JPS61216682A - Medium for production of antibiotic substance - Google Patents

Abstract

PURPOSE:To obtain a medium for the production of an antibiotic substance and capable of remarkably increasing the productivity of the antibiotic substance, by adding a silica alumina gel to a conventional medium for the production of antibiotic substance. CONSTITUTION:A conventional medium for the production of antibiotic substance and containing carbon source, nitrogen source, inorganic materials and various other necessary nutrients assimilable by the microorganisms to be cultured is added with a silica alumina gel at a concentration of usually 0.1-5% and a microbial strain capable of producing an antibiotic substance is cultured in said medium by conventional method.

Description

Detailed Description of the Invention Field 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). .

Conventional technology 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 and Shoshi Nakamura, Compendium of Antibiotics, 2nd Edition) , p. 1, 1977, University of Tokyo Press), and is currently used in many medicines, agricultural chemicals, animal feed additives, anthelmintics, etc.

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

(1) Medium containing effective precursors for antibiotic biosynthesis [
Journal of American Chemical Research (J, Am, Chem, Soc, ) Volume 68,
No. 1669 (1946)] (2) Medium containing a buffer solution effective for maintaining the pH of the culture solution within the optimal range [r Main (D@main) Archives of Microneurology (Arch, Mlcroblol,) No. 115
Vol., p. 169 (1977)] (3) A medium containing an effective surfactant to suppress foaming or to favor the release of antibiotics from the inside to the outside of the cells of antibiotic-producing microorganisms ( Edited by Yoshio Ueno and Satoshi Omura,
Microbial Pharmaceutical Chemistry, p. 176, 1979, Nankodo) (4) Nutrients in the culture medium (carbon source, nitrogen source, phosphate roots, etc.)
A culture medium containing the optimum concentration of each substance selected from among various substances (same as above). By combining these various compositions, a culture medium suitable for the production of each antibiotic is determined. Ru. However, this usually requires a huge amount of labor and a long time, and moreover, a medium that is advantageous for the production of one antibiotic is often disadvantageous for the production of another antibiotic. Therefore, it has been desired to provide a medium for antibiotic production that has high productivity and applicability to a wide range of antibiotic production.

Previously, the present inventor discovered that when antibiotics are produced by a conventional method using a medium containing zeolite, the production amount of many antibiotics can be significantly increased (M opening 57-
No. 206380).

Problems to be Solved by the Invention The purpose of the present invention is to provide a medium for cultivating microorganisms capable of producing antibiotics by a conventional method to produce and accumulate antibiotics in a simple and easy manner. The objective is to provide a medium that can be increased.

Means for Solving the Problems The culture medium of the present invention is characterized in that it contains a silica-alumina gel in addition to the compositions contained in conventionally used culture media. 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 it may be a strain that has specific nutritional requirements or resistance or sensitivity to specific substances. 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. In addition, the microorganisms are found in the natural world, such as soil,
It may be something that has existed in river water, seawater, air, etc., or it may be something that has been grown in advance in a medium other than the culture medium of the present invention.

The silica-alumina gel used in the present invention is a type of naturally occurring ore, and its scientific name is allophane (A11ophane).
It is a silica-alumina gel clay called ・).

According to mineralogy, such clays arise from crystalline silica-alumina metal salt ores through natural weathering. Therefore, it is clearly distinguishable from kaolinite (Kaolnit), zeolite (Zsolit), etc. in that it is amorphous.

In Japan, allophane is a major component of colloidal clay called Kanuma clay, Awa clay, Misojo, etc., but it is also found in Europe and America. Allophane is a hydrous silica alumina gel produced by leaching metal salts from volcanic glassy rock over a long period of natural weathering.These leached molecules are removed and fall, leaving many very small pores. There is. These fine pores exhibit excellent adsorption properties and are often used as adsorbents. In addition, although it is composed of hydrated silica alumina,
Its molecular structure is amorphous, such as mullite (3MJ
20,2810□) Kaolinite (AJ14(oH),
st,ol. ), it is chemically very active, as it easily reacts with hydrochloric acid and becomes gelatinous. The Myrica alumina gel used in the present invention can be used as is or purified from naturally occurring allophane. It can also be artificially synthesized and used.

Natural silica-alumina gel has different color tones and chemical compositions, probably because the type and amount of contaminants vary depending on the place of production and state of existence.
As a result, their physicochemical properties differ slightly. As an example, Table 1 shows the properties of allophane, which is one of the silica alumina gels used in the examples.

Table 1 It is well known that allophane clay has the ability to adsorb inorganic acids and moisture in the air, and has traditionally been used as a dehumidifier. It was not known that the production of substances could be increased. The silica-alumina gel used in the culture medium of the present invention may be used alone or in combination with zeolite, etc., as previously taught by the present inventors.

As the silica alumina gel used in the present invention, commercially available powder products may be used as they are, but
Use after heat treatment at 0°C for several hours tends to give good results in antibiotic production.

The concentration of silica-alumina gel to be included in the medium should be determined by considering its particle size, microorganisms used, and culture conditions, but a concentration of 0.1-5% is usually suitable.□ Production of certain antibiotics Conditions such as optimal temperature, time, pH, etc. for the known cough antibiotic production medium are as follows:
All you have to do is stop.

In addition to the silica-alumina gel, the culture medium of the present invention follows the example of conventional antibiotic production media and contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other desired nutrients that can be utilized by the microorganisms used. The type of this fermented medium 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 the silica-alumina gel include carbon sources such as glucose, chrycerol, fructose, maltose, mannitol, silose, galactose, lactose, ribose, starch, and their like. A variety of carbohydrates can be used, including 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 gluconic acid, glutamic acid, and alanine, alcohols such as methanol and ethanol, and normal/I! Various non-aromatic hydrocarbons such as fins, various vegetable or animal fats and oils, etc. can also be used.

Nitrogen sources include ammonia, ammonium chloride, heptammonium sulfate, ammonium nitrate, ammonium acetate,
Ammonium salts of various inorganic or organic acids such as ammonium lactate, urea, ibutone, Nz-amine, meat extract, yeast extract, dried yeast, hoon steep liquor, casein hydrolyzate, fishmeal or its digested product, soybean It is possible to use flour or its digested product, defatted soybean or its digested product, nitrogen-containing organic substances such as soybean hydrolyzate, and various amino acids such as glycine, glutamic acid, and alanine.

As the inorganic substance, various phosphates, magnesium sulfate, common salt, 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) Gurufus 2%, Peptone 0.5%, Yeast Processor 0.3%, Meat Extract 0.5%, NaC10,
,3%.

caco30-3% (pH 7-0) (23r lucose 1%, 4zotone 0.3%, meat extract 0.5%, N
aC) 0.3%, agar 1.2% (PH7,2) (3) Glycerin 1%, glucose 0.2%,
Soybean flour I It, NaCJ O, 3'Ji (pH 7
,5)(4) 2% starch, cornsteel delicacy
111゜Mg5O, 7H, OO, 1'% (pH 7,5
) (5) Dextrin 2%, Pezotone 0.5%, Soybean flour 1 t, KH2PO40.05 t, MgSO4.
7H200,05t (pH 7,0) (6) Glucose 4t, (NH4)2So40
．． 5th grade.

IG (, PO40, 02 Yu, Mg5O, 0,1 Chi, F
・so4・7H200,01%, Cu804-5H,
OO,0015tZn804'7H,Oo,ooi 4
．． Mn3O4・4H20G, 001To -c&c1
2-z+(,o 0-001% -CaCO30-
3'16 (pH 6,5) (7) f /l/ = r -X 2%, casamino acid ILs, 2HPO40,1%, Mg80
．．・7) 1,0 0.1chi, K (J 0.05%
.

ZnSO4-7H200, 01%, Mn3O4”5
H200,005St PO804-711,00,
Section 005, caco3o, a % (pH 7,5) 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 solution is usually in the range of 3-9, and the culture temperature is usually in the range of 20°C-40°C, which can bring about good results. The culture period is usually 1-10 days. Of course, when using 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 under the desired environment. Cultivate.

In addition, various additives such as effective precursors, various surfactants, various solvents,
Saturated or unsaturated fatty acids, etc. 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, if a microorganism capable of producing antibiotics is cultured using the medium of the present invention having a specific composition, the amount of antibiotic produced will be lower than that obtained using a conventional culture medium. However, depending on the microorganism used, it is true that the increase in production may not be observed in some cases. However, this cannot be said to impair the practical value of the present invention. This is because conditions such as the suitability of a particular medium according to the invention for a particular microorganism, the type and amount of silica-alumina gel to be used, etc. can be determined very easily by a simple experimental culture such as this. Those skilled in the art will readily appreciate that experimental cultivation of seeds is much simpler than the complex trial-and-error approach required for conventional determination of optimal medium composition.

As an experimental strain, the nanaomycin-producing bacterium Strezotomyces rosa subsp. nodoensis (8tr@to
m ass rosa aubsp, notoIns
lm) ATCC31135, tylosin-producing bacterium Streptomyces 7 larii (anus vomitus fradia)
) Human TCC19609 was used.

As a medium for culturing these strains to produce antibiotics, the following mediums 1 to 5, which are commonly used for culturing actinomycetes, were selected as experimental media. Silica alumina gel for each medium, brand name Shichika? (8@kado, manufactured by Shinayo Shiroenga Co., Ltd., 48 mesh) A medium containing 0.51,
and 0.5' of kaolin (Kanto Chemical Exhibition) in the medium of I.
A total of 6 types of media containing 16% of the total number of media were prepared, 100ml of each medium was dispensed into Sakaro flasks, and incubated at 27°C for 3 days for the production of nanaomycin and 5 days for the production of tylosin, as usual. Culture was performed with reciprocal shaking.

Medium composition 1. 2 glycerol, 1* Noctosoitone.

NaCJ O,3To (pH 7,0) ■, 2 t glycerol, 2 t soybean flour, Na07003% (pH 7,
0) ■, glucose 2%, peptone 0.5%, dry [11
Mother o, 3%, meat extract o, s s, NaCj! o
, :(ts.

CaCO30.3% (pH6.0) pi, f, II/course 1%, SuI'-chi2qb,
Peptone 0.5%, yeast extract 0.5 n, cac
o, 0.4 s tL-asnoglagic acid 0.3 s (
pH7,2)V. 2 liters of starch, 0.5 liters of glucose
Ammonium sulfate 082%, pore number sodium 0.3%
.

KH2O40,0596,MgSO4・7H,OO,0
5%.

Ga(:Os 0.3%, trace metal salt (iron, −+
ry try.

Zinc, copper, cobalt) each 3 / l (PHy, o) After the cultivation was completed, the amount of antibiotics produced and accumulated in the culture solution was measured, and the results shown in Table 2 were obtained. For comparison, the results of culturing in the same manner in a medium containing no kaol or kaolin are also shown.

Table 2 (Note) *Kaolin (Kanto Chemical System) was added.

From Table 2, it can be easily seen that the medium (2) containing 0.547 carr is suitable for the production of nanaomycin, and the medium (2) containing 0.547 carr for the production of tylosin. It is also clear from Table 2 that significantly more antibiotics are produced in the medium according to the invention than in the known medium. It can be seen that the chemical compound R used in the present invention is suitable as a material to be added to the medium in (2).

Examples of antibiotics suitable for production in the culture medium according to the invention include nanaomycin, tylosin, protyamycin, streptomycin, gentamicin 1 kanamycin, neoisin, butyrosin, penicillin, cephalosphorin, 77amycin, chenamycin. , chlortetracycline, vancomycin, ristomycin, candididine, nystatin, polymyxin, and other industrially important macrolytic antibiotics, polyene macrolytic P series, aminoglycosylated series, β-lactam antibiotics, tetracycline series, quinone series, and pezotyric antibiotics. Substances can be given.

The details of the mechanism by which the silica-alumina gel used in the present invention increases antibiotic production are currently not clear. However, the inventors
In the course of research to complete the present invention, it was revealed that the effect of silica-alumina gel to reduce the concentration of free phosphate radicals in the culture medium is closely related to the increase in antibiotic production. I found out. A test example is shown below.

Test Example The nanaomycin-producing bacterium Streptomyces rosa subsp. nodoensis (5tre t
ow a@s rosa 5ubap, noto@n
sig) Human TCC31135 was used. In addition, the following composition was used as a basal medium for nanaomycin production.

Glycerol 2%, Bactosoitone 1q6゜Na (J
0.351i (pH 7,0) Basal medium with this composition (
, one of the silica-alumina gels, Kakar)' (manufactured by Shinyo Shirorenga Co., Ltd., 48 mesh or less) was added in 4 combinations of 0 or 0.5, and KH2PO4 was added in 0 or 100μ1 air. A production medium was prepared. This 4
The test bacteria were inoculated into 2-capacity Sakaguchi flasks each containing 300 μl of seed production medium, and cultured with shaking at 27° C. for 3 days using a conventional method. At this time, nanaomycin was produced and accumulated in the culture solution as shown in Table 3.

Table 3 (Note) * Indicates the value measured immediately after inoculation.

** Indicates the production amount of the 38th.

The following is clear from Table 3:

Production of the first K, nanaomycin, is inhibited by phosphate radicals (compare experiments no. 1 and 3).

Second, when Nanakarr was added, the concentration of phosphate roots decreased and the production of nanaomycin increased (Experiments Nos. 1 and 2).
Thirdly, even under conditions where nanaomycin production was inhibited by adding phosphate roots, further addition of Nanaomycin reduced the concentration of phosphate roots and restored nanaomycin production (
Comparison with experiment numbers 3 and 4).

Fourth, seven cars? In the presence of 1oopl/lxl
of phosphoric acid does not result in a substantial increase in phosphoric acid nor does it cause a substantial decrease in nanaomycin production (compare experiments no. 2 and 4).

This is an example of nanaomycin production.
Even if we test the production of other suspension substances as an example, the results are the same! can get.

The reason why the concentration of free phosphate roots does not increase significantly even when phosphoric acid is added to the culture medium is due to the phosphate adsorption (or binding) effect of Secarr. This was confirmed by a clear decrease in the concentration of free phosphate roots after the boiling.

On the other hand, it is known that the production of not only nanaomycin but also many antibiotics, including tylosin, is significantly reduced under conditions where a certain concentration or more of free phosphate radicals is present in one culture solution. In this regard, see, for example, Martin (,T.P.

Martln), F'main (A, L* D@
main) , /? Criterological Review (B
aat@rlologl@alR@v1ew) Volume 44, Page 230 (1980). The concentration of free phosphate radicals that begins to show inhibition of antibiotic production is
It varies depending on the producing bacteria and culture medium. Furthermore, the detailed mechanism of inhibition is often unclear. Examples of antibiotics whose production is known to be inhibited by the presence of phosphate groups include nanaomycin, tylosin, streptomycin, penicillin, nakayandicidin, and polymycin. Many overlap with antibiotics suitable for production in the invention medium. From these facts and the results of the test examples, it is clear that when the free phosphate radicals are reduced in the culture medium of the present invention, the inhibition of antibiotic production caused by the free phosphate radicals in the conventional culture medium is substantially alleviated. I understand that there is something.

Example 1 Nanaomycin producing strain Streptomyces rosa subs. 7toensis (Str@to
nees roma 5ubsp, Botoens
la) ATCC31135 was used.

Glucose 2%, Pefton 0.5%, Meat extract 0.5
%, dry tlk yeast 0.3 *, NaC; l O,
Seed medium (pH 7) consisting of 5To, CaCO30, 3
.

Glycerol 2%, Nocto Soitone.

Na1l Q, 3'4. Seka l' (manufactured by Shirorenga Co., Ltd. for A, 48mets'/:>) Q, 597. 6d of the seed culture obtained as described above was inoculated into a Yosaka Lough flask containing 2!
When cultured with reciprocating shaking at 7°C for 5 days, nanaomycin (as shown in Table 4) was produced and accumulated in the culture solution.

Example 2 One strain of tylosin producing strain Strezoto Ises φ Fradlae A'I'CG as a seed fungus
1960G was used.

Glucose 2L Takufton 0.5%, Meat Extract 0.5%
, dry yeast 0.3 f&, NaC 10,5%, CaC
The seed culture was inoculated into a large test tube (2 x 20 tubes) containing a seed medium (pH 7,0310-) consisting of 2.
The cells were cultured with shaking at 7°C for 2 days.

Glucose t%, starch 2%, nzotone 0.5 t4
．． $mother z*x 0.59A, (aco 0.4
5001117 containing 1001114 (pH 7.2) of production medium (pH 7.2) consisting of
The seed culture solution 2 obtained as described above was inoculated into a Yosaka flask and cultured at 27° C. for 3 days with reciprocating shaking. As a result, the butyrosin shown in Table 4 was produced and accumulated in the culture solution.

Part 4! ! ! (Note) *Values converted to nanaomycin E are shown.

Effects of the Invention When microorganisms capable of producing antibiotics are cultured using the medium according to the present invention, the production amount can be significantly increased.

Claims (1)

[Claims] A medium containing a silica-alumina gel, which is used to culture microorganisms capable of producing antibiotics and to produce and accumulate antibiotics in the culture.