Note: Descriptions are shown in the official language in which they were submitted.
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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
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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.
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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
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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.
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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 %
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(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 %
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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.
