DE1022358B - Production and extraction of the antibiotic 101a - Google Patents

Description

Production and extraction of the antibiotic 101 a The invention relates focus on the manufacture of a new antibiotic, which is referred to herein as an antibiotic 101 a is designated.

According to the invention, the new antibiotic can be obtained by means of a fermentation process in which Streptomyces sp. 101 in an aqueous nutrient medium cultivated and, after this, given an essential antibiotic activity is obtained from it.

Streptomyces sp. 101 is a new species that emerged from a soil sample, taken in Dallas, Texas. A culture of the living Organism - was registered with the Fermentation Department of Northern Utilization Research Branch, of the U.S. Department of Agriculture, Peoria, Illinois, and the permanent collection incorporated as NRRL 2494. This new microorganism is different clearly through the formation of pigment grains both in the vegetative and in air mycelium. Only two other varieties, Streptomyces fulvissimus and Streptomyces rubrireticuli, also form pigment grains. However, both species produce antibiotics, which differ significantly from antibiotic 101 a. The first variety is valinomycin, which differs from 101 a in its colorless crystals. The other educates Streptin and oxystreptin (reticulin), both of which are distinguished by their insolubility in Differentiate acetone, chloroform and ether from 101a. Another characterization this species as well as various of its game and varieties, all of which produce 101 a, are listed below.

The macroscopic and microscopic appearance of Streptomyces sp. 101 on different media is of all in the -Manual of Determinative Bacteriology « von Bergy, 6th Edition, and in rActinomycetes and Their Antibiotics <; by Waksman and Lechevalier described cultures differently. Its suggested name is Streptomyces spectabilis.

Streptomyces sp. 101, as it was originally isolated from a soil sample, shows the properties of the cultures summarized in Table I. Each inoculation was carried out with an inoculum under development, grown in 100 ml tryptone yeast extract broth (containing 0.5% tryptone and 0.30 % yeast extract) on a reciprocating shaker table at 28 ° C. After 48 hours, the inoculum was mixed for 1 minute (Waring mixer) and then 0.2 ml each was placed on altar plates. The altar plates were incubated at 28 ° C. The determinations were made on the 4th and 7th days. Table I. Cultural characteristics of Streptomyces sp. 101 Medium Vegetative growth Growth in the air Remarks 1. Casein starch mottled orange to pale- traces of orange; Spren- backside orange sprinkled- orange; deep orange zones kelt to pale orange kelt to orange, hydro- lysis 2. sucrose. Altar to orange speckled to saw- orange speckled back of orange speckled C zapek nigorange kelt to orange-red Medium vegetative growth Growth in Air Remarks 3. Maltose-Tryptone-Agar cream-colored with orange-orange, different tones back side cream to orange- zones to cream and red orange changing tones 4. Bennett's agar yellow to orange-red traces orange-red to gray reverse side orange-yellow to and orange orange-red; yellow pig ment 5. Waksmans starch - cream colored with orange white with orange sprinkled - reverse cream colored to Agar A speckles celt to pale pink to orange, hydrolysis = - orange 6. Waksmans starch - cream-colored with orange pale orange, deeper zones on the reverse cream-colored red Agar B speckles especially around the circumference; spotted to cream colored pale pink to orange to orange; Hydrolysis -f- 7. Nutrient starch agar cream-colored against orange deep orange, paler zones on the reverse yellow-orange to on the circumference to pale pink brown-orange; yellow pig to orange ment; hydrolysis B. Peptone Iron Agar brown no reverse side brown; H, S dun- kelffarben The components of the aforementioned media are given in grams per liter of distilled water. The media were treated in an autoclave at a pressure of 1.05 kg / cm2 and 120 ° C. for 15 minutes.

1. Na caseinate 2.0; soluble starch 1.0, K, HP04, 0.2, MgS 04 - 7 H2 0 0.2, Fe S 0 ,, - 7 H2 0 traces, agar 15.

2. NaN03 2.0, K, HP04 1.0, MgS04-7 H2O 0.5, KCl 0.5, Fe SO4-7 H2O 0.01, sucrose 30.0, adjusted to pH 6.6, agar 15.0.

3. Maltose 10.0, Tryptone 5.0, K2HP04 0.5, NaCl 0.5, FeS04-7H20 Lanes, agar 15.0. (Tryptone: Pancreatic breakdown product of casein with a high tryptophan content.) 4.Yeast extract 1.0, beef extract 1.0, N - Z amine A (enzymatic casein breakdown product) 2.0, glucose 10.0, medium adjusted to pH 7.0, agar 15.0.

5. Corn starch 10.0, K, HP0, 0.3, MgCO3 1.0, NaCl 0.5, NaN03 1.0, medium adjusted to pH 7.0, agar 15.0. 6. Soluble starch 2.0, K2 H P 04 0.5, Mg S 0, - 7 H., 0 0.2, CaC12 0.05, NaN03 0.05, asparagine 0.05, FeS04 - 7 H2O traces, medium adjusted to pH 7.4, agar 20.0.

7. Beef Extract 3.0, Peptones (hydrolyzate of animal protein with low molecular weight) 5.0, soluble starch 2.0, medium adjusted to pH 7.0, Agar 15.0.

B. Peptone 15.0, Proteose Peptone (hydrolyzate of animal protein high molecular weight) 5.0, ferric ammonium citrate 0.5, dipotassium phosphate 1.0, Sodium thiosulfate 0.08, agar 15.0.

Streptomyces sp. When cultivated on the following media, 101 shows characteristic morphological and cultural properties. The amounts of the ingredients are given in grams per liter of distilled water. The media become 120 ° C and 1.05 kg for 15 minutes. cm2 sterilized in the autoclave.

Pure gelatin (Gelatin 120).

Nutrient gelatin (beef extract 3.0, peptone 5.0, gelatin 120, adjusted on p. 7,0). Nutrient agar (beef extract 3.0, peptone 5.0, adjusted to pH 7.0, Agar 15.0).

Nutrient broth (beef extract 3.0, peptone 5.0) d-glucose broth (Beef extract 3.0, peptone 5.0, glucose 10.0).

2-glucose agar (beef extract 3.0, peptone 5.0, glucose 10.0, adjusted to pH 7.0, agar 15.0. Tryptone Broth (Tryptone 10.0).

Waksman tyrosine agar [glucose 10.0, tyrosine 1.0, (NH4) 2SO4 0.5, K, HP0 ,, 0.5, pl, adjusted to 7.0, agar 15.0j.

Tyrosine broth (tyrosine 1.0 adjusted to pH 7.0). Litmus milk (Dried milk with litmus added). Nitrate nutrient broth (beef extract 3.0, peptone 5.0, KN03 1.0).

Synthetic nitrate nutrient broth (K2 H P O ,, 0.5 N a Cl 0.5, MgS0, - 7 H 2 O 0.2, Na - '; 0, 2.0, glucose 10.0). Calcium maleate: even (calcium maleate 10.0, NH, Cl 0.5, Ii @ H P 0, 0.5, adjust to pH 7.0, agar 18.0. Dilutions the organism, made from sterile soil, were based on Bennetsche and maltose Spread agar plates. Four different variants were found (after 6 days of incubation at 28 'C), one with the properties of the stem, d. H. an orange spotted one one very dark orange with no growth in air, one with white growth in the air and a colorless, d. H. with the same color as the medium. The same Variants were obtained when applying the orange-spotted variant and those with white growth in the air; dark orange and colorless variants by applying the dark orange form. The colorless variety remained stable. The cultural characteristics of the Variants with white growth in the air were the same as the orange-spotted ones Variant.

The culture characteristics of the varieties on different media are summarized in Table II, namely after 1 to 1 days of incubation at 28 ° C. Inoculation was carried out as for the cultures in Table 1. Table II Culture characteristics of Streptomyces sp. 101 varieties (Five varieties) (Two varieties) (Three varieties) Medium orange mottled dark orange colorless Pure gelatin liquefaction -t- dark- liquefaction -f- dark- liquefaction; - weak brown pigment, brown pigment, yellow pigment 1/2 depth of medium 1/2 depth of medium Nutrient gelatin liquefaction -j- dark- liquefaction + dark- liquefaction -j- weak brown pigment, brown pigment, yellow pigment 1/2 depth of medium 1/2 depth of medium Nitrate nutrient broth no reduction at 4; Re- no reduction no reduction production at 1. Synthetic nitrate broth reduction at 4; no reduction no reduction no reduction production at 1 Tryptone nutrient broth no indole formation at 3; no indole formation no indole formation Indole formation at 2 Casein starch agar strong hydrolysis, pink to strong hydrolysis, dark-strong hydrolysis, colorless orange, air growth, orange, vegetative vegetative growth some zoning, growth and back and forth Orange side on the back Nutrient starch agar strong hydrolysis, pink to strong hydrolysis, dark-strong hydrolysis, colorless orange, growth on orange, vegetative vegetative growth the air with zonal growth and back and forth dung, backside orange side Skimmed milk agar pink to orange to orange-orange vegetative wax- colorless vegetative flecked air growth, tum and reverse leather growth and reverse Backside orange, brown-brown pigment, neither side, strong hydrolysis nes pigment at 4, black hydrolysis che Hvdrolvse Peptone iron agar pink to orange-spotted dark vegetative wax- colorless vegetative Air growth, back with somewhat colorless growth and back side orange; H, S Dun- and back side H, S Dun- side H, S no dark- kelfarben kelfarben coloring Litmus milk, Bennets pH 7.0, speckled (compact pH 6.0, hard wrinkled v e- pH 5.0, traces of gray- Agar to cotton wool), orange-gray growth, red-white air growth pink air growth, orange back, yellow with feathery edge, Backside orange, yellow pigment, dark brown pigment pigment Maltose tryptose agar speckled cotton wool orange dark orange, vegetative colorless vegetative Air growth; Reverse growth with green growth, traces of a Side orange, brown pig-tones, orange-brown gray air growth, ment back, brown pigment wrinkled around the circumference, cre- ment m-colored back Czapek's sucrose speckled, cotton wool to com - cherry pink to orange veg - light gray air growth, pact, pink to peach-like growth with cream to light brown colored air growth, colorless zones on the back, slightly brown Orange pigment on the back The exploitation of carbon compounds by varieties of Streptomyces sp. 101 in synthetic medium is shown in Table III. The test tubes were incubated at 28 ° C and observations were made on the 7th day. The procedure of Pridham and Gottlieb, J. Bact., 56, pp. 107 to 114 (1948), with the following modifications: 1. 500 ml Erlenmeyer flasks containing 100 ml of sterile tryptone yeast extract broth were with a Inoculated soil culture of Streptomyces 101. The flasks were incubated at 28 ° C on a reciprocating shaker table. 2. After 48 hours, the supernatant liquid was decanted off, the vegetative culture was washed with 100 ml of sterile, distilled water and the supernatant liquid was decanted off again. 100 ml of sterile, distilled water were added to the washed culture. The flask was then incubated on a reciprocating shaker table at 28 ° C.

3. After a further 48 hours, the supernatant liquid was decanted off. The vegetative culture was, as described above and in the Warin mixer, mixed for 1 minute with 100 ml of sterile, distilled water. 4. The agar wedges were inoculated with 0.2 ml of the mixed inoculum. Table III Assimilation of carbon compounds through variation activities of Streptomyces 101 in synthetic culture medium by Pridham and Gottlieb 101 original variants of the original Carbon stains, compound g spotted dark color orange orange orange item Blind test .. (-) (-) (-) (-) d-Xylose ........ (-) -) (- @ -) (- @ -) 1-arabinose .... (-) (- @ -) -f- -I- Rhamnose ...... (-) (-) (-) (-) d-fructose .... - '-, d-galactose ..... (-f-) - @ - - @ - -; - d-glucose ..... (- @ -) -? - d-mannose .... -L -f- -! -, Maltose ....... -f- -i- -h- -r Sucrose. . . . . . . . . (-) (-) (-) (-) Lactose ....... (-) -E- (-) (-) Cellobiose ....... 7- + -1- Raffinose ....... - [- -; - -f- Dextrin ....... (- @ -) -t- -f- Inulin ......... (-) (-) (-) (-) Soluble starch .. (- ;-) - Glycerine ...... (-} -) @ - Dulcit ......... (-) (-) (-) (-) d-mannitol ...... (-E-) -j- -E- d-sorbitol ........ (-) (-) '- (-) Inositol. . . . . . . . . . ; - -f- Salicin .......... (-) (-) I (-) (--I-) Phenol ........ - - j - - Cresol ........ - - - - Na-formit ...... (-) (-) (-) (-) Na oxalate ...... (-) (-) (-) (-) Na-tartrate ...... (-) (@ -) (-) (-fi-) Na salicylate ... - - - - Na acetate ....... (@ -) (- ;-) (- @) () Na citrate ....... (-1-) (-1-) (@ -) (- @) Na-succinate ... (-E-) (- @ -) (-f-) (-f-) positive assimilation; (- @ -) = positive assimilation, weak growth; (-) = poor growth, no assimilation; - = no growth.

All varieties develop at temperatures of 24 to 37 ° C, the most favorable temperatures are between 24 and 28 ° C. Bennet's agar at 37 ° C produces a strong yellow pigment after 24 to 48 hours of incubation.

The microscopic examination shows very long straight chains of conidia, which rise monopodially from the aerial mycelium. These can be found in mushroom culture plates see Brown develop in situ. A typical feature of this organism is the formation of pigment grains in both the vegetative and aerial mycelium. on the fungal culture platelets according to Brown these appear exogenous and endogenous in zones of merging or merging mycelium. The grains are with the non-pigmented varieties are colorless and the colored varieties are orange-red.

For spotted specimens and wet specimens of non-spotted material, where the mycelium is separated, the grains appear in more or less ordered form Rows. In the case of spot preparations, the mycelium containing the grains is opaque and has a Diameter from 0.78 to 1.569. Solvent extracts from the pigment from agar slant cultures have antibiotic effectiveness. Colorless ones show the same antibiotic effectiveness and stained varieties on agar graticules.

101 a can be obtained by treating Streptomyces sp.101 in aqueous Culture medium cultivated under submerged aerobic conditions, the medium preferably contains both an assimilable carbohydrate and a nitrogen compound. Although a number of suitable media is available, one will, for reasons of economy, the highest yield of antibiotic material and easy isolation prefer certain culture media. The currently preferred sources of carbohydrates are Glucose, dextrin, molasses and starch and mixtures of these substances. Other suitable Sources are maltose, galactose, mannitol, soybean oil and the like. Preferred nitrogen suppliers are soybean meal, fish meal, cottonseed meal, kay soy (finely powdered defatted Soybean meal) and the like. Other suitable sources are peanut meal, brewer's yeast, corn gluten meal, Corn soaking water, etc.

The medium advantageously also contains inorganic nutrient salts such as potassium, Sodium, calcium phosphate or sulfate and the like. Important trace elements such as magnesium, Manganese, zinc, iron and the like can also be used in the culture medium for growing Streptomy ces sp. 101 should be included. Such trace elements are commonly used with the others Components of the medium introduced in the form of impurities.

The media used in the process can, in addition to the nutrient components also contain precursors that lead to valuable products. So you can for example, add an assimilable source of cobalt, if the emergence of cobaltamines (vitamin B1 and vitamin-B "like products) desires, and these by-products then isolate by conventional methods. Furthermore, one can use steroid precursors such as progesterone, Reichstein's compound S or S acetate, add to an 11-position oxidized Get steroid.

To get the best growth of Streptomyces sp. 101 should before inoculation with the microorganism, the culture medium on a pn between set about 6.5 to 7.5.

The preferred way of carrying out the fermentation is in the Production of large quantities of 101 a submerged aerobic culture. For production of Limited amounts of the antibiotic can be obtained in shake flask cultures or with Working surface cultures in bottles. If you have the culture in large containers or tanks, so it is recommended to use one in growth for inoculation to use the existing form of the microorganism in order to avoid a pronounced delay the formation of the antibiotic and the associated insufficient utilization of the Avoid attachment. It is therefore desirable to have a developing seed first of the microorganism by adding a relatively small amount of culture medium inoculated with material scraped from an agar slant, and if a young, vigorously developing inoculum has arisen, this aseptically into the large Containers or tanks transferred. The medium in which the vegetative seeds are produced can be the same as that used in making the antibiotic, or different from this.

The best yields of antibiotic 101 a are obtained at temperatures between about 24 and 37 ° C, preferably between about 28 and 342 C, during one Period of about 2 to about 6 days.

The method according to the invention is not limited to manufacture of the antibiotic 101 a with Streptomyces sp. 101. Of course you can too other strains producing this antibiotic, or variants use such as z. B. can be easily generated and isolated by routine isolation and / or modification methods, such as selection of cultured ones Microorganisms and modification of the same through physical action by means of X-rays and ultraviolet light or by means of mutagenic chemical agents, such as nitrogen mustard and colchicine.

The rate of formation and concentration of the antibiotic arm 101 A in the culture medium can easily be found during the growth period of the microorganism track, irccm Tran ccm culture medium samples taken on their antibictic Tests effectiveness against a microorganism that is known to be sensitive to the Antibiotic to the front, such as B. Myccbacterium ranae or Micrococcus pyogenes Variety aureus, using the standard agar diffusion test or the turbidimetric Exam applies. In general, the maximum production of the antibiotic is at Submerged aerobic culture about 2 to 6 days after inoculation of the culture medium achieved.

The antibiotic material can be extracted from the culture medium or adsorption can be obtained, e.g. B. by adsorbing the antibiotic on charcoal and eluted therefrom by suitable eluents. For technical production extraction with solvents is preferred as it takes less time, is cheaper and gives higher yields.

A preferred extraction method for obtaining the antibiotic effective material from the fermented nutrient medium is the nutrient medium at a pH between about 4.0 and 8.0 and then with one in water insoluble organic solvents such as lower alkyl acetates, e.g. B. amyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl acetate; low aliphatic ketones, z. B. methyl ethyl, methyl isobutyl and methyl isopropyl ketone; halogenated aiphatic Hydrocarbons such as methylene chloride, ethylene dichloride, carbon tetrachloride and chloroform; Hydrocarbons such as benzene, toluene, cyclohexane and methylcyclohexane. Ethyl acetate and methylene chloride «earths are particularly preferred. The hydrocarbons those for the weaker polar components, e.g. B. 101 a-4 and 101 a-5, more selective are to advantage together with one or more solvents of others Genus used.

The extract is then concentrated and dried to make the antibiotic Material. If the product thus obtained is in a suitable solvent such as cyclohexane, benzene, methylcyclohexane, toluene, ethyl acetate and the like. Dissolves and then with technical n-hexane (petroleum ether consisting essentially of n-hexane) added, the antibiotic material is obtained in the form of yellow crystals.

The antibiotic 101 a is a complex of closely related components, each of which has antibiotic effects. In most cultures one can find five components 101 a-1, 101 a-2, 101 a-3, 101 a-4 and 101 a-5. In some cultures Further components 101 a-6 and 101 a-7 were also found. Also was 101 a-2, a highly acylated component, by treatment with acetylation catalysts and converted into the other components by treatment with ammonia, resulting in Examples 10, 11 and 12 will be described.

In the drawing, FIG. 1A shows a paper chromatogram which was developed in a phosphate buffer solvent system for a typical fermentation product obtained according to Example 1 and the components 101 a-1, 101 a-2, 101 a-3, 101 a-4 and 101 a-5 shows. Figures 1B and 1C are paper chromatograms developed in a solvent system (benzene-methanol-water 2: 1: 1 by volume) with the same product. They show the breakdown into the components 101 a-1,101 a-2,101 a-3 and a mixture of 101 a-4 and 101 a-5. Figure 1 D is a quantitative paper chromatogram developed in a modified solvent system (benzene-methanol-water in a volume ratio of 1: 1: 2) at room temperature. It shows the same five components. 1E is a paper chromatogram of another culture prepared according to Example 1 and developed in the above solvent system and shows the presence of seven components. The R f values, ie the ratio of the distance covered by the component to the distance covered by the solvent for the seven components, were determined from these paper chromatographic analyzes. Table IV Component I Rf 101a-1 ......................... 0.65 101a-2 ......................... 0.35 101a-3 ......................... 0.75 101a-4 and 101a-5 ............. 0.85 101a-6 ......................... 0.25 101 a-7 .......................... 0.55 The close relationship between the chemical and physical properties of the components of 101 a results from the fact that they are not broken down in the usual solvent systems. As can be seen from the paper chromatographic analyzes in the following table, all components move in the same ratio in the various solvent systems. Table V Solvent system IR f n-butanol (810 org). . . . . . . . . . . . . . . . . . . 0.9 n-butanol (81%) - 0.25% p-toluene sulphate phosic acid ........................... 0.9 n-butanol-acetic acid-water (2: 1: 1) 0.9 n-butanol (81 ° / Qig) + 2 () 1;, piperidine .... 0.9 96 % water -f- 4 ° / p n-butanol. . . . . . . . . 0.85 96 % water + 4% n-butanol + 0.25% p-Toluenesulfonic acid ................. 0.9 0.1n NH40H with methyl isobutyl ketone saturated ......................... 0.8 Crystalline preparations of 101 a showed melting or decomposition points between about 155 and about 255 ° C; optical rotations [a] D between about 301 and 425 ° (c = 0.3 in absolute ethanol). With the Ouarz spectrophotometer model DU from Beckmann or the registration spectrophotometer from Cary one obtains absorption maxima in the ultraviolet at 435 and 245 mti in a 95% neutral ethanol solution. Elemental analysis for C "H47-s NOrs Calculated . . . . . . . . . . . . . . . . C 60.20. H 7.08. N2.07; found . . . . . . . . . . . . . . . . . . C 63.64, H 6.89, N 1.68. 101a is soluble in alcohols, methanol, ethanol, propanol, butanol, amyl alcohol and their isomeric forms, dodecyl alcohol, undecyl alcohol, decyl alcohol, nonyl alcohol, heptyl alcohol, hexyl alcohol and their isomeric forms; in lower alkyl acetates such as amyl acetate, ethyl acetate and the like; in lower alkyl ketones such as methyl ethyl ketone, methyl isobutyl ketone, ethyl isopropyl ketone and the like; in chlorinated aliphatic hydrocarbons such as methylene chloride, ethylene dichloride, chloroform and the like, in dioxane, dimethylacetamide, dimethylformamide (> 5 mg / ml); it is somewhat soluble in carbon tetrachloride, toluene (5 mg / ml); in ethers such as ethyl ether, butyl ether, propyl ether and their isomeric forms and the like; in technical hexane; Water (C as 1 mg; ml).

At room temperature 101 a is in the pH range from about 2.0 to about 6.0 Shelf life 3 to 4 days, at 80 ° C and pH 2.0 1 hour. In alkalis this is antibiotic Material is unstable and suffers a gradual loss of antibiotic effectiveness, if it is left to stand at room temperature at pH 7.8 for 2 or 3 days.

The infrared absorption spectrum (sodium chloride prism) of 101 a in mineral oil suspension according to FIG. 925, 875, 797, 735.

101 a is a neutral or weakly acidic material. With treatment it is hydrolyzed with dilute alkali. When implementing an ethanol solution this material with an aqueous methyl orange solution gives the heliantate of the complex; in the reaction with acylating agents the corresponding ones are obtained Acyl derivatives such as B. the acetyl and benzoyl derivatives.

101 a is characterized by its effectiveness against the following organisms: Table VI Antibacterial range of action from 101a-.minimum inhibitory concentration mcg, 'ml Test organism (l0la *) Mycobacterium tuberculosis H 37 Rv .... 0.16 Mycobacterium tuberculosis BCG ...... 0.16 Mycobacterium ranae. . . . . . . . . . . . . . . . . 0.78 Micrococcus pyogenes v. aureus ....... 0.39 Micrococcus pyogenes v. albus ......... 0.39 Streptococcus hemolyticus ............ 25 Streptococcus viridans ................ 3.12 Bacillus subtilis ..... .............. ... 0.39 Diplococcus pneumoniae ........... .... 6.25 Klebsiella pneumoniae. ....... ...... .. 3.12 Pseudomonas aeruginosa .............. 12.5 Salmonella typhosa. . . . . . . . . . . . . . . . . . . 25th Salmonella paratyphi. . ... . ... . . . . ... > 100 Pasteurella multocida. . . . . . . . . . . . . . . . . 1.56 Escherichia coli ...................... 25 Proteus vulgaris ...................... 100 Nocardia asteroides .................. 1000 (partially at 100) Blastomyces dermatitidis. . . . . . . . . . . . . > 1000 Coccidioides immitis. . . . . . . . . . . . . . . . . . > 1000 Geotrichum sp .................... .... 1000 Hormodendrum compactum ........... 1000 (partial Inhibition) '°) 101 a-1.370mcg'ml; 101 a-2,125rucg'ml; 101a-3, 430meg ml; 101a-4 and 101a-5 each about 20 rucg `ml. Test organism (101a *) Phialophora verrucosa ................ 1000 (partial Inhibition) Cryptococcus neoformans .............. 1000 Sporotrichumschenckii ................ 1000 (partial Inhibition) Monosporium apiospermum ........... 1000 (partial Inhibition) Trichophyton rubrum ................ 1000 Microsporon audouini ................. 1000 Candida albicans Ab. . . . . . . . . . . . . . . . . > 1000 Candida albicans Up ..................> 1000 Microsporon canis .................... 1000 Trichophyton interdigitale. .... ... ...> 1000 Trichophyton violaceum .............. 1000 *) 101 a-1, 370 mncg / ml; 101 a-2.125 mcg; 'ml; 101 a-3.430 mcg / ml; 101a-4 and 101a-5 about 20 mcglml each. The components of 101a can be separated by chromatography or by countercurrent extraction. For paper chromatography, as shown in FIGS. 1 A, 1 B, 1 C, 1 D and 1 E, various solvent systems have proven to be effective in separating. Paper chromatograms with isolated 101 a in the same solvent systems show the same seven components. Another solvent system that gives the same type of decomposition as the above system is chloroform saturated with water; furthermore cyclohexane-chloroform-water in a volume ratio of 1: 8: 2. In the latter system, strips buffered to pH 4.1 showed the following R f values after 2 hours of treatment in the aqueous phase and about 1 hour of development in the non-aqueous phase Table VII Component I Rj 101a-1 ........................... 0.37 101a = 2 ........................... 0.13 101a-3 ............................ 0.77 101 a-4 and 101 a-5. . . . . . . . . . . . . . . . . 0.88 The same or similar solvent systems can be used on columns to effect component separation. A good separation of all components with the exception of -4 and -5 is also achieved in this way. When using a column of infusor earth, good results were achieved with the solvent system of toluene-technical-n-hexane-methanol-water in a volume ratio of 67: 33: 60: 40. A portion of isolated 101 a, containing components 101 a-1, 101 a-2, 101 a-3, 101 a-4, 101 a-5, was placed on top of a column of infusory soil containing the stationary (lower) phase of the solvent system, and then with the mobile (upper) phase of the solvent system eluted. For this purpose, the isolated 101 a is dissolved in the stationary phase and the solution is mixed with 2 g of infusoric soil per ml. The two phases of the solvent system are obtained by allowing this system to separate into two phases. The column is filled in the ratio of 2 g of infusory soil per ml of the stationary or lower phase. When eluting with the mobile phase, components -4 and -5 go away together first, then components -3, -1 and -2 follow in the order given. Component -2 migrates very slowly and is sometimes advantageously washed out of the column using methylene chloride or a similar solvent after the other component fractions have been removed.

The complex can also only be partially dismantled, for which purpose the Countercurrent process according to Craig. Fig. 3 shows the distribution of the five components in the Craig fractions when used with a water-ethanol-cyclohexane-ethyl acetate solvent system (1: 1: 1: 1 parts by volume). You can see that a perfect separation of the components 101 a-1, 101 a-4 and 101 a-5 is achieved, but that the components 101 a-2 and 101 a-3 cannot be disassembled.

By combining a column separation with a Craig separation all five components are disassembled. So you can do the separation with the column Separate resulting mixture of components -4 and -5 according to the method of Craig or the mixture of components 101 obtained in the Craig separation Separate a-2 and 101 a-3 using the column.

Fig. 4 shows the movement of the Craig fractions on the paper strip when using the above solvent system benzene-methanol-water. Chromatogram A shows that the first Craig fraction contained only 101 a-2 and 101 a-3, chromatogram B shows that the second Craig fraction contained only 101 a-1. Chromatogram C shows the result with the third and fourth Craig factions. Since both 101a-4 and 101 a-5 have the same R f in the solvent system, a chromatogram is used for Illustration of both factions.

A typical quantitative distribution of the components is shown in Table VIII. The culture medium (1850 ml) was filtered and the clear liquid extracted with ethyl acetate. Table VIII Quantitative distribution of the components of 101a Amount of component in # Lg, 'ml or # tg / mg Stage, 101a-4 101 a-1 101. a-2 101a-3 and lor 101a-5 Filtered medium ... 48 28 85 70 Cake used 1) 12 <10 41 224 Medium used. 0 <10 tracks 0 Extract residue 2) ... 110 82 255 254 Projecting 2) ...... 84 <12I 237 546 1) 10 g of the cake was slurried in 20 ml of acetone, filtered and the acetone filtrate checked. 2) The ethyl acetate extract was 5 times the volume treated technical n-hexane and filtered. The main components of 101 a have the following physical properties Table IX Component analysis ° / ° C j ° / ° H ° / ° ° / ° acyl i 101 a-1. . . . . . . . . 58.84 7.22 1.70! 11.75 101 a-2. . . . . . . . . 59.72! 6.57 1.87 18.17 101a-3 ......... 59.07 6.99 2.03 9.84 101 a-4. . . . . . . . . 60.86 7.85 2.14 5.91 101 a-5. . . . . . . . . 63.11 7.16 1.92 6.13 Table X Specific rotation -24 solution melting point @aD medium - 454 ° I C13 182 to 184e - 618 "CH C13 195" 200 -; - 317- CHC13 168 " 171-- * CHCl3 115 "118 164 ° CH C13 102 "105- and the following range of antibacterial effects Table XI Antibacterial effect of 101a and its components Minimum inhibitory concentration, mcg / ml Test organism I 101a I 101a-1 I 101a-2 I 101a-3 101a-4 I 101a-5 Mycobacterium tuberculosis H 37 Rv. . . . . . . . . . . . . . . . 0.16 0.26 0.08 0.31 - - Mycobacterium tuberculosis BCG .................... 0.16 0.31 0.16 0.31 3.12 6.25 Mycobacterium ranae .............................. 0.78 0.78 0.39 - - - Micrococcus pyogenes v. aureus ..................... 0.39 0.39 0.39 3.12 12.5 6.25 Micrococcus pyogenes v. albus ...................... 0.39 0.2 0.2 0.78 6.25 6.25 Streptococcus hemolyticus .......................... 25 25 3.12 100 25 12.5 Streptococcus viridans ............................. 3.12 3.12 3.12 12.5 100 100 Bacillus subtilis ................................... 0.39 0.2 0.39 0.78 0 , 39 1.56 Diplococcus pneumoniae ............................ 6.25 12.5 1.56 100 50 1.56 Klebsiella pneumoniae ............................. 3.12 3.12 3.12 6.25 100 100 Pseudomonas aeruginosa ........................... 12.5 12.5 12.5 25 100 100 Salmonella typhosa ................................ 25 25 12.5 25 100 100 Salmonella paratyphi .............................. 100 100 100 100 100 100 Pasteurella multocida .............................. 1.56 1.56 0.78 1.56 50 50 Escherichia coli ................................... 25 12.5 25 25 100 100 Proteus vulgaris ................................ ... 100 100 25 100 100 100 Component 101a-2 was prepared according to Example 9, the other components according to Examples 13 and 14. Component 101a-2 was recrystallized from acetone, the remaining components from aqueous acetone (4 volumes of acetone to 1 volume of water). The recrystallized components show the following absorption maxima in the infrared spectrum in mineral oil suspension. Table XII Infrared absorption maima z-> Z-, i-> 101a-1: 3400, 1758, 1715, 1652 1642 1602 1584, 1540 1522, 1492, 1455, 1405, 1374, 1335, 1265, 1240, 1195, 1189, 1155, 1115, 1069, 1035, 1012, 988, 925, 875, 850, 835, 797, 750 740 725, 680. 101a-2: 3480 3310, 1740 1720 1706, 16_31, 1598 1585, 1550 536, 1510, 1459 437, 1406, 1375 1365, 1338, 1290 1281, 1252, 1198 1175, 114_9, 1120 1105, 1085 1070, 1037 Ö26 0 15, 1096, 972, 942 935, 916, 900, 975, 830, 800, 750 740 730 722, 685. 101a-3: 3410, 1756 720, 1651, 1615 590, 1535, 1510 1490, 1455, 1405, 1372, 1335, 1265, 1239, 1192, 1124, 1100, 1068, 1035, 1010, 990 75, 940, 924, 905, 877 855, 798 782, 750 737 726. 101a-4: 3420, 1760 1718, 1655 1602 1589, 1542, 1498, 1455, 1405, 1375 1365, 1339, 1275 1255, 1192, 1130, 1100 1089, 1060, 1035, 1008, 986 74, 900, 87-1, 825, 799 790 773, 740 730 710, 677. 101a-5: 3530 3480 3420, 1760, 1720, 1655, _1600, 1535, 1496, 1450, 1405, 1375 364, 1332, 1276 1 2 67, 1248 240, 1193, 1155, 1124, 1102, 1085 1072 1062, 1034, 1011, 990, 950, 925, 876, 852, 817, 798, 786 775, 752 745, 723 712 and ultraviolet absorption maxima and extinction coefficients in 95% ethanol. Table XIII Absorption maxima in the ultraviolet and visible light Component I max I a 101 a-1. . . . . . . . . . . . . . . . . . . . 245 40.83 266 33.85 320 14.01 432 10.97 101a-2 .................... 245 41.89 260 35.21 320 13.98 430 13.62 101a-3 .................... 245.5 44.35 260 38.97 320 15.31 430 12.19 101a-4 .................... 246 43.14 264 37.03 320 14.77 433 10.53 101a-5 .................... 245 38.22 273 34.69 320 19.04 437 9.19 These numbers were determined with acetone solutions, namely for 101 a - 2 in 100 ° o strength acetone and in the other cases in 800 ", acetone and 200; #, M'asser.

The components give positive reactions with FeCl and iodoform.

Antibiotic 101a and its components are valuable agents to combat many caused by bacterial infection in humans and animals Diseases. For this purpose one can use the antibiotic alone or in combination with a pharmaceutically acceptable carrier which is solid, powder or liquid can be use. The agent can be in the form of compressed tablets, effervescent tablets, Powder, granules, capsules (hard and oak gelatine capsules), aqueous solutions, Suspensions in edible oils or water or others for oral administration suitable forms are needed. Sterile ones are used for parenteral use liquid preparations. The medium can be a sterile solvent or suspending medium which is an injectable oil or hydrous hydrophilic colloids such as Na-carboxymethylcellulose, VIethylcellulose, polevinylpyrrolidone, gelatin, tragacanth, etc. contains. The effective one antibiotic material can be used with solid diluents or excipients the tableting be mixed, such as corn starch, lactose, talc, stearic acid, Magnesium stearate, gum resins and the like can also be used as a powder for the antibiotic Fill and use like that.

The antibiotic 101 a and its components and derivatives can also as a suspension in a fatty oil, such as coconut oil, arachid oil and the like, which are approximately 2 °; o Contains aluminum monostearate as a suspending agent, as such or in capsules administered. It can also be used in vaseline ointments, water-soluble ointment bases, such as Carbowaxe (solid polyethylene glycols with a molecular weight of 1500 and mixtures of polyethylene glycols with higher and lower molecular weight), to use locally as ointments. It can also be in water-in-oil or oil-in-water emulsions and lotions are incorporated. Another use case is in form given by nasal drops, lozenges and suppositories. For veterinary use Bougies, capsules, tablets, mastitis ointments, oil suspensions are particularly suitable etc.

As a result of its pronounced antibacterial effectiveness, the low Toxicity and the high achievable blood levels, the antibiotic is suitable 101 a and its derivatives for the treatment of various diseases. So can because of its unusually high effectiveness against M. tuberculosis H 37 Rv Treatment of tuberculosis can be used. Doing so will produce beneficial results obtained when one 101 a with other tuberculosis remedies, such as isonicotinic acid hydrazide, Streptomycin and; or dihydrostreptomycin, p-aminosalicylic acid and its salts, Ir-4-Amino-3-isooxyzolidone (Cvcloserin), combinations of isonicotinic hydrazide and p aminosalicylic acid and its salts, combinations of streptomycin, isonicotinic acid hydrazide and p-aminosalicylic acid and its salts, etc. are combined.

The antibiotic product obtainable according to the invention can also be used as Tuberculosis disinfectant used in hospitals and in the dairy industry will. For this purpose, it is the usual carriers, such as aerosols or detergent solutions, added. This can be used alone or in combination with sulfonamides, such as sulfadiazine, Sulfamerazine and sulfamethazine (in a ratio of about 1 part of the antibiotic to a total of 2 parts sulfonamide) or with other antibiotics, such as tetracycline, Oxvtetracycline, Chlortetracycline, Neomycin, polymycin, chloramphenicol, Penicillin G, 0 and V, novobiocin, bacitracin, streptothricin, circulin and erythromycin, happen. The antibiotic or its combinations with the aforementioned therapeutic agents are also suitable for the treatment of staphylococci and pneumococci Infections of the lungs and airways. Administration can be topical, oral or done parenterally. The antibiotic can also be used along with vitamins, such as Thiamine, riboflavin, ascorbic acid, niacinamide, pyridoxine, pantothenic acid, vitamin B12 and folic acid. It can also be therapeutically valuable with others Combine fabrics. In combination with various corticoids it becomes therapeutic Effect of 101 a and its components in the treatment of atopic and contact dermatitis, Neurodermatitis, pruritis, etc. greatly improved. Suitable corticoids are cortisone, Hydrocortisone and its esters; A 1-cortisone and @ 11 1-hydrocortisone and their esters in the 21-position, such as the acetates, cyclopentylpropionates and succinate as well as the water-soluble ones Salts thereof, ethyl-substituted cortisones and hydrocortisones and 6-methylhy drocortisone including its esters.

The antibiotic obtainable according to the invention can also be used in animals, such as poultry and cows, with the same effect as in humans. It can be the animal feed mixes, e.g. B. the poultry feed, in amounts of at least 0.10. /, Are added.

If the antibiotic is administered parenterally, for example, it is suitable It is also used to treat animals affected by Pasteurella multocida, the pathogen haemorrhagic blood poisoning, a feverish one that occurs when transporting cattle Infection. One can also use the antibiotic on its own or in Combination with other antibiotics or therapeutic agents as a dietary supplement Use to favor the growth of animals or poultry. As a result of its high Efficacy against S. pullorum, it is also valuable for combating through this Microorganism caused white diarrhea in cows.

The following examples illustrate the production, extraction, concentration, Purification and identification of the antibiotic 101 a and its components and Derivatives. Example 1 Production of Antibiotic 101a A. Seeds Streptomyces sp. 101 is distilled on a maltose tryptone agar slant culture (composition in g / 1 Water; Maltose 10, tryptone 5, K, HP04, 0.5; Fe S 04-7 H, 0, 0.1; Agar 15) 7 days cultivated at 28 ° C and the culture thus obtained as inoculum for a seed culture used.

B. Seed culture 50 ml of a sterile medium with the following composition: NZ-Amine A (enzymatic degradation product of casein) ........... 2.0 g Glucose ......................... 1.0 g Soy sauce (extract of hydrolysing ten soybeans and wheat) .... 1.0 ml K2 HP O.. . . . . . . . . . . . . . . . . . . . . . . . 0.25 g KH, P04 ..... ... ...... .......... 0.25 g Tap water . . . . . . . . . . . . . . . . to 100 ml is inoculated with the seed culture obtained according to regulation A and the medium is shaken for 40 hours at 28 ° C. on a rotating shaker at 250 revolutions / minute.

C. Fermentation The seed culture produced in accordance with regulation B was added in an amount of 1 percent by volume to 100 ml of a fermentation medium of the following composition: Kay Soy (defatted finely ground Soybean meal) ............... 1 g Glucose ......................... 2 g Brewer's yeast ......................... 0.25 g K Cl ............................ 0.3 g Ca Co ............................ 0.4 g Tap water . . . . . . . . . . . . . . . . to 100 ml and ferment this medium on a rotating shaker at 28 ° C for 5 days. The aeration was equivalent to a sulfite oxidation value of 0.3 millimoles of oxygen (liter / minute). The filtrate is effective against M. pyogelles v. aureus, Bacillus subtilis, M. tuberculosis H 37 Rv and saprophytic microbacteria.

Example 2 Production and isolation of 101a A. Pre-sowing culture 100 ml of a sterile pre-sowing medium containing the following components: NZ-Amine A .................... 20 g Glucose ......................... 10 g Soy sauce ....................... 10 ml KZHPO .. ........................ 2.5 g KH, PO4 ........................ 1.5 g Tap water, pFi 6.8 to 7.0, on 11 is with a culture of Streptomy ces sp. 101 produced on agar slants, inoculated as in Example 1, A and cultured for 3 days in the flask at 28 ° C. on a rotating shaker. B. Seed Culture A 191 volume container with a stirrer containing 12.51 of the following sterile medium: Kay Soy. . . . . . . . . . . . . . . . . . . . . . . . log Corn dextrin ...................... 20 g Brewer's yeast ......................... 2.5 g KC1 ............................ 3.0 g Ca C 03. . . . . . . . . . . . . . . . . . . . . . . . . . 4.0 g Corn steeping water. . . . . . . . . . . . . . . 10 ml Tap water . . . . . . ... . . . . .. on 1 1 is inoculated with 1 percent by volume of the pre-seed culture obtained according to A and cultivated at 28 ° C. for 10 days. C. Fermentation To two tanks each containing 2501 of the following medium Kay Soy .................... 10 g Corn dextrin .................. 20 g Brewer's yeast ..................... 2.5 g KCl ........................ 3.0 g Ca C 03. . . . . . . . . . . . . . . . . . . . . . 4.0 g Corn steeping water. . . . . . . . . . . 10 ml Tap water . ... . . . . ... on 1 1 Lard oil ...................... 300 ml tank Lard oil: 1 °; 'a stenol (Octadecanol). . ... ... . . ... 1550 ml / tank half of the seed culture B is added and the medium is fermented at 28 ° C. for 42 hours. The lard oil and lard oil -f- 10/0 stenol are added as required to prevent foaming.

D. Isolation After 42 hours the whole is filtered at harvest pR 7.4. The filtrate plus washing liquid of the mycelium (total volume 6001) is saturated with ethyl acetate and extracted with 0.2 times the volume of ethyl acetate. The solvent extract (121 l) is concentrated to 960 ml and evaporated to dryness in vacuo. 1Ian receives 34 g of a red glassy solid product (570; '0 yield). Purify the material in portions using a countercurrent partitioning technique (acetone-ethyl acetate-water, 1: 1: 1). After the purified material has been reprecipitated from ethyl acetate with technical grade n-hexane, 101 a is obtained in crystalline form. The product has a strength of 1000 mcg / mg in the Mr. 101a test. E. Tests The Mr. 101a test is carried out as follows: Mycobacterium ranae Upjohn culture collection No. 161 is cultivated on a Lowenstein-Jensen slant culture (KA Jensen, "Towards Standardization of Laboratory Methods", International Union against Tuberculosis, Vol. 24, pp. 102 and 103, 1954). A small amount of the culture is placed in a 250 ml flask or 50 ml of the following sterile medium Dextrose ......................... 10 g Beef extract (dried water riger extract from beef .... 4 g Peptone ........................... 4 g Yeast extract (dried aqueous Autolyzed yeast extract) ... 1 g Sodium chloride. . . . . . . . . . . . . . . . . . . 2.5 g Tween 80 (polyethylene derivative of Sorbitan monooleate). . . . . . . . . . . . . 10 ml Distilled water, before sterilization tion set to pa 7.0 ....... 1000 ml introduced and cultured on a reciprocating shaker for 24 hours at 37 ° C. 1 ml of the bacterial suspension, which in the Lumetron colorimeter (Photovolt Corp.) at 530 m [j, against a mean blank sample shows about 10 0/0 light permeability, is used to inoculate 100 ml test agar (brain-heart infusion broth, which per liter 200 g calf brain infusion, 250 g beef heart infusion, 10 g dextrose, 5 g NaCl, 2.5 g disodium phosphate and to which 1.50! `0 agar was added) is used, after which it is cooled to 48 ° C and kept at this temperature. 5 ml of the inoculated agar are left to solidify in Petri dishes and before use at room temperature.

Unknown solid preparations are in the ratio of 1 mg / ml in acetone dissolved and prepared using n! 10 phosphate buffer at pA 6 suitable dilutions. Fermentation liquor and other samples of processing solutions are also appropriately diluted with buffer solutions. The test takes place using the standard 12.7 mm filter paper discs to which 0.08 ml standard or Test solution incorporated. Contains the basic solution of a crystalline standard preparation 1 mg / ml in acetone. With n / 10 phosphate buffer, dilutions of 6.0 are made at pft 5, 10, 20, 40 and 80 mcg / ml. The disks are incubated at 37 ° C. overnight and the diameter of the inhibited zones was measured. The standard curve is on a semi-logarithmic basis Plotted on paper, namely the concentration in ftg ml as the abscissa and the zone diameter as the ordinate. The conversion factor is 4 lig per biological unit. A biological one Unit is the amount of antibiotic that has an inhibition zone of 20 mm during the test against a sensitive organism such as M. ranae (: 4b.-101 a-Test), S. aureus or B. subtilis using a 12.7 mm diameter paper disc results.

Example 3 Production and isolation of 101a A. Pre-sowing culture Five 500 ml Erlenmeyer flasks, each containing 100 ml of the following pre-sowing medium NZ-Amine A ...................... 20 g Glucose ........................... 10 g Soy sauce ......................... 10 ml K, HP 04 .......................... 2.5 g KH @ P04 .. ... ... . . ... . . ... . . ... . . 1.5 g Tap water ................. on 1 1 are obtained with a slant agar culture of Streptomyces sp. 101 inoculated and cultivated on a rotating shaker for 3 days at 28 ° C. B. Seed culture A tank containing 250 liters of the following medium: Kay Soy .......................... 10 g Corn dextrin ........................ 20 g Brewer's yeast ........................... 2.5 g KCl ............................... 3.0g CaCO3. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.0 g Corn steeping water. . . . . . . . . . . . . . . . . 10 ml Water ........................ to 1 1 is inoculated with the pre-seed culture obtained above and cultivated for 2 days at 28 ° C. with constant stirring and aeration. C. Fermentation After 2 days the contents of the seed tank are placed in a tank with a capacity of 7570 liters containing 50001 of the following medium. Corn dextrin. . . . . . . . . . . . . . . . . . . . . 20 g Brewer's yeast ........................ 2.5 g K C1 ............................ 3.0 g CaC03. . . . . . . . . . . . . . . . . . . . . . . . . 4.0 g Corn steeping water. . . . . . . . . . . . . . 10 nil Peanut flour .................... 10 g Water ..................... on 1 1 Lard oil ......................... 3,551,; tank Lard oil -E- 10l0 stenol. . . . . . . . . . . . . 45.429 1 / tank CLRS No. 10 (mixture of free Fatty acids + mono-, di- and tri- glyceride and unsaponifiables) ... 15.10 1l tank and fermented for 4 days at 28'C with constant stirring and aeration. The lard oil as well as lard oil + 10., ', stenol and CLRS are added as required to prevent foaming.

D. Isolation After 4 days of fermentation, the whole is filtered (at natural p "7.1) and the filtrate (54971) extracted with 1060 l (0.2 volume) of ethyl acetate. The extract is concentrated to 4200 ml under reduced pressure with 4200 ml of technical grade n-hexane are added and the precipitate obtained is dissolved in 3920 ml of methylene chloride, 3920 ml of technical grade n-hexane are added and the resinous red precipitate (preparation 3A) is removed by filtration n-hexane is added and the yellow crystalline precipitate is dried in vacuo, giving 104 g of 101a (preparation 3B) with a strength of about 920 mcg / mg.

The 0.5 g of the preparation is added to 10 ml of an aqueous ethanol solution 3B contains 30 ml of a saturated aqueous solution of methyl orange and holds the Mixture for 30 minutes at 50.degree. C., the helianthat salt of 101 a is obtained as a brown one Crystals. The crystalline material is removed by filtration and dried.

The resinous red precipitate (preparation 3A) is dissolved in 600 ml of acetone dissolved with moderate heating and stirring, then cool and at room temperature ditched. After 13 days the volume has decreased to about 500 ml. Of the The yellow precipitate that forms is filtered off with acetone and technical grade n-hexane washed and dried in vacuo. This gives 71 g of 101 a (preparation 3 C) with an efficiency of more than 2000 mcg / mg.

When the product (preparation 3 C) was chromatographed in a phosphate buffer system of pH 7.0 and a solvent system, it was found that components 101a-1, 101a-2 and 101a-3 were present. Example 4 Production and Isolation of 101a The procedure was as in Examples 3, A, B and C, but in a tank space of 56,800 liters and a medium which had the following composition per liter Kay Soy .......................... 20.0 g Corn dextrin. . . . . . . . . . . . . . . . . . . . . . . . 40.0 g Brewer's yeast ........................... 2.5 g KCl ............................... 3.0g CaCO3 ... . . . . . . . . . . . . ... . . . . . ... . . 12.0 g The temperature was maintained at 28 ° C and the harvest occurred after 84 to 88 hours. The medium from the three tanks was filtered and collected at p. 6.75 to 7.0 extracted with 30,280 l of ethyl acetate. The extract was gradually concentrated to 72 liters. The antibiotic was precipitated with twice the volume of technical grade n-hexane. The precipitate dissolved in boiling methylene chloride (with the exception of about 200 g of black tar which was discarded). The methylene chloride solution was mixed with the same volume of technical grade n-hexane, a solid product separating out, which after drying weighed 2665 g and per milligram was 132 pg 101 a-1, 120 @zg 101 a-2 and 141 pg 101 a -3 as indicated by paper chromatographic analysis (Preparation 4A). The supernatant liquid was admixed with a further 3 volumes of technical grade n-hexane, a further 1561 g being obtained which contained 345 g 101 a-1, 165 pg 101 a-2 and 665 g 101 a-3 in milligrams (preparation 4B ). The supernatant was filtered again after standing, whereby a further 178 g of solid material were obtained, which in milligrams 76 #ig 101 a-1, 2 #tg 101 a-2, 440 p, g 101 a-3 and 77 #tg 101 a-4 or 101 a-5 (Preparation 4C).

Example 5 Production and isolation of 101a A. The fermented liquid from two further 56 800-1 tanks, produced according to Example 4, was filtered and extracted with 18 1681 methylene chloride. The medium used had the following composition per liter: Soybean meal ..................... 20 g Brewer's yeast ............................ 2.5 g NaC1 .... .. ....... .. ....... ........ 3.0 g CaCO3 ....... .. .................... 0.59 Starch ............................. 40.0 g No CLRS was used. The fermentation took place for the first 65 hours at 28 ° C, then up to the 112th hour at 32 ° C, after which it was harvested. The extract was concentrated to 901 and 5 times the volume of technical grade n-hexane was added. The precipitate was collected on the filter and dried in vacuo. The yield was 22.157 kg (preparation 5A). The paper chromatographic analysis showed a content of 115 mcg 101 a-1, 105 mcg 101 a-2, 250 mcg 101 a-3 and a noticeable amount of 101 a-6 per milligram.

B. The liquid from two other 56 800-1 tanks, which are as in the example 4 was obtained, but cultivation was carried out at 32 ° C. for 114 hours filtered and extracted with methylene chloride 181681. The extract was filtered and treated further as in Example 5, A; 29.036 g of 101 a were obtained, that in the milligram 62.1 #tg 101 a-1, 62.6 #tg 101 a-2, 147.6 p.g 101 a-3 and 105.1 #Lg 101 a-5 (Preparation 5 B). Example 6 Preparation and Isolation of 101a The Fermentation Medium from further 56800-1 tanks, obtained as in Example 14, was filtered and kept at pH 4 to 5 extracted with 181681 methylene chloride. The extra lot was on one concentration with a boiling point of 60 ° C (one atmosphere) with a specific gravity of 0.99. It was found that the C H2 Cl, was contaminated with butyl acetate. The concentration was terminated when the boiling point in vacuo was 70'C. The concentrate was mixed with 7.5 times the volume of technical grade n-hexane. The precipitate thus obtained weighed 4.093 kg and contained 725 milligrams of active substance, which was as follows composed: 32.5 mcg 101a-1, 27.5 mcg 101a-2, 104.1 mcg 101 a-3 and some 101 a-4 and / or 101 a-5. Example 7 Preparation of 101a The culture solution of others 56800-1 tanks, produced as in example 4, but at 32'C and 136 hours of culture time, was filtered and extracted at pii 4 to 5 with 18 1681 methylene chloride. To Concentration of the extract, a separating aqueous layer was removed. The organic phase was further concentrated to 37.851. The concentrate was Poured into 5 times the volume of technical grade n-hexane and collected the precipitate and dried. It weighed 17.325 kg and contained 174 mcg / mg. The composition revealed quantitative paper chromatographic analysis results in 31 mcg / mg 101 a-1, 40 mcgimg 101 a-2 and 142 mcg / mg 101 a-3.

Example 8 Preparation of 101a-2 The above preparation was dissolved 3 C in a hot mixture of dioxane and benzene (3: 1) and obtained on cooling a fibrous crystalline material made with a mixture of dioxane and benzene (3: 1) was washed and dried in vacuo. The product was orange-yellow, dated Mp. 194 to 200 ° C, optical rotation [a] 24 = + 378- (0.25 ° / o in methanol), and shows a peak in the ultraviolet absorption spectrum at 435 m # t, Ei ° z, - 115. The product thus obtained (preparation 8A) had a strength of 3080 mcglmg.

The chromatographic analysis of the product in a phosphate buffer system of p ,, 7.0 showed that 101 a-2 predominated.

When triturated with benzene, the orange-yellow crystals (Preparation 8 A) were converted into another crystal form, the ratio of length to width is about 5 to 10: 1. Under polarized light it shows a chartreuse color and melts between 195 and 200`C. Upon drying, this material reverted to the appearance of the original yellow crystals. Example 9 Crystallization of 101a-2 A solution of 11.31 g of a product obtained according to Examples 3, A, B, C and D in 1131 1,4-dioxane was prepared. After adding an equal volume of technical grade hexane, 724 g of crude crystals of 101a-2 were obtained, which were recrystallized from dioxane and benzene. The crystals were dissolved in 7,251 dioxane and precipitated by adding an equal volume of benzene. The first crystallization gave 362 g (Preparation 9A). The mother liquors were concentrated to 21, and "purulent 283 g (preparation 9 B) were obtained by adding 5 times the volume of technical n-llexal".

The first mother liquors (technical dioxane n-hexane) were distilled off to 1-151. 191 of this solution was used to prepare a solution almost free of 101a-2 by precipitating the total amount of this component with 10 times the volume of technical grade n-hexane (preparation 9C). The remaining 1241 were mixed with 5 times the volume of technical grade hexane, with 7.860 kg of solid product with a content of 167 mcg; mg precipitated, which was composed as follows (paper chromatographic analysis): 335 mcg / 'mg 101a-1, 60 mcg.mg 101a-2 and 380 mcg'mg 101a-3 (preparation 9D).

Recrystallization from 101a = 2 Preparation 9A (362 g) was recrystallized from benzene (201) to give four fractions as follows 1st fraction, preparation 9E. . . . . . . . . . . . . . . 173 g 2nd fraction, preparation 9F. . . . . . . . . . . . . . . 62 g 3rd fraction, preparation 9 H. . . . . . . . . . . . . . . 41 g -1. Fraction, preparation 91. . . . . . . . . . . . . . . . 11 g Preparation 9E (benzene crystals) was then recrystallized from dioxane. When cooled rapidly, long hair-like crystals were obtained. If this was left to stand in the mother liquor or if the hot solution was slowly cooled, square plates formed. The result is the same when using peroxide-free dioxane. The square plates contain crystal dioxane in an amount of 1 to 1 '. Moles per mole 101a-2 (Preparation 9j).

Properties After the dryer. in the vacuum the long, fibrous crystals lost their shape, and masses were obtained which glow under the crossed Nicol prisms. If one is suspended in benzene or recrystallized from it, shorter and thicker needles are formed. When recrystallizing from butyl acetate, tufts of needles are obtained. 101 a-2 is soluble in Ühloroform, -% lethanol, dioxane, somewhat soluble in benzene, acetone and water and insoluble in petroleum ether. It does not take up bromine in C C14, the solution turns cherry red when heated. Chemical samples Attempt result control attempt conclusions Benedicts. . . . . . . . . . . . . . . . . . . . Negative glucose no reducing sugar Ninhydrin ...................... Negative glycylglycine no amino acids Biuret ......................... Negative Bactopeptone no peptide Molisch ... ... ... ...... ....... . Negative glucose not a carbohydrate Dear man ... .. ... ... . . ....... Negative - not a steroid Carpenter. . . . . . . . . . . . . . . . . . . Negative acetone no free ketones 2,4-Diliitrophenylhydrazine ..... . . . Negative acetone no free carbonyl groups Alcoholic AgN0 ............. Negative - no - C - = C - bond Ferric chloride. . . . . . . . . . . . . . . . . . . . . Positive phenol phenol or enol Iodoform. . . . . . . . . . . . . . . . . . . . . . . Positive __ -. CH _ C __ 0 Analysis results Preparation 9 E. C34 H47-49 N 013 * Calculated . . . . . . . . . . . . . . . . . C 60.2, H 7.08, N 2.07 (Molecular weight 678); found . . . . . . . . . . . . . . . . . C 59.72, H 6.57, N 1.87 Ash 0.0. Active hydrogen. . . . . . . . 6 Mo1 CH, Mol Hydrogenation at atmospheric printing with Adam's Kata- lyser ........ .. .... . .. 2.51, 2.57 11o1 H2. 1I01 Acyl ..... ................ 18,770110 or three 0 groups per molecule 11 CCH3 Methoxyl. . . . . . . . . . . . . . . . . -1.92 ° o or one 0 CH, group molecule C-Methvl. . . . . . . . . . . . . . . . . 23.65 ° -o or eleven C - C H3 groups? molecule a; D. . . . . . . . . . . . . . . . . . . . . - 618` (CH C13) @ "saponification number of preparations tion A (unsolved). .. ..... 169.5 Saponification number of preparations tion E (dioxane dissolved) ..... 226.3 Molecular weight (calculated) 678 Molecular weight of the dioxane solution .................. 810 Probable gross formula. . C34 H47-49 N 01s Melting point. . . . . . . . . . . . . 195 to 200 ° C When preparation 9E is recrystallized from acetone, crystals whose infrared and ultraviolet absorption spectra correspond to FIGS. 6 and 11 are obtained.

The close relationship of the components of 101 a results from the following examples: Example 10 Conversion of 101 a-2 by acetylation 1 g of 101 a-2 (preparation 9F) is dissolved in 20 ml of pyridine and refluxed for 4 hours with 3.3 ml of acetic anhydride cooked. The reaction mixture is kept in the refrigerator overnight and poured into 200 ml of cold water. 1 g of the yellow precipitate thus obtained is recrystallized, 564 mg of light yellow product (preparation 10A) having the following properties being obtained ialD . . . . . . . . . . . . ... . . . ... + 153.7 ° (c = 0.3 in 95 °, loigem ethanol) -; - 124 ° (c = 1 in CHC13) a412 MP-. . . . . . . . . . . . . . . . . . 16.5 a242 ml. . . . . . . . . . . . . . . . . . 63.5 and the following analysis data: C ........................ 60.02 H ................. ...... 6.42 N ........................ 1.36 acyl ............ ......... 30.09 Ash .................... 0.93 Analysis by paper chromatography shows that there are noticeable amounts of the component in Preparation 9F 101 a-7 are present, while the paper chromatogram indicates the presence of components 101 a-1, 101 a-2 and 101 a-3, 101 a-4, 101 a-5 and 101 a-7 in preparation 10A, under components 101a-1, 101a-2, 101a-3, 101a-4, 101a-5 and 101a-7 in the filtrate. Example 11 Conversion of 101 a-2 by p-toluenesulfonic acid and glacial acetic acid 1 g of 101a-2 (preparation 9F) is refluxed for 1 1/2 hours with 100 mg of sodium p-toluenesulfonic acid and 20 ml of glacial acetic acid. The solution is adjusted to pli3.3 and extracted twice with 30 ml of methylene chloride each time. The methylene chloride solution is fractionally precipitated by adding 1, 5, 10 and 20 volumes of technical grade n-hexane.

The paper chromatograms of the products thus obtained show only the Presence of component 101 a.

The preparations were combined and a countercurrent distribution subjected to that with 230 tubes and equal parts of cyclohexane, water, ethyl acetate and 95 °, oigem ethanol works. A single peak is obtained, that of the paper chromatogram only shows 101 a-1. It follows that the conversion of 101 a-2 to 101 a-1 was completed. Example 12 Conversion of 101 a-2 by ammonia 0.5 g of 101 a-2 (preparation 9F) are dissolved in 2 ml of 95 °; 'oigem ethanol and 25 minutes with 10 ml of concentrated Ammonium hydroxide stirred. Then with concentrated sulfuric acid to pII1.5 posed. On cooling to room temperature, the yellow aqueous separates out Phase out a dark brown oil. The oil is dissolved in 80 ml of acetone, whereby about Excrete 500 mg of a thoroughly white salt, which is filtered off. To the filtrate 210 ml of water are added and the solution is subjected to freeze-drying. The paper chromatogram shows the presence of all seven components of 101 a in the dried product.

Example 13 Chromatographic separation of 101a For 41 of the mixture: Toluene ......................... 1333 ml technical n-hexane ............. 667 ml Methanol ....................... 1200 ml Water ......................... 800 ml These solvents are combined, mixed well, and allowed to separate into two phases that are approximately the same volume.

The column is prepared as follows: 250 g of infusor soil are used Stirred for 1 hour with 500 ml of 6N hydrochloric acid, collected on the filter and mixed with distilled Washed water until the pII of the wash water is 5.5. Then it is washed with methanol and air dry. 180 g of this acid-washed infusor earth will be intimately mixed with 90 ml of the lower phase of the solvent system described above and into a tube of 25.4 mm internal width made of Pyrex glass with a stopper about 90 cm highly filled.

The fractionated sample was 1.0 g of 101 a from Example 6. This was dissolved in 10 ml of the upper phase of the system described, with 20 g of that with acid washed infusion soil mixed and the mixture packed on top of the column.

The column was then eluted with the upper phase and at intervals separated fractions (20 to 25 ml) from 20 minutes.

Fractions 1 to 25 contained 101 a-4 and 101 a-5, fractions 76 to 126 contained 101 a-3, fractions 148 to 193 contained 101 a-1.

The tape remaining in the column was shoveled out and the 101 a-2 eluted from the infusor earth with 300 ml of chloroform. After evaporation 121 mg of 101 a-2 (preparation 13 A) are obtained when dry.

Fractions 1 to 25 are combined and evaporated to dryness, about 1105 mg of a mixture of 101 a-4 and 101 a-5 are obtained (preparation 13B).

Fractions 76 to 126 are combined and evaporated, whereby 385 mg of solid 101 a-3 are obtained (preparation 13C).

Fractions 148 to 193 are combined and evaporated to give 275 mg of solid 101 a-1 (Preparation 13D).

Example 14 Separation of components 101 a-1 and 101 a-5 by countercurrent distribution A. A mixture of 101 a-4 and 101 obtained according to Example 13 (Preparation 13B) a-5 is broken down using the Craig countercurrent distribution method. That used System was 95 "" ', ethanol-ethyl acetate-cyclohexane-water (1: 1: 1: 1 by volume). A 5 g sample of the mixture was dissolved in 160 ml of the solvent system, and after 195 passes there were two colored bands. The first (K = 5.73) did not match the theoretical curve, which shows at least two substances. This tip contained 101 a-5 and a black amber impurity. The second (K = 2.04) corresponded to the theoretical curve, essentially pure 101 a-4 (Preparation 14A). B. Repeat the above experiment with a second sample of 5 g for 197 passes and evaporated to an aqueous paste a. This is extracted with methylene chloride and the methylene chloride is evaporated to dryness. This gives 1.61 g of biologically pure 101 a-4 (preparation 14B).

C. The 101 a-5 and 101 a-6 fractions from A and B are combined and the mixture containing 101 a-5 and the impurity is evaporated to dryness, again dissolved in 140 ml of the above-mentioned solvent system and again in Apparatus distributed by Craig. Using the return technique, there are 466 passes and again two colored bands became visible. The tape that 101 a-5 (K = 5.66), was evaporated to an aqueous paste and treated with methylene chloride extracted. 10 volumes of technical grade n-hexane were added and the mixture was filtered. The filtrate was evaporated to dryness to give 1.22 g of biologically pure 101 a-5 (Preparation 14C).

Claims (1)

PATENT CLAIM The use of Streptomyces sp. 101 and its variants and mutants for the production of the antibiotic 101 a in the usual biological way, extraction of the antibiotic from the fermentation medium and, if necessary, separation of the antibiotic into its components 101 a-1 to 101 a-7.