BACKGROUND OF THE INVENTION:
[0001] The present invention relates to an improvement in a method of producing polyether
type antibiotics on an industrial sc le.
[0002] As polyether type antibiotic,there have been known Monensin (sournal of American
Chemical Society, Vol.89, page 5757, 1 67), X-206 (Chemical Communications. 927, 1971),
Salinomycin (Br tish Patent No. 1,378,414), SY-1 substance (Japanese O.P.I. 8619
1/76), S' 2 substance (Japanese Patent Application No. 5762/77), 4-methylsalinomycin
(A 2808b substance) Japanese O.P.I. 9788/76, Losalocid (Journal of American Chemical
Society, Vol.73, 5295, 1951) Dianemycin ( Journal of Antibiotics, Vol. 22, page 161,
1969 ),
Nigericin ( Biochemical and Biophysical Research Communication, Vol.33, page 29, 1968
), A-204 A ( Journal of American Chemical Society, Vol. 95, 3399, 1973 )
and the like, and among these, Salinomvcin, 4-methvlsalino- mycin, SY-1, SY-2, SY-3,
SY-4, SY-5, SY-6, SY-7 and SY-8 substance are called Salinomycin type antibiotics
because they have similar chemical structures.
[0003] In this invention, the term "salinomycins" means each compound, or any mixture of
at least two compounds, selected from the group SY-7, SY-8 consisting of salinomycin,
SY-1, SY-2, SY-3, SY-4, SY-5, SY-6/and the like.
[0004] The present inventors already found that salinomycin, SY-1 and SY-2 were produced
in the culture of Streptomyces albus waxman and henrich No.80614 strain ( FERM-P.
No.419 ), and succeeded in isolating the antibiotics from the culture ( British Patent
No.1378414, Japanese Unexamined Patent Publication No. 86191/1976 and Japanese Patent
Application No. 5762/1977).
[0005] The inventors continued the study and found that, when the above strain is cultured
in a medium containing fatty acid or its precursor, it produces salinomycin, SY-1,
and SY-2
'in high yield, and also produces new compounds such as SY-3, SY-4, SY-5, SY-6, SY-7
and SY-8.
[0006] As described in said literatures, these are produced by culturing an antibiotic producing
microorganism belonging to the genus of Streptomyces. However, the yield of each antibiotic
produced by such known method is not always satisfactory.
SUMMARY OF THE INVENTION:
[0007] It is an object of the present invention to provide a method of producing polyether
type antibiotics in remarkably high yields with industrial advantages.
[0008] Another object of the present invention is to provide a method of producing Salinomycin
type antibiotics such as salinomycin, 4-methylsalinomycin, SY-1, SY-2, SY-3, SY-4,
SY-5, SY-6, SY-7 and SY-8 substances in high yield.
[0009] The foregoing and other objects of the present invention have been attained by culturing
a polyether type antibiotic-producing microorganism in a medium containing a fatty
acid or its precursor and ammonia or an ammonium salt or urea.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
[0010] In the special feature of the present invention, salinomycin type antibiotics such
as salinomycin, 4-methylsalinomycin, SY-1, SY-2, SY-3, SY-4, SY-5, SY-6, SY-7 and
SY-8 substances are produced by culturing a salinomycin type antibiotic-producing
microorganism in a medium containing a fatty acid or its precursor and ammonia or
an ammonium salt or urea.
[0011] The fatty acids used in the present invention are saturated or unsaturated fatty
acids, for example, acetic acid, propionic acid, caproic acid, capric acid, palmitic
acid, stearic acid, methacrylic acid, undecylic acid, and particularly linolic acid,
linolenic acid or oleic acid being preferable. The precursor of fatty acid means a
substance that is capable of giving said fatty acid .outside or inside the microorganism
cell, such as mono- di- or triglycerides of fatty acid, esters of fatty acid or salts
of fatty acid. Further, there can be used soy bean oil, safflower oil, cotton seed
oil, sesame oil, olive oil, rape oil, peanut oil, maize oil (corn oil), sunflower
oil and like vegetable oils, cod oil and like fish oils and lard and like animal fat-and-oils,
which contain said precursors. esters with The esters of fatty acid can be/C1-C18
alcohols of said fatty acid.
The salts of fatty acid can be ammonium salt and alkali metal salts and alkaline earth
metal salts of said fatty acid.
[0012] The addition amount is generally about 1-25
%, particularly about 12-20% based on the medium.
[0013] Ammonia is used in gaseous form or in the form of an aqueous solution. As ammonium
salt there are used ammonium salts of inorganic acids such as hydrochloric acid, sulfuric
acid, nitric acid and phosphoric acid or organic acids such as acetic acid, propionic
acid, higher fatty acid, oxalic acid, tartaric acid, hydrogentartaric acid, citric
acid, lactic acid and malic acid. The addition amount is generally about 0.1-1.0%
particularly about 0.3-0.5% based on the medium.
[0014] As for the time for addition of these additives, addition is effective so long as
production of potency of I the addition polyether type antibiotic continues, and
/is conducted at any time either before or after beginning of cultivation. Although
the cultivation conditions of the present invention can be selected in accordance
with the methods described in said known literatures, excepting that fatty acid or
its precursor is used as major carbon source and that ammonia or ammonium salt is
used as essential component of the medium, it is possible to raise the production
efficiency by varying the conditions depending on the kind of antibiotics.
[0015] The microorganismsused in the present invention include generally polyether type
antibiotics producing strains belonging to the genus of Streptomyces as well as the
strains described in said literatures and their natural or artificial mutants.
[0016] Separation and purification of the products can be carried out in accordance with
known methods. Since the objective substance is often contained mainly in the solid
portion containing the cells when the production amount of the objective substance
is abundant, it is desirable to vary the extraction step properly in order to heighten
the recovery percentage of the objective substance from the solid portion. It is possible
to use the objective compound in the state that it is contained in the solid portion
depending on the use purposes, without separating the objective substance from the
solid portion.
[0017] According to the present invention the production amount of polyether type antibiotics,
particularly Salinomycin type antibiotics can be remarkably increased. For example,
the yield of Salinomycin is generally 100-300 Y/ml in known method, whereas the yield
is about 10,000-20,000 y/ml in the medium containing fatty acid or its precursor,
and the yield is further increased to about 50,000-80,000 Y/ml when said medium contains
further ammonia or ammonium salt.
[0018] The preferable feature of the present invention to produce salinomycin type antibiotics
will be further illustrated.
[0019] According to the method of this invention, salinomycins mainly occur in the mycelial
mass,and it is preferrable to recover salinomycins from the mycelial mass. In addition
to salinoriiycin, SY-1, and SY-2, other new compounds, especially SY-3, SY-4, SY-5,
SY-6 and the like are obtained from the culture.
[0020] The strains used in this invention include Streptomyces albus No.80614 and its mutants
artificially or naturally produced, as well as the other Streptomyces strains capable
of producing salinomycins. However, some of the salinomycins can occasionally not
be detected in the culture, depending on the strain and fermentation conditions.
[0021] Fermentation conditions employed in this invention can be any one commonly used for
culturing Actinomycetes, except that main carbon source should be a fatty acid or
its precursor. Maximum production of salinomycins usually occurs after 150 to 260
hours The from the start of fermentation. /ratio of each of salinomycins produced
sometimes varies depending on the incubation time. Naturally, the composition of medium
and fermentation conditions should be decided for each strain and external conditions,
so that most desirable results are obtained.
[0022] Salinomycins can be isolated from the culture medium by utilizing the physico-chemical
properties of salinomycins. Because salinomycins are structurally related to each
other, the known extraction methods for salinomycin and SY-1 can be applied to the
isolation of salinomycins, however, as for salinomycin, because a large portion of
salinomycin is contained in mycelial mass, the extraction process is preferrably modified
so as to increase the recovery rate of salinomycin. For example, it is preferred to
adjust the whole fermentation broth to PH 2.0-6.0 to precipitate salinomycin, and
then to extract the mycelial mass together with the precipitate thus formed with an
organic solvent. Among the preferred solvents are acetone, ethyl acetate, butyl acetate,
n-hexane, chloroform and the like. After applying the solution to the absorptive materials
suitable for the absorption of salinomycins, the salinomycins are eluted by a suitable
solvent system.
[0023] The effluent is collected in 2-5 fractions according to the purpo-e. Separation and
purification of salinomycins are effected by the procedures utilizing the difference
between the properties of desired compound and imprities. Chromatograpy, solvent extraction
and the like are repeated for each fraction to give salinomycin, SY-1, SY-2, SY-3,
SY-4, SY-5, SY-6, SY-7, SY-8 anc the like, individually or as mixtures. The resulting
products can be further purified by recrystallization or chromatography. The properties
of salinomycins obtained by the process of this invention are shown in the following
table 1.

[0024] According to the preferred feature of this invention, salinomycin, SY-1, and SY-2,
especially salinomycin are obtained in surprisingly higher yield than the known process.
New compounds, SY-3 through -8 are also obtained according to this invention. They
are comparable to salinomycin in their activities against microorganisms and useful
as medicines. Also, because of their structural similarity to salinomycin, their usefulness
as veterinary medicines looks very likely.
REFERENCE EXAMPLE 1:
[0025] Streptomyces albus waxman and henrich No.80614 strain (FERM-P. No.419) is inoculated
to a medium containing glycerine 2.0%, peptone 0.5% and meat extract 0.5% and cultured
at 33°C for 48 hours under shaking. 1 litre of this culture liquid is inoculated to
100 litres of liquid medium ( 200 litre tank made of stainless steel ) containing
glucose 2%, starch 1%, soy bean powder 2.5%, beer yeast 0.4%, meat extract 0.1%, sodium
chloride 0.2% and antifoaming agent KM-68-2F ( product of Shinetsu Chemical Industry
Co., Ltd., silicone type ) 0.1% and cultured at 33°C for 144 hours under stirring
with the aeration volume of 100 1/m. There is obtained a culture liquid containing
.100-300 y/ml of Salinomycin.
REFERENCE EXAMPLE 2:
[0026] The 80614 strain which is the same strain as described in Example 1 is inoculated
to a medium containing glycerine 2.0%, peptone 0.25%, meat extract 0.5% and edible
salt 0.1%, and cultured at 33°C for 48 hours under shaking. This culture liquid in
an amount corresponding to 1% of the medium is inoculated to the medium containing
glucose 4.0%, soy bean powder 1.0%, beer yeast 1.0% and calcium carbonate 0.2%, and
cultured for 30 hours at 33°C under shaking, and this culture liquid is made the second-stage
pre-culture liquid. One litre of this second-stage pre-culture liquid is inoculated
to 100 litres of liquid medium containing soy bean oil 10%, glucose 1.0%, soy bean
powder 1.0%, calcium carbonate 0.5%, potassium secondary phosphate 0.01%, and cultured
for 210 hours at 33°C with an aeration volume of 100 1/m under stirring. There is
obtained a culture liquid containing 20,000 Y/ml of Salinomycin.
EXAMPLE 1:
(A) Production of salinomycin:
[0027] The 80614 strain which is the same strain as described in Example 1 is inoculated
to a medium containing glycerine 2.0%, peptone 0.25%, meat extract 0.5% and edible
salt 0.1%, and cultured at 33°C for 48 hours under shaking. This culture liquid in
an amount corresponding to 1% of the medium is inoculated to the medium containing
glucose 4.0%, soy bean powder 1.0%, beer yeast 1.0% and calcium carbonate 0.2%, and
cultured for 30 hours at 33°C under shaking, and this culture liquid is made the second-stage,
pre-culture liquid. One litre of this second-stage pre-culture liquid is inoculated
to 100 litres of liquid medium containing soy.bean oil 10%, glucose 1.0%, soy bean
powder 1.0%, calcium carbonate 0.5%, potassium secondary phosphate 0.01%, and cultured
for 210 hours at 33°C with an aeration volume of 100 1/m under stirrinq. There is
obtained a culture liquid containing 20,000 γ/ml of salinomycin(salinomycin only).
(B) Separation and purification of salinomycin, SY-1 and SY-2.
[0028] The culture liquid obtained in (A) is adjusted to PH 4.5-5.0 with dilute hydrochloride,
admixed with 4% by weight per volume of filter aid with stirring, and filtered. The
filtrate ( 80 litres ) is extracted with 50 litres of butyl acetate with stirring.
Mycelial mass is extracted with 30 litres of butyl acetate. Both butyl acetate solutions
are combined and washed with 20 litres of 5% aqueous sodium bicarbonate solution.
One litre of the washed butyl acetate solution is concentrated under vacuum to dryness
to give 250g of crude powder of salinomycin containing trace amount of SY-1 and SY-2.
[0029] Fifty grams of the crude salinomycin powder is dissolved in a ethyl acetate and applied
to/column ( 50g of active almina, commercial product of Wako Junyaku Co. ). After
washing the column with one litre of ethyl acetate, salinomycin and SY-1 are eluted
with a 100:5 mixture of ethyl acetate-methanol solution. The fractions containing
salinomycin and SY-1 are combined and concentrated. The concentrate is diluted in
50ml of chloroform-methanol solution ( 100:2 ) and applied to the column of 300g silica
gel ( Wakogel-200, Wako Junyaku Co. ) packed in the same solvent mixture. Elution
is carried out with the same solvent mixture to give pure salinomycin fraction and
pure SY-1 fraction. Each fraction is concentrated and crystallized from acetone-water
solution to give 5g of salinomycin and 30mg of SY-1 in pure crystals, respectively.
[0030] The almina column as mentioned above is further developed with a 100:15 mixture of
ethyl acetate-methanol to elute SY-2, which is separated and purified by silica gel
chromatography in the same manner as described above, and crystallized from acetone-water
solution to give 3mg of pure crystalline SY-2.
EXAMPLE 2:
(A) Salinomycin is cultured in the same manner as described in
Example 1.
(B) Recovery of salinomycins
[0031] The culture obtained in (A) is adjusted to pH 4.5-5.0, heated at 60°C for 10 minutes
with stirring, admixed with 4% by weight per volume of filter aid with stirring and
filtered. The resulting mycelial mass contains salinomycins and SY group compounds
in high yield.
(C) Separation of salinomycins complex
[0032] Mycelial mass obtained in (B) is extracted twice with 50 litres of ethyl acetate.
The extracts are combined and washed with 50 litres of 5% aqueous sodium bicarbonate
solution. Ethyl acetate phase is concentrated under vacuum to dryness to yield 7kg
of crude salinomycins complex containing SY group compounds.
(D) Separation and purification of salinomycin
[0033] Seven kg of crude powder obtained in (C) is dissolved in 100 litres of hexane, concentrated
under vacuum to 20 litres and left at 5°C to give 2.7kg of crude crystallin salinomycin.
After repeating this procedure, recrystallization of salinomycin from hexane gives
2.4kg of pure salinomycin sodium salt.
(E) Separation and purification of SY group compounds
[0034] The mother liquor separated from the precipitate in the process in (A) is concentrated
to dryness to give 4.6kg of complex contain-- ing a small amount of salinomycin and
SY group compounds. The complex is dissolved in 50 litres of hexane-ethyl acetate
solvent mixture (2:1) and applied to the column of 12kg of almina packed in the same
solvent system, developed with 20 litres of a 100:2 mixture of ethyl acetate-methanol
to elute SY-3, SY-7 and salinomycin. Two hundred g of the mixture of salinomycin and
SY-1 is separated by precipitation in the same manner as described in (D). The filtrate
containing SY-3 and SY-7 is concentrated to dryness to give 100g of crude powder.
[0035] One hundred g of the crude powder thus obtained is dissolved in 300ml of a 100:2
mixture of chloroform-methanol and applied to the column of 6kg of silica gel (Wakogel
C-200, Wako Junyaku Co.), and developed with the same solvent mixture. Each of SY-3
and SY-7 fractions is concentrated and applied to silica gel thin layer chromatogram
(Wakogel B-10 with thickness of 0.25 mm, solvent system; a 20:1 mixture of chloroform-methanol).
SY-3
'and SY-7 are collected by scraping off the areas with Rf values 0.45 and 0.80, respectivelly.
The silica gel holding each compound is eluted with a 20:1 mixture of chloroform-methanol
and the solvent mixture is removed by evaporation to give l8mg of SY-3 and 35mg of
SY-7 both in pure powders.
[0036] The above almina column is eluted with 20 litres of a 5:1 mixture of ethyl acetate-methanol,
and the effluent is concentrated under vacuum to give 200g of crude powder, which
contains the complex of SY-2, SY-4, SY-5, SY-6 and SY-8. These compounds are isolated
individually by silica gel column chromatography. Two hundred g of the crude powder
is dissolved in 500ml of a 100:2 mixture of chloroform-methanol, applied to the column
of 2kg silica gel (Wakogel C-200) packed in the same solvent mixture, and developed
with the same solvent mixture to give.each fraction containing SY-2, SY-4, SY-5, SY-6
and SY-8, respectively. Each fraction is concentrated to dryness to give 20g of SY-2,
300mg of SY-4, 250mg of SY-5, 800mg of SY-6 and 100mg of SY-8 as crude powders, respectively.
Crude SY-2 powders are crystallized from acetone water to give 15g of pure crystalline
SY-2.
[0037] Other crude powders are purified by thin layer chromatography of silica gel in the
same manner as employed in the separation and purification of SY-3 and SY-7 to give
26mg of SY-4, 20mg of SY-5, 80mg of SY-6 and 30mg of SY-8 each in pure powders.
EXAMPLE 3:
[0038] (A) The second-stage pre-culture liquid of Example 1 in an amount corresponding 10%
of the medium is inoculated to the medium containing glucose 4%, soy bean powder 3%,
defatted wheat germs 3.0%, calcium carbonate 0.2% and antifoaming agent KM-68-2F 0.1%,
and cultured for 24 hours at 33°C to give the third-stage pre-culture liquid.
[0039] Ten litres of the third-stage pre-culture liquid is inoculated to the medium containing
soy bean oil 16%, soy bean powder 0.5%, defatted wheat germs 1.0%, sodium chloride
0.2%, potassium chloride 0.2%, ammonium sulfate 0.3%, calcium secondary phosphate
0.02%, magnesium sulfate 0.01% and antifoaming agent KM-68-2F 0.1%, and cultured for
290 hours at 33°C with an aeration volume of 100 1/m under stirring. The production
amount of salinomycin at the end of cultivation is 60,000 Y/ml (salinomycin only).
- In this case the similar production amount is attained even when soy bean oil is
added in a small amount at the beginning and then the addition amount is increased.
No difference in production amount is observed between cases in which silicone type
and polyether type antifoaming agents are used.
[0040] (B)
'rreat the fermentation broth obtained in (A) according to the procedure of Example
2 (B) to give mycelial mass containig salinomycins in high yield.
[0041] (C) Treat the mycelial mass obtained in (B) according to the procedure of Example
2 (C) to give l6kg of the complex of SY-1, 2, 3, 4, 5, 6, 7 and 8.
[0042] (D) Dissolve l6kg of crude powder obtained in (C) in 100 litres of hexane and concentrate
under vacuum to 40 litres. Then treat the solution according to the same procedure
described in Example 2 (D) to give 7.lkg of crude crystallin salinomycin and then
6.3kg of pure salinomycin sodium salt.
[0043] (E) The mother liquor separated from crystals in the process in (D) is concentrated
to dryness to give 9.7kg of the complex containing SY-1 through -8. The complex is
dissolved in 100 litres of a 2:1 mixture of hexane-ethyl acetate, applied to the column
of 20kg of aluminain the same manner as described in Example 2 (E) and developed with
30 litres of a 100:2 mixture of ethyl acetate-methanol to elute SY-1, SY-3 and SY-7.
The effluent is treated in the same manner as described in Example 2 (E) to give 400g
of salinomycin-SY-1 mixture and 130g of crude powder containing SY-3 and SY-7.
[0044] One hundred and thirty g of said crude powder is dissolved in 400ml of chloroform-methanol
solution (100:2), treated by silica gel column chromatography (8kg of silica gel is
used) to give SY-3- and SY-7-fractions. Each fraction is chromatographed over silica
gel thin layer according to Example 2 (E), and after removing the solvent by evaporation,
30mg of SY-3 and 50mg of SY-7 are obtained as pure powders.
[0045] The above aluminacolumn is eluted with 40 litres of a 5:1 mixture of ethyl acetate-methanol
and the effluent is concentrated to dryness to give 250g of crude powder containing
the complex of SY-2, SY-4, SY-5, SY-6 and SY-7. The complex is dissolved in 600ml
of chloroform-methanol solution(100:2), and then treated by chromatography(20kg silica
gel) in the same manner as described above to give 40g of SY-2, 600mg each of SY-4
and SY-5, 1900mg of SY-6 and 300ing of SY-8 as crude powders, purification of which
according to the procedure of Example 2 (E) gives 30g of crystallin SY-2, 180mg of
SY-4 powder, 120mg of SY-5 powder, 350mg of SY-6 powder and 90mg of SY-8 powder, all
in pure form.
EXAMPLE 4:
[0046] The third-stage pre-culture liquid in Example 3 in an amount corresponding to 10%
of the medium is inoculated to the medium (50ml-) containing each fat-and-oil shown
in the following Table 12%, soy bean powder 0.5%, defatted wheat germs 1.0%, sodium
chloride 0.2%, potassium chloride 0.2%, calcium carbonate 0.5%, ammonium sulfate 0.3%,
potassium secondary phosphate,0.02% and magnesium sulfate 0.01%, and cultured at 33°C
for 216 hours under shaking. The production amounts of salinomycin at the end of cultivation
are shown in the following Table.

[0047] From the resulting culture, salinomycins are separated individually and purified
according to the procedures of Examples 1 - 3.