CA1292713C - Process for producing antibiotic salinomycin - Google Patents
Process for producing antibiotic salinomycinInfo
- Publication number
- CA1292713C CA1292713C CA000553599A CA553599A CA1292713C CA 1292713 C CA1292713 C CA 1292713C CA 000553599 A CA000553599 A CA 000553599A CA 553599 A CA553599 A CA 553599A CA 1292713 C CA1292713 C CA 1292713C
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- Canada
- Prior art keywords
- vitamin
- nutrient medium
- fermentation
- salinomycin
- microorganism
- Prior art date
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- Expired - Lifetime
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT
There is disclosed a biosynthesis process for producing antibiotic salinomycin by means of producing strains of the microorganism Streptomyces albus, which are derived from the strain ATCC 21838. The microorganism is cultivated under submersed aerobic conditions in a nutrient medium, containing an organic carbon source, a nitrogen source, mineral salts, the amino acid methionine and small quantities of vitamins, either D-biotin or pyridoxin (B6). By the presence of vitamins and methionine, a greater and more stable yield of the antibiotic is achieved more quickly, which makes the process shorter. As anti-foaming agents there are used vegetable oils and silicone type anti-foaming agents. From the fermented broth the antibiotic is isolated in a standard manner.
There is disclosed a biosynthesis process for producing antibiotic salinomycin by means of producing strains of the microorganism Streptomyces albus, which are derived from the strain ATCC 21838. The microorganism is cultivated under submersed aerobic conditions in a nutrient medium, containing an organic carbon source, a nitrogen source, mineral salts, the amino acid methionine and small quantities of vitamins, either D-biotin or pyridoxin (B6). By the presence of vitamins and methionine, a greater and more stable yield of the antibiotic is achieved more quickly, which makes the process shorter. As anti-foaming agents there are used vegetable oils and silicone type anti-foaming agents. From the fermented broth the antibiotic is isolated in a standard manner.
Description
7~3 PROCESS FOR PRODUCING ANTIBIOTIC SALINOMYCIN
The present invertion relates to an improved process for producing antibiotic salinomycin by fermentation by means of the strain of the microorganism Streptomyces albus, deposited in the collection of microorganisms American Type Culture Collection, Rockville, USA, No. ATCC 21838, and in the collection of microorganisms Fermentation Research Institute, Chibe, FERM-P No. 419, Japan.
It is well-known that salinomycin is produced by means of various strains of the microorganism Streptomyces albus in aerobic growth conditions in nutrient media containing an organic carbon source, such as starch, soya bean oil, glucose, a nitrogen source, such as ammonium sulfate, ammonium chloride, and mineral salts, such as CaC03, KH2P04, NaCl, MgS04.
Small quantities of the antibiotic salinomycin in the fermentation broth and high costs of the composition of the nutrient medium continually stimulate the search for better colutions with a greater yield and cheaper production.
A salimomycin molecule i~ very complicated and belongs to so-called polyether cyclic structures with a greater number of methyl and etyhl side radicals.
Chemically, polyether antibiotics are derivatives of polyhydroxy-fatty acids with a branched chain. The biosynthesis of salinomycin is probably started from acetate, propionate and butyrate precursors via the polyketide way. Of course, other similar ways are not excluded. Thus, different oils are the basis of nutrient media in cultivating said microorganism. Owing to the large quantity of oils in the nutrient medium, there is necessary ~;~''t27-13 a strong aeration and stirring, which gives rise to strong foaming, which can only be reduced by a special silicone type anti-foaming agent.
The antibiotic salinomycin and ~he microorganism ATCC 21838, which produces said antibiotic,were obtained for the first time in 1972 in the Japanese company Kakem Chemical Co. (JP patent 21553). Variants of the first process are patented, too. The yields, however, are very low, 100 to 500 /ug/ml.
~ater on, a novel sophisticated multi-stage process with novel mutants was patented, with yields even up to 60,000 ~g/ml. The nutrient media contained even up to 25 % of oil, which is very unusual for all known fermentations (EP patent 0 000 037). In said process a special silicone type antifoaming agent KM-68-2F
(Shinetsu Chemical Industry Co. Ltd.) was used.
In our research we have found that one part of the methyl side groups originates from amino acid methionine. Microorganisms obtain said acid in common nutrient media by means of soya bean meal hydrolysis. Our research has shown that the biosynthesis of salinomycin can be strongly stimulated by methionine. The stimulation is much stronger in the presence of vitamins, which are indispensable for trans-methylating and carboxylating enzyme reactionq. Said vitamins are preferably vitamin D-biotin and pyridoxin (vitamin B6).
On the basis of the above discovery, there was prepared a composition of a fermentation substrate comprising a carbon source ~soya bean oil, mollasses, starch), a nitrogen source (soya bean meal, (NH4)2SO4), inorganic salts (KCl, NaCl, CaCO3, KH2PO4), the amino acid DL-methionine and/or vitamin H, i.e.
D-biotin, or vitamin B6. With such a composition of the nutrient medium, there are achived greater yields, even for 100 %, in the same time and under the same conditions, whereas the foaming can be reduced by means of cheaper anti-foaming agents.
It is interesting that the simultaneous presence of both vitamins in the nutrient medium exhibits inhibitory action upon the biosynthesis of salinomycin.
The process for producing antibotic-salinomycin; by means of the fermentation of the microorganism Streptomyces albus ATCC 21839 is characterized in that said microorganism is cultivated under submersed aerobic conditions at a pH from 5.5 to 6.5 and a temperature from 26C to 29C at first in the vegetative phase and subsequently in the fermentation phase in a nutrient medium consisting of a carbon source, a nitrogen source, mineral salts, the amino acid DL-methionine from 0.01 to 0.15 % and/or vitamin D-biotin from 10 to 20 /ug/ml and/or vitamin B6 from 5 to 10 /ug/ml, provided tnat vitamin D-biotin and vitamin B6 are not simultaneously present in the nutrient medium.
In the vegetative phase it is cultivated for e.g. 40 to 60 hours and in the fermentation phase for e.g. 110 to 210 hours.
The following table shows the yields of salinomycin on laboratory scale.
Nutrient medium Yield: /ug/ml/200 h standard medium~ 3000 standard medium + 0.01 % methionine 4000 standard medium + 0.01 % methionine + 20 /ug/ml D biotin 6000 standard medium + 0.01 % methionine + 10 /ug/ml vitamin B6 4500 standard medium + 0.01 % methionine + 20 /ug/ml D-biotin + 10 /ug/ml vitamin B6 1700 compdsition of the medium as in Example 1 12.~ 713 In case of a stronger foaming of the fermentation medium, there are used different anti-foaming agents, such as vegetable oils, s~licone type anti-foaming agents etc.
The antibiotic is isolated from the fermented broth in a standard manner.
As mentioned above, the patent and other technical literature teaches that the foam can only be successfully destroyed with a special silicone type anti-foaming agent (KM-68-2P, Shinetsu Chemical Industry, Japan).
In the present process the composition of the medium is such that no strong foaming takes place; if, however, a foaming appears, it can be destroyed with other, cheaper anti-foaming agents.
The concentration of antibiotic salinomycin in the broth is determined by a bio-test method by means of the microorganism S. cereus.
The invention is illustrated by the following Examples and in no way limited thereto.
i3 Example 1 The microorganism Streptomyces albus 80614 (ATCC 21832) is cultivated on slant agar in a nutrient medium of the following composition:
meat extract 10 g yeast extract 4 g glucose ~4 g agar 20 g water ad1000 ml pH = 5.8 to 6.0; after sterilization 6.2 to 6.4.
The nutrient medium is previously sterilized at 120C for 20 minutes. To the nutrient medium thus prepared, a suspension of spores of Streptomyces albus is added and said suspension is incubated a 28C for 8 to 10 days. The sporulated mycelium, which grows up on the slant agar, is resuspended in 0.1 %
Tween 80 (10 ml). The suspension so prepared is added to a vegetative nutrient medium of the following composition:
glucose . 4 %
soya bean meal 1 %
yeast extract 0.5 %
ground CaC03 0.2 %
water ad 100 %
The pH of the nutrient medium after sterilization is 6.6 to 7Ø
The vegetative phase of microorganism growth lasts up to 48 hours at a temperature of 28C at 220 rpm and is used as inoculum;
the pH of inoculum is 6.4 to 6.8.
A fermentation nutrient medium is inoculated with S to 6 vol %
of the vegetative culture. The fermentation substrate has the following composition:
soya bean oil 10 %
soya bean meal 1 %
maize starch 0.5 %
lZ~Z'~i3 (NH4)2SO4 0.3 %
KCl 0.2 %
NaCl 0.2 %
KH2P04 0.02 %
3 0.5 %
MgS04 0.01 %
DL-methionine 0.01 %
water ~ ad 100 %
The fermentation substrate is sterilized at 120C for 30 minutes, the pH after the sterilization being 7.5. After the fermentation is completed, i.e. after 200 to 210 hours at 28C, the salinomycin content in the broth ;s 4000 /ug/ml. During the fermentation the concentration of the oil in the nutrient medium is continuousl~
kept between 3 and 4 % and the pH at 5.8 with a 10 % ammonia solution. The foam is destroyed with the silicone type anti-foaming agent 1510 Dow Corning.
Example 2 It is proceeded as in Example 1 with the exception that the fermentation nutrient medium additionally contains the vitamin D-biotin in the end concentration of 20 /ug/ml. After the fermentation is completed in 200 hours, the yield of salinomycin is 6000 yg/ml.
Example 3 It is proceeded as in Example 1 with the exception that the fermentation nutrient medium additionally contain~ vitamin B6 in a concentration of 10 /ug/ml. After the fermentation is completed in 200 hours, the yield of salinomycin is 4500 /ug/ml.
Example 4 It is proceeded as in Example 2, only that the composition of the fermentation nutrient medium is as follows:
soya bean oil 10 %
soya bean meal 1 %
corn steep liquor 1 %
molasses 1 %
DL-methionine 0.01 %
D-biotin 0.002 %
NH4Cl 0.11 %
3 0.5 %
NaCl 0.2 %
KCl ` 0.2 %
KH2P04 0.02 %
MgS04 0.01 %
water ~ ad 100-%
After the fermentation is completed in 190 hours, the yield of salinomycin is 8000 ~g/ml.
The present invertion relates to an improved process for producing antibiotic salinomycin by fermentation by means of the strain of the microorganism Streptomyces albus, deposited in the collection of microorganisms American Type Culture Collection, Rockville, USA, No. ATCC 21838, and in the collection of microorganisms Fermentation Research Institute, Chibe, FERM-P No. 419, Japan.
It is well-known that salinomycin is produced by means of various strains of the microorganism Streptomyces albus in aerobic growth conditions in nutrient media containing an organic carbon source, such as starch, soya bean oil, glucose, a nitrogen source, such as ammonium sulfate, ammonium chloride, and mineral salts, such as CaC03, KH2P04, NaCl, MgS04.
Small quantities of the antibiotic salinomycin in the fermentation broth and high costs of the composition of the nutrient medium continually stimulate the search for better colutions with a greater yield and cheaper production.
A salimomycin molecule i~ very complicated and belongs to so-called polyether cyclic structures with a greater number of methyl and etyhl side radicals.
Chemically, polyether antibiotics are derivatives of polyhydroxy-fatty acids with a branched chain. The biosynthesis of salinomycin is probably started from acetate, propionate and butyrate precursors via the polyketide way. Of course, other similar ways are not excluded. Thus, different oils are the basis of nutrient media in cultivating said microorganism. Owing to the large quantity of oils in the nutrient medium, there is necessary ~;~''t27-13 a strong aeration and stirring, which gives rise to strong foaming, which can only be reduced by a special silicone type anti-foaming agent.
The antibiotic salinomycin and ~he microorganism ATCC 21838, which produces said antibiotic,were obtained for the first time in 1972 in the Japanese company Kakem Chemical Co. (JP patent 21553). Variants of the first process are patented, too. The yields, however, are very low, 100 to 500 /ug/ml.
~ater on, a novel sophisticated multi-stage process with novel mutants was patented, with yields even up to 60,000 ~g/ml. The nutrient media contained even up to 25 % of oil, which is very unusual for all known fermentations (EP patent 0 000 037). In said process a special silicone type antifoaming agent KM-68-2F
(Shinetsu Chemical Industry Co. Ltd.) was used.
In our research we have found that one part of the methyl side groups originates from amino acid methionine. Microorganisms obtain said acid in common nutrient media by means of soya bean meal hydrolysis. Our research has shown that the biosynthesis of salinomycin can be strongly stimulated by methionine. The stimulation is much stronger in the presence of vitamins, which are indispensable for trans-methylating and carboxylating enzyme reactionq. Said vitamins are preferably vitamin D-biotin and pyridoxin (vitamin B6).
On the basis of the above discovery, there was prepared a composition of a fermentation substrate comprising a carbon source ~soya bean oil, mollasses, starch), a nitrogen source (soya bean meal, (NH4)2SO4), inorganic salts (KCl, NaCl, CaCO3, KH2PO4), the amino acid DL-methionine and/or vitamin H, i.e.
D-biotin, or vitamin B6. With such a composition of the nutrient medium, there are achived greater yields, even for 100 %, in the same time and under the same conditions, whereas the foaming can be reduced by means of cheaper anti-foaming agents.
It is interesting that the simultaneous presence of both vitamins in the nutrient medium exhibits inhibitory action upon the biosynthesis of salinomycin.
The process for producing antibotic-salinomycin; by means of the fermentation of the microorganism Streptomyces albus ATCC 21839 is characterized in that said microorganism is cultivated under submersed aerobic conditions at a pH from 5.5 to 6.5 and a temperature from 26C to 29C at first in the vegetative phase and subsequently in the fermentation phase in a nutrient medium consisting of a carbon source, a nitrogen source, mineral salts, the amino acid DL-methionine from 0.01 to 0.15 % and/or vitamin D-biotin from 10 to 20 /ug/ml and/or vitamin B6 from 5 to 10 /ug/ml, provided tnat vitamin D-biotin and vitamin B6 are not simultaneously present in the nutrient medium.
In the vegetative phase it is cultivated for e.g. 40 to 60 hours and in the fermentation phase for e.g. 110 to 210 hours.
The following table shows the yields of salinomycin on laboratory scale.
Nutrient medium Yield: /ug/ml/200 h standard medium~ 3000 standard medium + 0.01 % methionine 4000 standard medium + 0.01 % methionine + 20 /ug/ml D biotin 6000 standard medium + 0.01 % methionine + 10 /ug/ml vitamin B6 4500 standard medium + 0.01 % methionine + 20 /ug/ml D-biotin + 10 /ug/ml vitamin B6 1700 compdsition of the medium as in Example 1 12.~ 713 In case of a stronger foaming of the fermentation medium, there are used different anti-foaming agents, such as vegetable oils, s~licone type anti-foaming agents etc.
The antibiotic is isolated from the fermented broth in a standard manner.
As mentioned above, the patent and other technical literature teaches that the foam can only be successfully destroyed with a special silicone type anti-foaming agent (KM-68-2P, Shinetsu Chemical Industry, Japan).
In the present process the composition of the medium is such that no strong foaming takes place; if, however, a foaming appears, it can be destroyed with other, cheaper anti-foaming agents.
The concentration of antibiotic salinomycin in the broth is determined by a bio-test method by means of the microorganism S. cereus.
The invention is illustrated by the following Examples and in no way limited thereto.
i3 Example 1 The microorganism Streptomyces albus 80614 (ATCC 21832) is cultivated on slant agar in a nutrient medium of the following composition:
meat extract 10 g yeast extract 4 g glucose ~4 g agar 20 g water ad1000 ml pH = 5.8 to 6.0; after sterilization 6.2 to 6.4.
The nutrient medium is previously sterilized at 120C for 20 minutes. To the nutrient medium thus prepared, a suspension of spores of Streptomyces albus is added and said suspension is incubated a 28C for 8 to 10 days. The sporulated mycelium, which grows up on the slant agar, is resuspended in 0.1 %
Tween 80 (10 ml). The suspension so prepared is added to a vegetative nutrient medium of the following composition:
glucose . 4 %
soya bean meal 1 %
yeast extract 0.5 %
ground CaC03 0.2 %
water ad 100 %
The pH of the nutrient medium after sterilization is 6.6 to 7Ø
The vegetative phase of microorganism growth lasts up to 48 hours at a temperature of 28C at 220 rpm and is used as inoculum;
the pH of inoculum is 6.4 to 6.8.
A fermentation nutrient medium is inoculated with S to 6 vol %
of the vegetative culture. The fermentation substrate has the following composition:
soya bean oil 10 %
soya bean meal 1 %
maize starch 0.5 %
lZ~Z'~i3 (NH4)2SO4 0.3 %
KCl 0.2 %
NaCl 0.2 %
KH2P04 0.02 %
3 0.5 %
MgS04 0.01 %
DL-methionine 0.01 %
water ~ ad 100 %
The fermentation substrate is sterilized at 120C for 30 minutes, the pH after the sterilization being 7.5. After the fermentation is completed, i.e. after 200 to 210 hours at 28C, the salinomycin content in the broth ;s 4000 /ug/ml. During the fermentation the concentration of the oil in the nutrient medium is continuousl~
kept between 3 and 4 % and the pH at 5.8 with a 10 % ammonia solution. The foam is destroyed with the silicone type anti-foaming agent 1510 Dow Corning.
Example 2 It is proceeded as in Example 1 with the exception that the fermentation nutrient medium additionally contains the vitamin D-biotin in the end concentration of 20 /ug/ml. After the fermentation is completed in 200 hours, the yield of salinomycin is 6000 yg/ml.
Example 3 It is proceeded as in Example 1 with the exception that the fermentation nutrient medium additionally contain~ vitamin B6 in a concentration of 10 /ug/ml. After the fermentation is completed in 200 hours, the yield of salinomycin is 4500 /ug/ml.
Example 4 It is proceeded as in Example 2, only that the composition of the fermentation nutrient medium is as follows:
soya bean oil 10 %
soya bean meal 1 %
corn steep liquor 1 %
molasses 1 %
DL-methionine 0.01 %
D-biotin 0.002 %
NH4Cl 0.11 %
3 0.5 %
NaCl 0.2 %
KCl ` 0.2 %
KH2P04 0.02 %
MgS04 0.01 %
water ~ ad 100-%
After the fermentation is completed in 190 hours, the yield of salinomycin is 8000 ~g/ml.
Claims (5)
1. A process for producing antibiotic salinomycin by means of the fermentation of the microorganism Streptomyces albus ATCC 21839, characterized in that said microorganism is cultivated under submersed aerobic conditions at a pH from 5.5 to 6.5 and a temperature from 26°C to 29°C at first in the vegetative phase uns subsequently in the fermentation phase in a nutrient medium consisting of a carbon source, a nitrogen source, mineral salts, the amino acid DL-methionine from 0.01 to 0 15 % and/or vitamin D-biotin from 10 to 20 µg/ml and/or vitamin B6 from 5 to 10 µg/ml, provided that vitamin D-biotin and vitamin B6 are not simultaneously present in the nutrient medium.
2. Process according to claim 1, characterized in that as a carbon source there are used starch, vegetable oils, molasses.
3. Process according to claim 1, characterized in that as a nitrogen source there are used soya bean meal, (NH4)2SO4, corn steep liquor, NH4Cl.
4. Process according to claim 1, characterized in that as mineral salts there are used sulfates, phosphates, chlorides, carbonates of sodium, potassium, calcium, magnesium.
5. Process according to claim 1, characterized in that an anti-foaming agent is present and is a vegetable oil or a silicone type anti-foaming agent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| YUP2238/86 | 1986-12-24 | ||
| YU223886A YU43959B (en) | 1986-12-24 | 1986-12-24 | Process for obtaining salinomycine antibiotic |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1292713C true CA1292713C (en) | 1991-12-03 |
Family
ID=25557351
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000553599A Expired - Lifetime CA1292713C (en) | 1986-12-24 | 1987-12-04 | Process for producing antibiotic salinomycin |
Country Status (4)
| Country | Link |
|---|---|
| CA (1) | CA1292713C (en) |
| FI (1) | FI91084C (en) |
| SI (1) | SI8612238A8 (en) |
| YU (1) | YU43959B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102432623A (en) * | 2011-10-14 | 2012-05-02 | 山东鲁抗生物制造有限公司 | Solid-liquid separation method of salinomycin fermentation liquor |
-
1986
- 1986-12-24 SI SI8612238A patent/SI8612238A8/en unknown
- 1986-12-24 YU YU223886A patent/YU43959B/en unknown
-
1987
- 1987-12-02 FI FI875316A patent/FI91084C/en not_active IP Right Cessation
- 1987-12-04 CA CA000553599A patent/CA1292713C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102432623A (en) * | 2011-10-14 | 2012-05-02 | 山东鲁抗生物制造有限公司 | Solid-liquid separation method of salinomycin fermentation liquor |
Also Published As
| Publication number | Publication date |
|---|---|
| FI91084B (en) | 1994-01-31 |
| SI8612238A8 (en) | 1996-06-30 |
| YU223886A (en) | 1988-06-30 |
| FI91084C (en) | 1994-05-10 |
| YU43959B (en) | 1989-12-31 |
| FI875316A (en) | 1988-06-25 |
| FI875316A0 (en) | 1987-12-02 |
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