CS196263B2 - Fodder mixture for animals - Google Patents

Description

The invention relates to animal feed or beverage, or premixes containing an antibiotic.

In the literature on antibiotics, synergism is widely described:

The Journal of Anfibiotics 25, No. 6, 371 (1972); J. Chem. Soc. 19C, 1653 (1966); Bull. Soc.Chim. Belg. 68, 716 (1959); J. Amer. Chem. Soc. 82, 4414 (1960); Tetrahedron Letters 2687 (1971); J. Anfibiotics, Ser. A 14 (1961); Nature 187, 598 (1960); J. Chem. Soc. 2286 (1960); Antimicrobial Agents and Chemotherapy 360-365 (1964); Tetrahedron Letters 4231-4238 (1966); Organic Mass Spectrometry, 6: 151-166 (1972); Tetrahedron Letters 369-372 (1966) and J. Chem. Soc. 190, 1669-1766 (1966).

The novel synergistic antibiotic mixtures prepared according to the invention are coupled to other synergistic mixtures already described: micamycin, pristinamycin, ostrogrycin, streptogramin, P.A. 114, vernamycin and virginiamycin.

The invention relates to a compound feed (including beverages and premixtures) for animals containing conventional feed components. The subject of the invention is that such a feed composition comprises 10 to 100 ppm of antibiotics consisting of macrocyclic lactones 36 926 and 37 277 or a depsipeptide 37 952, obtained by culturing Actinoplanes auraticolor ATCC 31 011 micro2, or mixtures thereof.

The properties of the above macrocyclic lactones and depsipeptides are described in detail in the description of the invention. No. 5,638,949; 196 262 of the same applicant; there is also described in detail a process for the cultivation of said substances, which is carried out by culturing the microorganism Actinoplanes auranticolor, preferably in an aqueous medium at temperatures from 28 to 36 ° C. Such cultivation provides a mixture of compounds, the major components being referred to as compounds 37 277 (depsipeptides and 36 926 (macrocyclic lactone) and minor components as compounds 37 932 (depsipeptides and 35 763 (macrocyclic lactone)) These mixtures containing macrocyclic lactones and depsipeptides may be Separate and recovered from the fermentation broth by solvent extraction, countercurrent separation, column chromatography or a combination thereof The individual antibiotic components have significant antibiotic activity The crude antibiotic mixture or the combination of macrocyclic lactone and pure depsipeptide obtained from the crude mixture exhibit considerable synergistic antibiotic activity. , pure antibiotic components and mixtures of pure macrocyclic lactones and depsipeptides are

198263 potent growth promoters of chickens and pigs.

'Attached Figs. ¹ to 4 show the infra-i vena spectra of compounds 37277, 36926, 35763' and 37 932. These spectra are used for identification of these compounds. ·

Essential components of the - antibiotic compositions produced by Actinoplanes auranticolor ATCC 31011 include various macrocyclic lactones and depsipeptide antibiotic components. The composition of these antibiotic mixtures varies from fermentation to fermentation and is a function of time, pH, environmental composition, etc. Under the conditions set forth in the examples, the major antibiotic components in the antibiotic mixtures are 37 277 (depsipeptide) and 36 926 (macrocyclic lactone). minor antlbiotic components are compounds 37,932 '(depsipeptide) and 35,763 (macrocyclic lactone),

The compounds can be separated from the antibiotic mixtures and recovered from the fermented broth by a variety of methods such as solvent extraction, Crig countercurrent extraction, column chromatography, or a combination of these methods. Various organic solvents, such as chloroform, ethyl acetate and methyl isobutyl ketone, are useful for extraction of alpha-antibiotics from the culture medium. Solvent extraction is preferably carried out by twice extracting the culture medium at pH 7, with a volume of solvent of approximately V3 to V4 from which a mixture of antibiotics should be obtained. Depending on the volume of the broth, extraction devices such as separating funnels, mixing tanks and mechanical extraction devices such as centrifuges are useful for extraction purposes ^. A preferred method of separating and recovering the ingredients from the antibiotic compositions is as follows: the whole or clarified broth is adjusted to pH 7 and extracted twice with 1/3 to V2 volumes of methyl isobutyl ketone. The extract was concentrated in vacuo and the concentrate was defatted with heptane or petroleum ether. The defatted concentrate is then evaporated to dryness under vacuum. The solid residue was subjected to Craig countercurrent separation (6 levels) using toluene, 5 parts: ethanol, 2 parts: aqueous phosphate buffer, pH 4.5, 3 parts. After separating the layers into the upper and lower phases in counter current. divider. of the sequence is by thin-layer chromatography. The separated fractions are evaporated in vacuo.

The solids containing the dipipeptides were dissolved in chloroform, decolorized with charcoal and evaporated to dryness in vacuo. The residue obtained by evaporation of chloroform was dissolved in acetone. The solid precipitated by the addition of heptane was dissolved in a small amount of chloroform and applied to a column of buffered silica gel (pH 6) in chloroform: n-propanol (99: 1 v / v). The column is developed in the same solvent mixture under a pressure of 0.55 MPa. The individual fractions collected are chromatographed on a thin layer. The fractions containing the separated depsipeptides were combined, evaporated in vacuo and crystallized from acetone-heptam.

The countercurrent extraction fractions containing the macrocyclic lactones are combined, evaporated in vacuo and the residue taken up in ethyl acetate. Stir the solution with silica gel, filter and remove the solvent in vacuo. The residue is taken up again with ethyl acetate and precipitated by addition of hexane. The solids were dissolved in a small amount of chloroform and chromatographed under 0.55 MPa pressure on a puffed silica gel column (pH 6) in ethyl acetate; The evolving mixture has the composition ethyl acetate - tetrahydrofuran - hexane (80:

: 20: 20) and is saturated with aqueous phosphate buffer (pH 6). Separate fractions were monitored by thin layer chromatography. The fractions containing the separated macrocyclic lactones are pooled and evaporated under vacuum. The individual fractions are separated by further Craig countercurrent separation and / or column chromatography using different developing systems. By careful identification in each purification operation, it is ensured that the individual macrocyclic lactones are sufficiently isolated so that the liquid fractions can be evaporated to obtain pure compounds. ATCC 310100 produces at least four depsipeptides and at least four macrocyclic lactones. However, the primary components are depsipeptides: compounds number 37 277 (major) and 37 932 (minor), and macrocyclic lactones: compounds 36 926 (major) and 35 763 (minor).

The crude antibiotic mixtures directly obtained from the culture medium and the purified individual components have broad 'antibacterial' spectra. As microorganisms which cease to deplete in the presence of these antibiotics, Salmonella typhosa, Shigella dysenteriae, E-scherichia coli, Klebsiella, pneumoniae, Staphylococcus aurus, Streptococcus pyogenes, Streptococcus fecalis, Diplococus pneumoniaebacterium, Bacillus pneumoniae, Bacillus. Dipteriaej Clostridium septicum, Brucella abortus, Neisseria sicca, Lactobacillus acidbphilus and Pasteurella multocida.

Antibiotics prepared according to the invention, either as a crude mixture or in a form. The purified individual components or mixtures thereof can be used to combat various infections in humans and animals. In general, for these antibiotics, administration at daily oral doses of 0.5-1 g, or parenteral doses of from 0.5 to 1 g, is most appropriate. 100 to 500 mg depending on the type and severity of the infection and the weight of the organism being treated:

The compounds of the invention may be administered alone or in combination with pharmaceutically usable medicaments, and thus may be administered in single or multiple doses. .

Tablets for oral administration contain various fillers, such as sodium citrate, calcium carbonate and dicalcium phosphate, and various disintegrants such as starch, alginic acid and complex silicates may be added together, such as; for example (polyvinylpyrrolidone) sucrose, gelatin and gum arabic. Feeding agents are often used; such as magnesium stearate, sodium lauryl sulfonic acid, and talc for tabletting agents. Solid form preparations of a similar type may be used as fillers in soft and hard-filled gelatin. capsules; suitable materials are lactose and also high molecular weight. poles ethy lenglykply. Aqueous suspensions and / or elixirs are suitable for oral administration; their base component - may be combined with various sweeteners or instant substances, as well as 5 diluents such as water, ethanol, propylene glycol, glycerol and mixtures thereof.

Solutions of these antibiotics in sesame and

Peanut oil or in aqueous. The pylenglycol can be used for parental administration.

. Interestingly, the individual antibiotics of the present invention exhibit an antimicrohial effect which is largely bacteriostatic in nature. However, crude antibiotic mixtures or mixtures of purified dipeptides and purified macrocyclic lactones exhibit. it has a synergistic effect which is predominantly of a bactericidal nature.

Testing of the major macrocyclic lactones, compound 36 926 (Aj and major dépsipeptide, compound 37 277 (Being individually and in a mixture by dilution method in tubes against the following microorganisms ₎ showed a minimum inhibitory concentration in micrograms / ml / MIC:

Microorganism

Staph. aureus 005 1.56

Staph. aureus · 400 3.12

Strept. faecalis.>. 100

Neisseria siccas. 0.39

Treponemáhyodysenteriae '

Shard, pyogenes 0,39

Bacteriodes fragilis 35 613 25

Clostridium inocuum 6.25

Lactobacillus easei var. casei 3,12

B A + B Crude mixture

25 0,10> <0.10 - 12.5 0,78> <0.10 '12.5 1.56 0.20 <: 50 0.10 <0.10 - , - ~ 0.19 12.5 0.10. <0.10 50 0.73 0.78 > 100 0.20 0.39 6.25 · 0.20. ·· > 0.39

Comparable results were obtained when testing a minor macrocyclic lacquer; Compound 37 9.32 (B '), individually or at appropriate concentrations, i.e., A' =, B 'Φ Φ or, (A' B '). J Φ (A '+. B' j 'Φ • Φ (A + B) Φ' (A, + 'B);

Maximum syhergic efficacy of mixtures of pure macrocyclic lactones and depsipeptides is achieved; their ratio of about 1 to 2: 1; · Approximately such a ratio of ingredients occurs in vyf fermented nutrients / soils

A. auranticolor ATCC 31011 and in crude antibiotic mixtures isolated therefrom. This finding is in contradiction with the other described synergistic compositions where the synergistic factors appear in the fermented soils and crude mixtures in the suboptimal sponge. _ ·· '' ·, · ’'--y O * ·'

In vivo data on the protective effect achieved with oral and oral administration in mice experimentally infected with the strain Stahpylococcus aureus are given in Table I. ', ...... ·

TABLE I

PD 50 values (mg / kg) -S. aureus 01A005. Orally Subcutaneously

Compound 36 996

Compound 37 277

Compound 36,926 + 37,277 (1: 1)

Crude antibiotic mixture

Antibiotics made according to the present invention may be considered to be of particular interest as growth factors in poultry and animals due to their broad antibacterial spectrum, and further for the treatment of pig dysentery due to the remarkable effect against Treponema hyodysenteriae which is the anaerobic spirochete that causes this disease. .

· '- *> 200> 200> 200> 200

210 '60 150 to 200' / ‘. The effect on growth stimulation of crude antibiotic mixtures was investigated in young feed piglets in a 40-day experiment. Mean daily increment, feed consumption and feed efficiency improved significantly (p <<0.01) compared to no antibiotic control (Table II).

198143. β ' ^1.

TABLE II

Group

Average daily increment (kg)

Average daily feed consumption (kg)

Feed efficiency *) control without antibiotic 0.35 0.91 2.57 crude antibiotic mixture ppm 0.63 1.35 2.15 *) wt. parts feed on mass. part of weight gain

Comparable results can be obtained with other antibiotic compositions in the range of 10 to 100 ppm.

Comparable results can also be obtained by administering individual pure compounds 36,926, 37,932, and 37,277, or mixtures of these pure compounds, which are close to those of the crude antibiotic compositions.

The effect on stimulating the growth of livestock was demonstrated in hens in a battery experiment. Significant improvement (p <<0.1) in weight gain over control without antibiotic was observed on antibiotic diet. (Table III

TABLE III

Group

Average weight gain (g)

Average feed consumption (g)

Feed efficiency control without antibiotic 566 crude antibiotic mixture ppm 608

Comparable results can also be obtained with other crude antibiotic compositions above the range of 10 to 100 ppm.

Comparable results may also be obtained by administering single pure compounds 36,926, 37,932, and 37,277 or mixtures of pure compounds that approximate the composition of the crude antibiotic compositions.

The prophylactic effect of the antibiotics produced according to the invention was determined to be

939 1.66

949 1.56 pigs experimentally infected with infectious material causing pig dysenteria. Colon content and mucosal digestions were obtained from an epizootic of clinically diagnosed swine dysentery. Healthy pigs were infected with this material by direct vaccination. Feed containing antibiotic was administered for 28 days. The results are summarized in Table IV.

Group

TABLE IV

Morbidity Mortality rate (° / o) (° / o)

Average daily increment (kg) control 100 crude antibiotic mixture ppm 0

37.5 ppm; 0.25.0 ppm 0. 12.5 ppm 0

6.25 ppm 50

Comparable results can be obtained at doses above the dose range of 10 to 100 ppm 1 for other antibiotic mixtures with individual pure compounds 36,926, 37,932, and 37,277 or mixtures of crude antibiotic mixtures.

—0.13

0.66

0.57

0.68

0.61

0.47

Example 1

The antibiotic premlx to be used in the compound feed to increase non-ruminant food can be produced by the following formula: ·

190283

Ingredients: Maize percentage 78.40 Soya flour, 49% 18.50 Calcium phosphate 1.50 Limestone, 0.50 Iodide salt 0.50 Vitamin premix ^(a ' ^b) 0.50 Trace element premix ^(b) (a) Corresponds to the following vitamin levels in a ratio of 453 g; Vitamin A 2000 m.

Vitamin D 200 IU; Vitamin E 5 IU; vitamin Κ. 1 mg; niacin 12 mg; rlboflavln

1.5 mg; panthothenic acid 7 mg; choline chloride 500 mg; Vitamin B12 10 / tg.

(b) Corresponds to the following concentrations of trace elements in proportions to 1 million: manganese 120, iron 40, copper 4, iodine 2,4, cobalt 0,4, zinc 100.

The amount of antibiotic contained in each 0.5 kg can be varied as desired. A preferred antibiotic premix comprises 22.6 g of the antibiotic

SUBJECT

Animal feed composition containing the usual components of feeds comprising 10 to 100 ppm of antibiotic macrocyclic lactones of 36,926 and per kg, which may contain an antibiotic composition comprising the compound 35,763, 36,926, 37,277 and 37,932 produced fermentation with Actinoplanes auranticolor ATCC 31011, or compound 37 277 or compound 36 926 or compound 37 932. 0.5 kg of premix per 1000 kg of complete dose provides a final antibiotic concentration of 11 ppm.

Claims (1)

A liquid antibiotic premix concentrate suitable for addition to drinking water to enhance the feed efficiency of non-ruminants of the desired concentration can be prepared by dissolving an antibiotic mixture containing 35,763, 36,926, 37,277 and 37,932 produced under fermentation conditions by Actinoplanes auranticolor ATCC 31011 or 37 277 or compound 36 926 or compound 37 932. VYNALEZU 37 277 or a depsipeptide 37 932, obtained by culturing Actinoplanes auranticolor ATCC 31 011, or mixtures thereof.