JPS61247398A - Production of salinomycin - Google Patents

Abstract

PURPOSE:To collect salinomycin containing a new antibiotic in high yield, by cultivating a microorganism of the genus Streptomyces in a culture medium containing a fatty acid or a precursor therefor in producing the salinomycin by the fermentation method. CONSTITUTION:A microorganism, belonging to the genus Streptomyces, and having the ability to produce salinomycin, e.g. Streptomyces albus No. 80614 microorganism (FERM-P No. 419) is cultivated in a culture medium containing a fatty acid, e.g. inoleic acid, linolenic acid or oleic acid, or a fatty acid precursor, e.g. mono-, di- or triglyceride, as a main carbon source. Salinomycin is precipitated from the culture, and a solid part containing microbial cells is extracted to adsorb the salinomycin in an adsorbent. The adsorbed salinomycin is then eluted, purified and separated by the chromatography, etc. to afford the aimed salinomycin SY-1, SY-2 and new antibiotic SY-3-SY-8.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved method for producing salinomycins by fermentation.

In the present invention, salinomycins include salinomycin, SY-1, SY-2, SY-3, SY-4, SY-5
, BY-6, etc., or a mixture of two or more thereof.

Previously, the present inventors discovered that Streptomyces alps Waxman & Henrich No. 80614 (Feikoken Bacterium No. 419) produces and accumulates a novel antibiotic in its culture. succeeded in collecting salinomycin, SY-1 and SY-2 from the culture (British Patent No. 1378414, Japanese Patent Application Laid-open No. 1978-8).
6191 and Japanese Patent Application No. 52-5762).

As a result of further research by the present inventors, the present inventors found that by culturing this bacterium in a medium supplemented with fatty acids or their precursors, Salinomycin/, SY-1, and SY-2 were present in the culture.
BY-5
, SY-4, SY-5, SY-6, SY-7, SY-8
It was also discovered that new antibiotics such as

The present invention is based on this knowledge, and involves culturing Streptomyces bacteria capable of producing the antibiotic salinomycins in a medium containing fatty acids or their precursors, and collecting salinomycins from the culture. This is a unique method for producing salinomycins.

According to the method of the present invention, salinomycins are present in large quantities in the solid part containing the bacterial cells, so it is preferable to collect them from there. From the culture, salinomycin, SY-1 and S
In addition to Y-2, new antibiotics, especially BY-6, SY-4, and S
Y-5, SY-6, etc. can be collected.

In the present invention, the Streptomyces alpus No. 8
In addition to Bacterium No. 0614 or its artificial or natural sac variant,
Bacterial strains belonging to the genus Streptomyces and capable of producing salinomycin can generally be used. However, depending on the strain type and culture conditions, production of some salinomycins may not be observed.

The fatty acids used in the present invention are lower to higher saturated or unsaturated fatty acids, such as acetic acid, propionic acid, caproic acid, capric acid, lauric acid, palmitic acid, stearic acid, methacrylic acid, and undecylenic acid. ,
Particularly preferred are linoleic acid, linoleic acid, oleic acid, and the like.

In addition, the fatty acid precursor refers to a substance that can donate the above-mentioned fatty acids outside or inside the cells when culturing salinomycin-producing bacteria, such as mono-, di-, or triglycerides of fatty acids, and other esters. and salts. Oils and fats containing these substances, such as vegetable oils such as soybean oil, sesame oil, safflower oil, linseed oil, cottonseed oil, olive oil, and rapeseed oil, cod oil and other fish oils, and animal fats and oils such as lard can also be used. . The amount added is generally preferably about 8-25%, particularly about 12-20%, based on the medium.

The timing of addition of these additive substances is effective as long as the production of salinomycins continues to produce a titer, and may be before culturing or at an appropriate time after the start of culturing.

As the culture conditions of the present invention, conditions generally used for culturing actinomycetes can be used, except that fatty acids or their precursors are used as the main carbon source. Normally, the production and accumulation of salinomycins reaches its maximum at a culture time of 150 to 260 hours, but the production ratio of each component of salinomycins may vary depending on the culture time. It goes without saying that these medium compositions and culture conditions are appropriately selected depending on the type of bacterial strain used, external conditions, etc. so as to obtain preferable results.

Separation of salinomycins from the culture mixture is performed using the physicochemical properties of salinomycins. In other words, since the chemical structures of salinomycins are similar to each other, extraction may be carried out according to known methods applied to salinomycin and SY-1. Therefore, in order to increase the recovery rate of the target substance from the solid part, it is preferable to take this point into account and change the extraction process appropriately.For example, adjust the pH of the culture to 2.0 to 6.0. It is preferable to precipitate the salinomycin in the solution F, and then extract the precipitate and the solid portion containing the bacterial cells with an organic solvent.As a solvent, for example, acetone, ethyl acetate,
Butyl acetate, n-hexane, chloroform, etc. are used. This solvent phase is brought into contact with an appropriate adsorbent to adsorb salinomycins, and the salinomycins are eluted with an appropriate solvent.

After separating this eluate into two or several fractions depending on the purpose, using the difference in the properties of the target substance and impurities,
By repeating operations such as chromatography and solvent extraction for each fraction to purify and separate them, salinomycin, SY-1, SY-2, SY-3, SY-4, SY-
5, SY-6, SY-7, SY-8, etc. can be obtained individually or as a mixture. The product can be further purified by methods such as recrystallization or chromatography.

The properties of salinomycins obtained by the method of the present invention are summarized in the following table. Each data relates to the free form of the substance except for the molecular weight. Note that SY-3 and 5y-i analogues, SY-
4 are salinomycin analogues, SY-5, SY-6 and S
Y-8 is a 18.19-dihydrosalinomycin analog. The solvent system and the symbol ♦ in the table have the following meanings.

Solvent system a: chloroform-methanol (20: solvent system b
= Ethyl acetate-acetone (4:1) Solvent system C: Ethyl acetate ◆ Sprayed onto silica gel plates at room temperature.

Dissolve 6 g of vanillin in 10 mJ of methanol,
A reagent to which 5 ml of sulfuric acid Q was added was sprayed onto a silica gel plate while stirring, and heated to 60°C for 5 minutes.

Other physical properties are shown in the table below.

Table 2 Compound structure (free form): SY-1 SY-2 (Stereoisomer of SY-1) SY-4 SY-8 According to the present invention, as shown in the Examples, salinomycin,
sy- and SY-2 are obtained in extremely high yields compared to conventional methods. In addition, all of the substances below SY-5 obtained by the present invention are new substances, exhibit antibacterial activity almost equivalent to salinomycin, and can be used as medicines.Since they have a similar structure to salinomycin, they are also useful as veterinary drugs. be.

Reference example Glycerin 2.0%, peptone 0.5% and meat extract 0
．． Streptomyces e Alps Waxman & Henrich No. 80614 (
419) was inoculated and cultured with shaking at 66°C for 48 hours. 1 J of this culture solution was mixed with 2% glucose and 1 liter of starch.
%, soy flour 2.5%, beer yeast 0.4%, meat extract 0
．． 1%, sodium chloride 0.2% and antifoam agent KM-68
-2F (manufactured by Shin-Etsu Chemical, silicone type) 100ml of liquid medium (stainless steel 200A tank) containing 0.1% was inoculated and cultured with stirring at 36° C. for 144 hours under conditions of aeration rate of 10,047 minutes. As a result, salinomycin 100-6
A culture solution containing 00γ/G was obtained.

Example 1 (A) Production of salinomyne 7: glycerin 2%, peptone 0.25%, meat extract 0.5
% and 0.1% sodium chloride was inoculated with the same bacterium No. 806144 as in the reference example, and cultured with shaking at 66° C. for 48 hours. This culture solution was mixed with 4.0% glucose, 1.0% soybean flour, and 1.0% brewer's yeast at a ratio equivalent to 1%.
% and 0.2% calcium carbonate, cultured with shaking at 36°C for 30 hours, and this culture solution was used as the second preculture solution.

The second pre-culture solution 11 was mixed with soybean oil 10% and glucose 1.0.
%, soy flour 1.0%, sodium chloride 0.1%, calcium chloride 0.1%, calcium carbonate 0.5%, dibasic potassium phosphate 0.01% and antifoaming agent KM-68-2F 0.
The cells were inoculated into 100 J of a liquid medium containing 1% and cultured at 56° C. for 210 hours with stirring at an aeration rate of 1001/min.

@) Separation and purification of salinomycin, SY-1 and SY-2: The culture obtained in (A) was adjusted to pH 4.5-5°0 with dilute hydrochloric acid.
After adding 4% (weight/volume) of filter aid and stirring thoroughly, the mixture is filtered. F solution (801) is butyl acetate 50
! Add and stir to extract. On the other hand, the bacterial cells were extracted with acetone 601, and after distilling off the acetone, the residue was extracted with butyl acetate 601.
1 and combine it with the above-mentioned butyl acetate extract.

After washing the butyl acetate solution once with 5% sodium bicarbonate aqueous solution 201, take 11 and concentrate and dry it under reduced pressure to obtain 250 g of salinomycin crude powder containing trace amounts of SY-1 and SY-2. is obtained.

50 g of this salinomycin crude powder is dissolved in ethyl acetate and passed through an alumina column (Alumina 500I, activated alumina manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.). After washing with ethyl acetate 11, salinomycin and SY-1 are eluted with an ethyl acetate-methanol mixture (100:5), and the eluted sections of salinomycin 9 and SY-1 are collected and concentrated. Next, the concentrate was mixed with chloroform-methanol (100:2).
The solution was dissolved in 50 ml and passed through a silica gel column (300 g of silica gel, Wako T-Gel C-200 manufactured by Wako Tsumugi Co., Ltd.) that had been filled in advance with the same solvent. Continue elution with the same solvent, pure salinomycin and S
Collect and concentrate the elution area of Y-1. Crystallizing each concentrate with acetone-water yields 5 crystals of pure salinomycin.
Crystals 60rn9 of g and SY-1 are obtained.

When the above alumina column was further developed with a mixture of ethyl acetate and methanol (100:15), SY-2 was eluted, which was purified by silica gel column chromatography in the same manner as above, and purified with acetone-water. Upon crystallization, 6 quartz crystals of pure SY-2 are obtained.

Example 2 (A) High-unit production culture of salinomycin and production culture of salinomycin-related components are carried out in the same manner as in Example 1.

(B) Recovery of salinomycin and its related components: (A)
The culture obtained in step 1 is adjusted to pH 4.5 to 5.0 with dilute hydrochloric acid, and heated to 60° C. for 10 minutes while stirring.

Next, 4% (weight/volume) of filter aid was added, stirred thoroughly, and then filtered. The obtained bacterial cell portion contains salinomycin and its related components SY-1 to SY-8 in high yield.

(C) Separation of salinomycin complex containing components SY-1 to SY-8: The bacterial cell portion obtained in (B) was extracted twice with ethyl acetate 501, and the extracts were combined and added to a 5% aqueous sodium bicarbonate solution. 5
Wash once with 01. The ethyl acetate phase is concentrated and dried under reduced pressure to obtain 7 kg of a crude powder of Salinomyces/complex containing components SY-1 to SY-8.

(I)) Separation and purification of salinomycin: 7kl of crude powder obtained in (C)? Hexa 7100J! ! dissolved in
Concentrate to 201 under reduced pressure and refrigerate the concentrate at 5°C.
Crude crystals of salinomycin 27 to 9 are obtained. After repeating this operation and recrystallizing from hexane, 2.4 kg of pure salinomycin sodium salt is obtained.

(Smelling linomycin-related components SY-1 to SY-8
Separation and purification: When the crystal mother liquor obtained in (D) is concentrated and dried, a small amount of salinomycin and its related components SY-1 to 8Y
4.6 ml of salinomycin complex containing -8 is obtained.

This complex was mixed with hexane-ethyl acetate (2:1) 50
J and passed through a column of alumina (using alumina 12), which had been packed in advance with the same solvent, and 20! After washing with ethyl acetate-methanol (
When developed with 100:2) 201, part of salinomycin and components SY-1, SY-3 and SY-7 are eluted. From the eluate (salinomycosis/
After separating 200I of and 5y-i, other components 8Y
The mother liquor containing -3 and SY-7 is concentrated and dried to obtain 100 g of coarse powder.

100g of the obtained coarse powder was mixed with chloroform 1/-kc
100:2) in 500 ml of silica gel column (silica gel 6k
lil, Wakogel C-200 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) and developed with the same solvent. The resulting components SY-3 and S
Each of the elution areas of Y-7 was concentrated and further subjected to silica gel thin layer chromatography [Wako Gel B-10 manufactured by the same company, silica gel thickness 0.25 m, developing solvent: chloroform.
methanol (2o:1)), R2 value = o, 4
SY-3 with Rf value=0.8Q and SY-7 with Rf value=0.8Q are separated from the laminate. When the silica gel adsorbing each component is eluted with chloroform-methanol (20:1) and the solvent is removed by evaporation, 18 to 35 In of SY-5 and 35 In9 of SY-7 are obtained as pure powders, respectively.

The above alumina column was soaked in ethyl acetate-methanol (5
:1) 2OA! The eluate was concentrated under reduced pressure to obtain 200 g of a crude powder.

This coarse powder contains components 8Y-2, SY-4, SY-5, S
Y-6 and 3Y-8 exist as a complex. These components are separated by silica gel column chromatography. The obtained coarse powder 2009 was dissolved in chloroform-methanol (100:2) SOORILt, and the mixture was added to a silica gel column (silica gel 12 kfl, Wako Gel C-20) packed in advance using the same solvent.
0) and then developed with the same solvent, the component SY
-2, SY-4, SY-5, BY-3, and 8Y-8 are separated and eluted.

When the eluate of each component was concentrated, SY-2 was found to be 20
．． 9, SY-4 is 600■, SY-5 is 250~. S.Y.
-6 and SY-8 were obtained as coarse powders of 8001v and 11001n, respectively. Coarse powder of SY-2 in acetone
Crystallization from water gives 15 g of pure 8Y-2 crystals.

When the remaining components were treated by silica gel thin layer chromatography in the same manner as for the isolation and purification of components SY-3 and SY-7, SY-4 was purified as a pure powder.
61n9. 20 da of SY-5, 801 ng of SY-6 and 60 m9 of SY-8 were obtained. SY-2 is a columnar crystal,
SY-4 is obtained as needle-shaped crystals, and SY-8 is obtained as needle-shaped crystals.

Example 3 Production culture of mycin-related components: The second preculture solution of Example 1 was mixed with 4% glucose, 6.0% soybean flour, 3.0% defatted wheat germ, and 0.0% calcium carbonate.
2% and 0.1% antifoaming agent KM-68-2F at a rate of 10%, and cultured at 35°C for 24 hours to obtain a sixth preculture solution.

10J of this second pre-culture solution was mixed with 16% soybean oil and 0% soybean flour.
．． 5%, defatted wheat germ 1.0%, sodium chloride 0.2
%, potassium chloride 0.2%, calcium carbonate 0.5%,
Ammonium sulfate 0.6%, dibasic potassium phosphate 0.02
%, magnesium sulfate 0.01% and antifoaming agent KM-68
-2F Inoculated into medium 1001 containing 0.1%, and kept at 33°C.
The cells were cultured for 290 hours with stirring at an aeration rate of 100 l/min.

In this case, the same amount of production can be obtained by starting with a small amount of soybean oil and adding it at appropriate times along the way. Furthermore, there is no difference whether the antifoaming agent is silicone-based or polyether-based.

(B) When treated in the same manner as in Example 2 (B), a bacterial cell portion containing salinomycin and its related components SY-1 to SY-8 at a high yield is obtained.

(C) When treated in the same manner as in Example 2 (C), component S
16 kg of coarse powder of salinomycin complex containing Y-1 to SY-8 is obtained.

(D) After dissolving 9 in the crude powder 16 obtained in (C) above in 1001 hexane, it is concentrated to 401 under reduced pressure. The following processing is performed in the same manner as in Example 2 (D).

7.1 kg of crude crystals of salinomycin and then 6.3 kg of pure salinomycin sodium salt are obtained.

(E) When the crystal mother liquor obtained in (D) above is concentrated and dried, a small amount of salinomycin and its related components S
Salinomycin complex 97 containing Y-1 to SY-8
9 is obtained. This complex was converted into hexane-ethyl acetate (
2:1) 101:l? A part of salinomycin and components SY-1 and BY were dissolved in alumina and subjected to alumina column chromatography in the same manner as in Example 2 (E) using 20 kg of alumina and ethyl acetate-methanol (10:2) 501 as a developing solvent. -5 and 8Y-7 are eluted. After using the eluate in the same manner as in Example 2(E) to separate 400 g of salinomycin and 3Y-1, coarse powder 160I containing SY-3 and SY-7 is obtained.

160 g of the obtained crude powder was dissolved in 400 g of chloroform-methanol (100:2) and subjected to silica gel column chromatography (using 8 g of silica gel) in the same manner as in Example 2(E) to obtain components SY-6 and SY-7. was eluted and further separated by silica gel thin layer chromatography in the same manner. After the elution solvent was evaporated off, SY-3 was purified as 501n9 and SY-3 as pure powder.
7 is obtained as 501n9.

The above alumina column was soaked in ethyl acetate-methanol (5
:1) When eluting with 401 and removing the solvent, the component SY
250 g of coarse powder containing SY-2, SY-4, SY-5, SY-6 and SY-8 as a composite is obtained. 250.9 of this coarse powder was mixed with chloroform-methanol (100:
2) Dissolved in 600- and then similarly separated each component by silica gel column chromatography (using silica gel 20IK9), component SY-2 was 40/i, S
Y-4 is 600cm, SY-5 is 600m9. SY-6 and SY-8 were obtained as 1900n9 and 3001n9 crude powders, respectively, and purified in the same manner.
2 crystal 30.9, SY-4 powder 180m9, SY-
5 powder 120μ, 8M-6 powder 350mg and SY
90 μ of powder of -8 are obtained.

Example 4 The 6th preculture solution of Example B was added at a rate equivalent to 10%.
12% of each oil and fat listed in the table below, 0.5% soybean flour, 1.0% defatted wheat germ, 0.2% sodium chloride, 0.2% potassium chloride, 0.5% calcium carbonate, 0.3% ammonium sulfate. , a medium containing 0.02% potassium diphosphate and 0.01% magnesium sulfate (inoculated into 50 ml aliquots, 6
Culture with shaking at 6°C for 216 hours. The amount of salinomycin produced at the end of culture is shown below.

From each obtained culture, salinomycin and its related components SY-1 to SY- were extracted in the same manner as in Examples 1 to 3.
8 can be isolated and purified.

Claims (1)

[Claims] 1. Streptomyces bacteria capable of producing the antibiotic salinomycins are cultured in a medium containing fatty acids or their precursors, and salinomycins are collected from the culture. A method for producing salinomycins. 2. The method according to claim 1, characterized in that salinomycins are collected from the culture in a state contained in a solid part containing bacterial cells. 3. Antibiotics SY-1, SY-2, SY-3 from culture
, SY-4, SY-5 and/or SY-6.