DE2404958C2 - Metabolite A-27106 and process for its preparation - Google Patents

Description

d) an infrared absorption spectrum in chloroform according to the accompanying drawing, which forms part of this Claim is

e) a mass spectrum having a molecular ion peak at mle 854.44630 and characteristic peaks at mle 836.44129, 779.44690 761.44740 and as well as

0 has an Rf value of 0.49 in a silica gel thin-layer chromatogram in benzene / metha ^ l (7: 3),

and wherein its acid form is a white solid having a molecular weight of about 832 and one titratable group with a pKa value of 7.2.

2. A method for the preparation of the metabolite A-27106 according to claim 1, characterized in that one Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of carbon, Contains nitrogen and inorganic salts,

a) grows submerged under aerobic conditions in the presence of glucose and monensin until the Monensin by the organism in the culture medium in a substantial amount of the metabolite A-27106 has been converted, or

b) culturing submerged under aerobic conditions, separating the broth from the culture medium, lyophilizing the broth, the lyophilized broth rebuilt and rebuilt in an aqueous buffer medium Broth together with glucose and monensin until there is a substantial amount in the buffer medium Amount of metabolite A-27106 has arisen, whereupon the metabolite A-27106 is removed from the buffer medium wins, or

c) culturing submerged under aerobic conditions, separating the cells from the culture medium, the cells obtained by sonication, centrifugation and dialysis cleans the purified dialysate in an aqueous Buffer medium reacts with glucose and monensin until there is a substantial amount in the buffer medium on metabolite A-27106 and isolated the metabolite A-27106 therefrom.

3. Use of the metabolite A-27106 according to claim 1 in poultry or ruminant feed.

The invention relates to the metabolite A-27106 and to a process for its preparation as well as its use.

The subject of the invention is the metabolite A-27106 or its acid, ammonium, lithium, potassium, Rubidium or cesium forms, whereby this metabolite (Na salt) is a white crystalline solid,

Relatively well dissolves in lower alkanols, but is generally insoluble in lower alkanols and the like has further properties:

a) a molecular weight of 854, 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 C ₄₂ H ₇₁ O ₁₆ Na corresponding to the structural formula in claim 1,

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

ej is a mass spectrum with a molecular ion peak at mle 854.44630 and characteristic peaks at ml

e 836,44129, 779,44690, 761,44740 and
0 has an Rf value of 0.49 in a silica gel thin-layer chromatogram in benzene / methanol (7: 3),

and wherein its columnar form is a white solid with a molecular weight of about 832 and a filterable Group with a pKa value of 7.2.

The invention also relates to a process for the preparation of the metabolite A-27106, which consists in that Streptomyces candidus NRRL 5449 in a culture medium containing assimilable sources of Contains carbon, nitrogen and anot ^ ainic salts, submerged under aerobic conditions in the presence of glucose and monensin grows until the monensin is absorbed by the organism in the culture medium a substantial amount of the metabolite A-27106 has been converted.

Finally, the subject of the invention is the use of the metabolite A-27106 in poultry or ruminant feed. This achieves the prevention or treatment of coccidiosis in poultry /. '■ -. as well as an increase in the feed conversion of ruminant animals that developed a ruminant? Function by administering a propionate-increasing amount of the metabolite A-27106 to such animals

Coccidiosis is a known protozoal disease that results from infection by one or more types of Eimeria or Isospora (see Lund and Farr in "Diseases of Poultry," 5th Edition, Biester and Schwarte, Eds., Iowa State University Press, Ames, Ia., Pages 1056-1096, arises because of the great economic losses due to coccidiosis and the difficulty in using some known anti-coccidiosis remedies still a need for better agents for coccidiosis.

Ruminants are economically important animals and an increase in feed conversion is therefore always very important he wishes. The mechanism by which the predominant nutrient content (carbohydrates) of ruminant feed is processed is known. Microorganisms in the animal's rumen ferment carbohydrates to form Monosaccharides and build this «! Monosaccharides then turn into pyran compounds. The pyruvates will be metabolized by microbiological processes, creating acetates, butyrates or propionates, which are collectively referred to as volatile fatty acids (VFA).

The favorable influence given by the use of volatile fatty acids has already been discussed by McCullough, Feedstuffs, Jun 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. There will also be acetates and butyrate are used, but the use of propionates is more effective in comparison. If too little If propionate is present, ketosis can also develop in the animals. A corresponding cheap remedy should therefore stimulate the formation of propionates from carbohydrates in the animals, whereby the carbohydrates are better utilized and at the same time the ketosis infestation is reduced.

Monensin, from which the metabolite A-27106 can be produced, is one as factor A in US Pat. No. 3,501,568 antibiotic complex A3823. Monensin is also an anti-coccidial agent.

The bioactive agent of the present invention is arbitrarily referred to as metabolite A-27106. Of the Auauiutk Ä-27106 refers to the sodium salt. Other salts are the ammonium, lithium, Potassium, rubidium and 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, its sodium salt as follows Formula I has:

CH ₃

CH ₃

BO Na ⁺ OE

/ OH CH ₃ HIGH ₂

50

60 65

Monensin in the form of its sodium potatoes is produced by Streptomyces cinnamonensis, as described in U.S. Patent 35 01 568 is described.

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 produced by a new strain of Streptomyces candidus are formed. A culture of this new strain was made without restriction on its availability in 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, there under the number NRRL 5449.

Because of the uncertainty of taxomic studies in organisms of the group Streptomyces, there is KJassification a newly discovered organism always has a certain moment of uncertainty. In the most important Properties appears to be the organism NRRL 5449, which converts monensin to the metabolite A-27106, however, to be most similar to Streptomyces candidus (Krassilnikov) Waksman 1953. The nature of the culture was described by S. A. Waksman in The Actinomycetes, Volume II, Williams and Wilkins, Baltimore, 1961, sentence 187. The present organism is considered to be a new strain of the organism described. Even though S. candidus NRRL 5449 and the known strain each other in general mycelial morphology and Color as well as in the appearance of the spores, there are still enough differences between them make it necessary to designate the present organism as a new strain. So brings the published Organism, for example, milk does not curdle, 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, a culture sample grown from the known strain does not produce monensin Metaboiit Α-27Ί06 converts.

The organism that converts monensin to the metabolite A-27106 was derived from one on Mount Ararat in the Turkey collected soil sample isolated.

The taxonomic studies on S. candidus NRRL 5449 are carried out according to the International Cooperative Project for Description and Deposition of Streptomycetes and methods recommended by Shirling and Gottlieb, "Methods for Characterization of Streptomyces Species," International Bulletin of Systematic Bacteriology 16, 313-340 (1966) described method, and z>? Ar together with other supplementary studies. The abbreviation »ICP« at the beginning refers to media as used by Shirling and Gottlieb. The remaining media are in the Waksman literature already cited described. The color names are 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. In the Numbers given 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 stripe provisions are underlined, and the Maerz and Paul (Dictionary of Color, MacGraw-Hill, N.Y., 1950) are given in brackets.

The Sireptomycete NRRL 5449 is characterized by straight to wave-like sporophores and oval to

slightly cylindrical spores with an average size of 1.165 α x0.57 μ, in chains of 10 to 50. According to electron microscopic observations, the spores are smooth. Aerial mycelia are usually white. At 26 ° C, there is only a vegetative growth, and at 45 ⁰ C for no growth occurs. Maximum growth and maximum sporulation occurs at 30 to 37 ° C.

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

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

Good growth, pale yellow [1 ICl], no aerial mycelia or spores, no soluble pigment.

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

Good growth, pale yellow-green [lOCl], 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 [10B Ij, good aerial mycelia and spores, (W) white a, no soluble pigment.

Glycerine-glycine

Profuse growth, medium brown [11J4], rich sporulation and aerial mycelia, (W) white a and (Y) pale yellow 2db, slightly light brown soluble pigment.

Emer3on

Profuse waxing, greyish yellow [12H3J. 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. 5

Czap »k

Profuse growth, moderately orange-yellow [11J7J, abundant aerial mycelia and spores, (W) white a, none soluble pigment.

Glucose asparagine

Fair to good growth, pale yellow-green [10 sheets], no aerial mycelia or spores, no soluble pigment.

Calcium malate 15

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

Nutrient agar

Good waxing, pale yellow green [lOCl], no aerial mycelia or spores, no soluble pigment. 20th

The organism is treated according to standard procedures with regard to selected physiological properties examined. The observed properties and characteristics found for them are as follows:

Observed property characteristic

Effect on skimmed milk clearing, coagulation after 14 days

Ni '-. Atreduction positive ³⁰

Melanin production

Tryptone yeast extract broth weak

Tyrosine agar none

Gelatin liquefaction none after 21 days ³⁵

Temperature requirements 26 = C - vegetative growth only

30-37 ⁰ C - good vegetative growth, good aerial myce-

lien and spurs

43-55 ° C - no waxing '°

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

⁴⁵ + = consumption - good waxing

[+] = possible consumption - good to tolerable growth

[-] = questionable consumption - weak or no growth at all

= no consumption - no waxing 50

Carbon source assessment

of growing

55

Raffinose +

D-fructose [+]

Cellobiose [+]

L-arabinose [+ J ₆₀

D-mannitol [+]

Rhamnose [+]

Cellulose

Dextrose [+] to [-]

D-Xy! Ose [+]

Inositol +

-C (no carbohydrate) [-] to [+]

Any culture medium can be used to grow S. candidus NRRL 5449. However, for reasons of economy, an optimal yield and easy lujlierung of the product certain culture media are preferred. Preferred sources of carbohydrates for large scale fermentation are therefore for example invert sugar or corn syrup, but glucose, fructose, maltose, Use starch, inositol and the like. If one wants to convert monensin into the metabolite A-27106 in a fermentation broth at the same time as the growth of S. candidus NRRL 5449, then the medium for sufficient conversion also contain a source of glucose. If one leaves S. candidus NRRL 5449 against it grow only to form its enzyme system, which is later used to convert monensin into the metabolite A-27 ICu, then glucose does not necessarily have to be present during fermentation.

ίο Preferred nitrogen sources are peptones, soybean meal, amino acid mixtures and the like. To the Inorganic nutrient salts that can be added to the culture media include the usually soluble salts, the iron, sodium, potassium, ammonium, calcium, phosphate, chloride, carbonate and the like Deliver ions. Essential trace elements that are necessary for the growth and development of the organism should should also be present in the culture medium. Such trace elements usually come as a contaminant other components of the medium in quantities sufficient for the organism to grow.

The starting pH of the culture medium can be varied. Before inoculation with the organism however, the pH of the culture medium should expediently be adjusted to about pH 5.7 to about 7.5 depending on the medium used. As with other actinomycetes, the medium also in the present case gradually more alkaline as the fermentation progresses and can be of a Starting pH of about pH 5.9 during the growth period of the organism to a value of about increase pH 6.9 or above. The final pH is at least partially determined by the starting pH of the Medium, the buffers present in the medium and the length of time during which the organism will grow lets controlled. The pH can also be adjusted by adding an acid or a base, too good However, results are obtained without adjusting the pH.

Just like other Streptomyces strains, the organism NRRL 5449 must also be grown under aerobic conditions. Propagation or growth on a small scale is expedient on agar slants or agar plates, in shake flasks or flasks. For a large-scale pro a submerged aerobic culture in large tanks is preferred.

The fermentation medium in a sterile tank can be used to start the fermentation inoculate a suspended suspension. Because inoculation with a sporulated suspension is a growth delayed, however, a vegetative inoculum is preferred. The vegetative inoculum is made by a small volume of culture medium with the spore shape or with mycelium fragments of the organism be inoculates in order to achieve a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The same can be said of the growth of the vegetative inoculum

Use medium as later for large-scale production, but you can also use different media Media work. The organism S. candidus NRRL 5449 grows in a temperature range between about 26 ⁰ C and about

However, 40 ⁰ C. At maximum growth and sporulation maximum it is customary between about 32 and about 37 ⁰ C. As in aerobic submerged culture, it also blows in this case during the fermentation by the Kulturrwdium sterile air. For sufficient growth of the organism and a corresponding production of Metaboiit A-27106, the volume of air used in large-scale production of the substance should be above about 0.1 volume of air per minute per volume of culture medium. Optimal result nits are held with an air volume of at least one third to one half volume of air per minute and per volume of culture medium.

The fermentation time required to convert monensin into the metabolite A 27106 varies. For a sufficient amount of glucose must be present for the conversion of monensin to the metabolite A-27106. If there is enough glucose available and about 0.1 to 1.0 g of monensin per liter of medium is available. den, then the conversion of monensin to the metabolite Α-27ΊΟ6 takes place almost completely within about 36 to 72 hours. Optimal conversion occurs in the presence of an amount of about 0.5 monensin up to 0.7 grams per liter of medium. A sufficient amount of glucose is between about 2.0 and about 2.5 weight percent glucose based on the weight of the medium. Appropriate glucose test paper strips can be used to check the respective glucose concentration. If the glucose level drops to 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 does not have to be highly pure. A filtered fermentation broth can therefore be lyophilized, for example, and stored for at least two weeks before it has to be replenished with an aqueous buffer, for which about ¹ Å of the volume of the original broth is used. A sufficient The corresponding conversion is obtained after about 72 hours if 2.5 g of a preparation thus lyophilized in Recycled 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 produced from the separated fermentation cells. Let these cells store frozen for at least three months. The thawed cells can then be like bring them to life by suspending them in a buffer solution. The buffer discharge is further purified by beachalling and centrifugation. The purified cell broth separated in this way are then resuspended in a buffer and dialyzed. Using this method is obtained from 200 g of cells from the fermentation medium as much purified dialysate which is characterized glucose u i 25 mg

Monensin can convert to the metabolite A-27106.

The progress of the conversion can be followed by thin-layer chromatography. On silica gel (F-254) in benzene / methanol (7: 3) the Rf value for monensin is 0.62, and the Rf value for the metabolite A-27106 on the other hand is 0.49. A vanillin spray reagent can be used for detection. This reagent is prepared by adding fuming nitric acid (2 ml) to a solution of vanillin (3 g) in absolute Ethanol (100 ml) there.

The metabolite A-27106 is present in both the culture broth and in the mycelium. Appropriate techniques to isolate the metabolite A-27106 must therefore be designed so that they achieve maximum recovery of the product from both of the above media. For this purpose, the fermentation medium is, for example filtered, after which both the filtrate and the mycelial cake are extracted with suitable solvents, in order to arrive at the metabolite A-27106. From the solvents used for the extraction leaves win the desired product using conventional methods.

Alternatively, the solids contained in the culture, including the constituents of the medium and the mycelium, use as a source of the metabolite A-27106, but first of all the water the mycelium and the culture medium. For this purpose, the culture medium can be lyophilized, for example dry and mix with a feed. The solids can also be removed without total water removal Process into a thin sludge, which can be mixed with moist mixed feed and similar feedstuffs can add.

An Eiüzci-r.xifukiioris-isöiäiiünii procedure is not necessary. Filtering the finished culture medium using a filter aid is sufficient. The filter cake can be washed with a polar solvent such as Methanol, «radiate. The methanol extract is concentrated and then added to the original aqueous filtrate given. The combined solution thus obtained is extracted twice with half the volume of chloroform. the Chloroform extracts are concentrated in vacuo to a dark amber oil.

The Ö! decolorizes main in a column filled with activated carbon using Chloroform and about 20 g activated charcoal per gram of oil. The eluate is again concentrated under vacuum, whereby a pale yellow to colorless oil is obtained. This oil is dissolved in the minimum amount of chloroform required and then chromatograph the solution on a silica gel column using ethyl acetate as a solvent. The elution is monitored by thin-layer chromatography. Elution with ethyl acetate-methanol mixture yields the metabolite A-27106.

The metabolite A-27106 (sodium salt) is a white crystalline solid that forms at approx Melts from 170 to 175 ° 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 also changes Melting point, 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 ₇ ) O ₁₆ Na, which 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 practically no ultraviolet absorption above about 235 μΐη.

The infrared spectrum of the metabolite A-27106 in chloroform can be found in the attached drawing will. 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 ion peak and that also given Characteristic.

m / e
calculated

854.445230 836.45229 779.45490 761.44540

found

854.44630 836.44129 779.44690 761.447740

fragment

C ₄₂ H ₇₁ O ₁₆ Na
(M ⁺ )

C ₄₂ H ₆₉ O ₁₅ Na

(M ⁺ -H ₂ O)

C ₄₀ H ₆₈ O ₁₃ Na

(M ⁺ - [CH ₃ O + CO ₂ ])

C ₄₀ H ₆₆ O ₁₂ Na

(M ⁺ - [CH ₃ O + CO ₂ + H ₂ O])

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

In general, the metabolite A-27106 is slightly soluble in highly polar solvents, insoluble in non-polar solvents Solvents and variously soluble in solvents of medium polarity This is how A-27106 dissolves for example in lower aliphatic alcohols and partly in phenol, diethyl ether and acetone, and he 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 Figure 8 shows the presence of a titratable group with a pKa 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. The ammonium can be extracted from the acid and other alkali salts. The various metal forms behave somewhat similarly to alkali carboxylates and chelates.

To produce another form, the metabolite A-27106 (the sodium form) is in an aqueous Solvent, such as methanol / water, dissolved. An acid, for example, is then added to the solution obtained Hydrochloric acid to lower the pH to 5 or below. Then the methanol is under The vacuum is drawn off and the aqueous acidic product obtained is extracted with chloroform. The chloroform extract is dried and evaporated to the free acid form.

The acid form of the metabolite A-27106 thus obtained can be used as such or it can be further used modify them by titrating them with an aqueous alkali hydroxide or with aqueous ammonia to in this way to produce the corresponding 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 monensin to the metabolite A-27106 glucose is required and consumed, even in its absence metabolizing cells of 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, five hydroxyl sites would have to be present a glucosylmonensin may be present, which can be individually or collectively to form a simple ester, like an acetate. The presence of all of these five reactive sites arises by esterification attempts.

On the basis of the physical properties listed above, the following can be deduced for the metabolite A-27106 Propose structure of formula II:

O Na ⁺ OE

CH ₃
OH

O - (glucose)

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

The metabolite A-27106 is less toxic than monensin. After intraperitoneal administration of the metabolite A-27106 on groups of 6 mice each perished at a dose of 50 mg / kg of six mice one animal each, and when 100 mg / kg is administered, three out of six animals perish.

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

To prevent or treat coccidiosis in poultry, animals are given a non-toxic coccidiostatic Amount of metabolite A-27106 administered, preferably orally on a daily basis. For determination the appropriate concentration of A-27106 one must consider a number of factors that lead to however, the amount administered is generally between 0.005 and 0.05 percent by weight, based on aul Feed not mixed with active ingredient, and it is preferably between 0.01 and 0.04 percent by weight. Dei Metabolite A-27106 can be administered in a number of ways, but the easiest way is to use one Physiologically harmless carrier supplied, preferably with the feed administered to the animals.

The metabolite A-27106 also improves feed utilization in ruminants whose rumen function is developing is. Young ruminants, practically all animals that have not yet been weaned, are monogastric animals As soon as young ruminants start eating solid feed, the rumen function begins to develop none, and the microbiological population of the rumen increases. After the animal has fixed a certain amount of time: Has eaten food, its rumen function is fully developed and is maintained throughout the animal's life ten. Some economically important ruminants are cattle, sheep and goats.

The MeUbolit A-27106 increases feed efficiency when given orally to ruminants in large quantities

about 0.05 mg / kg / day to about 2.5 mg / kg / day administered. 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 metabolite A-27106 is by admixing it with animal feed. However, metabolite A-27106 can can also be administered in other forms, for example in the form of tablets, drinks, boIi or capsules. These different dosage forms can be formulated according to the usual methods used in animal pharmacy drive reach. Each individual 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 grown on agar slants made from Bennett's medium, whereby a well-defined colony is obtained. The colony is removed and made up with sterile deionized water (10 ml) slurried.

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

Up to parts quantity Corn distillers' solubles 25.0 g

Lactose 10.0 g 25

Maltose 10.0 g

FeSO ₄ · 7H ₂ O 0.01 g

MgSO ₄ · 7H ₂ O 2.0 g

I ₂ PO ₄ 2.0 g

CaCO ₃ 2.0 g 30

Deionized water up to 1.1 liters

Dk; four inoculated bottles are ^{incubated at 30 °} C. on a rotary shaker at 250 revolutions per minute for 24 hours. The vegetative medium obtained in this way (10 ml portions) is used to inoculate a total of 15 shake flasks (500 ml) which contain 100 ml of sterilized fermentation medium with the following composition:

Ingredients quantity

40

Beef extract 5 g Casein Fancreas Hydrolyzate Peptone 5 g

NaCl 5 g

Glycerin 15 g 45

CaCO ₃ 2 g

Deionized water up to 1 liter

The medium has a pH of 7.2 which is not adjusted. The inoculated medium will like 72 hours incubated as described above. 50

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

Components lot Polysiloxane ^ (antifoam agents) 5g Glycerin 375 g Dextrose 625 g Casein Pancreas Hydrolyzate Peptone 125 g Beef extract 125 g NaCl 125 g CaCO., 50 g deionized water up to 24 liters

The original pH of the medium is 7.0. The medium is then sterilized by treatment in an autoclave at 120 ^° C. for 30 minutes at a pressure of 1.05 to 1.41 bar / cm ^2. After sterilization, the pH of the medium is 7.6.

Purified monensin (25 g) is dissolved in ethanol (200 ml) and the resulting solution is added to the sterilized one Medium, whereupon the vegetative inoculum obtained from the second stage and prepared as described above (700 ml) is added

The fermentation medium is aerated with sterile air at a rate of about 1 liter per minute and stirred with conventional stirring at a stirring speed of 420 revolutions per minute. The inoculated medium is cultured at 30 ^° C. for about 114.5 hours.

ίο The course of the fermentation is determined by thin layer chromatography over silica gel as described above tracked in the first stage of fermentation only monensin is present. Then a second one gradually shows Mark the presence of the metabolite A-27106, and at the end all you see is the mark 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 Mycelial cake is extracted with methanol (about 5 liters) at room temperature. The methanol extract is filtered, whereupon the filtrate is concentrated in vacuo, the methanol is removed and so an aqueous concentrate receives

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

This oil is dissolved in chloroform and removed in a filled with 10 kg of activated charcoal (Pittsburg "12 x 40") Column which is eluted with chloroform (about 5 liters). The chloroform eluate is evaporated under vacuum, whereby about 500 ml of a colorless to pale yellow oil is obtained.

The decolorized oil is taken up in a minimal amount of chloroform and the whole is then chromatographed on a 25 kg column of silica gel (Grace, grade 62) in ethyl acetate. The elution is monitored by thin layer chromatography as described above. After the impurities have been removed with ethyl acetate, the metabolite A-27106 is eluted from the column with ethyl acetate-methanol (19: 1). The fractions containing the A-27106 are vereit: gt and evaporated to dryness under vacuum, thereby obtaining an amorphous, off-white product. The product is washed with hexane and dried, giving about 12.84 g of metabolite A-27106 (which shows a single spot on thin-layer chromatography).

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 as follows carried out:

Streptomyces cinnamonensis ATCC 15413 is left in the usual manner in 55 ml of medium in a 250 ml shake flask and grow into a vegetative inoculum under 47 hours of incubation (see US-PS 35 01 568). This vegetative inoculum becomes 220 ml of one in a 1 liter shake flask Medium given and incubated for 21 hours, whereby the seed inoculum for the fermenter is obtained.

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:

Ingredients quantity

j | Glucose 750.0 g

|| 55 soybean meal 625.0 g

| $ Soy! 500.0 g

'' Methyl oleate 500.0 g

; ·; Polysiloxane oil (antifoam agent) 5.0 g

Potassium chloride 2.5 g

iv 60 dipotassium hydrogen phosphate 2.5 g

ρ manganese chloride tetrahydrate 15.0 g

Hydrous iron sulfate 7.5 g

% Calcium carbonate 25.0 g

• 5 deionized water up to 24 liters

G; The medium obtained has a pH of 5.5, which can be increased by adding 10N potassium hydroxide (15 ml)

p \ pH 8.9 sets. The inoculated medium is incubated for 234 hours at 32 ^° C.

'=! - ■ After 42 hours, increase the aeration from 9.9 liters per minute to 22.6 liters per minute (0.35 to

ίο

0.8 cubic feet) and the agitation speed from 500 to 700 revolutions per minute. After 210 hours it is the formation of monensin is practically complete, as demonstrated by a thin-layer biopsy with Bacillus subti-Hs ATCC 6633 results as a detection organism

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

Ingredients quantity

Dextrose 750 g

Soybean flour 625 g

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 to 8.0 Ct-a by adding 215 ml of 5 η sodium hydroxide and the medium is sterilized. The medium is inoculated with then 'ne of rapidly growing vegetative culture of strep & jiyces candidus NRRL 5449 and incubated 137 hours at 30 ⁰ C. The fermentation is aerated with sterile air at a rate of 9.9 liters per minute. The fermentation medium is stirred with conventional stirrers, initially at a speed of rotation of 120 revolutions per minute, with 420 revolutions per minute after 16 hours and 500 revolutions per minute after 46 hours. After 44,66,5,89,97,113 and 127 hours each dextrose (400 g) is added. Calcium carbonate (175 g) is added after 72 and 99 hours.

The fermentation is monitored by thin-layer chromatography as described above. After 137 hours the formation of the metabolite A-27106 is practically over. The processing and purification of A-27106 takes place according to the procedure described in Example 2.

Example 4
Production of A-27106 by a granular enzyme preparation from 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, each 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 Centrifuged 10,000 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 chicroform 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: 1) gives 17 mg of metabolite A-27106, which is identical to the product obtained according to Example 2.

Example 5
Control of coccidiosis with the metabolite A-27106

75 one week old, healthy, incubator and battery rear crossbred male chickens of the heavy broiler type are used for this study. The chickens are divided into five groups of 15 animals each, which in turn are divided into three subgroups of 5 chickens each. Each subgroup is kept out of contact with the other subgroups. A first group is kept under favorable conditions as a health comparison for untreated animals. A second group is treated as the first group except that they are inoculated with coccidiosis by the oral administration of a dose containing 10 ⁵ sponsored oocytes of Eimeria tenella. The third, fourth and fifth groups of animals are bandaged in the same way as the second group, but deviating from this, their feed is modified by adding metabolite A-27106 in concentrations of 100, 150 and 200 ppm 24 hours before the mentioned 3rd vaccination

Seven days after inoculation, the chickens are weighed, sacrificed and checked for evidence of coccidial damage examined. The coccidiosis damage is assessed on an arbitrary scale between 0 and 4. The corresponding damage numbers indicate the number of animals for each value. The number "0" means that none Damage is caused by cofjcidiosis. The number "1" means that this is the lowest detectable coccidiosis damage acts. The number "2" denotes moderate damage, with only minor damage

or no bleeding at all and no serious tissue damage. The number "3" means no bleeding that oozes out of the caecum and severe tissue damage. The value »4« indicates a bleeding with a caecal nucleus of clotted blood and destroyed epithelial cells. Animals rated "4" or below can usually recover if they are no longer infected with coccidia.
Following the procedure described above, the following results are obtained:

10

15th

20th

30th

35

animal With E. tenella Metabolite Medium Assessment of the damage group inoculates A-27106 Weight (ppm) increase 0 12 3 4 in the feed per animal mg

no

Yes

Yes

Yes

Yes

100

ι cn

200

162 67

157 ι en

15th 0 0 0 0 0 0 0 0 15th 4th 1 0 3 7th 1 1 1 7th

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

After research indicates that the metabolite A-27106 is normally found in E. tenella kills the first attempt at spread in the host cell, prophylactic treatment is preferred.

Other studies with the metabolite A-27106 show that it 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 also in combination with monensin. These investigations lead to the result that A-27106 does not larger weight gains at 0.04% than at a dosage of 0.0i%, but the higher dose less caecal damage. At the higher dose, there were no toxic effects whatsoever.

Within the general range of effectiveness, combined administration of monensin and Metabolite A-27106 in the feed has 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 which is the combined single doses.

40

45

50

55

Example 6
Chicken feed modified with A-27106 to control coccidiosis

For the production of a balanced, high-energy feed with which chicken can be eaten more quickly Weight gain, one assumes the following components:

Components% kg

ground yellow corn

Soybean meal, extracted with solvent,

freed from the shell, 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 premix

(Vitamins A, D, E, K, and B ₁₂ ,

Choline, hiacin, pantothenic acid,

Riboflavin, biotin with glucose

as filler) trace mineral premix

(MnSO ₄ , ZnO, KJ, FeSO ₄ , CaCO ₃ ) 2-Amino-2-hydroxybutyric acid (hydroxy analog of methionine) Metabolite A-27106

50 454 31.08 281.7 6.5 59.0 5.0 45.4 4.0 36.3 1.8 16.3 C, 8 7.26 0.5 4.54

0.2 1.81 0.1 0.907 0.02 0.181

12th

The substances listed above are mixed with one another using the techniques customary in animal feed production. The chickens fed such feed and given water ad libitum are protected against coccidiosis. The weight gains thus obtained are comparable to those of Coccidiosis-free chickens that are fed a similar feed that has not been mixed with active ingredients.

Example 7 Improvement of feed conversion through A-27106

First of all, rumen fluid is obtained from an ox by means of an operation in the rumen pickled fistula. The ox is fed a high-grain feed, which has the following composition:

69.95% coarsely ground corn 10% wed «corn on the cob 8th% Soybean meal (50% protein) 5% Alfalfa flour 5% molasses 0.6% 1 »* _ rt *
naiiiHUii 0.5% Dicalcium phosphate 0.5% Calcium carbonate 0.3% salt 0.07% Vitamin A and D ₂ premix ¹ ) 0.05% Vitamin E premix ') 0.03% Trace mineral premix ³ )

') Content per 454 g: 2,000,000 IU of vitamin A; 227 200 IU vitamin D ₂ and 385.7 g soybean feed with 1% added oil.

² ) Distillers' solubles of dried corn kernels containing 20,000 IU of d-alpha-tocopheryl acetate per 454 g.

³ ) Manganese oxide, potassium iodide, cobalt carbonate, copper oxide and zinc sulfate content.

30th

E. The sample of rumen fluid is forced through four layers of cheesecloth and the filtrate is collected

man The granular residue retained by the cheesecloth is in a sufficient amount of a physiolo-; |

gischen buffer resuspended in order to bring it back to the original volume of the rumen fluid, vi

and this suspension is again strained. The buffer used has the following composition: j

35 %

g / liter of ingredients 4i

0.316 Na ₂ HPO ₄

0.152 KH ₂ PO ₄

2.260 NaHCO ₃ ⁴ ^

0.375 KCl

0.375 NaCl

0.112 MgSO ₄

0.038 CaCl ₂

0.008 FeSO ₄ · 7H ₂ O

0.004 MnSO ₄

0.004 ZnSO ₄ • 7H ₂ O

0.002 CuSO ₄ • 5H ₂ O

0.001 CaCl ₂

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

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

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 flasks each are also used A comparison is carried out at time zero and a further comparison after incubation for every hour carried out All test flasks are incubated for 16 hours at 38 ° C. After incubation, the pH is measured and 25% metaphosphoric acid (2 ml) is added to each flask. The samples are allowed to settle, and the The supernatant liquid is examined for propionate, acetate and butyrate compounds by gas chromatography. Active compounds make the propionate production rather than that of the comparisons greatly increased.

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

"β mg A-27106 / ml diluted propionate butyrate total amount

j | ter rumen fluid to VFA

% 5 2.15 0.73 1.03

\ i 1 1.42 0.96 0.98

fJ 0.7 1.02 0.91 1.07

ίί examples

υ Beef cattle feed improved with A-27106

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

15th . [| Components percent kg per ton

2Q i ^ uigciiiaiiiciici iviaia υ /, ο 6/5

100 50 99.56

with ϊ.ηςιιησ «ιτ» ϊτίι »1 p.Ytrahiprt S (W« protein

Sugar cane molasses 5 5

6 0.44

* α * · * 4VU4VI WIUWIlVJUUU ^ f AVtAAUItVIUHAt) A-TTVWVW V} ^ J

5 3 0.3

IC T IMllllllI ΓΎ UUU J_ / J ^T Wl ßSlHlOwli } V ₁ V / \ J ^ '

0.5 1.5

') Content per 454 g: 2,000,000 IU of vitamin A; 227 200 IU vitamin D ₂ and 385.7 g soybean feed with 1% added oil. 40 ² ) Distillers' solubles of dried corn kernels containing 20,000 IU of d-alpha-tocopheryl acetate per 454 g.

The feed mixture was compressed into pellets. With an average daily digestion amount of around 6.8 kg Feed per animal provides this feed about 300 mg A-27106 per animal and day.

Example 9
Acid form of the metabolite A-27106

Metabolite A-27106 (100 mg), prepared as the sodium form according to Example 1 and 2, is dissolved in 100 ml of methanol / 50 water (1: 1) dissolved. The solution obtained is titrated to pH 3 by the dropwise addition of 1 η hydrochloric acid. The methanol is then removed in vacuo.

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

55 The acid form of A-27106 is a white amorphous solid with a molecular weight of about 832. the The biological activity of the acid form roughly corresponds to that of the sodium form.

60 Example 10

Cesium form of the metabolite A-27106

The acid form of the metabolite A-27106 (97.3 mg), prepared according to Example 9, is dissolved in 100 ml of methanol / water (1: 1). The solution obtained is titrated to pH 7.1 by adding 1 η cesium hydroxide dropwise . The methanol is then stripped off under vacuum. The solution thus obtained is extracted twice with equal volumes of chloroform. The chloroform extract is dried (MgSO ₄ ) and evaporated in vacuo to give a white amorphous solid. Elemental analysis shows the presence of about IVA metal.

ground corn δ7.8 ground corn on the cob 10 dehydrated alfalfa flour, 17% protein 5 soybean meal stripped of its hulls, 9,956 solvent extracted, 50% protein Cane molasses 5 urea 0.6 Metabolite A-27106 0.044 Dicalcium phosphate, for animal feed 0.5 Calcium carbonate 0.5 Sodium chloride 0.3 Trace mineral premix 0.03 Vitamin A and D ₂ premix ¹ ) 0.07 Vitamin E premix ² ) 0.05 Calcium propionate 0.15

Example 11 Lithium form of the 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 which is a 5 has some crystal order but does not give a clean pattern in X-ray diffraction.

Example 12 Ammonium form of the metabolite A-27106 10

The procedure of Example 10 is repeated except that instead of cesium hydroxide however, ammonium hydroxide is used to produce the ammonium form of the metabolite A-27106. This Product has the general properties and capabilities of the other forms.

15th

1 sheet of drawings

15th

Claims (1)

Patent claims: 1. Metabolite A-27106 and its acid, ammonium, lithium, potassium, rubidium or cesium forms, this metabolite (sodium salt) being a white crystalline solid, comparatively well in lower Alkanols dissolves, but is generally insoluble in lower alkanes and has the following additional properties Has: a) a molecular weight of 854, determined by mass spectrometry, b) an approximate elemental composition of 58.78% carbon, 841% hydrogen, 27.85% oxygen and 3.63% sodium, c) an empirical formula C ₄₂ H ₇₁ O ₁₆ Na, corresponding to the structural formula CH ₃ OH Ö - (Gluer «)