Note: Descriptions are shown in the official language in which they were submitted.
~Z1~7~
METHOD OF PROMOTING GROWTH USING
ZINC-CONTAINING ANTI:BIOTIC AGENTS
This invention relates to the use and preparation of zinc
complexes of various polyether an-tibiotics as growth-promoting sub-
stances in food-producing animals such as cattle, sheep and hogs.
It is contemplated that the subject zinc complexes of polyether
antibiotics may be administered in feed compositions and feed
additive compositions, or as implants.
According to the present invention, there is provided a
process to prepare a biomass of a zinc complex of a selected
polyether antibiotic for promoting growth and enhancing feeding
efficiency in food-producing animals, said biomass being prepared
by: (a) fermenting a fermentation broth inoculated with a
Streptomyces microorganism capable of producing by fermentation of
the broth a polyether antibiotic selected fxom the group consisting
of linear monovalent polyethers, non-nitrogen containing divalent
polyethers, non-glycolic monovalent polyethers, mononitrogen-
containing divalent pyrrole ethers, polynitrogen-containing dival-
ent pyrrole ethers, glycolic monovalent monoglycoside polyethers,
ionomycin, aabomycin, disnerycin, duamycin, BL-580, K-41, SF-1195,
2~ M-4164A, A-32887, 30,504RP, 38,986, 44,161, 47,433, 47,434, and
47,224, for a period of time and under suitable fermentation
conditions in order to produce said polyether antibiotic in said
fermentation broth; (b) providing in said antibiotic-containing
fermentation broth a water-soluble zinc salt in an amount sufficient
to form a zinc complex of said polyether antibiotic, which complex
is insoluble in the fermentation broth; and (c) recovering said
biomass of insoluble material from said fermentation broth, said
2 -
~''
~20~5a:Z
biomass containing both a zinc complex of said polyether antibiotic
and insoluble zinc complexes of residual nitrogen-containing
compounds present in the fermentation broth.
Preferably the zinc complex of the polyether antibiotic
has a molecular weight in the range of 300-1800, and the polyether
antibiotic is selected from the group consisting of linear mono-
valent polyether antibiotics and non-nitrogen containing divalent
polyether antibiotics.
Polyether antibiotics can be generally characterized as
carboxylic acid ionophores which can be roduced by growing
Streptomyces type microorganisms in suitable nutrient media. These
polyether antibiotics have a basic structure generally consisting
essentially of the elements oxygen, hydrogen and carbon (and some-
times nitrogen) and have a molecular weight in the range of about
300 to about 1800, most often from about 400 to about 1200. They
have low solubility in water, are generally soluble in low molecular
weight alcohols, ethers, and ketones; and have at least one, and
usually one or two, carboxylic acid groups. A generally comprehen-
sive review of this class of antibiotics is set forth in Westley,
Adv. Appl. Microbiology 22, 177-233 (19771. As is mentioned there-
in, at least twenty different polyether antibiotics were known at
the time the article was written. Since then, additional polyether
antibiotics have been discovered.
In Westley (op. cit.), the known polyether antibiotics
are divided into four separate classes based on the ability
of the particular antibiotic to effect the transport of
- 2a -
-3- ~ 2~ 5 8 2
monovalent and divalent cations and based on the chemical
structure of the particular antibiotic. ~estley's classi~i-
cation system is adopted herein.
Westley defined Class la as monovalent polyether
antibiotics. In addition, the Class la polyether anti-
biotics have a generally linear configuration, i.e., the
carboxylic portion of the polyether molecule is attached
either directly or indirectly to a terminal ring structure,
and include about four to six tetrahydropyran and/or -furan
structures, and up to six total ring structures. Class la
includes monensin, laidlomycin, nigericin, grisorixin,
salinomycin, narasin, lonomycin, X-206, SY-l noborito-
mycins A and B, mutalomycin and alborixin. Class la anti-
biotics may also be described as "linear monovalent poly-
ether antibiotics".
According to Westley's system, monovalent mono~lyco-
side polyether antibiotics belong to Class lb. These poly-
ether antibiotics include a glycoside type structure, more
specifically, a 2,3,6-trideoxy-4-O-methyl-D-erythrohexa-
pyranose moiety, which is attached to the polyether mole-
cule such that a non-linear type molecule is formed, i.e.,
the carboxylic portion of the polyether molecule is attached
either directly or indirectly to a non-terminal rin~ struc-
ture or the molecule has a side chain ring structure, e.g.,
a 2,3,6-trideoxy-4-O-methyl-D-erythrohexapyranose moiety.
The polyether antibîotics of this class usually contain
about six or seven tetrahydropyran and/or -furan structures.
lZ075~2
--4--
Class 2a antibiotics as defined by Westley are di-
valent polye~hers, and have generally linear configura-
~ tion. They may contain from about two to about threetetrahydropyran and/or -furan structures, and up to about
three total ring structures. Nitrogen atoms are not
. present in the Class 2a molecules. Included within Class
2a are lasalocid and lysocellin. The Class 2a polyether
antibiotics are hereinafter sometimes designated "non-
nitrogen containing divalent polyether antibiotics".
Class 2b in Westley's system are divalent pyrrole
polyethers. In contrast to the other classes, the Class
2b antibiotics contain one or more nitrogen atoms.
Lasalocid is included in Class 2a as defined by
Westley. Lasalocid was discovered by Julius Berger et al
in media fermented with a _reptomyces microorganism
isolated from a sample of soil collected at Hyde Park,
Massachusetts. [Cf. Berger et al, J. Amer. Chem. Soc.
73, 5295-8 (1951)]. Originally this material was known
20 by the code name X-537A. About 1969 lasalocid was found
to possess coccidiostatic activity. Later this activity
was established for monensin, nigericin, salinomycin, and
narasin all of which belong to Class 1a.
The polyether antibiotics have usually been
recovered and employed in the form of their sodium salts.
For example, a process for recovering lasalocid from its
fermentation broth is disclosed in the Berger et al
article (op. cit.). In this process, the antibiotic or
its alkali metal salts are extracted into various organic
solvents with subsequent evaporation of the solvents in a
multi-step operation.
lZ1~75~Z
A process for the recovery of carriomycin from fer-
mentation beer is described by Imada et al in J. Antibiotics
; ~ 31, 7-14 (1978). In the disclosed process, fermented beer
containing the carriomycin antibiotic was adjusted in pH
with concentrated NaOH and acetone was then added. After
stirring the mixture for 1 hour at room temperature, mycelia
were filtered off and extracted again with acetone. The
extracts were combined and concéntrated in a vacuum until no
acetone remained. The concentrated aqueous solution was
extracted twice with equal volumes of ethyl acetate,
followed by drying with anhydrous Na25O4. The extracts were
concentrated in a vacuum and passed through a column of
activated charcoal, then the column was washed with ethyl
acetate. The fractions active against ~ aureus
FDA 209P were combined and the solvent was evaporated. To
the oily residue was added n-hexane. The resultant solid
material was collected by filtration and crystallized from
aqueous acetone. On recrystallization from aqueous acetone,
2~ crystals of the mixed sodium and potassium salts of
carriomycin were obtained, the mixture was dissolved in
aqueous a~etone, and the solution was extracted twice with
equal volumes of ethyl acetate. The extracts were dried
with anhydrous Na2SO4 and concentrated to dryness in a
vacuum. The resultant crystalline powder was recrystallized
from aqueous acetone to yield carriomycin free acid.
As is apparent from the above example, such processes
can be quite complicated and can require the use of
relatively large quantities of various organic solvents, at
least some of which may be quite expensive. In addition,
1~ -6- ~207S~
such solvent recovery processes inevitably will suffer anti-
biotic yield losses as well as losses of the various organic
solvents used in the process. There is thus a continuing
need for antibiotic preparation and recovery processes which
effectively and efficiently produce polyether antibiotics
in a form suitable for use zs feed additives.
Zinc complexes of polyether antibiotics can be
advantageously formed by adding water soluhle~zinc salts to
the ~ermentation broth in which such antibiotics have been
produced. When formed in a fermentation beer, the formation
of these complexes facilitates the recovery of-the po~yether
antibiotics from the fermentation beer in which the anti-
biotics have been produced by, among other things, avoiding
the necessity of using recovery methods which involve
extractions with organic solvents followed by their subse-
quent purification and reuse. The resulting broth-insoluble
zinc complexes of the antiblotics can then be recovered ~rom
the broth and employed, for instance, as feed efficiency
improving and growth-promoting agents for food-producing
mammals including cattle, sheep and swine. The zinc com-
plexes may be administered either as a feed additive or as
a subcutaneous i~plant.
An antibiotic-containing fermentation broth can be
prepared in conventional manner by fermenting a nutrient-
containing liqùid fermentation medium inoculated with a
Streptomyces microorganism capable of producing the desired
antibiotic. Suitable liquid fermentation media are gener-
ally aqueous dispersions containing a source of assimilable
nitrogen and carbohydrates. Nitrogen sources for use in
-7- ~20~5~
the fermentation media herein can include, for example,
yeast, yeast-derived products, corn meal, bean meal, e.g.,
soy bean meal, etc. Carbohydrate sources for use in the
fermentation media herein can include for example, sugar,
molasses, corn-steep liquor and the like. The fermentation
media can also contain a variety of optional ingredients,
i~ desired, such as for example, pH adjustment agents,
buffers, trace minerals, antifoam agents, filter aids, etc.
The antibiotic can be prepared by growing the
Streptomyces microorganism in an aerated, agitated, sub-
merged culture with the pH of the broth adiusted to about
neutral, i.e., from about 6.5 to 7.5. Fermentation can
generally be carried out at slightly elevated temperatures,
e.g., between about 25C and 35C. Incubation of the broth
can be carried out for a period of several days, e.g., from
about 4 to 6 days or longer if it is economically advantage-
- ous to do so.
The novel zinc complexes of the present invention can
be formed from any of the known polyether antibiotics which
include: linear monovalent and divalent polyethers (monen-
sin, nigericin, lasalocid, lysocellin, etc.); non-glycolic
monovalent monoglycoside polyethers (septamycin, dianemycin,
lenoremycin, carriomycin and antibiotic A-204); mononitrogen-
containing divalent pyrrole ethers (calcimycin, X-14547, etc.~;
polynitrogen-containing divalent pyrrole ethers (A-23187,
35c); glycolic monovalent monoglycoside polyethers ~ethero-
mycin, etc.); other polyether antibiotics including iono-
mycin, aabomycin, disnerycin, duamycin, BL-580, K-41, SF-
1195, M-4164A, A-32887, 30,504RP, 38,986, 44,161, 47,433,
_ _ ~, ~ _ _ . ... , , . . _ . . ... . .... .. . , .... ... .... , . . . . .. _ .... . . . ... . . . _ _ _ _
2075~lZ
47,434 and 47,224.
Detailed descriptions of these antibiotics are pre-
sented in succeeding paragraphs.
DESCRIPTION OF SPECIFIC ANTIB IOTICS
A more detailed description of members of Westley's
Class la polyether antibiotics is given below. These anti-
biotics have a generally linear configuration. Their zinc
complexes can be made as described herein.
Nonensin can be produced by inoculating the above
described fermentation medium with a Str~to~yces cinnamon-
ensis microorganism. Such a microorganism is on unrestricted
deposit under the number ATCC 15413 at the American Type
Culture Collection, 12301 Parklawn Drive, Rockville, Mary-
land 20852 (hereinafter referred to as the American Type
Culture Collection).
Monensin is characterized chemically as 2-15-ethyl-
tetrahydro-5-]tetrahydro-3-methyl-5-ltetrahydro-6-hydroxy-6-
(hydroxymethyl)-3,5-dimethyl-2H-pyran-2-yl]-2-furyl]-2-
furyl]-9-hydroxy-~-methoxy-a,y,2,8-tetramethyl-1,6-dioxa-
- spiro[4.5]~ecane-7-butyric acid. This material has the
following structural formula:
c~
Ct~ < ~C~
C~ o C~IO~
~O--C--C--C--C
c c~, H ~ c~l, , -
Monensin
-.. - ........ . .
-9- ~20~582
Monensin is described in greater detail in U.S. Patent
3,501,568 and U.S. Patent 3,794,732.
Nigericin can be produced by inoculating the fermen-
tation medium with a Streptomyces violaceoniger micro-
organism. Such a microorganism is on unrestricted deposit
at NRRL B1356 at the Northern Research and Development
Division, Agricultural Research Service, United States
Department of Agriculture, Peoria, Illinois (hereinafter
referred to as the Agricultural Research Service).
Nigericin is characterized chemically as a stereo-
isomer of tetrahydro-6-([9-methoxy-2,4,10-trimethyl-2-
[tetrahydro~5-methyl-5-ltetrahydro-3-methyl-5-[tetrahydro-
6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-2H-pyran-2-yl]-2-
furanyl]-2-furanyl]-1,6-dioxaspiro(4.5)dec-7-yll-methyl-
,3-dimethyl-2H-pyran-2-acetic acid). This antibiotic has
the following structural formula:
O CR, Cll, Ctl, CH,
~CII~
Nigericin
Nîgericin is also known by the names polyetherin A,
antibiotic X~464, antibiotic K178, helexin C and azolomycin
M. Nigericin (and its characteristics and preparation) is
described in greater detail in U.S. Patent 3,555,150; U.S.
Patent 3,794,732, Harned et al, Antibiotics and Chemotherapy,
Vol. 1, No. 9 ~December, 1951) pp. 594-596; Steinrauf et al,
Biochemical and Biophysical Research Communications, Vol.
,
-
` -10- ~20'75~2
33, No. 1 (1968) pp. 29-31 and Stempel et al, The Journal
of Antiblot _s, Vol. XXII, No. 8 (August, 1969) pp. 384-385.
Salinomycin can be produced by inoculating a fermen-
tation medium with a Streptom~ces albus microorganism which
is on deposit under number ATCC 21838 at the American Type
Culture Collection mentioned previously. Salinomycin was
reported by Miyazaki et al, J, Ant_biotics 27, 814-21 (1974)
as having the following structural formula:
, ' '
~I .Mo Me~ MD
~A ' 01l O ~ r ~ Mo J Et
~11 ~M~ 1:1 ~OH . Mo
Salinomycin
The above article sets forth methods of preparation and
properties of salinomycin and U.S. Patent 3,857,948 to
Tanaka et al al50 discloses methods for the preparation of
the salinomycin antibiotic.
Narasin (also known as 4-methylsalinomycin) can be
produced by inoculating a fermentation medium with a
Streptomyces aureofaciens microorganism which is on
unrestricted deposit at the Agricultural Research Service
mentioned previously under culture numbers NRRL 5758 and
.8092. The structure of narasin was reported by Berg et al,
J. Antibiot~cs 31, 1-6 (1978) as the following:
12075~Zr
Me ~OH 1 ~ OIJ
1~H H ~ ~H ~ ~ ~
C02H Me ~le Et OH Me
Narasin
-
The antibiotic is also the subject of U.S. Patent Nos.
4,035,481 and 4,038,384 to Berg et al:
The antibiotics noboritomycin A and B are the fermenta-
tion products of the microorganism ~ noboritoen-
sis which is on deposit at Agricultural Research Service
under the number NRRL 8123. A method for the preparation of
these antibiotics and their chemical structure was reported
by Keller-Juslen et al in J. Antibiotics 31, 820-828 (1978).
The antibiotics have the structural formula:
HO O Me Me Me ~3 ~
~ b Me Me k~ ~ ~ ~
~ ~ ~2~ ~ ~ U H O-Et
~ OH ~ I ~ Oll
Noboritomycin A & B
In noboritomycin A, R is methyl and in noboritomycin B, R is
ethyl.
The antibiotic grisorixin is produced from the
microorganism ~ griseus as reported by Gachon et
al, Chem. Comm.r 1421-1423 (1970) and J. Antibiotics 28,
345-350 (1975). As is disclosed in U.S. Patent No.
4,161,520 to Osborne et al, the microorganism is on deposit
~2~Z
-12-
at the Institut National de la Recherche Agronomique where
it has been assigned the designation INRA SAB 2142. Gris-
orixin is structurally very similar to nigericin, the only
difference being the presence of an additional oxygen in
nigericin. The structural formula for grisorixin is:
H3~
H02C-CH H ~ <~ 33
H3C _~ C~
H
Grisorixin
Various derivatives of grisorixin are disclosed by Gachon et
al, J. Antibiotics 28, 351-357 (1975).
Antibiotic X-206 was Eirst reported by Berger et al,
J. Am. Chem. Soc. 73, 5295-5298 (1951) and has the follow-
ing structure as reported by Blount et al, Chem. Comm., 927-
928 (1971):
H3C ~,~\ ~3 CH3 H3 CH3
H l ¦ H ~ 1 1 ¦ H ~\ )~ CH3
~C-C~O~C~Cl~C~\o~ ; I OH 1 /<~o CH3 /~ \2HS
X-206
Methods for preparation of the X-206 antibiotic as well as
further particulars as to its properties will be found in
U.S. Patent Nos. 3,839,557 to Raun and 3,794,732 to Raun.
The antibiotic lonomycin has the following struct~ral
30 formula as reported by Mitani et al, J. Antibiotics 31, 750-
~2075~2
MeO MeO t'~eO
~'~
HO O~l H OH
A method for producing the antibiotic is given by Omura et
al, J. Antibiotics 29, 15-20 (1976). The antibiotic was
also identified by Oshima et al, J. Antibiotics 29, 354-365
(1976) as DE-3936 and was determined to be identical to
emercid reported by Riche et al, J.C.S. Chem. Comm., 951-952
(1975) and to 31,559RP reported by Rhone Poulenc:
Japan Patent, Kokai 50~129, 796 (October 14, 1975). U.S.
Patent No. 3,950,514 to Sawada et al discloses the lonomycin
antibiotic as being produced by the Stre ~ ribosidicus
microorganism which has been deposited under number ATCC
31051 at the American Type Culture Collection.
The following structural formula was determined by
Gachon et al, J. Antibiotics 29, 603-610 (1976~ for the
antibiotic alborixin:
H }I B C~l H B El M~ b H
b C~l
}}COC
R = Me or H
Alborixin
, ,
~2~)75aZ
Certain characteristics of the~ antibiotic were presented in
the article by Delhomme et al, J. Antibiotics 29, 692-695
(1976). The alborixin antibiotic is produced from a
Streptomyces albus microorganism and as is disclosed in
U.S. Patent No. 4,161,520 to Osborne et al, the micro-
organism is on deposit at the Institut National de la
Recherche Agronomique and assigned the designation INRA SAB
3840.
Mutalomycin is produced by strain S11743/A of the
Streptomyces mutabilis microorganism which has been
deposited at the Agricultural Research Service under number
NRRL 8088. A method for preparing the antibiotic and its
physical and chemical properties were reported by Fehr et
al, J. Antibiotics 30, 903-907 (1977). The structural
formula of mutalomycin is:
2 0 H3C-o h3C ~ ~3
H ~; CH3 H CH3H H H CH3
HO ~ ~
H3C C02H
Mutalomycin
as reported by Fehr et al, J. Antibiotics 32~ 535-536 (1979).
The antibiotic laidlomycin has been described by
Kitame et al, J. Antibiotics 27, 884~887 (1974), the anti-
biotic being produced by the Streptomyces eurocidicus var.
asterocidicus microorganism which has been indexed as
~ .
species S-822 at the Department of Bacteriology, Tohoku ~
~1
~2~)'75~Z
-15-
University School of Medicinet Sendai, Japan. The chemical
structure of laidlomycin was reported by Westley, Adv. Appl.
Microbiology 22, 177-223 (1977) as being:
I 1 ~3 ~ ~3
H \ ~ 20H
H O
~O-C--C --C--C
C~3 H H CH3
Laidlomycin
The laidlomycin antibiotic also appears to be the
subject of U.S. Patent No. 4,016,256 to Ishida et al.
The antibiotic SY-1 is the fermentation product of a
albus microorganism, a culture of which has
been deposited at the American Type Culture Collection under
accession number ATCC 21838. As depicted in U.S. Patent No.
4,138,496 to Shibata et al, antibiotic SY-1 has the
following structural formula:
L`t.
co2
SY-1
The structure of antibiotic SY-1 is quite similar to that of
salinomycin, the only apparent structural difference bein~
that salinomycin contains a hydroxyl group on the ring
designated "C".
,i ~
~Z075~2
Lasalocid can be prepared by inoculating the fermenta~ion medium
with a Streptomyces lasaliensis microorganism. Lyophilized tubes of this
culture bearing the laboratory designation X-537A were originally deposited
at the Agricultural Research Service, USDA, Peoria, Illinois, under the iden-
tification number NRRL 3382. Replacement of NRRL 3382 has been made with cul-
ture given the identification number NRRL 3382R. Lasalocid may also be pro-
duced from a Streptomyces lasaliensis microorganism, a culture of which is
available from the American Type Culture Collection, Rockville, Maryland,
under the number ATCC 31180.
The antibiotic lasalocid has been chemically identified in United
States Patent 4,164,586 to l~'estley as 6-(7~R)-r5(S)-ethyl-5~5(R)-ethyltetra-
hydro-5-hydroxy-6(S)-methyl-2H-pyran-2(R)-yl)tetrahydro-3(S)-methyl-2(S)-
furyl]-4(S)-l,lydroxy-3~R),5-(S)~dimethyl-6-oxononyl)-2,3-cresotic acid. This
antibiotic has the following structural formula:
H0 ~ ~ oHH5
H3C 3
Lasalocid
A method for producing the antibiotic lysocellin was disclosed by
Liu et al in United States Patent No. 4,033,823. The method involves the cul-
tivation of a strain of Streptomyces longwoodensis which is on deposit at the
American Type Culture Collection under the designation ATCC 29251. The struc-
ture of lysocellin is as follows:
- 16 -
~.
~'
12~)'75~
CH CH
C~L/ ~ H3
CH3 C113 1 2 OH
CH3
Suitable methods for preparing the lysocellin antibiotic
are disclosed in the above-mentioned patent. The character-
istics of lysocellin were first discussed in the article by
Ebata et al, J. Antibiotics 28,118-121 (1975)~
~ dditional polyether antibiotics for forming the zinc
complexes of the subject invention include the antibiotics
septamycin, dianemycin, A-204, lenoremycin and carriomycin.
These latter antibiotics are non-glycolic, monovalent mono-
glycoside polyethers in Westley's Class 1b.
Septamycin is also known as A-28695 and is the sub-
ject of U.S. Patent Nos. 3,839,558 and 3,839,559 to Hamill
et al. As is set forth by Reller-Juslen et al, J. Anti-
biotics 28, 854-859 (1975)~ the antibiotic has the struc-
tural formula:
~o
Me,. . ,~ G > M~O Me ~: 0~:
7~, L
--~ 2 B
HO,
MB_ C _- H
c .
O 0- H
Septamy n
l~Z07S192
-18-
The antibiotic is produced from the cultivation of a
Str~tomy~es bygroscopicus microorganism which has been
deposited under number NRRL 5678 at the Agricultural
Research Service. The above-mentioned patents to Hamill et
al classified the septamycin producing microorganism as a
Streptomyces albus microorganism, a culture of which has
been deposited at the Agricultural Research Service under
accession number NRRL 3883. Further characteristics and a
method for producing the antibiotic are set forth in the
article by Keller-Juslen et al mentioned above.
Dianemycin is the fermentation product of a micro-
organism which is a strain of Streptomyces hygroscopicus
which is on unrestricted deposit as NRRL 3444 at the Agri-
cultural Research Service. Dianemycin was characterized by
Steinrauf et al, Biochemical and Bio~hysical Research
Communications 45, 1279-1283 (1971) as having the structure
formula:
H3C~0_CH3
O ~
3 ~ ~
l~C--C--C--C--C--C=C --C
~H
CH3 1~ CH3 0 H CH3
Dianemy~
U.S. Patent No. 3,577,531 to Gorman et al and U.S. Patent
No. 3,711,605 to Hamill et al disclose the description
preparation and characteristics of dianemycin.
.~.~
~207~
l g
The antibiotic A-204 is described and a method for
its preparation disclosed in U.S. Patent 3,705,23a to ~ Hamill et al and U.S. Patent 3,7~4,732 to Raun. The term
A-204 is used to designate the different components
obtained by fermentation in the presence of ~
. albus microorganism under aerobic conditions in a culture
medium containing assimilable sources of carbon, nitrogen
and inorganic salts. According to U.S. Patent 3,794,732
to Raun, the organism capable of producing antibiotic A-204
has been placed on permanent deposit, without restriction,
with the culture collection of the agricultural Research
Service, and is available to the public under culture number
NRRL 3384.
Component I of A-204 is the most important and the
most abundant. Component II constitutes about 5% of the
mixture of A-204 components produced and the other compon-
ents are obtained in smaller quantities. The structural
formula shown below is that of the acid form of A-204 I.
H3C-O \,
H3C /~ O lo I H3 0 H3C CH3 0 CH3 0-CH3
113C,~ ~ CH3
Cl~ H
CH3
A-204 I
_.
The antibiotic lenoremycin is the fermentation pro-
duct of a Streptom~ces hygroscopicus microorganism which is
30 deposited under number ATCC 21840 at the American Type
~Z~7~i2
-20-
Culture Collection. The antibiotic was described by Kubota
et al, J. Antibiotics 28, 931-934 (1975). The structure
reported by Liu et al, J. Antibiotics, 29, 21-28 (1976) i5
as follows:
c~,,~ ~
.. ~ o
~ ~
~* Me
Lenoremycin
The Liu article also stated that lenoremycin is identical to
the antibiotic A-130A described in Japanese patent
publication 7304558 of Shionogi. The antibiotic A-130A is
also the subject of U.S. Patent No. 3,903,264 to Oikawa et
al. The above structure is also reported in Blount et al,
Chem. Comm. 853-855 (1975) who designated the antibiotic as
Ro 21-6150.
The antibiotic carriomycin has the following struc-
tural formula as reported by Imada et al, J. Antibiotic
31, 7-14 (1978):
MeO~
J~o~ MeO C~k
30 " ~ _ =
Carrioymcin
-21- 1 2~ 7 S 8 2
The antibiotic is produced by strain ql-42082 of the
Streptomyces hxgroscopicus microorganism which has been
_
deposited at the Institute for Fermentation, Osaka, Japan,
under accession number IFO 13609 and at the American Type
Culture Collection under accession number ATCC 31080. The
carriomycin antibiotic is the subject of U.S. Patent No.
4,069,316 to Imada et al.
While the above description of the various known
non-glycolic polyether antibiotics have generally identified
the antibiotics as being single compounds, it should be
recognized that at least some of these polyether an~ibiotics
are produced as an antibiotic complex of structurally
related factors containing varying proportions of each
factor. As an example, the structure for A-204 ~et forth
previously is A-204 factor I which is produced in combina-
tion with other factors in ratios depending upon fermenta-
tion conditions. It should, therefore, ~e realized that
the present invention comprehends the zinc complexes of the
various factors of the non-glycolic ~olyether antibiotics
whether in combination with other factors or in their iso-
lated form as well as their use in promoting growth and
enhancing feed efficiency in food-producing mammals, more
par~icularly, in swine, and in ruminants including cattle
and sheep. Furthermore, zinc complexes of derivatives of
the previously mentioned non-glycolic polyether antibiotics
are also within the scope of the present in~ention. For
example, U.S. Patent No. 3,985,872 to Chamberlin is directed
to dihydro A-204 and U.S. Patent No. 3,907,832 to Hamill is
directed to monoether and monothioether derivatives of
- '
12075~92
-22-
A-204. Therefore, as used herein, the specific name of the
polyether antibiotic, e.g. A-204, encompasses all of the ~ factors of the antibiotics, e.g. A-204 I and II, as well as
isomers, homologs, and derivatives thereof.
For further particulars as to characteristics and
.. methods for the preparation of certain of the above poly-
ether antibiotics, reference is made to U.S. Patent No.
3,995,027 to Gale et al and the patents cited therein and to
10 U.S. Patent No. 3,794,732 to Raun and the patents and
articles cited therein.
The subclass 2b nitrogen-containing pyrrole ether
antibiotics include the antibiotic X-14547 (mononitrogen,
divalent) and the antibiotic A-23187 also known as calci-
mycin (polynitrogen-containing divalent). The pyrrole ether
antibiotic known under the code designation X-14547 is
characterized chemically as ~-(R),5(S)-dimethyl-6(R)-1-
ethyl-4-[4-(R)-(2)pyrrolylcarbonyl)-1(S)-ethyl-3a(R),4,5(R),
7a(R)-tetrahydroindan-5-yl]-1(E), 3(E)-butadienyl-tetra-
hydropyran-2-acetic acid. The antibiotic is produced by a
Streptoymces sp. X-14547 micr~organism, a culture of which
has been deposited under designation number NRRL 8167 at the
Agricultural Research Service. The X-14547 antibiotic has
the following structural formula:
~ , M e ~ H
30 0~
X-14547 Et
-23- ~2~75~
Further details of the characteristics of the X-14547 anti-
biotic and processes for its production and reco~ery are
disclosed in U.S. Patent No. 4,100,171 to Westley et al in
U.S. Patent No. 4,161,520 to Osborne èt al, and in the
articles by Liu et al~ ~. Antibiotics 32, 95-99 ~1979) and
.
Westley, J. Antibiotics 32, 100-107 (1979).
The pyrrole ether antibiotic known under the code
designation A-23187 (also known as calcimycin) is the sub-
ject of U.S. Patent No. 3,923,823 to Gale et al. The patent
discloses that the A-23187 antibiotic has an appreciable
affinity for Cd++, moderate affinity for Ni+~, Zn++, Co++
and Be++, and no apparent affinity for Hg++ and suggests
that because of its preferential binding of certain cations,
the antibiotic can be employed in applications wherein the
selective removal of particular cations is desired. It was
reported by Pfeiffer et al, Biochemistry, Vol. 15, No. 5,
935-943 (1976) that an A-23187 complex of zinc as well as
A-23187 complexes of other divalent cations, wa~ used to
investigate the selectivity of the antibiotic for divaient
cations over monovalent cations. However, no specific
utility for the zinc complex of A-23187 was taught or
sugges~ed by the above article. The use of the free acid
or calcium salt of the A-23187 antibiotic in a method of
enhancing the contractile force of the mammalian heart
muscle in a warm-blooded mam~al is disclosed in U.S. Patent
NoO 3,985,893 to Holland et al.
The A-23187 antibiotic is produced by culturing a
Stxeptomyces chartreusis microorganism. A culture of this
_ .
microorganism has been deposited in the collection of the
.... . . . . . .. .. ~ . . ~_ . . ... ..
12075~2
-24-
Agricultural Research Service under accession number NRRL
3882. The A ~23187 antibiotic has the structural formula:
", Me
0~-- N ~
C02H
A-23187
Further details of the characteristics of the antibiotic and
processes for its production and recovery are set forth in
U.S. Patent No. 3,923,823 to Gale et al.
The glycolic monovalent monoglycoside polyether
antibiotics include etheromycin (Westley Class 1b). The
antibiotic etheromycin (also known as C20-12 and CP 38295)
has the chemical structure:
~o ~
~ 1\o~0
H ~ .~0
OH MeO~
~ r
~10 ~ OH
OH H I tl
Etheromycin
This structure was published by Mitani et al in J. Anti-
biotics 31, 750-755 (1978) who al50 noted that etheromycin
is the same as the T-40517 antibiotic. According to Westley,
Ad. Appl. Microbiology 22, 177-223 (1977), etheromycin is
produced from the Streptom~ces ~ microorganism,
a culture of which is deposited under number ATCC 31050 at
. ~ ,
-25~ ~207582
the American Type Culture Collection. Additional details
concerning the etheromycin antibiotic can be found in the
previously mentioned U.S. Patent No. 4,129,578 to Celmer
et al.
Those polyether antibiotics for which structural
information is not yet available~, and which may be used to
make the novel zinc complexes of the subject invention in-
clude ionomycin; aabomycin; disnerycin; duamycin; BL-580;
K-41; SF-1195; M-4164A; A-32887; 30,504RP; 38,986; 44,161;
47,433; 47,434; and 47,224. Available information about
these antibiotics is presented below.
The antibiotic ionomycin is the fermentation product
of the Streptomyces conglobatus sp. nov. trejo microorg~nism
which has been deposited under accession number ATCC 31005
at the American Type Culture Collection. The antibiotic
has been characterized by Liu et al, J. Antibiotics 31,
815-819 (1978) which also exhibits a suitable method for
the preparation of the antibiotic. The ionomycin antibiotic
is also the subject of U.S. Patent No. 3,873,693 to Meyers
et al.
The isolation and characterization of the polyether
antibiotic K-41 was reported by Tsuji et al, J. Antibiotics
29, 10-14 (1976). The antibiotic is produced from a strain
of Streptomyces hygroscopicus microorganism deposited at
the ~ermentation Research Institute, Chiba, ~apan, with
deposit number FERM-P 1342. The above article reports that
the antibiotic is the subject of Japanese Patent 49-14692
(1974). A method utilizing the antibiotic K-41 in protect-
ing plants from mites is disclosed in U.S. Patent No.
-26- ~2~7582
4,148,881 to Ishiguro.
U.S. Patent No. 3,812,249 to J.H.E.J. Martin et al
is directed to the polyether antibiotics BL-580 a and ~.
These antibiotics are products of a Streptomyces hygroscop-
icus microorganism which has been deposited at the Agricul-
tural Research Service under deposit number NRRL 5647. The
above-mentioned patent discloses suitable methods for the
preparation of the BL-580 antibiotic. U.S. Pàtent No.
4,132,779 to Hertz et al discloses the antibiotic BL-580
zeta which is produced by a mutant strain o~ Streptomyces
hy~roscopicus derived by treatment of a natural section,
single colony isolate of S. hygroscopicus NRRL 5647 with
N-methyl-N'-nitro-N"-nitrosoguanidine. A culture of the
mutant strain has been deposited at the Agricultural
Research Service under accession number NRRL 11108.
The polyether antibiotic aabomycin X was recently
reported in the Supplement to "Index o Antibiotics from
Actinomyces" by ~r. Hamao Umezawa, J. Antibiotics 32, 79-
51 (1979) and is also apparently the subject of Japan
Kokai 77-90697 filed July 30, 1977, in the name of Shibata,
et al. The antibiotic is produced by the fermentation of
the microorganism Streptomyces hygroscopicus subsp. aabomy-
ceticus 325-17 which has been deposited at the American
Type Culture Collection under deposit number ATCC 21449.
The microorganism produces an antibiotic mixture which in-
cluaes the factors aabomycin X and aabomycin A. The anti-
biotic aabomycin A is the subject of U.S. Patent No.
3,657,422 to Misato et al.
.... . ... . . , .. . _ .. .. _ ..... ._ . _ . . . _ _ ..
-27- i2~75~
The antibiotic duamycin was described in Japanese
Patent 26719 (1970) to Kaken-~agaku, the patent being
abstracted in Chemical Abstracts 74, 21895p (1971). The
_ .
antibiotic SF-1195 was disclosed in Japanese Patent 49-
132212 (1974) to Sawada et al. Disnerycin was mentioned
in U.S. Pat~nt No. 4,159,322 to Cloyd as being a polycyclic
ether antibiotic of the same class as monensin, nigericin,
grisorixin, salinomycin, narasin and lasalocid. The anti-
biotic identified as M-4164A was described in Japan Kokai
Patent 50-12294 (1975) to Toyama et al.
The polyether antibiotic designated as Compound
38,986 is disclosed in U.S. Patents Nos. 4,022,885 and
4,048,304 to Celmer et al. The antibiotic is the product
of a Streptomyces flaveolus microorganism, a culture of
which has been deposited in the American Type Culture
Collection and given designation ATCC 31100.
The polyether antibiotic Compound 44,161 is produced
by cultivating a strain of Dactylosporangium salmoneum
Routien sp. nov., cultures of which have been deposited at
the American Type Culture Collection under accession numbers
ATCC 31222, 31223 and 31224. Additional details regarding
this antibiotic are contained in U.S. Patent No. 4,081,532
to Celmer et al.
The antibiotic A-32887 is the subject of U.S. Patents
Nos. 4,132,778 and 4,133,876 to Hamill et al. As is
described in these patents, the A-32887 antibiotic is
closely related to the K-41 antibiotic and is produced by
culturing a strain of Streptomyces albus which has been
30 deposited under designation NRRL 11109 at the Agricultural
_ .~ . . .. . . . . _. , _ _ . ..
-2~-
Research Service. lZ07582
The two polyether antibiotics disclosed in U.S.
Patent No. 4,148,882 to Celmer et al were given the designa-
tions Compounds 47,433 and 47,434. These antibiotics a~e
produced by a species of Actinomadura macer Huang sp. nov.,
a culture of which has been deposited at the American Type
Culture Collection and given the designation number ATCC
31286.
U.S. Patent No~ 3,989,820 to Florent et al is
directed to the antibiotic 30,504RP which is produced by
culturing a microorganism called Streptomyces gallinarius
DS 25881, a culture of which has been deposited at the
Agricultural Research Service under number NRRL 5785.
The polyether antibiotic given the designation
Compound 47,224 is produced by a strain of a Streptomyces
hygroscopicus microorganismO As is disclosed in U.S.
Patent No. 4,150,152 to Celmer et al, the microorganism
strain capable of producing Compound 47,224 has been
deposited at the American Type Culture Collection with the
accession number ATCC 31337.
While the above descriptions of the various known
polyether antibiotics have generally identifiea the anti-
biotics as being single compounds, it should be recognized
that at least some of the polyether antibiotics are produced
as an antibiotic complex of structurally related factors
containing varying proportions of each factor. As an
example, the structure for lasalocid set forth previously
is lasalocid factor A which is produced in combination with
: 30 factors B, C, D, and E in ratios depending upon fermenta-
_ _ _ _ __ _._ . ~ _ . _ . .... . ..... , . ... .. . . , . . . ... .. , .... . . . . .. ... . _ _._ . _ _
. -29 ~Z~5~
tion conditions. ~lomologs of lasalocid A are disclosed in
U.S. Patent No. 4J168,272 to Westley. An isomeric form of
lasalocid is also known from U.S. Patent ~o. 3,944,573 to
Westley. In addition, monensin is produced with factors B
and C as reported by Westley, Adv. Appl. Microbiology 22,
200 (1977) and narasin is produced with factors A, B and D
as is set forth in U.S. Patent No. 4,038,384 to Berg et al.
It should, therefore, be realized that the present invention
comprehends the zinc complexes of the various factors of
the polyether antibiotics whether in combination with other
factors or in their isolated form as well as their use in
promoting growth and enhancing feed efficiency in cattle,
sheep or swine.
Furthermore, inc complexes of derivatives o the
previously mentioned polyether antibiotics are also within
the scope of the present invention. For example, ~arious
derivatives of the lasalocid antibiotic are known from U.S.
Patent No. 3,715,372 to Stempel et al. In addition,
derivatives of monensin are disclosed in U.S. Patent No.
3,932,619 to Brannon et al which is directed to a metabolite
produced from monensin, U.S. Patent No. 3,832,258 to
Chamberlin which is directed to the deshydroxymethyl
derivative of monensin and U.S. Patent Nos. 4,141,907 and
4,141,404 to Nakatsukasa et al are directed to dl~oxynarasin.
Therefore, as used herein, the specific name of the poly-
ether antibiotic, e.g. lasalocid, encompasses all of the
factcrs of the antibiotic, e.g. lasalocid A, B, C, D, and
E, as well as isomers thereof, e~g. iso-~asalocid, and
3~ derivatives thereof.
-30- ~207582
For further particulars as to characteristics and
methods for the preparation of certain of the above poly-
ether antibiotics, reference is made to U.S. Patent No.
3,9gS,027 to Gale et al and the patents cited therein and
to U.S. Patent No. 3,794,732 to Raun and the patents and
articles cited therein.
It is also within the scope of the present invention
that the novel zinc complexes of the polyether antibiotics
described herein can be used in conjunction with other
active ingredients which are also useful for promoting growth
and enhancing feed efficiency in cattle, shaep or swine.
For example, the zinc complexes of polyether antibiotics may
have an enhanced effect when used in combination with estra-
diol.
To the extent necessary, the above-mentioned patents
and literature articles mentioned in describing the various
known polyether antibiotics and their uses are incorporated
herein by reference.
To obtain the useful zinc complexes, the polyether
antibiotic, generally in the form of its alkali metal,
alkaline earth metal or ammonium salt, is treated in situ
in the fermentation broth or beer by adding to the anti- -
biotic containing broth a water-soluble zinc salt. Addi-
tion of such a water-soluble zinc salt promotes the forma-
tion of a zinc complex o~ the polyether antibiotic. Such
a zinc complex of the antibiotic, along with zinc complexes
formed with residual nitrogen-containing compounds in the
broth such as amino acids, polypeptides, and proteins, are
insoluble in the fermentation broth liquid.
`` ~20758Z
The zinc ion~ from the added zinc salt apparently
form coordination bonds with the oxygen atoms of the spar-
ingly soluble polyether antibiotic. For example, the
structure of the zinc complex of lasalocid is believed to
be represented by the following:
~b Me Et Me
~ ~ - Zn~ t
~, , `
O
Et ~ ~
zinc Lasalocid Monohydrate
On the basis of the formation constants with ligands
20 such as citric acid, Iactic acid and tartaric acid, it is
believed that zinc ions form stronger bonds with oxygen-
containing compounds than do ions such as Mg++, Ca++, Ba~+,
Na+ and K+.
The zinc salt added to the fermentation broth can be
chosen from various water-soluble salts which ionize in the
fermentation broth. Such salts include, for example, zinc
chloride, zinc sulfate, zinc acetate, zinc benzoate, zinc
citrate, zinc lactate, etc. Water-soluble zinc salts are
generally those which can be dissolved to the extent of
about 1 per cent by weight or more in water at 20Co For
maximum production of the desired zinc complexes, the water-
soluble zinc salt should be added to the fermented broth in
an amount which is sufficient to fill substantially all~of
-32- ~Z075BZ
the possible zinc coordination sites of the pxoteins, poly-
peptides, amino acids and related compounds, in addition to
substantially all of the available coordination sites of the
antibiotic present. This is necessary because in general,
nitro~en atoms in the polypeptides, amino acids, etc., form
stronger coordination bonds with zinc than do the oxygen
atoms in the polyether antibiotic. Generally, therefore,
zinc salt is added to the fermentation broth in an amount
sufficien~ to provide a zinc content of from about 3 to 12
per cent, and preferably, from about 5 to 10 per cent by
weight of the dried precipitate recovered from the fermen-
tation broth as hereinafter more fully described.
The amount of soluble zinc salt to be added will
depend on the amount of nutrients added to the fermentation
broth during the course of the fermentation. The actual
amount of soluble zinc salt to be added to the broth
obtained from a given mash bill can be determined by simple
laboratory precipitations followed by zinc analyses on the
dried precipitates. When, for example, the preferred zinc
chloride salt is employed to form the desired zinc anti-
biotic complex, advantageously from about 4 to 10 ~allons
of a 67 weight per cent zinc chloride solution ~sp. gr.
1.8~3~, can be added to 1000 gallons of fermentation broth.
To form the zinc antibiotic complex in the ferm~nta-
tion broth, pH of the broth is advanta~eously adjusted to
about 6.5 to 7.5, and preferably, to about 6.8 to 7.2 after
addition of the soluble zinc salt to the fermentation broth.
The insoluble zinc complexes formed upon addition of
zinc salt can be readily separated from the fermentation
.. : .
~33~ 1207 5 e ~
broth or beer by conventional filtration or centrifugation
techniques. In this manner, a wet biomass containing the
zinc antibiotic complex is realized. This wet biomass is
resistant to wild fermentations because of its relatively
high zinc content. The wet biomass so obtained is easily
dried by spray drying or drum drying procedures, and this
zinc antibiotic-containing dried product can then be used
as a feed additive per se. If the an~ibiotic content of
the fermentation beer is lower than desired after comple-
tion of the fermentation, crude antibiotic in its sodium
salt form can be added to the fermentation beer prior to
the addition of the soluble zinc salt. In this manner,
the antibiotic content of the biomass composition to be
separated from the broth can be increased. To be suitable
as a feed additive, the dried biomass preferably contains
at least about 5 per cent by weight of the zinc antibiotic
complex, advantageously from about 10 per cent to 50 per
cent by weight of the zinc antibiotic complex.
Recovery of the zinc antibiotic complexes of the
present invention in the manner described herein provides
several important advantages over known antibiotic prepar-
ation and recovery processes. The present process, for
example, provides a means for recovering relatively high
yields of antibiotic in a salable feed additive product.
Further, the use of expensive extraction solvents and the
cost associated with the process losses of such solvents
are avoided. The present process also permits recovery of
salable feed values present in the mycelium of the Strepto-
~y~ microorganism used to produce the antibiotic. The
~3~~ 1 Z07 5 ~ X
present process further reduces the cost of waste disposal
operations needed in previous processes to deal with the
mycelial mat produced during fermentation. Use of this mat
as part of the feed additive product, in fact, re~uces the
cost of the carrier for the antibiotic material being
marketed.
The dried, antibiotic-containing biomass recovered
from the fermentation broth as hereinbefore dèscribed can
be added to conventional animal feed compositions as a
growth-promoting agent. Such feed compositions generally
contain whoLe or ground cereal or cereal byproducts as an
essential nutrient. The feed compositions can also contain
such optional additional materials as animal byproducts,
e.g., bone meal, fish meal, etc., carbohydrates, vitamins,
minerals and the like. The zinc antibiotic complexes of
the present inven~ion are generally employed in the feed
compositions to the extent of from about 50 grams per ton to
200 grams per ton, preferably from about 75 grams per ton to
125 grams per ton.
Purification of the zinc complexes of the present
invention so that the complexes are more suitable for
administration to humans can be accomplished in a variety
of manners. A presently preferred method for purification
of the zinc complexes from the recovered feed grade zinc
complex includes the steps of, after treatment of the
fermentation beer with a solu~le zinc salt, acidifying the
water slurry of the zinc complex with strong mineral acid
such as sulfuric acid to produce a relatively low pH, e.g.
a pH below about 4, preferably about 2 to about 3, and then
-35 ~ 207 5 82
extracting the acid form of the polyether antibiotic from
the slurry into a substantially water-insoluble organic
solvent such as butyl acetate.
Thereafter, a lower aliphatic alcohol such as
methanol is added to the organic solvent containing the
polyether antibiotic. The volume of alcohol added is
generally less than or about equal to the volume of organic
solvent, preferably about 0.25 to abo~t 1.0 volumes alcohol
to about 1.0 volume of organic solvent. A soluble zinc
salt such as zinc chloride dissolved in the same lower
aliphatic alcohol is then slowly added with vigorous agita-
tion to the organic solvent-alcohol mixture containing the
polyether antibiotic. Preferably, about 0.5 to 1.0 volumes
of the alcohol containing the zinc salt are added per volume
of mixture. The amount of zinc salt added should be suffi-
cient to convert essentially all of the contained antibiotic
to its zinc complexed form. The formed zinc complexes are
then filtered from the mixture, thoroughly washed and dried.
- If greater purification o~ the zinc complex is
desired, the above procedure can be modified to include
further purification steps. One such modification is, prior
to the addition of the lower aliphatic alcohol, adding an
aqueous solution containing an alkali metal hydroxide such
as potassium or sodium hydroxide to the organic solvent
containing the polyether antibiotic so that the antibiotic
is extracted into the aqueous solution. The antibiotic is
then re-extracted into the same organic solvent or a differ-
e~t water-insoluble organic solvent such as methyl tertiary-
butyl ether after acidification. These steps of the modi-
-36- 1 ZO 7 5 82
fied procedure can be repeated as many times as desired
until the proper degree of purification is achieved.
Thereafter, the polyether an~ibiotic is contacted with the
lower aliphatic alcohol and the previously mentioned pro-
cedure continued so as to yield the purified zinc complex
of the polyether antibiotic.
In the above description of the purification pro-
cedure and modification thereof, the amount of each of the
media, i.e., the organic solvent, aliphatic alcohol,
aqueous solution, etc., relative to the others when conduct-
ing the procedure may vary considerably, the primary con-
siderations being that sufficient media be utilized to
obtain a satisfactory yield of the zinc complex balanced
against the cost of the media and the capacity of the
- available equipment. Generally, the amount o~ a particular
medium used to treat another medium in any of the steps of
the above procedure is about 0.1 to 10 volumes, preferably
about Q.5 to about 5 volumes, for each volume treated.
Certain advantages are realized by the above pro-
cedure where the purified zinc complexes are recovered from
the feed grade complexes as opposed to recovery of the
purified complexes from virgin mycelia. Among others, the
feed grade complexes are filtered relatively easily from
the fermentation beer whereas filtering of virgin mycelia
is very slow and thus time-consuming. In addition, the
feed grade complexes tend to be more concentrated and thus
less organic solvent is required in conducting the purifica-
tion procedure and volume loss of solvent will be reduced.
- ~37~ 1 20 7 5 8 Z
Illustrated in the following examples are preparation
and recovery methods fox the zinc complexes of polyether
antibiotics as well as feed and feed additive compositions
including these zinc complexes and their usefulness as
growth-promoting agents for food-producing animals such as
cattle, sheep and swine. These examples are in no way to be
considered limiting of the present invention to compositions,
ingredients, and processes involving.that pàr`ticular material.
EXAMPLE I
A. Fermentation
_ _
About 450 ml of inoculum of Streptornyces lasaliensis
culture No. NRRL 3382R, obtained from the Agricultural
Research Service is introduced into 9,000 ml of fermentation
medium of the following composition:
Soybean Flour 2%
Brown Sugar 2%
Corn Steep Liquor 0.5~
K2HPO4 0.1%
Hodag Antifoam K-67 ~.05%
Water Balance
lno . oo~ :
: The fermentation is conducted in a 20-liter, stain-
less steel fermentor using the conditions listed below.
1. Amount of medium - 9.45 liters.
2. Temperature - 28C.
3. Air Flow - 9.0 liters per minute.
4. Mechanical agitation - One 13-cm diameter
-impeller rotating at 600 RPM.
~3~~ 1 20 7 5 8 Z
5. Bac~ pressure - about 16.7 psig.
6. Time of fermentation - 72 hours.
At the end of the fermentation the lasalocid assay
of the beer is 1.5 g per liter.
B. Recovery
Since the assay of the beer for lasalocid is low
compared to assays commonly obtained for antibiotics, the
beer i5 spiked with crude lasalocid which has been obtained
by extracting with butyl acetate a commercial product con-
taining approximately 81 grams of sodium lasalocid per
pound.
- Twenty-five grams of crude sodium lasalocid (78.S%
lasalocid) dissolved in 150 ml of methanol are added to
2000 ml of beer under constant agitation. After thorough
agitation, 12.5 ml of a zinc chloride solution (0.25 g Zn
per ml) are slowly added with agitation to the fermented
beer. The pH is adjusted to a value in the range 7.0~7.4.
After the treated beer has been agitated for about
30 minutes it is filtered, without filter aid, on a Buckner
funnel using No. 1 Whatman filter paper. The filtration
proceeds rapidly to give a firm cake which is dried in an
oven. The final dried product weighs 57 grams and has an
assay of 32.7% Iasalocid.
The calculated recovery from beer to dried product
is 82.5% derived from the following formula.
2 x 1 5 ~ 25 x 0.785 x 100% = 82.4%
"Hodag" and "l~atman" are registered trademarks.
'C
-39- ~Z075BZ
EXP.MPLE I I
Administration of zinc lasalocid growth-promoting
agent to cattle via cattle feed composition is illustrated
by this example. A cattle feed formulation having the
following composition is prepared:
Composition Concentration
Cracked Corn 68.5%
Alfalfa Meal ~5`.0%
Ground Cobs 10.0%
Soybean meal (50% protein) 15.0%
Mineral Mixture 1.0~
Salt 0.5%
100 . 0%
To such a composition is added enough of the zinc
lasalocid-containing dried product of Example I to provide
a feed composition containing about 100 grams of zinc lasa-
locid per ton of feed composition.
The zinc lasalocid-containing feed composition is
fed to cattle in amounts sufficient to provide from about
5 to 100 ppm of zinc lasalocid in the rumen fluid. Admin-
istration of the zinc lasalocid material in this manner
serves to promo~e cattle growth by enhancing the efficiency
with which the cattle so treated utilize their feed.
.
EXAMPLE III
The tendency of zinc lasalocid antibiotic to desir-
ably affect acetate/propionate ratios in rumen fluid from
cattle is demonstrated by means of an in vitro rumen fluid
analysis procedure. Rumen fluid i5 obtained from a steer
.. .. _ _ .
~Z()7582
-40-
which has a surgically installed fistula opening into the
rumen. The steer is maintained on a grain diet consisting
of the feed composition set forth in Example II. A sample
of rumen fluid is strained through four layers of cheese-
cloth and the eluate collected. An equal amount of buffer
solution with a pH of 7 is added to the rumen fluid. Ten
ml of the diluted rumen fluid is placed in ~lasks with 500
mg of the same feed shown above which has been finely ground.
Each of materials to be tested is weighed into a separate
test flask. Four control flasks are also employed. All of
the test flasks are incubated for 24 hours at 39C. At the
end of incubation, a pH is measured and one drop of mercuric
chloride is added to each flask. The samples are centri-
fuged at 3000 x g for 15 minutes and the supernatant is
analyzed by gas chromatographic methods for volatile fatty
acids.
Analyses for acetate, propionate and butyrate com-
pounds are performed. The results are statistically com-
pared with the results of the analyses of the control flasks.The acetic/propionic ratios are calculated for each treat-
ment. Treatments with propionate production significantly
~igher than the control are evidenced in this ratio expres-
sion by lesser numbers. These treatments are then regarded
as active treatments. Results of two such tests are set
forth in Tables I and II.
_, _ _, . ... ..... .... . . ....... ... ., . . . . . . . . . . . _, _ . _ _ .. _ . .. _
-41- 12 O~ S 82
~able I
Effect of Zinc Lasalocid on Acetate/Propionate
Ratios of In Vitro Ruminal Fluid _
Rumensin Zinc Lasalocid, ppm
Item* Control 5 ppm 5 ~ ~ 20 100
Acetate/propionate 1.90 1.06 1.47 1.30 1.16 1.~0
*Means of seven experiments, 3 reps/treatment
Table II
Effect of Zinc Lasalocid on Acetate/Propionate
_ _ Ratios of In Vitro Ruminal Fluid
Rumensin zinc Lasalocid, ppm
Item* Control 5 ppm 5 I0 20 100
Acetate/Pro~ionate 1.37 1.09 0.94 1.02
*Means of four experiments, 4 reps/treatment
The data in Tables I and II demonstrate that the pre-
sence of zinc lasalocid in the rumen fluid can beneficially
increase the production of propionate within the rumen rela-
tive to acetate production. Cattle wherein such a propionate
increa$e occurs are more efficiently able to utilize their
feed in the production of meat ana m-lk.
EXAMPLES IV-XVI
Other preferred zinc complexes of polyether anti-
biotics are produced and recovered and the resultant com-
plexes are used as gxowth-promoting agents in food producing
mammals. The polyether antibiotics utilized in the examples
are monensin, nigericin, salinomycin, narasin, noboritomycin
A and B/ lysocellin, grisorixin, X-206, lonomycin~ laidlo-
mycin, SY-l, mutalomycin and alborixin.
-42- ~207S8Z
Each of the zinc complexes is produced and recovered
by a process similar to that set forth in Example I except
that the appropriate microorganism is utilized instead of
the lasalocid producing microorganism. The recovered zinc
complex of each antibiotic is formulated into a feed compos-
ition similar to the composition set forth in Example II and
fed to cattle in amounts sufficient to provide from about 5
to 100 ppm of the zinc complex in the rumen fluid during
rumination. Positive effects are reali2ed for each poly-
ether antibiotic in its zinc complexed form in promoting
growth and feed efficiency in cattle. The results are set
forth below in tabular form, an "X" indicating that a posi-
tive effect is realized by the use of a particular zinc
complex.
Table III
Polyether , Cattle
Example Antibiotic Growth
Number Zinc Complex Promotion
20 IV Noboritomycin X
V Monensin
VI Laidlomycin X
VII Nigericin X
VIII Grisorixin X
IX Salinomycin X
X Narasin X
XI Lonomycin X ,
XII X-206 X
XIII Alborixin X
30 XIV SY-l X
' ^ -43-- ~20~58Z
Table III (Continued)
Polyether Cattle
Example Antibiotic Growth
Number Zinc Complex Promotion
. . .
XV Lysocellin X
XVI Mutalomycin X _
EXAMPLES XVII--XXXIX
, _
The following additional zinc antibiotic complexes
may also be administered to cattle as a growth promoting
agent: zinc carriomycin, and the zinc complexes of septa-
mycin, dianemycin, A-204, lenoremycin, X-14547, A-23187,
etheromycin, ionomycin, aabomycin, disnerycin, duamycin,
BL-580, K-41, SF 1195, M-4164A, A-32887, 30,504RP, 38,986,
44,161, 47,433, 47,434 and 47,224.
Each of the zinc complexes is produced and recovered
by a process similar to that set forth in Example I except
that the appropriate microorganism is utilized instead o~
the lasalocid producing microorganism. Specific processes
for obtaining the named antibiotics are set forth above.
Some of the recovered zinc complex of each antibiotic
is utiliæed in a feed composition and fed to food-producing
mammals. ALl of the above zinc polyether antibiotics pro-
vide similar results, each being active in food-producing
mammals for growth promotion. Similar results were also
obtained in myocardial stimulation in mammals.
While the present invention has been described with
reference to particular embodiments thereof, it will be
understood that numerous modifications may be made by those
skilled in the art without actually departing from the
spirit and scope of the invention.
