental example The strain used was a nanaomycin-producing bacterium, Streptomyces rosa var .
No.), cerulenin-producing bacteria, Cephalosborium,
Cephalosporium cerulens (J.Antibiot.)
(A Series) Volume 16, Page 236 (1963)] was used. As experimental media for culturing these bacterial strains to produce antibiotics, the following medium commonly used for culturing actinomycetes and the following mediums commonly used for culturing fungi were selected as experimental media.
Each medium contained 1% natural zeolite (Zolite, 40 ml), and the medium contained 1% synthetic zeolite A-3 (Wako Pure Chemical, 200 ml).
A total of 6 types of media were prepared, including %.
Dispense 10 ml of each medium into test tubes (2 x 20 cm) and incubate at 27°C for 3 days to produce nanaomycin.
For production of cerulenin, culture was carried out with reciprocating shaking for 2 days as usual. Medium composition: 2% glycerol, 2% soybean flour, 0.3% NaCl
(PH7.0) Glucose 2%, Peptone 0.5, Dry Yeast 0.3
%, meat extract 0.5%, NaCl 0.3%, CaCO3 _0.3 %
(PH6.8) Glucose 4%, Peptone 1% (PH7.0) Glucose 4%, NaNO ₃ 0.3%, KH ₂ PO ₄ 0.1
%, KCl0.05%, _MgSO4・_7H2O0.05 %,
FeSO ₄・7H ₂ O0.01% (PH6.7) Glycerol 3%, Glucose 1%, Peptone 0.5%, NaCl 0.2% (PH6.9) After culturing, remove the amount of antibiotics produced and accumulated in the culture solution. As a result of the measurement, the results shown in Table 1 were obtained. For comparison, a medium containing no zeolite was used.
The results of culturing in the same manner are also shown. [Table] used natural zeolite.
From Table 1, it can be easily seen that the medium containing 1% natural zeolite is suitable for the production of nanaomycin and the medium for the production of cerulenin. It is also clear from Table 1 that significantly more antibiotics are produced in the culture medium according to the invention than in the known culture medium.
Furthermore, it can be seen that natural zeolite is more suitable than synthetic zeolite A-3 as the type of zeolite to be added to the culture medium. Examples of antibiotics which are particularly suitable for production in the medium according to the invention include streptomycin, dihydrostreptomycin, penicillin, cephalosvorin, thienamycin, leucomycin, spiramycin, tylosin, tyalcomycin,
Bandolin, Kuzimycin, Erythromycin,
Aminoglycosides, β-lactams, macrolides, tetracyclines, and peptides that are currently widely used clinically, such as sillamycin, tetracycline, pacitracin, amphomycin, azureomycin, nanaomycin, chloramphenicol, and cerulenin. A large number of antibiotics can be mentioned, including antibiotics and the like.
The time, effort, and expense required for this type of experiment are
This is extremely small compared to what was previously required for selecting a culture medium for antibiotic production. Moreover, using such an extremely easily selected medium,
The production of antibiotics can be significantly increased. Example 1 A cerulenin-producing strain, Cephalosporium cerulens, was used as an inoculum.
caerulens) [J. Antibiotics (A series) Vol. 16, p. 236 (1963)] was used. Glucose 2%, peptone 0.5%, dry yeast 0.3%, meat extract 0.5%,
50ml containing 10ml of seed medium (PH7.0) consisting of 0.5% NaCl
Inoculate the inoculum into a large test tube (2 x 20 cm), and
The cells were cultured with shaking at ℃ for 2 days. 3% glycerol,
Seeds obtained as above were placed in a 500 ml Sakaguchi flask containing 100 ml of production medium (PH 7.0) consisting of 1% glucose, 0.5% peptone, 0.2% NaCl, and 1 or 2% natural zeolite (particle size 2 mm or less). When 1 ml of the culture solution was inoculated and cultured at 27°C for 2 days with reciprocal shaking,
Cerulenin was produced and accumulated in the culture solution as shown in Table 2. [ Table ] Example 2 Nanaomycin-producing strain, Streptomyces rosa var. notoensis ( S. rosa var.
No.) was used. 2% glucose, 1% meat extract,
Seed medium containing 0.5% NaCl, 0.3% _CaCO3 (PH5.0)
Inoculate the inoculum into a 500ml Sakaguchi flask containing 50ml of
It was grown for 30 hours at 37°C. 2 ml of this seed culture was transferred to a 500 ml Sakaguchi flask containing 50 ml of a medium (PH5.0) with the composition of the above medium supplemented with the substances shown in Table 3, and cultured at 37°C for 36 hours. Nanaomycin accumulated as shown in Table 3. [Table] Amount passed through sieve)
〃 1.0 450
No additives 370

Claims (1)

[Claims] 1. A medium containing zeolite for culturing microorganisms capable of producing antibiotics and producing and accumulating antibiotics in the culture.