CA1207582A - Method of promoting growth using zinc-containing antibiotic agents - Google Patents

Abstract

ABSTRACT
A process for obtaining improved growth in food-producing animals such as cattle, sheep and hogs, which comprises administering to said animals the zinc complexes of various monovalent and divalent polyether antibiotics which act as growth promoting agents. Soluble zinc salt is added to a fermentation beer containing the polyether anti-biotics to form an insoluble, recoverable biomass contain-ing the desired zinc antibiotic complexes used in the method of the subject invention. The zinc complexes may be admin-istered as a feed additive, or by subcutaneous implant, preferably on the ear of the animal.
The polyether antibiotics which may be used to make the subject zinc complexes include: linear monovalent and divalent polyethers (monensin, nigericin, lasalocid, lyso-cellin, etc.); non-glycolic monovalent monoglycoside poly-ethers (septamycin, dianemycin, lenoremycin, carriomycin and antibiotic A-204); mononitrogen-containing divalent pyrrole ethers (calcimycin, X-14547, etc.); polynitrogen-containing divalent pyrrole ethers (P.-23187, etc.); gly-colic monovalent monoglycoside polyethers (etheromycin, etc.); other polyether antibiotics including 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. The above zinc complexes are active agents for improving cardiovascular function in animals.

Description

~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 ~'' ~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 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

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

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 ~ ~

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

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 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 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:

~.
~' 12~)'75~

CH CH

C~L/ ~ H3 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 .

Septamy n l~Z07S192 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

_.
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 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 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 Agricultural Research Service under accession number NRRL
3882. The A ~23187 antibiotic has the structural formula:
", Me 0~-- N ~

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-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 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 , 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.

Claims (14)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. 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 from 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, 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 biomass containing both a zinc complex of said polyether antibiotic and insoluble zinc complexes of residual nitrogen-containing compounds present in the fermenta-tion broth.

2. A process according to claim 1 wherein the zinc complex of the polyether antibiotic has a molecular weight in the range of 300-1800.

3. A process according to claim 1 wherein the polyether antibiotic is selected from the group consisting of linear mono-valent polyether antibiotics and non-nitrogen containing divalent polyether antibiotics.

4. A process in accordance with claim 1 wherein the anti-biotic is selected from the group consisting of nigericin, salino-mycin, narasin, noboritomycin A and B, grisorixin, X-206, laidlo-mycin, SY-1, mutalomycin, alborixin and lonomycin.

5. A process in accordance with claim 1 wherein the polyether antibiotic is monensin.

6. A process in accordance with claim 1 wherein the polyether antibiotic is lasalocid.

7. A process in accordance with claim 1 wherein the polyether antibiotic is lysocellin.

8. A biomass of a zinc complex of a polyether antibiotic as defined in claim 1, whenever prepared according to the process of claim 1, or a chemically equivalent process.

9. A biomass of a zinc complex of a polyether antibiotic as defined in claim 2, whenever prepared according to the process of claim 2, or a chemically equivalent process.

10. A biomass of a zinc complex of a polyether antibiotic as defined in claim 3, whenever prepared according to the process of claim 3, or a chemically equivalent process.

11. A biomass of a zinc complex of a polyether antibiotic as definfed in claim 4, whenever prepared according to the process of claim 4, or a chemically equivalent process.

12. A biomass of a zinc complex of a polyether antibiotic as defined in claim 5, whenever prepared according to the process of claim 5, or a chemically equivalent process.

13. A biomass of a zinc complex of a polyether antibiotic as defined in claim 6, whenever prepared according to the process of claim 6, or a chemically equivalent process.

14. A biomass of a zinc complex of a polyether antibiotic as defined in claim 7, whenever prepared according to the process of claim 7, or a chemically equivalent process.