DE2404958A1 - METABOLIT A-27106 AND THE PROCESS FOR ITS MANUFACTURING - Google Patents

Description

Metabolite A-27106 and process for its preparation

The invention relates to the metabolite A-27106 and to a process for its production from monensin by submerged fermentation of S. candidus NRRL 5449.

The invention also relates to an animal feed that the Contains metabolite A-27106 as an active ingredient.

Finally, the invention relates to a method for controlling coccidiosis in poultry and a method to improve the feed yield in ruminants.

According to the invention, the metabolite A-27106 or its Acid, ammonium, lithium, potassium, rubidium or cesium forms are created, this metabolite being a white crystalline Is solid, dissolves relatively well in lower alkanols, but is generally insoluble in lower alkanols Alkanes, and has the following additional properties:

a) a molecular weight of 854, by mass spectrometry certainly,

b) an approximate elemental composition of 58.78% carbon, 8.51% hydrogen, 27.85% oxygen and 3.63% sodium,

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c) an empirical formula C. ₂ ^H 7iOig ^Na f

d) an infrared absorption spectrum in chloroform according to the attached drawing,

e) a mass spectrum with a molecular xon peak

m / e 854.44630 and characteristic peaks at m / e 836, 44120, 779,44690, 761,44740, as well as

f) an Rf value of 0.49 in the silica gel thin-layer chromatogram in benzene / methanol (7: 3),

and wherein its acid form 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 relates to a method for converting monensin into the metabolite A-27106, the consists in placing Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, Contains nitrogen and inorganic salts, submerged under aerobic conditions in the presence of glucose and Monensin grows until the organism in the culture medium converts the monensin into a substantial amount of the Metabolite A-27106 was converted.

The invention also relates to a method of preventing or treating coccidiosis in poultry by treating poultry administered an effective amount of an agent comprising the metabolite A-27106 and a physiologically acceptable carrier contains, as well as a method for increasing the feed conversion of ruminant animals, which one developed have ruminant function by giving such animals an oral propionate-increasing amount of the metabolite A-271O6.

Coccidiosis is a known protozoal disease that results from infection by one or more species of Eimeria or

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Isospora (see Lund and Farr in "Diseases of Poultry," 5th Edition, Biester and Schwarte, Eds., Iowa State university Press, Ames, Ia., Pp. 1056-1096. Because of the great economic losses caused by coccidiosis and the Difficulties in using some known coccidiosis agents, there is still a need for better ones Remedies against coccidiosis.

Ruminants are economically important animals, and one Increasing the feed conversion rate is therefore always very desirable. The mechanism by which the predominant nutrient content is processed (Carbohydrates) from ruminant feed is known. Microorganisms in the animal's rumen ferment charcoal '' hydrate with the formation of monosaccharides and build them up Monosaccharides then turn into pyruvate compounds. The pyruvates are metabolized by microbiological processes, creating acetates, butyrates or propionates, which are collectively referred to as volatile fatty acids (VFA).

The one given by using the volatile fatty acids. McCullough, Feedstuffs, June 19, 1971, p. 19; Eskeland et al., J. An. May be. 33: 282 (1971); and Church et al., "Digestive Physiology and Nutrition of Ruminats, "Volume 2, 1971, pages 622 and 625. Although acetates and butyrates are also used, however, the use of propionates is more effective in comparison. If there is not enough propionate, then ketosis can also develop in the animals. A suitably inexpensive remedy should be therefore stimulate the formation of propionates from carbohydrates in the animals, whereby the carbohydrates are better utilized and at the same time the ketosis attack is reduced.

Monensin, from which the metabolite A-27106 is made is described in US Pat. No. 3,501,568 as factor A of an antibiotic complex A3823. Monensin is also a Remedies for coccidiosis.

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The bioactive agent of the present invention is arbitrarily referred to as metabolite A-27106. The expression A-27106 refers to the sodium salt. Other salts are the ammonium, lithium, potassium, and rubidium Cesium salts, and also the acid form, are the subject of the present invention.

A starting material for the production of the metabolite A-27106 is monensin, the sodium salt of which has the following formula I:

CH ₃ CH ₃ 1 3 CH
I. 3 / \ Λ \ I B
\ / , C / \ D / E / f ^ ' H-C ChY ' + / O
H CH ₃ ³ I CH I ₁ Il MHW Q H-CO CH H j ^ -r
I. '-OH '*, P
HIGH ₂ CH / °

Monensin in the form of its sodium salt is produced by Streptomyces cinnamonensis as described in U.S. Patent 3,501,568 is.

The metabolite A-27106 arises either from monensin or the mycelial culture in which monensin is formed in the presence of glucose by an enzyme or by enzymes formed by a new strain of Streptomyces candidus will. A culture of this new tribe has been made available in the permanent culture collection without restriction the Northern Utilization Research and Development Division, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peorla, Illinois 61604, there under the No. NRRL 5449.

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Because of the uncertainty of taxomic studies on organisms of the Streptomyces group, there is always a certain element of uncertainty when classifying a newly discovered organism. The organism NRRL 5449, the monensin, appears to be the metabolite A-27106 in the most important properties transforms, however, to be most similar to Streptomyces candidus (Krassilnikov) Waksman 1953. The type of culture was published by S. A. Waksman in "The Actinomycetes", Volume II, Williams and Wilkins, Baltimore, 1961, sentence 187. The organism was also registered with the Institute of Microbiology of Rutgers University, New Brunswick, N.J., under reference IMRU 3416. The present organism will as a new strain of the described. Organism viewed. Although S. candidus NRRL 5449 and the known strain each other resemble the general mycelial morphology and color, as well as the appearance of the spores, there are enough between them Differences that make it necessary to designate the present organism as a new strain. So brings the published organism, for example, does not curdle milk, and it also liquefies gelatin only slowly, while the new strain forms curdled milk after 14 days and does not liquefy gelatin within 21 days. Most importantly, however, a culture sample propagated from the IMRU 3416 strain did not convert monensin to metabolite A-27106 converts.

The organism that converts momensin to the metabolite A-27106 is isolated from a soil sample collected on Mount Ararat in Turkey by suspending a certain amount of such soil in sterile distilled water and spreading the suspension on nutrient agar. The seeded agar plates are then ^{incubated at 25 to 35 °} C. until visible colonies can be determined. When the incubation period has ended, colonies of selected organisms are transferred to agar slants using a sterile platinum loop. One of the agar slants is then incubated to produce appropriate amounts of inoculum from the NRRL 5449 organism.

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The taxonomic studies on S. candidus NRRL 5449 are carried out according to the methods recommended by the International Cooperative Project for Description and Deposition of Streptomycetes and according to the methods of Shirling and Gottlieb, "Methods for Characterization of Streptomyces Species," International Bulletin of Systematic Bacteriology 16, 313- 340 (1966), together with other supplementary studies. ^{The abbreviation 11} ICP "at the beginning" refers to media as described by Shirling and Gottlieb. The remaining media are described in the literature by Waksman already cited. The color names are assigned according to Kelly and Judd in "The ISCC -NBS Method of Designating Colors and a Dictionary of Color Names ", US Department of Commerce Circular 553, 1955. The numbers in square brackets refer to the Tresner and Backus color scale, namely" System of Color Wheels for. Streptomyces Taxonomy ", Appl. Microbiol. 11, 335 (1963). The color strip determinations are underlined and the Maerz and Paul (" Dictionary of Color ", MacGraw-Hill, Ν.Υ., 195O) color blocks are given in brackets.

The Streptomycete NRRL 5449 is characterized by straight to wave-like sporophores and oval to slightly cylindrical spores with an average size of 1.165, u χ 0.57, u in chains of 10 to 50. According to electron microscopic observations, the spores are smooth. Aerial mycelia are usually white. At 26 ⁰ C, there is only one vegatativen waxes, and at 45 ⁰ C for no growth occurs. Maximum growth and maximum sporulation occurs at 30 to 37 ^° C.

Following a common practice is growing the microorganism NRRL 5449 examined on a range of media recognized in the study of Actinomycetes.

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The culture procedures are uniform and represent standard methods. The media used and those observed Cultural characteristics are as follows:

Good growth, pale yellow / 11Cl /, no aerial mycelia or Spores, not a soluble pigment.

Profuse growth, light yellow / 10J3 /, abundant aerial mycelia and spores, (W) white a, no soluble pigment.

Good. Waxing, pale yellow-green / 1OCl ₁ /, good aerial mycelia and spores, (W) white a, no soluble pigment. .

Good to copious growth, medium yellow / 10H4 /, good to copious aerial mycelia and spores, (W) white a., brown soluble pigment.

Abundant growth, medium orange-yellow / 1016 /, abundant aerial mycelia and spores, (Y) pale yellow 2db , slightly brown soluble pigment.

Good growth, pale yellow-green / 10Bl ₁ /, good aerial mycelia and spores, (W) white a, no soluble pigment.

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Glycerin-glycine

Abundant waxing, medium brown / 11J4 /, rich speculation and aerial mycelia, (W) white a and (Y) pale yellow 2db, light light brown soluble pigment.

Emerson

Abundant waxing, greyish yellow / 12H3 /, no aerial mycelia or spores, no soluble pigment.

Bennett

Good growth, light yellow / 1112 /, sparse aerial mycelia and Spores, (W) white a, no soluble pigment.

Czapek

Profuse growth, medium orange-yellow / 11J7 /, profuse Aerial mycelia and spores, (W) white a, no soluble pigment.

Glucose asparagine

Fair to good growth, pale yellow-green / 1OBi /, none Aerial mycelia or spores, no soluble pigment.

Calcium malate

Profuse waxing, greyish yellow / 11E4 /, abundant aerial mycelia and spores, (Y) pale yellow 2ba , light brown soluble pigment.

Nutrient agar

Good growth, pale yellow-green / lOCl /, no aerial mycelia or Spores, not a soluble pigment.

The organism is examined with regard to selected physiological properties according to standard methods. The watched Properties and characteristics found for them are as follows:

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Observed property

Characteristic

Effect on skimmed milk

Clear up, coagulate after 14 days

Nitrate reduction

positive

Me1anine production Tryptonic extract broth tyrosine agar

weak
no

Gelatin liquefaction

none after 21 days

Temperature requirements

26 C- only vegetative growth

30-37 C - good vegetative

Grow, good air milia and spores

43-55 ° C - no waxing

The following are the results of the study of coal consumption listed for the organism NRRL 5449. The following symbols are used to identify a growth:

+ = Consumption - good growth l_ + _ / = possible consumption - good to tolerable growth

l_ -_ / - consumption in question - weak or no growth at all

= no consumption - no waxing

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Carbon source

Raffinose D-fructose Cellobiose L-arabinose D-mannitol Rhamnose Cellulose Dextrose D-Xylose Inosit -C (no carbohydrate)

Assessment of growth

is / + _ /

Any culture medium can be used to grow S. candidus NRRL 5449. For reasons of economy, however, certain culture media are preferred for optimum yield and ease of isolation of the product. Preferred sources of carbohydrates for large-scale fermentation are therefore, for example, invert sugar or corn syrup, however, glucose, fructose, maltose, starch, inositol and the like can also be used. Would you like Monensin in the metabolite A-27106 in one fermentation broth at the same time convert with the growth of S. candidus NRRL 5449, then the medium must also for sufficient conversion contain a source of glucose. On the other hand, if S. candidus NRRL 5449 is only allowed to grow to form its enzyme system, which one later used to convert monensin to the metabolite A-27106, then glucose does not need during fermentation to be absolutely present. Preferred nitrogen sources are

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Peptones, soybean meal, mixtures of amino acids and the like. The inorganic nutrient salts that can be added to the culture media include the usually soluble salts that provide iron, sodium, potassium, ammonium , calcium, phosphate, chloride, carbonate and similar ions.

Essential trace elements necessary for growth and development of the organism should also be present in the culture medium. Such trace elements come normally present as impurities in other components of the medium in amounts necessary for the growth of the organism sufficient.

The starting pH of the culture medium can be varied. Before an inoculation with the organism, the pH value should of the culture medium, however, are expediently adjusted to about pH 5.7 to about 7.5, depending on the particular case medium used. As in the case of other actinomycetes, the medium in the present case also becomes as fermentation progresses Gradually more alkaline and can go from a starting pH of around pH 5.9 during the growing season of the organism rise to a value of about pH 6.9 or above. The final pH is at least partially through the starting pH of the medium that is present in the medium Buffer as well as the length of time during which the organism is allowed to grow controlled. The pH value can also be adjust by adding an acid or a base, but good results are obtained without adjusting the pH value.

Just like other Streptomyces species, you have to Grow organism NRRL 5449 under aerobic conditions. A multiplication or a growth on a small scale will expediently on agar slants or agar plates, in shake flasks or piston made. For a large-scale

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For production, preference is given to a submerged aerobic culture in large tanks.

The fermentation medium located in a sterile tank can be used to start the fermentation with a sporulated Inoculate suspension. Since an inoculation with a sporulated Suspension delays growth, but preference is given to a vegetative inoculum. The vegetative inoculum is made by inoculated a small volume of culture medium with the spore shape or with mycelium fragments of the organism in order to produce a fresh, actively growing culture of the organism to arrive. The vegetative inoculum is then transferred to a larger tank. The same medium can be used to grow the vegetative inoculum as it will be used later for large-scale production, however, you can also work with different media.

The organism S. candidus NRRL 5449 grows in a temperature range between approximately 26 ^° C. and approximately 40 ^° C. However, maximum growth and maximum sporulation occur between approximately 32 and approximately 37 ^° C.

As is usual with aerobic submerged culture processes, in the present case you blow through the fermentation during fermentation Culture medium sterile air. For a sufficient growth of the organism and a corresponding production of metabolite A-27106, the volume of air used in large-scale production of the substance should be greater than about 0.1 volume of air per minute per volume of culture medium. Optimal results are obtained with an air volume of at least one One third to one half volume of air per minute and per volume of culture medium.

The one responsible for the conversion of monensin into the metabolite A 27106 required fermentation time fluctuates. For the conversion

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of monensin in the metabolites A-27106 must be sufficient Amount of glucose to be present. If you have enough glucose available and about 0.1 to 1.0 g of monensin per liter of medium is present, then the conversion of monensin to the metabolite A-27106 proceeds practically completely within about 36 to 72 hours. Optimal conversion occurs in the presence of an amount of monensin of about 0.5 to 0.7 grams per liter Medium. A sufficient amount of glucose is between about 2.0 and about 2.5 weight percent glucose, by weight related to the medium. To check the respective glucose concentration appropriate glucose test paper strips can be used. If the glucose content falls below about 2%, then one should add glucose to maintain optimal concentration levels.

If the enzyme system S. candidus is used for the conversion of monensin into the metabolite A-27106, then the enzyme preparation must be used not be highly pure. A filtered fermentation broth can therefore, for example, be lyophilized and store for at least two weeks before they have to be replenished with an aqueous buffer, for which about 1/6 of the volume the original broth used. Sufficient conversion is obtained after about 72 hours when adding 2.5 g a preparation lyophilized in this way in the presence of 25 mg monensin.

The active enzyme system is present in both the filtered broth and the cells. A larger enzyme preparation Purity can be determined from the separated fermentation cells produce. These cells can be stored frozen for at least three months. The thawed cells can then be brought back to life by suspending them in a buffer solution. The buffer suspension leaves purify yourself further through sonication and centrifugation. The purified cell fragments thus separated are then

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resuspended in a buffer and dialyzed. using This method gives as much purified dialysate from 200 g cells from the fermentation medium that one thereby Can convert glycose and 25 mg monensin to the metabolite A-27106.

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

The metabolite A-27106 is in both the culture broth and present in the mycelium. Appropriate isolation techniques of the metabolite A-27106 must therefore be designed to maximize recovery of the product from both of the above allowed media. For this purpose, the fermentation medium for example, filtered, whereupon both the Fil-trike and the mycelium cake with suitable solvents extracted to arrive at the metabolite A-27106. From the solvents used for the extraction it can be Obtain the desired product using the usual methods.

Optionally, the solids contained in the culture can be found under Inclusion of the components of the medium and of the mycelium, use as a source of the metabolite A-27106, however separates the water from the mycelium and the culture medium beforehand. The culture medium can be used for this purpose, for example dry by lyophilization and mix with a feed. The solids can also be removed without total water removal Process into a thin sludge that can be added to moist mixed feed and similar feedstuffs.

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A single extraction-isolation procedure is not necessary. Filtering the finished culture medium using a Filter aid is already sufficient. The filter cake can be extracted with a polar solvent such as methanol. The methanol extract is concentrated and then added to the original aqueous filtrate. The so obtained combined The solution is extracted twice with half the volume of chloroform. The chloroform extracts become one under vacuum dark amber oil concentrated.

The oil obtained in this way is decolorized in a filled with activated charcoal Column using chloroform and about 20 g of activated carbon per gram of oil. The eluate is again concentrated in vacuo to give a pale yellow to colorless oil. This oil dissolves in the minimal required amount of chloroform and then chromatographed the solution on a silica gel column using of ethyl acetate as a solvent. The elution is monitored by thin rail chromatography. Impurities are eluted with ethyl acetate. Elution with ethyl acetate-methanol mixtures yields the metabolite A-27106.

The metabolite A-27106 (sodium salt) is a white crystalline solid that melts under blistering at about 170 to 175 ⁰ C. The metabolite A-27106 appears to form a hydrate or other solvate very easily. If the metabolite A 27106 is in a hydrated or solvated form, then its melting point also changes, and this compound then generally melts a few degrees below the value given above.

The elemental analysis of the metabolite A-27106 gives the following percentage composition: carbon 58.78, hydrogen 8.51, oxygen 27.85 and sodium 3.63. These values agree with an empirical formula ^C 42 ^H 7i ° i6 ^Na

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has the following theoretical composition in percent: carbon 59.00, hydrogen 8.37, oxygen 29.94 and sodium 2.42.

The metabolite A-27106 has virtually no ultraviolet absorption above about 235, u on.

The infrared absorption spectrum of the metabolite A-27106 in chloroform can be taken from the attached 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 the metabolite A-27106 shows the following molecular xon peak and the characteristics also given.

m / e

calculated found fragment

854.46230 854.44630 C ₄₃ H ₇₁ O ₁₆ Na

836.45229 836.44129 ^C 42 ^H 69 ° 15 ^Na

(M ⁺ -H ₂ O)

779.45490 779.44690 ΰ, -Η, -οΟ. "N / A

4U Thu 10

+ CO ₀ /)

761.44540 761.447740 ^C 4O ^H 66 ° 12 ^Na

(M ⁺ - / CH ₃ 0 + CO ₂ +

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The above values confirm the empirical formula given and a molecular weight of 854 for the sodium salt of the metabolite A-27106.

In general, the metabolite A-27106 is readily soluble in highly polar solvents, insoluble in non-polar solvents and variously soluble in solvents medium polarity. For example, A-27106 dissolves in lower aliphatic alcohols and partly in phenol, Diethyl ether and acetone, and it is relatively insoluble in liquid lower alkanes.

An electrometric titration of the metabolite A-27106 in the acid form in water at a starting pH of 8 shows the presence of a titratable group with a pKa value of 7.2.

The monosodium form described above is generally the natural form of the metabolite A-27106. The acid can easily be prepared from this sodium salt. From the Acid can be used to produce ammonium and other alkali salts. The different metal forms behave something similar to alkali carboxylates and chelates.

To make another form, metabolite A-27106 (the sodium form) is dissolved in an aqueous solvent such as methanol / water, solved. The solution obtained is then treated with an acid, for example hydrochloric acid, to bring the pH to 5 or below to humiliate. The methanol is then removed in vacuo and the aqueous acidic product obtained extracted with chloroform. The chloroform extract is dried and evaporated to the free acid form.

The acid form of the metabolite A-27106 obtained in this way can be used as such, or it can be further modified,

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by treating them with an aqueous alkali hydroxide or with aqueous ammonia titrated to the appropriate in this way To produce lithium, potassium, rubidium, cesium or ammonium salts. The acid, ammonium or alkali forms of the metabolite A-27106 are all biologically active.

The exact structure of the metabolite A-27106 is unknown. It is known that for the conversion of Moriensin to the metabolite A-27106 Glucose is required and consumed, even in the absence of metabolizing cells by S. candidus. The molecular weight of A-27106 corresponds to that of a glucosylmonensin.

In addition to the highly hindered tertiary hydroxyl group on ring E, there would have to be five hydroxyl sites on a glucosylmonensin be present, which can be individually or collectively with the formation of a simple ester, such as an acetate, should be left to esterify. The presence of all of these five reactive sites results from esterification experiments.

On the basis of the physical properties listed above, the following structure can be found for the metabolite A-2 7106 propose formula II:

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Since the structure is only postulated, the structure given above should of course only form a working hypothesis.

The metabolite Ä-27106 is less toxic than monensin. Do intraperitoneal administration of the metabolite A-27106 Groups of 6 mice each perish at a dose of 50 mg / kg of six mice and one animal each Administration of 100 mg / kg died of six animals three each.

In 'similar experiments, intraperitoneal administration leads of monensin to groups of 6 mice each, a dose of 10 mg / kg to death of one mouse out of six Mice, and at a dose of 20 mg / kg to death of three Mice out of a total of six.

Used to prevent or treat coccidiosis in poultry a non-toxic coccidiostatic amount of metabolite A-27106 is administered to the animals, preferably orally on a daily basis. To determine the appropriate concentration of A-27106 one must consider a number of factors, however, the amount to be administered is generally between 0.005 and 0.05 percent by weight, based on Feed not mixed with active ingredient, and it is preferably between 0.01 and 0.04 percent by weight. The metabolite A-271O6 can be administered in a number of ways, the simplest being the easiest however, if it is administered with a physiologically harmless carrier, preferably with that administered to the animals Lining.

The metabolite A-27106 also improves feed utilization in ruminants whose rumen function is developed. Young ruminants, practically all not yet weaned animals, represent monogastic animals. As soon as young ruminants start eating solid forage, begins

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rumen function develop and the microbiological population of the rumen increases. After the animal has eaten solid food for a certain time, its rumen function is fully developed and remains during the whole Preserve animal life. Some economically important ruminants are cattle, sheep and goats.

The metabolite A-27106 increases feed utilization when one administered orally to ruminants in amounts between about 0.05 mg / kg / day to about 2.5 mg / kg / day. The best results are obtained with amounts between about 0.1 mg / kg / day and about 1.5 mg / kg / day. A preferred method of administration for the metabolite A-27106 consists in mixing it with the animal feed. However, the metabolite A-27106 can can also be administered in other forms, for example in the form of tablets, drinks, boIi or capsules. A formulation these various dosage forms can be achieved by methods conventionally used in animal pharmacy. Each Dosage unit should contain such an amount of metabolite A-27106 that this directly results in the appropriate daily dose for the animal to be treated.

The invention is illustrated in more detail by means of the following examples.

Example 1 Production of A-27106 from monensin via S. candidus

S. candidus NRRL 5449 is left on made from Bennett's medium Grow agar slants, resulting in a well-defined colony. The colony is removed and covered with sterile slurried in deionized water (IO ml).

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lot G G G 25.0 G G 10.0 G G 10.0 liter 0.01 2.0 2.0 2.0 up to 1.1

The suspension obtained above is divided into four 500 ml shake flasks, each 100 ml vegetative Medium containing the following composition:

Components

Corn distillers' solubles

Lactose

Maltose

FeSO ₄ .7H ₂ O MgSO ₄ .7H ₂ O KH ₂ PO ₄ CaCO ₃ deionized water

Nadrisol: Distribution: National Distiller's Products Company, U.S.A.

The four inoculated bottles are kept at 30 C on a rotary shaker at 250 revolutions per minute for 24 hours long incubated. The vegetative medium obtained in this way (10 ml portions) is used for inoculating a total of 15 shake flasks (500 ml), the 100 ml sterilized Fermentation medium contain the following composition:

Components

Beef extract 5 g

Casein pancreas hydrolyzate peptone 5 g

NaCl 5g

Glycerin 15 g

CaCO ₃ 2g

Deionized water up to 1 liter

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The medium has a pH of 7.2 that has not been adjusted is. The inoculated medium is incubated for 72 hours as described above.

A production medium with the following composition is produced in a fermentor with a capacity of 40 liters:

Constituent amount

PolysiloxanesI antifoam agents 5 G Glycerin 375 G Dextrose 625 G Casein Pancreas Hydrolyzate Peptone 125 G Beef extract 125 g ■ NaCl 125 G CaCO ₃ 50 G deionized water up to 24 liter

The original pH of the medium is 7.0. The medium is then treated in an autoclave at 120 C during

Sterilized for 30 minutes at a pressure of 1.05 to 1.41 kg / cm. After sterilization, the pH of the medium is 7.6.

Purified monensin (25 g) is dissolved in ethanol (200 ml), and add the resulting solution to the sterilized medium, whereupon that obtained from the second step is added as above described prepared vegetative inoculum (700 ml) is added.

The fermentation medium is supplied with sterile air at a rate of about 1 liter (0.35 cubic feet) each Aerated minute and stirred with conventional stirring at a stirring speed of 420 revolutions per minute.

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The inoculated medium is grown at 30 ° C. for about 114.5 hours.

The course of fermentation is monitored by thin layer chromatography followed over silica gel as described above. In the first stage of fermentation, only monensin is present. Step by step, a second spot then shows the presence of the metabolite A-27106, and at the end you can only see that Spot for the metabolite A-27106.

Example 2 Isolation and Purification of A-27106

The fermentation broth prepared according to Example 1 is filtered using a filter aid. The mycelium cake extracted with methanol (about 5 liters) at room temperature. The methanol extract is filtered, whereupon the filtrate concentrated under vacuum, the methanol removed and an aqueous concentrate obtained.

This aqueous concentrate is combined with the original broth filtrate. The combined solution (about 22 liters) is extracted twice with 0.5 volumes of chloroform each time. The chloroform extracts are combined and placed under vacuum concentrated about 400 ml of a dark amber-colored oil.

This oil is dissolved in chloroform, it is removed in a column filled with 10 kg of activated carbon (Pittsburg "12 χ 40"), which are eluted with chloroform (about 5 liters). The chloroform eluate is evaporated in vacuo to give about 500 ml of a colorless to pale yellow oil.

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The decolorized Ql is in a minimal amount of chloroform added, and the whole thing is then chromatographed in a 25 kg column of silica gel (Grace, grade 62) in ethyl acetate, The elution is carried out by thin layer chromatography monitored as just described. After removing the impurities with ethyl acetate, the metabolite Ä-27106 is eluted from the column with ethyl acetate-methanol (19: 1). The fractions containing the A-27106 are pooled and evaporated to dryness under vacuum, whereby one an amorphous, almost white product is obtained. The product is washed with hexane and dried to give about 12.84 g of metabolite Ä-27106 (which shows a single spot in the thin-layer chromatogram).

Example 3 Production of A-27106 via S. cinnamonensis and S. candidus

Another method for producing the metabolite A-27106 in a fermenter culture is carried out as follows:

Streptomyces cinnamomensis ATCC 15413 is left in the usual way Way in 55 ml medium in a 250 3il pouring bottle and under 47 hours of incubation to form a vegetative inoculum (see OS-PS 3 501 568). This vegetative inoculum becomes to 220 ml of one in a 1 liter shake bottle Medium and incubated for 21 hours, which gives the seed inoculum for the fermenter »

The inoculum produced in this way is used as seed for a 40 liter fermenter, which is a heat-sterilized medium contains the following composition:

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Components

glucose

Soy bean flour

Soybean oil methyl oleate

Polysiloxane oil Antifoam agent Potassium chloride Dipotassium hydrogen phosphate Manganese chloride tetrahydrate hydrous iron sulfate calcium carbonate deionized water up to

lot
750.0 g 625.0 g

500.0 g 500.0 g

5.0 g

2.5 g

2.5 g

15.0 g

7.5 g

25.0 g 24 liters

The medium obtained has a pH of 5.5, which is adjusted to pH 8.9 by adding 10 η potassium hydroxide (15 ml). The inoculated medium is incubated for 234 hours at 32 ^° C.

After 42 hours the ventilation is increased from 9.9 liters per minute to 22.6 liters per minute (0.35 to 0.8 cubic feet) and the stirring speed from 500 to 700 revolutions per minute. After 210 hours there is monensin formation practically complete, as demonstrated by a thin-layer biopsy with Bacillus subtilis ATCC 6633 as the detection organism results.

After 234 hours, the contents of the fermenter are pasteurized to inactivate S. cinnamonensis. The fermenter will then added the following nutrients:

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Ingredients quantity

Dextrose 750 g

Soybean meal 625 σ

Manganese chloride tetrahydrate 155 g calcium carbonate 12.5 g

Iron 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 up to 24 liters

The pH is then adjusted by adding 215 ml
5 η sodium hydroxide to pH 8.0 and sterilized the medium. The medium is then enriched with a rapidly growing vegetative culture of Streptomyces candidus NRRL 5449
inoculated and ^{incubated at 30 0} C for 137 hours. The fermentation is aerated with sterile air at a rate of 9.9 liters per minute (0.35 cubic foot). The fermentation medium is stirred with conventional stirrers, namely, first at a speed of
120 revolutions per minute, after 16 hours
to 420 revolutions per minute, and after 40 hours
goes to 500 revolutions per minute. After 44, 66.5, 89, 97, 113 and 127 hours, dextrose (400 g)
admitted. Calcium carbonate (175 g) is added after 72 and 99 hours.

The fermentation is carried out by thin layer chromatography as described above supervised. After 137 hours, the formation of the metabolite A-27106 is practically complete. The work-up and purification of A-27106 is carried out according to the procedure described in Example 2.

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240Λ958

Example 4

Production of A-27106 by a granular enzyme preparation

by S. candidus

S. candidus NRRL 5449 is allowed to grow as described in Example 1 in a 100 liter batch. The cells are separated from the fermentation medium by vacuum filtration, divided into 200 g portions and frozen for storage. Two of these portions are thawed at room temperature and suspended in 0.05 M phosphate buffer (pH 5.8) to a final volume of 600 ml. The cell suspension obtained in this way is sonicated for 30 minutes, and the product obtained after sonication is at 10 for 30 minutes Centrifuged 000 revolutions per minute. The cell fragments obtained are suspended in 100 ml of the above-mentioned buffer. The suspension is dialyzed for 18 hours with 5 liters of quenched buffer of the type mentioned above. 50 ml of the lumpy dialysate are mixed with D-glucose (120 mg) and monensin (25 mg) in ethanol (2 ml). The reaction mixture is ^{stirred for 72 hours at 30 °} C. and then filtered. The filtrate is extracted with chloroform (100 ml). The chloroform extract is concentrated in vacuo and the concentrate is chromatographed on a silica gel column (10 g). After eluting with ethyl acetate, only a trace of monensin is obtained. Eluting with ethyl acetate-methanol (19 s 1) gives 17 mg of metabolite A-27106, which is identical to the product obtained according to Example 2.

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Example 5 Control of Coccidiosis with Metabolite A-2 7106

For this examination one uses 75 one week old, healthy, crossbred male chickens of the heavy broiler type reared in an incubator and battery. the Chickens are divided into five groups of 15 animals each, which in turn are divided into three subgroups of 5 chickens each divides. Each subgroup is kept out of contact with the other subgroups. A first group is held under favorable conditions as a health comparison for untreated animals. A second group becomes like the first Group treated, but inoculated with coccidiosis by oral administration of a dose that 10 sporulated oocytes of Simeria tenella contains. The third, fourth and fifth Groups of animals are treated like the second group, with the exception of this, however, 24 hours before the mentioned inoculation their feed by adding the metabolite Ä-27106 in concentrations modified by 100, 150 and 200 ppm.

Seven days after inoculation, the chickens are weighed, sacrificed and examined for signs of coccidial damage. The severe damage is assessed on an arbitrary scale between 0 and 4. The corresponding damage figures indicate the number of animals for each value. The number "0" means that there is no damage from eoccidiosis. The number "1" indicates that this is the lowest detectable coke-idiosis damage. The number "2" indicates moderate damage with little or no bleeding and no serious tissue damage. The number ^: '3 ^{; I} means bleeding that oozes out of the caecum and severe tissue damage. The value "4" indicates bleeding _f with a caecal core of clotted blood and destroyed epithelial cords. Animals that die with a value of "4" or below can usually eriiolate again if they are no longer infected with coccidia.

After the "/ experience" described above, one arrives at the following

Results; 409834/1125

Animal-
group With E.tenella
inoculates Metabolite
A-27106 (ppm)
in the feed Medium weight
increase per animal
in g 1 no 0 162 2 Yes 0 67 O 3 Yes 100 157 983 4th Yes 150 150 4/1 5 J ^a 200 166 125

15th

'0

C3 J> CO (Jl OO

The chickens treated with active ingredient with high levels of damage contain little or no blood in faeces and appear to be in good shape. Your good weight gain is seen as more telling than the damage values

After research suggests that the metabolite A-27106 normally kills E. tenella on the first attempt at spreading in the host cell, one is preferred prophylactic type of treatment.

Other studies with the metabolite A-27106 show that this is better tolerated by the animals and is also less toxic than monensin.

A number of other studies are being carried out on metabolite A-27106, both alone and in combination with monensin. These investigations lead to the result that, although A-27106 does not give any greater weight increases at 0.04% than at a dosage of 0.01%, the higher dose, however, results in less caecal damage. At the higher dose, there were no toxic effects whatsoever.

Combined administration of monensin and metabolite results in A-27106 within the general range of effectiveness in the feed at least an additional effect, but no increase in toxicity. The toxicity of the combination is lower than that of monensin alone at a dose equivalent to the combined single doses.

Example 6

Chicken feed modified with A-27106 to control coccidiosis

For the production of a balanced, high-energy feed that can be used to give chickens with rapid weight gain can feed, one assumes the following components:

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Components

ground yellow corn

Soybean meal, extracted with solvent, stripped of the hulls, finely ground,
50% protein

Animal fat (beef tallow)

dried fish meal with solubles (60% protein)

Distillers' solubles made from corn

Dicalcium phosphate, for animal feed

Calcium carbonate

Vitamin pre-mix

(Vitamins A, D, E, K, and B_ ₂ , choline, hiacin, '

Pantothenic acid, riboflavin, Biotin with glucose as a filler)

Trace mineral premix (MnSO ₄ , SnO, KJ, FeSO ^, CaCO ₃ )

2-amino-2-hydroxybutyric acid (Hydroxy analog of methionine)

Metabolite A-27106.

50

0 "2

kg (lbs.)

454

(1000)

31.08 281.7 (621.6) 6.5 59.0 (13O) 5.0 45.4 (100) 4.0 36.3 (80) 1.8 16.3 (36) 0.8 7.26 (16) 0.5 4.54 (1O)

1.81 (4)

0.1 0.907 0.02 0.181

The substances listed above are mixed with one another according to the techniques commonly used in animal feed production. The chickens fed with such feed, them
if you give water ad libitum, you are protected against coccidiosis. The resulting weight gains are comparable to those obtained from coccidiosis-free chickens
similar feed with no active ingredient added
fed.

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Example 7 Improvement in feed conversion by A-27106

First of all, rumen fluid is obtained from an ox, namely by means of a fistula surgically inserted into the rumen. The ox comes with a heavily cereal Fed feed, which has the following composition:

69.95% coarsely ground corn

%%? ground corn on the cob

8% soybean meal (50% protein)

5% alfalfa flour

5% molasses

0.6% urea

0.5% dicalcium phosphate

C, 5% calcium carbonate

0.3% salt

0.07% vitamin A and D ₀ premix

0.05% vitamin S premix ^ '

C, 03 1 trace mirieral premix

"" ■ 'Content per 454 g: 2,000,000 I.U.

Vitamin A; 227 200 I.U. Vitamin D, and 385.7 g soybean feed with 1% cSl added

Distillers' solubles of dried corn kernels that

Contains 20,000 I.U. d-alpha-tocopheryl acetate per 454 g

Content of manganese oxide, potassium iodide, cobalt carbonate, Copper oxide and zinc sulfate

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A sample of rumen fluid is forced through four layers of cheesecloth and the filtrate is collected. Of the granular residue retained by the cheesecloth is resuspended in a sufficient amount of a physiological buffer, to bring it back to the original volume of the rumen fluid, and this suspension becomes again strained. The buffer used has the following composition:

g / liter

0.316 0.152 2.260 0.375

0.375 0.112 0.038

Components

Na ₂ HPO ₄ KH ₂ PO ₄ NaHCO, KCl

NaCl

MgSO ₄

CaCl.,

0.008 0.004

FeSO ₄ .7H ₂ O MnSO ₁

0.004 0.002 0.001

ZnSO ₄ .

CuSO ₄ .5H ₂ O CaCl,

as described by Cheng et al. in J. Dairy Sci. 38, 1225, (1955).

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

The diluted rumen fluid is placed in a 25 ml flask together with 40 mg of the feed described above, a further 5 mg of soybean protein and the compound to be tested. Each treatment is repeated with four flasks. For comparison, two approaches with 4 comparison pistons each are carried out. A comparison is made at time zero and a further comparison after 16 hours of incubation. All test flasks are incubated for 16 hours at 38 ⁰ C. After incubation, the pH is measured and each flask is mixed with 25% metaphosphoric acid ( 2 ml). The samples are allowed to settle and the supernatant liquid is analyzed by gas chromatography on propionate, acetate and Butyratverbindungen. By active compounds, the propionate product xon is increased quite strongly compared to that of the controls.

The results obtained with the compound to be examined are compared statistically with the comparison results. The table below shows the ratio of the concentrations of volatile fatty acids (VFA) in those with metabolite A-27106 treated flasks versus the concentrations in the comparison flasks.

mg A-27106 / ml diluted rumen fluid

5 1 0.2

Example 1 8 Beef cattle feed improved with A-27106

A balanced, high-grain beef cattle feed is made as follows:

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Propionate Butyrate total quantity
to VFA 2.15 · 0.73 l, O3 1.42 0.96 0.98 1.02 0.91 l, O7

Components

finely ground corn ground corn on the cob

dehydrated alfalfa meal 17% protein

Soybean meal stripped of its hulls, with solvent extracted, 50% protein

Sugar cane molasses urea
Metabolite Ä-27106 dicalcium phosphate,

for feeding devices calcium carbonate sodium chloride
Trace natural premix

Vitamins A and D ₂ Pre-Vegetable ^J

2) Vitamin E Borgern! Sch calcium propionate

percent

67.8

10 kg per ton
(pounds per tone)

678
100

50

Cl35Sl C 200)

(ICO)

9, 956 99.56 (199.12) 5 50 (ICO) 0.6 6th (125 0.044 0.44 (0.88) 0.5 5 CIO} 0.5 5 (10) 0.3 3 (6) 0.03 0.3 CO ₅ -SJ 0.07 0.7 (1.4) - 0.05 0.5 AROUND 0.15 1.5 (3.0)

Content per 454 g: 2,000,000 IU of vitamin Aj 227 200 IU of vitamin D ₂ and 385.7 g of soybean feed with 1% added oil

Distillers' solubles of dried corn kernels containing 20,000 I.U. d-alpha-tocopheryl acetate per 454 g

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240495

The feed mixture is pressed into pellets. With a middle one Daily digestion of about 6.8 kg of feed per animal, this feed provides about 300 mg of A-27106 per animal and Taq.

Example 9
Acid form of the metabolite A-27106

Metabclit Ä-271G6 (100 mg), prepared as the sodium form according to Examples 1 and 2 , is dissolved in 100 ml of methanol / water
(1: i) solved. The solution obtained is titrated to pH 3 by adding 1 η hydrochloric acid dropwise. The methanol is then stripped off in vacuo.

The solution is then extracted twice with chloroform

(100 ml each). The chloroform extract is dried (MgSO ₄ ) and evaporated in vacuo to give 85 mg of the acid form of the metabolite A-27106.

The acid form of Ä-27106 is a white amorphous solid, whose molecular weight is about 832. The biological activity of the acid form roughly corresponds to that of the Sodium form.

Example 10
Cesium form of the metabolite A-271G6

The acid form of the metabolite A-27106 (97.3 rag), prepared according to Example 9, is dissolved in 100 ml of methane / water ils!). The solution obtained is titrated to pH 7.1 by adding dropwise I η casium hydroxide

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The methanol is then removed under vacuum. The solution thus obtained is extracted twice with the same Volumes of chloroform. The chloroform extract is dried (MgSO.) And evaporated in vacuo to give a white amorphous solid is obtained. Elemental analysis shows the presence of about 13% metal.

Example 11 Lithium Form of Metabolite A-27106

The procedure of Example 10 is repeated, but using lithium hydroxide instead of cesium hydroxide The lithium form of A-27106 obtained in this way is a white solid that has a certain crystal order, however, does not give a clean pattern in X-ray diffraction.

Example 12 Ammonium Form of Metabolite A-27106

The procedure of Example 10 is repeated, except that instead of cesium hydroxide, ammonium hydroxide is used is used, and so arrives at the ammonium form of the metabolite A-27106. This product has the general Properties and abilities of the other forms.

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Claims (10)

Claims 1. Metabolite A-27106 and its acid, ammonium, lithium, potassium, rubidium or cesium forms, where this metabolite is a white crystalline solid, dissolves relatively well in lower alkanoies, im but is generally insoluble in lower alkanes and has the following additional properties: a) a molecular weight of 854, by mass spectrometry certainly, b) an approximate elemental composition of 58.78% carbon, 8.51% hydrogen, 27.85% oxygen and 3.63% sodium, c) an empirical formula C ₄₂ ^ ₁ ⁰ , _g Na, d) an infrared absorption spectrum in chloroform according to the attached drawing, e) a mass spectrum with a molecular ion peak at m / e 854.44630 and characteristic peaks at m / e 836.44129, 779.44690 and 761.44740, as well as f) an Rf value of 0.49 in the silica gel thin-layer chromatogram in benzene / methanol (7: 3), and wherein its acid form is a white solid having a molecular weight of about 832 and a titratable Group with a pKa value of 7.2. 2. A method for converting monensin into the metabolite A-27106 according to claim 1, characterized 409834/1126 draws that Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of Contains carbon, nitrogen and inorganic salts, submerged under aerobic conditions in the presence of glucose and monensin grows until the monensin through the organism in the culture medium in a substantial Amount of the metabolite A-27106 was converted. 3. The method according to claim 2, characterized in that a stabilized culture medium is used which is obtained by sterilizing the culture medium in which the monensin is produced by cultivating Streptomyces cinnamonensis submerged under aerobic conditions. 4. A method for converting monensin to the metabolite A-27106 according to claim 1, characterized in that Streptomyces candidus NRRL 5449 is cultured submerged under aerobic conditions in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts, the broth from culture medium separates iyophiIisiert the broth, the lyophilized broth rebuilds in an aqueous buffer medium and long brings these reconstructed broth with glucose and monensin until the buffer medium a substantial amount of metabolite is formed a-27106, after which the metabolites Ä-27106 from the Buffer medium wins. 5. A method for converting monensin into the metabolites Ä-27106 according to claim I _f, characterized in that Streptomyces candidus NRRL 5449 in a culture medium which contains assimilable sources of carbon _f 4098 34 / 11-2 Containing nitrogen and inorganic salts, the cells grow submerged under aerobic conditions from the culture medium separates, purifies the cells obtained by sonication, centrifugation and dialysis, the purified dialysate reacts in an aqueous buffer medium with glucose and monensin until a substantial amount is in the buffer medium on metabolite A-27106, and metabolite A-27106 isolated from this. 6. Method of preventing or treating Poultry coccidiosis, characterized in that one poultry administered an effective amount of a preparation which has a metabolite according to claim 1 and a physiologically contains harmless carrier. 7. The method according to claim 6, characterized in that that one uses poultry feed as a carrier. 8. Method of improving the feed yield of ruminants that have an already developed rumen function have, characterized in that such animals orally a propionate-increasing amount of a metabolite of the in Claim 1 given type administered. 9. Feed for controlling coccidiosis, characterized in that that it contains poultry feed and so much of a metabolite according to claim 1 that the daily amount of feed a gives a non-toxic coccidiostatic dose of the metabolite. 10. Feed for fattening cattle, characterized in that cattle feed and so much metabolite according to claim 1 brings together, one of which is non-toxic PropioRat-increasing amount per animal and day results.