IL44065A - Metabolite a-27106 and process for its preparation and its use as coccidiostatic agent and feed additive - Google Patents

Abstract

1457796 Metabolite A-27106 ELI LILLY & CO 30 Jan 1974 [1 Feb 1973] 04414/74 Headings C2A and C2C The invention relates to Metabolite A-27106 and the acid, ammonium, lithium, potassium, rubidium or cesium forms thereof. The metabolite is a white crystalline solid with (a) a molecular weight of 854, as determined by mass spectrometry; (b) an elemental composition of 58À78% carbon, 8À51% hydrogen, 27À85% oxygen and 3À63% sodium; (c) an empirical formula of C 42 H 71 O 16 Na; (d) an infra-red absorption spectrum in chloroform as shown in the accompanying drawing; (e) a mass spectrum having a molecular-ion peak at m./e. 854À44630 and characteristic peaks at m./e. 836À44129, 779À44690, 761À44740; and (f) an Rf value of 0À49 on silica gel thin layer chromatography in benzene-methanol (7 : 3); and the acid form is a white solid having a molecular weight of 832 and a titratable group with a pKa value of 7À2. The following structure is proposed for Metabolite A-27106. Metabolite A-27106 is prepared by cultivating Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions in the presence of glucose and monensin until monensin is converted into metabolite A-27106. The metabolite may be mixed with a physiologically-acceptable carrier, e.g. poultry feed, and administered to poultry for preventing or treating coccidiosis therein. The metabolite may also be mixed with ruminant feed for increasing the efficiency of feed utilization of ruminant animals. [GB1457796A]

Description

44065/2 METABOLITE A-27106 AND PROCESS FOR ITS PREPARATION A DTUSE AS COCCIDIOSTATIC AGENT AND FEED ADDITIVE T3 4»in¾n lnaDn'? η ηπι A-27106 o'^ia Λon I lb/if The invention relates to metabolite A-27106 and the process for its preparation from monensin by submerged culture fermentation of S . candidus NRRL 5449.

The invention further relates to an animal feed containing metabolite A-27106 as the active ingredient.

The invention also relates to a method for control ling coccidiosis in poultry.

The invention additionally relates to a method of increasing efficiency of feed utilization of ruminant animals.

The invention provides metabolite A-27106, or the acid, ammonium, lithium, potassium, rubidium or cesium forms thereof, which metabolite is a white crystalline solid, relatively soluble in lower alkanols, but generally insolubl in lower alkanes and which has : a) a molecular weight of 854, as determined by mass spectro metry; b) an approximate elemental composition of 58.78% carbon, 8.51% hydrogen, 27.85% oxygen, and 3.63% sodium; c) an empirical formula of C42H7i°i6Na; d) an infrared absorption spectrum in chloroform as shown in the accompanying drawing; e) a mass spectrum which shows a molecular-ion peak at m/e 854.44630 and characteristic peaks at m/e 836.44129, 779.44690, 761.44740; and f) an Rf value of 0.49 on solica-gel thin-layer chromatography in benzene-methanol (7:3); and the acid form of which is a white solid having a molecular weight of about 832 and a titratable group with a pKa value of 7.2.

The invention also provides a process of converting monensin into metabolite A-27106 which comprises cultivating Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic conditions in the presence of glucose and monensin until monensin is converted into a substantial amount of metabolite A-27106 by said organism in said culture medium.

The invention further provides a method of preventing or treating coccidiosis in poultry which comprises administering to poultry an effective amount of a composition comprising metabolite A-27106 and a physiologically-acceptable carrier, and a method of increasing the efficiency of feed utilization of ruminant animals having a developed rumen function which comprises the oral administration to such animals of the propionate-increasing amount of metabolite A-27106.

The invention still further provides a feed adapted to control coccidiosis comprising poultry feed and an amount of metabolite A-27106 sufficient to provide a non-toxic, coccidiostatic dose of said product per bird per day, and a feed adapted to fatten cattle comprising cattle feed and sufficient metabolite A-27106 to provide a non-toxic, propionate-increasing amount per animal per day.

Coccidiosis is a well-known protozoan disease resulting from infection by one or more species of Eimeria or Isospora (for a summary, see Lund and Farr in "Diseases of Poultry" 5th ed., Biester and Schwarte, Eds., Iowa State University Press, Ames, la., pp. 1056-1096). In view of the great economic losses from coccidiosis and the difficulties attending the use of some known coccidiostats , the search for better coccidiostats continues. ^ Because ruminants are animals of economic importance, increasing ruminant feed-utilization efficiency is very desirable. The mechanism for utilization of the major nutritive portion (carbohydrates) of ruminant feed is well known. Microorganisms in the rumen of the animal ferment carbohydrates to produce monosaccharides and then degrade these monosaccharides to pyruvate compounds . Pyruvates are metabolized by microbiological processes to form acetates, butyrates or propionates, collectively known as volatile fatty acids (VFA) .

The relative efficiency of utilization of the VFA's is discussed by McCullough, Feedstuffs , June 19, 1971, page 19; Eskeland et al . , J. An. Sci. 33, 282 (1971); and Church et al . , "Digestive Physiology and Nutrition of Ruminants," Vol. 2, 1971, pp 622 and 625. Although acetates and butyrates are utilized, propionates are utilized with relatively better efficiency. Furthermore, when too little propionate is available, animals may develop ketosis. A beneficial drug, therefore, encourages animals to produce propionates from carbohydrates, thereby increasing carbohydrate-utilization efficiency and also reducing the incidence of ketosis.

Monensin, from which metabolite A-27106 can be prepared, was disclosed in U.S. Patent 3,501,568 as factor A of antibiotic A3823 complex. Monensin is also an anticoccidial agent .

The biologically active agent of the present process is arbitrarily designated as metabolite A-27106. The expression "A-27106" as used herein refers to the sodium salt.

Additional salts include the ammonium, lithium, potassium, rubidium and cesium salts and the acid form.

A starting material in the preparation of metabolite A-27106 is monensin, the sodium salt of which has the formula Formula I Monensin in its sodium form is produced by Streptomyces cinnamonensis as described in U.S. Patent 3,501,568.

Metabolite A-27106 is produced from either monensin or the mycelial culture in which monensin is produced, in the presence of glucose, by an enzyme or enzymes elaborated by a new strain of Streptomyces candidus . A culture of this new strain has been deposited, without restriction as to availability, with the permanent culture collection of the Northern Utilization Research and Development Division, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604, where it was assigned the accession number NRRL 5449.

Because of the uncertainty of taxonomic studies with the Streptomyces group of organisms, there is always an element of doubt associated with the classification of a newly discovered organism. In the most important characteristics, however, organism NRRL 5449 which converts monensin into metabolite A-27106 appears to resemble most nearly Streptomyces candidus (Krassilnikov) Waksman 1953. The type culture is described by S. A. Waksman in "The Actinomycetes , " Vol. II, Williams and Wilkins, Baltimore, 1961, p. 187.

The type organism is also on deposit at the Institute of Microbiology of Rutgers University, New Brunswick, N. J., under the accession number IMRU 3416. The present organism is considered a novel strain of the described organism.

Although S. candidus NRRL 5449 and the known strain resemble one another in general mycelial morphology and color and in spore appearance, sufficient differences exist between them to require that the organism described herein be characterized as a new strain. For example, the published organism does not coagulate milk and liquefies gelatin slowly, whereas the new strain forms curd after 14 days and does not liquefy gelatin in 21 days. Most importantly, a specimen culture propagated from strain IMRU 3416 did not convert monensin to metabolite A-27106.

The organism which converts monensin into metabolite A-27106 was isolated from a soil sample collected on Mount Ararat, Turkey, by suspending portions of the soil in sterile distilled water and streaking the suspension on nutrient agar. The seeded agar plates were incubated at 25-35°C. until visible colonies were observed. At the end of the incubation period, colonies of selected organisms were transferred by means of a sterile platinum loop to agar slants. One of the slants was then incubated to provide suitable quantities of inoculum of organism NRRL 5449.

Taxonomic studies of S. candidus NRRL 5449 were made using methods recommended for the International Cooperative Project for Description and Deposition of Streptomycetes in accordance with procedures described by Shirling and Gottlieb, "Methods for Characterization of Streptomyces Species," International Bulletin of Systematic Bacteriology 16, 313-340 (1966) , together with other supplementary tests. The prefix "ICP" refers to media described by Shirling and Gottlieb.

The remaining media are described by Waksman, cited. Color names were assigned according to Kelly and Judd in "The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names," U.S. Department of Commerce circular 553, 1955.

Figures in parenthesis refer to the Tresner and Backus color series, "System of Color Wheels for Streptomyces Taxonomy," Appl . Microbiol . 11, 335 (1963) . Color tab designations are underlined, and Maerz and Paul ("Dictionary of Color," McGraw-Hill, N.Y., 1950) color blocks are enclosed in brackets.

Streptomycete NRRL 5449 is characterized by straight to wavy sporophores and oval to slightly cylindrical spores, averaging 1.165μ x 0.57μ, in chains of from 10 to 50. Spores are smooth as observed by electron microscopy. Aerial mycelia are usually white. At 26 °C. only vegetative growth occurs, and at 45°C. no growth occurs. Maximum growth and sporulation occur at 30 to 37°C.

In accordance with standard practice, the growth of microorganism NRRL 5449 was studied on a variety of media that are accepted in the study of the Actinomycetes . Cultural procedures were uniform and standard. The media employed and the cultural characteristics observed are set forth below: ICP 1 Fair growth; pale yellow reverse [11C1] ; no aerial mycelia or spores; no soluble pigment.

ICP 2 Abundant growth; reverse light yellow [10J3] ; abundant aerial mycelia and spores ; (W) white a; no soluble pigment.

ICP 3 Good growth; reverse pale yellow green [10C1] ; good aerial mycelia and spores; (W) white a; no soluble pigment.

ICP 4 Good to abundant growth; reverse moderate yellow [10H4] ; good to abundant aerial mycelia and spores; (W) white a; brown soluble pigment.

ICP 5 Abundant growth; reverse moderate orange yellow [1016] ; abundant aerial mycelia and spores; (Y) pale yellow 2db; slight brown soluble pigment.

ICP 7 Good growth; reverse pale yellow green [10B1] ; good aerial mycelia and spores; (W) white a; no soluble pigment.

Glycerol-Glycine Abundant growth; medium brown reverse [11J4] ; abundant sporulation and aerial mycelia; (W) white a and (Y) pale yellow 2db; slight light brown soluble pigment. Emerson ' s Abundant growth; grayish yellow reverse [12H3] ; no aerial mycelia or spores; no soluble pigment.

Bennett ' s Good growth; light yellow reverse

[1112] ; scant aerial mycelia and spores; (W) white a; no soluble pigment.

Czapek ' s Abundant growth; moderate orange yellow [11J7] ; abundant aerial mycelia and spores; (W) white a; no soluble pigment.

Glucose-Asparagine Fair to good growth; pale yellow green reverse [10B1] ; no aerial mycelia or spores; no soluble pigment.

Calcium malate Abundant growth; reverse grayish yellow [11E4] ,- abundant aerial mycelia and spores; (Y) pale yellow 2ba; slight brown soluble pigment.

Nutrient Agar Good growth; reverse pale yellow green [10C1] ; no aerial mycelia or spores; no soluble pigment.

The organism was studied for selected physiological properties in accordance with standard procedures. The properties observed and characteristics found were as follows Property Observed Characteristic Action on skim milk clearing; curd after 14 days Nitrate reduction positive Melanin production Tryptone yeast extract broth slight Tyrosine agar none Gelatin none at 21 days liquefaction Temperature 26 °C . -vegetative growth only requirements 30-37 °C. -good vegetative growth; good aerial mycelia and spores 43-55°C.-no growth The results of carbon utilization tests carried out with organism NRRL 5449 are set forth below. The symbols used to indicate growth response are : + « Utilization - good growth [+] - Probable utilization - poor to fair growth [-] ■ Questionable utilization - little or no growth m No utilization - no growth.

Carbon source Response raffinose + D-fructose t+J cellobiose [+] L-arabinose [+] D-mannitol [+] rhamnose M cellulose dextrose [+] to [-] D-xylose [+] inositol + -C (no carbohydrate) [-] to [+] The culture medium employed to grow S. candidus NRRL 5449 can be any one of a number of media. However, for economy in production, optimal yield, and ease of product isolation, certain culture media are preferred. Thus, for example, among the preferred sources of carbohydrate in large-scale fermentation are invert sugar or corn syrup, although glucose, fructose, maltose, starch, inositol, and the like can also be employed. When monensin is to be converted to metabolite A-27106 in fermentation culture at the time S. candidus NRRL 5449 is grown, the medium must contain a source of glucose to achieve efficient conversion. When S. candidus NRRL 5449 is grown to produce its enzyme system for later use in converting monensin to metabolite A-27106, the presence of glucose during fermentation is optional. Preferred sources of nitrogen are peptones, soybean meal, amino acid mixtures and the like. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary soluble salts capable of yielding iron, sodium, potassium, ammonium, calcium, phosphate, chloride, carbonate, and like ions.

Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism.

The initial pH of the culture medium can be varied. Prior to inoculation with the organism, however, it is desirable to adjust the pH of the culture medium to between about pH 5.7 and about pH 7.5, depending upon the particular medium employed. As is the case with other Actinomycetes , the medium gradually becomes more alkaline as the fermentation proceeds and may rise from an initial pH of about pH 5.9 to about pH 6.9 or higher during the growth period of the organism. The final pH is controlled, at least in part, by the initial pH of the medium, the buffers present in the medium, and the duration of time the organism is permitted to grow. Although pH can be adjusted by addition of either acid or base, good results have been achieved with no adjustment of pH.

In common with other Streptomyces species, organism N RL 5449 requires aerobic growth conditions. Small-volume propagation is conveniently carried out on agar slants or plates, in shake flasks or in bottles. For large-scale production, submerged aerobic culture in large tanks is preferred.

The fermentation medium in a sterile tank can be inoculated with a sporulated suspension to initiate fermentation. However, since inoculation with a sporulated suspension involves a growth lag, a vegetative inoculum is preferable. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the growth of the vegetative inoculum can be the same as that used for large-scale production, but other media can be employed.

The organism S. candidus NRRL 5449 will grow over a temperature range between about 26°C. to about 40°C.

Maximum growth and sporulation, however, occur between about 32°C. and about 37°C.

As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium during fermentation. For efficient growth of the organism and production of metabolite A-27106, the volume of air employed in tank production of the substance should be above about 0.1 volume of air per minute per volume of culture medium.

Optimal yields are obtained when the volume of air used is at least one-third to one-half volume of air per minute per volume of culture medium.

The fermentation time needed to convert monensin to metabolite A-27106 varies. The presence of an adequate supply of glucose is essential for the conversion of monensin to metabolite A-27106. In general, when glucose is present in adequate amounts and monensin is present in about 0.1 to 1.0 grams per liter of medium, conversion of monensin to metabolite A-27106 is essentially complete by about 36 to 72 hours. Optimal conversion occurs when monensin is present in a range of from 0.5 to 0.7 grams per liter of medium. An adequate amount of glucose is from about 2.0 to about 2.5 percent of medium by weight. A glucose-sensing test paper may be used to check concentration levels. When glucose content drops below about two percent, glucose should be added to maintain concentration at optimum levels .

When the S. candidus enzyme system is used to effect conversion of monensin to metabolite A-27106, it is not essential that the enzyme preparation be highly purified. For example, filtered fermentation broth can be lyophilized and stored for at least as long as two weeks before reconstituting with an aqueous buffer, using approximately one-sixth the volume of original broth. Efficient conversion is achieved after about 72 hours when 2.5 g. of such a lyophilized preparation is reconstituted in the presence of 25 mg. of monensin.

The active enzyme system is present in both the filtered broth and the cells. An enzyme preparation of greater purity can be obtained from the separated fermentation cells. These cells may be frozen and stored for periods at least as long as three months. The thawed cells can be then reconstituted by suspending them in a buffer solution. The buffer suspension is further purified by sonication and centrifuga-tion. The purified cell debris thus separated is resuspended in buffer and is dialyzed. Using this method, 200 g. of cells from the fermentation medium give purified dialyzate sufficient to convert glucose and 25 mg. of monensin to metabolite A-27106.

Conversion progress can be monitored by thin-layer chromatography (tic). On silica gel (F-254, E-N Laboratories, Inc., Elmsford, N. Y.) in benzene-methanol (7:3), the Rf value for monensin is 0.62, whereas the Rf value for metabolite A-27106 is 0.49. A vanillin spray reagent can be used for detection. This reagent is prepared by adding fuming sulfuric acid (2 ml.) to a solution of vanillin (3 g.) in absolute ethanol (100 ml.) .

Metabolite A-27106 is present in both the culture broth and in the mycelia. Accordingly, techniques employed in the isolation of metabolite A-27106 are designed to permit maximum recovery of the product from either or both sources . Thus, for example, the fermentation medium is filtered, and both the filtrate and the mycelial cake are extracted with suitable solvents to obtain metabolite A-27106. The product is recovered from the extracting solvents by ordinary methods commonly employed in the art.

Alternatively, the culture solids, including medium constituents and mycelia, can be used without extraction or separation, but preferably with removal of water from the mycelia and culture medium, as a source of metabolite A-27106. For example, the culture medium can be dried by lyophilization and mixed into feed. Also, the solids can be converted without total removal of water to a thin slurry which is suitable for addition to wet mash and similar feeds .

No single extraction/isolation procedure is mandatory. In one satisfactory manner, the finished culture medium is filtered, using a filter aid. The filter cake is extracted with a polar solvent such as methanol. The methanol extract is concentrated and then added to the original aqueous filtrate. This combined solution is extracted twice with half volumes of chloroform. The chloroform extracts are evaporated under vacuum to give a dark amber oil.

This oil is decolorized over an activated-charcoal column, using chloroform and about 20 g. of activated charcoal per gram of oil. The eluate is again concentrated under vacuum to give a pale yellow to colorless oil. This oil, dissolved in a minimal amount of chloroform, is chromato-graphed on a silica-gel column, using ethyl acetate as a solvent. Elution is monitored by thin-layer chromatography. Impurities are eluted with ethyl acetate. Elution with ethyl acetate-methanol mixtures affords metabolite A-27106.

Metabolite A-27106 (sodium salt) is a white crystalline solid, melting with bubbling at about 170-175°C. Metabolite A-27106 appears to form a hydrate or other solvate very readily. When A-27106 is hydrated or solvated, its melting point varies, generally melting a few degrees below the indicated value.

Elemental analysis of metabolite A-27106 gave the following percentage composition: carbon, 58.78; hydrogen, 8.51; oxygen, 27.85; and sodium, 3.63. These values correlate with empirical formula C42H7i°i6Na wn;*-cn nas a theoretical percentage composition of carbon, 59.00; hydrogen, 8.37; oxygen, 29.94; and sodium, 2.42.

Metabolite A-27106 exhibits practically no ultraviolet absorption above about 235y.

The infrared absorption spectrum of metabolite A-27106 in chloroform is shown in Figure 1 of the accompanying drawing. The distinguishable absorption maxima of the spectrum are as follows: 3.1, 3.36, 6.39, 6.82, 7.1, 7.25, 7.9 (shoulder), 8.1, 8.3, 8.67, 8.8 (shoulder), 9.04, 9.22, 9.51, 9.66, 10.03, 10.26, 10.66, 11.23, 11.47, 11.83, and 12.15.

The mass spectrum of metabolite A-27106 shows a molecular-ion peak and other characteristic peaks as listed below: Calcd. Observed Fragment 20]) These findings confirm the assigned empirical formula and a molecular weight of 854 for the sodium salt of metabolite A-27106.

In general, metabolite A-27106 is readily soluble in highly polar solvents, is insoluble in nonpolar solvents, and varies in solubility in solvents of intermediate polarity. Illustratively, A-27106 is soluble in lower aliphatic alcohols, is partially soluble in phenol, diethyl ether and acetone, and is relatively insoluble in liquid lower alkanes.

Electrometric titration of metabolite A-27106 in the acid form in water at an initial pH of 8 revealed the presence of a titratable group with a pKa value of 7.2.

The monosodium form above-described is, in general, the natural form of metabolite A-27106. From the sodium salt, the acid is easily produced; from the acid, the ammonium and other alkali-metal salts are prepared. The various metallic forms behave somewhat like alkali-metal carboxylates and some- what like chelates .

In preparing another form, metabolite A-27106 (the sodium form) is dissolved in an aqueous solvent, such as methanol-water ; an acid such as, for example, hydrochloric acid is added to lower the pH to 5 or below. The methanol is removed under vacuum, and the resulting aqueous acid product is extracted with chloroform. The chloroform extract is dried and evaporated to give the free acid form.

This acid form of metabolite A-27106 can be used, or it can be further modified by titrating it with an aqueous alkali-metal hydroxide or aqueous ammonia to obtain the corresponding lithium, potassium, rubidium, cesium or ammonium forms. The acid, ammonium, and alkali-metal forms of metabolite A-27106 are all biologically active.

The exact structure of metabolite A-27106 is not known. It is known that in the conversion of monensin to metabolite A-27106, glucose is necessary and is consumed, even in the absence of metabolizing S. candidus cells. The molecular weight of A-27106 corresponds to that of a glucosyl monensin .

Other than the highly hindered tertiary hydroxyl on the E ring, there would be five hydroxyl sites on a glucosyl monensin, any or all of which should be susceptible of reaction to form a simple ester such as an acetate. The presence of all five such reactive sites has been demonstrated in esterification experiments.

Based on the physical characteristics hereinabove recited, the following structure can be proposed for metabolite A-27106: Formula II Since the structure is only postulated, it is to be understood that the structure presented above represents merely a working hypothesis .

Metabolite A-27106 is less toxic than monensin. In tests when metabolite A-27106 was administered intraperito-neally to groups of six mice each: at 50 mg./kg. one out of six died; at 100 mg./kg. three out of six died.

In similar tests, when monensin was administered intraperi-toneally to groups of six mice each: at 10 mg./kg. one out of six died; at 20 mg./kg. three out of six died.

For the prevention or treatment of coccidiosis in poultry, a non-toxic, coccidiostatic amount of metabolite A-27106 is administered to birds, preferably orally on a daily basis. Although a variety of factors must be considered in determining an appropriate concentration of A-27106, the rate of administration will be generally in the range of 0.005 to 0.05 percent by weight of unmedicated feed, and preferably in the range of 0.01 to 0.04 percent. Metabolite A-27106 can be supplied in many ways, but it is most conveniently supplied with a physiologically-acceptable carrier, preferably the feed ingested by the birds.

Metabolite A-27106 also improves feed utilization in ruminants which have a developed rumen function. Young ruminants, basically those still unweaned, function as mono-gastric animals. As young ruminants begin to eat solid food, the rumen function begins to develop, and the microbiological population of the rumen begins to increase. After the animal has eaten solid feed for a time, its rumen function reaches full development and continues to operate throughout the animal's life. Some economically important ruminant animals are cattle, sheep and goats.

Metabolite A-27106 is typically effective in increasing the efficiency of feed-utilization when administered to ruminants orally at rates of from about 0.05 mg./kg./ day to about 2.5 mg./kg./day. Most beneficial results are achieved at rates of from about 0.1 mg./kg. /day to about 1.5 mg./kg./day. A preferred method of administration of metabolite A-27106 is by mixing it with the animals' feed; however, it can be administered in other ways, for example, tablets, drenches, boluses, or capsules. Formulation of these various dosage forms can be accomplished by methods well known in the veterinary pharmaceutical art. Each individual dosage unit should contain a quantity of metabolite A-27106 directly related to the proper daily dose for the animal to be treated.

The following examples are provided.

EXAMPLE 1 Preparation of A-27106 from Monensin by S_. candidus S. candidus NRRL 5449 was grown on an agar slant prepared from Bennett's medium to give a well-defined colony.

The colony was removed and made up as a slurry with sterile deionized water (10 ml.) .

This slurry was divided among four 500-ml. shake flasks, each containing 100 ml. of vegetative medium of the following composition: Ingredient Amount Corn distillers' solubles* 25. g.

Lactose 10. g- Maltose 10. g.

MgS04.7H20 2. g.

KH2P04 2. g.

CaC03 2. g.

Deionized water q.s. 1.1 liter *Nadrisol, National Distiller's Products Company, U.S.A The four inoculated flasks were incubated at 30°C. on a rotary shaker at 250 r.p.m. for 24 hours. This vegetative medium (10-ml. portions) was used to inoculate each of 15 shake flasks (500 ml.) containing 100 ml. of sterilized fermentation medium of the following composition: Ingredient Amount Beef extract 5 g.

Casein pancreatic 5 g. hydrolysate peptone NaCl 5 g.

Glycerol 15 g.

CaC03 2 g.

Deionized water q.s. 1 liter The medium had a pH of 7.2 which was not adjusted. The inoculated medium was incubated for 72 hours as described above.

In a 40-liter fermentor a production medium of the following composition was prepared: Ingredient Amount Polysiloxane oil 5 g. antifoam agent Glycerol 375 g.

Dextrose 625 g.

Casein pancreatic 125 g. hydrolysate peptone Beef extract 125 g.

NaCl 125 g.

CaC03 50 g.

Deionized water q.s. 24 liters The initial pH of the medium was 7.0. The medium was then sterilized by autoclaving at 120 °C. for 30 minutes at 15-20 pounds per square inch pressure. After sterilization the pH of the medium was 7.6.

Purified monensin (25 g.) dissolved in ethanol (200 ml.) was added to the sterilized medium, and the second-stage vegetative inoculum (700 ml.) prepared as described above was introduced.

The fermentation medium was aerated with sterile air at a rate of 0.35 cubic feet (about one liter) per minute and was stirred with conventional agitators at 420 r.p.m. The inoculated medium was incubated at 30°C. for about 114.5 hours The course of the fermentation was followed by thin-layer chromatography on silica gel as described hereinabove. Early in the fermentation only monensin was present. Gradu-ally a second spot indicated the presence of metabolite A-27106; and finally, only the spot for metabolite A-27106 was present.

EXAMPLE 2 Isolation and Purification of A-27106 The fermentation broth, prepared as described in Example 1, was filtered, using a filter aid. The mycelial cake was extracted with methanol (about 5 1.) at room temperature. The methanol extract was filtered; the filtrate was concentrated under vacuum, removing the methanol and leaving an aqueous concentrate.

This aqueous concentrate was combined with the original broth filtrate. The combined solution (about 22 1.) was extracted twice with half-volumes of chloroform. The chloroform extracts were combined and concentrated under vacuum to about 400 ml. of a dark amber oil.

This oil, dissolved in chloroform, was decolorized over a 10-kg. carbon (Pittsburg "12 x 40") column, eluting with chloroform (about 5 1.). The chloroform eluate was evapo rated under vacuum to give about 500 ml . of a colorless to pale-yellow oil.

The decolorized oil , in a minimal amount of chloroform, was then chromatographed over a 25-kg. column of silica gel (Grace, grade 62) in ethyl acetate. Elution was monitored by thin-layer chromatography as described earlier. After impurities were removed with ethyl acetate, metabolite A-27106 was eluted from the column with ethyl acetate-methanol (19:1). The fractions containing A-27106 were combined and evaporated to dryness under vacuum to give an amorphous, almost white product. The product was washed with hexane and dried to give about 12.84 g. of metabolite A-27106 (one-spot material by tic) EXAMPLE 3 Production of A-27106 by S. cinnamonensis and S. candidus .

Another method of producing metabolite A-27106 in fermentor culture is illustrated by the following procedure Streptomyces cinnamonensis ATCC 15413 was conventionally grown (see U.S. Patent 3,501,568) in 55 ml. of medium in a 250-ml. shake flask, incubating for 47 hours to obtain a vegetative inoculum. This vegetative inoculum was added to 220 ml. of medium in a one-liter shake flask and was incubated for 21 hours to supply inoculum for seeding the fermentor .

The inoculum thus prepared was used to seed a 40-1. fermentor containing a heat-sterilized medium of the following composition: Ingredient Amount Glucose 750.0 g.

Soybean meal 625.0 g.

Soybean oil 500.0 g.

Methyl oleate 500.0 g.

Polysiloxane oil antifoam 5.0 g.

Potassium chloride 2.5 g.

Dipotassium hydrogen phosphate 2.5 g.

Manganous chloride tetrahydrate 15.0 g.

Hydrated ferric sulfate 7.5 g.

Calcium carbonate 25.0 g.

Deionized water q.s. 24 liters The resulting medium had a pH of 5.5 which was adjusted to pH 8.0 by addition of 10 N potassium hydroxide (15 ml.). The inoculated medium was incubated at 32°C. for 234 hours.

After 42 hours, aeration was increased from 0.35 to 0.8 cubic feet per minute, and agitation was increased from 500 to 700 r.p.m. Monensin production was essentially complete after 210 hours, as determined by thin-layer bioassay with Bacillus subtilis ATCC 6633 as the detection organism.

After 234 hours, the fermentor contents were pasteurized to inactivate S. cinnamonensis . The following nutrients were then added to the fermentor : Ingredient Amount Dextrose 750 g.

Soybean meal 625 g.

Manganeus chloride tetrahydrate 155 gg.

Calcium carbonate 12.5 g.

Ferric sulfate hexahydrate 7.5 g.

Potassium chloride 2.5 g.

Dipotassium hydrogen phosphate 2.5 g.

Methyl oleate 250 ml.

Soybean oil 250 ml.

Deionized water q.s. 24 liters The pH was adjusted to 8.0 with 215 ml. of 5 N NaOH, and the medium was sterilized. The medium was then inoculated with a rapidly growing vegetative culture of Streptomyces candidus NRRL 5449 and was incubated for 137 hours at 30°C. The fermentation was aerated with sterile air at a rate of 0.35 cubic foot per minute. The fermentation medium was stirred with conventional agitators first at 120 r.p.m., increasing after 16 hours to 420 r.p.m. and after 40 hours to 500 r.p.m. Dextrose (400 g.) was added at each of hours 44, 66.5, 89, 97, 113, and 127. Calcium carbonate (175 g.) was added at each of hours 72 and 99.

Thin-layer chromatography as described hereinabove was used to monitor the fermentation. After 137 hours production of metabolite A-27106 was essentially complete. Work-up and purification of A-27106 followed the procedures described in Example 2.

EXAMPLE 4 Production of A-27106 by a Particulate S. candidus Enzyme Preparation S. candidus NRRL 5449 was grown as described in Example 1, on a 100-liter scale. The cells were separated from the fermentation medium by vacuum filtration and were divided into 200-g. aliquots which were stored by freezing. Two of these aliquots were thawed at room temperature and were suspended in 0.05 M phosphate buffer (pH 5.8) to a final volume of 600 ml. This cell suspension was sonicated for 30 minutes, and the sonicate was centrifuged at 10,000 rpm for 30 minutes. The resulting cell debris was suspended in 100 ml. of the above-mentioned buffer; this suspension was dialyzed for 18 hours with 5 1. of chilled, above-mentioned buffer. To 50 ml. of the particulate dialyzate were added D-glucose (120 mg.) and monensin (25 mg.) in ethanol (2 ml.). The reaction mixture was stirred for 72 hours at 30eC. and then was filtered; the filtrate was extracted with chloroform (100 ml.). This chloroform extract, concentrated under vacuum was chromatographed on a silica gel column (10 g.) . Elution with ethyl acetate gave only a trace of monensin. Elution with ethyl acetate-methanol (19:1) gave 17 mg. of metabolite A-27106, identical to that obtained in Example 2.

EXAMPLE 5 Control of Coccidiosis with Metabolite A-27106 A group of 75 one-week-old, healthy, incubator and battery-reared, cross-bred male chicks, of a heavy broiler type was used. The birds were divided into five groups of 15 birds having three replicates of five-bird subgroups each. Each subgroup was reared out of contact with the other subgroups. A first group was held as an untreated healthy control under favorable conditions. A second group was treated as the first group, but was inoculated with coccidiosis by 5 " oral administration of a dose containing 10 sporulated oocysts of Eimeria tenella . The third, fourth and fifth groups were treated as the second group except that, 24 hours before the said inoculation, their feed was modified by addition of metabolite A-27106 in concentrations of 100, 150, and 200 parts per million (ppm) , respectively.

Seven days after inoculation, the birds were weighed, sacrificed and examined for evidence of coccidial lesions.

Coccidial involvement was expressed on an arbitrary scale of zero to four. Lesion scores indicate the number of birds at each level . Zero indicates no evidence of coccidiosis . One designates the least coccidial involvement that can be seen. Two indicates moderate involvement with little or no hemorrhage and no severe tissue damage. Three indicates hemorrhage, swelling of the caeca, and extensive tissue involvement. Four indicates hemorrhage, a caecal core of clotted blood and debrided epithelial cells. Birds scoring four or less usually recover if not further infected with coccidia.

Using the above-described procedures, the following results were observed: Metabolite Group Inoculated A-27106 Avg. Wt. of with (ppm) in Gain/Bird Birds £. tenella the diet in Grams 1 no 0 162 1 2 yes 0 67 3 yes 100 157 4 yes 150 150 5 yes 200 166 In the medicated birds with high lesion scores, fecal blood was scanty or absent, and the birds appeared to be in good condition. Their good weight gain was regarded as more meaningful than the lesion scores.

Since studies indicate that metabolite A-27106 usually kills E. tenella as it first attempts to establish itself in the host cell, a prophylactic regimen of treatment is preferable.

In other studies metabolite A-27106 was found to be more palatable to birds and also less toxic than was monensin Numerous other studies were carried out with metabo lite A-27106, both alone and in combination with monensin. These studies showed that although A-27106 gave no greater weight gains at 0.04 percent than at 0.01 percent, the higher rate resulted in fewer caecal lesions. There was no evidence of toxicity at the higher rate.

Within the general efficacy range, monensin and metabolite A-27106 combined in the diet show at least an additive result in efficacy, but not in toxicity. Toxicity of the combination is lower than that of monensin alone at a rate equal to the combined rates .

EXAMPLE 6 A-27106-Modified Chick Ration for Coccidiosis Control A balanced, high-energy ration adapted to feed chicks for rapid weight gain is prepared by the following recipe : Ingredient Ground yellow corn Soybean meal, solvent extracted dehulled, finely ground, 50 percent protein Animal fat (beef tallow) Dried fish meal, with solubles (60% protein) Distillers' solubles from corn Dicalcium phosphate, feed grade Calcium carbonate Vitamin premix (representing vitamins A,D,E,K, and B, ~ r choline, niacin, panto thenic acid, riboflavin, biotin with glucose bulking agent) Trace mineral premix (representing MnSO,, ZnO, KI, FeS04, CaC03) 2-Amino-4-hydroxybutyric acid (hydroxy analog of methionine) Metabolite A-27106 These substances are mixed in accordance with stan dard feed-mixing techniques . Chicks fed such a ration , with water ad libitum, are protected from exposure to coccidiosis weight gains are comparable to those of coccidiosis-free chicks fed a similar, unmedicated diet.

EXAMPLE 7 Feed-Utilization Efficiency Improved by A-27106 Rumen fluid is obtained from a steer with a surgically-installed fistula opening into the rumen. The steer is maintained on a high-grain ration, the composition of which follows : 69. 5% coarse ground corn 10 % ground corncobs 8 % soybean meal (50% protein) 5 % alfalfa meal 5 % molasses 0. 6 % urea 0. 5 % dicalcium phosphate 0. 5 % calcium carbonate 0. 3 % salt 0. 07% vitamin A and D2 premix* 0. 05% vitamin E premix** 0. 03% trace mineral premix*** Containing per pound: 2,000,000 I.U. of vitamin A; 227,200 I.U. of vitamin D2 and 385.7 g. of soybean feed with 1% oil added Corn distillers dried grains with solubles containing 20,000 I.U. of d-alpha tocopheryl acetate per pound Containing manganous oxide, potassium iodide, cobalt carbonate, copper oxide and zinc sulfate A sample of rumen fluid is strained through four layers of cheesecloth, and the filtrate is collected. The particulate matter retained by the cheesecloth is resuspended in enough physiological buffer to return it to the original volume of the rumen fluid, and this suspension is strained again. The buffer used has the following composition: g. /liter Ingredient 0.316 a2HP04 0.152 KH2P04 2.260 NaHC03 0.375 KC1 0.375 NaCl 0.112 MgS04 0.038 CaCl2 0.008 FeSO..7H 0 4 2 0.004 MnS04 0.004 ZnSO..7H 0 4 2 0.002 CuS04.5H20 0.001 CaCl2 as described by Cheng et a_l. in J. Dairy Sci . 38 , 1225, (1955).

The two filtrates are combined and allowed to stand until particulate matter separates to the top. The clear layer is separated, diluted with the same buffer (1:1) and then adjusted to pH 7.0.

The diluted rumen fluid (10 ml.) is placed in a 25-ml. flask with 40 mg. of the above-described feed, an additional 5 mg. of soybean protein, and the compound to be tested. Four replicate flasks are used per treatment. Two sets of four control flasks each are also employed. A zero-time control and an incubated 16-hour control are used. All test flasks are incubated for 16 hours at 38 °C. After incuba- tion the H is measured, and 25 percent metaphosphoric acid (2 ml.) is added to each flask. The samples are allowed to settle, and the supernatant is analyzed by gas chromatography for propionate, acetate, and butyrate compounds. Active compounds significantly increase propionate production over that of controls.

Test-compound results are statistically compared with the control results. The table below shows the ratio of volatile-fatty-acid concentrations in metabolite-A-27106-treated flasks to concentrations in control flasks. meg. A-27106/ml. diluted rumen fluid Propionate Butyrate Total VFA 5 2.15 0.73 1.03 1 1.42 0.96 0.98 0.2 1.02 0.91 1.07 EXAMPLE 8 A-27106-Improved Beef-Cattle Ration A balanced high-grain beef-cattle ration is pre pared as follows : Pounds Ingredient Percent Per Ton Finely ground corn 67.8 1356 Ground corn cob 10 200 Dehydrated alfalfa meal, 17 percent protein 5 100 Dehulled soybean meal, solvent extracted, 50 percent protein 9.956 199.12 Cane molasses 5 100 Urea 0.6 12 Metabolite A-27106 0.044 .88 Dicalcium phosphate, feed grade 0.5 10 Calcium carbonate 0.5 10 Sodium chloride 0.3 6 Trace mineral premix 0.03 0.6 Vitamin A dna D2 premix* 0.07 1.4 Vitamin E premix** 0.05 1.0 Calcium propionate 0.15 3.0 * Containing per pound: 2,000,000 I.U. of vitamin A; 227,200 I.U. of vitamin D- and 385.7 g. of soybean feed with 1% oil added ** Corn distillers dried grains with solubles containing 20,000 I.U. of d-alpha tocopheryl acetate per pound The mixed feed was compressed into pellets. At an average daily ingestion rate of 15 pounds of feed per animal, this feed supplies approximately 300 mg. of A-27106 per animal per day.

EXAMPLE 9 Acid Form of Metabolite A-27106 Metabolite A-27106 (100 mg.), prepared in the sodium form by procedures described in Examples 1 and 2, was dissolved in 100 ml. of methanol-water (1:1) . The resulting solution was titrated to pH 3 by dropwise addition of 1 N HCl. Methanol was then removed under vacuum.

The resulting solution was extracted twice with chloroform (100 ml. each) . The chloroform extract was dried (MgS04) and evaporated under vacuum to give 85 mg. of the acid form of metabolite A-27106.

The acid form of A-27106 is a white amorphous solid, having a molecular weight of about 832. The biological activity of the acid form is approximately the same as that of the sodium form.

EXAMPLE 10 Cesium Form of Metabolite A-27106 Metabolite A-27106 in the acid form (97.3 mg.), prepared as described in Example 9, was dissolved in 100 ml. of methanol-water (1:1) . The resulting solution was titrated to pH 7.1 by dropwise addition of 1 N cesium hydroxide.

Methanol was removed under vacuum. This solution was extracted twice with equal volumes of chloroform. The chloroform extract was dried (MgS04) and evaporated under vacuum to obtain a white amorphous solid. Elemental analysis showed the presence of about 13 percent metal.

Example 11 Lithium Form of Metabolite A-27106 The procedures of Example 10 were repeated, using lithium hydroxide instead of cesium hydroxide. The lithium form of A-27106 is a white solid exhibiting some organization tending toward crystallinity , but lacking a well-organized X-ray diffraction pattern.

EXAMPLE 12 Ammonium Form of Metabolite A-27106 The procedures of Example 10, using ammonium hydroxide instead of cesium hydroxide, are employed to produce the ammonium form of metabolite A-27106. This product has the general properties and utilities of the other forms.

Claims (16)

1. Metabolite A-27106, or the acid, ammonium, lithium, potassium, rubidium or cesium forms thereof, which metabolite is a white crystalline solid, relatively soluble in lower alkanols, but generally insoluble in lower alkanes and which has : a) a molecular weight of 854, as determined by mass spectrometry; b) an approximate elemental composition of 58.78% carbon, 8.51% hydrogen, 27.85% oxygen, and 3.63% sodium; c) an empirical formula of C42H7i°i6Na; d) an infrared absorption spectrum in chloroform as shown in the accompanying drawing; e) a mass spectrum which shows a molecular-ion peak at m/e 854.44630 and characteristic peaks at m/e 836.44129, 779.44690, 761.44740; and f) an Rf value of 0.49 on silica-gel thin-layer chromatography in benzene-methanol (7:3); and the acid form of which is a white solid having a molecular weight of about 832 and a titratable group with a pKa value of 7.2.

2. A process of converting monensin into metabolite A-27106 of Claim 1 which comprises cultivating Strepto-myces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic conditions in the presence of glucose and monensin until monensin is converted into a substantial amount of metabolite A-27106 by said organism in said culture medium .

3. The process of claim 2 wherein the culture medium includes a sterilized culture medium, the sterilized culture medium being obtained by sterilizing the culture medium in which the monensin was prepared by cultivating Streptomyces cinnamonensis under submerged aerobic conditions.

4. A process of converting monensin to metabolite A-27106 as defined in Claim 1 which comprises cultivating Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic conditions, separating the broth from said culture medium, lyophilizing said broth, reconstituting said lyophilized broth in an aqueous buffer medium and contacting said reconstituted broth with glucose and monensin until a substantial amount of metabolite A-27106 is produced in said buffer medium, and recovering metabolite A-27106 from said buffer medium.

5. A process of converting monensin to metabolite A-27106 as defined in claim 1 which comprises cultivating Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic conditions; separating the cells from said culture medium; purifying said cells by sonication, centrifugation, and dialysis; reacting said purified dialyzate in an aqueous buffer medium with glucose and monensin until a substantial amount of metabolite A-27106 is produced in said buffer medium; and recovering metabolite A-27106 therefrom.

6. A method of preventing or treating coccidiosis in poultry which comprises administering to poultry an effective amount of a composition comprising a metabolite as defined in claim 1 and a physiologically-acceptable carrier.

7. The method of Claim 6 wherein the carrier is a poultry feed.

8. A method of increasing the efficiency of feed utilization of ruminant animals having a developed rumen function which comprises the oral administration to such animals of a propionate-increasing amount of a metabolite as defined in claim 1.

9. A feed adapted to control coccidiosis comprising poultry feed and an amount of a metabolite as defined in Claim 1 sufficient such that a daily feeding provides a nontoxic, coccidiostatic dose of the metabolite.

10. A feed adapted to fatten cattle comprising cattle feed and sufficient metabolite as defined in claim 1 to provide a non-toxic, propionate-increasing amount per animal per day.

11. Metabolite A-27106 or its pharmaceutically-acceptable salts substantially as herein described with particular reference to any one of Examples 1,2,3,4,9,10,11 and 12.

12. A process for preparing metabolite A-27106 or its pharmaceutically acceptable salts substantially as herein described with particular reference to any one of Examples 1,2,3,4,9,10,11 and 12.

13. A method of preventing or treating coccidiosis in poultry substantially as herein described with particular reference to Example 5.

14. A feed adapted to control coccidiosis substantially as herein described with particular reference to Example 6.

15. A method of increasing the efficiency of feed utilization of ruminant animals substantially as herein described with particular reference to Example 7.

16. A feed adapted to fatten cattle substantially as herein described with particular reference to Example 8. AGENTS FOR APPLICANTS