CH621687A5 - Process for producing the novel antibiotic A-28086 complex and its use for increasing the feed intake of ruminants - Google Patents

Description

The invention relates to a method for producing the new antibiotic A-28086 complex, from which the factors A, B and D are derived. The antibiotic compounds are interesting antibacterial, anti-fungal, antiviral, anticoccidial, insecticide and acaricide agents as well as agents against pleuropneumia-like organisms and agents for improving feed utilization in ruminants. The new antibiotic A-28086 complex as such, the factors A and D as well as the physiologically acceptable salts thereof and the C2-C0-acyl esters can be used to increase the feed intake in ruminants.

According to the invention there is provided a process for the preparation of the new antibiotic A-28086 complex with the factors A, B and D, which consists in that Streptomyces aureofaciens NRRL 5758 or Streptomyces aureofaciens NRRL 8092 in a culture medium, the assimilable sources of carbohydrate, nitrogen and also contains inorganic salts, submersed under aerobic fermentation conditions until these organisms have produced a substantial amount of antibiotic activity in the culture medium and separate the Antibioticum A-28086 complex from the culture medium.

After recrystallization from acetone water, the antibiotic A-28086 factor A is a white crystalline compound which dissolves in lower alcohols, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but is only slightly soluble in hexane and dissolves in

Water does not dissolve, melts at around 98 to 100 ° C, solidifies again and then melts again at around 195 to 200 ° C and has the following additional properties:

a) a molecular weight of 764, determined by spectrometry 5,

b) an approximate elemental composition of 66.69% carbon, 9.85% hydrogen and 23.10% oxygen,

c) an empirical formula Cj! 3Hr, On, determined by mass io spectrometry,

d) a specific rotation of -54 ° (c = 0.2, methanol) measured at 25 ° C,

e) an infrared absorption spectrum in chloroform with the following distinguishable absorption maxima: 2.85, 3.34,

is 5.83, 6.82, 7.22, 7.53 (weak), 7.78 (weak), 8.75 (strong), 8.95 (strong), 9.15, 9.50 (strong ), 9.55 (strong), 9.60, 9.85, 10.15, 10.45 and 10.70 (weak) microns,

f) only a final absorption at less than 200 m [j, in the ultraviolet spectrum in ethanol,

20 g) a nuclear magnetic resonance spectrum in deuterochloroform with the following properties: 5 6.01, 4.21, 4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3.57, 3 , 55, 2.83, 2.76, 2.74, 2.68, 2.66, 2.58, 2.56, 2.30, 2.22, 2.17, 2.10, 2.05 , 1.96, 1.90, 1.85, 1.70, 1.62, 1.60, 1.47, 1.39, 1.31, 1.25, 1.18.0.95, 0 , 93.0.90, 25 0.88, 0.85, 0.77, 0.75, 0.73, 0.68 and 0.66 ppm,

h) a titratable group with a pKa of 7.9 in 80 percent aqueous dimethylformamide,

i) a characteristic X-ray diffraction spectrum for the powder (Cu ++ radiation, 1.5405 X, nickel filter) with the following

30 interplanar distances in Angström (d):

d

Relative intensity

12.00

100

10.10

50

9.25

90

8.00

40

7.50

15

6.92

90

6.40

40

5.98

05

5.68

15

5.20

40

4.98

40

4.62

40

4.21

20th

3.48

10th

j) an Rr value of 0.24 in thin-layer chromatography over silica gel using a 3: 2 solvent system composed of benzene-ethyl acetate and Bacillus subtilis 65 ATCC 6633 as detection organism,

k) the following Rf values in the paper chromatographic systems specified below using Bacillus subtilis ATCC 6633 as the detection organism:

3rd

621687

R values

Solvent system

Rf values

Solvent system

0.11 Water saturated with methyl isobutyl ketone (MIBK)

0.41 Water saturated with MIBK plus 2% p-toluenesulfonic acid and 1% piperidine

0.54 water: methanol: acetone (12: 3: 1)

adjusted to pH 10.5 with NH4OH and then reduced to pH 7.5 with H3P04

0.48 1% MIBK, 0.5% NH4OH in water

0.15 17.4 g K2HP04, 30 ml ethanol per liter

water

0.24 Benzene saturated with water

0.24 water

0.75 water: MIBK: ethyl acetate (98: 1: 1)

10th

15

0.16 Water saturated with MIKB plus 2% p-toluenesulfonic acid and 1% piperidine

0.46 water: methanol: acetone (12: 3: 1)

adjusted to pH 10.5 with NH4OH and then reduced to pH 7.5 with H3P04

0.36 1% MIBK, 0.5% NH4OH in water

0.51 Benzene saturated with water

0.11 water

0.61 water: MIBK: ethyl acetate (98: 1: 1)

1) an acid function capable of forming salts and ester derivatives and m) at least one esterifiable hydroxyl group,

or the above antibiotic is in the form of its C2-Cu acyl ester derivatives and physiologically acceptable salts.

After recrystallization from acetone water, the antibiotic A-28086 factor B is a white crystalline compound that dissolves in lower alcohols, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but is only slightly soluble in hexane and does not dissolve in water solves and has the following additional properties:

a) a melting point of about 150-153 ° C,

b) a molecular weight of 762, determined by mass spectrometry with high resolution,

c) an empirical formula C43H70On, determined by high resolution mass spectrometry,

d) an infrared absorption spectrum in chloroform with the following distinguishable absorption maxima: 2.82, 3.30, 5.77, 5.85, 6.80, 7.20, 7.50 (weak), 7.72 (weak), 7 , 80 (weak), 8.57 (strong), 8.68, 8.90 (strong), 9.10, 9.50, 9.83 (strong), 9.90, 10.10, 10.17 (strong), 10.43 (weak), 10.80 (weak), 11.20 (weak), 11.35 (weak), 11.73 (weak) and 12.03 (weak) microns,

e) an absorption maximum in ethanol in the ultraviolet spectrum at 220 mu (E = 137.5, e = - 10 477),

f) a nuclear magnetic resonance spectrum in deuterochloroform with the following properties: 5 7.20, 7.09, 6.26, 6.15, 4.19, 4.12, 4.05, 3.95, 3.89, 3, 78, 3.62, 3.59, 3.52, 3.48, 2.81, 2.73, 2.63, 2.54, 2.52, 1.99, 1.91, 1.84, 1.71, 1.67, 1.64, 1.55, 1.43, 1.33, 1.18, 1.11, 0.96, 0.94, 0.90, 0.87, 0, 84, 0.77, 0.74 and 0.68 ppm,

g) an Rr value of 0.42 in a thin layer chromatogram over silica gel using a 3: 2 mixture of benzene and ethyl acetate and Bacillus subtilis ATCC 6633 as detection organism,

h) in the paper chromatographic systems specified below using Bacillus subtilis ATCC 6633 as the detection organism the following Rr values:

i) one used for the formation of salts and ester derivatives

20 capable acid function,

j) two keto functions and k) at least one hydroxyl group,

or the above antibiotic is in the form of its physiologically acceptable salts.

25 Antibiotic A-28086 factor D after recrystallization from acetone water is a white crystalline compound that is soluble in methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but only weakly dissolves in hexane and in

30 water is insoluble, melts at about 96 to 98 ° C and has the following additional properties:

a) a molecular weight of 778, determined by high-resolution mass spectrometry,

b) an approximate elementary composition of

35 67.59% carbon, 9.38% hydrogen and 22.77% oxygen,

c) an empirical formula C ^ H ^ O,! determined by high resolution mass spectrometry,

d) a specific rotation of -56 ° (c = 0.1, metha-

40 noi), determined at 25 ° C,

e) an infrared absorption spectrum in chloroform with the following observable absorption maxima: 2.89, 3.39, 3.43, 3.50, 5.88, 6.90, 7.27, 7.60, 7.84, 9.00 , 9.26, 9.62, 10.31, 10.58, 11.10 and 11.49 microns,

45 f) no observable ultraviolet absorption in 95 percent aqueous ethanol,

g) in chloroform, a magnetic resonance spectrum with the following properties: 6.00, 4.20, 4.10, 4.00, 3.98, 3.92, 3.86, 3.83, 3.79, 3 , 67, 3.64, 3.57, 3.54, 2.88, 2.81,

50 2.71, 2.62, 2.58, 2.48, 2.43, 2.37, 2.29, 2.21, 2.15, 2.10, 2.04, 1.97, 1 , 89, 1.83, 1.76, 1.68, 1.61, 1.58, 1.55, 1.47, 1.39, 1.30, 1.25, 1.18, 0.95 , 0.90, 0.88, 0.84, 0.74 and 0.68 ppm,

h) in 80% aqueous dimethylformamide, a titratable group with a pK. value of 8.67,

55 i) as a powder, the following characteristic X-ray diffraction spectrum (Cu ++ radiation, 1.5405 X, nickel filter) with the following interplanar distances in angstroms (d):

60

65

621687

4th

d Relative intensity

12.40

100

10.20

70

8.85

90

7.80

30th

6.80

10th

6.30

100

5.70

20th

5.35

20th

5.10

20th

4.90

10th

4.65

20th

4.45

40

4.20

30th

3.30

10th

3.15

10th

2.99

05

2.77

05

2.28

05

j) in thin-layer chromatography systems on silica gel using Bacillus subtilis ATCC 6633 as the detection organism, the following Rt values:

Rr values solvent system

0.26 benzene-ethyl acetate (3: 2)

0.66 ethyl acetate-diethylamine (95: 5)

k) in paper chromatography systems using Bacillus subtilis ATCC 6633 as the detection organism, the following Rr values:

Rt values solvent system

1) an acid function capable of forming salts and ester derivatives and m) at least one esterifiable hydroxyl group,

or the above antibiotic is in the form of its C2-C6-acyl-5 esters and physiologically acceptable salts.

A number of antibacterial agents already exist, but there is still a need for new and better antibiotics. A problem with the current antibiotic therapy is the fact that antibiotics differ in their effectiveness against pathogenic organisms. Another problem is the development of organism strains that are resistant to standard antibiotics. Finally, another problem is the fact that individual patients often suffer from severe reactions to 15 specific antibiotics due to hypersensitivity and / or toxic effects. Because of these problems in current therapy, there is still a need for new antibiotics.

In addition to new antibiotics for the treatment of human diseases, better antibiotics are also needed in veterinary medicine. An important aspect here is the need for better antibiotics to promote the growth of poultry and pets. Such growth is promoted, for example, by reducing disease and improving feed utilization.

A well-known disease of economic importance in veterinary medicine, particularly in the poultry industry, is protocoenal coccidiosis. Coccidiosis occurs when infected with one or more species of Eimeria or Isospora (for a summary, see Lund and Farr in Diseases of Poultry, 5th Edition, Biester and Schwarte, Eds., Iowa State University Press, Ames, la., 1965, pages 35 1056-1096. Because of the heavy economic losses due to coccidiosis and because of the disadvantages of some known anticoccidial agents, better anticoccidial agents are still being sought.

Another disease in animals is enteritis, 40 which can also cause serious economic losses for animal breeders. Enteritis occurs in chickens, pigs, cattle and sheep and is mainly due to anaerobic bacteria, especially Clostridium perfringens, and viruses. Entero-45 toxemia in ruminants, an example of which is overeating in sheep, is a condition caused by infection with C. perfringens.

Promoting the growth of ruminants such as cattle is another economically interesting goal of veterinary medicine. Of particular interest is promoting growth, which can be achieved by improving feed conversion. The mechanism for utilizing the predominant nutritional portion (carbohydrates) of ruminant feed is known. Microorganisms present in the rumen 55 of the animals break down carbohydrates to form monosaccharides, and these monosaccharides are then converted into pyruvate compounds. The pyruvates are metabo-60 Iized by microbiological processes to form acetates, butyrates or propionates, which are collectively known as volatile fatty acids (VFA). For a more detailed discussion, see Leng "Physiology of Digestion and Metabolism in the Ruminant", Phillipson et al. Eds., Oriel Press, Newcastle-upon-Tyne, England (1970) pages 408-410. 65 The relative efficiency of VFA utilization is described by McCullough in Feedstuffs, June 18, 1971, page 19, Eskeland et al. in J. An. Be. 33, 282 (1971) and Church et al. in "Digestive Physiology and Nutrition of Ruminants", Volume 2

0.10

With methyl isobutyl ketone (MIBK)

saturated water

0.26

With MIBK plus 2% p-toluenesulfonic acid

and 1% piperidine saturated water

0.36

Water: methanol: acetone (12: 3: 1)

adjusted to pH 10.5 with NH4OH

and then with H, PO, to pH 7.5

degraded

0.29

1% MIBK, 0.5% NH4OH in water

0.25

17.4 g K2HP04, 30 ml ethanol per liter

water

0.26

Benzene saturated with water

0.09

water

0.64

Water: MIBK: ethyl acetate (98: 1: 1)

5

621687

(1971), pages 622 and 625. While acetates and butyrates are also used, the propionates are used far more efficiently. In addition, the animals can develop a ketosis if too little propionate is available. A correspondingly favorable compound therefore stimulates the animals to form a higher proportion of propionates from carbohydrates, which improves carbohydrate utilization and reduces the amount of ketosis.

Antibiotic A-28086 factors A, B and D belongs to a new member of a group of polyether antibiotics. Examples of representatives from this group include Monensin (US Pat. No. 3,501,568), Dianemycin [R. L. Hamill, M. M. Hoehn, G. E. Pittenger, J. Chamberlin and M. Gorman, J. Antiniotics 22, 161 (1969)], Nigericin [L. K. Steinrauf, Mary Pinkerton and J. W. Chamberlin, Biochem. Biophys. Res. Comm. 33,29 (1968)] and Salino-mycin [JA-PS 47-25392, application number 19620/1971, Derwent No. 76960T, US-PS 3 857 948, and H. Kinashi, N. Otake, H. Yonehara, S Sato and Y. Saito, Tetrahedron Lett. 49, 4955-4958 (1973)].

The indication of antibiotic complex, as used in the fermentation chemistry and also in the present description, does not refer to a chemical complex, but to a mixture of individual antibiotic factors formed at the same time. The ratio of the individual factors formed in an antibiotic complex fluctuates, as is known to the person skilled in the art with the formation of antibiotics by fermentation, depending on the fermentation conditions used in each case.

The Antibioticum A-28086 complex is formed by cultivating the new strain Streptomyces aureofaciens NRRL 5758 submerged under aerobic fermentation conditions until a substantial amount of antibiotic activity has developed. Antibiotic complex A-28086 is also formed by growing another new strain, Streptomyces aureofaciens NRRL 8092. The antibiotic complex produced either by S. aureofaciens NRRL 5758 or by S. aureofaciens NRRL 8092 is preferably extracted from the fermentation broth and the mycelium by polar organic solvents. The extracted antibiotic mixture can be obtained by concentrating the solvents, adding the concentrates to excess petroleum ether to precipitate impurities, filtering off and evaporating the filtrates, thereby obtaining a mixture of the antibiotic A-28086. The antibiotic mixture is then usually further purified and can be separated into the individual factors by column chromatography.

The A-28086 compounds inhibit the growth of animal and plant-pathogenic organisms. This inhibitory effect is based in part on the fact that the compounds A-28086 have an anticoccidial effect. The compounds A-28086 are also antibacterial, antiviral, active against PPLO (pleuropneumonia-like organisms), insecticid and acaricid and increase feed utilization in ruminants.

The following infrared absorption spectra in chloroform are shown in the attached drawing:

Figure 1 Antibiotic A-28086 factor A.

Figure 2 Antibiotic A-28086 factor B

Fig. 3 Antibiotic A-28086 factor A acetylester derivative

Figure 4 Antibiotic A-28086 factor A propionyl ester derivative

Fig. 5 Antibiotic A-28086 factor A butyryl ester derivative

Figure 6 Antibiotic A-28086 factor A valerylester derivative

Figure 7 Antibiotic A-28086 factor A caproyl ester derivative

Fig. 8 Antibiotic A-28086 factor D.

The A-28086 factors are structurally related. At least four antibiotic factors are formed simultaneously during fermentation and are obtained in the form of a mixture. The factors are separated from one another, whereby the factors A, B and D can be isolated as individual compounds, as described below. The mixture of the factors of A-28086 is soluble in most organic solvents, but insoluble in water.

The following describes the physical and spectral properties of A-28086 factors A, B and D:

Antibiotic A-28086 factor A crystallizes from acetone water. Factor A of antibiotic A-28086 melts at about 98-100 ° C, whereupon it solidifies again and then melts again at about 195-200 ° C. The elementary analysis of factor A shows the following average percentage composition: carbon 66.69%, hydrogen 9.85%, oxygen 23.10%.

C43H72On is suggested for factor A as an empirical formula.

Factor A has an apparent molecular weight of 764 as determined by mass spectrometry.

The infrared spectrum of factor A in chloroform is shown in Fig. 1 of the accompanying drawing. The following absorption maxima are obtained: 2.85, 3.34, 5.83, 6.82, 7.22, 7.53 (weak), 7.78 (weak), 8.75 (strong), 8.95 (strong), 9.15, 9.50 (strong), 9.55 (strong), 9.60, 9.85, 10.15, 10.45 and 10.70 (weak) microns.

The ultraviolet spectrum of factor A in ethanol only shows an end absorption below 220 mn "

The nuclear magnetic resonance spectrum of A-28086 factor A in deuterochloroform shows the following characteristics: 5 6.01, 4.21, 4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3, 57, 3.55, 2.83, 2.76, 2.74, 2.68, 2.66, 2.58, 2.56, 2.30, 2.22, 2.17, 2.10, 2.05, 1.96, 1.90, 1.85, 1.70, 1.62, 1.60, 1.47, 1.39, 1.31, 1.25, 1.18, 0, 95, 0.93, 0.90, 0.88, 0.85, 0.77 0.75, 0.73, 0.68 and 0.66 ppm.

Antibiotic A-28086 factor A recrystallized from acetone water gives the following characteristic X-ray diffraction spectrum as powder (Cu ++ radiation, 1.5405 X, nickel filter, d = interplanar distance in angstroms):

d

Relative intensity

12.00

100

10.10

50

9.25

90

8.00

40

7.50

15

6.92

90

6.40

40

5.98

05

5.68

15

5.20

40

4.98

40

4.62

40

4.21

20th

3.48

10th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

621687

6

The specific rotation of the antibiotic A-28086 factor A is -54 ° (c = 0.2, methanol), measured at a temperature of 25 ° C. This specific rotation is an average based on several determinations.

Electrometric titration of factor A in 80 percent aqueous dimethylformamide shows the presence of a titratable group with a pKa of 7.9.

The antibiotic A-28086 factor A dissolves in a number of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but is only slightly soluble in non-polar organic solvents such as hexane and insoluble in water.

The antibiotic A-28086 factor A has an acid function capable of forming salts and ester derivatives and at least one esterifiable hydroxyl group.

On the basis of the physical properties described above, a structure can be proposed for the antibiotic A-28086 factor A. After the structure determination is only postulated, however, it is pointed out 20 that this structural proposal is only a working hypothesis. The antibiotic A-28086 factor A should therefore have the structure of the following formula I:

10th

15

HCH

Formula I.

chs

Antibiotic A-28086 factor B is a white crystalline compound (from acetone water) that melts at around 150 to 153 ° C.

The factor B has an apparent molecular weight of 762 due to a high-resolution mass spectrometric determination, and the empirical formula C ^ H ^ On is proposed for this.

The infrared spectrum of factor B in chloroform is shown in Fig. II of the accompanying drawing. The following absorption maxima can be determined: 2.82, 3.30, 5.77, 5.85, 6.80, 7.20, 7.50 (weak), 7.72 (weak), 7.80 (weak ), 8.57 (strong), 8.68, 8.90 (strong), 9.10, 9.50, 9.83 (strong), 9.90, 10.10, 10.17 (strong), 10.43 (weak), 10.80 (weak), 11.20 (weak), 11.35 (weak), 11.73 (weak) and 12.03 (weak) microns.

The ultraviolet spectrum of factor B in ethanol shows an absorption maximum at 220 mp, (E = 137.5, e = 10 477).

The nuclear magnetic resonance spectrum of A-28086 of factor B in deuterochloroform gives the following characteristics: S 7, 20, 7, 9, 6.26, 6.15, 4.19, 4.12, 4.05, 3.95, 3 , 89, 3.78, 3.62, 3.59, 3.52, 3.48, 2.81, 2.73, 2.63, 2.54, 2.52, 1.99, 1.91 , 1.84, 1.71, 1.67, 1.64, 1.55, 1.43, 1.33, 1.18, 1.11, 0.96, 0.94, 0.90, 0 , 87, 0.84, 0.77, 0.74 and 0.68 ppm.

Antibiotic A-28086 Factor B is soluble in a range of 45 organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but it is only slightly soluble in non-polar organic solvents such as hexane and is in Water insoluble.

50 Although the chemical structure of antibiotic A-28086 factor B has not yet been elucidated, the available physical data indicate that factor B has a single carboxylic acid group, two keto groups and one or more hydroxyl groups.

55 When manufactured via S. aureofaciens NRRL 5758, the antibiotic A-28086 factor D is formed as a lower factor. However, if it is produced via S. aureofaciens NRRL 8092, then the antibiotic A-28086 factor D is present in an amount of up to 10%, based on the antibiotic activity obtained in 60.

Antibiotic A-28086 factor D is a white crystalline material (from water acetone) with a melting point of around 96-98 ° C. A-28086 Factor D has an apparent molecular weight of 778 as determined by 65 high resolution mass spectrometry.

The elementary composition of the maximum in the mass spectrum of the sodium salt of A-28086 factor D gives a value of 800 5050 (calculated for C44H73OuNa

7

621687

= 800 5050). In the mass spectrum, the free acid of A-28086 factor D shows a small maximum at 778 and a larger maximum at 760 5117 (calculated for C44H72O10 = 760 5125). The value m / e of 760 obtained in the mass spectrum of the free acid stems from the water loss of the molecular ion. The molecular ion composition of the free acid of the antibiotic A-28086 factor D is therefore C ^ H ^ Ou-

The empirical formula C44H740h is therefore proposed for A-28086 factor D. The elementary analysis of factor D shows the following percentage composition: carbon 67.59%, hydrogen 9.38%, oxygen 22.77%.

The theoretical percentage composition for C44H740h is as follows: carbon 67.87%, hydrogen 9.51%, oxygen 22.77%.

The infrared absorption spectrum of A-28086 factor D (FIG. 8) contains the following observable absorption maxima: 2.89, 3.39, 3.43, 3.50, 5.88, 6.90, 7.27, 7.60, 7.84, 9.00, 9.26, 9.62, 10.31, 10.58, 11.10 and 11.49 microns.

Antibiotic A-28086 factor D shows no ultraviolet absorption in 95 percent aqueous ethanol.

The nuclear magnetic resonance spectrum of the antibiotic A-28086 factor D in deuterochloroform gives the following characteristics: 5 6.00, 4.20.4.10, 4.00, 3.98, 3.92, 3.86, 3.83, 3 , 79, 3.67, 3.64, 3.57, 3.54, 2.88, 2.81, 2.71, 2.62, 2.58, 2.48, 2.43, 2.37 , 2.29, 2.21, 2.15, 2.10, 2.04, 1.97, 1.89, 1.83, 1.76, 1.68, 1.61, 1.58, 1 , 55, 1.47, 1.39, 1.30, 1.25, 1.18, 0.95, 0.90, 0.88, 0.84, 0.74 and 0.68 ppm.

The antibiotic A-28086 factor D after recrystallization from acetone water in powder form gives the following characteristic X-ray diffraction spectrum (Cu ++ radiation, 1.5405 X, nickel filter, d = interplaner distance in angstroms):

to

15

20th

d

Relative intensity

5.70

20th

5.35

20th

5.10

20th

4.90

10th

4.65

20th

4.45

40

4.20

30th

3.30

10th

3.15

10th

2.99

05

2.77

05

2.28

05

d

Relative intensity

12.40

100

10.20

70

8.85

90

7.80

30th

6.80

10th

6.30

100

The specific rotation of the antibiotic A-28086 factor-25 tors D is -56 ° (c-0.1, methanol), measured at a temperature of 25 ° C.

The electrometric titration of A-28086 factor D in 80 percent aqueous dimethylformamide shows the presence of a titratable group with a pKa of 30 8.67.

Antibioticum 28086 factor D is soluble in a number of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, in non-polar organic solvents such as hexane, but is only slightly soluble and insoluble in water.

The antibiotic A-28086 factor D has an acid function capable of forming salts and ester derivatives and has at least one esterifiable hydroxyl-4o group.

Based on the physical properties given above, a structure can be proposed for the antibiotic A-28086 factor D. However, since this structural proposal is only postulated, the structure indicated should only be regarded as a working hypothesis. The structure assumed for A-28086 factor D corresponds to the following formula II:

cha

/ \ fH

CHa 0 f — CHa-CHs

Q / ~ \ /

r! Formula II

/

: * ~ oh cha

Here the substituent Rt is CH3 and the substituent R2 is CoH3, or the substituent R4 is C2H5 and the substituent R is CH3.

621687

8th

The R £ values of the antibiotic A-28086 factors A, B and D in various paper chromatography systems using Bacillus subtilis ATCC 6633 as the detection organism are shown in Table I below.

TABLE I

Rf values

Factor A factor B factor D

Solvent system

0.11 0.09 0.10 Water saturated with methyl isobutyl ketone (MIKB)

0.41 0.16 0.26 With MIBK plus 2% p-toluenesulfonic acid and 1%

Piperidine saturated water

0.54 0.46 0.36 water: methanol: acetone (12: 3: 1) with NH40H

adjusted to pH 10.5 and then lowered to pH 7.5 with H3P04

0.48 0.36 0.29 1% MIBK, 0.5% NH40H in water

0.15 0.33 0.25 17.4 g K2HP04, 30 ml ethanol per liter water

0.24 0.51 0.26 Benzene saturated with water

0.24 0.11 0.09 water

0.75 0.61 0.64 water: MIBK: ethyl acetate (98: 1: 1)

Table II shows the Rt values for antibiotic A-28086 factors A, B and D in two thin-layer chromatography systems on silica gel (precoated plates, E. Merk, Darmstadt, F-254, layer thickness 0.25 mm), for which purpose B. subtilis ATCC 6633 is again used as the detection organism.

30th

TABLE II

Factor A

Rr values factor B

Factor D

Solvent system

0.24 0.54

0.42 0.34

0.26 benzene-ethyl acetate (3: 2)

0.66 ethyl acetate-diethylamine (95: 5)

Together with the antibiotic A-28086 complex, another substance is formed, which is arbitrarily referred to as A-28086-I. Although A-28086-I is not microbiologically active, it is structurally related to the factors of antibiotic A-28086. A-18086-I is a white crystalline compound (from acetone-water) with a melting point of about 160 to 162 ° C. Comparative studies of the NMR spectra and other properties of A-28086-I with synthetically produced A-28086 Factor A methyl ester provide evidence that A-28086-I is the methyl ester of A-28086 Factor A or a closely related compound , acts like a stereoisomer.

A-28086-I originally precipitates together with the active factors of antibiotic A-28086, but can easily be separated from it by silica gel chromatography. A-28086-I has an approximate Rr value of 0.53 in the thin layer chromatogram on silica gel using ethyl acetate as eluent and vanillin as spray (3% vanillin in methanol + 0.5 ml concentrated sulfuric acid per 100 ml solution) for detection. tion. After spraying with vanillin and heating results

A-28086-I bruises, while the factors of Antibioticum A-28086 give bright pink spots that quickly turn dark brownish blue.

50 The new antibiotic 28086 factors A, B and D and the specified acyl esters of factors A and D are capable of forming salts. Physiologically acceptable salts, which are also pharmacologically acceptable salts, are salts in which the toxicity of the compound in relation to the non-salt form to warm-blooded animals does not increase overall. Typical and suitable alkali and alkaline earth salts of A-28086 factors A and B are, for example, the sodium, potassium, lithium, cesium, rubidium, 60 barium, calcium and magnesium salts. Suitable amine salts of A-28086 factors A and B include the ammonium salts and the primary, secondary and tertiary Cj-Q-alkylammonium and hydroxy-C2-C4-alkyl-ammonium salts. Examples of amine salts are those obtained by reacting A-28086 factors A and B with ammonium hydroxide, methylamine, sec-butylamine, isopropylamine, diethylamine, diisopropylamine, ethanolamine, triethylamine, 3-amino-l-propanol and the like arise.

9

621687

The cationic alkali and alkaline earth salts of A-28086 factors A, B and D and the corresponding C2-C6 acyl ester derivatives of factors A and D can be prepared by methods which are customary for the preparation of cationic salts. For this purpose, for example, the free acid of the antibiotic factor or corresponding ester derivative is dissolved in a suitable solvent, such as warm methanol or ethanol, and then a solution containing the stoichiometric amount of the desired inorganic base in aqueous methanol is added to this solution. The salt formed in this way can be isolated in a conventional manner, for example by filtering off or evaporating the solvent.

The salts of organic amines can be prepared in a similar manner. For example, the gaseous or liquid amine can be added to a solution containing the antibiotic factor in a suitable solvent, such as acetone, after which the solvent and the excess amine can be removed by evaporation.

It is known in veterinary medicine that the form of the antibiotic is not significant when treating an animal with the antibiotic. In most cases, the active ingredient is changed to forms that are different from the administered forms by the conditions prevailing in the animal. The salt form in which the active ingredient can be administered is therefore not significant for the treatment process. However, the salt form can be chosen for reasons of economy, ease of use and favorable toxicity.

The antibiotic A-28086 factor A forms C2-C0-acyl ester. Esterification occurs e.g. on the hydroxyl groups of A-28086 factor A after treatment with a C2-C, acid anhydride or acid chloride. Such esters are best made by reacting A-28086 factor A at room temperature with, for example, the corresponding acid anhydride. These ester derivatives are also suitable as antibiotics and agents for improving feed utilization.

The properties of the above-mentioned acyl esters of A-28086 factor A are described in more detail below. The A-28086 factor A acetylester is a white crystalline compound (from acetone-water) with a melting point of approx. 100 - 103 ° C. The acetylester derivative of A-28086 factor A has an empirical formula of C45H74012 and a molecular weight of about 807 based on the empirical formula proposed for A-28086 factor A. The elementary analysis of the acetylic ester of factor A shows the following percentage composition:

C45H74012:

Calculated: C 66.97 H 9.24 O 23.79

found: C 67.67 H 8.71 O 23.13

The infrared spectrum of A-28086 factor A acetylester in chloroform is shown in Fig. 3 of the accompanying drawing. The following absorption maxima can be observed: 2.85, 3.36, 3.38 (strong), 5.80, 6.83, 7.25, 7.52, (strong), 7.60 (weak), 7 , 80 (strong), 8.45 (strong), 8.80 (strong), 8.95 (strong), 9.10 (strong), 9.20, 9.63, 9.80 (strong), 10 , 12 (weak), 10.25 (weak) and 10.50 microns.

The ultraviolet spectrum of A-28086 factor A acetylester in ethanol shows only end absorption.

The electrometric titration of A-28086 factor A acetylester in 80 percent aqueous dimethylformamide shows the presence of a titratable group with a pKa of 8.5.

The A-28086 factor A propionyl ester is a white crystalline compound (from acetone-water) with a melting point of about 96-98 ° C. The propionyl ester of A-28086 has an empirical formula of C40H7 "O12 and a molecular weight of approximately 821, based on the empirical formula proposed for A-28086 factor A. The elemental analysis of factor A propionyl ester gives the following percentage composition:

5 C4eH7e012:

calculated: C 67.29 H 9.33 O 23.38 found: C 66.06 H 9.17 O 23.41 The infrared spectrum of the propionyl ester of A-28086 factor A in chloroform is shown in FIG. 4 of the attached drawing . The following absorption maxima can be observed: 2.85, 3.33, 3.38 (strong), 3.45 (strong), 5.75 (strong), 5.82, 6.81, 7.22, 7, 30 (strong), 7.50 (weak), 7.60 (weak), 7.80, 8.43, 8.75 (strong), 8.90, 9.05, 9.15 (strong), 9 , 50 (strong), 9.63, 9.83 (weak), 10.05 (strong), 15 10.13.10.20 (strong), 10.45 and 16.68 microns.

The ultraviolet spectrum of the propionyl ester of A-28086 factor A in ethanol shows only end absorption.

The butyryl ester of the antibiotic A-28086 factor A is a white crystalline compound (from acetone-water) 20 with a melting point of about 96-98 ° C. The butyryl ester of A-28086 factor A has the empirical formula C47H78Oj2, a molecular weight of about 835 and an approximate composition of 67.60% carbon, 9.41% hydrogen and 22.99% oxygen, based on the 25 for A. -28086 factor A proposed empirical formula. The infrared spectrum for the butyl acrylate of A-28086 factor A in chloroform is shown in Fig. 5 of the accompanying drawing. The following absorption maxima can be observed: 2.89, 3.40, 3.45, 3.51, 5.85, 5.92 (strong), 30 5.97 (strong), 6.90, 7.30, 7.84 (weak), 8.55, 8.85 (weak), 9.01 (strong), 9.26, 9.76, 9.95, 10.31 and 10.64 microns.

The valerylester of the antibiotic A-28086 factor A is a white crystalline compound (from acetone-water) with a melting point of approximately 173 to 175 ° C. The Vale-35 rylester of A-28086 factor A has the empirical formula C4gHS0O12, a molecular weight of about 849 and an approximate composition of 67.89% carbon, 9.50% hydrogen and 22.61% oxygen, based on the for A-28086 factor A suggested empirical formula. 40 The infrared spectrum for the valerylester of A-28086 factor A in chloroform is shown in Fig. 6 of the accompanying drawing. The following absorption maxima can be observed: 2.90, 3.40, 3.45, 3.51, 5.87, 5.92 (strong), 5.99 (strong), 6.91, 7.30, 7 , 69 (weak), 7.87 (weak), 8.16, 45 8.58, 8.85 (weak), 9.26, 9.76, 10.00 (weak), 10.31 and 10, 64 microns.

The caproyl ester of the antibiotic A-28086 factor A is a white crystalline compound (from acetone-water) with a melting point of about 163-167 ° C. The caproyl-50 ester of A-28086 factor A has the empirical formula Cj ,, H8201; ,, a molecular weight of approximately 863 and an approximate composition of 68.18% carbon, 9.58% hydrogen and 22.24% oxygen, on the basis of the empirical For-55 mei proposed for A-28086 factor A.

The infrared spectrum for the caproyl ester of A-28086 factor A in chloroform is shown in Fig. 7 of the accompanying drawing. The following absorption maxima can be observed: 2.90, 3.40, 3.45, 3.51, 5.87, 5.92 (strong), 60 5.97 (strong), 6.90, 7.30, 7.66 (weak), 7.84 (weak), 8.16, 8.58, 8.85 (weak), 9.05 (strong), 9.17, 9.72, 9.95, 10, 29 and 10.62 microns.

The acylation of A-28086 factor A results in the following changes in the magnetic nuclear magnetic resonance spectrum: The carbinyl resonance occurring at about 4 ppm is shifted down by about 5.3 ppm (the exact position varies somewhat with the various acyl derivatives), and the signals of the vinyl proton are also shifted

621687

10th

ben. This is characteristic of the change as it emerges from the following partial structure:

H

OH. H

RCOX

The substituent R here is C1-C5-alkyl.

The above partial structure fully agrees with ^ -homonu-clear decoupling experiments and the nuclear magnetic detection of 13C for the acetyl and propionyl ester derivatives.

The C2-C0 acyl esters of A-28086 factor A are soluble in a number of organic solvents, such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform, acetone and benzene, but only weakly dissolve in non-polar organic solvents such as hexane and are insoluble in water.

Each of the C2-C0 acyl esters of A-28086 has an acid function capable of forming salts and ester derivatives.

The new antibiotics are made by growing an A-28086 producing strain of Streptomyces aureofaciens submers under aerobic conditions in a suitable culture medium until substantial antibiotic activity is formed. The antibiotics can be obtained using various isolation and purification techniques that are common in the art.

One of the new organisms suitable for the production of Antibiotics A-28086 was isolated from a soil sample that was found on the Ararat mountain in Turkey. This organism is classified as a strain of Streptomyces aureofaciens Duggar, as described by E. B. Shirling and D. Gottlieb in "Cooperative Description of Type Cultures of 5 Streptomyces III, Additional Species Descriptions from First and Second Studios," Intern. Bull. Systematic Bacteriol. 18, 297-392 (1968). This classification is based on methods used for the International Streptomyces project [E. B. Shirling and D. Gottlieb “Methods for Cha-io racterization of Streptomyces species”, Intern. Bull. Systematic Bacteriol. 16, 313-340 (1966)] together with certain supplementary examinations. The color names are assigned according to the ISCC-NBS method (KL Kelly and DB Judd, "The ISCC-NBS Method of 15 Designating Color and a Dictionary of Color Names", US Dept. of Commerce, Circ. 553, 1955, Washington , DC). The numbers given in parenthesis refer to the Tresner and Backus color charts [Tresner, H.D. and S.J. Backus, “System of Color Wheels for Streptomyces 20 Taxonomy”, Appi. Microbiol. 11, 335-338 (1963)]. The color stripe determinations are underlined. The corresponding values according to the Maerz and Paul color tables (A. Maerz and M. R. Paul, "Dictionary of Color", McGraw-Hill Book Co., New York, N.Y., 1950) are given in brackets 25. The appropriate cultures are grown at 30 ° C for 14 days unless otherwise stated.

Characterization of the A-28086 producing strain NRRL 5758

30th

morphology

The sporulating aerial hyphae consist of hair hooks, loops and open spirals. A morphology typical of rectus flexibilis was also observed. The spores are short and cylindrical and are in chains of 10 to 50 spores. The spores measure 1.3 n X 1.75 [i with a range from 1.3 | x to 1.95 (i X 1.3 [x. The spore surface is smooth due to an electron microscopic observation.

Cultural characteristics of NRRL 5758 on different media

medium

Characteristics

ISP ß2 (yeast extract-malt extract)

ISP ß3 (oatmeal)

ISP ß4 (inorganic salts-starch agar)

ISP ß5 (glycerin-asparagine agar)

Tomato paste oatmeal agar

Glycerin-glycine agar

Glycose-asparagine agar

Abundant growth, undersides massive yellow [11K3], sufficient to good aerial mycelium and sufficient to good sporulation, white (W) 13 ba and dark gray (Gy) 3 ih, no soluble pigment

Good growth, greyish-yellow underside [11B2], sufficient aerial mycelium (Gy), dark gray, 3 ih, slightly brown soluble pigment

Abundant growth, underside slightly yellow-brown [12E5], aerial mycelium and spores (W) purple-like white 13 ba to (Gy) light gray d, no soluble pigment

Good growth, underside pale yellow-green [10B1], good aerial mycelium and good spores (Gy), yellowish gray 2 de to light grayish reddish brown 5 fe, no soluble pigment

Abundant growth, underside light yellowish brown [13H7], sufficient to good aerial mycelium and corresponding spores (W) white a to (Gy) medium gray g, very light brown soluble pigment

Abundant growth, underside dark grayish yellow [12J6], good aerial mycelium and corresponding spores (Y) pale yellow 2 db, no soluble pigment

Abundant growth, underside greyish greenish yellow [12E2] abundant aerial mycelium and corresponding spores (Gy) yellowish gray 2 de, very light brown soluble pigment

11

621687

Cultural characteristics of NRRL 5758 on different media

(Continuation)

medium

Characteristics

Nutrient agar

Good growth, greyish yellow underside [12B2], aerial mycelium and spores,

due to poor growth no color determination, no soluble pigment

Bennett agar

Abundant growth, greyish yellow underside [12K3], scanty air

mycelium and corresponding spores (Gy) yellowish gray 2 de, no soluble

pigment

Calcium maleate agar

Good growth, underside greyish brown [15C8], no aerial mycelium and

no sporulation, brown soluble pigment, the surface clears up

Inoculum

Czapek solution agar

Poor growth, no color due to this poor growth

determination

Emerson agar

Abundant growth, greyish yellow underside [11J5], no aerial mycelium

and no spores, no soluble pigment

Tyrosine agar

Abundant growth, underside light olive brown [14C4], abundant air

mycelium and corresponding spores from (W) b (center) to (Gy) light brown

light gray 3 feet (on the border), very light brown soluble pigment

Trypton yeast agar

Sparse growth, no color determination

The organism NRRL 5758 was examined according to standard methods with regard to certain physiological properties. The following properties were examined and the following characteristics were found:

Observed properties

Characteristics

Effect on milk

The milk is peptonized, you can see a white growth ring that

clarified area is tan-yellow, the pH is 5.7

Nitrate reduction

positive

Nutritional gelatin

30% liquefaction after 14 days

Melanin pigment production:

Tyrosine slant agar

Extremely weakly positive (pigment after 4 days)

Difco peptone yeast extract iron slant agar

negative

T rypton yeast extract broth

negative

Temperature requirements (ISP medium ß2

Good growth at 26 to 30 ° C, excellent growth at 30 to 37 ° C

Yeast extract-malt extract slants)

and sporulation, slightly vegetative growth at 45 ° C, reddish soluble

Pigment.

Carbon utilization studies were also carried out using the organism NRRL 5758, and these results are shown below. The symbols used have the following meanings:

+ = good growth, positive recovery 60

(+) = poor to sufficient growth (-) = weak growth, possibly no recovery

- = no growth, no utilization

65

621687

12

Carbon source

behavior

D-glucose

+

L-arabinose

+

D-xylose

+

D-fructose

+

Sucrose

-

D-mannitol

-

i-inositol

+

Rhamnose

+

Raffinose

-

-C control

(no carbohydrate)

Through a series of natural selections followed by a chemical mutation, S. aureofaciens NRRL 5758 also derived a second new A-28086 producing organism. This organism is called NRRL

8092, and it is also classified as a strain of Streptomyces aureofaciens Duggar. The classification is based on studies of the organism NRRL 8092 using the classification methods described above for 5 Streptomyces and similar growth conditions. The characteristics observed in these studies for the organism NRRL 8092 are evident from the following explanations.

10 Characterization of the A-28086 producing strain NRRL 8092

morphology

The culture 15 occasionally forms loops on medium ISP ß7 (tyrosine agar), but predominantly short straight sporophores are formed. The spore chains consist of less than 10 spores per chain, normally 4 to 7 spores per chain.

Short straight spore chains can be observed in the following media: ISP ß3, Czapek solution agar and ISP ß5. An abundant coremia can be observed on Emerson agar. Examinations in the electron microscope are also carried out on tyrosine agar (ISP ß7) and glucose-asparagine agar. The spores are smooth and 1.2 to 2.0 p, long 25 and have a diameter of about 1.0 [i. The average spore size is 1.6 n X 1.0 yes.

Cultural characteristics of NRRL 8092 on different media

medium

Characteristics

ISP ß2 (yeast extract-malt extract)

ISP ß3 (oatmeal)

ISP ß4 (inorganic salts-starch agar)

ISP ß5 (glycerin-asparagine agar)

Tomatopaste Oatmeal Agar

Glycerin-glycine agar

Glucose-asparagine agar

Nutrient agar

Bennett agar

Calcium malate agar

Czapek solution agar

Emerson agar

Tyrosine agar

Trypton yeast agar

Sufficient growth, underside light yellow-brown [12H8], sufficient aerial mycelium, poor sporulation, aerial mycelium pale gray [11 AI], no soluble pigment

Sparse growth, hyaline underside, no aerial mycelium, no soluble pigment

Moderate growth, greyish-yellow underside [11B2], sparse aerial mycelium and sparse sporulation, aerial mycelium pale yellow-gray [10A1], no soluble pigment

Moderate growth, underside pale yellow [10F2], sufficient aerial mycelium, sparse sporulation, aerial mycelium white [10A1], no soluble pigment

Moderate growth, greyish green-yellow underside, sufficient aerial mycelium, massive sporulation, light pale gray [53 A2], no soluble pigment

Abundant growth, underside greyish-yellow [11E4], moderate aerial mycelium, white [10A1], no sporulation, no soluble pigment

Moderate growth, underside pale yellow [10F2], moderate aerial mycelium and massive sporulation, white [10A1], no soluble pigment

Sparse growth, underside pale yellow [10B2], no aerial mycelium, no soluble pigment

Passable growth, underside medium-yellow-pink [11A7], very sparse aerial mycelium, no sporulation, no soluble pigment

Extremely sparse growth, hyaline, no aerial mycelium, no soluble pigment

Extremely sparse growth, hyaline underside, no aerial mycelium, no soluble pigment

Moderate growth, greyish-yellow underside [1115], spotted aerial mycelium, no sporulation, no soluble pigment

Moderate growth, underside pale yellow-brown [12H6), moderate aerial mycelium, slightly pale gray edge [53A2], center almost white, moderate sporulation, no soluble pigment

Extremely sparse growth, hyaline, no aerial mycelium, no soluble pigment.

13

621687

The organism NRRL 8092 is also examined according to standard methods with regard to certain physiological properties. The following properties are observed and the following characteristics are found:

Observed properties

Characteristics

Effect on milk

Nitrate reduction nutritional gelatin

Melanin pigment production on: Tyrosine slant agar Trypton yeast extract broth Carrot Potato

Temperature requirements (ISP Medium ß2 yeast extract-malt extract slants)

Peptonized (90%), pale yellow growth ring, cleared area pale yellow, pH reaction 4.6

positive

50% hydrolyzed within 14 days

Extremely weak positive negative

Abundant growth, pale yellow, no aerial mycelium Abundant growth, greyish white, no aerial mycelium, no change

Abundant growth at 25 ° C, sufficient aerial mycelium, underside light brown, no soluble pigment

Abundant growth at 30 ° C, sufficient aerial mycelium, underside light brown, no soluble pigment

Abundant growth at 37 ° C, sufficient aerial mycelium, brown underside, no soluble brown pigment

Abundant growth at 40 ° C, sparse aerial mycelium, underside reddish brown, deep reddish brown soluble pigment

Sufficient growth at 45 ° C, no aerial mycelium, underside reddish brown, medium reddish brown pigment.

The organism NRRL 8092 is also examined for its carbon utilization, and the results obtained are summarized below. The symbols used for this have the following meanings:

+ = good growth, positive recovery (+) = poor to sufficient growth (-) = weak growth, possibly no recovery

- = no growth, no utilization

Certain characteristics of the A-28086 producing strains of S. aureofaciens differ from the 40 characteristics of the organism described by Shirling and Gottlieb. These differences are shown in Table III below.

45

Carbon source

behavior

50

D-glucose

+

L-arabinose

+

D-xylose

+

55

D-fructose

+

Sucrose

-

D-mannitol

-

60

i-isosit

+

Rhamnose

+

Raffinose

-

65

-C control

(no carbohydrate)

621687

14

TABLE III

Carbon utilization

NRRL 5758

NRRL 8092

published description

Sucrose i-inositol

Rhamnose

Gelatin liquefaction effect on milk

+

+

30% in 14 days

Peptonized milk, white growth rings

+

+

50% in 14 days

Peptonized milk, pale yellow growth rings

+

limited or no limited or variable peptonization (often none), limited growth and limited coagulation

The characteristics of the organism NRRL 5758, which are different from the characteristics of the organism NRRL 8092, are summarized in Table IV below.

TABLE IV

Characteristics

NRRL 5758

NRRL 8092

Vegetative color sporulation

Growth on:

Calcium malate inorganic salt starch

Bennett agar yellow on several media a few spiral sporophores on media made of tomato paste: oatmeal and inorganic salt starch sufficient growth, brown, clarification of moderate sporulation, aerial mycelium purple-white to gray

Underside pale yellow chemical to pale yellow on several media short straight sporophores with occasional loops sparse growth, clear, no clarification moderate sporulation, aerial mycelium pale yellow-gray

Underside pink

Streptomyces aureofaciens suitable for the production of A-28086 antibiotics have been deposited and added to the culture collection of Northern Marketing and Nutrition Research Division, U.S. Dept. of Agriculture, Agricultural Research Service, Paoria, Illinois, 61604, and they are freely available from there under numbers NRRL 5758 and NRRL 8092.

Any culture medium can be used to grow Streptomyces aureofaciens NRRL 5758 and Streptomyces aureofaciens NRRL 8092. However, certain culture media are preferred for reasons of cheap production, optimum yield and easy isolation of the product. For large-scale fermentation, tapioca dextrin and sucrose, for example, are therefore preferred as carbohydrate sources, but glucose, corn starch, fructose, mannose, maltose, lactose and the like can also be used. Other suitable carbon sources are corn oil, peanut oil, soybean oil and fish oil. A preferred nitrogen source is an enzyme hydrolyzed casein, but peptones, soybean meal, cottonseed meal, amino acids such as glutamic acid and the like can also be used. The inorganic nutrient salts which can be added to the culture media include the usual soluble salts which provide sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate and similar ions.

The essential trace elements required for the growth and development of the organisms can also be present in the culture medium. Such trace elements normally appear as impurities in other components of the medium in amounts that meet the needs necessary for the growth of the organism. If the problem of foaming arises in large-scale fermentation, then it is advisable to add small amounts (namely 0.2 ml / l) of an antifoam, such as polypropylene glycol, to such fermentation media.

Antibiotic A-28086 produce-55 strains of Streptomyces aureofaciens can be improved by adding a small amount of an oil such as soybean oil, although this is not essential.

Submerged fermentation under aerobic conditions in tanks is preferred for the production of significant quantities of Antibiotics A-28086. Small amounts of Antibiotics A-28086 can also be made by shaking flasks. Because of the time delay that occurs in the usual production of antibiotics by inoculating large tanks with the spore shape of the organism 65, a vegetative inoculum is preferably assumed. The vegetative inoculum is e.g. prepared by inoculating a small amount of culture medium with the spore form or with mycelium fragments of the organism, where

15

621687

by maintaining a fresh, actively growing culture of the organism. The vegetative inoculum can then be transferred to a larger tank. The same medium can be used to grow the vegetative inoculum as for larger fermentations, but one can also work with other media.

The organisms producing A-28086 can be grown at temperatures between about 20 and about 40 ° C. Optimal production of A-28086 apparently occurs at temperatures of around 27 to 30 ° C.

As is customary in aerobic submerged culture processes, air is preferably also blown through the culture medium in the process according to the invention. For a sufficient growth of the organism one should work with the tank production with an air volume of preferably over 0.1 volume of air per volume of culture medium per minute. For effective production of the A-28086 antibiotics, the volume of air used in tank production should preferably be over 0.25 volumes of air per volume of culture medium per minute. The formation of antibiotic is not reduced by higher concentrations of dissolved oxygen.

Antibiotic production can be monitored during fermentation by examining samples of the broth or extracts of mycelium solids against organisms known to be sensitive to the antibiotics for their antibiotic activity. One such test organism suitable for examining the new antibiotics is Bacillus subtilis ATCC 6633. The bioassay is best carried out using paper papers on agar plates.

The initial pH of the non-inoculated culture varies depending on the medium used. The pH should generally be between 6.0 and 7.5. The pH at the end of the fermentation at which the product is harvested is usually somewhat higher, and in particular is 6.5 to 8.0.

Antibiotic activity is generally seen on the second day of fermentation. A maximum production of antibiotic activity can occur between about the sixth and tenth day.

After production under submerged aerobic fermentation conditions, the A-28086 antibiotics described above can be obtained from the fermentation medium in the usual way. The antibiotic activity formed during the fermentation of an A-28086 producing organism occurs both in the mycelium mass and in the filtered broth. The A-28086 antibiotics can therefore be obtained to the maximum by a preferred combination of methods, which include filtration, extraction and absorption chromatography. A preferred solvent for separating the A-28086 antibiotics from either the whole or the filtered fermentation broth is ethyl acetate, but other solvents normally used for this purpose are also sufficient for this purpose.

A particularly expedient method for separating the A-28086 factors A, B and D consists in lowering the pH of the entire fermentation broth to about pH 3.0. At this pH value of 3.0, A-28086 factors A, B and D can be easily separated from the mycelial mass by filtration. Another convenient method for separating the A-28086 factors is to add a bicarbonate, such as sodium bicarbonate, to the total broth in amounts of about 1 g per liter. The A-28086 factors can also be separated from the mycelial mass in salt form. Methanol is preferred as the solvent for separating the antibiotics from the mycelial mass, but other lower alcohols and ketones can also be used for this purpose.

The A-28086 antibiotics can advantageously also be obtained by azeotropic distillation. To do this, e.g. the aqueous fermentation broth with an organic solvent that forms a suitable azeotrope with water. This mixture of solvent and fermentation broth can then be azeotroped to remove at least half of the water from the broth, leaving a mixture of water and solvent with the A-28086 antibiotics dissolved in the organic solvent. Insoluble by-products can be separated off in a suitable manner, for example by filtering or centrifuging. The A-28086 antibiotics can then be obtained from the organic solution in a known manner, for example by evaporation of the solvent, precipitation with the addition of a non-solvent or extraction.

Organic solvents which form an azeotrope suitable for carrying out such a recovery process are, above all, butyl alcohol, amyl alcohol, hexyl alcohol, benzyl alcohol, butyl acetate, amyl acetate, 1,2-dichloroethane, 3-pentanone, 2-hexanone, benzene, cyclohexa-non , Toluene, the xylenes and the like.

Extraction by azeotropic distillation offers a particular advantage in large-scale fermentation processes. Both water and solvent, both of which are removed overhead in the azeotrope, can be separated from one another by known processes and then recycled for further use. The water removed in this way is free of impurities and therefore does not have to be treated as waste water before it is passed on. The solvent removed can be returned to the process.

The further purification of the A-28086 antibiotics generally consists in an additional extraction and adsorption process. For this purpose, adsorbable materials are expediently used, such as silica gel, activated carbon, Florisil (magnesium silicate, Floridin Co., P.O. Box 989, Tallahassee, Fla.) And the like.

The solids of the culture broth, including the constituents of the medium and of the mycelium, can optionally also be used as the source of the A-28086 antibiotics without extraction or separation, but preferably the water is first separated off. The culture medium can be dried after production of the A-28086 antibiotic activity, for example by lyophilization, and mixed directly with a feed premix.

However, after production of the A-28086 activity in the culture medium, the mycelium can be separated and dried, whereby a product is obtained which can be used directly as a feed premix. If the mycelium is separated for this purpose, the whole can be filtered better by adding calcium carbonate (about 10 grams per liter), and at the same time a better dried product is obtained.

Under the conditions used up to now, the strains of Streptomyces aureofaciens described above, which are designated with the numbers NRRL 5758 and NRRL 8092, produce in particular the antibiotic A-28086 factor A as the predominant factor. The ratio of the factors varies depending on the fermentation conditions used, but factor A generally accounts for more than 99% of the total antibiotic activity derived from NRRL 5758 and approximately 90% of the total antibiotic activity derived from NRRL 8092. A-28086 factor B accounts for the majority of the remaining antibiotic activity from NRRL 5758,

5

10th

IS

20th

25th

30th

35

40

45

50

55

60

65

621687

and the factor D is only present in a smaller amount. However, A-28086 factor D, on the other hand, usually accounts for about 8 to 10% of the total antibiotic activity obtained from NRRL 8092, and factor B is only present in a minor amount here.

Antibiotics A-28086 factors A, B and D can be separated from one another in a known manner and isolated as individual compounds, for example by column chromatography, thin-layer chromatography and the like. To separate the factors A, B and D, one uses, for example, column chromatography on silica gel, the column being eluted with varying solvent mixtures, such as mixtures of benzene and ethyl acetate. When preferred solvent mixtures of benzene and ethyl acetate are used to elute a silica gel column, factor B is usually eluted first, and factors A and D come later. The easiest way to monitor the progress of the elution is by the thin-layer chromatographic method described above.

The A-28086 compounds inhibit the growth of bacteria and fungi, which are pathogens for animals and plants. The relative microbiological activities of A-28086 factors A and B are shown in Table V below.

The values contained therein are determined by the usual disk diffusion method (for this purpose, 6 mm disks are immersed in solutions containing 1 mg of active ingredient per ml of solution, and the separate disks obtained in this way are then placed on agar plates which have been inoculated with the respective test organisms) .

TABLE V

Inhibition zone (mm) test organism A-28086 A-28086

Factor A factor B

16

TABLE VI

Test organism

MIC (ng / ml)

5

Actinomyces bovis

<0.5

Clostridium inoeuum

<0.5

10th

Clostridium perfringens

<0.5

Clostridium ramosum

4.0

Clostridium septicum

<0.5

Clostridium septicum bovine

<0.5

15

Eubacterium aerofaciens

1.0

Peptococcus anaerobius

<0.5

Peptostreptococcus intermedius

16.0

20th

Propionibacterium acnes 44

• 16.0

Propionibacterium acnes 79

2.0

Bacteroides fragilis ssp. fragilis

8.0

25th

Bacteroides fragilis ssp. thetaiotacmicron

8.0

Bacteroides fragilis ssp. vulgatis No. 1563

8.0

30th

Bacteroides fragilis ssp. vulgatis No. 1211

2.0

Fusobacterium symbiosum

<0.5

35

Fusobacterium necrophorum

8.0

Veillonella alcalescens

16.0

Staphylococcus aureus 19th

Bacillus subtilis ATCC 6633 28

Sarcina lutea ATCC 9341 26

Mycobacterium avium ATCC

7992 12

Saccharomyces pastorianum

ATCC 2366 17th

Candida tropicalis 14

Fusarium moniliform 12

21 31 16

12 14

not examined

An important consideration is the fact that the A-28086 compounds inhibit the growth of anaerobic bacteria. The minimum inhibitory concentrations (MIC) at which A-28086 factor A inhibits various anaerobic bacteria are determined using the standard agar dilution test and the values obtained are summarized in Table VI. The final values are determined after 24 hours of incubation.

The C2-CG acyl esters of A-28086 factor A are 40 antibacterial and antifungal. These derivatives show an increased gram-positive effectiveness. The relative gram-positive effect of certain such derivatives is compared with the effectiveness of factor A. The compounds are examined by a turbidometric test on a semi-automated system (Autoturb® microbiological test system, Elanco), as described by N.R. Kuzel and F.W. Kavanaugh in J. Pharmaceut. Be. 60 (5), 764 (1971). The following experimental parameters are used to investigate the antibiotics A-28086: Staphylococcus aureus (H-Heatley) NRRL B-314 in a nutrient broth medium (pH 7), incubating for four hours at 37 ° C. The compounds to be examined and the standard are dissolved in methanol-water (10:90). The standard, namely A-28086 factor A, 55 is given in concentrations of 2, 3.4 and 5 mg / ml in the turntable of the autoturb device. The compounds to be investigated are diluted to such an extent that they contain about 3 or 4 mg of activity per ml when inputted into the turntable. The investigated compounds give 6o the following relative activities compared to the standard:

65

17th

621687

Relative G + activity link

Another interesting effect is the antimicrobial activity of the compounds A-28086, namely the activity against Mycoplasma. Mycoplasma species, which are also referred to as Pleuropneumonia-like (PPLO) organisms, are pathogens in humans and in various animals. Agents that work against PPLO organisms are particularly needed by the poultry industry. By in vitro broth dilution studies with antibiotic A-28086 factor A compared to different types of mycoplasma, the following minimum inhibitory concentrations (MIC values) are summarized in Table VII:

TABLE VII

organism

M. gallisepticum

12.5

M. hyorhinis

12.5

M. synoviae

6.25

M. hypopneumoniae

6.25

M. hyosynoviae

6.25

Solutions with only 10 milligrams of Antibioticum A-28086 per milliliter of solution are sufficient to disinfect surfaces to protect animals from various types of mycoplasma.

The A-28086 compounds are also antiviral agents. A-28086 factor A acts against poliovirus type III, vaccinia virus, herpes virus and Semliki forest virus, as shown by in vitro plate suppression experiments, which are described by Siminoff in Applied Microbiology 9 (1), 66-72 (1961 ) experiments described are similar. A-28086 factor A is also effective against the transmissible gastroenteritis virus, Newcastle disease virus and bovine rhinotracheitis infectious virus, as shown by similar tissue culture studies.

Compounds A-28086 can therefore be administered orally, topically or parenterally to combat viruses in mammals. Suitable dosage levels for preventing or treating a viral disease are between about 1 and about 5 mg / kg of animal body weight, depending on the virus and the question of whether the active ingredient should be used prophylactically or therapeutically.

Solutions containing an A-28086 compound, preferably together with a surfactant, can also be used to decontaminate the in vitro habitat on which viruses such as polio or herpes are present. Solutions containing about 1 to about 1500 mg / ml of an A-28086 compound are sufficient to combat viruses.

The acute toxicity LD50 of the antibiotic A-28086 factor A is 7.15 mg / kg after intraperitoneal administration to the mouse.

The compounds A-28086 are also insecticide-5 cide and acaricide. Compounds A-28086 are effective at only 500 ppm against insects such as the Mexican bean beetle, the spurge beetle and the house fly, and they are also effective against mites such as the leaf spider mite. In addition, the compounds A-28086 act in an application amount of 0.1 ppm against mosquito larvae.

An important property of the compounds A-28086 is their anticoccidial activity. Feeding trials, for example, show that 15 young chickens can gain weight more easily by feeding them a diet containing only 0.003% of an A-28086 compound, and drug concentrations of only 0.005% can prevent mortality and the number of injuries reduce in chickens afflicted with coccidiosis 20. The results obtained in experiments using factor A on chickens infested with various Eimeria species are shown in Tables VIII to X below.

MIC (mg / ml)

A28086 factor A (standard)

1

A28086-A acetylester derivative

2.5

A28086-A-propionyl ester derivative

7

A28086-A-n-butyl ester derivative

8th

A28086-A-n-V alerylester derivative

14

A28086-A-n-caproyl ester derivative

20th

621687 18

TABLE Vili

Effectiveness of antibiotic A-28086 factor A against Eimeria Tenella

Active ingredient concentration in the feed in% by weight

Number of chickens% mortality% weight gain

Average damage value Percentage reduction in the damage value

0.04

10th

0

50

0

100

0.02

10th

0

82

0.2

95

0.01

10th

0

94

0.2

95

Infected controls

20th

35

68

3.65

-

Normal controls

20th

0

100

-

-

0.02

20th

0

84

0

100

0.01

20th

0

96

0

100

0.005

20th

0

91

2.8

27th

0.0025

20th

10th

87

3.85

0

Infected controls

20th

40

74

3.85

-

Normal controls

20th

0

100

-

-

TABLE IX

Efficacy of antibiotic A-28086 factor A against various species of Eimeria 1

infecting organisms

Active ingredient concentration in the feed in% by weight

percentage mortality percentage weight gain

Mean damage value Percentage reduction in the damage value of total oocytes / animal that passed through (1 X 106)

percentage reduction

Eimeria acervulina

0.005 0.0025

0 0

87 82

0.75 1.25

53

6.15 4.91

83 86

infected controls

-

0

63

1.60

-

36.32

-

Eimeria maxima

0.005

0

85

0.2

85

1.95

40

infected controls

-

0

65

1.3

-

3.24

-

Eimeria brunetti

0.005

0

88

1.2

__

2.31

58

infected controls

-

0

62

1.7

-

5.55

-

1 four repetitions, 5 fried chicken each

19th

TABLE X

Efficacy of antibiotic A-28086 factor A against mixed species of Eimeria 1

621687

Infecting organisms

Active substance concentration in% by weight

percentage mortality percentage weight gain

% Reduction in intestinal damage

medium

Caecal damage percentage reduction

medium

E. necatrix and E. tenella infected controls

0.005

0 20

94 52

0.7 1.7

59

1.75 3.5

50

E. tenella, E. necatrix, E. maxima, E. brunetti, E. acervulina and

Controls infected with E. mivati

0.01

0 50

91 12

0.4 1.9

79

0.9 3.3

73

1 four repetitions, 5 fried chicken each

To prevent or treat poultry coccidiosis, a non-toxic anti-coccidial amount of Compound A-28086 may be administered to the animals, preferably orally on a daily basis. Connection A-28086 can be supplied in a number of ways. However, the simplest type of administration is in connection with a physiologically acceptable carrier, preferably the respective animal feed. While various factors need to be considered to determine the appropriate concentration of Compound A-28086, the amounts to be administered are generally from 0.003 to 0.04 percent by weight, based on the non-drug feed, and are preferably 0.005 to 0.02 Weight percent of the feed.

Another important property of Compounds A-28086 is their ability to improve feed utilization efficiency in animals. For example, the use of A-28086 compounds improves the rationing efficiency of ruminants with a rumen function.

As mentioned above, the efficiency of carbohydrate utilization in ruminants is improved by treatments which stimulate the animal's rumen flora to produce propionate compounds rather than to produce acetate or butyrate compounds. The efficiency of feed conversion can be monitored by observing the production and concentration of the propionate compounds in the rumen using the following methods:

A rumen fluid is removed from an ox through a surgically inserted fistula that extends into the rumen. The ox is fed with a high-quality grain feed. A sample of the rumen fluid is strained through a four-cloth cheesecloth and the resulting filtrate is collected. The lumpy material retained by the cheesecloth is then resuspended in sufficient physiological buffer to restore the original volume of the rumen fluid.

30 holds, and this suspension is filtered again. The buffer used for this has the following composition:

Ingredients g / liter

35

Well, HP04

0.316

kh2po4

0.152

NaHCOa

2,260

40

KCl

0.375

NaCl

0.375

MgS04

0.112

45

CaCl2. 2H, 0

0.050

FeS04. 7H2H

0.008

MnS04. H20

0.004

50

ZnS04. 7H20

0.004

CuS04. 5HaO

0.002

CoC12. 6HLO

0.001

This buffer is from Cheng et al. in J. Dairy Sei. 38, 1225-1230 (1955).

The two filtrates are combined, after which they are left to stand until solid material separates out at the top. The clear layer is separated off, diluted with the same buffer (1: 1) and then adjusted to a pH of 6.8 to 7.0.

The diluted rumen fluid (10 ml) is placed in a 25 ml flask together with 40 mg of the feed described above, a further 65 milligrams of soybean protein and the compound to be examined. Each treatment is carried out in four flasks. Two sets of four comparison pistons are also used. Man

621687

20th

works with a comparison to time zero and a comparison after 16 hours of incubation. All test flasks are incubated at 38 ° C for 16 hours. After incubation, the pH is measured, and 25% methaphosphoric acid 5 is added to each flask

(2 ml). The contents of the flask are then allowed to settle and the supernatants are analyzed by gas chromatography for the propionate, acetate and butyrate compounds contained therein. The active ingredients according to the invention greatly increase the production of propionate according to the comparisons.

The results obtained with the new active ingredients are compared statistically with the results of the comparisons. Table XI shows the ratio of the concentrations of volatile fatty acids in the treated flask 15 and the concentrations in the comparison flask.

TABLE XI

Treatment to control ratio

connection

ppm in the rumen fluid

Mole percent acetate

Mole percent butyrate

Mole percent propionate total VFA mmol / liter

A-28086 factor

A

1

0.94

0.87

'1.34

1.26

A-28086 factor

B

1

0.92

0.96

1.15

1.29

A-28086 factor acetylester

A

1

0.79

0.76

2.04

1.04

A-28086 factor propionyl ester

A

1

0.90

0.68

1.73

1.25

A-28086 factor butyl esters

A

1

0.91

0.81

1.56

1.19

A-28086 factor valerylester

A

1

0.92

0.79

1.55

1.18

A-28086 factor caproyl ester

A

1

0.86

0.97

1.57

0.99

table feed with A-28086 factor A. Each portion (100 ml) of the rumen fluid 45 removed is mixed with 10 percent metaphosphoric acid (100 ml). The samples are then allowed to settle and the supernatants are analyzed by gas chromatography to determine the propionic acid concentration.

The test results shown in Table XII are the mean percentage increases in the rumen propionic acid concentration, and mean values from 5 analyzes over a 14-day test period. The comparisons contain about 20 mole percent propionic acid.

TABLE XII

A-28086 factor A percentage increase versus

(mg / day) the controls

60 25 17.4

100 54.0

In vivo experiments also show 65 that A-28086 factor A increases the feed utilization efficiency in sheep. The results of these tests, which are carried out over a period of 56 days, are shown in Table XIII below.

from each treatment group you feed with an identical

The effectiveness of carbohydrate utilization is further demonstrated on the basis of in vivo investigations that are carried out on animals in whose rumen fistulas are created, via which samples of the rumen contents can be taken.

The experiment listed in Table XII is performed using adult fistulated oxen, each weighing approximately 554 kg. Two oxen are fed normal food, and four oxen

21st

621687

TABLE XIII

A-28086 Factor A number in g per ton of animal feed

Average daily weight gain in kg per day

Feed intake in kg per day

Ratio of feed intake to weight gain percentage improvement

9

18th

control

13 13 17

0.20 0.17 0.19

1.50 1.43 1.61

7.40 8.31 8.38

12 1

Compounds A-28086 factor D are antiviral agents. A-28086 factor D is effective against Maryland B virus, poliovirus type III, COE virus, herpes virus and Semliki forest virus, as shown by the in vitro plate suppression experiment described by Siminoff in Applied Microbiology 9 (1), 66-72 (1961). A-28086 factor D is also effective against the transmissible gastroenteritis virus, new castle disease virus and infectious bovine rhinotracheitis virus, as evidenced by similar tissue culture studies.

The fact that Compounds A-28086 Factor D are effective against both viruses and anaerobic bacteria makes these compounds particularly suitable for the treatment or prevention of enteritis in chickens, pigs, cattle and sheep. The compound A-28086 factor D is also suitable for the treatment of enterotoxemia in ruminants.

The anticoccidial activity of the new compounds A-28086 factor D is an important property. The anticoccidial effect of A-28086 factor D against Eimeria tenella is shown by in vitro experiments. To do this, use the following procedure [for details, see L. R. McDougald and R. B. Galloway in Exper. Parasitology 34, 189-196 (1973)]:

Using conventional techniques, host cell cultures are prepared using Leighton tubes, plastic dishes or plates or other suitable culture containers. After growing for 2 to 3 days in a suitable culture medium (Eeagle minimal essential medium, lactalbumin hydrolyzate in Earle or Hank salt solution, medium 199 and the like), a suitable monolayer usually forms which can be infected and treated with active substance. Primary cultures of chicken kidney cells are used for these studies.

Eimeria tenella live oocysts are obtained from the feces of infected chickens, and these are cleaned and sterilized before use. The oocysts are then sporulated by incubation at room temperature, by passing air through the suspension or by gently shaking this suspension on a rotary shaker or other suitable device. Potassium dichromate or sulfuric acid (or other chemicals) can be used to prevent bacteria or mold growth during sporulation.

The infectious sporozoites can be excised from the oocysts by a combination of mechanical destruction of the wall of the oocyst, subsequent treatment with trypsin and treatment with bile salts, which initiates the self-liberation of the sporozoites from the sporocysts.

Cell cultures are infected by introducing living sporozoites into the culture vessel. The chemicals to be examined can be introduced at the same time or later. The active substance to be investigated is introduced in the desired concentration in 10% dimethylformamide in physiological saline. The endpoint of the trial is inhibition of non-sexual stages of development and is determined by microscopic examination of cultures which are fixed and stained 96 hours after infection. The results are reported as effective (A), ineffective (N) or cycotoxic (C) depending on the presence or absence of second generation schizonts and any apparent toxicity to the cell mono-25 layer.

The anticoccidial activity obtained for A-28086 factor D in this study is summarized in Table XIV below.

30th

TABLE XIV

Anticoccidial effectiveness of antibiotic A-28086 factor D.

35

40

45

50

Concentration of A-28086 factor D (mg / ml)

rating

5

C.

1

C.

0.2

c

0.4

A, C *

0.03

A

0.02

A

0.01

A

0.008

N

* Result from two attempts.

Another interesting feature of Compounds 55 A-28086 Factor D is their ability to improve feed utilization efficiency in ruminants that have developed rumen function. The mechanism for utilizing or utilizing the predominant nutritional part (carbohydrates) of ruminant feed is exactly known. Microorganisms present in the rumen of the animal break down carbohydrates to form monosaccharides, and these monosaccharides are then converted into pyruvate compounds. The pyruvates are metabolized by microbiological processes with the formation of acetates, butyrates or propionates, which are collectively known as volatile fatty acids (VFA). For a more detailed discussion of these relationships, see Leng in “Physiology of Digestion

621687

22

and Metabolismi in the Ruminant, «Phillipson et al. Eds., Oriel Press, Newcastle upon Tyne, England (1970), pages 408-410.

The relative efficiency of VFA utilization is described by McCullough in Feedstuffs, June 19, 1971, page 19, Eskeland et al. in "Digestive Physiology and Nutrition of Ruminants", Volume 2, (1971), pages 622 and 625. The acetates and butyrates are also used, but the utilization of the propionates is higher. If too little propionate is present, a so-called ketosis also develops in the animals. A suitable compound therefore stimulates the animals to form a higher proportion of propionates from carbohydrates, which increases the utilization efficiency for carbohydrates and at the same time reduces the risk of ketosis.

The effectiveness of such an active ingredient can be determined by determining its influence on the production and concentration of the propionate compounds in the rumen by the same method as is used above in connection with factor A.

The test results obtained are compared with comparison results of statistical values. Table XV shows the ratio of the concentrations of volatile fatty acids in flasks treated with A-28086 factor D and the concentrations in the comparison flasks.

10th

15

20th

25th

TABLE XV

A-28086 D mg / ml

Mole percent acetate

Mole percent butyrate

Mole percent propionate total VFA mmol / liter

1.0 0.3

0.8715 0.8976

0.7973 0.8726

1.7981 1.5871

1.1979 1.0630

The invention is illustrated by the following examples.

example 1

A. Schiittel bottle fermentation of A-28086 using S. aureofaciens NRRL 5758

A culture of Streptomyces aureofaciens NRRL 5758 is prepared and kept on sloping agar of the following composition:

Components

The amount of serum suspension of the spores and the mycelium fragments 40 is lyophilized to 6 pellets.

A lyophilized pellet produced in this way is used to inoculate 50 ml of a vegetative medium of the following composition:

Agar

20th

G

Dextrin

10th

g Casein hydrolyzed with enzyme

2nd

G

Bovine extract

2nd

G

Yeast extract

2nd

G

Distilled water q. s. on

1

liter

45

50

55

Components

amount

glucose

20th

G

Soybean meal

15

G

Corn steep liquor

10th

G

CaC03

2nd

G

Tap water q. s. on

1

liter

The slant is inoculated with Streptomyces aureofaciens NRRL 5758, after which the inoculated slant is incubated at 30 ° C. for 6 to 10 days. The mature slanted culture is covered with bovine serum and scraped off with a sterile loop to detach the spores. The rings obtained

The inoculated vegetative medium is placed in a 250 ml Erlenmeyer flask and incubated at a temperature of 30 ° C. over a period of 72 hours on a 60 shaker which rotates in an arc about 5 cm in diameter at 250 revolutions per minute.

B. Tank fermentation of A-28086 using S. aureofaciens NRRL 5758 65. To form a larger volume of inoculum, use 10 ml of the above-described incubated vegetative medium to inoculate 400 ml of a second vegetative growth medium that has the same additive.

23

621687

collection as the vegetative medium has. This second medium, located in a 2 liter flask, is incubated at a temperature of 30 ° C. over a period of 24 hours on a shaker which rotates in an arc of about 5 cm in diameter at a speed of 250 revolutions per minute.

The vegetative medium (liters) obtained in this second stage is used to inoculate 100 liters of sterile production medium of the following composition:

Ingredients amount

Tapioca extract

60.0

g / liter casein hydrolyzed with enzyme

8.0

g / liter

molasses

15.0

g / liter

MgS04. 7H20

0.5

g / liter

CaC03

2.0

g / liter of refined soybean oil

5.0

g / liter

Deionized water q. s. on

1

liter

After sterilizing for 30 minutes in an autoclave at a temperature of 120 ° C. and a pressure of 1.05 to 1.41 kg / cm 2, the medium has a pH of 6.7. The inoculated production medium is then left to ferment in a 165 liter fermentation tank for 10 days at a temperature of 29 ° C. The fermentation medium is aerated with sterile air at a rate of 0.4 volumes of air per volume of culture medium and per minute. The medium is stirred with conventional stirrers at a stirring speed of 250 revolutions per minute.

Example 2

Antibiotics A-28086 are prepared by the same procedure as in Example 1, but using a piston production medium of the following composition:

Ingredients amount

glucose

10th

g / liter of edible molasses

20th

g / liter

Peptone

5

g / liter

CaC03

2nd

g / liter

Tap water q. s. on

1

liter

Example 3

Separation of the antibiotic A-28086 complex produced by S. aureofaciens NRRL 5758

The entire fermentation broth (132 liters) obtained by the procedure described in Example 1 is filtered using a filter aid (Hyflo Supercel, namely a diatomaceous earth, Johns-Manville Products Corp.), whereby 97 liters of filtered broth are obtained. The filtered broth is extracted with an approximately equal volume of ethyl acetate. The ethyl acetate extract is separated from the aqueous phase and concentrated to a volume of about 500 ml. The concentrated ethyl acetate extract obtained in this way is added to a large excess of petroleum ether (Skelly Solve F; about 10 liters) in order to precipitate out and separate unwanted material. The separated filtrate is evaporated in vacuo to give the broth portion of the Antibioticum A-28086 complex (6.9 g).

The portion of the antibiotic A-28086 complex present in the mycelium is obtained by extracting the filtered mycelium twice with about half the volume of methanol (62 liters and 59 liters). The two methanol extracts are combined and concentrated under vacuum to remove the methanol. After this concentration, about 10 liters of an aqueous phase bleach back. The aqueous phase is adjusted to about pH 7.5 with dilute sodium hydroxide. The solution obtained is extracted twice with approximately equal volumes of ethyl acetate (9 liters and 10 liters). The ethyl acetate extracts are combined and then concentrated to a volume of approximately 400 ml. The concentrated ethyl acetate extract thus obtained is added to a large excess of petroleum ether to remove unwanted materials, which is done in exactly the same way as for the concentrated filtered broth extract. The portion of the A-28086 antibiotic complex present in the mycelium from the filtrate in this way weighs 20.6 g.

Example 4

Isolation of the individual factors A and B from A-28086

The mycelium portion of the A-28086 antibiotic complex (235 g, prepared according to Example 3) is dissolved in about 80 ml of benzene. The benzene solution is applied to a silica gel column (9X130 cm, 8 liters, Matheson silica gel type 62). The column is eluted with varying mixtures of benzene and ethyl acetate. The progress of the elution is monitored by thin layer chromatography. Using a solvent system consisting of benzene and ethyl acetate (90:10), factor B is first eluted and isolated as a single factor. Factor B (43 g) recrystallized from acetone water melts at 150 to 153 ° C.

It is further eluted with mixtures of benzene and ethyl acetate, but gradually increasing the amount of ethyl acetate. The factor A is eluted. The various fractions containing factor A are combined and evaporated to a residue under vacuum. The residue thus obtained is dissolved in acetone (about 150 ml) and water (about 150 ml) is added to the acetone solution. The pH of the solution obtained is then adjusted to pH 3 by adding 1N hydrochloric acid. The acidified mixture is stirred for about 1 hour, during which time a precipitate forms. The precipitate is separated off by filtration and recrystallized from acetone (approximately 150 ml) with the subsequent addition of water (approximately 60 ml). The product obtained is dried under vacuum overnight, giving factor A (about 6.6 g). After partial evaporation of the acetone from the filtrate, a second crop of factor A (about 1.2 g) is obtained.

Example 5 A-28086 Factor A Acetylester

Antibioticum A-28086 factor A (7.4 g) is dissolved in pyridine (150 ml). Acetic anhydride (50 ml) is added to the solution. The solution obtained is mixed thoroughly, after which it is left overnight at room temperature

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

621687

24th

lets stand. Water (200 ml) is then added with thorough mixing. The mixture thus obtained is left to stand at room temperature for 4 hours. The white solid that precipitates is separated off by filtration, washed with water and air-dried. The solid obtained is dissolved in acetone (100 ml) and the acetone solution is evaporated to dryness in vacuo (the above procedure is repeated three times). After recrystallizing this solid from acetone (100 ml) and water (50 ml), A-28086 factor A acetylester derivative (6.14 g) is obtained, which melts at 100-103 ° C.

Examples 6-9

a) According to the process described in Example 5, by reacting factor A with propionic anhydride in the presence of pyridine antibiotic A-28086, factor A propionyl ester derivative is produced, which melts at 96-98 ° C.

b) According to the process described in Example 5, by reacting factor A with n-butyric anhydride in the presence of pyridine antibiotic A-28086, factor A n-butyryl ester derivative is produced, which melts at 79-81 ° C.

c) The process described in Example 5 is followed by reacting factor A with caproic acid anhydride in the presence of pyridine antibiotic A-28086 and factor A n-caproyl ester derivative which melts at 163-167 ° C.

d) According to the process described in Example 5, reacting factor A with valeric acid anhydride in the presence of pyridine antibiotic A-28086 produces factor n n-valerylester derivative which melts at 173-175 ° C.

Example 10

Preparation of A-28086 Factor A sodium salt

Antibioticum A-28086 factor A (500 mg) is dissolved in acetone (50 ml). Water (50 ml) is added to the solution obtained, after which the pH of 5N sodium hydroxide is adjusted to 10.5 to 11. The solution is stirred for 1 hour and then extracted with ethyl acetate. The ethyl acetate extract is evaporated to dryness in vacuo. The residue is precipitated from acetone-water solution, giving 378 mg of A-28086 factor A sodium salt, which melts at 120-123 ° C.

Examples 11-15

a) By reacting Antibioticum A-28086 factor A (500 mg) with saturated barium hydroxide using the method described in Example 10

369 mg A-28086 factor A barium salt that melts at 188-190 ° C.

b) By reacting Antibioticum A-28086 factor A (500 mg) with 5 N potassium hydroxide according to the method described in Example 10, 363 mg of A-28086 factor A potassium salt are obtained, which melts at 165-167 ° C.

c) By reacting Antibioticum A-28086 factor A (500 mg) with 1N cesium hydroxide according to the method described in Example 10, 540 mg of A-28086 factor A cesium salt is obtained, which melts at 190-210 ° C.

d) By reacting Antibioticum A-28086 factor B with 5N sodium hydroxide according to the method described in Example 10, Antibioticum A-28086 factor B sodium salt is obtained.

Example 16

Shake bottle fermentation of A-28086 using S. aureofaciens NRRL 8092

A culture of Streptomyces aureofaciens NRRL 8092 is prepared and kept on sloping agar of the following composition:

Components

Amount k2hpo4

2 g

MgS04. 7H20

0.25 g nh4no3

2 g

CaC03

2.5 g

FeS04. 7H20

0.001 g

MnCl2. 7HaO

0.001 g

ZnS04. 7H20

0.001 g

glucose

10 g

Agar

20 g

Deionized water q. s. on

1 liter pH (not adjusted)

7.7

The slant agar is inoculated with Streptomyces aureofaciens NRRL 8092, after which the inoculated slant is incubated at 30 ° C. for about 7 days. The mature slant culture is covered with sterile bovine serum and scraped off with a sterile loop, whereby a spore and mycelium suspension is obtained from the slant culture. The suspension obtained is lyophilized to a maximum of 6 pellets.

A lyophilized pellet produced in this way is used to inoculate 50 ml of a vegetative medium of the following composition:

Components

amount

glucose

20 g

Soybean flour

15 g

Corn steep liquor

10 g

CaC03

2 g

Tap water q. s. on

1 liter

The pH is adjusted to pH 6.5 with dilute NaOH.

The inoculated vegetative medium in a 250 ml Erlenmeyer flask is incubated at a temperature of 30 ° C. over a period of 48 hours on a rotary shaker which rotates under an arc of 5 cm at a speed of 250 revolutions per minute.

The incubated vegetative medium (0.5 ml, 1%) described above is used to inoculate 50 ml of a fermentation medium of the following composition:

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

25th

621687

Ingredients amount

Tapiocadextrin1)

60.0

G

Casein hydrolyzed with enzyme2)

6.0

G

Enzymatic hydrolyzate of casein3)

2.0

G

CaCOa

2.0

G

MgS04. 7H20

0.5

G

Rummelasse (Blackstrap Molasses)

15.0

G

Refined soy

5.0

ml

Tap water q. s. on

1

Liter pH (not adjusted)

6.6

*) Staley Dextrin No. 11, A.E. Staley Co, Decatur III.

2) Amber EHC, Amber Laboratories, Juneau, Wisc.

3) NZ Amin A, Sheffield Chemical Co., Norwich, N.Y.

Example 17

Tank fermentation of A-28086 using S. aureofaciens NRRL 8092

The method used in Example 16 for the shake flask fermentation of A-28086 is also used for tank fermentation. To form a larger volume of inoculum, 10 ml of the incubated vegetative medium are used to inoculate 400 ml of a second vegetative medium, which has the same composition as the first vegetative medium. This second medium is incubated in a 2 liter Erlenmeyer flask at 30 ° C. over a period of 24 hours on a rotary shaker at a speed of 250 revolutions per minute and under an arc of about 5 cm.

The new compounds increase the proportion of propionates and thus the efficiency of feed utilization when they are administered orally to ruminants in amounts of about 0.05 to about 5.0 mg per kg and day. The best results are obtained with an administration of about 0.1 to about 2.5 mg of active ingredient per kg and day. A preferred method for administering the active substances mentioned is mixing with the animal feed, but the active substances can also be administered in other ways, for example in the form of tablets, drinkers, boluses or capsules. These various dosage forms can be formulated in a manner known in veterinary medicine.

Each individual dosage unit should contain an amount of active ingredient that corresponds directly to the respective daily dose with which the animal is to be treated.

The A-28086 can be used in conjunction with fattening feed containing a beef feed and 1 to 30 g of active ingredient per ton. As is well known, cattle feed differs from other feed such as poultry feed. Cattle feed contains raw feed such as cotton seed pods or corn silage. Beef feed also contains a high percentage, often 15 to 25%, of non-proteinaceous nitrogen sources. A commonly used non-protein type nitrogen source is urea.

As mentioned above, the new A-28086 compounds are antiviral agents and they are also effective against anaerobic bacteria, particularly Clostridium perfringens. The A-28086 compounds are therefore suitable for the treatment or prevention of enteritis in chickens, pigs,

Cattle and sheep. The new compounds A-28086 can also be used for the treatment of enterotoxemia in ruminants.

The resulting incubated vegetative medium of the 5 second stage (800 ml) is used to inoculate 100 liters of sterile fermentation medium of the following composition:

Ingredients amount

Tapiocadextrin *)

60.0

g / liter

Casein hydrolyzed with enzyme 2)

6.0

g / liter

Enzymatic hydrolyzate of casein 3)

2.0

g / liter

CaC03

2.0

g / liter

MgS04. 7H20

0.5

g / liter

Rummelasse (Blackstrap Molasses)

15.0

g / liter

Refined soy

5.0

g / liter

Tap water q. s. on

1

liter

*) Staley Dextrin No. 11, A.E. Staley Co., Decatur, III.

2) Amber EHC, Amber Laboratories, Juneau, Wisc.

3) NZ Amin A, Sheffield Chemical Co., Norwich, N.Y.

After sterilization in an autoclave at a temperature of 121 ° C. over a period of 30 minutes at a pressure of 1.05 to 1.41 kg / cm 2, the medium has a pH of 6.8 ± 0.1 . The inoculated production medium is left to ferment in a 165 liter fermentation tank for 19 to 12 days at a temperature of 28 ± 1 ° C 35. The fermentation medium is aerated with sterile air at a rate of 0.4 volumes of air per volume of culture medium per minute. The medium is stirred with conventional stirrers at a speed of 300 revolutions per minute.

40

Example 18

Antibiotics A-28086 are prepared by the process described in Example 17, but using a shake bottle tank production medium of the following composition:

Ingredients amount

50

Tapiocadextrin 2)

30.0

g / liter

glucose

15.0

g / liter

Casein hydrolyzed with enzyme 2)

3.0

g / liter

55

Enzymatic hydrolyzate of casein 3)

1.0

g / liter

Yeast extract

2.5

g / liter

CaC03

2.0

g / liter

60

MgS04. 7H20

1.0

g / liter

Rummelasse (Blackstrap Molasses)

15.0

g / liter

Refined soy

5.0

ml / liter

65

Tap water q. s. on

1

liter

1) Staley Dextrin No. 11, A.E. Staley Co., Decatur, III.

2) Amber EHC, Amber Laboratories, Juneau, Wisc.

3) NZ Amin A, Sheffield Chemical Co., Norwich, N.Y.

621 687

26

After sterilization in an autoclave, the pH of the medium is adjusted to 6.4 in the manner described in Example 17.

Example 19

Separation of the A-28086 antibiotic complex produced by S. aureofaciens NRRL 8092

The total fermentation broth (60 liters) obtained by the method of Example 17 is adjusted to pH 3 by adding dilute hydrochloric acid. The solution obtained is filtered using a filter aid (Hyflo Su-percel, namely a diatomaceous earth, Johns-Manville Products Corp.). The separated mycelium cake is extracted with 30 liters of methanol, whereupon the extract is mixed with 1.56 kg of sodium bicarbonate. After this extract has been separated off, the mycelium cake is extracted again with a further 30 liters of methanol. The two methanol extracts are combined and concentrated under vacuum to remove the methanol. The aqueous solution obtained (about 7 liters) is adjusted to pH 7.5 with dilute hydrochloric acid. This solution is then extracted twice with ethyl acetate (7 liters each). The ethyl acetate extracts are combined and concentrated in vacuo to an oily residue. The oily residue is dissolved in 1500 ml of acetone. Water (1500 ml) is added to the acetone solution. The solution obtained is adjusted to pH 3 with dilute hydrochloric acid and stirred for 1 hour. The resulting precipitate is filtered off and then dissolved in acetone (1500 ml), whereupon the solution is mixed with water (400 ml). The solution obtained is then left to stand for 16 hours until it crystallizes. The resulting crystals are filtered off and dried under vacuum to give 74 g of crude crystalline product containing A-28086 factors A and B and other crystalline impurities.

The above crystalline product (40 g) is dissolved in about 250 ml of benzene. The benzene solution is then placed on a silica gel column (9X120 mm, silica gel from Grace-Davidson, grade 62). The column is in turn with each

40 1 of the following solvents eluted:

1) benzene

2) Benzene: ethyl acetate

(9:

: D

3) Benzene: ethyl acetate

(4:

: 1)

4) Benzene: ethyl acetate

(7:

: 3)

5) Benzene: ethyl acetate

(1:

: 1)

6) Ethyl acetate

7) methanol

Fractions of 1 liter each are collected. Each fraction is examined by a corresponding experiment against Bacillus subtilis and evaluated by thin-layer chromatography to identify the eluted compounds. The A-28086-I is mixed with a mixture of benzene and. Ethyl acetate (4: 1) eluted. A-28086 factor B is eluted with a mixture of benzene and ethyl acetate (7: 3). A-28086 factors A and D are eluted in the fractions obtained with the mixtures of benzene and ethyl acetate (7: 3 and 1: 1), namely fractions 119 to 156. These fractions are combined and evaporated to dryness in vacuo. The residue obtained is dissolved in acetone (500 ml). Water (500 ml) is added to the acetone solution, whereupon the pH of the aqueous solution obtained is adjusted to pH 3 with dilute hydrochloric acid and the solution is stirred for 1 hour. The resulting precipitate is filtered off and extracted from acetone (500 ml) and water (180 ml)

recrystallized. The resulting crystals are filtered off and dried under vacuum, giving 20.1 g of a mixture of A-28086 factors A and D.

5

Example 20

Separation and cleaning of the individual factors A and B

The crystalline mixture of io factors A and D of A-28086 (18.8 g) obtained according to Example 18 is dissolved in benzene (50 ml). The benzene solution is applied to a silica gel column (7 × 100 cm column, E. Merk silica gel, grade 60, finer than 0.062 mm). The column is eluted at a flow rate of 90 ml per hour in order using the following eluents:

D

12 liters of benzene

2)

12 liters of benzene:

: Ethyl acetate

(9:

D

3)

12 liters of benzene:

: Ethyl acetate

(4:

1)

4)

12 liters of benzene

: Ethyl acetate

(7

: 3)

5)

10 liters of methanol

The elution process is followed by thin layer chromatography with cellulose (Merk. Darmstadt, cellulose on an aluminum support) and bioautographically using B. subtilis. The following solvent systems are used: water: methanol: acetone (12: 3: 1), the pH of the solution being adjusted first to 30 pH 10.5 with ammonium hydroxide and then to pH 7.5 with hydrochloric acid.

Fractions of 1 to 2 liters are collected until activity is found, and then fractions of 200 ml are collected. The fractions containing only A-28086 factor D are combined and evaporated in vacuo to a residue. The residue is recrystallized from acetone-water (1: 1). The crystals are separated and dried under vacuum, giving 140 mg of crystalline A-28086 factor D. Those fractions which contain A-28086 factor D with a trace A-28086 factor A-40 are treated in the same way to obtain a further 150 mg of crystalline A-28086 factor D which contains a small amount of A-28086 factor A.

The fractions containing only A-28086 factor A are also treated in the same manner as that by which 4.7 g of crystalline A-28086 factor A are obtained.

Example 21

A-28086 antibiotics are prepared according to the procedure described in Example 16, but using a sloping medium of the following composition:

Composition amount

55 beef extract 2.00 g

Dextrin 20.00 g

Yeast extract 2.00 g go Enzymatic hydrolyzate of casein 4.00 g

CoCl2. 6H.O 0.02 g

Agar 20.00 g

Deionized water q. s. to 1 liter 65 n

The pH is 7.0 with KOH

set.

27

621687

The inoculated slant is incubated at a temperature of 28 ° C for about 7 days.

Example 22

Antibiotics A-28086 are prepared according to the procedure described in Example 17, but using a flask production medium of the following composition:

Ingredients amount

Tapiocadextrin l)

80

g / liter

Casein hydrolyzed with enzyme 2)

15.0

g / liter

Rummelasse (Blackstrap Molasses)

15.0

g / liter

Calcium carbonate

2.0

g / liter

Ammonium sulfate

1.0

g / liter

Magnesium sulfate

0.5

g / liter

Refined soy

4.6

g / liter

1) Staley Dextrin No. 11, A.E. Staley Co., Decatur, III.

2) Arab EHC, Amber Laboratories, Juneau, Wisc.

Example 23

A-28086 antibiotics are produced by the process described in Example 21, but using a vegetative medium of the following composition as the third stage in between:

Ingredients amount

Cerelose

20.0

g / liter

Corn steep liquor (wet weight)

10.0

g / liter

Soybean meal

15.0

g / liter

CaC03

2.0

g / liter

yeast

2.0

g / liter

Beet molasses

5.0

g / liter

Example 24 Beef feed containing A-28086

A balanced high-grain cattle feed is made from the following ingredients:

Components

%

kg

Finely ground corn

67.8

615.6

Ground corn cobs

10th

90.7

Dehydrated alfalfa flour 17% protein

5

45.4

Flour from extracted soybeans, extracted with solvent, 50% protein

9.9956

90.76

Raw sugar molasses

5

45.4

urea

0.6

5.44

Components

%

kg

A-28086 factor A.

0.0044

0.04

Dicalcium phosphate, feed quality

0.5

4.54

Calcium carbonate

0.5

4.54

Sodium chloride

0.3

2.72

Trace mineral premix

0.03

0.27

Premix of vitamins A and D2 1)

0.07

0.63

Premix of vitamin E2)

0.05

0.45

Calicum propionate

0.15

1.36

!) Contains per kg: 4405300 I.E. Vitamin A, 5004400 IU

Vitamin D2 and 849.5 g soybean feed with 1% added oil. 2) Dried corn stills with solubles that

440530 I.E. Contain d-alpha-tocopheryl acetate per kg.

The feed mixture is pressed into pellets. With an average daily intake of 6.8 kg of feed per animal, this feed gives you about 300 mg of A-28086 factor A per animal and day.

Example 25 A-28086 Factor D Acetylester

Antibioticum A-28086 factor D is dissolved in pyridine. The solution is mixed with a stoichiometric amount of acetic anhydride. The solution obtained is mixed thoroughly, after which it is left to stand overnight at room temperature. The solution obtained is then mixed with an excess of water, whereupon everything is mixed thoroughly and the mixture is left to stand for several hours at room temperature. The resulting precipitate is filtered off, washed with water and dried. The solid obtained is dissolved in acetone and evaporated to dryness in vacuo, giving A-28086 factor D acetylester.

Examples 26-29

a) According to the method described in Example 26, A-28086 factor D is reacted with propionic anhydride in the presence of pyridine to give antibiotic A-28086 factor D propionyl ester derivative.

b) According to the process described in Example 26, A-28086 factor D is reacted with n-butyric anhydride in the presence of pyridine to antibiotic A-28086 factor D n-butyl acrylate derivative.

c) According to the method described in Example 26, A-28086 factor D is reacted with caproic anhydride in the presence of pyridine to antibiotic A-28086 factor D n-Ca-proylester derivative.

d) According to the process described in Example 26, A-28086 factor D is reacted with valeric anhydride in the presence of pyridine to antibiotic A-28086 factor D n-valerylester derivative.

Example 30

Preparation of A-28086 factor D sodium salt

Antibioticum A-28086 factor D is dissolved in acetone. The solution is mixed with an equivalent amount of water, after which enough 5 N sodium hydroxide is added that

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

621687

28

the pH adjusts to around 11. The resulting solution is stirred for about 1 hour and then extracted with ethyl acetate. The ethyl acetate extract is evaporated in vacuo to give A-28086 factor D sodium salt.

Examples 31-33

a) According to the process described in Example 31, A-28086 factor D is converted with 5N potassium hydroxide to antibiotic A-28086 factor D potassium salt.

b) According to the method described in Example 31, A-28086 factor D is converted with saturated barium hydroxide to antibiotic A-28086 factor D barium salt.

c) According to the method described in Example 30, A-28086 factor D is converted with 1N cesium hydroxide to antibiotic A-28086 factor D cesium salt.

Example 34 A-28086 factor D cattle feed

A balanced high-grain cattle feed is made from the following ingredients:

Components

%

kg

Finely ground corn

5 Ground corn cobs

Dehydrated alfalfa flour 17% protein

I0 Flour from extracted soybeans, extracted with solvent, 50% protein

Raw sugar molasses

15 urea

25th

30th

A-28086 factor A.

Dicalcium phosphate, feed quality

Calcium carbonate

Sodium chloride

Trace mineral premix

Premix of vitamins A and D, ï)

Premix of vitamin E 2)

Calcium propionate

67.8 10

9.9956 5

0.6

0.0044

0.5 0.5 0.3 0.03

0.07 0.05 0.25

615.6 90.76

45.4

90.7 45.4 5.44 0.040

4.54 4.54 2.72 0.27

0.63 0.45 1.36

1) Contains per kg: 4405300 I.U. Vitamin A, 5004400 IU

Vitamin D2 and 849.5 g soybean feed with 1% added oil.

2) Dried corn stills with solubles containing 440530 I.E. Contain d-alpha-tocopheryl acetate per kg.

35

The mixed feed is pressed into pellets. With an average daily feed quantity of 6.8 kg of feed per animal, this feed provides about 300 mg of A-28086

Factor D per animal and day.

v

8 sheets of drawing egg

Claims (6)

621687 1. Method of the new antibiotic A-28086 complex with the factors A, B and D or the physiologically acceptable salts thereof or the corresponding C2-Cc-acyl esters to increase the feed intake in ruminants. 2. Use according to claim 1 of factor A of the antibiotic A-28086 complex or the physiologically acceptable salts or the C2-Cu-Acy] esters thereof. 2nd PATENT CLAIMS 3. Use according to claim 1 of factor D of the antibiotic A-28086 complex or the physiologically acceptable salts or the C2-C (i-acyl esters thereof. 4. A process for the preparation of the new antibiotic A-28086 complex with the factors A, B and D, characterized in that Streptomyces aureofaciens NRRL 5758 or Streptomyces aureofaciens NRRL 8092 in a culture medium, the assimilable sources of carbohydrate, nitrogen and inorganic salts contains, submersed under aerobic fermentation conditions until these organisms have produced a substantial amount of antibiotic activity in the culture medium and separate the Antibioticum A-28086 complex from the culture medium. 5. A process for the preparation of the new factors A, B and D of the antibiotic A-28086 complex or the production of the physiologically acceptable salts of these factors, characterized in that the antibiotic A-28086 complex with the Factors A, B and D are generated and separated from the culture medium and the factors A, B and D are then isolated and, if necessary, converted into the physiologically acceptable salts. 6. A process for the preparation of C2-Cc-acyl esters of the new factors A, B and D of the antibiotic A-28086 complex, characterized in that the factors A, B and D are prepared by the process according to claim 5 and then converted into the corresponding C2-Cβ-acyl esters by reaction with an acylating agent.