DE2652677C2 - Antibiotics 890A ↓ 1 ↓ and 890A ↓ 3 ↓, processes for their preparation, and antibacterial agents containing these antibiotics - Google Patents

Description

The discovery of the anubiotic properties of penicillin has generated a great deal of interest in the field. which has led to the discovery of many other antibiotic substances; such antibiotics are e.g. B. Others Penicillins, Cephalosporins, Streptomycin, Bacitracin, Tetracyclines, Chloramphenicol, Erythromycins and like that. In general, the antibacterial activity of any of these antibiotics does not extend to certain clinically important pathogenic bacteria. Some of them are mainly only effective against gram-positive species of bacteria. In the course of the widespread use of previously known antibiotics for the treatment of bacterial infections, the acquired resistance has led to the emergence of difficult resistance problems.

Hence, the deficiencies of the known antibiotics need further research to find other antibiotics stimulated against a wider range of pathogens as well as resistant strains special microorganisms are active.

The invention has for its object to provide new and valuable antibiotics available in effectively inhibit the growth of various gram-negative and gram-positive microorganisms.

This object is achieved by providing what is described in claims 1, 3 and 5 Antibiotics 890Ai and 89OA3 and their mixtures and the agents described in claim 7. the Production of these antibiotics takes place according to the method set out in claims 2, 4 and 6.

On the basis of extensive classifying studies it has been determined that the strains of microorganisms used in the context of the invention belong to the species Streptomyces flavogriseus and these are in the culture collection of Merck & Co., Inc., Rahway, NJ, as M A-4434a and M A -4600a. A culture of each of these strains has been permanently deposited in the culture collection of the Northern Regional Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, US Department of Agriculture, Peoria, I 11. and has been given the identification number NRRL 8139 respectively.

Streptomyces flavogriseus NRRL 8139 produces both antibiotics 850Ai and 890A3, which are practically pure Form can be isolated from the fermentation liquid. Streptomyces flavogriseus NRRL 8140 produces the antibiotic 890Ai with no detectable amounts of 89OAi.

The characteristic morphological features and cultural features of Streptomyces flavogriseus NRRL 8139 are summarized in the following overview.

morphology

The sporophores are branching, straight to winding spore chains that form tufts. The chains have a length of more than 10 spores. The spores are spherical to oval - 0.9 μίτι x 1.2 μπι (at 970x magnification).

Cultural features

Oatmeal agar

Vegetative growth - back side - yellowish-brownish, parchment-like growth;

Aerial mycelium - light gray with a medium gray border

Soluble pigment - none.
Czapek Dox agar (sucrose nitrate agar)

Vegetative growth - back side - brown with a dark brown margin;

Aerial mycelium - medium gray, velvety;

Soluble pigment - light browning of the medium.
Egg albumin agar

Vegetative growth - back - yellowish-brownish with a brown border;

Aerial mycelium - medium gray, mixed with yellowish gray (2dc) and grayish yellow (2db);

Soluble pigment - light yellowish-brownish.
Glycerine asparagine agar

Vegetative growth - back - yellowish-brownish, flat, spreading;

Aerial mycelium - velvety, light gray with a strong yellowish tone to gray (2dc);

Soluble pigment - none.
Inorganic Salts Starch Agar

Vegetative growth - back side - brown;

Aerial mycelium - medium gray, velvety;

Soluble pigment - light yellowish-brownish.
Yeast extract dextrose + salt agar

Vegetative growth - back - brown with a very dark brown margin;

Aerial mycelium - dark gray, mixed with light gray, velvety;

Soluble pigment - none.
Yeast Extract-Malt Extract Agar

Vegetative growth - back side - dark brown;

Aerial mycelium - dark gray, velvety;

Soluble pigment - none.
Skimmed milk agar

Vegetative growth - brownish;

Aerial mycelium - scanty, greyish;

Soluble pigment - slight browning of the medium;

Casein hydrolysis - good.

i-l litmus milk

^J ; Vegetative growth - moderate growth ring, dark brownish;

■ aerial mycelium - none;

Color - purple;

: ⁵ coagulation and / or peptonization - complete peptonization; becomes alkaline, pH 8.2.

; Skimmed milk

Vegetative growth - moderate growth ring, brownish; Aerial mycelium - none; Soluble pigment - brownish;

"■ '" Coagulation and / or peptonization - complete peptonization; becomes alkaline, pH 8.0.

Tyrosine agar

Vegetative growth - back side - dark brown; Aerial mycelium - dark gray;

Soluble pigment - weak browning of the medium; "; ^1? Decomposition of tyrosine - none.

Peptone Iron Yeast Extract Agar

{■ [' Vegetative growth - brownish;

Aerial mycelium - scanty, greyish; Soluble pigment - none; - "Melanin - none;

HjS development - none. Nutrient agar

Vegetative growth - back side - light grayish brown with a darker grayish brown border; Aerial mycelium - light gray with a dark gray border; - Soluble pigment - none.

Nutrient starch agar

Vegetative growth - brownish with a gray border; Aerial mycelium - medium gray with a dark gray border; Soluble pigment - none; Hydrolysis of starch - good.

Nutrient gelatin agar

Vegetative growth - colorless with a dark gray border; Aerial mycelium - greyish-white; Soluble pigment - none; ^{; s} liquefaction of gelatin - good.

Potato wad

Vegetative growth - good growth, severely wrinkled; Aerial mycelium - gray to greenish gray; Soluble pigment - slight browning of the medium. ■ "'Loeffler's blood serum

^: Vegetative growth - cream colored;

Aerial mycelium - none; Soluble pigment - none; Liquefaction - none. J * Vertical tube of gelatin Vegetative growth - cream colored; Aerial mycelium - none; Soluble pigment - none; Liquefaction of gelatin - good.

Unless otherwise stated, all of the features given above were determined after incubation at 28 ^° C. for three weeks. The media used in these studies were approximately neutral (pH 6.8 to 7.2). The color designations used in the description correspond to the definitions in "Color Harmony Manual", 4th Aullage (1958), published by the Container Corporation of America, Chicago, Illinois.

Streptomyces llavogriseus NRRL 8139 was also tested for its ability to metabolize various carbohydrates to exploit or assimilate. For this purpose, the microorganism was kept at 28 ° C for three weeks grown on a basic synthetic medium containing the carbohydrate in question in a concentration of 1% contained. The media used in these studies were approximately neutral (pH 6.8 to 7.2). Table I. '■ "shows the utilization of these carbohydrates by Streptomyces fiavogriseus NRRL 8139, where + is a good one Growth, ± means poor growth and - means no growth on the carbohydrate in question.

Table I.

Glucose + fructose +

Arabinose + inositol

Cellulose - lactose +

continuation

Xylose + raffinose

Maltose + cream +

Mannitol + sucrose ±

Mannose +

For the extent of growth with temperature change and the oxygen requirement of the microorganism the following applies:

Temperature range (yeast extract-dextrose + salt agar); 28 ⁰ C - good

37 ⁰ C - good vegetative growth; no aerial hyphae

50 ⁰ C - No growth Oxygen Demand (Nadelimpfkultur in yeast extract-dextrose salt agar); aerobic.

The characteristic morphological features and cultural features of Streptomyces flavogriseus NRRL 8140 are compiled in the following overview.

morphology

The sporophores are branching, straight to tortuous chains of spores that form tufts. the Chains are more than 10 spurs in length. The spores are round to oval - 0.9 μm x 1.2 μm (at 970x magnification).

Cultural features

Oatmeal agar

Vegetative growth - back - yellowish-brownish, with a dark brown border; Aerial mycelium - light gray with a medium gray border;

Soluble pigment - none. Czapek Dox agar (sucrose nitrate agar) Vegetative growth - back side - brown with a dark brown margin; Aerial mycelium - medium gray, velvety;

Soluble pigment - none. Egg albumin agar

Vegetative growth - back side - yellowish-brownish with strongly yellow-brownish sections; Aerial mycelium - medium gray, greyish-white, and yellowish-yellow (2dc) sections;

Soluble pigment - very light brownish. Glycerine asparagine agar Vegetative growth - yellowish-brownish; Aerial mycelium - scanty, greyish;

Soluble pigment - none. Inorganic Salts Starch Agar Vegetative growth - reverse side - greyish cream-colored; Aerial mycelium - medium gray, velvety;

Soluble pigment - none; Yeast extract de-xtrose + salt agar Vegetative growth - back side - dark brown; Aerial mycelium - dark gray, mixed with light gray, velvety;

Soluble pigment - none. Yeast Extract-Malt Extract Agar Vegetative growth - back side - dark brown; Aerial mycelium - dark gray, velvety;

Soluble pigment - none. Peptone Iron Yeast Extract Agar Vegetative growth - brownish; Aerial mycelium - none; Soluble pigment - none; Melanin - none;

H2S development - none. Nutrient agar

Vegetative growth - light brownish; Aerial mycelium - none; Soluble pigment - none.

Starch agar Vegetative growth - cream colored; Aerial mycelium - none; Soluble pigment - none; Hydrolysis of starch - good.

Nutrient gelatine agar vegetative growth - cream colored; Aerial mycelium - none;

Soluble pigment - none; '"Liquefaction of gelatin - good.

Vertical tube of gelatin Vegetative growth - brownish; Aerial mycelium - none; Soluble pigment - none;

^: "Liquefaction of Geiaiine - completely.

Skimmed milk

Vegetative growth - brownish; Aerial mycelium - none;

Soluble pigment - none; ^: "Hydrolysis of casein - good.

Litmus milk

Vegetative growth - brownish growth ring; Aerial mycelium - none; Color - brownish-purple;

^: "Coagulation and / or peptonization - complete peptonization; becomes alkaline, pH 8.0.

Skimmed milk

Vegetative growth - brownish, moderate growth ring; Aerial mycelium - none; Soluble pigment - light brown;

■ "Coagulation and / or peptonization - complete peptonization; becomes alkaline, pH 8.5.

Potato plugs Vegetative growth - good, brownish; Aerial mycelium - very sparse, whitish;

Soluble pigment - none. • 5 Loeffler's blood serum Vegetative growth - cream colored; Aerial mycelium - none; Soluble pigment - none;

Liquefaction - none. ^J "tyrosine agar

Vegetative growth - brownish; Aerial mycelium - none; Soluble pigment - slight browning of the medium; Decomposition of tyrosine - very weak.

Unless otherwise noted, all of the above observations were made after three weeks of incubation at 28 ° C. The media used in these studies were approximately neutral (pH 6.8 to 7.2). The color names used in the description correspond to the definitions in »Color Harmony Manual ", 4th edition (1958), published by Container Corporation of America, Chicago, Illinois.

^; "Streptomyces flavogriseus NRRL 8140 was also examined for its ability to utilize or assimilate various carbohydrates. For this purpose, the microorganism was grown for three weeks at 28 ° C. on a basic synthetic medium containing the carbohydrate in question at a concentration of 1% The media used in these investigations were approximately neutral (pH 6.8 to 7.2). Table II shows the utilization of these carbohydrates by Streptomyces flavogriseus NRRJL 8140, with + good growth, ± poor growth and - no growth on the concerned carbohydrate means.

Table II

Glucose + maltose +

Arabinose + mannitol +

Cellulose - mannose +

Fructose + raffinose ±

Inositol ± creamer ±

Lactose + sucrose ±

Xvlose +

For the extent of growth when the temperature changes and the oxygen demand of the microorganism the following applies:

Temperature range (yeast extract dextrose + salt agar);

28 ⁰ C - good

37 ° C - moderate vegetative growth; no aerial hyphae

50 ⁰ C - no growth
Oxygen demand

aerobic.

The new antibiotics 890Ai and 89OA3 according to the invention are used in the under controlled conditions carried out aerobic fermentation generated suitable aqueous nutrient media with strains of the organism Streptomyces flavogriseus NRRL 8139 or 8140 have been inoculated. For the manufacture of 890Ai and Aqueous media, such as those used to generate other antibiotics, are suitable for 89OA3. Such media contain C, N sources and inorganic salts that are assimilated by the microorganism will.

In general, sugars, e.g. B. dextrose, glucose, fructose, maltose, sucrose, xylose, mannitol and the like, and starches such as dextrin or cereals, e.g. B. oats, rye, corn starch, corn flour, alone or along with other sources of assimilable carbon, can be used in the nutrient medium. the the exact amount of carbohydrates in the medium depends partly on the other components of the Medium; in general, however, the amount of carbohydrates will vary between about 1 and 6 percent by weight of the carbohydrate Medium. These carbon sources can be used individually, or several such carbon sources can be used in combined with the medium. In general, many protein substances can be used as N-sources in the fermentation process be used. Suitable N sources are e.g. B. yeast hydrolysates, primary yeast, soybean meal, Cottonseed flour, casein hydrolysates, corn steep liquor, soluble ingredients in sludge or Tomato puree and the like. The N sources are used alone or in combination with one another in amounts of about 0.2 to 6 wt .-% of the aqueous medium applied.

The inorganic nutrient salts that can be added to the culture media include the usual ones Salts containing sodium, potassium, ammonium, calcium, magnesium, phosphate, sulfate, chlorine, carbonate ions and deliver other ions. This subheading also includes trace metals such as cobalt, manganese and iron.

The culture media described in the examples are for illustration purposes only; you can do the most diverse Use media.

The fermentation is carried out at temperatures of about 20 to 37 ° C; to get the best results however, the fermentation is preferably carried out at temperatures of about 23 to 28 ° C. Of the initial pH of the nutrient medium for growing Streptomyces flavogriseus N RRL 8139 or 8140 and for producing antibiotics 890Ai and 89OA3 can vary in the range of 6.0 to 8.0.

The fermentation to produce the antibiotic is expediently carried out on a small scale by inoculation a suitable nutrient medium with the culture producing the antibiotic and continuing the fermentation after transferring to a production medium for several days in a shaker at a constant temperature of around 28 ° C.

The fermentation is started in a sterilized flask filled with nutrient medium via an or several stages of vaccine development. Any suitable combination can be used as the nutrient medium for the inoculation stage from C and N sources. The inoculation flask is placed in a constant temperature chamber for a day shaken at about 28 ° C until the culture has grown satisfactorily, and some of the resulting Culture is used to inoculate either a two-stage inoculum or the production medium. Intermediate inoculation flasks, when used, are designed in essentially the same way d. H. part of the contents of the flask from the last inoculation stage is used to convert the production medium inoculate. The inoculated flasks are shaken at constant temperature for several days, and at the end During the incubation period, the contents of the flask are centrifuged or filtered.

For work carried out on a large scale, fermentation is preferably carried out in suitable fermenters, which are provided with a stirrer and a system for aerating the fermentation medium. After this Method, the nutrient medium is made up in the fermenter and heated to temperatures up to sterilized around 120 ° C. After cooling, the sterilized medium is inoculated with a previously grown inoculates the culture and the fermentation for a time, e.g. B. 1 to 6 days, with stirring and / or aeration of the nutrient medium and maintaining a temperature of about 24 to 28 ° C. This method of manufacture The antibiotics 890 A1 and 890 A3 are particularly suitable for the production of large quantities of antibiotics.

Physical and chemical properties of the antibiotics 89OAi and 890A3
Properties of the 890Ai antibiotic

The antibiotic 890Ai is an acidic substance that becomes positive during electrophoresis at a neutral pH value Pole wanders.

The sodium salt of the antibiotic 890Ai is in the form in which it is freeze-dried from aqueous Solution obtained is a highly water-soluble white powder.

The ultraviolet absorption spectrum shows an A _max . at 299.5 do and an X _mm at 242 nm. A solution of the sodium salt of the antibiotic 890 Ai in water at neutral pH shows an E% of for a pure sample

208 at 300 nm; the ratio of the absorbance values at 300 nm and 245 nm is 4.25, and the ratio AWA21U is 1.41. More than 92% of the absorption at 300 nm can be extinguished by reaction with hydroxylamine and a similar decrease is also observed upon reaction with cysteine.

The circular dichroism spectrum of 890Ai shows a positive maximum at 290.5 nm with a specific Ellipticity of 5270 degrees ml per dm g, a zero point of ellipticity at 250 nm and a negative one Minimum at 214 nm with a specific ellipticity of -10 910 degrees ■ m 'per dm - g.

In the table below, the 100 MHz NMR signals for the sodium salt of 890Ai and D2O are related to Sodium 2,2-dimethyl-2-silapentane-5-sulfonate, hereinafter abbreviated as DSS, indicated as internal standard, the chemical shifts are given in ppm and the coupling constants in Hz; the apparent Multiple values are also given.

1.35 (D. J = 6.5); 1.98 (s); 3.63 (d of d, J = 5.2 and J = 9.8); ~ 4.02 to 4.26 (m); 3.18 (d of d, J = ~ 11 and J = 10); 3.41 (t, J = 6); 2.97 (ti of t, J = 3.5 and J = 6).

The antibiotic strength of 890Au, as determined by Vibrio percolans ATCC 8461, is 250 units each HAEAiuo unit has been defined.

Properties of the antibiotic 890A.1

The antibiotic 890Aj is an acidic substance that becomes positive during electrophoresis at a neutral pH value Pole wanders.

The sodium salt is in the state in which it is obtained from aqueous solution by freeze-drying, a white powder.

The ultraviolet absorption spectrum has a maximum at 300.5 nm and a minimum at 243 nm Solution of the sodium salt of the antibiotic 890Aj in water at neutral pH shows an E% of 375 300 nm for a pure sample; the ratio of the absorbances at 300 nm and 245 nm is 3.54. More than 90% the absorption at 300 nm can be extinguished by reaction with hydroxylamine; a similar Decrease is observed in the reaction with cysteine.

The circular dichroism spectrum of 890Aj has a positive maximum at 294 nm with a specific Ellipticity of 9127 degrees ml per dm g, a zero point of ellipticity at 249 nm and a specific ellipticity from -15 053 degrees ml per dm g at 220 nm.

The table below shows the 100 MHz NMR signals for the sodium salt of 890Ai in D ₂ O with respect to DSS as an internal standard, as well as the chemical shifts in ppm. the coupling constants in Hz as well as the apparent multiple values are given.

1.29 (d, J = 6.5); 1.98 (s); 3.42 (d of d, J = 5 and J = 2.4); ~ 4.01 to 4.28 (m); 3.14 (d of d, J = 5 and J = 9); 3.39 (t, J = 6.5); 2.92 (d of t, J = ~ 4 and J = 6).

The antibiotic potency of 890Aj as determined on Vibrio percolans ATCC 8461 is 31 units each HAEAjoo unit.

Mass spectral analysis of the 890Ai and 890A3

The mass spectral data for 890Ai and 890A ₃ are determined on trimethylsilyl derivatives which are prepared from the ammonium salts of antibiotics with bis-trimethylsilyltrifluoroacetamide in dimethylformamide. The conversion of the sodium salts of the antibiotics into the ammonium salts takes place using the ammonium salt of an acidic ion exchange resin.

In the trimethylsilylation of 890Ai and 890A ₃ , three different derivatives are formed, namely a di- and a tri-trimethylsilyl derivative (molecular weights 458 and 530, respectively) and a small amount of a tetra-trimethylsilyl derivative of a hydrolyzed product (molecular weight 620), in which the / J-lactam ring is open.

The values of the most important mass spectrum fragments are given below:

Di-trimethylsilyl derivative: 443.1495; 301.1034; 300.0962; 241.0590 and 86 0610

Tri-trimethylsilyl derivative: 515.1931; 373.1422 and 158.1000.

Tetra-trimethylsilyl derivative (only a low resolution signal is observed): 620 and 605.

Antibiotics 890Ai and 89OA ₃ are believed to be isomers of the following molecular structure: CH ₃ -CH (OH) -

I.,> - S i.M-> IH, MH «" f'M.

Antibiotics 890Ai and 890A ₃ are further characterized by the following antibiotic spectrum profiles:

•> —S — CH ₂ -CH ₂ -NH-C-CH ₃

The test to determine the antibiotic spectrum profile is performed by placing a droplet of 0.015 ml on the surface of a 100 x 15 mm Petri dish, the 5 ml inoculated Nutrient agar and 0.2% yeast extract contains the antibiotic 890Ai in a concentration of 33 y / ml and examined the antibiotic 890A3 at a concentration of 182 μg / ml. The results, expressed as The diameter of the inhibition zone in millimeters can be found in Table III.

Table St.

Antibacterial spectrum profile (ASP) of antibiotics 890Ai and 890A3 in vitro

Microorganism

Merck No.

ATCC No.

Inhibition zone diameter, mm 890Ai 8OA3

Bacillus sp.
Proteus vulgaris Pseudornonas aeruginosa Serratia marcescens Staphylococcus aureus Bacillus subtilis Sarcina lutea
Staphylococcus aureus, Streptococcus faecalis, Alcaligenes faecalis, Bruceila bronchiseptica, Salmonella gallinarum, Vibrio percolans, Xanthomonas vesicatoria, Proteus vulgaris, Escherichia coli, Pseudomonas stutzeri, Klebsiella pneumoniae, Aerobacter aerogenes, Erwinia atroseptica, Pseudomonasnebuginosa.

Escherichia coli in synthetic medium

Streptococcus faecium Streptococcus agalactiae

Vibrio percolans resistant to Ceph. C)

Proteus vulgaris (Episome) Proteus mirabilis

Vibrio percolans + 2 x units / ml penicillinase

The antibiotic 890Ai shows in vivo activity against gram-negative and gram-positive organisms and is therefore suitable for combating bacterial infections in animals and humans. To determine the In vivo activity is the antibiotic 890Ai in a solution of 0.15 molar NaCl and 0.01 molar sodium phosphate Dissolved with a pH of 7.0 and with this solution to five fourfold concentrations of the antibiotic diluted for examination. Female white Swiss mice with an average Weight of about 21 g are infected intraperitoneally with the test organism suspended in meat broth. The number of injected organisms is determined using standardized plate counting methods. At the time of infection and 6 hours later, some of the mice are treated intraperitoneally with the antibiotic. Five mice are used for each concentration of the antibiotic tested. Another two mice, who have not been infected are treated with the antibiotic to determine if the amount of the injected agent was toxic. Each test includes controls of five mice for each of the different ones Dilutions of the infecting culture to calculate the number of organisms present on 50% of the infecting

MB- 633 - 990 - 213 40 37 MB-1012 - 6 538 P. - 4617 21 24 MB- 979 - 6 633 - 0 0 - 9 341 8 461 26th 28 - - 35 36 - 21 100 38 40 - - 38 44 MB- 698 11 607 28 34 MB- 753 - 17th 17th - - 27 34 - 4,466 22nd 27 MB-1287 - 33 33 - 9 742 34 40 MB-815 9 637 26th 26th - - 34 34 MB-1418 - 27 32 - - 10 32 MB-1264 _ 27 33 MB-835 - 26th 30th - 8 461 20th 30th MB-2824 0 32 - 34 36 - 27 29 MB-2820 18th 24 MB-2875 36 30th MB-2566 15th 34 MB-2112 32 38 MB-3126 23 30th 10 35

th, untreated mice are lethal (LD50). This calculation is made based on survival data on the seventh day after infection, and at that time the amount of antibiotic is also calculated calculated that should protect 50% of infected mice (EDsg) -

All mice infected in this way and not treated with the antibiotic die within 48 hours after infection. The effectiveness of the antibiotic 89UAi with a strength of 5.5 units μg against Salmonella schottmuelleri MB-2837 is given below:

EDso * 2 cans

Society

ml ¹ ) type thinning ml

Units / dosage units / ug ^b )

908 ip 1:32 14 3

') 1 unit / ml - that concentration which produces an inhibition zone of 25 mm against Vibrio pcrcolans ATCC 8461.

^b ) The ug value is calculated on the basis of the planned purity of 89OA, at 5500 units / mg.

The antibiotics 890Ai and 890A.1 are valuable antibiotics that are used against a wide variety of gram-positive and gram-negative bacteria are active and are therefore used in both human and veterinary medicine. The compounds according to the invention can be used alone or in combination together as antibacterial drugs to be used to treat infections caused by gram-positive or gram-negative bacteria, e.g. B. against Staphylococcus aurcus, Proteus mirabilis, Escherichia coli, Klebsieila pneumoniae and Enterobacter cloacae. The antibacterial agents according to of the invention can also be used as an additive to animal feed, for preserving food and as a disinfectant. For example, they can be used in aqueous compositions in concentrations of about 0.1 up to 100 parts of antibiotic per million parts of solution, or preferably in concentrations of about 1 to 10 Parts antibiotic per million parts solution used to prevent the growth of harmful bacteria on medical and dental equipment to destroy and inhibit, as well as bactericides for technical purposes, e.g. B. in water-based paints, in the white water of paper mills and to inhibit the growth of harmful bacteria.

The antibiotics according to the invention can be used in a wide variety of pharmaceutical preparations as the sole active ingredients or in combination with one or more other antibiotics or with one or more pharmacologically active substances. As an example for the former case, a Aminocyclitol antibiotic, gentamicin, administered together with the antibiotics according to the invention in order to minimize the possibility of the occurrence of resistant organisms. as Example of the latter case can be diphenoxylate and atropine in dosage forms for the treatment of Combine gastrointestinal diseases. The antibiotics can be in the form of capsules, tablets, powders, liquid solutions or as suspensions or elixirs. You can do it orally, locally, intravenously or intramuscularly. Furthermore, the antibiotics 890Ai and 890A3 can be used in combination can be applied to each other.

Tablets and capsules for oral administration can be in unit dosage form and conventional Extenders such as binders, e.g. B. syrup, acacia resin, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone, Fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine, lubricants, e.g. B. magnesium stearate, talc, polyethylene glycol, silicon dioxide, disintegrating agents, e.g. B. potato starch or contain permitted wetting agents such as sodium lauryl sulfate. The tablets can be coated by methods known per se. Liquid preparations to be administered orally can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs, etc., or as a dry product that can be mixed up be prepared with water or other suitable vehicle before use. Such liquid preparations conventional additives such as suspending agents, e.g. B. sorbitol syrup, methyl cellulose, glucose / sugar syrup, Gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible Fats, emulsifiers, e.g. B. lecithin, sorbitan monooleate or acacia resin, non-aqueous vehicles such as edible oils, e.g. B. almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol, preservatives, e.g. B. p-Hydroxybenzoic acid methyl or propyl ester or sorbic acid contain. Suppositories contain conventional suppository bases such as cocoa butter or other glycerides.

Means for injection may be in unit dose form in ampoules or in multiple dose form in containers with the addition of preservatives. The funds can take the form of Suspensions, solutions, emulsions in oily or aqueous vehicles and formulating agents, we contain suspending, stabilizing and / or dispersing agents. The active ingredient can also be in powder form and prior to use with a suitable carrier, e.g. B. sterile, pyrogen-free water, be turned on.

The funds can also be used in for absorption through the mucous membranes of the nose and throat or the Bronchial tissue of suitable shape can be prepared, e.g. B. as a powder or liquid spray or inhalant, Pastilles, throat paints, etc. For the treatment of the eyes or ears, the preparations can be used individually Capsules, provided in liquid or semi-liquid form, or used as drops, etc.

For local application, they can be used in hydrophobic or hydrophilic bases as ointments, creams, lotions, Paints, powders, etc. are present.

In addition to a carrier, the agents according to the invention can contain other constituents, such as stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, viscosity-influencing agents or flavorings and the like.

In veterinary medicine, such as in the treatment of chickens, cows, sheep, pigs and the like, can the means z. B. formulated as preparations for injecting into the breast, the long-term effect are turned off or quickly release the active ingredient.

The correct dose depends largely on the condition of the animal to be treated, the Weight of the same and the type of infection, the method and frequency of administration, the parenteral being Administration for general infections and oral administration for intestinal infections is preferred.

For the treatment of bacterial infections in humans, the compounds according to the invention are administered orally or parenterally according to conventional methods for treatment with antibiotics in amounts of about 2 to 600, preferably about 5 to 100 mg / kg / day administered and preferably divided into individual doses, the z. B. be given three to four times a day. Unit dosage forms can be used that contain z. B. contain 25, 330, 400 or 1000 mg of active ingredient together with physiologically acceptable carriers or extenders. The dose units are in the form of liquid preparations, such as solutions or Suspensions, or as solid substances in tablets or capsules. The optimal dose depends on the type and severity of the infection in each individual case, with smaller doses being used for treating children; all such design rules are known to the person skilled in the art.

The invention of course also encompasses the nontoxic, pharmaceutically acceptable salts from 890Ai and 890 A3, e.g. B. the pharmacologically acceptable salts with inorganic or organic Bases arise. These include B. metal salts, those of alkali or alkaline earth metal hydroxides, carbonates or bicarbonates are derived, such as. B. sodium, potassium, ammonium and calcium salts, as well as salts of primary, secondary or tertiary amines, such as monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines ^ lower dialkanolamines, lower alkylenediamines, Ν, Ν-diaralkyl-lower-alkylenediamines, aralkylamines ^ amino-substituted lower alkanols, by lower Ν, Ν-dialkylamino groups substituted lower alkanols, amino-, polyamino- and guanidino-substituted lower alkanoic acids and nitrogen-containing heterocyclic amines. Typical examples are salts derived from sodium hydroxide, ammonium hydroxide, sodium carbonate, sodium carbonate, potassium carbonate, potassium hydroxide, calcium carbonate, Trimethylamine, triethylamine, piperidine, N-ethylpiperidine, morpholine, quinine, lysine, protamine, arginine, Procaine, ethanolamine, morphine, benzylamine, ethylenediamine, Ν, Ν'-dibenzylethylenediamine, diethanolamine, Piperazine, dimethylaminoethanol, 2-amino-2-methylpropanol- (l), theophylline or N-methylglucamine are derived.

The salts of the compounds according to the invention can be prepared by known methods. So you get z. B. the monosalts, such as the monosodium salt, by reacting 1 equivalent of sodium hydroxide with 1 equivalent of the product (I) in a suitable solvent. Mixed salts with divalent cations can also be prepared by adding 1 mol of a divalent base with 1 mol of Product (I) and 1 equivalent of another acid combined. Salts can also be made by one equivalent of a base with a divalent cation, such as calcium hydroxide, with 1 equivalent of the Product (I) implements. The salts according to the invention are pharmacologically acceptable, non-toxic Derivatives which can be used as active ingredients in suitable pharmaceutical unit dosage forms. They can also be combined with other drugs to obtain funds with a wide range of activities.

The fermentation liquids obtained by the process according to the invention, which contain the antibiotics 890Ai and 890A3 contain activities ranging from about 2 to 170 units / ml when after Disk diffusion method using Vibrio percolans (ATCC 8461). Antibiotic preparations of 890Ai and 890A3 with an activity of at least 5 units / mg can be purified and the antibiotics are obtained by various methods. One such method is that the antibiotics 890Ai and 890A3 are adsorbed on strongly basic anion exchange resins. examples for such strongly basic anion exchange resins are those having a styrene-divinylbenzene backbone, e.g. B. that in the polystyrene core quaternized ammonium resin Dowex-1 Χ 2 in the chloride form. Other representative Members of this class of strongly basic ion exchange resins are: Duolite A-40, A-42, A-101, A-102 and A-114: Amberlite IRA-400, IRA-401, and IRA-410. You can also use weakly basic anion exchange resins, like Amberlite IR-68.

The adsorbed antibiotic can be easily removed from the anion exchange resin with aqueous salt solutions elute. The eluate obtained in this way can optionally be further purified using other purification methods will. The eluate can be purified by passing it through a column loaded with an acrylic acid ester polymer of medium polarity, such as XAD-7 or 8, or through a column loaded with a non-polar, hydrophobic, crosslinked polystyrene-divinylbenzene polymer is charged, such as XAD-I, 2 and 4, preferably XAD-2. This procedure partially eliminates antibiotics 890Ai and 89OA3 separated. The fractions rich in 890Ai and the fractions rich in 890A3 are combined with each other. These pooled fractions can be further purified.

Antibiotics 890Ai and 890A3 that have been purified further are obtained by gel filtration through polyacrylic acid amides! with a pore size that excludes molecules with molecular weights greater than 1800, such as Bio-Gel P-2. Other gels such as Sephadex G-10 can also be used for desalting.

The preferred method of removing antibiotics 890Ai and 890Aj in high purity from one Fermentation liquid can be obtained, consists in centrifuging or filtering off the solid

abut, the adsorption and the elution on or from an anion exchange resin, such as Dowex-1 x 2 in the Chloride form, with 3 to 5 percent saline solution, which makes the antibiotic both concentrated and concentrated partially purified, passing through a column of suitably prepared XAD-2, which delays the antibiotics and thereby purifies them and removes the egg from the Dowex-1 x 2 . «Is desalinated. Furthermore, the antibiotic 890A3 is delayed more than the antibiotic 890Ai, so that the two antibiotics are partially separated from one another. The fractions containing 890Ai and 890Aj are united and further purified. By chromatography on Dowex-1 x 2 (particle sizes below 37 µm) and Eluting with NaCl and / or N H4CI results in a product that is free of most UV-absorbing impurities (the NHCl is used in order to give the eluent a certain buffering capacity

1 »vfjhaben); and desalting on Bio-Gel P-2 or Sephadex G-IO removes the salt introduced during the chromatography on Dowex-1 x 2 and further reduces the amount of remaining impurities in the antibiotic preparations.

When treating low strength fermentation liquors according to the procedure described above significant amounts (more than 50%) of external impurities can remain in the end product.

Ii ben. These impurities can occur through an additional process step of chromatography Dowex-1 x 2 (particle sizes below 37 μΐη) and eluting with a solution that Contains sodium chloride and 50% methanol. If the antibiotic is isolated from fermentation liquors of low strength, it is also advisable to follow-up a second stage of chromatography on XAD-2. This achieves additional cleaning, residual salts are removed and the antibiotics 890Ai

: t> and 890Aj are partially separated from one another, in particular 890 A3 is eluted later than 890Ai, and the two substances can be distinguished from one another by their different ratios of bioactivity to HAEA300. Those fractions which have a ratio of bioactivity against Vibrio percolans ATCC 8461 to HAEA300 of 250 contain the antibiotic 89OAi, and those fractions which have a ratio of bioactivity against Vibrio percolans ATCC 8461 to HAEA300 of 31 contain it

:? Antibiotic 890A ₃ .

Fractions rich in antibiotic 890A ₃ can be treated with penicillinase to remove residual traces of antibiotic 890Ai and further purified by chromatography on Dowex-1 x 2 and Sephadex G-10. When working on a laboratory scale (less than 20 l sample volume), all chromatographic

<M graphic process steps with the exception of the chromatography on XAD-2 carried out in a cold room at 2 to 5 ° C. The chromatography on XAD-2 is carried out at room temperature, unless otherwise stated. The pH of the antibiotic solutions is adjusted to 7 to 8 by carefully adding dilute sodium hydroxide or hydrochloric acid before they are stored. Aqueous solutions are stored in a refrigerator or, preferably, in ice water and 50 percent methanolic solutions at -20 ⁰ C.

i> The cleaning procedure for antibiotics 890Ai and 890Aj is illustrated by flow diagrams 1 and 2 below, respectively.

Flow diagram 1 Purification of the antibiotic 890Aι

89OAi and 890A3

containing

Fermentation

liquid wedge

(1) cooling

(2) centrifugation

(3) Addition of EDTA

XAD-2
Eluate

J =

(1) focus

(2) Adsorb on XAD-2

(3) Elute with water

(1) focus

(2) Adsorb on XAD-2

(3) Elute with water

(1) Adsorbing the 890A | fractions on Dowex-1 x 4 (<37 μτη)

Centrifuged

Fermentation

liquid

Eluate from Dowex-1 x 2 (150-300 μπι)

Second XAD-2 elual

(2) Elute with 0.07 molar NaCl +

0.005 molar NH ₄ CI + 0.001 molar NH ₃

Dowex 1x4 eluate

(1) focus

(2) Adsorb on Bio-Gel P-2

(3) Elute with water

Bio-Gel P-2 eluate

(1) Adsorb on Dowex-1 x 2 (150-300 μπι)

(2) Elute with 5% NaCl and 0.01 molar Tris-HCl + 25 µmolar EDTA

The 890Ai factions will

united and according to the flow chart

cleaned

Sephadax G-10 Eulat

(1) focus

(2) Adsorb on Sephadex G-10

(3) Elute with water

(1) Concentration

(2) freeze drying

50% CHjOH-

Eluate from Dowex-1 x 2

890Ai

(1) focus

(2) Addition of 1 space part CHjOH

(3) Adsorb on Dowex-1 x 2 (<37 μΐη)

(4) Elute with 0.05 molar NaCl + 0.005 molar NH ₄ CI + 0.001 molar NH-, in 50% CHjOH

(1) Concentration

(2) freeze drying

Different procedure

Flow diagram 2

Cleaning the antibiotic 890A ₃

Obtained in the process according to flow diagram I,

Pooled XAD fractions containing _{89OA 3}

(1) Adsorb on Dowex-1 x 4 (<37 μm)

(2) Elute with 0.07 molar NaCl

+ 0.005 molar N HjCl + 0.0001 molar NHj

Dowex 1 x 4 egg

(1) Concentration »

(2) Adsorb on Bio-Gel P-2

(3) Elute with water

Bio-Gel P-2-Elual

Sephadex G-IO eluate

(1) focus

(2) Adsorb on Sephadex G-IO

(3) Elute with water + 0.02 molar NHi

(I) focus

50% CH ₃ OH-

Elual from
Dowex-i x 2

(1) Treating with penicillinase

(2) Adsorption on Dowex-1 x 2 «37 μτη)

(3) Elute with 0.05 molar NaCl + 0.005 molar NH ₄ Cl + 0.0001 molar NH ₃ in 50% CH ₃ OH

(2) freeze drying

Analysis methods For the antibiotics 890Ai and 890A ₃
I. Microbiological analysis

An agar plate-disc diffusion method is used using either Vibrio percolans ATCC 8461 or Salmonella galinarium MB-1287 is used as the test organism. A purified sample of the antibiotic 890Ai is used as the standard.

Plates containing Vibrio percolans ATCC 8461 are made as follows:
A freeze-dried culture of Vibrio percolans ATCC 8461 is suspended in 15 ml of a sterile medium which contains 8 g Difco nutrient broth / 1 and 2 g yeast extract / i in distilled water and referred to as “nutrient broth yeast extract” (hereinafter abbreviated as N BYE) will. The culture is incubated overnight in a rotating shaker at 28 ° C. This culture is used to inoculate the surface of slants containing 1.5% agar in NB YE and the inoculated slants are incubated overnight at 28 ° C and then stored in the refrigerator.

The chilled slant tubes made from a single freeze-dried culture are used for a Period of up to 4 weeks after its production, used as follows: An eyelet with inoculum from the The inclined tube is dispersed in 50 ml NBYE in a 250 ml Erlenmeyer flask. The culture is over Incubated overnight in a rotating shaker at 28 ° C and then diluted to a density that corresponds to a Results in 50 percent light transmission at 660 nm. 33.2 ml of this diluted culture becomes one

Vnliimpn \ tr \ r \ I 1 NDVC ^ ,. _nA <. _n «^» * 4 ~~ IC _n * «u ^ i« ι r λ £ ο r * ui · ...:. j

-w. «...., .. .w ·· ■ ι ι, L» » ι ~ tu5v3i> ia, ud3 i_> g r-igai cittiiait uuu am tu \ _ gciiaiidi miu.

The inoculated, agar-containing medium is placed in plastic Petri dishes of 100 x 15 mm in quantities of 5 ml each Peel, chilled, and held at 2 to 4 ° C for 5 days prior to use.

The plates containing Salmonella gallinarium MB-1287 are prepared as follows:
A sealed tube containing frozen and freeze-dried cells of Salmonella gallinarium MB-1287 in skimmed milk and sealed under vacuum is opened and inoculated into 15 ml of brain-heart broth. The cells are allowed to grow overnight at 37 ° C. without shaking, and the culture is inoculated into inclined tubes containing 0.8% BBL nutrient medium + 0.2% Difco yeast extract + 1.5% agar. After overnight growth at 37 ° C, one loop of the culture is transferred from the slant into a flask containing 50 ml of 0.8 percent nutrient broth + 0.2 percent yeast extract. The flask is shaken overnight and 1 l of 0.8% nutrient broth + 0.2% yeast extract + 1.5% agar, which has previously been sterilized and cooled to 48 ° C., is inoculated with 20 ml of this culture. The inoculated NB YE agar is immediately poured into plastic Petri dishes of 100 x 15 mm (5 ml per dish) and the plates are kept at 0 to 3 ° C until use.

Filter paper discs with a diameter of 12.7 mm are dipped into the solution to be analyzed and placed on the Agar applied. Another method is to load the discs by adding 0.1 ml of the The solution is pipetted onto a dry disk and the disk is then placed on the agar. After suitable Incubation (for Salmonella gallinarium MB-1287 9 to 18 hours at 37 ° C, for Vibrio percolans ATCC8461 12

up to 24 hours at 25 ° C) the diameter of the inhibition zone is measured.

If necessary, dilutions of the solutions to be analyzed are made in 0.05 molar potassium phosphate buffer (pH 7.4; hereinafter abbreviated as KPB) or made in demineralized water.

The strength is calculated as follows: A slope is determined by taking the zone diameter a solution of the antibiotic 890Ai or 890Aj and a four-fold dilution of this solution (in KPB) measures. Two slices of each concentration are analyzed on a single plate, and the middle one Zone size is determined at each concentration. The slope is equal to half the difference in the Zone size mean values. The strength is then calculated according to the following equation:

Strength (units / ml) =

(Strength of the standard) x dilution x 10.

gradient

In the above equation, D is the mean diameter of the zones formed by the unknown sample, Ds is the mean diameter of the standard zones, and "dilution" is the degree to which the unknown sample was diluted prior to analysis. If no standard is used, D _{b is} assumed to be 25 mm and the size (thickness of the standard) to be 1 unit / ml if Vibrio percolans ATCC 8461 is used as the test organism. Neat 890Ai is determined to have a strength of 250 units per unit of hydroxylamine-erasable absorbance at 300 nm when used as a standard. Accordingly, the strength of the antibiotic 890Aj is 31 units per unit of the extinction at 300 nm that can be canceled by hydroxylamine.

For analyzes on plates with Salmonella gallinarium MB-1287, the slope is determined in the same way as with Vibrio percolans ATCC 8461 determined. If no standard is used, only relative strengths will be used calculated. If no slope is measured, a value of 2.3 mm is assumed. The strength calculations are carried out as in the analysis with Vibrio percolans ATCC 8461. If not an 89OA | standard is used, one can work with a control of penicillin G at 250 μg / ml to demonstrate that the sensitivity of the organisms is in the normal range.

H. Analysis method for the determination of »890 analysis units

The conventional disc-plate analysis method is used with discs with a diameter of 12.7 mm. One works with routinely poured 10 ml per plate, inoculated with Vibrio percolans (ATCC 8461) and uses cephaloridin as the standard. Four concentrations of cephaloridin, namely 3.12, 6.25, 12.5 and 25 μg / ml, form the standard, with the concentration of 12.5 μg / ml being used as the reference solution will. The zone diameters on a 5 ml plate for the standard are as follows:

Concentration, (ig / ml zone diameter mm

3.12 16.8

6.25 22.3

12.5 25.0

^{27-29 6}

One unit is defined as that amount of the antibiotic that forms an inhibition zone on a 5 ml plate of 25 mm. Therefore, in this analysis, a concentration of cephaloridin of 12.5 μg / ml is considered to be a Unit considered equivalent to 890A. Since the slope of the line for cephaloridin is 4.0, the Calculations of the strength of a sample were made using a slope of 4.0.

III. Hydroxylamine reaction

The two antibiotics 890Ai and 890A.1 react with hydroxylamine to form a substance that the Extinction at 300 nm greatly reduced. This provides a basis for the quantitative analysis on the antibiotics 890Ai and 890Aj.

The solution to be analyzed is adjusted to 0.05 molar in a potassium phosphate solution (pH 7.4) by 1/20 volume part of a solution of 0.8 molar K3HPO4 and 0.2 molar KH2PO4 is added. Then you bet 1/100 Volume 1 molar hydroxylamine hydrochloride and determined at intervals of 1/2 to 2 minutes Absorbance at 300 nm. The reaction is carried out at 22 to 28 ° C. You take a kinetic reaction first order and determines the half-life from the decrease in absorbance during the first 10 Minutes. From this half-life, the point in time is estimated beyond which no further decrease in the Absorbance should be observed, and the observations are continued beyond this period, If no further decrease is observed beyond this point in time, the total decrease becomes the Extinction (corrected for the dilution effect and the extinction of the hydroxylamine) as »by hydroxylamine erasable absorbance at 300 nm «(hereinafter abbreviated as HAEAwi). If over

Beyond this line point a decrease in the absorbance takes place, the rate of decrease in the Background absorbance was calculated and the observed decrease at that point in time for the decrease in Corrected background absorbance assuming the decrease in background absorbance runs linearly over time. The corrected value is then recorded as HAEAjoo.

The number of H AEAjoo units is equal to the value of HAEA3oo, multiplied by the volume in ml.

In the examples below, methods are illustrated by which the products according to the invention can be obtained.

example 1
Shake flask production of a mixture containing the antibiotics 890Ai and 89OA3

A portion of a slant culture of Streptomyces flavogriseus MA-4434a is used to make a with to inoculate a baffle provided 250 ml Erlenmeyer inoculation flask, the 50 ml medium A of the following Composition includes:

Medium A 10.0 G Dextrose 10.0 so Yeast autolysate (type pH) 0.05 G MgSO ₄ · 7H ₃ O 2.0 ml Phosphate buffer *) 1000 ml distilled water pH 6.5 91.0 g
95th floor
1000 ml ') Phosphorus solution
KH ^ PO ₄
Na _: H PO ₄
distilled water

This inoculation flask is shaken for 2 days at 28 ° C. in a shaking machine running at 220 rpm (stroke 5.1 cm), after which the culture has grown satisfactorily.
Three 250 ml Erlenmeyer flasks, each containing 40 ml of medium B of the following composition:

Medium B

Tomato puree 20.0 g

Whole grain oats (ground) 20.0 g

distilled water 1000 ml

pH adjusted to 7.0 with NaOH,

the flasks are inoculated with 2 ml (5%) of the culture medium from the inoculation flask. These three production flasks are shaken for up to 4 days at 28 ^° C. in a shaking machine operating at 220 rpm (stroke 5.1 cm). After 3 days, the supernatant liquid centrifuged off from the fermentation liquid gives a zone of 22 mm inhibition against Proteus vulgaris MB 838 and a zone of 15 mm against Salmonella gallinarum MB 1287 when using 6.35 mm analysis discs on standard analysis plates.

Example 2
Shake flask production of a mixture containing the antibiotics 890Ai and 89OA3

A portion of a slant culture of Streptomyces flavogriseus M A-4434a is used to make a with to inoculate a baffle provided 250 ml Erlenmeyer inoculation flask, the 50 ml medium A of the following Composition includes:

Medium A 10.0 g Dextrose 10.0 g Yeast autolysate (type pH) 0.05 g MgSO-i. 7H ₂ O 2.0 ml Phosphate buffer *) 1000 ml distilled water 91.0 g
95th floor
1000 ml. *) Phosphate buffer solution
KI ^ PO ₄
Na-I IPOj
distilled water

This inoculation flask is placed for one day at 28 ° C. in a shaking machine operating at 220 rpm (stroke 5.1 cm), after which the culture is satisfactory. The culture is then kept for 2 days for use

C held. G

Three 2000 m! Erlenmeyer flasks containing 200 ml of medium B each containing the following composition: ff

th: $ 5

Medium B 20.0 g Tomato puree 20.0 g Whole grain oats (ground) 1000 ml distilled water

pH adjusted to 7.0 with NaOH,

are inoculated with 7 ml (3.5%) of the culture from the inoculation flask per flask. These three production pistons will be Shaken for 4 days at 28 ° C. in a foaming machine operating at 220 rpm (stroke 5.1 cm).

The analysis of centrifuged parts using 12.7 mm discs on standard analysis plates and the measurement of the pH values gives the following results:

age V. percolans,
ATCC 8461 S. gallinarum
MB 1287 pH 3 days
4 days 22 mm
26 mm 16 mm
18 mm 6.1
7.5 After 4 days the Pistons harvested. Example 3

Shake flask production and processing of a mixture containing the antibiotics 890Ai and 890Aj

A stirrer with a freeze-dried culture of Streptomyces flavogriseus M A-4434a is opened aseptically and the contents suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition contains:

Davis salts 0.5 g Sodium citrate 7.0 g K ₂ HPOj 3.0 g KH ₂ PO ₄ 1.0 g (NH ₄ ) JSO ₄ 0.1 g MgSO ₄ · 7H ₂ O 1000 ml distilled water

This suspension is used to inoculate slant tubes and plates of various media. from One of these plates is selected from a natural isolate and placed on an inclined tube with medium A of the following Painted composition:

Medium A

Dextrose 10.0 g

Peptone 5.0 g

Yeast extract 3.0 g

NaCl 12.705 g

KCl 0.72 g

FeSO ₄ ■ (NH _{4 I 2} SO ₄ · 6H ₂ O 0.0351 g

MgCl ₂ · 6H ₂ O 5.32 g

CaCl ₂ · 2H ₂ O 0.728 g

distilled water 1000 ml

pH 7.4 (before sterilization)

Agar 25.0 g.

This inoculated slant is incubated for 7 days and, after spore formation, a spore suspension is prepared with 0.8 ml of sterile Davis salts. Four medium A slants are made with this suspension second generation inoculated. The inoculated slant tubes are incubated for one week at 28 ° C and then stored at 4 to 6 ° C until used 14 days later. Half the surface culture of one This slanted tube of the second generation is used to inoculate 250 ml Erlenmeyer flasks with a deflector, each containing 50 ml of medium B of the following composition:

Medium B »0.0 g Yeast autolysate 10.0 g glucose 2.0 ml Phosphate buffer *) 50 mg MgSO ₄ • 7H ₂ O 1000 ml distilled water set to 6.5 pH with HCl or NaOH 91.0 E.
95.0 g
1000 ml. *) Phosphate buffer solution
KH.POj
Na ₂ HPO,
dcslillicrics water

Three seed flasks are inoculated so as to obtain enough inoculum for 12 production flasks, each with 2 I, which are fermented in this approach. The seed flasks are shaken for one day at 28 ⁰ C in a 220 U / min rotating shaker (stroke cm 5.1). The flasks are removed from the shaker and stored at 4 ° C for one day. The contents of these inoculation flasks are used to inoculate 12 Erlenmeyer production flasks without deflectors of 21 each (8 ml inoculum per flask) , which contain 250 ml production medium C of the following composition:

Medium C 20.0 g Tomato puree 20.0 g Whole grain oats 1000 ml distilled water

pH adjusted to 7.0 with NaOH.

After inoculation, the production flasks are incubated for 4 days at 2 ° C. with shaking in a shaking machine operating at 220 rpm (stroke 5.1 cm). The flasks are then harvested and the activity is determined on analysis plates against Salmonella gallinarum MB 1287 and Vibrio percolans ATCC 8461 using 12.7 mm analysis disks which are immersed in centrifuged fermentation fluid samples.

Analysis during harvest

Cultural age, h 96 Ph 7.4 Salmonella gallinarum Inhibition zone, mm 23 Vibrio percolans ATCC 8461 Inhibition zone, mm 29

The fermentation liquid is filtered and the pH of the filtrate is increased to 7.7 with dilute hydrochloric acid 7.0 brought. 2.65 1 of the filtrate adjusted in this way are transferred to 135 ml at a rate of 20 ml / min 1RA68 resin in the chloride form (weakly basic, cross-linked acrylic acid polymer anion exchange resin) adsorbed with the effluent being collected as a fraction. The adsorbate is washed with 250 ml of water and eluted in six 100 ml fractions with 5% NaCl. All fractions are made according to the disk-plate method analyzed with the help of Vibrio percolans ATCC 8461 and Salmonella gallinarum MB1287. These Analyzes show that eluate fractions No. 1 to 3 contain 45% of the bioactivity applied to the resin.

The eluate fractions No. 1 to 3 are combined to form 310 ml of solution. This solution is mixed with 15 g of sodium chloride and cooled to 2 ° C. The cold solution is adjusted with HCI to a pH of 3.0 and immediately extracted with alcohol 1 ^c ι ml n-Buty!. The extraction is repeated twice with a further 150 ml of n-butyl alcohol. The n-butyl alcohol phases are back-extracted three times in succession with 75 ml of water each time. During the extraction, the aqueous phases are kept at a pH of 7 by adding dilute sodium hydroxide solution. All fractions are analyzed against Vibrio percolans ATCC 8461 and Salmonella gallinarum M B-1287; the results are given below as a percentage of the initial bioactivity:

26th fraction 52 677 Output
watery drainage
n-butyl alcohol drain Extraction aqueous reverse extract number 1
No. 2
No. 3 100%
12%
0%
0%
0% No. I.
No. 2
No. 3 25%
37%
5%

The first and second back extract are individually freeze-dried. The freeze-dried solids are combined to give 984 mg of product

Example 4

Preparation of a mixture, which contains the antibiotics 890Ai and 890A3, and its processing

A tube with a freeze-dried culture of Streptomyces flavogriseus M A-4434a is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Davis salts 0.5 g Sodium citrate 7.0 g K ₂ HPO ₄ 3.0 g KH ₂ PO ₄ 1.0 g (NH ₄ J ₂ SO * 0.1 g MgSO ₄ • 7H ₂ O 1000 ml. distilled water

This suspension is used to inoculate four slant tubes, which are a medium of the following Composition include:

Medium A

Dextrose 10.0 G Peptone 5.0 G Yeast extract 3.0 G NaCl 12,705 G KCl 0.72 G FeSO ₄ · (NH _{4 I 2} SO ₄ · 6H ₂ O 0.0351 G MgCl ₂ 6H ₂ O 5.32 G CaCl ₂ · 2H ₂ O 0.728 G distilled water 1000 ml pH 7.4 (before sterilization) Agar 25.0 G-

The inoculated slants are incubated for one week at 27-28 ° C and then stored at 4 to 6 ⁰ C until 10 days later be used. Half of the surface culture of one of these oblique tubes is used to inoculate 250 ml Erlenmeyer flasks equipped with a deflector, each containing 50 ml of medium B of the following composition:

Medium B 10, floor Yeast autolysate 10.0 g glucose 2.0 ml Phosphate buffer *) 91.0 g
95.0 g
1000 ml. ·) Phosphate buffer solution
KH ₂ PO ₄
Na ₂ HPO ₄
distilled water

continuation

MgSO ₄ · 7H ₂ O 50 mg

distilled water 1000 ml

pH adjusted to 6.5 with HCl or NaOH.

Three inoculation flasks are inoculated in such a way that enough inoculum is obtained for 12 production flasks of 21 each, which i fermented in this approach. The inoculation flasks are rotated at 220 rpm for one day at 28 ° C the shaker (stroke 5.1 cm) shaken. The flasks are removed from the shaking machine and a Stored well at 4 ° C. The contents of this inoculation flask are used to make 12 Erlenmeyer production flasks without Deflecting organs to inoculate 2 1 each (7 ml inoculum per flask), the 250 ml production medium C the following Composition include:

Medium C 20.0 g Tomato puree 10.0 g Primary yeast 20.0 g Dextrin 1000 ml demineralized water

pH adjusted to 7.3 with NaOH.

After inoculation, the production flasks are run at 220 rpm for 4 days and 5 hours at 24 ° C running shaker (stroke 5.1 cm) incubated. After this period the cobs are harvested, and the activity against Salmonella gallinarum MB-1287 and Vibrio percolans ATCC 8461 is using from analysis plates and 12.7 mm analysis disks, which are in centrifuged fermentation liquid samples be dived. The results are as follows:

Analysis during harvest 101 Cultural age, h 7.1 ph Salmonella gallinarum 31 Inhibition zone, mm Vibrio percolans 43 Inhibition zone, mm

The fermentation liquid is centrifuged. 22 mg are added to the 2.2 l of the clear centrifugate (pH 6.8) (Ethylenedinitrilo) tetraacetic acid and adjusts the pH to 7.2. The adjusted to the stated pH Centrifugate at a rate of 15 ml / min on 150 ml of Dowex 1 x 2 resin in the chloride form adsorbed, collecting the effluent as a fraction. The adsorbate is demineralized with 150 ml Washed water containing 10 jig / ml (ethylenedinitrilo) -tetraacetic acid, and with 5% NaCl-

¹ ^ solution cluierl. which contain 10 μg / ml (ethylenedinitrilo) -tetΓaacetic acid, five fractions of 75 ml each being collected. All fractions are analyzed against Vibrio percolans ATCC 8461. The results as a percentage of the Anlimgsbioaktiviiäi are as follows:

Fraction extraction

Output 100%
Procedure 0

5% NaCi eluate fractions No. 2 to 4 75%
Water displacement washing liquid 1%

50 ml of fraction no. 3 of the 5% NaCl eluate are adjusted to a pH of 5.2 with dilute hydrochloric acid and passed through a 100 ml cone of XAD-2 that was previously filled with five times its volume 60% by volume aqueous acetone solution, five times its volume of demineralized water and the "Five times its volume in a solution of 5 parts by weight of NaCl in 100 parts by volume of demineralized Water that is 25 ^ molar of neutral EDTA, and then five times its volume of demineralized Water has been washed. The resin is mixed with demineralized water, which is 10 μg / ml Contains (ethylenedinitrilo) tetraacetic acid, developing two fractions of 25 ml each and five fractions collects 10 ml each and monitors the process until it reacts negatively to acidic silver nitrate. At the end the fifth 10 ml fraction will give a negative silver nitrate reaction. The development with demineralized Water is continued until four 100 ml fractions have been collected. The resin is made with 60% aqueous acetone solution containing 4 μg / ml (ethylenedinitrilo) -tetraacetic acid, developed to two Fractions of 50 ml each have been collected. All fractions are against Vibrio percolans ATCC 8461

analyzed; the results are as follows:

Initial sample Volume 50 ml Extraction 1007 ,, Parliamentary group 1 25th 0 2 25th 0 3 10 0 4th 10 0 5 10 1 6th 10 4.5 7th 10 7th 8th 100 60 9 100 6th 10 100 1 11th 100 0 12th 50 1 13th 50 0

Fraction # 8 is concentrated to 10 ml and freeze-dried to 34.2 mg of solids.

33.2 mg of the freeze-dried solids are taken up in 1.5 ml of 1% aqueous n-butyl alcohol solution, and the solution is placed in a 1.4 x 81.5 cm column of Bio-Gel P-2 (37 to 74 μΐη), which has previously been brought into equilibrium with 1% aqueous n-butyl alcohol solution. The gel comes with the same solvent developed at a rate of 1 ml / min, fractions of 2 ml catches. Each of Fractions No. 29 to 46 is analyzed against Vibrio percolans ATCC 8461. The highest Bioactivity is observed in fractions # 36 to 44 with a maximum in fraction # 39. the Fractions No. 38 to 41 are combined and freeze-dried to give 4.0 mg of antibiotic.

Example 5

Shake flask production of a mixture,
which contains the antibiotics 890Ai and 890Ai, and its processing

A tube with a freeze-dried culture of Streptomyces flavogriseus M A-4434a is opened aseptically and the contents suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition contains:

Davis salts 0.5 g Sodium citrate 7.0 g K ₂ HPO ₄ 3.0 g KH2PO4 1.0 g (NH ₄ ) JSO ₄ 0.1 g MgSO ₄ • 7H ₂ O innn «-.1
1 UW Uli

This suspension is used to inoculate four slant tubes with Medium A, which is the following Composition has:

Medium A

Glycerin 20.0 g

Primary yeast 5.0 g

Fish meal 15.0 g

distilled water 1000 ml

Agar 20.0 g

pH adjusted to 7.2 with NaOH.

The inoculated slants are incubated for one week at 27-28 ° C and then lifted until use at 4 to 6 ⁰ C.
A third of the surface culture of two oblique tubes is used to connect six with a deflector

see inoculating 250 ml Erlennieyer flasks, each containing 50 ml of medium B of the following composition:

Medium B 10, floor Yeast autolysate 10.0 g glucose 2.0 ml Phosphate buffer *) 50 mg MgSO ₄ • 7H ₂ O 1000 ml distilled water set to 6.5 pH with HCl or NaOH *) Phosphate powder solution 91.0 g KIhPO ₄ 95.0 g Nü.iMFOj 1000 ml. ücsiillicrtcs water

The inoculation flask is shaken for one day at 27 to 28 ° C. in a schiittel machine running at 220 rpm (stroke 5.1 cm). The flask and its contents are ^{kept at 4 °} C. for one day at rest.

22 Erlenmeyer production flasks of 2 l each, each holding 300 ml of medium, are each flask with 8 ml of the Inoculated culture from the inoculation flask. The medium C has the following composition:

Medium C 20.0 g Tomato puree 10.0 g Primary yeast 20.0 g Dextrin 5.0 mg CoCI: 6H ₂ O 1000 ml distilled water

pH adjusted to 7.2 to 7.4 with NaOH.

After inoculation, the production flasks are run at 212 rpm for 4 days and 5 hours at 24 ° C running shaker (stroke 5.1 cm) incubated. The flasks are harvested and the activity is recorded with Using standard analysis plates against Salmonella gallinarum MB-1287 and Vibrio percolans ATCC 8461 analyzed by 1.27 mm analytical disks placed in centrifuged fermentation fluid samples be dived. If necessary, the samples are diluted with 0.02 molar phosphate buffer (pH 7.0). The results are as follows:

Cultural age, h 101

pH 7.1

Salmonella gallinarum
Inhibition zone, mm 31

Vibrio percolans, dilution 1/10
Inhibition zone, mm 24.5

890 analysis units 46

4 liters of the complete fermentation liquor containing 23 units / ml are cooled to 3 ° C and poured into Shares of 200 m! each centrifuged for 5 minutes at 9000 rpm. The combined supernatants (3.6 l) are mixed with 1 ml of 0.1 molar neutral EDTA solution and 10 ml of 1 molar Tris-HCl buffer solution (pH 7.5) offset. The 0.10 molar neutral EDTA solution is made by dissolving 0.1 mol of the disodium salt of Ethylene diamine tetraacetic acid in 1 liter of demineralized water and adjustment of the pH value with sodium hydroxide solution manufactured on 7.

The centrifuged fermentation liquid is in a column on a 5.1 cm x 20 cm bed of Dowex-1 x 2 (Cl "), particle size 150 to 300 μΐη, adsorbed before the centrifuged liquid is applied washed with 1 liter of demineralized water. The flow rate during adsorption is 50 to 80 ml / min. After the adsorption has ended, the column is filled with 500 ml of demineralized water, which are 25 jimolar of neutral EDTA, washed and then with 1300 ml of 5% NaCl in demineralized water, which is 25 ^ molar of neutral EDTA, eluted. Calculated from the first infusion of the saline solution, nine fractions are caught. Fractions No. 1 to 4 contain 100 to 115 ml per fraction, the fractions No. 5 to 9 each comprise 170 to 185 ml. The fractions are tested for their bioactivity against Salmonella gallinarum MB-1287 analyzed. The bioactivity appears in fractions No. 2 to 9 with a maximum at parliamentary groups 4 and 5.

The ultraviolet absorbance of appropriate dilutions of each fraction is measured with a spectrophotometer measured and the ratio of bioactivity to absorbance at 220 nm (A: 2o) calculated for each fraction. Those fractions in which the ratio of bioactivity to A220 is greater than half of the maximum value

tes are united for further processing. So the fractions No. 4 to 7 are combined, the 30% of the contain abandoned bioactivity.

The combined NaCl eluate is concentrated to 100 ml under reduced pressure, the temperature at Evaporation is kept below 35 ° C.

The pH of the concentrate is adjusted to 5.2 by adding 3 ml of 1 molar hydrochloric acid and that Concentrate placed in a 3.5 cm wide and 57 cm long column with XAD-2, which was previously filled with 3 160% iger aqueous acetone solution, 3 l of demineralized water, 3 l of a solution of 5 parts by weight of common salt per 100 Parts of the volume of demineralized water, which is 25 ^ molar of neutral EDTA, and 1 l of saturated saline solution has been washed. After washing, the column is transferred to a cold room at 3 ° C held before the concentrate is dispensed.

The applied concentrate is drained to the level of the bed of the adsorbent, and the active fractions are with 1.3 1 25 ^ molar neutral EDTA solution in demineralized water eluted. From the first feed of the concentrate into the column, fractions of 10 ml each are counted caught.

Every fifth fraction is tested for bioactivity against Salmonella gallinarum using the plate method ΜΒΊ287 is analyzed and the A22o-Wcrtc are determined at suitable dilutions. Those factions in which the ratio of bioactivity to A220 is greater than half of the maximum value, are combined. In this way, the fractions No. 48 to 95 are combined, which 50% of the abandoned activity, corresponding to 17% of the activity of the filtered fermentation liquid.

The combined XAD-2 fractions are concentrated to 50 ml and placed in a column of Dowex-1 × 4 (CD with particle sizes below 37 μm, in which the dimensions of the resin bed are 1.3 × 13 cm ml of demineralized water and eluted with 400 ml of a solution in demineralized water which is 0.08 molar in NaCl, 0.005 molar in NH ₄ CI and 0.0001 molar in NH ₃ at a flow rate of 0.6 ml / min fractions of 10 ml each are collected. An activity equivalent to 100% of the activity given is eluted in fractions No. 23 to 30; the maximum of the activity occurs in fraction No. 26. Fractions No. 26 and No. 27 have the highest ratio of absorbance at 300 nm to absorbance at 220 nm (A30C / A220) and these two fractions are combined for desalting.The total activity in fractions No. 26 and 27 is 7390 units or 59% of the total Abandoned Activity. The United Frak Options no. 26 and 27 are mixed with 5 μΐ 0.01 molar neutral EDTA solution.

The combined fractions are reduced to 1.95 ml in a rotary evaporator under reduced pressure concentrated, and the concentrate is placed in a column of Bio-Gel P-2 (37 to 74 μm) in which the Dimensions of the adsorbent bed are 2.2 x 80 cm. The sample is made up in proportions of 1 ml and 2 ml demineralized water and then washed at a rate of 0.7 ml / min with cmmineralized Water elutes. Fractions of 2.1 to 2.2 ml are collected. The main maximum of the UV absorbance is found in fractions No. 89 to 100, which contain 74% of the extinction units determined at 300 nm (A.iim units). Fractions No. 92 to 98 are combined to make 2 ml concentrated and freeze-dried.

This product group contains 18.6 A.ioo units, which is 5.8% of that in the starting fermentation liquid contained bioactivity. The fractions No. 89 to 91 and No. 99 to 100 are further according to Example 6 cleaned.

Example 6
(Shake flask) production of the antibiotic 890Ai

A tube with a freeze-dried KuItU ₁ of Streptomyces flavogriseus MA-4434a is opened aseptically and the contents are suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following composition:

Davis salts 0 5 " 7.0 g K2HPO4 3.0 g KH ₂ PO. 1.0 g (NH ₄ I ₂ SO. 0.1 g MgSO .. 7H ₂ O 1000 ml. distilled water

This suspension is used to inoculate four slant tubes with Medium A, which is the following Composition has:

Medium A

Glycerin 20.0 g

Primary yeast 5.0 g

Fish meal 15.0 g

continuation

distilled water 1000 ml

Agar 20.0 g

pH adjusted to 7.2 with NaOH.

The inoculated oblique tubes are incubated for one week at 27 to 28 ° C and then until used canceled at 4 to 6 ° C.

A third of the culture of these three oblique tubes is used to make nine baffles Inoculate 250 ml Erlenmeyer flasks, each containing 50 ml of medium B of the following composition:

Medium B

Yeast autolysate 10.0 g

Glucose 10.0 g
Phosphate buffer *) 2.0 ml

MgSO ₄ • 7H ₂ O 50 mg

distilled water 1000 ml

pH adjusted to 6.5 with HCl or NaOH

*) PhosphaipulTcrlösuni;

■ KHjPOj 91.0g

NajllPOj 95.0 g

full water 1000 ml.

The inoculation flask is placed for one day at 27 to 28 ° C in a shaking machine running at 220 rpm (Hub 5.1 cm) shaken. The flask and its contents are kept at 4 ° C. for one day.

33 Erlenmeyer production flasks of 2 l each, each containing 250 ml of medium C, are each flask with 8 ml of the Inoculated culture from the inoculation flask. Medium C has the following composition:

Medium C 20.0 g Tomato puree 10.0 g Primary yeast 20.0 g Dextrin 5.0 mg CoCI ₂ • 6H ₂ O 1000 ml distilled water

pH adjusted to 7.2 to 7.4 with NaOH.

After inoculation, the production flasks are ^{incubated for 4 days at 24 °} C. in a shaking machine running at 212 rpm (stroke 5.1 cm). The flasks are harvested and the activity is analyzed with standard analysis plates against Salmonella gallinarum MB-1287 and Vibrio percolans ATCC 8461 using 1.27 mm analysis disks which are immersed in centrifuged fermentation fluid samples. If necessary, the samples are diluted with 0.02 molar phosphate buffer (pH 7.0). The results are as follows:

Cultural age, h 96

pH 7.2

Salmonella gallinarum
Inhibition zone, mm 29.5

Vibrio percolans, dilution 1/10
Inhibition zone, mm 31

890 analysis units 40

7 l of the complete fermentation liquid are cooled to 3 ° C. and in portions of 200 ml for 15 minutes centrifuged at 9000 rpm.

The combined supernatant liquids are mixed with 7 ml of 0.1 molar neutral EDTA solution, and the entire sample is fed at a flow rate of 40 ml / min on a column with Dowex-1 x 2 (CD, particle sizes 150 to 300 μm, Bed dimensions 5.1 x 25 cm, adsorbed. The column is washed with 500 ml of demineralized water which contains 5 ml of 1 molar Tris-HCl buffer (pH 7.0) and is 25% of neutral EDTA Antibiotic is eluted with 1 l of demineralized water with a saline content of 50 g and the column is then washed with 300 ml of demineralized water. From the first occurrence of salt in the column, fractions of 100 ml are collected. The bioactivity appears in the fractions No. 1 to 10 with a maximum in fraction No. 2. The fractions with the highest ratios of bioactivity to A ₂₂ u are pooled, thus fractions No. 2 to 5

combined, which contain 17% of the abandoned activity.

The combined fractions are made 110 ml in a rotary evaporator under reduced pressure concentrated, and the nH value is adjusted to 5.8 by adding 4.2 ml of 1 molar hydrochloric acid. That so adjusted concentrate is fed into an XAD-2 column with bed dimensions of 3.8 x 50 cm, the first with 3 liters of 60 percent by volume aqueous acetone solution, then with 3 liters of demineralized water, with 3 liters Saline solution (5 parts by weight per 100 parts by volume) and 11 parts by weight saline solution (25 parts by weight per 100 parts by volume) has been washed in demineralized water. The concentrate is left up to the level of the adsorbent bed expire. The antibiotic is demineralized at a flow rate of 15 ml / min Water elutes. Counting from the first application of the sample, there are 22 fractions of 100 ml each caught. The bioactivity appears in the fractions No. 6 to 22 with a maximum in the fractions No. 9 and i0. The fractions whose ratio of bioactivity to A220 is greater than 0.3 times the maximum value are united with one another. In this way, fractions 9 to 20 become the next Processing united. Fractions 89, 90, 91.99 and 100 from Bio-Gel are added to these fractions P-2 column of Example 5 was added. This mixture is reduced in the rotary evaporator Pressure reduced to 70 ml.

The concentrate is adsorbed on a column with Dowex-1 Χ 4 (CI "), particle sizes below 37 μm, bed dimensions 2.2 x 27 cm. The column is washed with 50 ml of demineralized water and the antibiotic with 2 l of sodium chloride 0.070 molar, NH4Cl 0.005 molar and NH ₃ 0.0001 molar solutions are eluted in demineralized water at a flow rate of 2 ml / min, thereby collecting fractions of 9.5 ml each.

The main peak of the antibiotic is eluted in fractions no. 142 to 163. The parliamentary groups No. 146 to 157 contain the material with the highest A.ioo / A2so ratios and are used for further processing united.

The combined fractions No. 146 to 157 are added in a rotary evaporator under reduced pressure 3 ml concentrated, and the concentrate is by adding 5 μί 1 molar Nri.i solution to a pH of 6.5 set. The concentrate is placed in a column (2.2 x 70 cm) with Bio-Gel P-2 (37 to 74 μπι), which has previously been washed with 20 ml of 5 molar saline solution and 500 ml of demineralized water. As soon as the concentrate has run off up to the level of the adsorbent bed, the column is switched on twice Rinsed 1 ml of demineralized water each time, whereupon it is allowed to run off again up to the level of the bed. the The column is then eluted with demineralized water at a flow rate of 0.6 ml / min, with fractions of 2.9 ml each being collected.

The main peak in antibiotic activity is found in fractions Nos. 63 to 70eluie. The factions Nos. 64 and 65 have the highest ajoo / ajjo ratios and are combined for further processing. These two fractions contain 44% of the UV absorption at 300 nm, which is added to the Bio-Gel P-2 cone had been.

The combined fractions No. 64 and 65 are in the rotary evaporator under reduced pressure 2 ml concentrated and then frozen for 8 hours in a 14 ml ampule with screw cap and * .u 2.25 mg practical 890Ai pure antibiotic freeze-dried (45.5 Ajoo units).

Example 7
(Shake flask) production of the antibiotic 890Ai

A tube with a freeze-dried culture of Streptomyces fiavogriseus MA-4434a is placed aseptic opened and the contents suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following Composition includes:

Davis salts 0.5 g Sodium citrate 7.0 g K ₂ HPO ₄ 3.0 g KH ₂ PO ₄ 1.0 g (NH ^) ₂ SO ₄ 0.1 g MgSO ₄ · 7H ₂ O 1000 ml distilled water

This suspension is used to inoculate four inclined tubes with Medium A, which is the following Composition has:

Medium A 20.0 g Glycerin 5.0 g Primary yeast 15.0 g Fish meal 1000 ml distilled water 20.0 g Agar

pH adjusted to 7.2 with NaOH.

The inoculated slant tubes are incubated for one week at 27 to 28 ° C and then until used canceled at 4 to 6 ° C (no longer than 21 days).

A third of the culture from four oblique tubes is used to make 12 baffles Inoculate ml Erlenmeyer flasks, each containing 50 ml of medium B of the following composition:

Medium B 10, floor Yeast autolysate 10.0 g glucose 2.0 ml Phosphate buffer *) 50 mg MgSO ₄ • 7H ₂ O 1000 ml distilled water set to 6.5 pH with HCl or NaOH "» 1.0 g
95th floor
1 (XX) ml. ") Phosphate powder solution
KH ^ PO ₄
Na.HPOj
distilled water

The inoculation flask is shaken for one day at 27 to 28 ° C. in a schiittel machine running at 220 rpm (stroke 5.1 cm). The flask and its contents are ^{kept at 4 °} C. for one day at rest.

Erienmeyer production flasks of 2 l each, each containing 200 ml of medium C, are each filled with 8 ml of the culture inoculated from the inoculation flask. Medium C has the following composition:

Medium C

Tomato puree 20.0 g

Primary yeast 10.0 g

Dextrin 20.0 g

CoCl ₃ • 6H ₂ O 5.0 mg

distilled water 1000 ml
pH adjusted to 7.2 to 7.4 with NaOH.

After inoculation, the production flasks are run at 212 rpm for 4 days and 5 hours at 24 ° C running shaker (stroke 5.1 cm) incubated. The flasks are harvested and the activity is recorded with Using standard analysis plates against Salmonella gallinarum MB-1287 and Vibrio percolans ATCC 8461 analyzed by 1.27 mm analytical disks placed in centrifuged fermentation fluid samples be dived. If necessary, the samples are diluted with 0.02 molar phosphate buffer (pH 7.0). The results are as follows:

Cultural age, h 101

PH 7.2
Salmonella gallinarum

Inhibition zone, mm 35
Vibrio percolans, dilution 1/10

Inhibition zone, mm 34

890 analysis units 121

The entire 7.0 l of the complete fermentation liquid is cooled to 3 ° C. and added in portions ml centrifuged at 9000 rpm for 15 minutes. The combined supernatant liquids are with 1.7 ml of 0.1 molar neutral EDTA solution are added, and the batch is kept at 3 ° C.

The fermentation described above is repeated under the same conditions with the difference, that the 44 2-liter Erlenmeyer production flasks are inoculated with 7 ml of the culture from the inoculation flask. pH and analysis results are as follows:

Cultural age, h 101

PH 7.3
Salmonella gallinarum

Inhibition zone, mm 38
Vibrio percolans, dilution 1/10

Inhibition zone, mm 39

890 analysis units 92.8

26th

The total of 7.41 of the liquid obtained in this fermentation is cooled to 3 ° C and in portions centrifuged from 200 ml for 15 minutes at 9000 rpm. To the combined supernatant fluids 1.8 ml of a 0.1 molar EDTA solution are added.

The supernatants of the fermentation liquors that have been obtained in the two fermentation experiments of this example are combined to a total of 13 l, which has a strength of 80 units / ml as determined by analysis against Vibrio Percolans ATCC 8461.

The combined supernatant liquid is at a flow rate of 60 ml / min through a Column with Dowex-1 x 2 (Cl ") (particle size 150 to 300 μπι, bed dimensions 4.7 cm x 50 cm) passed. The The column is washed with 1 l of demineralized water and the antibiotic with 5 l of saline solution (5 parts by weight of table salt per 100 parts by volume), the 0.01-molar Tris-HCl buffer (pH 7.0) and 25 ^ molar of neutral EDTA are eluted. There are fractions of 220 ml each with a flow rate of 50 ml / min collected and analyzed against Salmonella gallinarum MB-1287 by the plate method.

The antibiotic activity appears in the fractions No. 3 to 26 with a maximum in the fractions No. 5 and 6. Fractions No. 5 to 9, which have the highest ratio of bioactivity to Av> o, are used for further processing combined. The pH of the combined fractions is 7.8, and the pooled fractions contain 24% of the initial bioactivity, determined by the plate method against Salmonella gallinarum MB-1287

The combined fractions Nos. 5 to 9 are added to a rotary evaporator under reduced pressure Concentrated 150 ml and placed in a column (bed dimensions 4.9 cm x 47 cm) of XAD-2, which was previously with 5,160 percent by volume aqueous acetone solution, 51 demineralized water and 51 a solution of 50 g common salt per liter of demineralized water has been washed. The sample is poured into the Entered the column, the column being allowed to drain up to the level of the bed each time. After finished The samples are given in three portions of 20 ml of demineralized water each time and up to drained to the level of the bed. Then the sample is at a flow rate of Eluted 20 ml / min with demineralized water. All operations with the XAD column are carried out at Room temperature (24 ° C) carried out, and the collected fractions are immediately immediately im Chilled ice bath. Fractions from 95 to 230 ml are collected.

The antibiotic activity appears in fractions No. 2 to? 1, determined by the plate method against Salmonella gallinarum MB-1287, and the maximum is contained in fractions Nos. 5 to 7 (the ones counted by the first abandonment of demineralized water, ranging from 510 to 895 ml of deseluted volume). the Fractions Nos. 6 to 21 in which the proportions of bioactivity to Ajju are within 50% of that observed Maximum values are combined. The total bioactivity recovered is 280,000 units or 112% of apparent cessation of activity.

The combined fractions No. 6 to 21 are added in a rotary evaporator under reduced pressure Concentrated to 68 ml and then diluted to 112 ml with demineralized water. The concentrate is in a Column (bed dimensions 2.2 cm x 21 cm) of Dowex-I x 4 (CI ") with particle sizes below 37 μπι entered. The column is washed with 20 ml of demineralized water and the antibiotics are washed with a Flow rate of 1.6 ml / min with a 0.07 molar of sodium chloride, 0.005 molar of N HjCl and eluted on NH3 0.0001 molar solution in demineralized water. Thereby fractions of 8 ml caught.

The main maximum of the absorbance at 300 nm appears in fractions No. 153 to 182 with a Maximum in fraction # 162. Fractions # 157 to 179 that had the highest ratios of absorbance at 300 nm to absorbance at 245 nm are combined for further processing. The United Fractions have a total of 780 absorption units at 300 nm.

The combined fractions are concentrated to 7 ml in a rotary evaporator under reduced pressure, and the pH is adjusted to 7.5 by adding 20 μl 1 molar sodium hydroxide solution. The solution will be further concentrated to 5 ml and placed in a column (2.2 x 75 cm) of Bio-Gel P-2 (37 to 74 μm). the The sample is washed into the column with two portions of 1 ml demineralized water each and with eluted with demineralized water at a flow rate of 0.6 ml / min. First will be ten Fractions of 3.3 ml each, then sixty fractions of 2.65 ml each and finally ten fractions of 2.0 ml each caught.

The main maximum of the extinction at 300 nm appears in fractions no. 60 to 68. The pH values of the Maximum fractions are reduced to a range of 7.5 by adding 1.5 to 2.5 μΙ 0.1 molar sodium hydroxide solution set to 8.0. Fractions No. 62, 63, 64 and 65 are individually frozen in 14 ml glass ampoules and Freeze-dried and stored in vacuo at -20 ° C. These four fractions contain a total of 606 absorption units at 300 nm.

In order to obtain the antibiotic 890A.1 free from the antibiotic 890Ai, one uses the proportionally increased resistance of the antibiotic 890Ai to degradation by penicillinase as follows from: The Bio-Gel fraction no. 63 is combined with the fractions no. 61, 66 and 67 from the Bio-Gel column. These four combined fractions have a total of 235 absorbance units at 300 nm. Set 0.2 ml a 1 molar Tris-HCl buffer solution (pH 7.5) and 0.2 ml of penicillinase to [containing 500,000 units per ml (1000 LU / ml), where the term LU refers to Levy units; 1000 LU inactivate 500000 units Penicillin G]. After 113 minutes at 23 ° C., a further 0.2 ml of penicillinase is added. After further Another 0.2 ml penicillinase is added for 7 hours at room temperature, and after a further 2 hours the reaction mixture is cooled in an ice bath at room temperature. The mixture is then made by adding 5 ml of demineralized water diluted to 15 ml. The absorbance of the diluted reacted preparation is 6.3 at 300 nm, of which 2.64 units can be erased by reaction with cysteine.

The reaction mixture is applied to a column of Dowex 1 x 4 (CP), particle size below 37 _u m, Bettab-

dimensions 2.15 x 40 cm, adsorbed. The antibiotic 890A3 is eluted at a flow rate of 3 ml / min with a 0.07-molar sodium chloride, 0.005-molar NHiCl and _{0.0901-molar NH 3} solution in demineralized water. Fractions of 13.8 ml each are collected.

The main maximum of the absorbance at 300 nm appears in fractions No. 185 to 203. The fractions No. 187 to 200 have the highest A3U0 / A245 ratios and are combined for further processing. The united parliamentary groups have a total of 31 A3oo units.

The combined fractions are concentrated to 4 ml in a rotary evaporator under reduced pressure, and the concentrate is placed in a column (2.2 x 70 cm) of Bio-Gel P-2 (37 to 74 μm). That Antibiotic 89OA3 is mixed with demineralized water at a flow rate of 0.6 ml / min tared. 30 fractions of 3.3 ml each and then 50 fractions of 2.65 ml each are collected.

The main peak of the absorbance at 300 nm appears in fractions No. 64 to 74 with a maximum in Fraction No. 70. Fractions No. 66 to 70 are combined; they contain 19 Aioo units or 61% of the discontinued units. The combined samples are in the rotating evaporator under reduced The pressure is reduced to 1.5 ml and the concentrate is frozen and freeze-dried. 5.4 mg are obtained Solids containing antibiotic 890Aj and remaining Sa! * E.

Example 8
Manufacture of antibiotics 890Ai and 890A.1

A tube with a freeze-dried culture of Streptomyces flavogriseus MA-4434a is placed aseptic opened and the contents suspended in a tube containing 0.8 ml of sterile Davis saline solution of the following Composition includes:

Davis salts 0.5 g Sodium citrate 7.0 g K ₂ HPO ₄ 3.0 g KH2PO4 1.0 g (NH ₄ I ₂ SO ₄ 0.1 g MgSO ₄ • 7H ₂ O 1000 ml distilled water

This suspension is used to inoculate four slant tubes with Medium A, which is the following Composition has:

Medium A

Glycerin 20.0 g

Primary yeast 5.0 g

Fish meal 15.0 g

distilled water lOCO ml

Agar 20.0 g

pH adjusted to 7.2 with NaOH.

The inoculated slants are incubated for one week at 27 to 28 ° C and then stored at 4 to 6 ° C until use (no longer than 21 days).
10 ml medium B with the following composition:

Medium B

Yeast autolysate 10.0 g

Glucose 10.0 g

Phosphate buffer *) 2.0 ml

MgSO .. 7H ₂ O 50 mg

distilled water 1000 ml

pH adjusted to 6.5 with HCI or NaOH.

*) Phosphate powder solution

Na.iHPOj 95.0 g

distilled water K) (K) ml,

are aseptically transferred to one of these oblique tubes, the spores and aerial mycelium become in suspension scraped, and 3.3 ml of this suspension are used in order to

See the Erlenmeyer flask containing 500 ml of medium B. This inoculation flask will last for 36 hours 28 ° C in a shaker operating at 160 rpm (stroke 5.1 cm), whereupon the culture is has developed satisfactorily.

The culture from this inoculum is used to power a 189 liter stainless steel fermenter inoculate containing 160 l of medium B. This fermenter is 24 hours at 28 ° C while stirring with a Speed of 150 rpm operated with an air flow of 85 l / min. An anti-foaming agent (Polyglycol 2000) is used as required, but not in a concentration greater than 0.1%. The pH levels are the following:

Age, h 0 12

pH 6.3 6.35

43 l of the culture of this seed fermenter are used to make a 756 l stainless steel fermenter Inoculate steel containing 467 J Medium C of the following composition:

Medium C 20.0 g Tomato puree 10.0 g Primary yeast 20.0 g Dextrin 5.0 mg CoCl ₂ • 6H ₂ O 1000 ml distilled water

pH adjusted to 7.2 to 7.4 with NaOH.

This fermenter is 92 hours at 25 ° C with stirring at a speed of 146 rpm an air flow of 255! / min operated. If necessary, additional anti-foaming agent (polyglycol 2000), but not more than 0.1%, was added. There are antibacterial analyzes against Salmonella gallinarum MB-1287 and Vibrio percolans ATCC 8461 with the following results:

Dude, H 0 12th 24 36 48 60 72 84 92 pH 6.6 6.7 6.65 6.3 6.0 6.4 6.15 6.5 6.5 MB-1287, mm - - - S. - 19th 26th 28 32 ATCC 8461, mm (1 to 10) - - - S. - 21 24 26th 30th 890A units / ml _ _ _ N / A N / A 6.8 13.5 24.9 24.3

The 890A units / ml are, as described above in section »II. Analysis method for determining the 890 analysis units « described, determined.

4731 fermentation liquids are cooled to 5 ° C and centrifuged in a centrifuge. 22.7 kg of diatomaceous earth are added to 378.5 liters of the supernatant liquid and the suspension is passed through a 45.7 cm filter press filtered. The 344.4 l of filtrate are adsorbed on a column, the 26.5 l of Dowex-1 x 2 (CF) with particle sizes from 150 to 300 μπι contains, and the column is washed with 37.85 l of demineralized water. That Mixture of the antibiotics 890Ai and 890A3 is made with 113.5 15% saline solution, the 0.01 molar of Tris-HCl buffer (pH 7.0) and 25 ^ molar of neutral EDTA, eluted in demineralized water. It will Fractions of 18.9 1 each collected. The mixture of antibiotics 890Ai and 890 A3 appears in the fractions No. 3 to 6 with a maximum strength in the fractions No. 4 and 5. The fractions No. 4.5 and 6, the 8% the activity of the filtered fermentation liquid are combined with each other and reduced under reduced Pressure concentrated to 7.6 liters.

The 7.6 l of concentrate are placed in a column that contains 37.85 l of XAD-2 and previously with 189 l 60% aqueous acetone, then with 1891 demineralized water and finally with 18915% saline solution has been washed. The adsorbate of this concentrate is demineralized with 142 l eluted. Three fractions of 9.51 and six fractions of 18.91 are collected. The activity appears in the fractions No. 1 to 6 with a maximum strength in the fraction No. 3. The fractions No. 4 and 5, the 64% of the activity given up in the XAD column or containing 370,000 units are combined.

Fractions Nos. 4 and 5 are concentrated to 120 ml by evaporation under reduced pressure. Of the The pH is adjusted to 6.5 and the concentrate is added to a column (7 x 50 cm) of XAD-2, which beforehand with 8 liters of 60% aqueous acetone solution, then with 4 liters of demineralized water and finally with 8 liters 5% saline solution has been washed in demineralized water. The sample is raised to the level of the Let the adsorbent bed run off and the column three times with 20 ml of demineralized water each time washed and drained to bed level each time. Then the antibiotic comes with a Flow rate of 40 l / min eluted with 7 l of demineralized water. There will be eight parliamentary groups collected at 200 ml each and then 14 fractions of 400 ml each. The antibiotic activity appears in the Fractions No. 4 to 19, which contain 77% of the bioactivity introduced into the column (determined against Salmonella gallinarum MB-1287); a maximum of the activity is found in fractions no. 6 to 8.

The ratio of the bioactivity of the fractions against Vibrio percolans ATCC 8461 to that is determined HAEA3oo value of these fractions. Those fractions where the ratio is about 250 contain

the antibiotic 890Ai, and those fractions where the ratio is about 31 contain the Antibiotic 890Ai. Accordingly, Fractions Nos. 7, 8 and 9, most of which are antibiotic 890Ai included, united for further processing, the following under the heading »a) Purification of the antibiotic 890Ai «is described. Fractions No. 10, 11, 12 and 13, which are mainly the antibiotic > 890A3 are in turn combined and processed further, as described below under the heading "B) Cleaning the antibiotic 890A3" is described.

a) Purification of the antibiotic 890Ai

κ »The combined fractions Nos. 7, 8 and 9 from the second XAD-2 column, the 480 which were extinguishable by hydroxylamine Absorbance units at 300 nm are concentrated to 100 ml under reduced pressure. The sample is placed in a column with Dowex-1 x 2 (CO (particle size of the resin less than 37 μm, bed dimensions 2.15 x 40 cm) and with 4 l one of 0.075 molar NH4Cl and 0.001 molar NH3 Solution eluted in demineralized water. There are fractions of 12 ml with a flow velocity wedge of 2 mi / min.

The antibiotic activity as determined by analysis against Salmonella gallinarum MB-1287 appears in the Fractions No. 119 to 146 with a maximum in fractions No. 134 to 140. Every third fraction in the In the bioactivity range, the ultraviolet absorbances are determined at 300 nm, 245 nm and 220 nm, and on several fractions on either side of the maximum becomes the extinction which can be canceled by hydroxylamine

; ii measured at 300 nm. Fractions Nos. 129 to 141, which have the highest ratios of by hydroxylamine have extinctable 300 nm absorbance to UV absorbance at 220 nm are combined with one another.

The combined fractions No. 129 to 141 are concentrated to 4 ml and mixed with 0.1 ml of 1 molar ammonia solution offset. The concentrate is placed in a column of Bio-Gel P-2 (37 to 74 μm, bed dimensions 2.2 x 70 cm) and eluted with demineralized water at a flow rate of 1 ml / min.

: j Fractions of 3.1 ml each are collected.

The main maximum of the UV absorbance at 300 nm appears in fractions No. 40 to 47. The fractions Nos. 42, 43 and 44 have the highest ratios of hydroxylamine-erasable 300 nm absorbance to UV absorbance at 220 nm and are combined. These combined groups contain a total of 207 Absorbance units at 300 nm, of which 133.4 are extinct by hydroxylamine, which is a recovery

Mi of 44% corresponds to the extinction introduced into the column, which can be extinguished by hydroxlamine.

The combined Bio-Gel P-2 fractions are diluted with an equal volume of methanol and transferred to a Column with Dowex-1 x 2 (Cl ") (particle sizes below 37 μΐη, bed dimensions 2.15 cm x 40 cm) abandoned, which has previously been brought into equilibrium with 50% by volume methanol. The antibiotic comes with a Flow rate of 2 ml / min with 2 liters of 0.065 molar NH4Cl and 0.001 molar NH3 Solution eluted in 50% methanol. Fractions of 12.5 ml each are collected.

The main maximum of the absorbance at 300 nm appears in fractions no. 59 to 72. Fractions no. 62 to 68, which have the highest A3oo / Aj45 ratios, are pooled. The united factions contain 93.7 extinction units at 300 nm, 76.3 of which can be erased by hydroxylamine. this corresponds to a recovery of 57% of the abandoned, by hydroxylamine extinguishable absorption in the Maximum fractions.

These combined fractions are made 3.4 ml on a rotary evaporator under reduced pressure concentrated and treated with 100 μΙ 1 molar NHi solution. The concentrate adjusted in this way is placed in a column (2.15 cm X 70 cm) of Sephadex G-10 entered previously with 20 ml of saturated saline and 20 ml 12% ammonium chloride solution and then washed with 500 ml of demineralized water.

j> The sample is eluted with demineralized water at a flow rate of 2.15 ml / min. It four fractions of 10.3 ml and then 56 fractions of 3.23 ml are collected. The first maximum of the Ultraviolet absorption at 300 nm containing the desalted antibiotic 890Ai appears in the fractions No. 25 to 47. Fractions No. 28 to 41 are combined and have a pH of 3.5. The pH is adjusted to 7.3 by adding 8 μΐ 1 molar NH3 solution. The combined fractions contain 28 Absorbance units at 300 nm, 18 of which can be erased by hydroxylamine.

2 rn! these pooled fractions are taken as reference samples and the remainder becomes 2.0 ml constricted. The pH of the concentrate is adjusted to 7.4 by adding 0.7 μΐ 1 molar NH3 solution. 50 μΐ of the concentrate are removed and the remainder is frozen in a 14 ml glass ampoule and closed 4.32 mg of solids freeze-dried containing the antibiotic 890Ai and residual salts.

b) Purification of the antibiotic 890As

The fractions no. 10 to 13 obtained from the second XAD-2 column are concentrated to 40 ml and in a column (bed dimensions 2.15 cm x 40 cm) of Dowex-1 x 4 (Cl ") with particle sizes below 37 µm a - (, 1, given. The antibiotic is mixed with 3 l of a 0.075-molar NH4Cl and 0.001-molar NH3 solution in demineralized water eluted at a flow rate of 3 ml / min. There will be factions too 9 ml each collected. The antibiotic activity determined against Vibrio percolans ATCC 8461 appears in the Fractions No. 165 to 234 with a maximum activity in Fractions No. 195 to 201.

Fractions No. 186 to 213 were combined; they contain a total of 472 A3oo units, of which 312 , 0 can be erased by reaction with hydroxylamine.

The combined fractions are concentrated to 4.2 ml in a rotary evaporator under reduced pressure. The concentrate is placed in a column with Bio-Gel P-2 (37 to 74 µm, bed dimensions 2, i 5 x 60 cm). The sample is allowed to drain up to the level of the bed, twice with 1 ml of demineralized

Washed with water and allowed to run down to bed level each time. Then the column moves at a speed of 1 ml / min eluted with demineralized water, collecting fractions of 2.6 ml each.

The antibiotic 890A3 is eluted in fractions Nos. 38 to 48; the maximum activity, determined by the extinction at 300 nm, which can be erased by hydroxylamine, occurs in fraction 41. The parliamentary groups No. 40 to 43 are united; they contain 260 ajoo units, 173 of which can be erased with hydroxylamine.

In order to remove the remaining contamination of the antibiotic 890A3 by the antibiotic 890Ai, the combined desalted fractions No. 40 to 43 with 50 μΐ penicillinase [containing 500,000 units per ml (1000 LU / ml, where the term LU refers to Levy units; 1000 Lu inactivate 500000 Units of penicillin G)] and 0.1 ml of 1 molar Tris-HCl buffer solution (pH 7.5).

The reaction mixture is allowed to stand for 6 hours at 23 ° C. and then 3 ml of demineralized water are used 15 ml of methanol to. This mixture is in a column (2.15 x 44 cm bed dimensions) of Dowex-1 x 2 (Cl ") (particle size below 37 μπι) entered, which is filled in 50% methanol and filled with 21 50% by volume Methanol has been washed. The antibiotic 890Aj is eluted with 2150% methanol, that of sodium chloride 0.05 molar, 0.005 molar in NH4Cl and 0.0001 molar in NH3. Fractions of 9.2 ml each are collected. The main maximum of the UV absorbance, measured at 300 nm, occurs in fractions no. 74 to 88. the Fractions No. 78 to 85 are combined. After driving off the methanol under reduced pressure the total value of extinction units of these fractions at 300 nm is 97.6, of which 87.5 by reaction are extinguishable with hydroxylamine.

The combined fractions Nos. 70 to 85 are collected in a rotary evaporator under reduced pressure concentrated to 3.92 ml, and the concentrate is transferred to a column of Sephadex G-10 (bed dimensions 2.15 x 70 cm) entered, washed with 4 ml of 4-molar ammonia solution and then with 0.15-molar ammonia solution has been equilibrated in demineralized water. After washing twice with The antibiotic 890Aj is released per 1 ml of 0.02 molar NH3 solution at a flow rate of 0.8 ml / min eluted with 0.02 molar NH3 solution. First 49 fractions of 2.45 ml each and then Collect 20 fractions of 3.33 ml each. The main maximum of the absorbance at 300 nm appears in the Fractions No. 35 to 53. Fractions No. 38 to 46, which have the highest A3oo / Aj4s values, are used for the further processing combined. The combined fractions have a total of 65 extinction units 300 nm. The combined fractions are added to a rotary evaporator under reduced pressure Concentrated 2.84 ml, divided into two equal parts and quick-frozen and separately in 14 ml glass ampoules freeze dried. This is how you get the antibiotic 890A3. A sample contains 32.0 A3oo units in 0.88 mg, the other sample contains 31.9 A3oo units in 0.82 mg. The former sample, which contains 32.0 Awo units, shows the following peaks in the NMR analysis:

1.29 (d, J = 6.5); 1.98 (s); 3.42 (d of d, J = 5 and J = 2.4); -4.01-4.28 (m);
3.14 (d of d, J = 5 and J = 9); 3.39 (t, J = 6.5); 3.92 (d of t, J = ~ 4 and J = 6).

In the table above, the 100 MHz NMR signals for the sodium salt of 890A.1 in D2O are relative to DSS specified as internal standard; the chemical shifts are in ppm and the coupling constants given in Hz, multiple values are also given.

Example 9
(Shake flask) production of the antibiotic 890Ai

A tube with a freeze-dried culture of Streptomyces flavogriseus MA-4600a is placed aseptic opened and the contents suspended in a tube, the 1.5 ml of sterile medium A of the following composition contains:

Medium A 10.0 g Yeast extract in λ «
ιυ, υ jt glucose 0.05 mg MgSO ₄ · 7H ₂ O 2.0 ml Phosphate buffer *) 1000 ml distilled water 91.0 g
95.0 g
1000 ml. *) Phosphate powder solution
KH ^ PO,
Na ₂ HPO ₄
distilled water

This suspension is used to transfer a 250 ml Erlenmeyer inoculation flask fitted with three baffles to inoculate, which contains 54 ml of inoculation medium B of the following composition:

Medium B 10.0 g Autolyzed yeast 10.0 g glucose

W)

continuation

MgSO .. ■ 7H ₂ O 0.05 g

Phosphate buffer *) 2.0 ml

distilled water 1000 ml

·) Phosphorus powder solution

KIIjPO ₄ 91.0g

Na ₃ HPO ₄ 95.0 g

distilled water 1000 ml.

The inoculation flask is closed with a cotton plug and placed in a rotary shaker for 30 hours at 28 ± 1 ° C (Stroke 5.1 cm) shaken at 220 rpm.

50 Erlenmeyer production flasks without baffle, each having a capacity of 250 ml and each containing 40 ml production medium C, are each filled with 1 ml of the fermentation liquid obtained from the inoculation flask i> inoculates. The production flasks are closed with cotton plugs.

Medium C 20.0 g Tomato puree 10.0 g Primary yeast 20.0 κ Dextrin 5.0 mg CoCl: 6H: O 1000 ml distilled water

pH adjusted to 7.2 to 7.4 with NaOH.

After inoculation, the production flasks are kept for 3 days at 28 ± IT with shaking in one with :: 0 1 mm working rotary dish machine (stroke 5.1 cm; incubated. The KoIUr, are aul their aimviui Analyzed against standard analysis plates with Vibrio percolans ATCC 8461 using 1.27 cm discs, which are immersed in centrifuged fermentation liquid samples. The samples are 0.05 molar Phosphate buffer solution (pH 7.4) diluted. The results are as follows:

Cultural age, h 72 pH 6.4 Vibrio percolans, 1/100 dilution Inhibition zone, mm 23

890 analysis units / ml 103

The 890A units / m! are determined as described in section »II. Analytical method for determining the 890 analysis units «.

The entire fermentation liquid is mixed in 200 ml portions in polycarbonate bottles for 15 minutes each 9000 rpm centrifuging, 1600 ml of combined supernatant liquids having a strength of Receives 104 units / ml. To this end, 0.5 ml of neutral EDTA solution is added.

The centrifuged fermentation liquid is at a flow rate of 6 to 20 ml / min a column with Dowex-1 x 2 (CP) (150 to 300 μπι, bed dimensions 3.8 x 22 cm) adsorbed. The column is washed with 100 ml of demineralized water and at a flow rate of 6 ml / min eluted with 1 l of demineralized water which contains 50 g of sodium chloride and 0.02 molar of Tris-HCl buffer (pH 7.0) and 25 ^ molar of neutral EDTA. Fractions of 10 ml each are collected.

The antibiotic 890 Ai appears in fractions No. 13 to 81 with a maximum in fractions No. 25 to 33, counting from the first infusion of the salt eluate. The factions No. 24 to 41, which are the highest Bioactivity to A220 ratios are pooled for further processing. The United Fractions contain a total of 29,000 units or 17% of the abandoned bioactivity.

The Dowex eluate is concentrated to 10 ml, the pH is adjusted to 6.5 with dilute hydrochloric acid and the Concentrate in a column with XAD-2 (bed dimensions 3.3 x 36 cm) entered, each with 2 1 60% aqueous acetone, demineralized water and a solution of 5 parts by weight of common salt in 100 Parts of the room have been washed with demineralized water. The sample is at a flow rate eluted at 6 ml / min with demineralized water, collecting fractions from 40 to 260 ml will.

The antibiotic activity appears in fractions No. 6 to 14, which eluted from 220 to 2560 ml of the Sufficient in volume. The maximum is contained in fractions nos. 9 and 10, which range from 370 to 590 ml of des the eluted volume. Fractions No. 9 to 12, which comprise 370 to 1060 ml of the eluted volume, have the highest ratios ΗΑΕΑ.κκ) / Α22ο and are combined for further processing. These fractions have 36,600 units, which is 126% of the apparent abandoned activity.

The combined fractions Nos. 9 to 12 are concentrated to 100 ml, and the concentrate is with a Flow rate of 2 ml / min in a column with Dowex-1 x 4 (Cl ") entered, in which the Adsorbent has particle sizes below 37 μΓη and bed dimensions of 2.2 x 41 cm. The column

it is washed with 50 ml of demineralized water and eluted at a rate of 2 ml / min with a _{solution in demineralized water that is 0.07 molar in sodium chloride, 0.005 molar in Nh 4} Cl and 0.0001 molar in NH3. Starting with the first application of the eluent, 10.8 ml fractions are collected.

The main peak of the activity of the antibiotic 890Ai appears in fractions Nos. 181 to 217 with a Maximum in the fraction No. 198. The fractions No. 186 to 210, the total of 114 absorption units at 300 nm are combined.

The combined fractions are concentrated to 4.0 ml and the pH value by adding 16 μΐ 1 molar Sodium hydroxide solution adjusted to 7.3 The concentrate is placed in a column with Bio-Gel P-2 (37 to 74 μm, bed dimensions 2.15 X 70 cm) and washed three times with 1 ml of demineralized water each time. the Column is eluted with demineralized water at a rate of 0.96 ml / min 3.85 ml fractions collected.

The main peak of antibiotic 890Ai activity appears in fractions Nos. 24 to 44 with a Maximum in fractions # 33 and 34. Fractions # 27 through 38 that had the highest A3oo / A245 ratios have, are combined for freeze-drying. These united fractions contain a total of 72 A3oo units.

To carry out the freeze-drying, the combined fractions are concentrated to 3.0 ml, and the The pH of the concentrate is adjusted to 7.5 by adding 10 ml of 0.1 molar sodium hydroxide solution. The sample is divided into two parts of 1.50 ml each, which are then quick-frozen separately from 14 ml glass ampoules with screw caps and freeze-dried. Each sample contains 1.73 mg of 89OAi, equivalent to 35.8 Ajoo units. 2ft

Agents containing the antibiotics according to the invention can be in various dosage forms, e.g. B. be given in solid or liquid oral dosage form. The antibiotics 890 A; and 890A3 can be used separately or in combination. The agents described below can be prepared in the same way either with the antibiotics 890Ai or 890A3 on their own or with both antibiotics together. The agents can, regardless of whether they are liquid or solid, contain 0.1 to 99% active ingredient per unit dose:>; the preferred range is about 10 to 60%. The agent generally contains about 25 to 1000 mg of active ingredient, based on the total weight of the agent; however, dose levels in the range from about 250 to 1000 mg are generally used. For parenteral administration, the unit dose usually consists of the pure compound in sterile aqueous solution or in the form of a powder intended to be dissolved. Typical blends can be made by the following procedures: Mt

Capsules per capsule

Antibiotic 89 ¹ OAi 400 mg

Lactose, U.S.P., approximately 475 mg each.

In the example above, the active ingredient and diluent become a uniform mixture mixed, which is then filled into hard gelatin capsules by hand or by machine. The mixing and Filling is preferably carried out in a room with a relative humidity of less than 40%.

Tablets per tablet Antibiotic 890Ai 330 mg Calcium phosphate 192 mg Lactose, U.S.P. 190 mg Cornstarch 80 mg Magnesium stearate 8 mg

800 mg.

In the example above, the active ingredient is the calcium phosphate, the lactose and about half of the Corn starch mixed. The mixture is granulated with a 15% corn starch pulp, coarsely sieved and then again sieved through a sieve with a 1.19 mm aperture. After adding the rest of the corn starch and the Magnesium stearate, the mixture is compressed into tablets which have a diameter of about 1.27 cm and weigh 800 mg each.

Another method is to mix the active ingredient with the calcium phosphate, the lactose and the Half of the cornstarch. The mixture is compressed, tablet-like in a high-performance press Mass processed. These are crushed into grains with a diameter of about 1.2 mm. Then sets the rest of the corn starch and the magnesium stearate are added and the mixture is compressed into tablets of about 1.27 cm in diameter and 800 mg in weight

Lyo form (for injection;

per ampoule

Antibiotic 89OAi 250 mg

With water for injections, U.S.P., S ml. filled up

In the above example, the active ingredient is used in the specified ratio in a sufficient amount of water resolved for injections. The solution is filtered through Selas candles or membrane filters to sterilize it. The solution is distributed in sterile ampoules. The ampoules are frozen with their contents, and that Water is removed aseptically by freeze drying. Those containing the sterile dry solid Ampoules are closed aseptically.

To regenerate a solution for parenteral administration, 5 ml of sterile water for injections are added to the contents of an ampoule.

Oral liquid forms

1000 ml each

Antibiotic 890A, 1.0 g Sucrose 600.0 g

Glucose 250.0 g

Sodium benzoate 1.0 g

Concentrated orange oil 0.2 ml With Purified Water, U.S.P., 1000.0 ml. filled up

The sucrose and glucose are dissolved in about 400 ml of water with heating. The solution will be cooled and first mixed with sodium benzoate and then with concentrated orange oil. The solution will be made up to about 900 ml with water, whereupon the antibiotic is added. The solution is obtained by filtering clarified through a coarse filter.

34

Claims (7)

Patent claims: 1. Antibiotic 890Ai, characterized by the following parameters: (a) Saurer Sioli, which migrates to the positive pole during electrophoresis at neutral pH; (b) the sodium salt is in the form in which it is obtained from aqueous solution by freeze-drying, a highly water-soluble, white powder; (C) the ultraviolet absorption spectrum shows an X _mm . at 299.5 nm and an X _m ". at 242 nm; a solution of the sodium salt in water at neutral pH shows an E% of 208 at 300 nm for a pure sample; the ratio of the absorbance values at 300 nm and 245 nm is 4.25, and the ratio A300 / A210 is 1.41 ; more than 92% of the absorbance at 300 nm can be extinguished by reaction with hydroxylamine, and a similar decrease is observed with reaction with cysteine; (d) the circular dichroism spectrum shows a positive maximum at 290.5 nm with a specific ellipticity of 5270 degrees - ml per dm - g, a zero point of ellipticity at 250 nm and a negative minimum at 214 nm with a specific ellipticity of -10 910 Degree · ml per dm · g; (e) 100 MHz NMR signals for the sodium salt in D ₂ O (sodium 2,2-dimethyl-2-silapentane-5-sulfonate as internal standard; chemical shifts in ppm; coupling constants in Hz; apparent multiple values given) : 1.35 (d, J = 6.5); 1.98 (s); 3.63 (d of d, J = 5.2 and J = 9.8); ~ 4.02 to 4.26 (m);
3.18 (d of d, J = ~ 11 and J = 10); 3.41 (t, J = 6); 2.97 (d of t, J = 3.5 and J = 6). 2. A process for the preparation of the antibiotic 890 Ai of claim 1, characterized in that Streptomyces flavogriseus NRRL 8139 or NRRL 8140 is cultured under submerged aerobic conditions in an aqueous nutrient medium containing assimilable C and N sources, the solids are centrifuged from the fermentation liquid or filtered off, adsorbed on a basic anion exchange resin, eluted with aqueous salt solution, .'einigt in a manner known per se and the antibiotic 890 Ai separated by chromatography from the accompanying antibiotic 890A _3. 3. Antibiotic 890A ₃ , characterized by the following parameters: (a) Acid that migrates to the positive pole during electrophoresis at neutral pH; (b) the sodium salt is in the form in which it is obtained from aqueous solution by freeze-drying, a white powder; (c) the ultraviolet absorption spectrum has an A " _mv . at 300.5 nm and an X _m ". at 243 nm; a solution of the sodium salt in water at neutral pH shows an E% of 375 at 300 nm for a pure sample; the ratio of the absorbances at 300 nm and 245 nm is 3.54; more than 90% of the absorption at 300 nm can be extinguished by reaction with hydroxylamine; a similar decrease is observed with the reaction with cysteine; (d) the circular dichroism spectrum shows a positive maximum at 294 nm with a specific ellipticity of 9127 degrees ml per dm g, a zero point of the ellipticity at 249 nm and a specific ellipticity of -15,053 degrees ml per dm g at 220 nm; (e) 100 MHz NMR signals for the sodium salt in DjO (sodium 2,2-dimethyl-2-silapentane-5-sulfonate as internal standard; chemical shifts in ppm; Coupling constants in Hz; apparent multiples specified): 1.29 (d, J = 6.5); 1.98 (s); 3.42 (d of d, J = 5 and J = 2.4); ~ 4.01 to 4.28 (m);
3.14 (d of d, J = 5 and J = 9); 3.39 (t, J = 6.5); 2.92 (d of t, J = ~ 4 and J = 6). 4. A process for the preparation of the antibiotic 890A ₃ of claim 3, characterized in that Streptomyces flavogriseus NRRL 8139 is cultured under submerged aerobic conditions in an aqueous nutrient medium containing assimilable C and N cheeses, the solids are centrifuged or filtered off from the fermentation broth adsorbed on a basic anion exchange resin, eluted with aqueous salt solution, purified in a manner known per se and the antibiotic 890Aj from the accompanying antibiotic 890A | separates chromatographically. 5. Mixture of the antibiotic 890A, of claim 1 and the antibiotic 890A ₃ of claim 3. 6. Process for the preparation of the mixture of antibiotics 890A | and 89OA ₃ of claim 5, characterized in that Streptomyces llavogriseus NRRL 8139 is cultured under submerged aerobic conditions in an aqueous nutrient medium containing assimilable C and N sources, the solids are centrifuged or filtered off from the fermentation broth, they are adsorbed on a basic anion exchange resin, eluted with aqueous salt solution and purified in a manner known per se. 7. Agent containing an antibacterially effective amount of the antibiotic 890Ai of claim 1 or of the antibiotic 890A ₃ of claim 3 or the mixture of claim 5 and / or pharmaceutically acceptable salts thereof and a non-toxic, pharmaceutically acceptable carrier.