PL108561B1 - Method of producing antibiotic mixture a-35512,containing agents a,b,c,e,f,g and h - Google Patents

Description

The subject of the invention is a method for the preparation of the A-35512 antibiotic mixture containing microbiologically active and related factors A, B, C, E, F, G and H. Antibiotics A-35512 are antimicrobial and growth stimulating agents and increase absorption In addition, factor B of antibiotics A-35512 is useful in the treatment of tooth decay and acne. A-35512 antibiotics are closely related glycopeptide antibiotics. Factor B of the antibiotic A-35512, the most well-characterized component of the antibiotic complex A-35512, belongs to the group of peptide-containing antibiotics, such as vancomycin (US Patent No. 3,067,099), antibiotics A-4696 A, B and C (U.S. Patent No. 3,952,095), avoparcin (U.S. Patent No. 3,855,410), ristomycin A (N. Lomakina, 7th International Symposium of Chemistry of Natural Products, Riga, 1970, - p. 625) and ristocetin A (US Patent No. 2,990,329). Antibiotics A-35512 differs from the above known antibiotics, including mobility in different chromatographic systems and amino acid and sugar composition. Although many antibacterial agents are known today, there is still a need to search for new and better antibiotics. One of the problems of modern antibiotic therapy is the fact that different antibiotics are effective against pathogenic microorganisms. On the other hand, strains of microorganisms become resistant to the effects of currently used antibiotics. In addition, individual patients often experience severe side effects after administration of certain antibiotics, such as hypersensitivity and / or toxic effects. Therefore, it is advantageous to produce new antibiotics which are active in microbial diseases. is to obtain feed additives that increase the efficiency of feeding animals and poultry. Increasing the efficiency of feeding is of increasing importance. Diethylstilbestrol and other estrogens are agents that increase the feeding efficiency of many animals and birds. However, there is a risk that the remnants of these feed additives will end up in the meat intended for consumption. Therefore, there is a real economic need to search for new ways to increase the efficiency of the organic feed resources available for the production of meat from ruminants and poultry. The use of the A-35512 antibiotic mixture or the individual agents and their derivatives is an advance in this. The method according to the invention produces the antibiotic mixture A-35512, containing the factors A, B, C * E, F, GiH. The method according to the invention consists in growing the new strain Streptomyces candidus NRRL 8156 in a nutrient containing digestible sources of carbohydrates, nitrogen and inorganic salts, under subsurface fermentation with aeration, until significant antibiotic activity is achieved. Antibiotic mixture A-35512 possibly separates from the broth and, if necessary, the factors A, B, C, E, F, G and H are isolated from it. The number and relationship of the individual factors in the antibiotic mixture depends on on the fermentation conditions used. The antibiotic substances obtained by the process of the present invention are named A-35512 antibiotics. The essential ingredient in this mixture is factor B. The various factors of the A-35512 mixture are separated from the mixture in the form of hydrochlorides. Each of the factors can be converted into a free base, diffusing chloride ions, by means of, for example, chromatography. on the base of the ion exchange resin. In addition to specific bases and hydrochlorides, also useful are the pharmaceutically acceptable salts of the individual A "35512" compounds. Abbreviation for "Compound A-35512" to the A p, C, E and H compounds A-35512 and their pharmaceutically acceptable The individual factors of A-35512 according to the invention are closely related compounds. As an antibiotic mixture, A-35512 is isolated from the fermentation broth as many as seven antibiotics. The individual factors are separated and the factors A, B, C, B are isolated. and H as previously described. A-35512 mixture is soluble in water, partially dissolved in alcohols such as methanol and ethanol, but insoluble in other organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane. The accompanying figures show the infrared spectra of potassium bromide pellets of the individual factors A-35512, where Figure 1 shows the spectrum of factor A-35512-A dichloride; Figure 2 is the spectrum of factor A-35512-B dihydrochloride; 3 is the spectrum of the hydrochloride of factor A-35512-H; Fig. 4 is the spectrum of factor A-35512-C dihydrochloride and Fig. 5 is the spectrum of factor A-3S512-E hydrochloride. Factor A-35512-A is a white, amorphous, basic substance with the following elemental composition: C - 54.29%, H - 5.19%, N - 5.58%, O - ± 33.76%, Q - 1.69%, Factor A-35512-A dihydrochloride is white, amorphous, hygroscopic a substance with the following average elemental composition: C - 51.03%, H - 5.10%, K - 4.75%, O - 34.20%, Cl - 4.80%. Infrared spectrum, in a tablet from potassium bromide, factor A-35512-A dihydrochloride is shown in Fig. 1. The most important absorption bands occur at the following frequencies (cm-1): 3405 (strong), 3300 (bend), 2950 (weak) * 1750 (weak), 167Ó (sime), 1625 (bend), 1602 (sime), 1520 (sime), 1470 (weak), 1440 (weak), 1345 (bend), 1312 (medium), 1225 (average), 1180 (weak), 1080 (strong) and 1020 (break). In the ultraviolet absorption spectrum of A-35512-A there is a maximum abso in acid and inert methanol at 282 mm (2T = 11,700) and in basic methanol at 292 mm (Z = 14,000), the molar extinction coefficient being calculated for a molecular weight of 2000. There is also a band of final absorption in the ultraviolet spectrum at 225 nm. In "nuclear carbon magnetic resonance spectrum ** Q /" in heavy water, factor A- 3351 & A dihydrochloride: shows the signals listed in Table 1. 4 Table 1 1 No. and 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 1 n 1 PPm 2 175.3 173.0 172.1 1 171.4 170 , 9 170.5 169.5 159.0 157.9 156.2 155.6 155.3 154.4 136.3 136.0 135.1 133.5 129.6 129.1 128.7 127.5 126.0 124.3 122.1 109.9 107.4 101.7 1 77.6 76.3 75.5 74.8 74.5 73.4 72.8 72.0 70.9 69.6 67 , 4 65.4 61.7 56.7 55.5 54.7 24.6 19.1 17.9 17.2 and 16.7 16.2 height (%) ~ "" 0.8 2.1 2 , 0 1.5 2.7 2.3 1.6 1.3 23 2.6 2.3 2.4 1.1 1.4 i, o ¦¦¦¦¦¦- M 1.2 1.6 1.7 1.8 1.0 1.4 2.8 1.6 W 1.6 0.9 3.8 4.6 2.6 2.5 2.4 1 3.7 6.0 4.4 7.0 2.8 90.9 * 1.7 3.1 1.7 1.3 0.8 0.9 1.7 2.0 1.9 3.2 \ 3.0 *) signal used as standard of dioxane Factor A-35512-A dihydrochloride shows the following values of specific condensation: [a]] = -100 ° M (C = 1, water) and [a] _l = -400 ° (C = 1, water). electrometric measurements of factor A-35512-A dihydrochloride in a 66% aqueous solution of dimethylformamide indicate the presence of four titratable groups with pKa values of about 7 , 35, 9.09i ss 19.49 and 12.44 (initial pH value was 6 * 2) 108 561 5 Probable molecular weight of factor A-35512-A dihydrochloride is titrated around 2106. Factor A-35512-A dihydrochloride it is soluble in water, partially soluble in alcohols such as methanol and ethanol, and insoluble in other organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane. A-35512-A is stable for 72 hours in aqueous solutions having a pH of about 3 to about 10. Amino acid analysis of the hydrolyzed acid of factor A-35512-A dihydrochloride shows the presence of at least five amino acid residues, one of which is glycine. Factor A-35512-B is a white, amorphous, basic substance with a similar empirical formula C ^. ^ H101J105N8.fO46.48Cl and the following approximate elemental composition: C - 53.97%, H - 4.75% N - 5.25 %, O - 34.29%, ClR - 1.59%. In particular, the given results of elemental analysis are consistent with the preferred empirical formula C98H104N49-047C1 (calculated: C - 53.60, H - 4.75, N - 574 O - * 34.30, Cl - 1.61). Another preferred sum formula is CwH103NaO47Cl (calculated: C - 54.00 H - 4.75, N - 5.15, O - 34.50, Cl - 1.60). In the ultraviolet absorption spectrum of factor A-35512 - -B there is a maximum in acid and neutral methanol at 282 nm (Z = 15.000) and in basic methanol at 292 nm (27 = 16.000), with the molar extinction coefficient calculated for a molecular weight of 2000. In the ultraviolet spectrum there is also a final absorption band at 225 nm. The A-35512-B factor shows the following specific conductivity values: [a] £ = —123 ° (c = 1, water) and [a] "= —446 * (c = 1, water). Electrometric titration factor A-35512-B in a 66% aqueous solution of dimethylformamide indicates the presence of four titratable groups with pKa values of around 7.15, 8.81, 20.20, 12.00 and the possible presence of a group with a pKa greater than 13 , 50. The probable molecular weight of factor A-35512-B according to a titration is around 2143. The dihydrochloride of factor A-35512-B is after crystallization. From a 50% aqueous solution of methanol, a white crystalline substance. Although factor A-35512-B dihydrochloride is hygroscopic and does not have a pronounced melting point, the thermogram shows a weight loss starting at 25 ° C and a 7.4% loss at 121 ° C and decomposition at temperature. 135 # C. Factor A-35512-B dihydrochloride has the following elemental composition: C - 52.57%, H - 4.80%, N - 5.66%, O - 32.86%, Cl - 4.51 The given results of elemental analysis are in particular consistent with another alternative empirical formula CwH ^ NiC ^ Cl. 2HCl (calculated: C - 51.93, H - 4.65, N - 5.57, O - 33.20, Cl - 4.65). In the ultraviolet absorption spectrum of factor A-35512-B dihydrochloride there is a maximum in acidic and neutral methanol at 282 nm (Z1 = 12,000) and basic methanol at 292 nm (E - 14,000), the molar extinction coefficient was calculated for the molecular weight of 2000. There is also a final absorption band at 225 nm in the ultraviolet spectrum. Factor A-35512-B dihydrochloride in the list they have the correct values of proper skewness: [«] !! = —128 ° 5 (c = 1, water) and [a] II = —475 ° (c = 1, water). Electrometric titration of factor A-35512-B dihydrochloride in 66% aqueous dimethylformamide solution indicates the presence of four A titratable group with pKa values of around 7.15, 8.87, 10.30 and 12.10 and the possible presence of a group with a pKa greater than 13.1 The likely molecular weight of factor A-35512-B dihydrochloride is based on the titration 1C around 2027. In the 13C nuclear magnetic resonance spectrum of factor A-35512-B dihydrochloride in D20, there are signals presented in Table 2 Table 2: 1 No. * 2 ~~ 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 1 \ 40 41 42 43 | ppm '2 ¦ 173.0 171.9 171.6 171.0 170.8 169.6 159.0 157.9 157.5 156.6 155.6 155.3 154.9 154.3 151.7 144 , 3 136.7 136.2 135.4 135.2 133.6 133.3 129.8 129.3 128.8; 127.6 126.1 ', .124.2 / 122.4 122.0 120.7 116.5 109.5 108.2 107.7 104.5 101.8 100.9 98.2 76.9 76 , 1. 74.1 | height (%) r * - r- 4i -¦ 3.7, 33 5.8 5.0 3.6 4.1 4.4 3.7 4.8 6.1 4.2 3.3 4.2 3.3 3.1 3.5 4.9 4.0 4.4 4.2 4.1 in 3.0 2.6 and 1.5 3.9, 5.6 M 4.4 3.3 2 , 7 0.8 W 2.7 17 * 2.9 1.6 1.0 1.2 1.8 2.0 \ 108 561 7 cd Table 2 1 1 44 45 46 47 48 49 50 51 52 53 54 55 56 57. 58 59 60 2 73.5 72.7 72.3 71.0 70.3 69.7 67.4 64.6 62.0 58.0 56.8 55.4 54.3 24.5 17.9 17 , 2 16.3 3 2.7 2.4 4.0 7.1 ¦ 2.5 2.5 74.7 * 1.2 W 1.3 1.7 3.9 2.5 2.0 3, 0 2.0 2.5 | *) signal of dioxane used as a standard Factor A-35512-B dihydrochloride, crystallized from a mixture of methanol - water, has an X-ray spectrum, made by the powder method (Cu + + radiation, 1.5405 X nickel filter, d is the interplanar distances in Angstroms) with the characteristics shown in Table 3 Table 3 | | relative intensity 1 17.15 12.90 10.85 9.25 8.87 8.22 7.86 6.93 6.20 5.62 5.04 4.02 3.54 | 100 80 70 70 60 50 50 40 40 40 05 02 02 | The infrared spectrum of factor A-35512-B dihydrochloride, made in a potassium bromide tablet, is shown in Fig. 2. The most important absorption bands appear at the following frequencies (cm-1): 3420 (strong), 3300 (bend), 2950 (weak) , 1752 (weak), 1675 (strong), 1630 (bend), 1605 (strong), 1520 (strong), 1470 (weak), 1440 (weak), 1410 (weak), 1345 (bend), 1312 (medium no), 1225 (moderate), 1180 (weak), 1135 (weak), 1080 (strong) and 1020 (weak) Amino acid analysis of acid-hydrolyzed factor A-35512-B shows that at least one furnace is present. amino acid residues, one of which is glycine. The four remaining amino acid residues for factor A-35512-B form a complex and appear to be identical to those found in factor A-35512-A. One of these amino acid residues is probably structured by the formula shown in the figure. Analysis of the acid hydrolysis products indicates that factor A-35512-B dihydrochloride contains the following 8 sugars: glucose, fucose, mannose, rhamnose and 3-amino-2, 3,6-triodesoxy-3-C-methyl-L-xyl-hexopyranose. The mild acid hydrolysis removes glucose, fucose, mannose, and rhamnose to give the characteristic aglycone derivative. Factor A-35512-B dihydrochloride has at least one esterifiable hydroxyl group. Factor A dihydrochloride 35512-B is soluble in water, partially soluble in alcohols such as methanol and ethanol, and insoluble in other less polar organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane. Factor A-35512-B dihydrochloride is stable for up to 72 hours in aqueous solutions with a pH of about 3 to about 10. Factor A-35512-C is a white, amorphous, basic substance with the following composition approx. tariff: C -. 53.93%, H - 5.15%, N - 5.80%, O - 32.35%, Cl — 1.90%. The given results of the elemental analysis are in particular consistent with the preferred empirical formula C ^ H ^ NgOjgCl. Factor A-35512-C has an approximate molecular weight of 1862. 25 Factor A-35512-C dihydrochloride is a white, amorphous compound with the following approximate elemental composition: C - 51.76%, H - 5.07%, N - 5, 61%, O - 30.29%, Cl - 4.88%. Factor A-35512-C dihydrochloride has a similar empirical formula: C83-85H97_101 N8037_39C13. Elemental analysis corresponds in particular to another favorable empirical formula: C84H97N8038C1 • • 2HCl (calculated: C - 52.2, H - 5.1, N - 5.8, O - 31.5, 35 Cl —5.4%). Infrared spectrum of factor A-35512-C dihydrochloride, made in a potassium bromide tablet is shown in Fig. 4. The most important absorption bands occur at the following frequencies (cm-1): 3370 (strong), 3280 (bend), 3040 (bend), 2980 (bend), 2920 (weak), 1740 (weak), 1658 (strong), 1620 (weak), 1589 (medium), 1503 (strong), 1460 (weak), 1428 (medium), 1385 (weak), - 1330 (weak), 1295 (medium), 1210 (strong) , 1162 (moderate), 1120 (weak), 1060 (strong) and 1005 (moderate): In the ultraviolet absorption spectrum of factor A-35512-C dihydrochloride there is a maximum in acid and neutral methanol at 282 nm (Z = 14,600) and in basic methanol at 292 nm (£ = 16,400), with the molar extinction coefficient calculated for the molecular weight of 2000. In the 13C carbon nuclear magnetic resonance spectrum performed in D20, factor A- -35512-C dihydrochloride shows the signals listed in the table 4. 55 Table4 | No. 1 1 1 2 3 4 5 6. ppm 2 172.9 172.2 171.5 171.0 169.6. 158.6 ..... height (%) 3: 0.1 2.5 1 2.0; . 2.2. 3.9 2.0. .IN. 1108 561 9 continued table 4] 1 7 8 9 10 11 12 13 14 15 16 17 18 19 | 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39. 40. . 41 42 1 43 44 1 2 1 157.8 J 156.5 156.1 155.6 154.6 151.1 143.3 136.0 135.4 133.2 128.7 126.5 124.6 • r 129.3 121.5 120.1 118.0 116.5 107.8 104.5 101.7 94.5 75.9 74.3 73.4 72.1 70.9 68.7 67.4 64, 3 62.2 56.1 55.2 54.2 24.3 17.9 17.1 16.2 | 3 3.0 2.1 2.8 4.2 3.9 1.5 1.4 3.2 2.7 3.7 2.3 3.1 2.1 2.7 2.2 1.2 1 , 7 1.2 2.7 1.9 1.9 1.0 3.0 2.0 2.3 3.5 4.3 2.9 71.7 * 1.3 1.6 1.4 3, 5 2.1 1.9 2.2 2.0 2.0 | ') the signal of the dioxane used as a standard Factor A-35512-C dihydrochloride shows the following values of specific capacitance: [a] "= -161 ° (c = 1.05, water) and [a] ™ 5 = -614 ° ( c = 1.05, water) Electrometric titration of factor A-35512-C dihydrochloride in a 66% aqueous solution of dimethylformamide shows the presence of three titratable groups with pKa values of around 7.30, 8.92 and 10.99 and Possible presence of two or more groups with pKa values above 11.5 The probable molecular weight of A-35512-C dihydrochloride, calculated from the titration, is around 1982. Amino acid analysis of acid-hydrolyzed factor A-35512-C dihydrochloride shows that the factor contains at least five amino acid residues, one of which is probably glycine. The remaining four amino acid residues have not yet been identified. Factor A-35512-C dihydrochloride is soluble in water, dimethyl sulfoxide and aqueous solution. dimethylformamide, partially soluble in alcohols such as methanol and ethanol, and insoluble in other less polar organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane. Factor A dihydrochloride -35512-C is stable for 146 hours in aqueous solutions with a pH of about 3 to about 10. Factor A-35512-E is a white, amorphous, basic substance with the following elemental composition: C - 54.84% , H - 4.73%, N - 5.26%, O - 32.67%, Cl - 1.72%. Factor A-35512-E hydrochloride is a white, amorphous substance with the following formula elemental: C - 52.67%, H - 4.59%, N - 5.55%, O - 33.51%, Cl - 3.62%. 15 The infrared spectrum of factor A-35512-E hydrochloride, made in a potassium bromide tablet, is shown in Fig. 5. The most important absorption bands occur at the following frequencies (cm-1): 3360 (strong), 3220 (bent), 2900 (weak), 1725 (weak), 20 1650 (strong), 1580 (medium), 1498 (strong), 1450 (weak), 1419 (weak), 1295 (average), 1205 (average), 1172 (average) no), 1110 (weak), 1060 (strong) and 1000 (weak). In the ultraviolet absorption spectrum of A-35512-E hydrochloride, the following absorption maxima occur: at 270 nm (bend) and 359 nm (Z = 16,216) in neutral methanol, at 286 nm (L = 18,018) and 310 nm (break) in acid methanol and at 270 nm (break) and 300 nm (27 = 16,216) in basic methanol, the molar extinction coefficient of - 30 for a molecular weight of 2000. Factor A-35512-E hydrochloride has a specific concreteness [a] "= -108 ° (c = 1, water). Electrometric titration of the hydrochloride factor and A-35512-E in a 66% aqueous solution of dimethylformamide shows the presence of three titratable groups with pKa values of around '6.30, 9.09 and 11.62 and the possible presence of one or two further groups with pKa values above about 12.5 (initial pH value was 5.45). 40 The probable molecular weight of factor A-35512-E hydrochloride, calculated from the titration, is around 2018. Amino acid analysis of the acid-hydrolyzed factor A-35512-E hydrochloride shows that factor A-35512-E hydrochloride contains six unidentified amino acids. Factor A-35512-E hydrochloride is soluble in water, partially soluble in alcohols such as methanol and ethanol, but soluble in most organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate , toluene, hexane, acetonitrile and dioxane. Factor A-35512-H is a white, amorphous, basic substance with the following elemental composition: C - 53.76%, H - 5.32%, N - 5.53%, O - 33.48%, Cl - 1.59%. The A-35512-H factor has the following approximate empirical formula: C85_87H103_io7N8038-4oCl, the preferred formula 60 total C86H104N8O39Cl and approximate molecular weight 1908. Factor A-35512-hydrochloride hydrochloride it is white, without the form, hygroscopic substance with the following elemental composition: C - 53.10, H - 5.37%, 65 N - 5.35%, O - 30.12%, Cl - 3.78%. 108 561 u 12 The infrared spectrum of factor A- -35512-H hydrochloride, made in a potassium bromide tablet, is shown in Fig. 3. The most important bands appear at the following frequencies (cm-1): 3410 (strong), 3240 (bend), 2940 (weak 1670 (strong), 1630 (bending), 1605 (strong), 1520 (strong), 1470 (weak), 1442 (weak), 1400 (weak), 1345 (bending), 1310 (medium), 1225 (moderate), 1180 (weak), 1080 (strong) and 1020 (curve). In the ultraviolet absorption spectrum of A-35512-H hydrochloride there is a maximum in neutral and acid methanol at 282 nm (27 = 12.500) and a maximum ¬mum in basic methanol at 292 nm (27 = 14,000), with the molar extinction coefficient calculated for a molecular weight of 2000. There is also a final absorption band at 225 nm in the ultraviolet spectrum. nuclear analysis of 13C carbon of factor A-35512-H hydrochloride, performed in D20, there are signals presented in Table 5. Table 5 1 No. 1 2 3 4 .-: 5 6 7 8 9 10 11 12 13 14; 15 16 17 18 19. 20 21 22 23 24 25 26 27 28 29 30 31 32 ¦ 33 34 35 36 37 38 PPm '2. 177.2 171.6 170.9 169.6 158.9 157.6 156.6 155.6 155.4 154.3 151.3 137.7 136.7 136.0 135.3 133.5 129, 4 127.3 126.1 124.2 122.6 107.6 101.8 76.2 73.5 72.3 71.0 69.7 67.4 61.6 56.8 55.4 1 55.0 24.5 17.9 17.2 16.3 height (%) "* 1.2.7. 5.2 5.8 4.7 3.1 43; ¦ '3.8 • •. 4.1 • 3.8. ¦ 2.4 ¦ ¦. 1.6 2.0 2.2 ¦4.0 1.9 5.0 3.7 1.3 3.2 6.9 4.1 2.7. 1.8 2.8 4.4 7.4 12.2 4.6 73.1 * 3.5 1.8 2.8 1.5 2.6 4.6 2.6 3.6 | 20 25 SO 35 40 45 50 55 Electrometric titration of A-35512-H hydrochloride in a 66% aqueous dimethylformamide solution shows the presence of five titratable groups with pKa values of about 5.0, 7.46, 9.80, 11.43 and 13.02 (initial value pH = 5.93) Probable molecular weight of A-35512-H hydrochloride, calculated from the titration, is around 1660. Amino acid analysis of acid-hydrolyzed factor A-35512 hydrochloride -H shows that the factor has at least five amino acid residues, one of which is glycine The remaining four amino acid residues for factor A-35512-H appear to be identical to those found in factors A-35512-A and B. Factor A-35512-H hydrochloride is water-soluble, partially soluble in alcohols such as methanol and ethanol and insoluble in most organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane; acetonitrile and dioxane. Factor A-35512-H hydrochloride is stable for 72 hours in aqueous solutions with a pH of about 3 to about 10. Factor A-35512-A, B, C, E, H and minor F factors and G can be conveniently separated by paper chromatography using a 15: 10: 3: 12 mixture of n-butanol, pyridine, acetic acid and water for development. The preferred method of development is bioautography using Sarcina Lutea. Approximate Rp values for A-35512 factors are given in Table 6 Table 6 A-35512 Factor A.2HC1 Factor B.2HC1 Factor C.2HC1 Factor E.HC1 Factor F Factor G H.HC1 Factor RF Value | 0.21 1 0.34 0.46 0.64 0.81 0.93 0.15. In many systems, factor A-35512-H had chromatographic characteristics very similar to that of the antibiotic AM374 described in U.S. Patent No. 3,700,768. Antibiotic agent A-35512-H can be distinguished from antibiotic AM374 by at least two paper chromatography systems. Table 7 shows the Rp values of A-35512-H (dihydrochloride) and the antibiotic AM374 in the above two arrangements. Table 7 *) Dioxane signal used as a standard Factor A-35512-H hydrochloride shows specificity [a] £ = —123.5 ° (c = 1, water). Solvent composition Acetic acid, 1 N hydrochloric acid (3: 1) n-propanol, NH4OH, water (6: 3: 1) RF value | A-35512-H 0.47 0.11 AM374 0.58 0.20 65 The individual factors of A-35512 can be separated by high-resolution liquid chromatography (HP-108 561 13 LC) using polyamide as stationary phase (type ZIPAX from Dupont ), and as the mobile phase, 1.33 molar aqueous solution of monobasic potassium phosphate, and used to detect ultraviolet measurements at 250 nm. Table 8 gives the retention times for the individual factors A-35512 for an example high-resolution liquid chromatography, carried out under the following conditions: column dimensions about 0.3 x 15 mm, carrier - ZIPAX polyamide by Dupont, solvent - solution of 327 g KH2PO04 in 1800 ml of water, flow rate - 0 * 5 ml / min., writing speed - 5 mm / hour, pressure - about 105 kg / cm2 Table 8 Factor A-35512 ABCEF '.... ..' G. "': H Retention time in min. 9.375 1 13.125 26.25 5.625 7 , 5 7.5 7.5 | In addition to the individual free bases and the hydrochlorides of factors A-35512, factors A, B, C, E and H are also obtained in the form of the other pharmaceutically acceptable acid addition salts. The term "pharmaceutically acceptable" denotes salts whose toxicity to warm-blooded animals does not exceed that of the free factors A-35512. Suitable and representative salts of factors A-35512 A, B, C, E and H are the salts obtained by conventional reaction with organic and inorganic acids, such as sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, embonic, mucic, D-glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, lauric , stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic and the like. According to the present invention, new antibiotics are produced by culturing the A-35512 producing strain Streptomyces candidus NRRL 8156 under sub-surface fermentation, under surface fermentation, a suitable nutrient, until a considerable amount of antibiotics is obtained. Antibiotics are isolated by various isolation and purification techniques. ia, used in fermentation processes. A new microorganism useful for the production of A-35512 antibiotics was isolated from soil samples collected at Eniwetok Atoll. The microorganism has been classified as a new strain of Streptomyces candidus (Krasilnikow) Waksman as described by E. B. Shirling and D. Gottlieb in the Cooperative Description of Type Cultures of Streptomyces. II. Species Descriptions from Second Study ". Intern. J. Systematic Bacteriol., 18 (4), 279-392 (1968) and SA Waksman" The Actinomycetes. Vol. 2 Classification, Identification and Descriptions of Genera and Species * ", Williams and Wilkins Co., Baltimore, 1961. The above classification is based on the methods recommended by the International Streptomyces Project (EBShirling and D. Gottlieb, "Methods for Characterization of Streptomyces Species", Intern. Bull. Systematic Bacteriol., 16, 313. —340, 1966) and on other supplementary tests. Colors were described according to the ISCC-NBS method, according to 14 KL Kelly and DB Judd, "The ISCC-NBS Method of Determining Colors and a Dictionary of Color Names". US Department of Commerce Cir 553, Washington, D. C, 1955. The symbols in normal brackets refer to the color series according to Tresner and Backus (HD Tresner and SJ Backus, "System of Color Wheels for Streptomyces Taxonomy". Appl. Microbiol., 11, 335 —338, 1963), and the color markings of the tables are colored salty. In square brackets there are 10 color symbols according to Maerz and Paul (A. Maerz and AL R. Paul, "Dictionary of Color", Mc Graw-Hill, New York NY, 1950). Cultures were carried out at 30 ° C for a period of time. 14 days, unless otherwise stated. Characteristics of the strain producing antibiotics 15 A-35512 Morphology / Long, wavy sporophores are produced. Chain spores, 10-50 units, are cylindrical in shape and dimensions 0.7 to 1.05 x 1.4 to 3.5 microns Spore-like structures are produced on various media. In some! 20 cases bifurcated or bundled hyphae are observed. The surface of the spores is smooth when viewed under an electron microscope. The cultivation characteristics of the different media are presented in Table 9. Table 9 25 Medium | 1 ISP No. 2 (yeast agar and sweet extract) ISP No. 3 (oatmeal agar) 1 ISP No. 4 (starch agar 1) with inorganic salts) and ISP No. 5 (aspartic agar with glycerin) 'Emerson agar Modified Bennett agar Characteristics 2 Profuse growth, grayish-yellow underside (11J5), abundant aerial mycelium and sporulation, white (w) a; no soluble pigments Growth moderate, pale yellow-green underside (10B1); nice aerial mycelium and sporulation, white (w) 13ba; no soluble pigments, spore-like bodies are observed. Growth good; underside amber-white (10C1), good air mycelium and sporulation, yellowish-gray (CY) 2dc; light brown soluble pigments; spore-like bodies are observed "Growth good; underside pale yellow-green (10B2); good airborne and sporulation mycelium, white (in); no soluble pigments ^ mycelium appears to be bundles l Growth good; lightly olive bottom (14L6); no air mycelium and sporulation; light-brown soluble segments Abundant growth; underside moderately yellow (11J6); abundant aerial mycelium and sporulation, white (w) a; no soluble pigments | 10S 561 15 16 Table 9 | 1 1 2 [Czapek Agar Orange Paste and Oatmeal Agar 1 Nutrient Agar 1 Glucose Aspartic Agar 1 Tryptone Agar and yeast extract 1 Tyrosine agar and ¦ ...- ¦ 1 Agar with glycerin and glycine 1 Agar with calcium malate Li: -::: _ '1 Growth good; bottom pale green (10B1); good aerial and sporangia mycelium Tearing, white (w) b; no soluble pigments; mycelium seems to be in bundles Abundant growth; underside of gray yellow (1115); abundant aerial mycelium and sporulation, white (w) a; mycelium rolls off the agar surface; no soluble pigments Moderate to good growth; underside pale yellow green (18D2); good aerial mycelium and sporulation, white (w), light-brown soluble pigments Growth good; pale yellow green (17E1); good aerial mycelium and sporulation, white (w) 13b; no soluble pigments | Growth pronounced; white underside 1 (10A1); clear aerial mycelium and sporulation, white (in) ) b; no soluble pigments Growth good, white bottom 1 (10B2); good aerial mycelium and sporulation, white (w) a; brown soluble pigments; spore-like bodies are observed Growth good; white bottom (10B2) ; good air mycelium and sporulation, white (w) b; no soluble pigments Good growth; white underside (10B1); good air mycelium and sporulation, white (w) a; no soluble pigments ; the substrate becomes transparent around the contaminated surface | Dr. the organism was examined for selected physiological properties using standard protocol. The properties were found as listed in Table 10. Table 10 Property Observed Effects on Milk Nitrate Reduction Characteristics Coagulation with some transitions within 14 days 10 15 20 55 60 65 | Property observed Manufacture of mallein pigment ;. peptone iron agar slants; L tyrosine agar slants | nutrient solution containing tryptone and yeast extract Gelatine liquefaction Temperature requirements (ISP medium No. 2 - sap with yeast and malt extract 1 | Characteristics ¦-¦ • no weak Pigmentation after 7 days; none Complete within 14 days 26-30 ° C - good growth and sporulation 37 ° C - poor growth; no aerial mycelium and spores 40 ° C - poor vegetative growth 45 ° C - no growth Table 11 shows the results of the carbon uptake tests by microorganisms NRRL 8156; The meaning of the symbols used is as follows: + - —¦ good growth, absorption (+) - '• poor to clear growth (-) ¦—- poor growth, probably no absorption 30 -— - no growth, no absorption Table 11 35 40 45 Carbon source None (negative control) D-glucose (positive control) L-arabirose Sucrose i-Inosit D-mannft D-fructose ¦¦ * ¦..Raffinose ramnose D-xylose Assay (-) + "(-) '¦' '" (-) "' + + + (- (-) (+) Some characteristics of szc zep S. eandinus NRRL'8156, producing antibiotics A-35512, differs from the characteristics of the microorganism described by Elwood B. Shirling and David Gottlieb in Cooperative Descrip- tion of Type Cultures of Streptomyces III. Additjonal Species Descriptions from First and Second Studies ", Intern. J. Systematic Bacteriol., 18 (2), 69 - 189 (1968) and by Waksman SA in" The Actinomycetes. Classification, Identifications and Descriptions of Genera and Species " , Vol. 2, The Williams and Wilkins Co., Baltimore, Md., 1961. The differences are summarized in Table 12. The Streptomyces candidus strain producing antibiotics A-35512 has been deposited with the Northern Regional Research Center, U. pS strain collection. Department of Agri-culture, Agricurtural Research Service ,. Peoria, Illinois, 61604 * from which this collection is- commonly available under number NRRL 8156. r Any of the many canes can be used as a growth medium for Streptomyces candidus NRRL 8156-10S 561 17 Table 12 Carbon uptake L-arabinose Rhamnose and-Inositolising gelatine Effects on milk NRRL 8156 - - + Total within 14 days Coagulation with some transitions within 14 days Published description + + = Slow No coagulation, good perponization of the yields. However, for reasons of economy and in view of optimal yield and ease of isolating the product, certain culture media are preferred. For example, sucrose is a preferred source of carbohydrate in industrial production, although glucose, tapioca dextrin, fructose, mannitol, maltose, lactose and the like may also be used. Soluble meat peptone is a preferred source of nitrogen, but also soybean flour may be used. meat, amino acids such as glutamic acid and related products. Nutritional inorganic nutrient salts include commercial soluble salts that are sources of zinc, sodium, magnesium, calcium, ammonium, chlorine, carbonates, sulfates, nitrates, and similar ions. Nutrient can also include essential elements. trace, necessary for the growth and development of the microorganism. Such elements are usually found as impurities in other nutrients in sufficient amounts to meet the growth needs of the microorganism. If desired, a small amount (0.2 ml / liter) of an anti-foaming agent such as polypropylene glycol is added if necessary. In large-scale fermentation, foaming becomes a problem .- ".... For the production of large amounts of the A-35512 antibiotics, subsurface fermentation with aeration is preferred. Small amounts of A-35512 antibiotics may be obtained in a tricuspid flask culture. Due to the delay in antibiotic production, generally associated with inoculation of large fermenters with sporulating form of the microorganism, the use of a vegetative inoculum is preferred. The vegetative inoculum is prepared by inoculating a small volume of the culture medium with spore forms or mycelial fragments of the microorganism to obtain a fresh, actively growing culture of the microorganism. The vegetative inoculum is then transferred to larger fermenters. The microorganism that produces the antibiotics A-35512 can be grown at temperatures around 20-40 ° C. The optimal yield of A-35512 antibiotics is achieved at a temperature of 30-34 ° C. As is usual in subsurface cultivation with aeration, sterile air is passed through the medium. In order to achieve efficient microbial growth in industrial fermenters, preferably more than 0.1 volumes of air per minute per minute per volume of nutrient are passed through the medium. For the efficient production of A-35512 antibiotics it is preferable to pass about 0.25 times the volume of air per minute. The process of producing A-35512 antibiotics during fermentation can be controlled by analyzing samples of the wort or extracts for the antibiotic content. A microorganism known to be sensitive to the antibiotics it produces. Such microorganisms include Bacillus subtilis ATCC 6633. The biological assay is preferably carried out using paper discs on agar plates containing low-nutrient agar. After fermentation, the A-35512 antibiotics can be extracted from the fermentation broth known in practice by means of practical methods. . Antibiotics, produced by the fermentation of the microorganism producing A-35512, (They are found almost exclusively in the broth. Therefore, the best efficiency in isolating A-35512 antibiotics is obtained by first draining the mycelium mass. The filtered wort can be purified to form a mixture. A-35512, using a variety of methods A preferred method is to adsorb on a polyamide column followed by elution with water and an aqueous alcohol solution Eluate fractions containing antibiotics are combined to give the A-35512 mixture. The same technique can also be combined using the same technique. individual fractions based on the results of thin-layer chromatography to obtain purified factor A-35512-B and enriched mixtures of other factors, A-35512 Further purification of individual factors A-35512 consists in additional adsorption and elution. As adsorption materials, oxide can be successfully used aluminum, silica gel, ion exchangers and the like. Agents A-35 512 are present in the fermentation broth in the form of hydrochlorides. In a preferred separation process on the polyamide material, the A-35512-B agent and the mixture of the remaining agents are obtained in the form of hydrochlorides. Each of the individual agents can be converted into a free base, free of iodine chlorine, using, for example, a weakly base exchange chromatography. Alternatively, the solid components of the culture, including nutrients and mycelium, can be used as a source of the A-35512 antibiotics, omitting the extraction or separation operation, but preferably after removal of the water, e.g., after fermentation is complete, the wort containing the antibiotics. A- -35512 can be freeze-dried and used directly as an admixture in fodder. Antibiotic mixture A-35512 and anti-agents. Table 13 Staphylococeus aureus 3055 test strain Staphylococeus aureus 3074 Streptococeus faecalis MIC 0 factor A-35512-A .2HC1 6.25 25.0 6.25 factor A-35512- -B 2HC1 3.12 6.25 3.12 factor A-35512- -C.2HC1 6.25 6.25 3.12 factor A-35512 - -H.HCl 25.0 25.0 12.5 10 15 20 25 30 35 40 45 50 55 6019 108 561 Table 14 20 Staphylococcus aureus 3055 test strain (penicillin G sensitive) Staphylococcus aureus 3074 (penicillin G resistant) Staphylococcus aureus 3130 (methycyline resistant) Streptococcus pyogenes (group A) Streptococcus sp. 9960 (group D) Diplococcus pneumoniae (Park I) Zone diameter in mm 1 factor A-35512-A- • 2HC! 13.4 13.2 14.4 17.0 15.5 - Factor A-35512-B- • 2HC1 18.4 16.8 18.4 19.6 19.4 -. factor A-35512-C- • 2HC1 0 0 0 10.5 0 11.0 factor A-35512-E- • HCl 14.4 14.5 15.4 17.0 16.4 20.0 factor A-35512 -H- • HCl 11.9 11.6 12.6 16.0 13.8 - biotic A, B, C, E and H inhibit the growth of various pathogenic microorganisms, especially gram-positive bacteria. Table 13 summarizes the minimum inhibitory concentration (MIC) values at which factors A-35512-A, B, C and H inhibit the growth of selected bacteria in a standard agar-diluted test. A-35512 antibiotics inhibit the growth of microorganisms that are resistant to the action. other antibiotics. Table 14 shows the results of tests for the activity of factors A-35512-A, B, C, H and E against selected microorganisms. The tests were carried out using the standard disc-plate method (30 µg / disc) and the activity was expressed as the size (diameter in mm) of the inhibition zones observed. Table 15 shows the results of an additional agar dilution test, illustrating the factor A dihydrochloride activity -35512-B against various strains of Streptococci, Group A and Diplococcus pneumoniae. The results are expressed as minimum inhibitory concentration (MIC) values Table 15 Test organism Streptococcus pyogenes C203 Streptococcus pyogenes 10389 Streptococcus pyogenes 12344 Streptococcus pyogenes 12961 Streptococcus pyogenes 663-72 Streptococcus pyogenes 664-72 Streptococcus pyogenes 664-72 Streptococcus pyogenes 664-72 Streptococcus pyogenes 664-72 19035 Streptococcus pyogenes M6517 Streptococcus pyogenes D27781 Diplococcus pneumoniae Park I Diplococcus pneumoniae Type 14 MIC (// g / ml) 1 Factor A-35512-B 1 1 8 1 0.5 0.5 1 0.5 0.5 0.5 1 2 2 Table 16 shows the results of an additional agar dilution test showing the activity of factor A-35512-B dichloride against various Streptococci strains, Group D. Table 16 Test organism Streptococcus sp. D282 Streptococcus sp. 9901 Streptococcus sp. 9913 Streptococcus sp. 9933 Streptococcus sp. 9960 Streptococcus sp. 12253F Streptococcus sp. Shrigley Streptococcus sp. Mitis Streptococcus sp. sp. 238 Streptococcus sp. SS992 MIC (// g / ml) Factor A-35512-B 2 2 4 4 4 4 4 4 2 4 Table 17 gives the results of an additional agar dilution test, illustrating the activity of the dihydrochloride factor A-35512-B against other gram-positive microorganisms Table 17 Test organism Staphylococcus aureus 3123 * Staphylococcus aureus H43 * * Staphylococcus aureus 3124 ** Staphylococcus aureus 3126 ** Staphylococcus aureus aureus 3127 ** Staphylococcus aureus aureus 3127 ** 3074 ** Staphylococcus aureus 3130 *** Staphylococcus aureus 3131 **** Staphylococcus aureus 3133 *** Staphylococcus aureus 3136 *** Staphylococcus aureus 3125 *** 1. Staphylococcus epidermis 3064 * * MIC (// g / ml) Factor A-35512-B 4 4 4 2 4 2 4 4 4 2 2 4 2 | 108 561 21 22 cont Table 17 Staphylococcus epidermis 3078 test organism ** Bacillus subtilis ATCC 6633 Sarcina lutea PCI-10D1-FDA MIC 0 * g / ml) Factor A-35512-B 1 1 2 1 * - penicyline G sensitive ** - penicyline G resistant ** • - resistant to penicyline G and methicine **** - resistant to penicyline G, methicine and clindemycin. A-35512 antibiotics also inhibit the growth of some anaerobic bacteria. Table 18 shows the results of testing the activity of factor A-35512-B dihydrochloride against various anaerobic bacteria using the standard agar dilution test. intermedius Propionibacterium acnes Bacteroides fragilis ssp. fragilis Ul Fusobacterium necrophorum MIC Gug / ml) 1 Factor A-35512-B 0.5 2.0 4.0 2.0 1.0 1.0 2.0 4.0 0.5 64.0 8.0 Factor A-35512-B dihydrochloride exhibits in vivo antibacterial activity against a variety of experimentally induced bacterial infections. Infected mice were administered two subcutaneous doses of factor A-35512-B and the observed activity was designated ED50 *. ED30, this is the mg / kg dose, protecting 50% of the test animals (Warren Age et al., J. Bacteriol., 81 233-235, 1961). The ED50 values obtained for factor A-35512-B dihydrochloride are given in Table 19. Table 19 Test organism Streptococcus pyogenes C203 Diplococcus pneumoniae Parks Staphylococcus aureus 3055 ED50 1.75 4.3 6.9 Infection rate (intraperitoneal) 260xLD50 60.8xLD50 206xLD50 Factor A-35512-B dihydrochloride also protects guinea pigs contaminated with the anaerobic organism Clostridium chauvoci, which causes rustling in bovine animals. Tested according to USDA Supplemental 10 15 20 SO 40 45 50 55 Assay Method for Potency Testing Clostridium chauvoci containing Products (SAM 200, US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services Laboratories, Ames. Iowa 50010, April 7, 1975, 9 CFR 113. 91). The test results are given in Table 20. Table 20 Factor Factor A-35512-B 10 mg / kg * Factor A-35512-B 1 mg / kg * Control Sample Dead to total ratio 0/10 3/10 10/10 Percentage of protection 100 70 0. * '- intramuscularly A-35512 antibiotics are relatively non-toxic, e.g. LD50 for factor A-35512-A dihydrochloride is greater than 8000 mg / kg when administered subcutaneously to Cox-Harlan IRC mice The LD50 value for factor A-35512-B dihydrochloride is approximately 1356 mg / kg when administered intraperitoneally to mice and 1000-1250 mg / kg when administered intravenously. Factor A-35512-C dihydrochloride and factor A-35512- hydrochloride E show acute toxicity when administered to mice at doses greater than 300 mg / kg. Another valuable property of the mixture of A-35512 and its components is the ability to increase the feeding efficiency of animals. This has been found in ruminants with a developed lather function. It is known that the efficiency of carbohydrate uptake in rewormers increases as a result of an action that stimulates an increase in the production of the bacterial flora, i.e. propionates rather than acetates or butyrates. Church et al. In Digestive Physiology and Nutrition of Ruminants, Vol. 2, 1971, pp. 622 and 625. Feed efficiency can be determined in vivo in cattle with an erectile fistula using the method outlined by Arthur P. Rann in in U.S. Patent No. 3,794,732, especially in Example 8. Table 21 shows the results of such a test for factor A-35512-B dihydrochloride. The stated propionic acid concentration is therefore the average of five analyzes carried out over a period of 21 days. control test with the factor A-35512-B (50 g / tonne) Table 21 Number of animals 5 5 Average mole percent of pro acid of vertical 19.3 26.0 Percentage of 1 mol of growth 1 in relation to the control sample - 6.7 1108 561 23 Mixture A-35512 and its components increase the propionate concentration and thus the feed efficiency when fed in feeding doses daily doses of 0.15-10.0 mg / Ig. Better results are obtained when administered at doses of 1.0-2.0 mg / kg / day. A preferred method of administering the A-35512 mixture or an individual compound is by mixing it with the animal feed. These antibiotics can also be administered by other means, such as tablets, throat infusions or capsules. The foregoing pharmaceutical forms are prepared by methods well known in the field of veterinary pharmaceutical pharmacy. Each single dose of the drug should contain a mixture of A-35512 or a component thereof in an amount corresponding to the daily dose administered to the animal. Mixture A-35512 and its components are also useful as growth factors for animals. In the broiler chickens test, factor A-35512-B dihydrochloride, added to the feed at a rate of 20 g / ton, increased weight gain and feed efficiency. Table 22 shows the results of such a test, carried out in a multi-storey lair, in which four groups of 8 birds were used for each of the two time periods. Table 23 presents the results of the test carried out in the cage nesting house with the use of 12 groups of 80 birds. Table 22 Control test Factor A-35512-B what is 20 or 380 394 l I & 3.68 g ¦a 1.850 1.776 4, 00 Table 23 l :: Pr 'Kontrolnk "Factor A-25S12-B CO b0 20 t'n.'. V ••". • 'Weight gain according to 1898 1956 * Na aiaoog 3.06 Feed efficiency 2.250 2.177 j% improvement * ¦ against the control 2.81 | 10 15 35 W 40 24 No. 2. The infected slant is incubated at 30 ° C for about 7 days. A mature culture on slants is covered with 5 ml of water and a large sterile pipette is scraped off. 2.5 ml aliquots of the resulting spore suspension are used to infect 30 ml of vegetative nutrient solution with the following composition: Ingredient Quantity Triptych Soybean (Baltimore Biological, Laboratories, Cockeyville, Md). 30 g Demineralised water up to 1 liter. The contaminated vegetative medium is incubated in 250 ml Erlenmayer flasks at 30 ° C for 48 hours, on a 250 revolutions per minute spinner with 50 mm inclination. After the incubation is completed, 0.5 ml of vegetative medium is used to contaminate 50 ml of production medium with the following composition: 25 Ingredient Tapioca dextrin Glucose NH4NO3 KCl MgSO4 FeCl2.4H20 ZnCl2 KH2P04 L-glutamic acid CaalalizedCitruline water Quantity in g / liter 25.0 10.0 2.5 1.5 U 0.03 0.03 0.1 W 0.1 5.0 to 1 liter ¦i_ ZH5 - Mixture A-35512 and its components work effectively in increasing the growth of birds when fed with a feed of about 100 g per ton. feed. Given; the examples further illustrate the method according to the invention. and Example I. "Fermentation of A-35512 in swing flasks. Freeze-dried pellets of Streptomyces candidus NRRL 8156 are dissolved in 1-2 ml of sterile water and the solution is banned from agar slants containing Bacto yeast and slime extract (ISP No. 2). , manufactured by Difco Laboratories, Detroit, Michigan). The contaminated stacks are incubated at 30 ° C for approximately 7 days. The mature cultures on the slopes are covered with 2 ml of water and a sterile pipette is scraped off a large portion. 0.1 ml portions. the aqueous spore suspension is used to infect the next agar slant from ISP 50 55 60 65. The banned production medium is incubated in 250 ml Erlenmayer flasks at 32 ° C for 8-10 days on a 250-speed rotating jig. minute and 50 mm deflections. Fermentation of A-35512 in fermenters. In order to obtain more inoculum, 20 ml of vegetative medium obtained as described above is used to order 400 ml of 2nd stage vegetative medium with the same composition as a clade of vegetative medium. Stage II medium is incubated in 2-liter flasks for 24 hours at 32 ° C, on a rotating shaker with 250 revolutions per minute and a swing of 50 mm. 800 ml of stage II vegetative medium is used to prohibit 100 liters of sterile production medium. Zak-. The production nutrient solution is fermented in a fermentor with a capacity of 165 liters for about 8-10 days, August, v§J & giving 0.25 air volumes per minute per nutrient solution and mixed, usually using a stirrer with 200 revolutions per minute. Example II. The incubated vegetative medium prepared as in example I can be stored for later use by keeping it under liquid nitrogen vapor. To this end, a 2 "ml suspension of dispersant with the following composition is placed in a test tube measuring 13 x 100 ml, sterilized and fitted with a screw cap: Ingredient Amount Glycerin 20% Lactose 10% Demineralised water 70% To 2 ml of a vegetative culture incubated for 48 hours, prepared as previously described, is added to the suspension and the mixture is frozen and held under vapor in a container containing liquid nitrogen. The nutrient inoculum stored in this way is thawed before use, by immersing the vial in a water bath at 43 ° C. 1 ml of the thawed inoculum is used to forbid 50 ml of a vegetative medium of the composition given in example 1. The contaminated vegetative medium is used as described in example I, for fermentation in swing flasks or to obtain more inoculum for fermentation in fermenters. Example III The fermentation process is carried out as described in in example I, using the production medium for cultivation in flasks or fermenters with the following composition: s - "Ingredient Amount / g / liter Melt dextrin 75.0 15 Molasses 40.0 ¦ - Soluble peptin mass 15.0 'MgS04.7H2O 0, 5 CaCQ3:. 2.0 Water up to 1.0 liters 20 Example. Isolation of the A-3551Z Antibiotic Mixture The entire fermentation broth (946 liters) obtained as described in Example I is filtered using filtration aid (Hyflo Super-cel 25 diatomaceous earth from Johns-Manvjlle Products Corp.). The pH of the broth is 6.8-7.2. The clear effluent is passed through a column containing Amberlite XAD-4 resins (manufactured by Rohm and Haas Co.) using 10 ml of resin per 100 ml of wort lumen. The successive fractions are tested for biological activity using the standard Sarcina rutea disc analysis. The biologically inactive fractions are discarded. The columns are washed with water in an amount equal to 1/8 of the volume of the wort, at a rate of 150 ml / minute. Inactive rinse water is discarded. The product is then eluted with 600 liters of 50% aqueous methanol at a rate of 200 ml / minute. The eluates containing the A-35512 antibiotic mixture are concentrated under reduced pressure to a volume of about 15 liters. One liter contains about 200 grams of the antibiotic mixture A-35512. Example 5: Isolation of Individual Factors from Antibiotic Mixture A-35512. About 3000 grams of Antibiotic Mixture A-35512: dissolved in 15 liters of methanol prepared as described in Example IV, are chromatographed on a 45th column. filled with 100 liters of polyamide produced by Woelnu, the column is eluted with demineralised * water at a rate of about 80-120 ml / minute. The collected fractions are examined using cellulose thin-layer chromatography or paper chromatography, 50 using a mixture of n-butanol, acetic acid, pyridine for development and water (15: 10: 3: 12) and to develop a bioautograph against Sarcina lutea. The first 100 liters of eluate is discarded and the flow rate set at about 160-200 ml / minute. 12 55 tailcoats with a volume of 12 liters each are collected. The eluent is then changed and eluted in a gradient of methanol and water using the following procedure. The tank, containing 360 liters of methanol, is connected by a siphon connection to the tank containing 120 liters of water. The solvent mixture is mixed and applied to the columns. 24 fractions of 24 liters each are collected, eluting at a rate of 200-300 ml / min. Based on the results of bioautography, the 26 general fractions are combined into groups and evaporated under reduced pressure to give factor A dihydrochloride -35512- B and the following enriched mixtures of other factors * as shown in Table 24 Table 24 Fractions 1—10 11—24 15—31 32 = 44 Volume (liters) 120 216 168 312 Factor A + HBB + CC, E, P, G Amount 192 g 269 g 590 g 224 g Example VI. Purification of Factor A-35512-B. Partially purified 400 g of factor A-35512-B dihydrochloride obtained according to Example V are dissolved in 1.2 liters of 50% aqueous methanol and chromatographed on an aluminum oxide column. The column is prepared as follows. 10 kg of acidic alumina (from Woelrn) is mixed with a 50% aqueous methanol solution. The mixture is allowed to stand, the supernatant is decanted and discarded. The sword alumina is re-mixed with a 50% aqueous methanol solution and packed into a 13.5 cm liquid column, then washed with a 50% aqueous methanol solution until a clear eluate is obtained. The product is eluted with a 50% aqueous solution of methanol at a rate of about 8- ^ 1 ml / minute, collecting fractions of about 24-3 .mu.ml. Subsequent fractions are examined by thin layer chromatography, using bioautography to develop; ak this is described in Example V. The individual fractions are combined from the results to give purified factor A-35512-B dihydrochloride. The results are shown in Table 25. Table 25 - • ¦ 1 Fractions 17-21 22-29 30-37 Amount "" ": | 9.6 g- 72.0 g H 7.0 g | Each of the given fractions was crystallized at temperature 4 ° C from a concentrated 50% aqueous solution of methanol. The obtained factor A-35512-B dihydrochloride contains approximately 4.6% chlorine. The pH value of a solution of factor A-35512-B dichloride in 66% aqueous solution of dimethylformamidia is approximately 6 , 5 Example 7 Preparation of factor A-35512-B, free from ionic chlorine. 1 g of purified factor A-35512-B dihydrochloride, obtained as described in example VI, is dissolved in water and passed through the column of - measures 2.5x18 cm, filled with an ion exchanger; Bio-Rad AG3-4x in hydroxide form, at a rate of 0.5 ml / minute, using demineralised water for elution. The first 5 ml of eluate is discarded. The next 50 ml of eluate is evaporated off to dry under reduced pressure.: and get 0.76 g of non-chlorine ion - factor A-35512-B in the form of a white powder containing approximately 1.59% chlorine. The pH value of a solution of this preparation in a 66% aqueous solution of dimethylformamide is 9.13. * -.'- 108 S61 27 28 Example VIII. Purification of factor A-35512-A Partially purified 1 g of factor A-35512-A dihydrochloride, fractions 1-10 of example V, is dissolved in 5 ml of 50% aqueous methanol. The solution is chromatographed on a 3 × 16 cm column filled with acid alumina (M. Woelm) prepared as described in Example V. Elution is carried out with 50% aqueous methanol at 0.5 ml / minute. Fractions of 5 ml are collected and each is analyzed using the thin film bioautograph as described in example V. Based on the above determinations, fractions 7-15 are combined, concentrated under reduced pressure to a low volume and lyophilized. 0.3 g of factor A-35512-A dihydrochloride with a chlorine content of 4.71% is obtained. Example IX. Production of factor A-35512-A, which does not contain ionic chlorine. 200 mg of factor A-35512-A dihydrochloride, obtained as in Example VIII, is dissolved in 10 ml of water and this solution is passed through a 1.5x10 cm column containing the Bio-Rad AG3 ion exchanger. 4 x in the form of hydroxide, at a rate of 0.5 ml / minute, using demineralized water for ehiation. The first 10 ml of the eluate is discarded, the next 20 ml are concentrated under reduced pressure to a low volume and lyophilized. 115 mg of factor A-35512-A are obtained, containing no ionic chlorine. Example X. Purification of factor A-35512-C Partially purified 15 g of factor A-35512-C dihydrochloride (fraction 25-31 from example V) is dissolved. In demineralized water (40 ml). The solution is applied to a 4 × 115 cm column, filled with polyamide (MN, <0.07 mm, Brinkman Instruments Inc.), prepared in water and washed overnight with water. Demineralised water is used at a rate of about 3 ml for ehiation. / minute. The first 250 ml of the eluate is discarded and then 24 ml fractions are collected. The successive fractions are monitored by thin layer chromatography on cellulose-coated aluminum plates (produced by E. Merck, Germany). For development, the solvent system II-pre-butanol, pyridine, acetic acid, water (10: 10: 3: 8) is used, and for bioautography with Bacillus subtilis. Based on the results of thin-layer chromatography, fractions 1-33 are combined , concentrated in vacuo to 150 ml and lyophilized. Two further aliquots of partially purified factor A-35512-C dichloride were chromatographed using the conditions described above. In both cases fractions 1-33 were combined, concentrated under reduced pressure to a volume of 150 ml and freeze-dried. The three portions of the lyophilisate from the three columns weighed a total of 12.3 g. Factor A-35512-C dihydrochloride was further purified by chromatography on another polyamide column, operating as previously described but collecting 15 ml fractions and using a flow rate of 1 ml / minute. Subsequent fractions are investigated by means of thin layer chromatography with bioautography. Fractions 36 * 58 are combined, concentrated under reduced pressure to a volume of 150 ml and lyophilized. 5.3 g of purified factor A-35512-C dihydrochloride is obtained. For final purification, factor A-35512-. C dihydrochloride is chromatographed on a 5 × 41 column containing acid alumina (Volm). The carrier is suspended and washed with 50% aqueous methanol until the eluate is clear, then 5.3 g of factor A * 35512-C are applied to the column. 5 It is eluted with 50% aqueous methanol solution at 1 ml / minute. Fractions of 12 ml are collected and analyzed by thin layer chromatography combined with bioautography as described previously. Frakqe 22-74 are combined, concentrated under vacuum to 250 ml and lyophilized. 3.86 g of factor A-35512-C dihydrochloride are obtained. Example XI. - Preparation of A-35512-C, chlorine free 200 mg of A-35512-C dihydrochloride, prepared as in Example X, is chromatographed on a weakly basic ion exchanger using the procedure described in Example IX ; 156 mg of A-35512-C is obtained, containing no ionic chlorine. The obtained preparation contains about 1.90% chlorine. Example XII. Purification of Factor A-35512-E The partially purified factor A-35512-E hydrochloride (8.1 g), prepared according to Example V (fractions 32-44), is dissolved in 40 ml of demineralised water. The solution is applied to a column with dimensions of 5 X 110 cm, filled with polyamide. MN "<0" 07 (Brinkman Instruments Inc.), prepared in water washed overnight with water. For eluaq, I use deionized water, the ehiation rate is 20 ml for 15 minutes. 20 ml Si fractions are collected. After collecting 118 fractions, the solvent is changed to 0% aqueous methanol. The successive fractions are analyzed by bioautograph thin layer chromatography as described in example X. Fractions 148-193 containing factor A-35512- E is combined, concentrated under reduced pressure to 150 ml and freeze-dried. This gives 2.7 g of factor A-35512-E hydrochloride. Part (615 mg) of the above partially purified factor A-35512-E hydrochloride is dissolved in 5 ml of a 40 50% aqueous methanol solution and applied to a 1.5 x column. 50 cm, filled with acid alumina (Woelm). The carrier is suspended and washed with 50% aqueous methanol until a clear eluate is obtained. The same solvent is eluted at 1 ml / minute and 10 ml fractions are collected. The successive fractions are analyzed by bioautography thin-layer chromatography. Fractions 5-8 are combined and concentrated under reduced pressure to a volume of about 10 ml, then 50 ml of demineralised water 50 are added and lyophilized. 480 mg of the hydrochloride of the factor A-35512-E are obtained. EXAMPLE XIII. Preparation of A-35512-E, chlorine-free factor ... 200 mg of A-35512-E hydrochloride, prepared according to example XII, is chromatographed on a weakly basic ion exchanger using the procedure described in the example IX. 170 mg of factor A-35512-E is obtained, which does not contain ionic chlorine. The obtained preparation contains approximately 172% chlorine. 60 Example XIV. Purification of factor A-35512-H. The partially purified factor A-35512-H hydrochloride (30 g), obtained according to example V (fractions 1-10), is dissolved in a minimum amount of a 7: 3 mixture of methanol and water. The solution is applied to a column 55, 3 × 60 cm in size, filled with acid aluminum oxide (Woelm), charged in methanol and washed with methanol, until the solvent discharges is clear. Methanol was used for elution at a flow rate of 4 ml / minute. Fractions with a volume of 24 ml are collected. After 59 fractions were collected, the solvent was changed to a 1: 1 mixture of methanol and water. The successive fractions are analyzed by thin layer chromatography using a mixture of chloroform, methanol and ammonium hydroxide (2: 3: 1) as the developing system and the chromatograms are developed by bioautography in an alkaline environment using Bacillus subtilis as a test strain. Frakqe 51-118 is combined and evaporated under reduced pressure. 6.4 g of purified factor A-35512-H hydrochloride are obtained. Example XV. Production of A-35512-H factor, which does not contain ionic chlorine. 200 mg of factor A-35512-H hydrochloride, prepared in accordance with Example XIV, is chromatographed on a weakly basic ion exchanger using the procedure described in Example IX. 143 mg of factor A-35512-H containing no ionic chlorine are obtained. The obtained preparation contains about 1.59% of chlorine. characterized in that the strain Streptomyces candidus NRRL 8156 is grown in a nutrient containing an assimilable source of carbohydrates, nitrogen and inorganic salts under subsurface fermentation with aeration until a significant antibiotic activity is obtained and the antibiotic mixture A is released 35512 from the wort, and optionally, the factors A, B, C, E, F, G and H are isolated from the antibiotic mixture A-35512. 25 2000 1800 1600 1400 1200 1000 800 600 (CM "1) 4011 250 FIG. I FIG. 2108 561 7 8 9 10 12 U 181820 25 30 40 FIG. 3 7 8 9 10 12 14 1818 20 25 30 40 FIG. 4 25 3 4 5 8 7 8 9 10 12 U 181620 2530 40 r 1 1 1 1 1 1 1 1 1 1 — ri 1 — i 4000 3500 3000 2500 2000 1800 1600 1400 1200 ICM "1) 1000 800 600 4C0 250 FIG. 5 HOOC NH2 HO -v-0 Cl H, N COOH Model LZG Z ~ d 3, z. 790/1400/80, n. 90 + 20 copies Price PLN 45 PL

Claims (1)

Claim 1. A method of producing the A-35512 antibiotic mixture containing factors A, B, C, E, F, G and H, characterized in that the Streptomyces candidus NRRL 8156 strain is grown in a medium containing an assimilable source of carbohydrates, nitrogen and inorganic salts, under the conditions of subsurface fermentation with aeration, until a significant antibiotic activity is obtained and, if necessary, the antibiotic mixture A-35512 is separated from the wort, and then the factors A, B, C, E, F are separated, G and H from the A-35512 antibiotic mixture. 25 2000 1800 1600 1400 1200 1000 800 600 (CM "1) 4011 250 FIG. I FIG. 2108 561 7 8 9 10 12 U 181820 25 30 40 FIG. 3 7 8 9 10 12 14 1818 20 25 30 40 FIG. 4 25 3 4 5 8 7 8 9 10 12 U 181620 2530 40 r 1 1 1 1 1 1 1 1 1 1 — ri 1 — i 4000 3500 3000 2500 2000 1800 1600 1400 1200 ICM "1) 1000 800 600 4C0 250 FIG. 5 HOOC NH2 HO -v-0 Cl H, N COOH Model LZG Z ~ d 3, z. 790/1400/80, n. 90 + 20 copies Price PLN 45 PL