DE2057033C2 - Antibiotic thiopeptin A? 4? and its use in feed additives - Google Patents

Description

2. Use of thiopeptin A4 according to claim I in feed additives.

The invention relates to a component of the antibiotic thiopeptin, namely the component thiopeptin A4. Thiopeptin A4 can be in diluted form, as raw concentrates and in pure crystalline form. The thiopeptin A ₄ has antibacterial activity against a variety of microorganisms and is useful as an additive for animal feed.

_{The thiopeptin A 4} according to the invention can be characterized as indicated in claim 1. The approximate empirical formula and the parameter / were submitted after the filing date.

The thiopeptin A ₄ was found in a fermentation broth in which Streptomyces tateyamcnsis ATCC 389 was cultivated in an aqueous nutrient medium under the conditions described in DE-OS 19 29 355, the thiopeptin components Ai, A2, A] and B and method related to their production. For this purpose, according to DE-OS 19 29 355 Streptomyces tateyamensis in the aqueous nutrient medium under submerscn aerobic conditions are treated. That which is useful for breeding Streptomyces tateyamensis Nutrient medium for the production of thiopeptin contains both a carbon source, for example a assimilable carbohydrate, as well as a nitrogen source, for example an assimilable nitrogen compound or a proteinaceous material. examples for useful carbon sources are starch, glucose, sucrose and glycerine. Examples of useful Nitrogen sources are meat extract, peptor .. glutin meal, soybean meal, cottonseed meal, corn steep liquor, dry yeast, yeast extract, ammonium sulfate, sodium nitrate, casamino acid and urea. It combinations of these carbon

Is and / or nitrogen swelling can be used. Inorganic salts capable of forming ions such as potassium, sodium, calcium, phosphate and sulfate can be added to the culture medium. Trace elements such as magnesium, manganese, zinc, "nd iron can also be added to the culture medium. Such trace metals can be used as impurities be delivered incidentally with the addition of the constituents of the medium. It should therefore be pointed out that the addition of such trace metals or inorganic see salts effectively done when of the antibiotics Cumulative microorganism producing them as a component of the culture medium requires vitamins such as inositol. Vitamin Bu, Isoasvorbic acid and Biotin can dem medium used for cultivation as well can be added.

To obtain the highest possible yield of thiopeptin from the medium described above the use of sulfur compounds of an organic or inorganic nature is required. examples for such sulfur compounds are N-acetyl-DL-methionine. Methionine. 2-naphthol-6.8-disulfonic acid. Sodium sulfosalicylate, sodium cetyl sulfate, taurine, thionine. Methyl orange and sodium sulfate The cheapest sulfur compounds are N-acetyl-DL-methionine and sodium sulphate Sodium sulphate is used for economic reasons.

Optimal yields of thiopeptin are obtained when the cultivation is carried out at a temperature which enables satisfactory growth of the

Microorganism and between about

24 ° C and about 37 ° C, preferably between about 27 ° C and about 32 "C., the cultivation period being about 2 to about 6 days.

After the cultivation has been carried out. can

according to DE-OS 19 29 355 a variety of processes can be used to isolate and purify thiopeptin. Suitable methods are, for example solvent extraction, chromatography and crystallization from solvents.

According to an expedient recovery process, the thiopeptin is obtained from the entire fermentation broth in that the mycelial cake after conventional methods, e.g. B. by filtration or centrifugation, separated and then an extraction by means of a suitable organic solvent such as acetone or methanol. acetone is the cheaper extraction solvent. The extract obtained can by customary methods concentrated to give a residue is, in turn, an extraction with a suitable organic solvents such as ethyl acetate or chloroform is subjected. An impure one crystalline mass of thiopeptin can thereby be obtained

20th

25th

will get the ''?: if Extr & k 'concentrated to a residue which is then cooled to room temperature. The impure crystalline mass of thiopieptine can also be obtained by concentrating the obtained extract to a residue, which is then in turn in an organic solvent such as η-hexane or Pctrolätha ge! W>v> in order to then bring about a precipitation. The Kompiex the Thiopcptinantibiotika-forming components according to DE-OS 19 29 355 by ^'0 be separated, that the impure crystalline sreihmg SRA; obtained as described above is dissolved in a suitable organic solvent such as chloroform, and the solution is then subjected to column chromatography with a suitable organic solvent system, with chloroform and methanol being preferably used.

It has now been found, surprisingly, that the fractions obtained by the above-described process in DE-OS 19 29 355 from a silica column with a mixture of chloroform and methanol (50: 1) contained another component, which was separated off by ascending paper chromatography, a mixture of ethylacelate, n-hexane and 2N ammonia (4: 1: 1) and other solvent systems being used which are capable of separating the thiopeptin A ₄ component.

The component according to the invention can be separated off by ^Μ chromatography such as paper and thin-layer chromatography. The development system contains the same solvents as are used to separate the other four components of thiopi: ptine. If a mixture of chloroform and methanol in a volume ratio of 50: 1 is used, the fractions thiopeptin Ai, Aj, A) and A4 are successively recovered from the column filled with silica gel on which the thiopeptin compound had been adsorbed. The fractions of a mixture of thiopeptin A components are also obtained. In this case, the same recovery procedures as used above can be used repeatedly. The thiopeptin-A component can be crystallized by evaporating the fraction to dryness and dissolving the residue in acetone.

The antibiotic thiopeptin A4 according to the invention has an antibacterial effect. It can be used favorably in feed additives use because it is completely excreted after 48 hours and not even in traces remains behind the Oi'ganen (see »Antimicrobial Agents and Chemotherapy. 19/2. Be'en 496-503). Thiopeptin A < proves to be superior to known antibiotics such as tylosin, as it is e.g. B. no cross resistance to erythromycin shows.

When using Thiopeptin A ₄ , for example, feeds can be prepared that contain a basal animal feed ration in addition to the thiop <: ptin-A ₄ component. Feeds best contain the Thiopt: p (in-A4 component in an amount of 0.1 ppm up to 100 ppm. The thiopeptin A4 can also be contained in a feed or feed additive in the form of a mycelium _{cake containing thiopeptin A 4} , which was obtained in the fermentative production of the thiopeptin A ₄ -K ') components.

The following example explains the preparation of the thiopeptin A ₄ component.

50

55

60

example

The thiopeptin account would thereby manufactured that Streptomyc-ea taiejamensis ATCC Ji 389 under submerged aerobic conditions in one aqueous nutrient medium was grown.

A. Fermentation

1. An inoculum of Streptomyces tateyamfcrcis was inoculated onto a sterile medium which contained the following ingredients in 100 ml of water:

Potato starch

Cottonseed flour

Corn steep water

Calcium carbonate

Potassium hydrogen phosphate

Disodium hydrogen phosphate

12H ₂ O

2.0%

2.0%

1.0%

0.3%

2.18%

1.43%

The inoculated broth was ^{incubated at 30 °} C. for 2 days. This culture was then placed in a 30 l fermentation apparatus made of stainless steel which contained 20 l of the aforementioned Medi: ms. The sterile fermenter was incubated for 48 hours at 30 ° C. with 201 sterile air being passed through it per minute. A rotary shaker at 300 rpm was used.

The fermentation was carried out under the same conditions and by the same procedure as as described in Section 1, but using a sterile medium that is described in 100 ml of water contained the following components:

Potato starch 4.0% Cottonseed flour 2.0% Corn steep water 1.0% dry yeast 1.0% Calcium carbonate 0.3 ^c .i; Potassium dihydrogen phosphate 2.18% Disodium hydrogen phosphate · 12H ₂ O 1.43% N-acetyl-DL-methionine 0.17%

3. The fermentation was carried out again under the same conditions and according to the same Procedure carried out as in Section 1 above, but using a medium which contained the following components in 100 ml of water:

Potato starch 2.0% Cottonseed flour 2.0% Corn steep water 1.0% dry yeast 1.0% Calcium carbonate 0.3% Potassium dihydrogen phosphate 2.18% Disodium hydrogen phosphate · 12H ₂ O 1.43% Sodium sulfate 0.5%

B. Obtaining Thiopeptin

The recovery of Thiopeptin has been achieved in that the culture broth is filtered and 1.8 k £ of the mycelial cake obtained with 7 I of a mixture of chloroform and methanol (2:. 1 by volume) were extracted, wherein the GemiscM to 50 ⁰ C. was heated. This extraction procedure was repeated three times, and the

20 d7 033

combined extracts were filtered. D.mn was the filter is concentrated to dryness, and the The residue was dissolved in 200 ml of chloroform, hot Addition of 200 ml of Denzene formed Precipitate which was removed from the first solution mixture by filtration. The one by constricting of the obtained filtrate remaining residue was in about ten times the amount n-llexan dissolved to form a crystalline material, consisting of the three thiopepiine components. to build.

2. Thiopeptin was obtained by filtering the cooling liquid and extracting the so obtained mycelial cake with acetone. The acetone extract was concentrated to a residue, which in turn was extracted with ethyl acetate. The crude crystalline thiopeptin was thereby obtained that the extract obtained and the product were cooled.

C. Isolation of Thiopeptin A _{4 According to the Invention}

The raw materials of the thiopeptin A and B components. those from the fermentation broth were obtained in the thiopeptin Α components by dissolving in chloroform and Chromatograph on silica with a chloroform: methanol (19: 1) mixture separated. The thiopeptin A-i component contained in the fractions recovered from the column. was separated and purified by column chromatography on silica gel with a chloroform: methanol (50: I) Solvent system.

The thiopeptin Ai component is a pale yellow crystalline material which melts and decomposes at 236 to 245 "C. Using a mixture of ethyl acetate, η-hexane and 2N-ammonia (4: 1: I) as the development system, its Rf Value determined to be 0.57. The optical rotation was [<x] ' _D ³ = -81.5 (c = 1.0 in chloroform). This component had a molecular weight of 1854. Determined by the vapor pressure method. The following analysis values were obtained: C = 50.13: H = 5.30: N = 15.22: and S = 12.02. The ultraviolet absorption spectrum shows in Fig. I shoulders at 235 to 250 mji. 295 μm. 303 to 308 m *. * .. Fig. 2 is an infrared absorption spectrum of this component in Nujol and shows maximum absorption bands at the following wavelengths: 3340, 3330, 1735, 1655, 1585, 1530, 1490, 1420, 1345, 1305, 1250, 1210, 1160, 1110, 1090, 1065, 1025, 1000,975 .950. 920,895,880,860,840,768,725, 705,680 and 660 cm- '. It is soluble in dioxane. Dimethyl sulfoxide, dimethyl form amide. Pyridine and chloroform, moderately soluble in methanol, acetone and ethyl acetate: and insoluble in ether, benzene. Petroleum ether and water. This component shows a positive reaction in the permanganate test, and negative reactions in the ninhydrin, biuret, Fehling and ferric chloride tests. It is stable for one hour at 60 ° C in a pH range of about 2.0 to about 8.0.

Investigation of the antibiotic effectiveness of thiopeptin A <:

The antibiotic effectiveness of Thiopeptin A4 against a number of organisms listed below have been investigated in these Try to find the results in the table below! put together became the activity of the compounds expressed as the minimum inhibitory concentration (MIC). The MlC was named after the Determine usual agar dilution method. l>! e MK values are given as the concentration eggs component in ng-ml. which the growth of the () i'ganism prevented.

Table I.

The MK of the I hiopeptin Aj component

Si-iplnlncoceus aurciis 209P 0.06

MacillusMihtilis 0.25

liacilliis mcgaicrium 0.25

Sarcinalutca 0.01

Corsnebactenum xerosis 0.25

Escheriehiacoli -128.0

Pseudomonas aeruginosa> 128.0

Mycobacteriuiii phlei 8.0

To determine the acute toxicity of thiopeptin

* i) A; -Ki; !! iponentc at de * M: nis umrrlp pine 5% iue Suspension of the antibiotic '' in sodium carboxymethylcellulose administered intraperitoneally to the mice.

The LDio value was determined to be over 250 mg / kg.

For use as a growth-promoting additive in animal feed, the thiopeptin A4 component can be used alone or in combination with other thiopeptin components. The thiopeptin Ai component is expediently kept in less pure forms, for example in the form of the dried, raw mycelium cake together with the diets with which the animals are usually fed. The proportion of this antibiotic. de ^r animal feed mixtures may be added, kanr; vary within wide limits, depending on the properties of the antibiotic and the animals to which the rations containing the thiopeptin A <component. should be fed alone or in combination. Antibiotic amounts between about 0.1 ppm and 100 ppm are favorable. No antibiotic residues were found in the serum or tissue of the animals examined. Even if trace amounts of the antibiotic residues within the existing analytical limits could be present in the abdominal cavity, it should be noted that in view of the fact that the antibiotic residues could not be found in the meat of the animals and detectable amounts of active antibiotic residues quickly disappeared when antibiotic feeding was stopped a few days before slaughter. the antibiotic residues do not lead to any undesirable effects in humans. It should also be noted that these antibiotic additives, when added to the feed rations in the correct amounts, do not cause any undesirable effects in the animals and cause increased or accelerated growth.

The following description explains the composition of basal rations, which the new Component was added and the effect on the growth of chickens when thiopeptin A4 alone was added.

a) Groups of 40 chickens each were weighed at three and ten weeks, and the individual weights were each determined. The control group was fed the basal ration selected from the following named substances existed. The test group was given the basal ration with a content of 10 and 20 g Thiopeptin A ^ component fed per ton.

I The composition of the nasal ration, which up to / ur the third week was as follows:

Hcsijiullcilc

Soybean meal

Fischnunl

alfalfa

Alkaline phosphate

Calcium carbonate

Sodium chloride

(Some of the vitamins Λ. D and F.

Ciemisch the B vitamins

Traces of inorganic compounds. Preparation with 25% amprolium Preparation with 0.2% pyrimethamine

52.46 14.0 (1 20.00 8.00 3rd (H) 1.00 0.70 0.45 0.05 0.10 0.10 0.09 0.05

When two ppm I hiopeptin Λ., - component is added the amount of maize was reduced to 52..16%. 2. From the fourth week .ib the following ration was fed:

Components

Soybean meal

Fischm.hl

alfalfa

Calcium phosphate

Calcium carbonate

Sodium chloride

Ciemisch the vitamins A. D and E Mixture of the B vitamins

Traces of inorganic compounds. Preparation with 25% amprolium. Preparation with 0.2% pyrimethamine

55.26 20.00 14.00 5.00 3, (W 1.00 0.90 0.45 0.05 0.10 0.10 0.09 0.05

When 2 ppm thiopeptin A _{4 was added to} this ration, the amount of corn was reduced to 55.16%.

b) The individual weights of groups of 150 chickens each were determined after 4 and 10 weeks. The control group was fed the basal ration which had the following composition. The test group was fed the basal ration, which contained 10 g per ton of thiopeptin A ₄ .

1. The basal ration. that was fed for four weeks, contained the following components:

Components%

Soybean meal

Fish meal

alfalfa

Calcium phosphate

Calcium carbonate

Sodium chloride

Preparation with 25% amprolium mixture of vitamins A and D.

Mixture of the B vitamins

Traces of inorganic compounds DL-methionine

45.00 20.00 23.00 7.00 2.40 0.70 1.30 0.25 0.13 0.05 0.09 0.03 0.05

I ppm thiopeptin A ₄ component was added to this Masal ration.

2. The basal ration. those from the fifth week on was made up of the following components :

licshiiulleilt:

Corn

MiIo

Soybean meal

Fish meal

removed rice bran

fat

alfalfa

C iiiCiiirnCtiTf »Oiliii

Calcium phosphate

Sodium chloride

Preparation with 25% amprolium

Ethoxyquine preparation

Traces of inorganic compounds Mixtures of vitamins A and D Mixtures of B vitamins

DL-methionine

40.00 20.00 20.00 5.00 6.00 4.00 2.40 i .2Ci 0.80 0.25 0.OS 0 D5 0.03 0.05 0.09 0.05

1 ppm thiopeptin A _{4 was} added to this ration.

Tables 2 and "> show the effect of the thiopeptin A ₄ component when used in amounts of 2 ppm and 1 ppm, respectively
became.

Table 2

added to basal rations a) and b)

Basal ration thiopeptin \ _Λ 2 ppm

Average
Initial weight (g)

average
Weight according to
6 weeks (g)
average
Final weight (g)

Table 3

36.4

36.0

928.2 1006.4

2071.2 2194.0

Basal ration Thiopeptin A ₄ 1 ppm

Average

Initial weight (g) 43 43

average

Final weight (g) 1961 2183

For this purpose 2 sheets of drawings

230 261/26

Claims (1)

Patent claims: 1. Thiopeptin A4, characterized in that it a) the elemental analysis gives the following values: C 50.13; H 530; N 15.22 and S 1Z02; b) has a molecular weight of 1854, determined by the vapor pressure method, from which the approximate empirical formula can be calculated: C77 to C78; H98 to H99; N20 to N21; O20 to 0 ·>ι; S ₇ ; c) is a pale yellow crystalline substance with melting and decomposition points between 236 ^{° C and} 245 ° C; d) an optical rotation of [oc] J? = - 81 ^ ° (c = 1.0 in chloroform); e) an Rf value of 0.57 in a mixture Ethyl acetate, η-hexane and 2N ammonia in a volume ratio of 4: 1: 1; f) in the UV absorptiunnspekiruui shoulders at 235 results in up to 250 ηιμ, 295 ΐημ and 303 to 308 πιμ; g) maximum in the IR absorption spectrum in Nujol Absorption bands at the following wavelengths result: 3340, 3330, 1735, 1655, 1585, 1530. 1490, 1420, 1345, 1305.! 250. 1210, 1160, 1110. 1090, 1065, 1025, 1000, 975, 950, 920, 895, 880, 860,840,768,725,705,680 and 660 cm- '; h) soluble in dioxane, dimethyl sulfoxide, dimethylformamide. Pyridine and chloroform; moderate soluble in methanol, acetone and ethyl acetate: and insoluble in ether. Benzene. Petroleum ether and Water is; i) the hydrolysis with 6 ρ hydrochloric acid at 110 ^° C. for 24 hours based on 2 moles of alanine, 0.9 t moles of threonine, 1.03 moles of valine and 037 moles of cysteine; j) is stable at 60 ° C for one hour at a pH range of 2.0 to 8.0.